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Genes May 2023The reference gene expression is not always stable under different experimental conditions, and screening of suitable reference genes is a prerequisite in quantitative...
The reference gene expression is not always stable under different experimental conditions, and screening of suitable reference genes is a prerequisite in quantitative real-time polymerase chain reaction (qRT-PCR). In this study, we investigated gene selection, and the most stable reference gene for the Chinese mitten crab () was screened under the stimulation of and copper ions, respectively. Ten candidate reference genes were selected, including arginine kinase (), ubiquitin-conjugating enzyme E2b (), glutathione S-transferase (), glyceraldehyde-3-phosphate dehydrogenase (), elongation factor 1α (), α-tubulin (), heat shock protein 90 (), β-actin (), elongation factor 2 () and phosphoglucomutase 2 (). Expression levels of these reference genes were detected under the stimulation of at different times (0 h, 6 h, 12 h, 24 h, 48 h and 72 h) and copper ions in different concentrations (11.08 mg/L, 2.77 mg/L, 0.69 mg/L and 0.17 mg/L). Four types of analytical software, namely geNorm, BestKeeper, NormFinder and Ref-Finder, were applied to evaluate the reference gene stability. The results showed that the stability of the 10 candidate reference genes was in the following order: > > > > > > > > > under stimulation. It was > > > > > > > > > under copper ion stimulation. The expression of Peroxiredoxin4 () was detected when the most stable and least stable internal reference genes were selected, respectively. The results showed that reference genes with different stability had great influence on the accurate results of the target gene expression. In the Chinese mitten crab (), AK and EF-1α were the most suitable reference genes under the stimulation of . Under the stimulation of copper ions, and were the most suitable reference genes. This study provided important information for further research on immune genes in or copper ion stimulation.
Topics: Peptide Elongation Factor 1; Copper; Actins; Peptide Elongation Factor 2; Gene Expression Profiling
PubMed: 37239459
DOI: 10.3390/genes14051099 -
Therapeutic Advances in Rare Disease 2023Phosphoglucomutase-1-congenital disorder of glycosylation (PGM1-CDG) (OMIM: 614921) is a rare autosomal recessive inherited metabolic disease caused by the deficiency of...
Phosphoglucomutase-1-congenital disorder of glycosylation (PGM1-CDG) (OMIM: 614921) is a rare autosomal recessive inherited metabolic disease caused by the deficiency of the PGM1 enzyme. Like other CDGs, PGM1-CDG has a multisystemic presentation. The most common clinical findings include liver involvement, rhabdomyolysis, hypoglycemia, and cardiac involvement. Phenotypic severity can vary, though cardiac presentation is usually part of the most severe phenotype, often resulting in early death. Unlike the majority of CDGs, PGM1-CDG has a treatment: oral D-galactose (D-gal) supplementation, which significantly improves many aspects of the disorder. Here, we describe five PGM1-CDG patients treated with D-gal and report both on novel clinical symptoms in PGM1-CDG as well as the effects of the D-gal treatment. D-gal resulted in notable clinical improvement in four patients, though the efficacy of treatment varied between the patients. Furthermore, there was a significant improvement or normalization in transferrin glycosylation, liver transaminases and coagulation factors in three patients, creatine kinase (CK) levels in two, while hypoglycemia resolved in two patients. One patient discontinued the treatment due to urinary frequency and lack of clinical improvement. Furthermore, one patient experienced recurrent episodes of rhabdomyolysis and tachycardia even on higher doses of therapy. D-gal also failed to improve the cardiac function, which was initially abnormal in three patients, and remains the biggest challenge in treating PGM1-CDG. Together, our findings expand the phenotype of PGM1-CDG and underline the importance of developing novel therapies that would specifically treat the cardiac phenotype in PGM1-CDG.
PubMed: 37181075
DOI: 10.1177/26330040221150269 -
International Journal of Molecular... May 2023Phosphoglucomutase 1 (PGM1) is a key enzyme for the regulation of energy metabolism from glycogen and glycolysis, as it catalyzes the interconversion of glucose...
