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Animals : An Open Access Journal From... Nov 2023(EHP) is highly contagious and can cause hepatopancreatic microsporidiosis (HPM), which is typically characterized by the slow growth of shrimp. In this study, the...
(EHP) is highly contagious and can cause hepatopancreatic microsporidiosis (HPM), which is typically characterized by the slow growth of shrimp. In this study, the differences in histology, metabolism, oxidative stress and growth between healthy and EHP-infected were analyzed using an EHP challenge experiment. Histology showed that EHP caused lesions in the hepatic tubules of , such as hepatic tubular atrophy and epithelial cell shedding, with mature spores. Meanwhile, white feces may appear when the infection is severe. Furthermore, the content of total protein, glycogen, ATP and glucose in the EHP challenge group was significantly reduced. The qPCR results showed that EHP infection changed the expression of key genes in glucose metabolism, among which hexokinase (), phosphofructokinase (), pyruvatekinase (), citrate synthase () and isocitric dehydrogenase () were significantly down-regulated, while phosphoenolpyruvate carboxykinase (), fructose bisphosphatase () and glucose-6-phosphatase () were significantly up-regulated. Obviously, the expression of growth-related genes was disordered. Simultaneously, the antioxidant genes manganese superoxide dismutase (), catalase (), glutathione peroxidase (), glutathione-S-transferases () and nuclear factor E2-related factor2 () were up-regulated to varying degrees in the EHP challenge group, and EHP infection induced significant increases in the oxidative damage products lipid peroxide (LPO) and malondialdehyde (MDA). Ultimately, the shrimp weight of the challenge group was 6.85 ± 0.86 g, which was significantly lower than that of the control group (8.95 ± 0.75 g). Taken together, we speculate that EHP changes the substance metabolism and growth process by causing oxidative damage to the hepatopancreas, which may lead to the growth retardation of .
PubMed: 38067012
DOI: 10.3390/ani13233661 -
Theriogenology Feb 2024If a mechanism of more efficient glycolysis depending on pyruvate is present in stallion spermatozoa, detrimental effects of higher glucose concentrations that are...
If a mechanism of more efficient glycolysis depending on pyruvate is present in stallion spermatozoa, detrimental effects of higher glucose concentrations that are common in current commercial extenders could be counteracted. To test this hypothesis, spermatozoa were incubated in a 67 mM Glucose modified Tyrode's media in the presence of 1- or 10-mM pyruvate and in the Tyrode's basal media which contains 5 mM glucose. Spermatozoa incubated for 3 h at 37 °C in 67 mM Tyrode's media with 10 mM pyruvate showed increased motility in comparison with aliquots incubated in Tyrode's 5 mM glucose and Tyrode's 67 mM glucose (57.1 ± 3.5 and 58.1 ± 1.9 to 73.0 ± 1.1 %; P < 0.01). Spermatozoa incubated in Tyrode's with 67 mM glucose 10 mM pyruvate maintained the viability along the incubation (64.03 ± 15.4 vs 61.3 ± 10.2), while spermatozoa incubated in 67 mM Glucose-Tyrode's showed a decrease in viability (38.01 ± 11.2, P < 0.01). 40 mM oxamate, an inhibitor of the lactate dehydrogenase LDH, reduced sperm viability (P < 0.05, from 76 ± 5 in 67 mM Glucose/10 mM pyruvate to 68.0 ± 4.3 %, P < 0.05). Apoptotic markers increased in the presence of oxamate. (P < 0.01). UHPLC/MS/MS showed that 10 mM pyruvate increased pyruvate, lactate, ATP and NAD while phosphoenolpyruvate decreased. The mechanisms that explain the improvement of in presence of 10 mM pyruvate involve the conversion of lactate to pyruvate and increased NAD enhancing the efficiency of the glycolysis.
Topics: Male; Animals; Horses; Pyruvic Acid; Semen; NAD; Tandem Mass Spectrometry; Sperm Motility; Spermatozoa; Lactates; Glucose
PubMed: 38029686
DOI: 10.1016/j.theriogenology.2023.11.019 -
Immunity, Inflammation and Disease Nov 2023What is highlighted in this study refers to the role and molecular mechanism of long noncoding RNA (lncRNA) X-inactive specific transcript (XIST) in cells with insulin...
