Glyceraldehyde-3-phosphate Research Articles

Biliverdin Reductase B Protects Against Primaquine Induced Hemolysis - Identification of a Novel Anti-Oxidant Pathway in Murine Erythrocytes

Background: Biliverdin Reductase B (BlvrB) is the fourth most abundant enzyme in red blood cells (RBCs) and is widely expressed in other tissues. In normal heme catabolism, porphyrin is converted to biliverdin-alpha (by hemoxygenase (HO)) and then reduced to bilirubin-alpha (by biliverdin reductase A (BlvrA)). Bilirubin-alpha is then glucuronidated and excreted. The large body of literature investigating heme metabolism, bilirubin-based pathologies, and the oxidative biology of bilirubin/biliverdin in adults focuses almost exclusively on BlvrA and the alpha isomers of biliverdin and bilirubin. However, porphyrin is a tetrapyrrole with 4 distinct meso-carbon bridges, each of which can be broken to generate a distinct biliverdin isomer (alpha, beta, gamma, or delta). Although HO is exquisitely specific for the alpha-bond (generating only biliverdin-alpha), chemical coupled oxidation of heme yields all four biliverdin isomers. BlvrB (and not BlvrA) can reduce each of non-alpha biliverdin isomers into their corresponding bilirubin isomers (bilirubin-beta is prevalent in neonatal jaundice and kernicterus). BlvrB also reduces flavins and additional quinone metabolites. Despite BlvrB's abundance and evolutionary conservation, its only known function is a subtle effect on hematopoietic lineage fate (megakaryocyte vs. erythroid). BlvrB has no known function in mature RBCs. We hypothesized that BlvrB plays an important antioxidant role that would only become obvious when other redundant anti-oxidant pathways were limited. Methods: A novel BlvrB knockout mouse was generated on a C57BL/6 background. A model of primaquine (PQ) induced oxidative hemolytic anemia in G6PD deficient mice was used as a clinically relevant scenario in which compromised antioxidant biology leads to treatment limiting pharmacotoxicity in RBCs. BlvrB-KO mice were crossed with two strains of mice that had murine G6PD replaced with either the A- deficient variant of human G6PD (hG6PD(A-)) or the non-deficient form of human G6PD (hG6PD(ND)). Four mouse strains (i.e., hG6PD(ND) and hG6PD(A-) mice with or without BlvrB deletion) received either a 4-day course of PQ or control PBS injections. Anemia was monitored by hematocrit (HCT). Reticulocyte count (RET) was monitored by flow cytometry as CD71+, thiazole orange+ erythrocytes. Peripheral blood was subjected to high resolution metabolomics to elucidate biochemical changes. Results: All mouse strains were healthy at baseline, with normal measured hematological parameters (i.e., Hct 40, RET 1.5%). No change in HCT or RET was observed in hG6PD(ND) or BlvrB-KOxhG6PD(ND) mice receiving PQ or in any strain receiving PBS. PQ induced hemolytic anemia in hG6PD(A-) mice, with an average HCT of 32.7 (18% drop) with RET of 3.1%. A significant exacerbation of PQ induced hemolysis was observed in BlvrB-KOxhG6PD(A-) mice that had an average HCT of 22.5 (44% drop) with RET of 5.3%. (n= 11 mice per group, p <0.001 between groups). Blood from PQ-treated hG6PD(A-)xBlvrBKO mice showed defective glycolysis at the level of glyceraldehyde 3-phosphate dehydrogenase, increased levels of multiple long-chain acylcarnitines, altered redox metabolism, and specific increases of hypoxanthine and sphingosine-1-phosphate (S1P). Three separate experiments were performed with similar results. Discussion: These data demonstrate that BlvrB deletion exacerbates oxidative injury and suggest that BlvrB provides a novel and hitherto undescribed antioxidant pathway in RBCs. Because approximately half a billion humans exhibit some form of G6PD deficiency, these data are also directly relevant to a large population of humans, which were modeled using hG6PD(A-) mice. These studies also define the metabolic lesion in peripheral blood. For example, GAPDH is well known to be highly sensitive to inactivation by oxidative damage, resulting in impaired glycolysis. Changes in hypoxanthine and S1P are known to be associated with decreased RBC function and survival. Finally, altered carnitine metabolism is associated with repair of lipid peroxidation in RBCs via the Lands Cycle. Although the mechanism by which BlvrB facilitates antioxidation is not yet clear, we hypothesize it involves redox cycling of non-alpha isomers of biliverdin and bilirubin. However, the mechanisms may be through reduction of flavins and/or other endogenous substrates. Ongoing studies are testing these hypotheses.

Functional and Multi-Omics Signatures of Mitapivat Efficacy upon Activation of Pyruvate Kinase in Red Blood Cells from Patients with Sickle Cell Disease

