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Current Research in Food Science 2024Alcohol liver disease (ALD) is a liver disease caused by long-term heavy drinking. Glucosamine (GLC) is an amino monosaccharide that plays a very important role in the...
Alcohol liver disease (ALD) is a liver disease caused by long-term heavy drinking. Glucosamine (GLC) is an amino monosaccharide that plays a very important role in the synthesis of human and animal cartilage. GLC is commonly used in the treatment of mild to moderate osteoarthritis and has good anti-inflammatory and antioxidant properties. In this study, alcoholic injury models were constructed in mice and human normal hepatocyte L02 cells to explore the protective effect and mechanism of GLC on ALD. Mice were given GLC by gavage for 30 days. Liver injury models of both mice and L02 cells were produced by ethanol. Detecting the levels of liver injury biomarkers, lipid metabolism, oxidative stress biomarkers, and inflammatory factors through different reagent kits. Exploring oxidative and inflammatory pathways in mouse liver tissue through Western blot and RT-PCR. The results showed that GLC can significantly inhibit the abnormal increase of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), triglycerides (TG), total cholesterol (TC), very low density lipoprotein (VLDL), low-density lipoprotein cholesterol (LDL-C), and can significantly improve the level of high-density lipoprotein cholesterol (HDL-C). In addition, GLC intervention significantly improved alcohol induced hepatic oxidative stress by reducing the levels of malondialdehyde (MDA) and, increasing the levels of glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD) in the liver. Further mechanisms suggest that GLC can inhibit the expression of ethanol metabolism enzyme cytochrome P4502E1 (CYP2E1), activate the antioxidant pathway Keap1/Nrf2/HO-1, down-regulate the phosphorylation of MAPK and NF-κB signaling pathways, and thus reduce the expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Therefore, GLC may be a significant candidate functional food for attenuating alcohol induced acute liver injury.
PubMed: 38420347
DOI: 10.1016/j.crfs.2024.100699 -
Nature Communications Feb 2024Oxidative (or respiratory) burst confers host defense against pathogens by generating reactive species, including reactive nitrogen species (RNS). The microbial...
Oxidative (or respiratory) burst confers host defense against pathogens by generating reactive species, including reactive nitrogen species (RNS). The microbial infection-induced excessive RNS damages many biological molecules via S-nitrosothiol (SNO) accumulation. However, the mechanism by which the host enables innate immunity activation during oxidative burst remains largely unknown. Here, we demonstrate that S-nitrosoglutathione (GSNO), the main endogenous SNO, attenuates innate immune responses against herpes simplex virus-1 (HSV-1) and Listeria monocytogenes infections. Mechanistically, GSNO induces the S-nitrosylation of stimulator of interferon genes (STING) at Cys257, inhibiting its binding to the second messenger cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Alcohol dehydrogenase 5 (ADH5), the key enzyme that metabolizes GSNO to decrease cellular SNOs, facilitates STING activation by inhibiting S-nitrosylation. Concordantly, Adh5 deficiency show defective STING-dependent immune responses upon microbial challenge and facilitates viral replication. Thus, cellular oxidative burst-induced RNS attenuates the STING-mediated innate immune responses to microbial infection, while ADH5 licenses STING activation by maintaining cellular SNO homeostasis.
Topics: S-Nitrosothiols; Membrane Proteins; Herpesvirus 1, Human; Immunity, Innate; Homeostasis; Aldehyde Oxidoreductases
PubMed: 38409248
DOI: 10.1038/s41467-024-46212-z -
Microbial Cell Factories Feb 2024Komagataella phaffii (a.k.a. Pichia pastoris) harbors a glutamate utilization pathway in which synthesis of glutamate dehydrogenase 2 and phosphoenolpyruvate...
BACKGROUND
Komagataella phaffii (a.k.a. Pichia pastoris) harbors a glutamate utilization pathway in which synthesis of glutamate dehydrogenase 2 and phosphoenolpyruvate carboxykinase (PEPCK) is induced by glutamate. Glutamate-inducible synthesis of these enzymes is regulated by Rtg1p, a cytosolic, basic helix-loop-helix protein. Here, we report food-grade monosodium glutamate (MSG)-inducible recombinant protein production from K. phaffii PEPCK promoter (P) using green fluorescent protein (GFP) and receptor binding domain of SARS-CoV-2 virus (RBD) as model proteins.