BACKGROUND
What is highlighted in this study refers to the role and molecular mechanism of long noncoding RNA (lncRNA) X-inactive specific transcript (XIST) in cells with insulin resistance (IR).
METHODS
In this study, LX-2 cells were applied to establish IR model in vitro. The expressions of lncRNA XIST, phosphoenolpyruvate carboxykinase (PEPCK,) and glucose-6-phosphatase (G6Pase) were quantified by quantitative reverse transcription polymerase chain reaction. The 2-deoxy-d-glucose-6-phosphate (2-DG6P) level was detected utilizing 2-deoxy-d-glucose (2-DG) uptake measurement kit. Western blot was adopted to measure the protein expressions of insulin-like growth factor-1 receptor (IGF-1R), G6Pase, PEPCK, and phosphatidylinositol 3-kinase (PI3K)/Akt pathway-related genes. StarBase was used to predict the targeting relationship between lncRNA XIST or IGF-1R with miR-182-5p, the results of which were verified by dual-luciferase reporter, RNA pull-down, and RNA immunoprecipitation assays. Rescue experiments were conducted to investigate the effect of miR-182-5p on IR cells. Next, low-expressed lncRNA XIST and high-expressed miR-182-5p were observed in IR cells.
RESULTS
Upregulation of lncRNA XIST increased IGF-1R and 2-DG6P levels, decreased G6Pase and PEPCK expressions, and promoted PI3K/Akt pathway activation in IR cells. LncRNA XIST sponged miR-182-5p which targeted IGF-1R. MiR-182-5p mimic reversed the above effects of lncRNA XIST overexpression on IR cells.
CONCLUSIONS
In conclusion, lncRNA XIST/miR-182-5p axis alleviates hepatic IR in vitro via IGF-1R/PI3K/Akt signaling pathway, which could be the promising therapeutic target.
Topics: Humans; Insulin Resistance; MicroRNAs; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA, Long Noncoding; Hepatocytes
PubMed: 38018594
DOI: 10.1002/iid3.969 -
Journal of Translational Medicine Nov 2023N6-methyladenosine (m6A) is the most prevalent RNA modification. Although hnRNPA2B1, as a reader of m6A modification, has been reported to promote tumorigenesis in a few...
BACKGROUND
N6-methyladenosine (m6A) is the most prevalent RNA modification. Although hnRNPA2B1, as a reader of m6A modification, has been reported to promote tumorigenesis in a few types of tumors, its role in hepatocellular carcinoma (HCC) and the underlying molecular mechanism remains unclear.
METHODS
Multiple public databases were used to analyze the expression of hnRNPA2B1 in HCC and its correlation with survival prognosis. We employed a CRISPR-Cas9 sgRNA editing strategy to knockout hnRNPA2B1 expression in HCC cells. The biological function of hnRNPA2B1 in vitro in HCC cells was measured by CCK8, colony formation, migration, and invasion assay. The tumorigenic function of hnRNPA2B1 in vivo was determined by a subcutaneous tumor formation experiment and a HCC mouse model via tail injection of several plasmids into the mouse within 5s-7s. RNA binding protein immunoprecipitation (RIP) experiment using hnRNPA2B1 was performed to test the target genes of hnRNPA2B1 and methylated RNA immunoprecipitation (MeRIP) assay was performed to explore the m6A methylated mRNA of target genes.
RESULTS
hnRNPA2B1 highly expressed in HCC tissues, correlated with high grades and poor prognosis. Its knockout reduced HCC cell proliferation, migration, and invasion in vitro, while overexpression promoted these processes. hnRNPA2B1-knockout cells inhibited tumor formation in graft experiments. In HCC mice, endogenous knockout attenuated hepatocarcinogenesis. RNA-seq showed downregulated gluconeogenesis with high hnRNPA2B1 expression. hnRNPA2B1 negatively correlated with PCK1, a key enzyme. RIP assay revealed hnRNPA2B1 binding to PCK1 mRNA. hnRNPA2B1 knockout increased m6A-methylation of PCK1 mRNA. Interestingly, PCK1 knockout partially counteracted tumor inhibition by hnRNPA2B1 knockout in mice.