2,3-diphosphoglycerate (DPG) in red blood cells (RBCs) stabilizes deoxyhemoglobin to promote hemoglobin (Hb) oxygen off-loading and counteract hypoxia. In sickle cell disease (SCD), mutation of glutamate 6 to valine in the beta chain of Hb results in HbS that polymerizes, causing RBCs to sickle upon deoxygenation in the tissues. Elevated DPG promotes sickling by stabilizing HbS fibers. DPG is an intermediate substrate in the glycolytic pathway in which pyruvate kinase (PK) is a rate-limiting enzyme. In a previous Phase 1 study (NCT04000165, Xu et al, 2022), we established proof-of-concept for activating PK as a viable anti-sickling therapeutic approach; mitapivat, an investigational oral allosteric activator of PK, decreased DPG and increased adenosine triphosphate (ATP) levels, improved hematologic and sickling parameters in patients (pts) with SCD (HbSS). Here, we investigated the changes in the metabolome, lipidome and proteome of RBCs from patients with SCD currently enrolled in an open label, Phase 1/2 study (NCT04610866) that evaluates long-term safety and tolerability of mitapivat in pts with SCD. Fifteen pts with HbSS (aged 25 - 57 years, 10 males) were evaluated. All pts started mitapivat at 50 mg twice daily (BID), escalating after 4 wks to 100 mg BID; dose adjustments were performed for safety and tolerability, per PI discretion. At the time of data cutoff (March 23, 2023), all 15 pts completed the core period of 24 wks, 14 pts completed 48 wks ,10 pts completed 72 wks and 6 pts completed 92 wks. Leuko-depleted RBCs (LDRBCs) obtained from fresh whole blood in EDTA at baseline (V1, prior to drug initiation) and various longitudinal time points over the course of the study were flash-frozen in liquid nitrogen and kept frozen at -80°C until analysis. Clinical measurements of DPG, ATP, red cell PK (PKR) protein and activity levels, oxygen affinity (p50) and sickling kinetics (t50) were accompanied by multi-omics (metabolomics, lipidomics, proteomics) characterization of the LDRBCs. In total, 150 (6x12, 4x10, 4x8 and 1x6) timepoint samples were analyzed (Fig 1A). Multi-omics analyses were performed through a stepwise extraction of metabolites, lipids and proteins on 50 µl of packed LDRBC pellets. Extracts were separated via ultra-high-pressure liquid chromatography coupled to mass spectrometry, prior to peak picking and identification of ddMS2 data as previously described (Thomas et al 2020). Statistical analyses were conducted using MetaboAnalyst v 5.0 and Rstudio upon normalization of relative quantitative data to visit 0 levels for each variable. Network analysis and pathway analysis were performed in OmicsNet v 2.0. Mitapivat therapy had significant effects on the metabolome, lipidome and proteome of SCD RBCs. Overall, the temporal trends of omics responses to mitapivat treatment across visits was evident, especially when focusing on the top 50 metabolites/lipids or proteins by time series ANOVA. Specifically, results suggested a positive correlation between mass spectrometry-based quantitation of RBC mitapivat levels and DPG consumption, ATP accumulation, PKR activity (measured via enzymatic assays), and PKR protein levels, with internal consistency between omics data and antibody-based or enzymatic assays (Fig. 1A). The treatment also promoted activation of the Lands cycle, transient elevation of lysophosphatidylcholines and oxylipins, and depletion in free fatty acids within the first 6 months, suggestive of an effect on membrane lipid remodeling. SCD RBCs abnormally retain mitochondria; here, we showed a consistent decrease in the levels of residual mitochondrial proteins in RBCs within 4 wks of mitapivat treatment (Fig. 1B). In keeping with the predicted activation of PKR upon mitapivat treatment, we observed decreases in DPG and increases in ATP, with elevation in glycolytic fluxes, as inferred from longitudinal changes in glycolytic intermediates at the hexose and triose phosphate steps, as well as lactate. Unsupervised analyses of omics data confirmed the specificity of mitapivat on targeting late glycolysis, with glycolytic metabolites ranking as the top correlates to parameters of oxygen affinity (p50) and sickling kinetics (t50) during mitapivat treatment. Multi-omics analysis of RBCs from HbSS patients treated with mitapivat identified benefits for glycolysis, DPG consumption and ATP generation, as well as activation of the Lands cycle.

Integrative Placental Multi-Omics Analysis Reveals Perturbed Pathways and Potential Prognostic Biomarkers in Gestational Hypertension

Gestational hypertension (GH) is a severe complication that occurs after 20 weeks of pregnancy; however, its molecular mechanisms are not yet fully understood. Through this case-control discovery phase study, we aimed to find disease-specific candidate placental microRNAs (miRNAs) and metabolite markers for differentiating GH by integrating next-generation sequencing and metabolomics multi-omics analysis of placenta. Using small RNA sequencing and metabolomics of placental tissues of healthy pregnant (HP, n=24) and GH subjects (n=20), the transcriptome and metabolome were characterized in both groups. The study identified a total of 44 downregulated placental miRNAs which includes three novel, three mature and 38 precursor miRNAs. Six miRNAs including three mature (hsa-miR-181a-5p, hsa-miR-498-5p, and hsa-miR-26b-5p) and three novel (NC_000016.10_1061, NC_000005.10_475, and NC_000001.11_53) were considered for final target prediction and functional annotation. Integrative analysis of differentially expressed miRNAs and metabolites yielded five pathways such as purine, glutathione, glycerophospholipid, inositol phosphate and β-alanine to be significantly perturbed in GH. We present fourteen genes (LPCAT1, LPCAT2, DGKH, PISD, GPAT2, PTEN, SACM1L, PGM2, AMPD3, AK7, AK3, CNDP1, IDH2, and ODC1) and eight metabolites (xanthosine, xanthine, spermine, glycine, CDP-Choline, glyceraldehyde 3-phosphate, β-alanine, and histidine) with potential to distinguish GH and HP. The differential expression of miRNAs, their target genes, altered metabolites and metabolic pathways in GH patients were identified for the first time in our study. Further, the altered miRNAs and metabolites were integrated to build their inter-connectivity network. The findings obtained from our study may be used as a valuable source to further unravel the molecular pathways associated with GH and also for the evaluation of prognostic markers.

