Phosphoglucose Isomerase Research Articles

Conditioned media from AICAR-treated skeletal muscle cells increases neuronal differentiation of adult neural progenitor cells

Exercise has profound benefits for brain function in animals and humans. In rodents, voluntary wheel running increases the production of new neurons and upregulates neurotrophin levels in the hippocampus, as well as improving synaptic plasticity, memory function and mood. The underlying cellular mechanisms, however, remain unresolved. Recent research indicates that peripheral organs such as skeletal muscle, liver and adipose tissue secrete factors during physical activity that may influence neuronal function. Here we used an in vitro cell assay and proteomic analysis to investigate the effects of proteins secreted from skeletal muscle cells on adult hippocampal neural progenitor cell (aNPC) differentiation. We also sought to identify the relevant molecules driving these effects. Specifically, we treated rat L6 skeletal muscle cells with the AMP-kinase (AMPK) agonist 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) or vehicle (distilled water). We then collected the conditioned media (CM) and fractionated it using high-performance liquid chromatography (HPLC). Treatment of aNPCs with a specific fraction of the AICAR-CM upregulated expression of doublecortin (DCX) and Tuj1, markers of immature neurons. Proteomic analysis of this fraction identified proteins known to be involved in energy metabolism, cell migration, adhesion and neurogenesis. Culturing differentiating aNPCs in the presence of one of the factors, glycolytic enzyme glucose-6-phosphate isomerase (GPI), or AICAR-CM, increased the proportion of neuronal (Tuj1+) and astrocytic, glial fibrillary acidic protein (GFAP+) cells. Our study provides further evidence that proteins secreted from skeletal muscle cells may serve as a critical communication link to the brain through factors that enhance neural differentiation.

Repurposing of an old drug: In vitro and in vivo efficacies of buparvaquone against Echinococcus multilocularis

The metacestode stage of the fox tapeworm Echinococcus multilocularis causes the lethal disease alveolar echinococcosis. Current chemotherapeutic treatment options are based on benzimidazoles (albendazole and mebendazole), which are insufficient and hence alternative drugs are needed. In this study, we screened the 400 compounds of the Medicines for Malaria Venture (MMV) Pathogen Box against E. multilocularis metacestodes. For the screen, we employed the phosphoglucose isomerase (PGI) assay which assesses drug-induced damage on metacestodes, and identified ten new compounds with activity against the parasite. The anti-theilerial drug MMV689480 (buparvaquone) and MMV671636 (ELQ-400) were the most promising compounds, with an IC50 of 2.87 μM and 0.02 μM respectively against in vitro cultured E. multilocularis metacestodes. Both drugs suggested a therapeutic window based on their cytotoxicity against mammalian cells. Transmission electron microscopy revealed that treatment with buparvaquone impaired parasite mitochondria early on and additional tests showed that buparvaquone had a reduced activity under anaerobic conditions. Furthermore, we established a system to assess mitochondrial respiration in isolated E. multilocularis cells in real time using the Seahorse XFp Analyzer and demonstrated inhibition of the cytochrome bc1 complex by buparvaquone. Mice with secondary alveolar echinococcosis were treated with buparvaquone (100 mg/kg per dose, three doses per week, four weeks of treatment), but the drug failed to reduce the parasite burden in vivo. Future studies will reveal whether improved formulations of buparvaquone could increase its effectivity.

Molecular diagnosis of unexplained haemolytic anaemia using targeted next-generation sequencing panel revealed (p.Ala337Thr) novel mutation in GPI gene in two Indian patients

Glucose-6-phosphate isomerase (GPI) deficiency is an autosomal recessive genetic disorder causing congenital haemolytic anaemia (CHA). Diagnosis of GPI deficiency by the biochemical method is unpredicted. Molecular diagnosis by identifying genetic...