CONCLUSION
Our study indicated that hnRNPA2B1 is highly expressed in HCC and correlated with a poor prognosis. hnRNPA2B1 promotes the tumorigenesis and progression of HCC both in vitro and in vivo. Moreover, hnRNPA2B1 downregulates the expression of PCK1 mRNA via a m6A methylation manner. More importantly, the ability of hnRNPA2B1 to induce tumorigenesis and progression in HCC is dependent on its ability to decrease the expression of PCK1. Therefore, this study suggested that hnRNPA2B1 might be a diagnostic marker of poor prognosis of HCC and a potential therapeutic target for HCC patients.
Topics: Animals; Humans; Mice; Carcinogenesis; Carcinoma, Hepatocellular; Cell Line, Tumor; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Methylation; Phosphoenolpyruvate Carboxykinase (GTP); RNA; RNA, Guide, CRISPR-Cas Systems; RNA, Messenger; RNA-Binding Proteins
PubMed: 38017546
DOI: 10.1186/s12967-023-04704-4 -
Pathogens (Basel, Switzerland) Oct 2023Glucose metabolism is critical for the African trypanosome, , serving as the lone source of ATP production for the bloodstream form (BSF) parasite in the glucose-rich...
Glucose metabolism is critical for the African trypanosome, , serving as the lone source of ATP production for the bloodstream form (BSF) parasite in the glucose-rich environment of the host blood. Recently, phosphonate inhibitors of human enolase (ENO), the enzyme responsible for the interconversion of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP) in glycolysis or PEP to 2-PG in gluconeogenesis, have been developed for the treatment of glioblastoma multiforme (GBM). Here, we have tested these agents against ENO (ENO) and found the compounds to be potent enzyme inhibitors and trypanocides. For example, (1-hydroxy-2-oxopyrrolidin-3-yl) phosphonic acid (deoxy-SF2312) was a potent enzyme inhibitor (IC value of 0.60 ± 0.23 µM), while a six-membered ring-bearing phosphonate, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX), was less potent (IC value of 2.1 ± 1.1 µM). An analog with a larger seven-membered ring, (1-hydroxy-2-oxoazepan-3-yl) phosphonic acid (HEPTA), was not active. Molecular docking simulations revealed that deoxy-SF2312 and HEX had binding affinities of -6.8 and -7.5 kcal/mol, respectively, while the larger HEPTA did not bind as well, with a binding of affinity of -4.8 kcal/mol. None of these compounds were toxic to BSF parasites; however, modification of enzyme-active phosphonates through the addition of pivaloyloxymethyl (POM) groups improved activity against , with POM-modified (1,5-dihydroxy-2-oxopyrrolidin-3-yl) phosphonic acid (POMSF) and POMHEX having EC values of 0.45 ± 0.10 and 0.61 ± 0.08 µM, respectively. These findings suggest that HEX is a promising lead against and that further development of prodrug HEX analogs is warranted.
PubMed: 38003754
DOI: 10.3390/pathogens12111290 -
Animals : An Open Access Journal From... Nov 2023Palmitic (C16:0), α-linolenic acid (C18:3n-3 ), and propionate regulate bovine pyruvate carboxylase () and phosphoenolpyruvate carboxykinase () expression in vitro. The...
Effects of an Hourly Bolus Postruminal Infusion of Flaxseed Oil or Palm Oil on Circulating Fatty Acid Concentrations and Hepatic Expression of Pyruvate Carboxylase and Phosphoenolpyruvate Carboxykinase in Dairy Cattle.
Palmitic (C16:0), α-linolenic acid (C18:3n-3 ), and propionate regulate bovine pyruvate carboxylase () and phosphoenolpyruvate carboxykinase () expression in vitro. The objective of this experiment was to determine the impact of C16:0, C18:3n-3 , propionate, and acetate postruminal infusions on hepatic and expression. We hypothesized that circulating fatty acids alter hepatic and in lactating dairy cows. Acetate, propionate, palm oil, and flaxseed oil were supplied postruminally to lactating cows ( = 4) using two 4 × 4 Latin square studies. For Experiment 1, cows were infused on an hourly basis with either a bolus of propionate, acetate, or the combination of propionate and palm oil, or acetate and palm oil, and Experiment 2 was similar, but flaxseed oil replaced palm oil. Flaxseed infusions increased plasma concentration and the molar percent of C18:3n-3 and decreased C16:0 but did not affect or expression. Palm infusions did not affect blood metabolites or the hepatic expression of or . The lack of responses to short-chain fatty acid infusions and changes in circulating long-chain fatty acids in mature cattle are not suitable models to study the effects of α-linolenic acid and propionate on bovine and expression previously observed in vitro.