Application of serum peptidomics for Parkinson's disease in SNCA-A30P mice

Intraneuronal inclusions of alpha-synuclein (α-synuclein, α-syn) are commonly found in the brain of patients with Parkinson's disease (PD). The pathogenesis of the abundant α-syn protein in the blood has been extensively studied to understand its properties better. In recent years, peptidome analysis has received increasing attention. In this study, we identified and analyzed serum peptides from wild-type (WT) and the (Thy-1)-h[A30P] alpha-synuclein transgenic mice (SNCA-A30P mice) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). One thousand eight hundred fifty-six peptides from 771 proteins were analyzed. Among them, 151 peptides from 107 proteins were significantly differentially expressed. The glycoprotein VI platelet pathway (GP6) was the pathway's most significant differentially expressed signaling pathway. Cleavage sites of the differentially expressed peptides may reflect protease distribution and activity. We selected the most significantly differentially expressed peptide, VGGDPI, and found that it contained cathepsin K (Ctsk) and trypsin-1 cleavage sites, suggesting that Ctsk and trypsin-1 may be key peptidases in PD. α-syn is a protein associated with the pathogenesis of PD. mutations in several genes, including SNCA, which encodes α-syn, are associated with the development of PD. Bioinformatics analysis of the physiological pathways related to SNCA genes and apoptosis genes found the five most markedly up-regulated proteins: formin homology 2 domain-containing 1 (FHOD1), insulin receptor substrate 1(IRS1), TRPM8 channel-associated factor 1 (TCAF1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and interleukin-16 (IL-16). Therefore, the differentially expressed peptides in the five precursor protein domains may be potential bioactive peptides associated with α-syn and apoptosis. This study provides a validated peptidomics profile of SNCA-A30P mice and identifies potentially bioactive peptides linked to α-syn and apoptosis.

Probing milk extracellular vesicles for intestinal delivery of RNA therapies

BackgroundOral delivery remains unattainable for nucleic acid therapies. Many nanoparticle-based drug delivery systems have been investigated for this, but most suffer from poor gut stability, poor mucus diffusion and/or inefficient epithelial uptake. Extracellular vesicles from bovine milk (mEVs) possess desirable characteristics for oral delivery of nucleic acid therapies since they both survive digestion and traverse the intestinal mucosa.ResultsUsing novel tools, we comprehensively examine the intestinal delivery of mEVs, probing whether they could be used as, or inform the design of, nanoparticles for oral nucleic acid therapies. We show that mEVs efficiently translocate across the Caco-2 intestinal model, which is not compromised by treatment with simulated intestinal fluids. For the first time, we also demonstrate transport of mEVs in novel 3D ‘apical-out’ and monolayer-based human intestinal epithelial organoids (IEOs). Importantly, mEVs loaded with small interfering RNA (siRNA) induced (glyceraldehyde 3-phosphate dehydrogenase, GAPDH) gene silencing in macrophages. Using inflammatory bowel disease (IBD) as an example application, we show that administration of anti-tumour necrosis factor alpha (TNFα) siRNA-loaded mEVs reduced inflammation in a IBD rat model.ConclusionsTogether, this work demonstrates that mEVs could either act as natural and safe systems for oral delivery or nucleic acid therapies, or inform the design of synthetic systems for such application.Graphical

AMPK Controls Glycolytic Compensation in Gvhd-Causing T Cells

Background: The heightened metabolic requirements of alloreactive T cells following allogeneic stem cell transplantation represents a promising area for therapeutic intervention. We have shown that during the initiation of acute graft-versus-host-disease (aGVHD), murine T cells upregulate activity of AMP-activated protein kinase (AMPK), a cellular energy sensor that regulates oxidative and mitochondrial metabolism. Further, transfer of allogeneic T cells deficient in AMPK (AMPK KO) increased recipient survival and decreased disease severity while still preserving leukemia clearance. In the current studies, we demonstrate a novel role for AMPK in controlling glycolytic compensation, both in vitro and vivo, and provide a mechanism as to why cells lacking AMPK are unable to perform this vital function. Together, these data highlight the potential clinical utility in targeting metabolic decision-making to curtail T cell-mediated inflammatory responses, with significant therapeutic implications for the prevention and treatment of aGVHD. Methods: AMPK-deficient murine T cells from AMPKa1/a2 dKO C57BL/6J mice were transplanted into allogeneic B6D2F1 recipients and recovered on day 7 post-transplant. To delete AMPK in primary human T cells, T cells were isolated from healthy donors and electroporated with Cas 9 ribonucleoprotein complexes targeting the AMPKa1 locus. Glycolytic compensation was assessed by measuring extracellular acidification rates (ECAR) using the Agilent Seahorse analyzer and a mitochondrial stress kit. Intracellular cytokine expression for IFNgamma was assessed in day 7 murine T cells following re-stimulation with F1 splenocytes in the presence of Brefeldin A. Aldolase A and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) enzymatic assays were run on cell lysates with Biovision-Abcam kits according to manufacturer instructions. To measure GAPDH activity, T cells were positively selected for CD45.1 from day 7 recipient spleens, followed by cell lysis and detection of enzymatic activity. Results: AMPK KO murine T cells recovered on day 7 were unable to mediate a compensatory increase in glycolysis following inhibition of the electron transport chain (Figure 1A), (-6.499±5.156 vs 29.11±7.064, p<0.0001). Human T cells lacking AMPK gave similar results, with glycolytic compensation impaired both in vitro and following human T cell expansion in vivo in a xenogeneic model of GVHD. Immunoprecipitation of proteins from day 7 murine T cells, using an antibody specific to phosphorylated AMPK targets, recovered lower levels of multiple glycolytic enzymes including aldolase, enolase, pyruvate kinase M, and GAPDH (Figure 1B). Functionally, murine T cells lacking AMPK exhibited impaired aldolase activity following anti-CD3/CD28 stimulation and a decrease in GAPDH activity on day 7 post-transplant. Importantly, these changes in glycolysis correlated with an impaired ability of AMPK KO T cells to produce significant amounts of IFNγ upon antigenic re-stimulation with F1 splenocytes. Discussion: Our results demonstrate that in addition to influencing oxidative metabolism, AMPK also plays a role in glycolytic metabolism during GVHD in both murine and human T cells. Further, our data suggest that the more prominent lesion in human T cells post-transplant was in glycolysis , a result supported by the novel observation that a set of glycolysis-related proteins are immunoprecipitated together at much lower rates in the absence of AMPK. These findings suggest a model in which AMPK phosphorylation provides a nidus for important interactions among glycolytic enzymes and that in the absence of AMPK, these interactions are lessened, and glycolysis is consequently reduced. Important to our understanding of GVHD pathogenesis, rates of T cell glycolysis can be directly linked to IFNγ production, where reduced glycolytic activity frees GAPDH to sequester IFNγ transcripts and thereby limit IFNγ protein translation. Altogether, these results document a prominent role for AMPK in controlling T cell glycolytic activity during GVHD development and offer an additional mechanistic explanation for the decreased severity of GVHD seen following transplantation of AMPK KO T cells. Consistently similar results in human xenogeneic T cells provides a further strong rationale for development of AMPK inhibition as a clinically translatable strategy for GVHD prevention and treatment.

