Resolving the metabolism of monolignols and other lignin-related aromatic compounds in Xanthomonas citri.

Lignin, a major plant cell wall component, has an important role in plant-defense mechanisms against pathogens and is a promising renewable carbon source to produce bio-based chemicals. However, our understanding of microbial metabolism is incomplete regarding certain lignin-related compounds like p-coumaryl and sinapyl alcohols. Here, we reveal peripheral pathways for the catabolism of the three main lignin precursors (p-coumaryl, coniferyl, and sinapyl alcohols) in the plant pathogen Xanthomonas citri. Our study demonstrates all the necessary enzymatic steps for funneling these monolignols into the tricarboxylic acid cycle, concurrently uncovering aryl aldehyde reductases that likely protect the pathogen from aldehydes toxicity. It also shows that lignin-related aromatic compounds activate transcriptional responses related to chemotaxis and flagellar-dependent motility, which might play an important role during plant infection. Together our findings provide foundational knowledge to support biotechnological advances for both plant diseases treatments and conversion of lignin-derived compounds into bio-based chemicals.

Phylogenetic diversity of putative nickel-containing carbon monoxide dehydrogenase-encoding prokaryotes in the human gut microbiome.

Although the production of carbon monoxide (CO) within the human body has been detected, only two CO-utilizing prokaryotes (CO utilizers) have been reported in the human gut. Therefore, the phylogenetic diversity of the human gut CO-utilizing prokaryotes remains unclear. Here, we unveiled more than a thousand representative genomes containing genes for putative nickel-containing CO dehydrogenase (pCODH), an essential enzyme for CO utilization. The taxonomy of genomes encoding pCODH was expanded to include 8 phyla, comprising 82 genera and 248 species. In contrast, putative molybdenum-containing CODH genes were not detected in the human gut microbial genomes. pCODH transcripts were detected in 97.3 % (n=110) of public metatranscriptome datasets derived from healthy human faeces, suggesting the ubiquitous presence of prokaryotes bearing transcriptionally active pCODH genes in the human gut. More than half of the pCODH-encoding genomes contain a set of genes for the autotrophic Wood-Ljungdahl pathway (WLP). However, 79 % of these genomes commonly lack a key gene for the WLP, which encodes the enzyme that synthesizes formate from CO2, suggesting that potential human gut CO-utilizing prokaryotes share a degenerated gene set for WLP. In the other half of the pCODH-encoding genomes, seven genes, including putative genes for flavin adenine dinucleotide-dependent NAD(P) oxidoreductase (FNOR), ABC transporter and Fe-hydrogenase, were found adjacent to the pCODH gene. None of the putative genes associated with CO-oxidizing respiratory machinery, such as energy-converting hydrogenase genes, were found in pCODH-encoding genomes. This suggests that the human gut CO utilization is not for CO removal, but potentially for fixation and/or biosynthesis, consistent with the harmless yet continuous production of CO in the human gut. Our findings reveal the diversity and distribution of prokaryotes with pCODH in the human gut microbiome, suggesting their potential contribution to microbial ecosystems in human gut environments.

Improving the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one in Mycolicibacterium smegmatis.

The 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) is a C22 steroid synthon of pharmaceutical interest that can be produced as a lateral end-product of the catabolism of natural sterols (e.g., cholesterol or phytosterols). This work studies the role of an aldehyde dehydrogenase coded by the MSMEG_6563 gene of Mycolicibacterium smegmatis, named msRed, in 4-HBC production. This gene is located contiguously to the MSMEG_6561 encoding the aldolase msSal which catalyses the retroaldol elimination of acetyl-CoA of the metabolite intermediate 22-hydroxy-3-oxo-cholest-4-ene-24-carboxyl-CoA to deliver 3-oxo-4-pregnene-20-carboxyl aldehyde (3-OPA). We have demonstrated that msRed reduces 3-OPA to 4-HBC. Moreover, the role of msOpccR reductase encoded by MSMEG_1623 was also explored confirming that it also performs the reduction of 3-OPA into 4-HBC, but less efficiently than msRed. To obtain a M. smegmatis 4-HBC producer strain we deleted MSMEG_5903 (hsd4A) gene in strain MS6039-5941 (ΔkshB1, ΔkstD1) that produces 4-androstene-3,17-dione (AD) from natural sterols (cholesterol or phytosterols). The triple MS6039-5941-5903 mutant was able to produce 9 g/L of 4-HBC from 14 g/L of phytosterols in 2 L bioreactor, showing a productivity of 0.140 g/L h-1. To improve the metabolic flux of sterols towards the production of 4-HBC we have cloned and overexpressed the msSal and msRed enzymes in the MS6039-5941-5903 mutant rendering a production titter of 12.7 g/L with a productivity of 0.185 g/L h-1, and demonstrating that the new recombinant strain has a great potential for its industrial application.