PubMed: 38003190
DOI: 10.3390/ani13223572 -
Antioxidants (Basel, Switzerland) Oct 2023Rapeseed seeding dates are largely delayed under the rice-rape rotation system, but how rapeseeds adapt to the delayed environment remains unclear. Here, five seeding...
Rapeseed seeding dates are largely delayed under the rice-rape rotation system, but how rapeseeds adapt to the delayed environment remains unclear. Here, five seeding dates (20 October, 30 October, 10 November, 20 November and 30 November, T1 to T5) were set and the dynamic differences between two late-seeding-tolerant (LST) and two late-seeding-sensitive (LSS) rapeseed cultivars were investigated in a field experiment. The growth was significantly repressed and the foldchange (LST/LSS) of yield increased from 1.50-T1 to 2.64-T5 with the delay in seeding. Both LST cultivars showed higher plant coverage than the LSS cultivars according to visible/hyperspectral imaging and the vegetation index acquired from an unmanned aerial vehicle. Fluorescence imaging, DAB and NBT staining showed that the LSS cultivars suffered more stress damage than the LST cultivars. Antioxidant enzymes (SOD, POD, CAT, APX) and osmoregulation substances (proline, soluble sugar, soluble protein) were decreased with the delay in seeding, while the LST cultivar levels were higher than those of the LSS cultivars. A comparative analysis of transcriptomes and metabolomes showed that 55 pathways involving 123 differentially expressed genes (DEGs) and 107 differentially accumulated metabolites (DAMs) participated in late seeding tolerance regulation, while 39 pathways involving 60 DEGs and 68 DAMs were related to sensitivity. Levanbiose, α-isopropylmalate, s-ribosyl-L-homocysteine, lauroyl-CoA and argino-succinate were differentially accumulated in both cultivars, while genes including isocitrate dehydrogenase, pyruvate kinase, phosphoenolpyruvate carboxykinase and newgene_7532 were also largely regulated. This study revealed the dynamic regulation mechanisms of rapeseeds on late seeding conditions, which showed considerable potential for the genetic improvement of rapeseed.
PubMed: 38001769
DOI: 10.3390/antiox12111915 -
Human & Experimental Toxicology 2023: Adipose tissue is a dynamic endocrine organ that plays a key role in regulating metabolic homeostasis. Previous studies confirmed that bisphenol A (BPA) or fructose...
: Adipose tissue is a dynamic endocrine organ that plays a key role in regulating metabolic homeostasis. Previous studies confirmed that bisphenol A (BPA) or fructose can interfere with the function of adipose tissue. Nonetheless, knowledge on how exposure to BPA and fructose impacts energy metabolism in adipose tissue remains limited.: To determine impact of combined chronic exposure to low-dose bisphenol A and fructose on serum adipocytokines and the energy target metabolome in white adipose tissue.: 57 energy metabolic intermediates in adipose tissue and 7 adipocytokines in serum from Sprague Dawley rats were examined after combined exposure to two levels of BPA (lower dose: 0.25, and higher dose: 25 μg/kg every other day) and 5% fructose for 6 months.: combined exposure to lower-dose BPA and fructose significantly increased omentin-1, pyruvic acid, adenosine triphosphate (ATP), adenosine monophosphate (AMP), inosine monophosphate (IMP), inosine, and l-lactate; however, these parameters were not significantly affected by higher-dose BPA combined with fructose. Interestingly, the level of succinate (an intermediate of the citric acid cycle) increased dose-dependently in adipose tissue, and the level of apelin 13 (a versatile adipocytokine) decreased dose-dependently in serum after combined exposure to BPA and fructose. Phosphoenolpyruvic acid, phenyl-lactate, and ornithine were significantly correlated with asprosin, omentin-1, apelin, apelin 13, and adiponectin, while l-tyrosine was significantly correlated with irisin and a-FABP under combined exposure to BPA and fructose.: these findings indicated that lower-dose BPA combined with fructose could amplify the impact on glycolysis, energy storage, and purine nucleotide biosynthesis in adipose tissue, and adipocytokines, such as omentin-1 and apelin 13, may be related to metabolic interference induced by BPA and fructose exposure.