Molecular diagnostic assay for pre-harvest detection of Tilletia indica infection in wheat plants.

The current study describes a new diagnostic method for the rapid and accurate detection of Tilletia indica, the pathogen accountable for causing Karnal bunt (KB) disease in wheat. This method uses quantitative real-time polymerase chain reaction (qPCR) and a primer set derived from glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of T. indica to identify the presence of the pathogen. The qPCR assay using this primer set was found highly sensitive, with a limit of detection (LOD) value of 4 pg of T. indica DNA. This level of sensitivity allows for the detection of the pathogen even in cases of different growth stages of wheat, where no visible symptoms of infection on the wheat plants can be seen by naked eyes. The study also validated the qPCR assay on ten different wheat cultivars. Overall, this study presents a valuable molecular tool for rapid, specific and sensitive detection of KB fungus in wheat host. This method has practical applications in disease management, screening of wheat genotypes against KB and can aid in the development of strategies to mitigate the impact of Karnal bunt disease on wheat production.

Luobuma Leaf Spot Disease Caused by Alternaria tenuissima in China.

Luobuma (Apocynum venetum and Poacynum hendersonni) is widely cultivated for environmental conservation, medicinal purposes and the textile industry. In 2018, a severe leaf spot disease that attacked the leaves of Luobuma was observed in plants cultivated in Yuzhong County, Gansu Province, China. Symptoms of the disease appeared as white or off-white spots surrounded by brown margins on the leaves of A. venetum. The spots expanded and covered a large area of the leaf, presenting as "cankers" with progression of the disease, leading to leaf death. The initial symptoms of the disease on P. hendersonni were similar to the symptoms of A. venetum, with a larger disease spot than A. venetum, and the spot was black and thicker. The aim of this study was to identify the fungal species and evaluate the effectiveness of fungicides (hymexazol and zhongshengmycin) against the pathogen in vitro. The fungi species that caused the new disease was identified as Alternaria tenuissima based on the morphological characteristics, pathogenicity tests, and phylogenetic analysis of the internal transcribed spacer (ITS) region, glyceraldehyde 3-phosphate dehydrogenase (gpd), translation elongation factor 1-alpha (TEF) and the histone 3 (H3) gene sequences. The findings showed that hymexazol fungicide can be used to control leaf spot disease. This is the first report on Luobuma leaf spot disease caused by A. tenuissima in China.

103 In Utero Exposure to Maternal Heat Stress Impairs Leydig Cell Development and Function in the Boar

Abstract Due to the nearly exclusive use of artificial insemination, a typical U.S. boar sires ~8,398 progeny per year which is a 99.7% greater gene transmission rate compared with a sow (~23.1 progeny/year). Therefore, the boar has the greatest cumulative impact on swine reproductive performance and drives genetic progress in the herd. However, boar fertility is threatened by climate change. While boar studs are cooled to prevent heat stress, it is cost prohibitive to efficiently cool the six million pregnant sows that are exposed annually, leading to in utero heat stress (IUHS) of developing progeny. The IUHS of the boar reduces the number of pre-spermatogonia in neonatal testes and decimates subsequent semen quality by reducing both total sperm count (24% decrease) and increasing the proportion of abnormal sperm (42% increase). These data suggest that IUHS programs a lifetime of testis dysfunction reducing the breeding potential of boars. However, the biological mechanisms mediating this effect are undefined. The objective of this study was to determine how IUHS affects Leydig cell development and the testicular proteome. Pregnant sows were exposed to either heat stress (28 to 38°C; n = 18) or thermal neutral (17 to 22 °C; n = 18) conditions from day 27 to 57 of gestation. Neonatal boars exposed to IUHS (n = 23) or in utero thermal neutral (IUTN; n = 29) conditions were castrated at 3 days of age. Testes were preserved for histological analysis and proteomics. Leydig cells within IUHS testes were hypertrophic compared with IUTN controls (147.3 ± 1.6 µm2 versus 132.8 ± 1.4 µm2; P < 0.0001). Moreover, IUHS decreased the number of Leydig cells (Per microscopy field) compared with IUTN males (72.7 ± 2.0 Leydig cells/field versus 93.5 ± 1.8 Leydig cells/field). The testicular proteome was interrogated in a subset of animals (n = 8/treatment). A total of 519 proteins (32% of those detected) were differentially abundant between IUHS and IUTN testes (278 proteins down-regulated, 241 proteins up-regulated) including proteins important for Sertoli (e.g., insulin-like growth factor binding protein), Leydig (e.g., CYP19), and germ (e.g., glyceraldehyde 3-phosphate dehydrogenase) cell function. Notably, gene ontology analysis revealed that both steroid & cholesterol biosynthesis pathways were enriched (P < 0.0001) indicating differential expression of proteins needed for testicular steroidogenesis. Notably, CYP19 (enzyme expressed in porcine Leydig cells that converts testosterone to 17β-estradiol) was down-regulated in IUHS testes. Therefore, these data suggest that IUHS leads to Leydig cell dysfunction in boars. The importance of Leydig cells to male reproduction is unequivocal; thus, IUHS represents a mounting threat to male fertility, especially in the face of climate change.