Proteomic and metabolomic analysis of the cellular biomarkers related to inhibitors tolerance in Zymomonas mobilis ZM4

BackgroundToxic compounds present in both the hydrolysate and pyrolysate of lignocellulosic biomass severely hinder the further conversion of lignocellulose-derived fermentable sugars into useful chemicals by common biocatalysts like Zymomonas mobilis, which has remarkable advantages over yeast. Although the extra detoxification treatment prior to fermentation process can help biocatalysts to eliminate the inhibitory environment, it is not environment friendly and cost effective for industrial application. As also reported by previous studies, an ideal and holistic approach to solve this issue is to develop microbial strains with inhibitor tolerance. However, previously engineered strains had the limitation that they could not cope well with the synergistic effect of multiple inhibitors as they are resistant only to a single inhibitor. Hence, understanding the universal cellular responses of Z. mobilis to various inhibitors may guide the designing of rational strategies to obtain more robust engineered strains for biofuel production from lignocellulosic biomass.ResultsQuantitative proteomics and metabolomics approaches were used to determine the cellular responses of Z. mobilis ZM4 to representative biomass-derived inhibitors like formic acid, acetic acid, furfural, 5-hydroxymethylfurfural, and phenol. The differentially expressed proteins identified under the challenge of single and combined inhibitors were involved in cell wall/membrane biogenesis, energy production, DNA replication, DNA recombination, DNA repair, DNA transcription, RNA translation, posttranslational modification, biosynthesis of amino acids, central carbon metabolism, etc. Metabolomics analysis showed that the up- or down-regulation pattern of metabolites was changed consistently with that of relevant proteins.ConclusionFifteen up-regulated proteins (e.g., Isopropylmalate isomerase LeuC, transcription-repair-coupling factor Mfd, and phosphoglucose isomerase PGI) and thirteen down-regulated proteins (e.g., TonB-dependent transporter ZMO1522, transcription termination factor Rho, and S1/P1 nuclease ZMO0127) were identified as candidate proteins related to all the stress conditions, implying that these proteins are potential biomarkers for the improvement of Z. mobilis ZM4 to resist complex biomass-derived inhibitors. These data can be used to generate a database of inhibitor-tolerance biomarkers, which could provide a basis for engineering Z. mobilis that would be able to grow in the presence of multiple inhibitors and directly ferment the biomass-derived sugars into biofuels.

Higher autocrine motility factor/glucose-6-phosphate isomerase expression is associated with tumorigenesis and poorer prognosis in gastric cancer.

BackgroundGlucose-6-phosphate isomerase (GPI) is a glycolytic-related enzyme that inter-converts glucose-6-phosphate and fructose-6-phosphate in the cytoplasm. This protein is also secreted into the extracellular matrix by cancer cells and is, therefore, also called autocrine motility factor (AMF).MethodsTo clarify the roles of AMF/GPI in gastric cancer (GC), we collected 335 GC tissues and the corresponding adjacent noncancerous tissues, performed immunohistochemical studies, and analyzed the relationship between AMF/GPI expression and the patients’ clinicopathologic features.ResultsAMF/GPI expression was found to be significantly higher in the GC group than in the corresponding noncancerous tissue group (P<0.001). Additionally, AMF/GPI expression positively associated with a higher TNM stage and poorer prognosis in patients. Through Kaplan–Meier analysis and according to the Oncomine database, we found that AMF/GPI was overexpressed in GC tissues compared to normal mucosa, and the patients with higher AMF/GPI expression had poorer outcomes. We used AMF/GPI-silenced GC cell lines to observe how changes in AMP/GPI affect cellular phenotypes. AMF/GPI knockdown suppressed proliferation, migration, invasion, and glycolysis, and induced apoptosis in GC cells.ConclusionThese findings suggest that AMF/GPI overexpression is involved in carcinogenesis and promotes the aggressive phenotypes of GC cells.

Discovery and Functional Characterization of a Yeast Sugar Alcohol Phosphatase.

Sugar alcohols (polyols) exist widely in nature. While some specific sugar alcohol phosphatases are known, there is no known phosphatase for some important sugar alcohols (e.g., sorbitol-6-phosphate). Using liquid chromatography-mass spectrometry-based metabolomics, we screened yeast strains with putative phosphatases of unknown function deleted. We show that the yeast gene YNL010W, which has close homologues in all fungi species and some plants, encodes a sugar alcohol phosphatase. We term this enzyme, which hydrolyzes sorbitol-6-phosphate, ribitol-5-phosphate, and (d)-glycerol-3-phosphate, polyol phosphatase 1 or PYP1. Polyol phosphates are structural analogs of the enediol intermediate of phosphoglucose isomerase (Pgi). We find that sorbitol-6-phosphate and ribitol-5-phosphate inhibit Pgi and that Pyp1 activity is important for yeast to maintain Pgi activity in the presence of environmental sugar alcohols. Pyp1 expression is strongly positively correlated with yeast growth rate, presumably because faster growth requires greater glycolytic and accordingly Pgi flux. Thus, yeast express the previously uncharacterized enzyme Pyp1 to prevent inhibition of glycolysis by sugar alcohol phosphates. Pyp1 may be useful for engineering sugar alcohol production.