Glycometabolic reprogramming-induced XRCC1 lactylation confers therapeutic resistance in ALDH1A3-overexpressing glioblastoma.

Patients with high ALDH1A3-expressing glioblastoma (ALDH1A3hi GBM) show limited benefit from postoperative chemoradiotherapy. Understanding the mechanisms underlying such resistance in these patients is crucial for the development of new treatments. Here, we show that the interaction between ALDH1A3 and PKM2 enhances the latter's tetramerization and promotes lactate accumulation in glioblastoma stem cells (GSCs). By scanning the lactylated proteome in lactate-accumulating GSCs, we show that XRCC1 undergoes lactylation at lysine 247 (K247). Lactylated XRCC1 shows a stronger affinity for importin α, allowing for greater nuclear transposition of XRCC1 and enhanced DNA repair. Through high-throughput screening of a small-molecule library, we show that D34-919 potently disrupts the ALDH1A3-PKM2 interaction, preventing the ALDH1A3-mediated enhancement of PKM2 tetramerization. In vitro and in vivo treatment with D34-919 enhanced chemoradiotherapy-induced apoptosis of GBM cells. Together, our findings show that ALDH1A3-mediated PKM2 tetramerization is a potential therapeutic target to improve the response to chemoradiotherapy in ALDH1A3hi GBM.

ALDH1A3-acetaldehyde metabolism potentiates transcriptional heterogeneity in melanoma.

Cancer cellular heterogeneity and therapy resistance arise substantially from metabolic and transcriptional adaptations, but how these are interconnected is poorly understood. Here, we show that, in melanoma, the cancer stem cell marker aldehyde dehydrogenase 1A3 (ALDH1A3) forms an enzymatic partnership with acetyl-coenzyme A (CoA) synthetase 2 (ACSS2) in the nucleus to couple high glucose metabolic flux with acetyl-histone H3 modification of neural crest (NC) lineage and glucose metabolism genes. Importantly, we show that acetaldehyde is a metabolite source for acetyl-histone H3 modification in an ALDH1A3-dependent manner, providing a physiologic function for this highly volatile and toxic metabolite. In a zebrafish melanoma residual disease model, an ALDH1-high subpopulation emerges following BRAF inhibitor treatment, and targeting these with an ALDH1 suicide inhibitor, nifuroxazide, delays or prevents BRAF inhibitor drug-resistant relapse. Our work reveals that the ALDH1A3-ACSS2 couple directly coordinates nuclear acetaldehyde-acetyl-CoA metabolism with specific chromatin-based gene regulation and represents a potential therapeutic vulnerability in melanoma.

Beneficial base substitutions in Escherichia coli fucO gene for enhancement of glycolic acid production.