RESULTS
P-RBD/GFP expression cassette was integrated at two different sites in the genome to improve recombinant protein yield from P. The traditional, methanol-inducible alcohol oxidase 1 promoter (P) was used as the benchmark. Initial studies carried out with MSG as the inducer resulted in low recombinant protein yield. A new strategy employing MSG/ethanol mixed feeding improved biomass generation as well as recombinant protein yield. Cell density of 100-120 A units/ml was achieved after 72 h of induction in shake flask cultivations, resulting in recombinant protein yield from P that is comparable or even higher than that from P.
CONCLUSIONS
We have designed an induction medium for recombinant protein production from K. phaffii P in shake flask cultivations. It consists of 1.0% yeast extract, 2.0% peptone, 0.17% yeast nitrogen base with ammonium sulfate, 100 mM potassium phosphate (pH 6.0), 0.4 mg/L biotin, 2.0% MSG, and 2% ethanol. Substitution of ammonium sulphate with 0.5% urea is optional. Carbon source was replenished every 24 h during 72 h induction period. Under these conditions, GFP and RBD yields from P equaled and even surpassed those from P. Compared to the traditional methanol-inducible expression system, the inducers of glutamate-inducible expression system are non-toxic and their metabolism does not generate toxic metabolites such as formaldehyde and hydrogen peroxide. This study sets the stage for MSG-inducible, industrial scale recombinant protein production from K. phaffii P in bioreactors.
Topics: Methanol; Sodium Glutamate; Recombinant Proteins; Glutamates; Saccharomycetales; Ethanol; Pichia
PubMed: 38402195
DOI: 10.1186/s12934-024-02340-1 -
Polymers Feb 2024Bioactive materials may be applied in tissue regeneration, and an example of such materials are wound dressings, which are used to accelerate skin healing, especially...
Bioactive materials may be applied in tissue regeneration, and an example of such materials are wound dressings, which are used to accelerate skin healing, especially after trauma. Here, we proposed a novel dressing enriched by a bioactive component. The aim of our study was to prepare and characterize poly(vinyl alcohol) films modified with strontium titanate nanoparticles. The physicochemical properties of films were studied, such as surface free energy and surface roughness, as well as the mechanical properties of materials. Moreover, different biological studies were carried out, like in vitro hemo- and cyto-compatibility, biocidal activity, and anti-biofilm formation. Also, the degradation of the materials' utilization possibilities and enzymatic activity in compost were checked. The decrease of surface free energy, increase of roughness, and improvement of mechanical strength were found after the addition of nanoparticles. All developed films were cyto-compatible, and did not induce a hemolytic effect on the human erythrocytes. The PVA films containing the highest concentration of STO (20%) reduced the proliferation of , , and significantly. Also, all films were characterized by surface anti-biofilm activity, as they significantly lowered the bacterial biofilm abundance and its dehydrogenase activity. The films were degraded by the compost microorganism. However, PVA with the addition of 20%STO was more difficult to degrade. Based on our results, for wound dressing application, we suggest using bioactive films based on PVA + 20%STO, as they were characterized by high antibacterial properties, favorable physicochemical characteristics, and good biocompatibility with human cells.
PubMed: 38399862
DOI: 10.3390/polym16040484 -
Microorganisms Feb 2024is an extremely thermophilic bacterium capable of growing at 90 °C and producing ethanol, which is catalyzed by an alcohol dehydrogenase (ADH). The gene encoding ADH...
is an extremely thermophilic bacterium capable of growing at 90 °C and producing ethanol, which is catalyzed by an alcohol dehydrogenase (ADH). The gene encoding ADH (ADH) was cloned, sequenced and over-expressed. The gene sequence (1164 bp) was obtained by sequencing all fragments of the gene, which were amplified from the genomic DNA. The deduced amino acid sequence showed high identity to iron-containing ADHs from other species and harbored typical iron- and NADP-binding motifs, Asp195His199His268His282 and Gly39Gly40Gly41Ser42, respectively. Structural modeling showed that the N-terminal domain of ADH contains an α/β-dinucleotide-binding motif and that its C-terminal domain is an α-helix-rich region containing the iron-binding motif. The recombinant ADH was soluble, active, and thermostable, with a subunit size of 43 ± 1 kDa revealed by SDS-PAGE analyses. The recombinant ADH (69 ± 2 U/mg) was shown to have similar properties to the native enzyme. The optimal pH values for alcohol oxidation and aldehyde reduction were 11.0 and 8.0, respectively. It was also thermostable, with a half-life of 5 h at 70 °C. The successful expression of the recombinant ADH in significantly enhanced the yield of enzyme production and thus will facilitate further investigation of the catalytic mechanisms of iron-containing ADHs.
PubMed: 38399715
DOI: 10.3390/microorganisms12020311 -
Biomolecules Jan 2024Holm oak () is considered to be one of the major structural elements of Mediterranean forests and the agrosilvopastoral Spanish "dehesa", making it an outstanding...