Changes in secondary metabolites, organic acids and soluble sugars during the development of plum fruit cv. 'Furongli' (Prunus salicina Lindl).

Organic acids, sugars and pigments are key components that determine the taste and flavor of plum fruit. However, metabolism of organic acid and sugar is not fully understood during the development of plum fruit cv. 'Furongli'. Mature fruit of 'Furongli' has the highest content of anthocyanins and the lowest content of total phenol compounds and flavonoids. Malate is the predominant organic acid anion in 'Furongli' fruit, followed by citrate and isocitrate. Glucose was the predominant sugar form, followed by fructose and sucrose. Correlation analysis indicated that malate content increased with increasing phosphoenolpyruvate carboxylase (PEPC) activity and decreasing nicotinamide adenine dinucleotide-malate dehydrogenase (NAD-MDH) activity. Citrate and isocitrate content increased with increasing PEPC and aconitase (ACO) activities, respectively. Both acid invertase and neutral invertase had higher activities at the early stage than later stage of fruit development. Fructose content decreased with increasing phosphoglucoisomerase (PGI) activity, whereas glucose content increased with decreasing hexokinase (HK) activity. Dynamics in organic acid anions were not solely controlled by a single enzyme but regulated by the integrated activity of enzymes such as nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME), NAD-ME, PEPC, ACO and NADP-isocitrate dehydrogenase. Sugar metabolism enzymes such as PGI, invertase and HK may play vital roles in the regulation of individual sugar metabolic processes. © 2018 Society of Chemical Industry.

Inhibition of Glycolysis Reduces Disease Severity in an Autoimmune Model of Rheumatoid Arthritis.

The K/BxN mouse is a spontaneous model of arthritis driven by T cell receptor transgenic CD4+ T cells from the KRN strain that are activated by glucose-6-phosphate isomerase (GPI) peptides presented by the H-2g7 allele from the NOD strain. It is a model of autoimmune seropositive arthritis because the production of anti-GPI IgG is necessary and sufficient for joint pathology. The production of high levels of anti-GPI IgG requires on the expansion of CD4+ follicular helper T (Tfh) cells. The metabolic requirements of this expansion have never been characterized. Based on the therapeutic effects of the combination of metformin and 2-deoxyglucose (2DG) in lupus models that normalized the expansion of effector CD4+ T cells. We showed that the CD4+ T cells and to a lesser extent, the B cells from K/BxN mice are more metabolically active than the KRN controls. Accordingly, preventive inhibition of glycolysis with 2DG significantly reduced joint inflammation and the activation of both adaptive and innate immune cells, as well as the production of pathogenic autoantibodies. However, contrary to the lupus-prone mice, the addition of metformin had little beneficial effect, suggesting that glycolysis is the major driver of immune activation in this model. We propose that K/BxN mice are another model in which autoreactive Tfh cells are highly glycolytic and that their function can be limited by inhibiting glucose metabolism.

Plastidial Phosphoglucose Isomerase Is an Important Determinant of Seed Yield through Its Involvement in Gibberellin-Mediated Reproductive Development and Storage Reserve Biosynthesis in Arabidopsis.