Microbial production of glycolic acid (GA) from ethylene glycol is extensively used in a variety of industries because ethylene glycol is not only an inexpensive raw material but also the main component of industrial wastes. In this study, we produced GA from ethylene glycol using Escherichia coli overexpressing the endogenous 1,2-propanediol oxidoreductase (fucO) and lactaldehyde dehydrogenase (aldA) genes. To increase GA productivity, we screened a random mutant library generated using an error-prone polymerase chain reaction of fucO and obtained FucO mutants MF2-9 and MF6-9 with enhanced GA production in Lysogeny Broth medium containing 800 mM ethylene glycol. MF2-9 contained three amino acid substitutions (D23E, E222K, and G363S) and two synonymous mutations (coding DNA [c.] 93G > A and c.1131T > C) in fucO. MF6-9 contained one amino acid substitution (L377H) in FucO. An amino acid substitution (L377H) and a single synonymous mutation (c.1131T > C) in fucO contributed to the enhancement in GA production. Notably, cell lysates from E. coli harboring a synonymous mutation (c.1131T > C) or amino acid substitution (L377H) in fucO showed that only AldA activity was 1.3-fold higher than that of the cell lysate from E. coli harboring the wild-type fucO. We confirmed that c.1131T > C and L377H mutations increased aldA expression in E. coli. Analysis of mRNA levels and simulation of mRNA stabilization indicated that base substitutions at positions c.1130T, which corresponds to L377H amino acid substitution, and c.1131T increased aldA expression due to partial destabilization of the mRNA. These findings will be useful for the large-scale microbial production of GA from industrial waste.

Non-canonical NF-κB signaling limits the tolerogenic ß-catenin-Raldh2 axis in gut dendritic cells to exacerbate intestinal pathologies.

Dendritic cell (DC) dysfunction is known to exacerbate intestinal pathologies, but the mechanisms compromising DC-mediated immune regulation in this context remain unclear. Here, we show that intestinal dendritic cells from a mouse model of experimental colitis exhibit significant levels of noncanonical NF-κB signaling, which activates the RelB:p52 heterodimer. Genetic inactivation of this pathway in DCs alleviates intestinal pathologies in mice suffering from colitis. Deficiency of RelB:p52 diminishes transcription of Axin1, a critical component of the ß-catenin destruction complex, reinforcing ß-catenin-dependent expression of Raldh2, which imparts tolerogenic DC attributes by promoting retinoic acid synthesis. DC-specific impairment of noncanonical NF-κB signaling leads to increased colonic numbers of Tregs and IgA+ B cells, which promote luminal IgA production and foster eubiosis. Experimentally introduced ß-catenin haploinsufficiency in DCs with deficient noncanonical NF-κB signaling moderates Raldh2 activity, reinstating colitogenic sensitivity in mice. Finally, inflammatory bowel-disease patients also display a deleterious noncanonical NF-κB signaling signature in intestinal DCs. In sum, we establish how noncanonical NF-κB signaling in dendritic cells can subvert retinoic acid synthesis to fuel intestinal inflammation.

Metabolic engineering of Komagataella phaffii for the efficient utilization of methanol.

BACKGROUND: Komagataella phaffii, a type of methanotrophic yeast, can use methanol, a favorable non-sugar substrate in eco-friendly bio-manufacturing. The dissimilation pathway in K. phaffii leads to the loss of carbon atoms in the form of CO2. However, the ΔFLD strain, engineered to lack formaldehyde dehydrogenase-an essential enzyme in the dissimilation pathway-displayed growth defects when exposed to a methanol-containing medium. RESULTS: Inhibiting the dissimilation pathway triggers an excessive accumulation of formaldehyde and a decline in the intracellular NAD+/NADH ratio. Here, we designed dual-enzyme complex with the alcohol oxidase1/dihydroxyacetone synthase1 (Aox1/Das1), enhancing the regeneration of the formaldehyde receptor xylulose-5-phosphate (Xu5P). This strategy mitigated the harmful effects of formaldehyde accumulation and associated toxicity to cells. Concurrently, we elevated the NAD+/NADH ratio by overexpressing isocitrate dehydrogenase in the TCA cycle, promoting intracellular redox homeostasis. The OD600 of the optimized combination of the above strategies, strain DF02-1, was 4.28 times higher than that of the control strain DF00 (ΔFLD, HIS4+) under 1% methanol. Subsequently, the heterologous expression of methanol oxidase Mox from Hansenula polymorpha in strain DF02-1 resulted in the recombinant strain DF02-4, which displayed a growth at an OD600 4.08 times higher than that the control strain DF00 in medium containing 3% methanol. CONCLUSIONS: The reduction of formaldehyde accumulation, the increase of NAD+/NADH ratio, and the enhancement of methanol oxidation effectively improved the efficient utilization of a high methanol concentration by strain ΔFLD strain lacking formaldehyde dehydrogenase. The modification strategies implemented in this study collectively serve as a foundational framework for advancing the efficient utilization of methanol in K. phaffii.