Differential heat-response characteristics of two plastid isoforms of triose phosphate isomerase in tomato.

Heat stress causes dysfunction of the carbon-assimilation metabolism. As a member of Calvin-Benson-Bassham (CBB) cycle, the chloroplast triose phosphate isomerases (TPI) catalyse the interconversion of glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). The tomato (Solanum lycopersicum) genome contains two individual SlTPI genes, Solyc10g054870 and Solyc01g111120, which encode the chloroplast-located proteins SlTPI1 and SlTPI2, respectively. The tpi1 and tpi2 single mutants had no visible phenotypes, but the leaves of their double mutant lines tpi1tpi2 had obviously reduced TPI activity and displayed chlorotic variegation, dysplasic chloroplasts and lower carbon-assimilation efficiency. In addition to altering carbon metabolism, proteomic data showed that the loss of both SlTPI1 and SlTPI2 severely affected photosystem proteins, reducing photosynthetic capacity. None of these phenotypes was evident in the tpi1 or tpi2 single mutants, suggesting that SlTPI1 and SlTPI2 are functionally redundant. However, the two proteins differed in their responses to heat stress; the protein encoded by the heat-induced SlTPI2 showed a higher level of thermotolerance than that encoded by the heat-suppressed SlTPI1. Notably, heat-induced transcription factors, SlWRKY21 and SlHSFA2/7, which negatively regulated SlTPI1 expression and positively regulated SlTPI2 expression, respectively. Our findings thus reveal that SlTPI1 and SlTPI2 have different thermostabilities and expression patterns in response to heat stress, which have the potential to be applied in thermotolerance strategies in crops.

The Carbon Flow Shifts from Primary to Secondary Metabolism during Xylem Vessel Cell Differentiation inArabidopsis thaliana.

Xylem vessel cell differentiation is characterized by the deposition of a secondary cell wall (SCW) containing cellulose, hemicellulose and lignin. VASCULAR-RELATED NAC-DOMAIN7 (VND7), a plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factor, is a master regulator of xylem vessel cell differentiation in Arabidopsis (Arabidopsis thaliana). Previous metabolome analysis using the VND7-inducible system in tobacco BY-2 cells successfully revealed significant quantitative changes in primary metabolites during xylem vessel cell differentiation. However, the flow of primary metabolites is not yet well understood. Here, we performed a metabolomic analysis of VND7-inducible Arabidopsis T87 suspension cells. Capillary electrophoresis-time-of-flight mass spectrometry quantified 57 metabolites, and subsequent data analysis highlighted active changes in the levels of UDP-glucose and phenylalanine, which are building blocks of cellulose and lignin, respectively. In a metabolic flow analysis using stable carbon 13 (13C) isotope, the 13C-labeling ratio specifically increased in 3-phosphoglycerate after 12 h of VND7 induction, followed by an increase in shikimate after 24 h of induction, while the inflow of 13C into lactate from pyruvate was significantly inhibited, indicating an active shift of carbon flow from glycolysis to the shikimate pathway during xylem vessel cell differentiation. In support of this notion, most glycolytic genes involved in the downstream of glyceraldehyde 3-phosphate were downregulated following the induction of xylem vessel cell differentiation, whereas genes for the shikimate pathway and phenylalanine biosynthesis were upregulated. These findings provide evidence for the active shift of carbon flow from primary metabolic pathways to the SCW polymer biosynthetic pathway at specific points during xylem vessel cell differentiation.

Evaluation of potential reference genes in the biting midge Culicoides sonorensis for real-time quantitative PCR analyses

Studies examining differentially expressed genes and gene silencing by RNA interference (RNAi) require a set of stably expressed reference genes for accurate normalization. The biting midge Culicoides sonorensis is an important vector of livestock pathogens and is often used as a model species for biting midge research. Here, we examine the stable expression of six candidate reference genes in C. sonorensis: actin, β-tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein subunit (RPS) 18, vacuolar ATPase subunit A (VhaA), and elongation factor 1-beta (EF1b). Gene expression was assessed under seven conditions, including cells treated with double-stranded RNA (dsRNA), 3rd and 4th instar larvae treated with dsRNA, six developmental stages, four adult female body parts or tissue groups, and females injected with bluetongue virus or vesicular stomatitis virus. Stable gene expression was assessed using RefFinder, NormFinder, geNorm, and BestKeeper. The ranked results for each analysis tool under each condition and a comprehensive ranking for each condition are presented. The data show that optimal reference genes vary between conditions and that just two reference genes were necessary for each condition. These findings provide reference genes for use under these conditions in future studies using real-time quantitative PCR to evaluate gene expression in C. sonorensis.

First report of Anthracnose caused by Colletotrichum theobromicola on Cyclamen persicum in South Carolina, USA.