The plastid-localized phosphoglucose isomerase isoform PGI1 is an important determinant of growth in Arabidopsis thaliana, likely due to its involvement in the biosynthesis of plastidial isoprenoid-derived hormones. Here, we investigated whether PGI1 also influences seed yields. PGI1 is strongly expressed in maturing seed embryos and vascular tissues. PGI1-null pgi1-2 plants had ∼60% lower seed yields than wild-type plants, with reduced numbers of inflorescences and thus fewer siliques and seeds per plant. These traits were associated with low bioactive gibberellin (GA) contents. Accordingly, wild-type phenotypes were restored by exogenous GA application. pgi1-2 seeds were lighter and accumulated ∼50% less fatty acids (FAs) and ∼35% less protein than wild-type seeds. Seeds of cytokinin-deficient plants overexpressing CYTOKININ OXIDASE/DEHYDROGENASE1 (35S:AtCKX1) and GA-deficient ga20ox1 ga20ox2 mutants did not accumulate low levels of FAs, and exogenous application of the cytokinin 6-benzylaminopurine and GAs did not rescue the reduced weight and FA content of pgi1-2 seeds. Seeds from reciprocal crosses between pgi1-2 and wild-type plants accumulated wild-type levels of FAs and proteins. Therefore, PGI1 is an important determinant of Arabidopsis seed yield due to its involvement in two processes: GA-mediated reproductive development and the metabolic conversion of plastidial glucose-6-phosphate to storage reserves in the embryo.

Immunophenotype and Transcriptome Profile of Patients With Multiple Sclerosis Treated With Fingolimod: Setting Up a Model for Prediction of Response in a 2-Year Translational Study.

Fingolimod is a functional sphingosine-1-phosphate antagonist approved for the treatment of multiple sclerosis (MS). Fingolimod affects lymphocyte subpopulations and regulates gene expression in the lymphocyte transcriptome. Translational studies are necessary to identify cellular and molecular biomarkers that might be used to predict the clinical response to the drug. In MS patients, we aimed to clarify the differential effects of fingolimod on T, B, and natural killer (NK) cell subsets and to identify differentially expressed genes in responders and non-responders (NRs) to treatment. Samples were obtained from relapsing-remitting multiple sclerosis patients before and 6 months after starting fingolimod. Forty-eight lymphocyte subpopulations were measured by flow cytometry based on surface and intracellular marker analysis. Transcriptome sequencing by next-generation technologies was used to define the gene expression profiling in lymphocytes at the same time points. NEDA-3 (no evidence of disease activity) and NEDA-4 scores were measured for all patients at 1 and 2 years after beginning fingolimod treatment to investigate an association with cellular and molecular characteristics. Fingolimod affects practically all lymphocyte subpopulations and exerts a strong effect on genetic transcription switching toward an anti-inflammatory and antioxidant response. Fingolimod induces a differential effect in lymphocyte subpopulations after 6 months of treatment in responder and NR patients. Patients who achieved a good response to the drug compared to NR patients exhibited higher percentages of NK bright cells and plasmablasts, higher levels of FOXP3, glucose phosphate isomerase, lower levels of FCRL1, and lower Expanded Disability Status Scale at baseline. The combination of these possible markers enabled us to build a probabilistic linear model to predict the clinical response to fingolimod. MS patients responsive to fingolimod exhibit a recognizable distribution of lymphocyte subpopulations and a different pretreatment gene expression signature that might be useful as a biomarker.

The secreted glycolytic enzyme GPI/AMF stimulates glioblastoma cell migration and invasion in an autocrine fashion but can have anti-proliferative effects.

Aerobic glycolysis confers several advantages to tumor cells, including shunting of metabolites into anabolic pathways. In glioblastoma cells, hypoxia induces a flux shift from the pentose phosphate pathway toward glycolysis and a switch from proliferation to migration. The mechanistic link between glycolysis and migration is poorly understood. Since glucose-6-phosphate isomerase (GPI) is identical to the secreted cytokine autocrine motility factor (AMF), we investigated whether GPI/AMF regulates glioblastoma cell invasion. The expression and hypoxic regulation of GPI/AMF and its receptor AMFR were analyzed in glioblastoma tissue and cell lines. Functional effects were studied in vitro and in xenograft models. High GPI/AMF expression in glioblastomas was found to be associated with a worse patient prognosis, and levels were highest in hypoxic pseudopalisades. Hypoxia upregulated both GPI/AMF and AMFR expression as well as GPI/AMF secretion in vitro. GPI/AMF stimulated cell migration in an autocrine fashion, and GPI/AMF expression was upregulated in migratory cells but reduced in rapidly proliferating cells. Knockdown or inhibition of GPI/AMF reduced glioblastoma cell migration but in part stimulated proliferation. In a highly invasive orthotopic glioblastoma model, GPI/AMF knockdown reduced tumor cell invasion but did not prolong survival. In a highly proliferative model, knockdown tumors were even larger and more proliferative than controls; however, perivascular invasion, provoked by simultaneous bevacizumab treatment, was reduced. GPI/AMF is a potent motogen for glioblastoma cells, explaining in part the association between glycolysis and migration. Targeting GPI/AMF is, however, problematic, since beneficial anti-invasive effects may be outweighed by unintended mitogenic effects. 1.Increased glycolysis is linked with increased cell migration and invasion in glioblastoma cells. 2.The glycolysis enzyme GPI/AMF may serve as a target for antimetabolic and anti-invasive therapy. 3.Despite reducing tumor invasion, GPI/AMF targeting may have unwanted growth stimulatory effects.