Phosphoglucomutase 1 (PGM1) is a key enzyme for the regulation of energy metabolism from glycogen and glycolysis, as it catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate. PGM1 deficiency is an autosomal recessive disorder characterized by a highly heterogenous clinical spectrum, including hypoglycemia, cleft palate, liver dysfunction, growth delay, exercise intolerance, and dilated cardiomyopathy. Abnormal protein glycosylation has been observed in this disease. Oral supplementation with D-galactose efficiently restores protein glycosylation by replenishing the lacking pool of UDP-galactose, and rescues some symptoms, such as hypoglycemia, hepatopathy, and growth delay. However, D-galactose effects on skeletal muscle and heart symptoms remain unclear. In this study, we established an in vitro muscle model for PGM1 deficiency to investigate the role of PGM1 and the effect of D-galactose on nucleotide sugars and energy metabolism. Genome-editing of C2C12 myoblasts via CRISPR/Cas9 resulted in (mouse homologue of human , according to updated nomenclature) knockout clones, which showed impaired maturation to myotubes. No difference was found for steady-state levels of nucleotide sugars, while dynamic flux analysis based on C6-galactose suggested a block in the use of galactose for energy production in knockout myoblasts. Subsequent analyses revealed a lower basal respiration and mitochondrial ATP production capacity in the knockout myoblasts and myotubes, which were not restored by D-galactose. In conclusion, an in vitro mouse muscle cell model has been established to study the muscle-specific metabolic mechanisms in PGM1 deficiency, which suggested that galactose was unable to restore the reduced energy production capacity.
Topics: Animals; Mice; Galactose; Glucose; Homeostasis; Hypoglycemia; Muscle Fibers, Skeletal; Muscle, Skeletal; Nucleotides; Phosphates; Phosphoglucomutase
PubMed: 37175952
DOI: 10.3390/ijms24098247 -
Vaccine May 2023Brucellosis remains one of the most worldwide distributed zoonosis inflicting serious economical and human health problems in many areas of the world. The disease is...
Brucellosis remains one of the most worldwide distributed zoonosis inflicting serious economical and human health problems in many areas of the world. The disease is caused by different species of the genus Brucella that have different tropisms towards different mammals being the most relevant for human health Brucella abortus, Brucella melitensis and Brucella suis that infect cows, goats/sheep, and swine respectively. For B. melitensis, considered the species with more zoonotic potential and highly aggressive for animals, only one vaccine is available to date in the market: Rev 1. This attenuated strain has the disadvantage that is has a very high residual virulence for animals and humans and, for this reason, it is applied by ocular instillation which is technically challenging in many productive settings. For this reason, the search for new vaccines for caprine and ovine brucellosis is an active topic of research. We describe here the construction of a novel highly attenuated vaccine strain (Bm Delta-pgm) that confers excellent levels of protection against B. melitensis in the mouse model of infection. This strain is a clean deletion of the phosphoglucomutase (pgm) gene that codes for a protein that catalyzes the conversion of glucose-6-P to glucose-1-P, which is used as a precursor for the biosynthesis of many polysaccharides, including the O-antigen of the lipopolysaccharide and cyclic beta glucans. Our results indicate that vaccination with Bm Delta-pgm induces a robust memory cellular immune response but no antibody production against the O-antigen. Cross protection experiments show that this new vaccine protects against B. abortus and B. suis raising the possibility that Bm Delta-pgm could be used as a universal vaccine for the most important Brucella species.
Topics: Female; Mice; Animals; Sheep; Cattle; Humans; Swine; Brucella melitensis; Phosphoglucomutase; Goats; O Antigens; Brucella Vaccine; Brucellosis; Brucella abortus
PubMed: 37149444
DOI: 10.1016/j.vaccine.2023.04.076 -
Applied Microbiology and Biotechnology May 2023α-Arbutin has been widely used as a skin-whitening ingredient. Previously, we successfully produced α-arbutin via whole-cell biocatalysis and found that the conversion...
α-Arbutin has been widely used as a skin-whitening ingredient. Previously, we successfully produced α-arbutin via whole-cell biocatalysis and found that the conversion rate of sucrose to α-arbutin was low (~45%). To overcome this issue, herein, we knocked out the genes of enzymes related to the sucrose hydrolysis, including sacB, sacC, levB, and sacA. The sucrose consumption was reduced by 17.4% in 24 h, and the sucrose conversion rate was increased to 51.5%. Furthermore, we developed an inducible protein degradation system with Lon protease isolated from Mesoplasma florum (MfLon) and proteolytic tag to control the PfkA activity, so that more fructose-6-phosphate (F6P) can be converted into glucose-1-phosphate (Glc1P) for α-arbutin synthesis, which can reduce the addition of sucrose and increase the sucrose conversion efficiency. Finally, the pathway of F6P to Glc1P was enhanced by integrating another copy of glucose 6-phosphate isomerase (Pgi) and phosphoglucomutase (PgcA); a high α-arbutin titer (~120 g/L) was obtained. The sucrose conversion rate was increased to 60.4% (mol/mol). In this study, the substrate utilization rate was boosted due to the attenuation of its hydrolysis and the assistance of the intracellular enzymes that converted the side product back into the substrate for α-arbutin synthesis. This strategy provides a new idea for the whole-cell biocatalytic synthesis of other products using sucrose as substrate, especially valuable glycosides.Key points The genes of sucrose metabolic pathway were knocked out to reduce the sucrose consumption. The by-product fructose was reused to synthesize α-arbutin. The optimized whole-cell system improved sucrose conversion by 15.3%.