S-nitrosoglutathione reductase alleviates morphine analgesic tolerance by restricting PKCα S-nitrosation.

Morphine, a typical opiate, is widely used for controlling pain but can lead to various side effects with long-term use, including addiction, analgesic tolerance, and hyperalgesia. At present, however, the mechanisms underlying the development of morphine analgesic tolerance are not fully understood. This tolerance is influenced by various opioid receptor and kinase protein modifications, such as phosphorylation and ubiquitination. Here, we established a murine morphine tolerance model to investigate whether and how S-nitrosoglutathione reductase (GSNOR) is involved in morphine tolerance. Repeated administration of morphine resulted in the down-regulation of GSNOR, which increased excessive total protein S-nitrosation in the prefrontal cortex. Knockout or chemical inhibition of GSNOR promoted the development of morphine analgesic tolerance and neuron-specific overexpression of GSNOR alleviated morphine analgesic tolerance. Mechanistically, GSNOR deficiency enhanced S-nitrosation of cellular protein kinase alpha (PKCα) at the Cys78 and Cys132 sites, leading to inhibition of PKCα kinase activity, which ultimately promoted the development of morphine analgesic tolerance. Our study highlighted the significant role of GSNOR as a key regulator of PKCα S-nitrosation and its involvement in morphine analgesic tolerance, thus providing a potential therapeutic target for morphine tolerance.

Structural identification and comprehension of human ALDH1L1-Gossypol complex.

The folate metabolism enzyme ALDH1L1 catalyzed 10-formyltetrahydrofolate to tetrahydrofolate and CO2. Non-small cell lung cancer cells (NSCLC) strongly express ALDH1L1. Gossypol binds to an allosteric site and disrupts the folate metabolism by preventing NADP+ binding. The Cryo-EM structures of tetrameric C-terminal aldehyde dehydrogenase human ALDH1L1 complex with gossypol were examined. Gossypol-bound ALDH1L1 interfered with NADP+ by shifting the allosteric site of the structural conformation, producing a closed-form NADP+ binding site. In addition, the inhibition activity of ALDH1L1 was targeted with gossypol in NSCLC. The gossypol treatment had anti-cancer effects on NSCLC by blocking NADPH and ATP production. These findings emphasize the structure characterizing ALDH1L1 with gossypol.

Distinct DNA repair mechanisms prevent formaldehyde toxicity during development, reproduction and aging.

Formaldehyde (FA) is a recognized environmental and metabolic toxin implicated in cancer development and aging. Inherited mutations in the FA-detoxifying enzymes ADH5 and ALDH2 genes lead to FA overload in the severe multisystem AMeD syndrome. FA accumulation causes genome damage including DNA-protein-, inter- and intra-strand crosslinks and oxidative lesions. However, the influence of distinct DNA repair systems on organismal FA resistance remains elusive. We have here investigated the consequence of a range of DNA repair mutants in a model of endogenous FA overload generated by downregulating the orthologs of human ADH5 and ALDH2 in C. elegans. We have focused on the distinct components of nucleotide excision repair (NER) during developmental growth, reproduction and aging. Our results reveal three distinct modes of repair of FA-induced DNA damage: Transcription-coupled repair (TCR) operating NER-independently during developmental growth or through NER during adulthood, and, in concert with global-genome (GG-) NER, in the germline and early embryonic development. Additionally, we show that the Cockayne syndrome B (CSB) factor is involved in the resolution of FA-induced DNA-protein crosslinks, and that the antioxidant and FA quencher N-acetyl-l-cysteine (NAC) reverses the sensitivity of detoxification and DNA repair defects during development, suggesting a therapeutic intervention to revert FA-pathogenic consequences.