Biostimulant Potential of Humic Acids Extracted From an Amendment Obtained via Combination of Olive Mill Wastewaters (OMW) and a Pre-treated Organic Material Derived From Municipal Solid Waste (MSW).

Olive mill wastewaters (OMW) contain significant levels of phenolic compounds with antimicrobial/phytotoxic activity and high amounts of undecomposed organic matter that may exert negative effects on soil biology. Among OMW detoxification techniques, those focusing on oxidative degradation of phenolic compounds are relevant. The composting (bio-oxidation) process in particular, exploits exothermic oxidation reactions by microorganisms to transform the organic matrix of OMW into an amendment biologically stable and feasible to use in agriculture. This process consists of an active phase during which organic compounds are rapidly decomposed, and a curing phase characterized by a slow breakdown of the remaining materials with the formation of humic substances (HS) as by-products. In this study, bio-oxidation of OMW was performed using a pre-treated organic material derived from municipal solid waste (MSW). The obtained amendment (OMWF) was stable and in accordance with the legislative parameters of mixed organic amendments. HS were then extracted from OMWF and MSW (control amendment, Amd-C), and differences in structural properties of their humic acid (HA) fraction were highlighted via spectroscopy (Fourier Transform Infrared) and Dynamic Light Scattering. To assay a potential use of HA as biostimulants for crops, 12-day old Zea Mays L. plants were supplied with HA at 0.5 mg and 1 mg C L-1 for 2 days. HA from both amendments increased plant growth, but HA from OMWF was more effective at both dosages (plus 35–37%). Also, HA from OMWF enhanced both nitrogen assimilation and glycolysis by increasing the activity of nitrate reductase (∼1.8–1.9 fold), phosphoglucose isomerase (PGI) (∼1.8–2 fold) and pyruvate kinase (PK) (∼1.5–1.8 fold), while HA from Amd-C targeted glycolysis preferentially. HA from OMWF, however, significantly stimulated plant nutrition only at lower dosage, perhaps because certain undetermined compounds from detoxified OMW and incorporated in HA altered the root membrane permeability, thus preventing the increase of nutrient uptake. Conversely, HA from Amd-C increased nutrient accumulation in maize at both dosages. In conclusion, our results indicate that the amendment obtained via OMW composting using MSW had a reduced pollution load in terms of phenolic compounds, and HA extracted from OMWF could be used as valuable biostimulants during maize cultivation.

Differential Modulation of Gene Expression Encoding Hepatic and Renal Xenobiotic Metabolizing Enzymes by an Aspalathin-Enriched Rooibos Extract and Aspalathin.

Modulation of the expression of hepatic and renal genes encoding xenobiotic metabolizing enzymes by an aspalathin-enriched green rooibos (Aspalathus linearis) extract (GRE) was investigated in the liver and kidneys of F344 rats following dietary exposure of 28 d, as well as selected xenobiotic metabolizing genes in rat primary hepatocytes. In the liver, GRE upregulated genes (p < 0.05) encoding aldehyde dehydrogenase, glucose phosphate isomerase, and cytochrome P450 while 17β-hydroxysteroid dehydrogenase 2 (Hsd17β2) was downregulated. In primary hepatocytes, GRE lacked any effect, while aspalathin downregulated Hsd17β2, mimicking the effect of GRE in vivo, and upregulated catechol-O-methyl transferase and marginally (p < 0.1) cytochrome P450 2e1. In the kidneys, GRE upregulated (p < 0.05) genes encoding the phase II xenobiotic metabolism enzymes, glutathione-S-transferase mµ and microsomal glutathione-S-transferase, while downregulating genes encoding the ATP binding cassette transporter, cytochrome P450, gamma glutamyltransferase 1, and N-acetyltransferase 1. Differential modulation of the expression of xenobiotic metabolizing genes in vivo and in vitro by GRE is dose-related, duration of exposure, the tissue type, and interactions between specific polyphenol and/or combinations thereof. Aspalathin is likely to be responsible for the downregulation of estradiol and testosterone catabolism by GRE in the liver. The differential gene expression by GRE in the liver and kidneys could, depending on the duration exposure and dose utilized, determine the safe use of such an extract in humans for specific health and/or disease outcomes.