Holm oak () is considered to be one of the major structural elements of Mediterranean forests and the agrosilvopastoral Spanish "dehesa", making it an outstanding example of ecological and socioeconomic sustainability in forest ecosystems. The exotic is one of the most aggressive pathogens of woody species and, together with drought, is considered to be one of the main drivers of holm oak decline. The effect of and response to inoculation were studied in the offspring of mother trees from two Andalusian populations, Cordoba and Huelva. At the two locations, acorns collected from both symptomatic (damaged) and asymptomatic (apparently healthy) trees were sampled. Damage symptoms, mortality, and chlorophyll fluorescence were evaluated in seedlings inoculated under humid and drought conditions. The effect and response depended on the population and were more apparent in Huelva than in Cordoba. An integrated proteomic and metabolomic analysis revealed the involvement of different metabolic pathways in response to the pathogen in both populations, including amino acid metabolism pathways in Huelva, and terpenoid and flavonoid biosynthesis in Cordoba. However, no differential response was observed between seedlings inoculated under humid and drought conditions. A protective mechanism of the photosynthetic apparatus was activated in response to defective photosynthetic activity in inoculated plants, which seemed to be more efficient in the Cordoba population. In addition, enzymes and metabolites of the phenylpropanoid and flavonoid biosynthesis pathways may have conferred higher resistance in the Cordoba population. Some enzymes are proposed as markers of resilience, among which glyoxalase I, glutathione reductase, thioredoxin reductase, and cinnamyl alcohol dehydrogenase are candidates.
Topics: Ecosystem; Quercus; Droughts; Phytophthora; Proteomics; Trees; Seedlings; Flavonoids
PubMed: 38397397
DOI: 10.3390/biom14020160 -
International Journal of Molecular... Feb 2024Hepatocellular carcinoma (HCC), the main pathological type of liver cancer, is related to risk factors such as viral hepatitis, alcohol intake, and non-alcoholic fatty...
Hepatocellular carcinoma (HCC), the main pathological type of liver cancer, is related to risk factors such as viral hepatitis, alcohol intake, and non-alcoholic fatty liver disease (NAFLD). The constitutive activation of the PI3K/AKT signaling pathway is common in HCC and has essential involvement in tumor progression. The serine/threonine kinase AKT has several downstream substrates, which have been implicated in the regulation of cellular metabolism. However, the contribution of each of the three AKT isoforms, i.e., AKT1, AKT2 and AKT3, to HCC metabolism has not been comprehensively investigated. In this study, we analyzed the functional role of AKT1, AKT2 and AKT3 in HCC metabolism. The overexpression of activated AKT1, AKT2 and AKT3 isoforms in the human HCC cell lines Hep3B and Huh7 resulted in higher oxygen consumption rate (OCR), ATP production, maximal respiration and spare respiratory capacity in comparison to vector-transduced cells. Vice versa, lentiviral vector-mediated knockdowns of each AKT isoform reduced OCR in both cell lines. Reduced OCR rates observed in the three AKT isoform knockdowns were associated with reduced extracellular acidification rates (ECAR) and reduced lactate production in both analyzed cell lines. Mechanistically, the downregulation of OCR by AKT isoform knockdowns correlated with an increased phosphorylation of the pyruvate dehydrogenase on Ser232, which negatively regulates the activity of this crucial gatekeeper of mitochondrial respiration. In summary, our data indicate that each of the three AKT isoforms is able to upregulate OCR, ECAR and lactate production independently of each other in human HCC cells through the regulation of the pyruvate dehydrogenase.
Topics: Humans; Carcinoma, Hepatocellular; Cell Line, Tumor; Lactic Acid; Liver Neoplasms; Oxidoreductases; Oxygen; Phosphatidylinositol 3-Kinases; Protein Isoforms; Proto-Oncogene Proteins c-akt; Pyruvates
PubMed: 38396845
DOI: 10.3390/ijms25042168 -
Journal of Fungi (Basel, Switzerland) Feb 2024The medium-chain dehydrogenase/reductase (MDR) superfamily contains many members that are widely present in organisms and play important roles in growth, metabolism, and...
Genome-Wide Identification and Characterization of the Medium-Chain Dehydrogenase/Reductase Superfamily of and Its Involvement in the Regulation of Fluconazole Resistance.
