-
Cellular Physiology and Biochemistry :... Apr 2022Bioreactor-based bioartificial liver support systems have had limited success in a translational setting and at preclinical stages. None of the existing systems monitor...
BACKGROUND/AIMS
Bioreactor-based bioartificial liver support systems have had limited success in a translational setting and at preclinical stages. None of the existing systems monitor the metabolic pathways of glycolysis, glycogen synthesis, the urea cycle, and cytochrome peroxidase oxidative reabsorption. Herein, we designed a bioreactor that mimics the human liver microenvironment in vivo and monitors different hepatic metabolic pathways in order to help establish in vitro culture conditions for improved glycolysis, glycogen synthesis, the urea cycle, cytochrome peroxidase oxidative reabsorption and improved hepatic functions in a miniature bioartificial liver. An abnormality in such pathways negatively influences survivability and hepatic functions, including spontaneous liver regeneration.
METHODS
We investigated the metabolic functions of primary mouse adult hepatocytes cultured in a three-dimensional configuration under direct oxygenation conditions (5%, 10%, 20%, and 40% O) for 14 days in the bioreactor. We analyzed the expression of the genes of hepatic metabolic pathways, such as glycolysis (glucokinase, phosphofructokinase, and pyruvate kinase), glycogen synthesis (glycogen synthetase, UTP glucose-1-phosphate uridylylisomerase, phosphoglucomutase, and glycogen phosphorylase), the urea cycle (arginase, ornithine carbomoyltransferase, fumarate hydratase), oxidative reabsorption (peroxidase), and cytochrome peroxides (catalase and superoxide dismutase), and compared it with the level in vivo. The metabolic mini-map was used to represent the above-mentioned metabolic genes.
RESULTS
Increased urea secretion under normoxia and hyperoxia conditions (20% and 40% O, respectively) was observed, while albumin secretion was decreased in hyperoxic cultures. Lactate formation was up to 15 mg/L/h/10 cells, 2 mg/L/h/10 cells, and 0.2 mg/L/h/10 cells in 5%, 20%, and 40% O conditions, respectively while glucose consumption was enhanced under hypoxic conditions (5% and 10% O). Cellular membrane integrity was estimated by lactate dehydrogenase assay and was found to be negligible in only 20% and 40% O conditions. The expression of the phase II enzyme UDP-glucuronosyltransferase was only upregulated in 20% oxygenation.
CONCLUSION
Taken together, 20% O was found to be an optimal condition for the long-term culture (up to 14 days) of hepatocytes that promoted the expression of genes in metabolic pathways such as glycolysis, glycogen synthesis, the urea cycle, and cytochrome peroxidase oxidative reabsorption, and improved hepatic functions in a miniature bioreactor for bioartificial liver construction.
Topics: Animals; Bioreactors; Cytochrome-c Peroxidase; Glycogen; Glycolysis; Liver; Mice; Oxidative Stress; Urea
PubMed: 35462472
DOI: 10.33594/000000513 -
Bioengineered Apr 2022Long non-coding RNAs (lncRNAs) have been widely studied and play crucial roles in cervical cancer (CC) progression. Here, we investigated the function and mechanism of...
Long non-coding RNAs (lncRNAs) have been widely studied and play crucial roles in cervical cancer (CC) progression. Here, we investigated the function and mechanism of lncRNA PGM5-AS1 action in CC cells. Using real-time quantitative polymerase chain reaction or western blotting, PGM5-AS1 and decorin (DCN) were downregulated in CC tissues and cells, whereas miR-4284 was upregulated. Luciferase assay, RNA pull-down assay, and western blotting showed that PGM5-AS1 could sponge miR-4284 to upregulate DCN expression in CC cells. Additionally, cell functional experiments showed that PGM5-AS1 overexpression led to decreased proliferation, migration, and invasion of CC cells. However, the inhibitory effect of PGM5-AS1 overexpression on CC cells was partly relieved by DCN knockdown because of the targeting interaction between PGM5-AS1, miR-4284, and DCN. In summary, this study identified that PGM5-AS1 negatively regulates CC cell malignancy by targeting miR-4284/DCN.
Topics: Cell Line, Tumor; Cell Proliferation; Cytoskeletal Proteins; Decorin; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Phosphoglucomutase; RNA, Long Noncoding; Uterine Cervical Neoplasms
PubMed: 35420507
DOI: 10.1080/21655979.2022.2062088 -
Microorganisms Mar 2022Hyperoside (quercetin 3--galactoside) exhibits many biological functions, along with higher bioactivities than quercetin. In this study, three UDP-dependent...