Alterations in the liver of intrauterine growth retarded piglets may predispose to development of insulin resistance and obesity in later life.

Intrauterine growth retardation (IUGR) leads to increased predisposition to metabolic syndrome in adult life but the mechanisms remain obscure. Considering a significant number of functional similarities, IUGR piglets appear to be a good model to study the development of this syndrome in humans. The aim of the present study was to investigate the ultrastructure and proteomic profile of the liver in IUGR pig neonates to discover early markers of predisposition to obesity and insulin resistance. In our study intestine and liver tissue samples were investigated in 7 day old IUGR and normal body weight (NBW) littermate piglets using histometry, mass spectrometry, in-tissue cytometry analysis and confocal microscopy. Compared to NBW, the liver in IUGR neonates was characterized by a significantly enhanced ratio of Kupffer cells to hepatocytes and insulin receptor abundance as well as higher percentages of cells expressing receptors for adipokines (resistin and adiponectin), increased expression of TNF-α (as marker of inflammation), and increased expression of insulin receptor and uncoupling protein 3 (UCP3). Moreover, NBW and IUGR differed in proteomic profile, including protein metabolism (proteasomes, cathepsin D, phermitin, phosphoglucomutase), carbohydrate metabolism (hexokinase 1, phosphoglucokinase, galactokinase, aldolase B, glucose-6-phosphate isomerase), oxidative stress and chromatin organization and DNA uptake (histones, lamin a/c). Reduction of hepatocyte numbers concomitant with significant modifications of expression of key hormones and enzymes for protein and carbohydrate metabolism in IUGR neonates may predispose to insulin resistance and obesity in adult life.

Re-engineering of an Escherichia coli K-12 strain for the efficient production of recombinant human Interferon Gamma

The Escherichia coli phosphoglucose isomerase (pgi) mutant strain GALG20 was developed previously from wild-type K12 strain MG1655 for increased plasmid yield. To investigate the potential effects of the pgi deletion/higher plasmid levels on recombinant human Interferon Gamma (IFN-γ) production, a detailed network of the central metabolic pathway (100 metabolites, 114 reactions) of GALG20 and MG1655 was constructed. Elementary mode analysis (EMA) was then performed to compare the phenotypic spaces of both the strains and to check the effect of the pgi deletion on flux efficiency of each metabolic reaction. The results suggested that pgi deletion increases amino acid biosynthesis and flux efficiency towards IFN-γ synthesis by 11%. To further confirm the qualitative prediction that the pgi mutation favours recombinant human IFN-γ expression, GALG20 and MG1655 were lysogenised, transformed with a plasmid coding for IFN-γ and tested alongside with BL21(DE3) for their expression capabilities in shake flask experiments using complex media. IFN-γ gene expression was analysed by quantifying plasmid and mRNA copy number per cell and IFN-γ protein production level. Specific IFN-γ yields confirmed the in silico metabolic network predictions, with GALG20(DE3) producing 3.0-fold and 1.5-fold more IFN-γ as compared to MG1655(DE3) and BL21(DE3), respectively. Most of the total IFN-γ was expressed as inclusion bodies across the three strains: 95% in GALG20(DE3), 97% in BL21(DE3) and 72% in MG1655(DE3). The copy number of mRNA coding for IFN-γ was found to be higher in GALG20(DE3) as compared to the other two strains. Overall, these findings show that GALG20(DE3) has the potential to become an excellent protein expression strain.

A High-throughput workflow for CRISPR/Cas9 mediated combinatorial promoter replacements and phenotype characterization in yeast.