The medium-chain dehydrogenase/reductase (MDR) superfamily contains many members that are widely present in organisms and play important roles in growth, metabolism, and stress resistance but have not been studied in . In this study, bioinformatics and RNA sequencing methods were used to analyze the MDR superfamily of and its regulatory effect on fluconazole resistance. A phylogenetic tree was constructed using , , and and 73 were identified, all of which contained NADPH-binding motifs. contained 20 that were unevenly distributed across six chromosomes. () had similar 3D structures but varied greatly in their genetic evolution at different phylum levels. RNA-seq and gene expression analyses revealed that the fluconazole-resistant strain upregulates xylitol dehydrogenase, and downregulated alcohol dehydrogenase and sorbitol dehydrogenase concluded that the fluconazole-resistant strain was less selective toward carbon sources and had higher adaptability to the environment. Overall, our study contributes to our understanding of , providing a basis for further analysis of the genes associated with drug resistance in .
PubMed: 38392795
DOI: 10.3390/jof10020123 -
Biology Feb 2024Ammonium (NH) toxicity is ubiquitous in plants. To investigate the underlying mechanisms of this toxicity and bicarbonate (HCO)-dependent alleviation, wheat plants were...
Ammonium (NH) toxicity is ubiquitous in plants. To investigate the underlying mechanisms of this toxicity and bicarbonate (HCO)-dependent alleviation, wheat plants were hydroponically cultivated in half-strength Hoagland nutrient solution containing 7.5 mM NO (CK), 7.5 mM NH (SA), or 7.5 mM NH + 3 mM HCO (AC). Transcriptomic analysis revealed that compared to CK, SA treatment at 48 h significantly upregulated the expression of genes encoding fermentation enzymes (pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), and lactate dehydrogenase (LDH)) and oxygen consumption enzymes (respiratory burst oxidase homologs, dioxygenases, and alternative oxidases), downregulated the expression of genes encoding oxygen transporters (PIP-type aquaporins, non-symbiotic hemoglobins), and those involved in energy metabolism, including tricarboxylic acid (TCA) cycle enzymes and ATP synthases, but upregulated the glycolytic enzymes in the roots and downregulated the expression of genes involved in the cell cycle and elongation. The physiological assay showed that SA treatment significantly increased PDC, ADH, and LDH activity by 36.69%, 43.66%, and 61.60%, respectively; root ethanol concentration by 62.95%; and lactate efflux by 23.20%, and significantly decreased the concentrations of pyruvate and most TCA cycle intermediates, the complex V activity, ATP content, and ATP/ADP ratio. As a consequence, SA significantly inhibited root growth. AC treatment reversed the changes caused by SA and alleviated the inhibition of root growth. In conclusion, NH treatment alone may cause hypoxic stress in the roots, inhibit energy generation, suppress cell division and elongation, and ultimately inhibit root growth, and adding HCO remarkably alleviates the NH-induced inhibitory effects on root growth largely by attenuating the hypoxic stress.
PubMed: 38392319
DOI: 10.3390/biology13020101 -
Frontiers in Plant Science 2023With current trends in global climate change, both flooding episodes and higher levels of CO have been key factors to impact plant growth and stress tolerance. Very...
INTRODUCTION
With current trends in global climate change, both flooding episodes and higher levels of CO have been key factors to impact plant growth and stress tolerance. Very little is known about how both factors can influence the microbiome diversity and function, especially in tolerant soybean cultivars. This work aims to (i) elucidate the impact of flooding stress and increased levels of CO on the plant defenses and (ii) understand the microbiome diversity during flooding stress and elevated CO (eCO).
METHODS
We used next-generation sequencing and bioinformatic methods to show the impact of natural flooding and eCO on the microbiome architecture of soybean plants' below- (soil) and above-ground organs (root and shoot). We used high throughput rhizospheric extra-cellular enzymes and molecular analysis of plant defense-related genes to understand microbial diversity in plant responses during eCO and flooding.
RESULTS
Results revealed that bacterial and fungal diversity was substantially higher in combined flooding and eCO treatments than in non-flooding control. Microbial diversity was soil>root>shoot in response to flooding and eCO. We found that sole treatment of eCO and flooding had significant abundances of , and . Whereas the combination of flooding and eCO2 conditions showed a significant abundance of and . Rhizospheric extra-cellular enzyme activities were significantly higher in eCO than flooding or its combination with eCO. Plant defense responses were significantly regulated by the oxidative stress enzyme activities and gene expression of and in floodings and eCO treatments in soybean plant root or shoot parts.
CONCLUSION
This work suggests that climatic-induced changes in eCO and submergence can reshape microbiome structure and host defenses, essential in plant breeding and developing stress-tolerant crops. This work can help in identifying core-microbiome species that are unique to flooding stress environments and increasing eCO.
PubMed: 38389716
DOI: 10.3389/fpls.2023.1295674