Topics: Biocatalysis; Arbutin; Sucrose; Metabolic Engineering; Glycosides
PubMed: 37000229
DOI: 10.1007/s00253-023-12496-2 -
Biochemical and Biophysical Research... May 2023PGM1 is an essential enzyme for glucose metabolism and is involved in cell viability, proliferation, and metabolism. However, the regulatory role of PGMI in glioma...
PGM1 is an essential enzyme for glucose metabolism and is involved in cell viability, proliferation, and metabolism. However, the regulatory role of PGMI in glioma progression and the relation between gliomas and PGM1 expression are still unclear. This study aimed to explore the role of PGM1 in glycolysis and oxidative phosphorylation in glioma. Correlation and enrichment analyses of PGM1 in glioma cells were explored in TCGA database and two hospital cohorts. The cell viability, glycolysis, and oxidative phosphorylation were investigated in PGM1 knock-down and overexpression situations. Higher PGM1 expression in glioma patients was associated with a poor survival rate. However, knock-down of PGM1 reduced glioma cell viability, glycolysis, and oxidative phosphorylation under low glucose condition. Moreover, it suppressed tumor growth in vivo. On the other hand, PGM1 overexpression promoted glioma cell viability, glycolysis, and oxidative phosphorylation under low glucose condition by a Myc positive feedback loop. Glioma patients with higher PGM1 expression were associated with poor survival rates. Additionally, PGM1 could promote glioma cell viability, glycolysis, and oxidative phosphorylation under low glucose condition via a myc-positive feedback loop, suggesting PGM1 could be a potential therapeutic target for gliomas.
Topics: Humans; Signal Transduction; Oxidative Phosphorylation; Proto-Oncogene Proteins c-myc; Cell Survival; Cell Line, Tumor; Glioma; Glucose; Glycolysis; Cell Proliferation
PubMed: 36947965
DOI: 10.1016/j.bbrc.2023.03.034 -
Journal of Experimental Botany Mar 2023This article comments on: . 2023. Stress response requires an efficient glycogen and central carbon metabolism connection by phosphoglucomutases in cyanobacteria....
This article comments on: . 2023. Stress response requires an efficient glycogen and central carbon metabolism connection by phosphoglucomutases in cyanobacteria. Journal of Experimental Botany , 1532–1550
Topics: Phosphoglucomutase; Synechocystis; Bacterial Proteins
PubMed: 36913621
DOI: 10.1093/jxb/erac513 -
JIMD Reports Mar 2023We report successful heart transplantation in a phosphoglucomutase 1 deficient (PGM1-CDG) patient. She presented with facial dysmorphism, bifid uvula and structural...
We report successful heart transplantation in a phosphoglucomutase 1 deficient (PGM1-CDG) patient. She presented with facial dysmorphism, bifid uvula and structural heart defects. Newborn screening was positive for classic galactosemia. The patient was on a galactose-free diet for 8 months. Eventually, whole exome sequencing excluded the galactosemia and revealed PGM1-CDG. Oral D-galactose therapy was started. Rapid deterioration of the progressive dilated cardiomyopathy prompted heart transplantation at the age of 12 months. Cardiac function was stable in the first 18 months of follow-up, and hematologic, hepatic, and endocrine laboratory findings improved during D-galactose therapy. The latter therapy improves several systemic symptoms and biochemical abnormalities in PGM1-CDG but does not correct the heart failure related to cardiomyopathy. Heart transplantation has so far only been described in DOLK-CDG.
PubMed: 36873091
DOI: 10.1002/jmd2.12350 -
Journal of Inherited Metabolic Disease Mar 2023
Topics: Humans; Phenotype; Phosphoglucomutase; Genetic Association Studies; Congenital Disorders of Glycosylation
PubMed: 36843136
DOI: 10.1002/jimd.12601