Florist's cyclamen (Cyclamen persicum) is an herbaceous perennial native to the Mediterranean region and has become an increasingly popular plant around the world. Leaves of these plants are cordate-shaped with varying green and silver patterns. Flowers vary in color from white through different shades of pink, lavender, and red. In September 2022, symptoms of anthracnose including leaf spots and chlorosis, wilting, dieback, and crown and bulb rot were observed on 20 to 30% of approximately 1,000 cyclamen plants in an ornamental production nursery in Sumter County, SC. Tissue samples surrounding the necrotic crowns were excised and sterilized in 10% bleach for 1 min, rinsed in sterile water, placed onto acidified potato dextrose agar (APDA), and incubated at 25°C with 24-h photoperiod. A total of five Colletotrichum isolates, 22-0729-A, 22-0729-B, 22-0729-C, 22-0729-D, and 22-0729-E were obtained by transferring hyphal tips to new plates. The morphology of these five isolates was identical, observed as gray and black with aerial gray-white mycelia and orange-colored spore masses. Conidia (n=50) measured 19.4 ± 5.1 mm (11.7 to 27.1 mm) in length and 5.1 ± 0.8 mm (3.7 to 7.9 mm) in width. Conidia were tapered with rounded ends. Setae and irregular appressoria were infrequently observed in aged cultures (> 60-day-old). These morphological features resembled those of members of the Colletotrichum gloeosporioides species complex (Rojas et al. 2010; Weir et al. 2012). Sequence of the internal transcript spacer (ITS) region of a representative isolate 22-0729-E (GenBank accession No. OQ413075) is 99.8% (532 / 533 nt) and 100% (533 / 533 nt) identical to those of the ex-neotype of Co. theobromicola CBS124945 (JX010294) and the ex-epitype of Co. fragariae (= Co. theobromicola) CBS 142.31 (JX010286), respectively. Its glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene sequence is 99.6% (272 / 273 nt) identical to those of CBS124945 (JX010006) and CBS 142.31 (JX010024). Its actin (ACT) gene sequence shares 99.7% (281 / 282 nt) and 100% (282 / 282 nt) identities with those of CBS124945 (JX009444) and CBS 142.31 (JX009516), respectively. Lastly, its beta-tubulin 2 (TUB2) gene sequence is 99.6% (704 / 707 nt) and 100% (707 / 707 nt) identical to those of CBS124945 (JX010447) and CBS 142.31 (JX010373), respectively. The causal agent causing anthracnose on cyclamen in SC was identified as Co. theobromicola. To confirm the pathogenicity, cyclamen 'Verano Red' plants grown in 2.5-inch pots were used in two pathogenicity assays using different inoculation methods. In the first assay, three plants were inoculated by spraying a conidial suspension (1 × 106 conidia per ml; 30 ml per plant) of isolate 22-0729-E onto the foliage. Three non-inoculated control plants were sprayed with distilled water. All six plants were placed in a plastic tray with wet paper towels. The tray was placed at 22°C for an 8-h photoperiod and covered for 7 days to maintain humidity. Early symptoms including small spots, marginal necrosis, and chlorosis were observed on leaves and flowers 8 days after inoculation (DAI) and the entire aboveground tissues of inoculated plants were blighted 13 to 21 DAI. Non-inoculated plants remained asymptomatic. In the second assay, sterile toothpicks were used to slightly wound the crown and bulb surface of three plants and secure a mycelial APDA plug of isolate 22-0729-E (5×5 mm2) onto each wound (three wounds per plant). Three control plants were wounded in the same manner, while sterile APDA plugs were used in place of mycelial plugs. All six plants were maintained in the same manner as in the first assay. Apparent leaf yellowing and wilting symptoms appeared as early as 13 DAI. On 21 to 28 DAI, severe crown rot on inoculated plants caused the entire foliage to collapse. At least one third of the inner crown and bulb tissues of each inoculated plant were rotten, while those of non-inoculated plants appeared healthy. Each assay was repeated once. Colletotrichum isolates resembling morphological characters of 22-0729-E were recovered from leaves and inner crown tissues of all inoculated plants in both assays, respectively, but not from non-inoculated control plants. Anthracnose diseases on Cyclamen persicum caused by Co. theobromicola (syn. Co. fragariae) have been reported in NC, USA (Lui et al. 2011) and Israel (Sharma et al. 2016). This is the first report of anthracnose on cyclamen in SC, USA. Colletotrichum gloeosporioides (teleomorph Glomerella cingulate) species complex on cyclamen has also been reported in Argentina (Wright et al. 2006), South Africa, and several other U.S. states (Farr and Rossman 2022). However, it remains unknown whether these previous reports in fact attributed to Co. theobromicola due to lack of molecular identification (Weir et al. 2012). Colletotrichum theobromicola can cause diseases on at least 30 other agricultural and horticultural crops such as strawberry, cacao, and boxwood (Farr and Rossman 2022). It may pose a threat to cyclamen in greenhouse and nursery productions. Therefore, management strategies are warranted in the future.

First Report of Anthracnose Caused by Colletotrichum ciggaro on Lonicera macranthoides Hand.-Mazz. in China.