Topics: Rats; Animals; Fructose; Rats, Sprague-Dawley; Adipokines; Apelin; Adipose Tissue; Benzhydryl Compounds; Adipose Tissue, White; Metabolome; Lactates
PubMed: 37990541
DOI: 10.1177/09603271231217992 -
Carbohydrate Polymers Jan 2024Glycosaminoglycan (GAG) mimics carrying phosphate rather than sulfate anionic groups have been poorly investigated, in spite of their interesting perspectives. While...
Glycosaminoglycan (GAG) mimics carrying phosphate rather than sulfate anionic groups have been poorly investigated, in spite of their interesting perspectives. While some GAG-mimicking phosphorylated polymers have been reported, to the best of our knowledge no phosphorylated polysaccharides having the same backbone of natural sulfated GAGs have been accessed yet. To fill this gap, in this work two standard phosphorylation protocols and two recently reported procedures have been screened on a set of polysaccharide species composed by microbial sourced chondroitin and three partially protected, semi-synthetic derivatives thereof. A detailed structural characterization by H, C and P NMR spectroscopy revealed the higher versatility of the innovative, biomimetic reaction employing monopotassium salt of phosphoenolpyruvate (PEPK) with respect to standard phosphorylating agents (phosphoric acid or phosphorus oxychloride). Indeed, PEP-K and HPO gave similar results in the regioselective phosphorylation of the primary hydroxyls of unprotected chondroitin, while only the former reacted on partially protected chondroitin derivatives in a controlled, regioselective fashion, affording chondroitin phosphate (CP) polysaccharides with different derivatization patterns. The reported results represent the first, key steps towards the systematic semi-synthesis of phosphorylated GAGs as a new class of GAG mimics and to the evaluation of their biological activities in comparison with native sulfated GAGs.
Topics: Glycosaminoglycans; Chondroitin; Phosphorylation; Polysaccharides; Phosphates; Chondroitin Sulfates
PubMed: 37985053
DOI: 10.1016/j.carbpol.2023.121517 -
Bio-protocol Nov 2023While site-specific translational encoding of phosphoserine (pSer) into proteins in via genetic code expansion (GCE) technologies has transformed our ability to study...
While site-specific translational encoding of phosphoserine (pSer) into proteins in via genetic code expansion (GCE) technologies has transformed our ability to study phospho-protein structure and function, recombinant phospho-proteins can be dephosphorylated during expression/purification, and their exposure to cellular-like environments such as cell lysates results in rapid reversion back to the non-phosphorylated form. To help overcome these challenges, we developed an efficient and scalable GCE expression system enabling site-specific incorporation of a non-hydrolyzable phosphoserine (nhpSer) mimic into proteins of interest. This nhpSer mimic, with the γ-oxygen of phosphoserine replaced by a methylene (CH) group, is impervious to hydrolysis and recapitulates phosphoserine function even when phosphomimetics aspartate and glutamate do not. Key to this expression system is the co-expression of a biosynthetic pathway that converts the central metabolite phosphoenolpyruvate into non-hydrolyzable phosphoserine (nhpSer) amino acid, which provides a > 40-fold improvement in expression yields compared to media supplementation by increasing bioavailability of nhpSer and enables scalability of expressions. This "PermaPhos" expression system uses the E. coli BL21(DE3) Δ strain and three plasmids that express (i) the protein of interest, (ii) the GCE machinery for translational installation of nhpSer at UAG amber stop codons, and (iii) the nhpSer biosynthetic pathway. Successful expression requires efficient transformation of all three plasmids simultaneously into the expression host, and IPTG is used to induce expression of all components. Permanently phosphorylated proteins made in are particularly useful for discovering phosphorylation-dependent protein-protein interaction networks from cell lysates or transfected cells. Key features • Protocol builds on the nhpSer GCE system by Rogerson et al. (2015), but with a > 40-fold improvement in yields enabled by the nhpSer biosynthetic pathway. • Protein expression uses standard Terrific Broth (TB) media and requires three days to complete. • C-terminal purification tags on target protein are recommended to avoid co-purification of prematurely truncated protein with full-length nhpSer-containing protein. • Phos-tag gel electrophoresis provides a convenient method to confirm accurate nhpSer encoding, as it can distinguish between non-phosphorylated, pSer- and nhpSer-containing variants.
PubMed: 37969748
DOI: 10.21769/BioProtoc.4861