Hyperoside (quercetin 3--galactoside) exhibits many biological functions, along with higher bioactivities than quercetin. In this study, three UDP-dependent glycosyltransferases (UGTs) were screened for efficient hyperoside synthesis from quercetin. The highest hyperoside production of 58.5 mg·L was obtained in a recombinant co-expressing UGT from (PhUGT) and UDP-glucose epimerase (GalE, a key enzyme catalyzing the conversion of UDP-glucose to UDP-galactose) from . When additional enzymes (phosphoglucomutase (Pgm) and UDP-glucose pyrophosphorylase (GalU)) were introduced into the recombinant , the increased flux toward UDP-glucose synthesis led to enhanced UDP-galactose-derived hyperoside synthesis. The efficiency of the recombinant strain was further improved by increasing the copy number of the PhUGT, which is a limiting step in the bioconversion. Through the optimization of the fermentation conditions, the production of hyperoside increased from 245.6 to 411.2 mg·L. The production was also conducted using a substrate-fed batch fermentation, and the maximal hyperoside production was 831.6 mg·L, with a molar conversion ratio of 90.2% and a specific productivity of 27.7 mg·L·h after 30 h of fermentation. The efficient hyperoside synthesis pathway described here can be used widely for the glycosylation of other flavonoids and bioactive substances.
PubMed: 35336203
DOI: 10.3390/microorganisms10030628 -
Foods (Basel, Switzerland) Mar 2022The effects of slaughter age (2 vs. 9 years) and postmortem time (6, 8, 10, 12, 24, 48 and 72 h) on the meat quality and protein changes of the longissimus lumborum...
The effects of slaughter age (2 vs. 9 years) and postmortem time (6, 8, 10, 12, 24, 48 and 72 h) on the meat quality and protein changes of the longissimus lumborum muscles of the Algerian Sahraoui dromedary were investigated. Muscles of young dromedaries evidenced a slower acidification process and a significantly higher myofibrillar fragmentation index throughout the postmortem time. The SDS-PAGE of sarcoplasmic and myofibrillar proteins revealed that meat from young dromedaries was characterized by the lowest percentage of myoglobin (p < 0.001) and the highest percentage of desmin (p < 0.01). During postmortem time, a decrease was found for phosphoglucomutase (p < 0.01), α-actinin (p < 0.05) and desmin (p < 0.01) in meat from young dromedaries. Western blot revealed an intense degradation of troponin T in younger dromedaries, with an earlier appearance of the 28 kDa polypeptide highlighting differences in the proteolytic potential between dromedaries of different ages. Principal component analysis showed that meat from young dromedaries, starting from 24 h postmortem, was located in a zone of the plot characterized by higher levels of the myofibrillar fragmentation index, 30 kDa polypeptide and enolase, overall confirming greater proteolysis in younger animals. Data suggest that the investigation of the muscle proteome is necessary to set targeted interventions to improve the aging process of dromedary meat cuts.
PubMed: 35267365
DOI: 10.3390/foods11050732 -
Frontiers of Chemical Science and... 2022is one of the most important microbial cell factories, but infection by bacteriophages in the environment may have a huge impact on its application in industrial...
UNLABELLED
is one of the most important microbial cell factories, but infection by bacteriophages in the environment may have a huge impact on its application in industrial production. Here, we developed a mobile CRISPR-Cas9 based anti-phage system for bacteriophages defense in . Two conjugative plasmids pGM1 (phosphoglucomutase 1) and pGM2 carrying one and two guide RNAs, respectively, were designed to defend against a filamentous phage. The results showed that the pGM1 and pGM2 could decrease the phage infection rate to 1.6% and 0.2% respectively in infected cells. For preventing phage infection in , the pGM2 decreased the phage infection rate to 0.1 %, while pGM1 failed to block phage infection. Sequence verification revealed that point mutations in protospacer or protospacer adjacent motif sequences of the phage genome caused loss of the defense function. These results support the potential application of MCBAS in cell factories to defend against phage infections.
ELECTRONIC SUPPLEMENTARY MATERIAL
Supplementary material is available in the online version of this article at 10.1007/s11705-022-2141-7 and is accessible for authorized users.
PubMed: 35251747
DOI: 10.1007/s11705-022-2141-7 -
Computational and Structural... 2022, a perennial herb in the Araceae family, is a cash crop that can produce a large amount of konjac glucomannan. To explore mechanisms underlying such large genomes in...
, a perennial herb in the Araceae family, is a cash crop that can produce a large amount of konjac glucomannan. To explore mechanisms underlying such large genomes in the genus as well as the gene regulation of glucomannan biosynthesis, we present a chromosome-level genome assembly of with a total genome size of 5.60 Gb and a contig N50 of 1.20 Mb. Comparative genomic analysis reveals that has undergone two whole-genome duplication (WGD) events in quick succession. Two recent bursts of transposable elements are identified in the genome, which contribute greatly to the large genome size. Our transcriptomic analysis of the developmental corms characterizes key genes involved in the biosynthesis of glucomannan and related starches. High expression of cellulose synthase-like A, Cellulose synthase-like D, mannan-synthesis related 1, GDP-mannose pyrophosphorylase and phosphomannomutase fructokinase contributes to glucomannan synthesis during the corm expansion period while high expression of starch synthase, starch branching enzyme and phosphoglucomutase is responsible for starch synthesis in the late corm development stage. In conclusion, we generate a high-quality genome of with different sequencing technologies. The expansion of transposable elements has caused the large genome of this species. And the identified key genes in the glucomannan biosynthesis provide valuable candidates for molecular breeding of this crop in the future.