Lonicera macranthoides Hand.-Mazz. is a traditional medicinal plant that is cultivated in Hunan, Yunnan, and Guizhou Provinces in China. In June 2020, a new leaf spot disease was observed on this plant in Longhui County, Shaoyang City, Hunan Province, China, where 14,000 hm2 of L. macranthoides had been planted. About 20% of the total cultivated area exhibited symptoms. Brown spots appeared on the leaves during the early stage and gradually expanded into irregular lesions, which became necrotic and dry. The whole plant withered and died in severe cases. To isolate the pathogen, the infected leaves were collected from different fields and washed with flowing sterile water. The small lesions were then cut and surface sterilized with 75% alcohol for 45 s followed by a 3 min treatment in 3% sodium hypochlorite. The lesions were rinsed five times in sterile water, incubated on potato dextrose agar (PDA) plates and cultured for 3-5 d at 28℃. In total, eleven isolates were obtained, and eight of them were Colletotrichum (isolation frequency 73%). Three representative isolates (JYH1, JYH2, and JYH5) were selected for further study. The fungus grew as circular white colonies, which then became grey. The older colonies looked like cotton and had dense aerial hyphae. The conidia were aseptate, transparent, cylindric, and thin walled, which measured 11.54 to 22.64 × 3.55 to 4.75 μm (n=100). Six genetic regions were amplified and sequenced to further confirm the identity of fungus. They included β-tubulin (TUB2), the internal transcribed spacer (ITS), actin (ACT), chitin synthase (CHS), calmodulin (CAL) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The sequences were submitted to GenBank (ITS: OQ746331; ON954583; OQ746334; TUB2: OQ772278; ON960155; OQ772279; CHS: OQ772280; ON960156; OQ772281; ACT: OQ772282; ON960157; OQ772283; CAL: OQ772284; ON960158; OQ772285; GAPDH: OQ772286; ON960159; OQ772287). The construction of a 6-gene joint phylogenetic tree analysis showed that the three isolates unambiguously clustered with Colletotrichum kahawae subsp. ciggaro strains C022-1 (GenBank: KJ001120.1, KJ001124.1, KJ001109.1, KJ001102.1, KJ001106.1, KJ001113.1) and R019 (GenBank: JN715847.1, KC860023.1, KC859980.1, KC859954.1, KC859972.1, KC859997.1), which was recently reclassified as C. cigarro (Cabral et al. 2020). Three representative isolates were used for the pathogenicity test on the young leaves of the whole plant. A sterile pin was used to prick the leaf epidermis, and 6 × 6 mm mycelial blocks that had been cultured on PDA for 7 d were placed on the leaf wounds. The controls were treated in the same manner except that sterile blocks of PDA were used. There were three replicates per treatment. All the plants used in the experiment were maintained at 28°C in a climate chamber. There was a 12 h photoperiod, and the chamber was kept at 80% relative humidity. Dark brown spots appeared at the sites of inoculation on the plants after 5 days. All the strains that were re-isolated from the lesions shared the same morphological characteristics and had the same type of colonies as the pathogen Colletotrichum ciggaro. Thus, Koch's postulates were fulfilled. C. ciggaro had been shown to cause anthracnose on Olea europaea L. (Weir et al. 2012), Mangifera indica L. (Ismail et al. 2015), Citrus reticulata L. (Perrone et al. 2016) and Areca catechu L. (Zhang et al. 2020). To our knowledge, this is the first report of C. ciggaro causing anthracnose on L. macranthoides in China and worldwide. This research provides a basis for further research to control epidemics of this disease.

Triosephosphate Isomerase: The Crippling Effect of the P168A/I172A Substitution at the Heart of an Enzyme Active Site.

The P168 and I172 side chains sit at the heart of the active site of triosephosphate isomerase (TIM) and play important roles in the catalysis of the isomerization reaction. The phosphodianion of substrate glyceraldehyde 3-phosphate (GAP) drives a conformational change at the TIM that creates a steric interaction with the P168 side chain that is relieved by the movement of P168 that carries the basic E167 side chain into a clamp that consists of the hydrophobic I172 and L232 side chains. The P168A/I172A substitution at TIM from Trypanosoma brucei brucei (TbbTIM) causes a large 120,000-fold decrease in kcat for isomerization of GAP that eliminates most of the difference in the reactivity of TIM compared to the small amine base quinuclidinone for deprotonation of catalyst-bound GAP. The I172A substitution causes a > 2-unit decrease in the pKa of the E167 carboxylic acid in a complex to the intermediate analog PGA, but the P168A substitution at the I172A variant has no further effect on this pKa. The P168A/I172A substitutions cause a 5-fold decrease in Km for the isomerization of GAP from a 0.9 kcal/mol stabilization of the substrate Michaelis complexes. The results show that the P168 and I172 side chains play a dual role in destabilizing the ground-state Michaelis complex to GAP and in promoting stabilization of the transition state for substrate isomerization. This is consistent with an important role for these side chains in an induced fit reaction mechanism [Richard, J. P. (2022) Enabling Role of Ligand-Driven Conformational Changes in Enzyme Evolution. Biochemistry 61, 1533-1542].

Evaluation of the storage longevity, postharvest quality, and sugar metabolism-related gene expression in two lines (N1 and N3) of Chinese cabbage during long-term storage

The quality and taste of ‘Beijing New 3′ (N3) and Beijing New 1′ (N1) Chinese cabbage was assessed using physiological indicators monitored during storage and by transcriptome profiling conducted at 0, 25, and 100 d of storage. Results indicated that N3 had better storage longevity and postharvest quality, relative to N1. Electrolyte leakage values in N3 increased at a slower rate than they did in N1 cabbage during the latter stages of storage (125 days), while ethylene levels and respiratory rates were also lower in N3 cabbage than in N1 cabbage during the entire period of storage. The N3 variety had a higher sugar content (mainly glucose and fructose) during storage than the N1 variety. The expression level of genes involved in sugar metabolism, such as hexokinase (HK), sucrose synthase (SUS), phosphofructokinase (PFK), fructose bisphosphate aldolase (FBA), PCKA (Phosphoenolpyruvate carboxykinase), ALDO (aldose), GAPDH (glyceraldehyde phosphate dehydrogenase), and ENO (Enolase) were higher in N3 than in N1, while the expression level of GPI (Glycosylphosphatidylinositol) and PGM (Phosphoglucomutase) was lower. These results provide valuable information on the quality and taste of Chinese cabbage, especially in relation to sugar metabolism, and indicated that N3 had better postharvest storage properties than N1.