Due to the rapidly increasing sequence information on gene variants generated by evolution and our improved abilities to engineer novel biological activities, microbial cells can be evolved for the production of a growing spectrum of compounds. For high productivity, efficient carbon channeling towards the end product is a key element. In large scale production systems the genetic modifications that ensure optimal performance cannot be dependent on plasmid-based regulators, but need to be engineered stably into the host genome. Here we describe a CRISPR/Cas9 mediated high-throughput workflow for combinatorial and multiplexed replacement of native promoters with synthetic promoters and the following high-throughput phenotype characterization in the yeast Saccharomyces cerevisiae. The workflow is demonstrated with three central metabolic genes, ZWF1, PGI1 and TKL1 encoding a glucose-6-phosphate dehydrogenase, phosphoglucose isomerase and transketolase, respectively. The synthetic promoter donor DNA libraries were generated by PCR and transformed to yeast cells. A 50% efficiency was achieved for simultaneous replacement at three individual loci using short 60-bp flanking homology sequences in the donor promoters. Phenotypic strain characterization was validated and demonstrated using liquid handling automation and 150 µl cultivation volume in 96-well plate format. The established workflow offers a robust platform for automated engineering and improvement of yeast strains.

Comparative transcriptome sequencing of the intestine reveals differentially expressed genes in Pelteobagrus vachellii

To better understand the molecular mechanisms underlying digestion and absorption in the intestine of the darkbarbel catfish (Pelteobagrus vachellii), we studied the intestinal transcriptome between day and night-time. In this study, two triplicate groups of P. vachellii (1.05 ± 0.36 g) were fed either at 08:00 (D24 group), control) or 20:00 (W24 group) with the same amount of commercial feed. A significant increase of the apparent digestibility (AD) of protein and lipid was observed in the night group (p < 0.05). Comparison of gene expression levels revealed that 1,125 genes were significantly up-regulated and 364 genes were significantly down-regulated in the night group. Among them, 54 digestion- and absorption-related genes, apolipoprotein A-I, trypsin 1b, solute carrier family 26 member 6 b, sodium/glucose cotransporter 1, cholecystokinin B, maltase-glucoamylase, diacylglycerol O-acyltransferase 2, ATP-binding cassette subfamily A member 1-like and phospholipase A2, etc., were up-regulated. Additionally, in the night group, 18 carbohydrate metabolism-related genes, 17 protein metabolism-related genes and 16 lipid metabolism-related genes were up-regulated, including fatty acid-binding protein, diacylglycerol O-acyltransferase 2, adipose triglyceride lipase, phospholipase A2, glucose phosphate isomerase, glycogen phosphorylase, arginase-2, d-amino-acid oxidase, glutamate dehydrogenase, glycine n-acyltransferase, tryptophan 2,3-dioxygenase, kynureninase, cysteine dioxygenase type 1 and glycine dehydrogenase. The results indicated that a shift in feeding time from day to night may improve the digestion and absorption of proteins and lipids. Moreover, the amino acid metabolism, triglyceride synthesis, gluconeogenesis may be also influenced by the shifted feeding time.

Use of Laser Assisted Optical Rotational Cell Analyzer (LoRRca MaxSis) in the Diagnosis of RBC Membrane Disorders, Enzyme Defects, and Congenital Dyserythropoietic Anemias: A Monocentric Study on 202 Patients.