PubMed: 35242290
DOI: 10.1016/j.csbj.2022.02.009 -
Genes Jan 2022The polychaete lives exclusively on the walls of deep-sea hydrothermal chimneys along the East Pacific Rise (EPR), and displays specific adaptations to withstand the...
The polychaete lives exclusively on the walls of deep-sea hydrothermal chimneys along the East Pacific Rise (EPR), and displays specific adaptations to withstand the high temperatures and hypoxia associated with this highly variable habitat. Previous studies have revealed the existence of a balanced polymorphism on the enzyme phosphoglucomutase associated with thermal variations, where allozymes 90 and 100 exhibit different optimal activities and thermostabilities. Exploration of the mutational landscape of phosphoglucomutase 1 revealed the maintenance of four highly divergent allelic lineages encoding the three most frequent electromorphs over the geographic range of . This polymorphism is only governed by two linked amino acid replacements, located in exon 3 (E155Q and E190Q). A two-niche model of selection, including 'cold' and 'hot' conditions, represents the most likely scenario for the long-term persistence of these isoforms. Using directed mutagenesis and the expression of the three recombinant variants allowed us to test the additive effect of these two mutations on the biochemical properties of this enzyme. Our results are coherent with those previously obtained from native proteins, and reveal a thermodynamic trade-off between protein thermostability and catalysis, which is likely to have maintained these functional phenotypes prior to the geographic separation of populations across the Equator about 1.2 million years ago.
Topics: Alleles; Animals; Mutation; Phosphoglucomutase; Polychaeta; Polymorphism, Genetic
PubMed: 35205251
DOI: 10.3390/genes13020206 -
Pathogens (Basel, Switzerland) Dec 2021Phosphoglucomutase 1 (PGM1) catalyzes the conversion between glucose-1-phosphate and glucose-6-phosphate in the glycolysis/glucogenesis pathway. PGM1s are typically...
Phosphoglucomutase 1 (PGM1) catalyzes the conversion between glucose-1-phosphate and glucose-6-phosphate in the glycolysis/glucogenesis pathway. PGM1s are typically cytosolic enzymes in organisms lacking chloroplasts. However, the protozoan parasites possess two tandemly duplicated genes evolved by a gene duplication after their split from other apicomplexans. Moreover, the downstream PGM1 isoform contains an N-terminal signal peptide, predicting a non-cytosolic location. Here we expressed recombinant proteins of the two PGM1 isoforms from the zoonotic , namely CpPGM1A and CpPGM1B, and confirmed their enzyme activity. Both isoforms followed Michaelis-Menten kinetics towards glucose-1-phosphate ( = 0.17 and 0.13 mM, = 7.30 and 2.76 μmol/min/mg, respectively). and genes were expressed in oocysts, sporozoites and intracellular parasites at a similar pattern of expression, however was expressed at much higher levels than . Immunofluorescence assay showed that CpPGM1A was present in the cytosol of sporozoites, however this was enriched towards the plasma membranes in the intracellular parasites; whereas CpPGM1B was mainly present under sporozoite pellicle, although relocated to the parasitophorous vacuole membrane in the intracellular development. These observations indicated that CpPGM1A played a house-keeping function, while CpPGM1B played a different biological role that remains to be defined by future investigations.
PubMed: 35055969
DOI: 10.3390/pathogens11010021 -
Biomedicines Jan 2022infection is associated with several gastric diseases, including gastritis, peptic ulcer, gastric adenocarcinoma and mucosa-associated lymphatic tissue (MALT) lymphoma....
infection is associated with several gastric diseases, including gastritis, peptic ulcer, gastric adenocarcinoma and mucosa-associated lymphatic tissue (MALT) lymphoma. Due to the prevalence and severeness of infection, a thorough understanding of this pathogen is necessary. Lipopolysaccharide, one of the major virulence factors of , can exert immunomodulating and immunostimulating functions on the host. In this study, the and genes were under investigation. These two genes potentially encode GDP-D-mannose dehydratase (GMD) and phosphomannomutase (PMM)/phosphoglucomutase (PGM), respectively, and are involved in the biosynthesis of fucose. and knockout mutants were generated; both mutants displayed a truncated LPS, suggesting that the encoded enzymes are not only involved in fucose production but are also important for LPS construction. In addition, these two gene knockout mutants exhibited retarded growth, increased surface hydrophobicity and autoaggregation as well as being more sensitive to the detergent SDS and the antibiotic novobiocin. Furthermore, the LPS-defective mutants also had significantly reduced bacterial infection, adhesion and internalization in the in vitro cell line model. Moreover, disruptions of the and genes in altered protein sorting into outer membrane vesicles. The critical roles of and in LPS biosynthesis, bacterial fitness and pathogenesis make them attractive candidates for drug inventions against infection.
PubMed: 35052824
DOI: 10.3390/biomedicines10010145 -
Journal of Clinical Immunology Apr 2022
Topics: Hematopoietic Stem Cell Transplantation; Humans; Immunologic Deficiency Syndromes; Phosphoglucomutase
PubMed: 35040011
DOI: 10.1007/s10875-021-01196-z