The fructose-bisphosphate, Aldolase A (ALDOA), facilitates DNA-PKcs and ATM kinase activity to regulate DNA double-strand break repair

Glucose metabolism and DNA repair are fundamental cellular processes frequently dysregulated in cancer. In this study, we define a direct role for the glycolytic Aldolase A (ALDOA) protein in DNA double-strand break (DSB) repair. ALDOA is a fructose biphosphate Aldolase that catalyses fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), during glycolysis. Here, we show that upon DNA damage induced by ionising radiation (IR), ALDOA translocates from the cytoplasm into the nucleus, where it partially co-localises with the DNA DSB marker γ-H2AX. DNA damage was shown to be elevated in ALDOA-depleted cells prior to IR and following IR the damage was repaired more slowly. Consistent with this, cells depleted of ALDOA exhibited decreased DNA DSB repair via non-homologous end-joining and homologous recombination. In support of the defective repair observed in its absence, ALDOA was found to associate with the major DSB repair effector kinases, DNA-dependent Protein Kinase (DNA-PK) and Ataxia Telangiectasia Mutated (ATM) and their autophosphorylation was decreased when ALDOA was depleted. Together, these data establish a role for an essential metabolic protein, ALDOA in DNA DSB repair and suggests that targeting ALDOA may enable the concurrent targeting of cancer metabolism and DNA repair to induce tumour cell death.

Effects of simultaneous Rubisco, glyceraldehyde-3-phosphate dehydrogenase, and triosephosphate isomerase overexpression on photosynthesis in rice

ABSTRACT Rubisco overexpression does not lead to substantial photosynthesis improvement. One of the possible causes is the over-accumulation of 3-phosphoglycerate in the Calvin–Benson cycle. We have previously suggested that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI), involved in 3-phosphoglycerate metabolism, have control over the Calvin–Benson cycle metabolism and photosynthesis. In the present study, Rubisco, GADPH, and TPI were simultaneously overexpressed in rice plants through crossing to improve photosynthesis and its N use efficiency. The percentage of N allocated to Rubisco and GAPDH and TPI levels in the transgenic plant increased 1.1-, 4.9-, and 2.1-fold, respectively, compared to those in wild-type plants, showing overexpression of these enzymes. However, there were no marked increases in CO2 assimilation rates at different CO2 levels and capacities of Rubisco carboxylation, regeneration of Rubisco substrate, and triose phosphate use calculated using the CO2 assimilation rates in the transgenic plants. The ratios of these photosynthetic capacities to total leaf-N level tended to be slightly lower in the transgenic plants than those in the wild-type plants, implying a marginal decrease in photosynthetic N use efficiency. In the transgenic rice plants substantially overexpressing GAPDH and TPI, photosynthesis at elevated CO2 levels, which is assumed to be limited factors other than Rubisco, was only slightly improved. Therefore, it is suggested that simultaneous Rubisco, GAPDH, and TPI overexpression do not improve photosynthesis and its N use efficiency, as GAPDH and TPI overexpression do not improve the 3-phosphoglycerate metabolism sufficiently to alleviate its over-accumulation caused by Rubisco overexpression.

Analysis of the stability of the reference genes GAPDH, SDHA and RPL-19 in sheep from a semi-arid region infected by gastrointestinal nematodes

Analyzing the stability of reference genes already described as universal is an important methodology to lead gene expression analysis because different studies have shown that the expression of universal reference genes may vary between experimental treatments. In this sense, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Succinate dehydrogenase complex subunit A (SDHA) and Ribosomal Protein L-19 (RPL-19) reference genes (already described in other studies with sheep from different regions, breeds and infectious agents or in organisms evolutionarily close to sheep) were investigated in the abomasum, small and large intestines of resistant and susceptible crossbred sheep groups to gastrointestinal nematode infections in the Semi-arid region in Northeast of Brazil. The animals were naturally infected to determine the resistance or susceptibility status by counting eggs per gram (EPG) of feces from the gastrointestinal tract after 33 weeks of observations of infection evolution. Relative gene expression was performed by RT-qPCR methodology using Sybr green and relative gene expression stability was tested by different software programs such as REST, BestKeeper, geNorm and Normfinder. Our results showed the susceptible animals had increase in egg counts per gram of feces than resistant animals (p < 0.001), and both groups showed a mixed infection by nematodes of the genus Haemonchus, Trichostrongylus, Oesophagostomum and Trichuris. Furthermore, we show the importance of analyzing different genes in different software programs and the importance to choose ideal reference genes. In this sense, GAPDH was the most stable gene in the abomasum, whereas SDHA was the most stable in the small and large intestines. In addition, we discuss about variables which can interfere in relative expression such as breed, species, climate and tissue. However, utilizing other reference genes already described in other studies with the same and different variables should be performed.

Revert total protein normalization method offers a reliable loading control for mitochondrial samples following TBI

Owing to evidence that mitochondrial dysfunction plays a dominant role in the traumatic brain injury (TBI) pathophysiology, the Western blot (WB) based immunoblotting method is widely employed to identify changes in the mitochondrial protein expressions after neurotrauma. In WB method, the housekeeping proteins (HKPs) expression is routinely used as an internal control for sample normalization. However, the traditionally employed HKPs can be susceptible to complex cascades of TBI pathogenesis, leading to their inconsistent expression. Remarkably, our data illustrated here that mitochondrial HKPs, including Voltage-dependent anion channels (VDAC), Complex-IV, Cytochrome C and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) yielded altered expressions following penetrating TBI (PTBI) as compared to Sham. Therefore, our goal was to identify more precise normalization procedure in WB. Adult male Sprague Dawley rats (N = 6 rats/group) were used to perform PTBI, and the novel REVERT Total Protein (RTP) method was used to quantify mitochondrial protein load consistency between samples at 6 h and 24 h post-injury. Notably, the RTP method displayed superior protein normalization compared to HKPs method with higher sensitivity at both time-points between experimental groups. Our data favors application of RTP based normalization to accurately quantify protein expression where inconsistent HKPs may be evident in neuroscience research.