Chronic hemolytic anemias are a group of heterogeneous diseases mainly due to abnormalities of red cell (RBC) membrane and metabolism. The more common RBC membrane disorders, classified on the basis of blood smear morphology, are hereditary spherocytosis (HS), elliptocytosis, and hereditary stomatocytoses (HSt). Among RBC enzymopathies, the most frequent is pyruvate kinase (PK) deficiency, followed by glucose-6-phosphate isomerase, pyrimidine 5′ nucleotidase P5′N, and other rare enzymes defects. Because of the rarity and heterogeneity of these diseases, diagnosis may be often challenging despite the availability of a variety of laboratory tests. The ektacytometer laser-assisted optical rotational cell analyser (LoRRca MaxSis), able to assess the RBC deformability in osmotic gradient conditions (Osmoscan analysis), is a useful diagnostic tool for RBC membrane disorders and in particular for the identification of hereditary stomatocytosis. Few data are so far available in other hemolytic anemias. We evaluated the diagnostic power of LoRRca MaxSis in a large series of 140 patients affected by RBC membrane disorders, 37 by enzymopathies, and 16 by congenital diserythropoietic anemia type II. Moreover, nine patients with paroxysmal nocturnal hemoglobinuria (PNH) were also investigated. All the hereditary spherocytoses, regardless the biochemical defect, showed altered Osmoscan curves, with a decreased Elongation Index (EI) max and right shifted Omin; hereditary elliptocytosis (HE) displayed a trapezoidal curve and decreased EImax. Dehydrated hereditary stomatocytosis (DHSt) caused by PIEZO1 mutations was characterized by left-shifted curve, whereas KCNN4 mutations were associated with a normal curve. Congenital diserythropoietic anemia type II and RBC enzymopathies had Osmoscan curve within the normal range except for glucosephosphate isomerase (GPI) deficient cases who displayed an enlarged curve associated with significantly increased Ohyper, offering a new diagnostic tool for this rare enzyme defect. The Osmoscan analysis performed by LoRRca MaxSis represents a useful and feasible first step screening test for specialized centers involved in the diagnosis of hemolytic anemias. However, the results should be interpreted by caution because different factors (i.e., splenectomy or coexistent diseases) may interfere with the analysis; additional tests or molecular investigations are therefore needed to confirm the diagnosis.

Changes in respiration and structure of non-heading Chinese cabbage seeds during gradual artificial aging

Abstract The aging of seeds is a major problem in the agricultural sector. It is also a complex biological question that remains unclear for many crop seeds. In this experiment, we investigated how different ageing levels influence seeds germination, storage components, structure, respiration metabolism, and whether respiration is a promising indicator that can non-destructively evaluate seed aging. First, we obtained non-heading Chinese cabbage seeds with varying ageing levels by applying artificial aging treatments over a period of six days (0 d–6 d). Subsequently, respiration related indexes and ultrastructural observations were conducted. Our results showed that the respiratory rate remains low when seeds have a low water content. With the increased water content, the activities of respiratory-related enzymes, phosphohexose isomerase (PGI) and malate dehydrogenase (MDH), reached maximum levels when seeds were aged for two days. Respiration rates peaked one day later, followed by a rapid decline with increasing age. The ageing process consumed a large amount of storage components (e.g. soluble sugar, sucrose, and starch), leading to the degradation of mitochondria, proteasomes, fat bodies and cell nuclei in the aged seeds, which resulted in the reduction of respiratory rates. This, in turn, negatively affected seed germination. When seeds germinated (1 h–6 h, 1 d–6 d) under standard germination condition, the germination rate and respiratory rate showed significant correlation. Especially for 5 h and 6 h, the correlation coefficients reached 0.954 and 0.976 respectively. Measured at those times, respiratory rate should be a promising indicator for seed aging.

Electrostatic interactions in natural enzymes: What can we learn for enzyme design?

The goal of this work is to understand the intrinsic properties that give natural enzymes their catalytic power and to learn how to build these properties in silico, for the design and expression of artificial enzymes that catalyze reactions that do not occur in nature. Partial Order Optimum Likelihood (POOL) is a machine learning method developed by us to predict residues important for function, using the 3D structure of the query protein. The input features to POOL are based on computed electrostatic and chemical properties from THEMATICS. These input features are effectively measures of the strength of coupling between protonation events. POOL is used to characterize the properties of natural enzymes that are necessary for efficient catalysis. Catalytic sites in proteins are characterized by networks of strongly coupled protonation states; these networks impart the necessary electrostatic and proton‐transfer properties to the active residues in the first layer around the reacting substrate molecule(s). Typically these networks include first‐, second‐, and sometimes third‐layer residues. POOL‐predicted, multi‐layer active sites with significant participation by distal residues have been verified experimentally by single‐point site‐directed mutagenesis and kinetics assays for Ps. putida nitrile hydratase, human phosphoglucose isomerase, E. coli replicative DNA polymerase Pol III, E. coli Y family DNA polymerase DinB, and E. coli ornithine transcarbamoylase. In designed enzymes, such as retroaldolases, the residue‐specific input features to POOL – measures of the strength of coupling between protonation equilibria – rise as the enzymes evolve to higher rates of catalytic turnover. An approach to build these properties into the initial designs is proposed.Support or Funding InformationNSF MCB‐1517290 (MJO &amp; PJB) and a National Institute of Justice Fellowship (TAC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.