Coupling CO2 Reduction and Acetyl-CoA Formation: The Role of a CO Capturing Tunnel in Enzymatic Catalysis.

The bifunctional CO-dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex couples the reduction of CO2 to the condensation of CO with a methyl moiety and CoA to acetyl-CoA. Catalysis occurs at two sites connected by a tunnel transporting the CO. In this study, we investigated how the bifunctional complex and its tunnel support catalysis using the CODH/ACS from Carboxydothermus hydrogenoformans as a model. Although CODH/ACS adapted to form a stable bifunctional complex with a secluded substrate tunnel, catalysis and CO transport is even more efficient when two monofunctional enzymes are coupled. Efficient CO channeling appears to be ensured by hydrophobic binding sites for CO, which act in a bucket-brigade fashion rather than as a simple tube. Tunnel remodeling showed that opening the tunnel increased activity but impaired directed transport of CO. Constricting the tunnel impaired activity and CO transport, suggesting that the tunnel evolved to sequester CO rather than to maximize turnover.

S-nitrosylation may inhibit the activity of COP1 in plant photomorphogenesis.

Protein S-nitrosylation, which is defined by the covalent attachment of nitric oxide (NO) to the thiol group of cysteine residues, is known to play critical roles in plant development and stress responses. NO promotes seedling photomorphogenesis and NO emission is enhanced by light. However, the function of protein S-nitrosylation in plant photomorphogenesis is largely unknown. E3 ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) and transcription factor ELONGATED HYPOCOTYL 5 (HY5) antagonistically regulate seedling photomorphogenesis. COP1 inhibits plant photomorphogenesis by targeting photomorphogenic promoters like HY5 for 26S proteasome degradation. Here, we report that COP1 is S-nitrosylated in vitro. Mass spectrometry analyses revealed that two evolutionarily well conserved residues, cysteine 425 and cysteine 607, in the WD40 domain of COP1 are S-nitrosylated. S-nitrosylated glutathione (GSNO) is an important physiological NO donor for protein S-nitrosylation. The Arabidopsis (Arabidopsis thaliana) gsnor1-3 mutant, which accumulates higher level of GSNO, accumulated higher HY5 levels than wildtype (WT), indicating that COP1 activity is inhibited. Protein S-nitrosylation can be reversed by Thioredoxin-h5 (TRXh5) in plants. Indeed, COP1 interacts directly with TRXh5 and its close homolog TRXh3. Moreover, catalase 3 (CAT3) acts as a transnitrosylase that transfers NO to its target proteins like GSNO reductase (GSNOR). We found that CAT3 interacts with COP1 in plants. Taken together, our data indicate that the activity of COP1 is likely inhibited by NO via S-nitrosylation to promote the accumulation of HY5 and photomorphogenesis.

Pharmacologic Targeting of Histone H3K27 Acetylation/BRD4-dependent Induction of ALDH1A3 for Early-phase Drug Tolerance of Gastric Cancer.

Anticancer drug-tolerant persister (DTP) cells at an early phase of chemotherapy reshape refractory tumors. Aldehyde dehydrogenase 1 family member A3 (ALDH1A3) is commonly upregulated by various anticancer drugs in gastric cancer patient-derived cells (PDC) and promotes tumor growth. However, the mechanism underlying the generation of ALDH1A3-positive DTP cells remains elusive. Here, we investigated the mechanism of ALDH1A3 expression and a combination therapy targeting gastric cancer DTP cells. We found that gastric cancer tissues treated with neoadjuvant chemotherapy showed high ALDH1A3 expression. Chromatin immunoprecipitation (ChIP)-PCR and ChIP sequencing analyses revealed that histone H3 lysine 27 acetylation was enriched in the ALDH1A3 promoter in 5-fluorouracil (5-FU)-tolerant persister PDCs. By chemical library screening, we found that the bromodomain and extraterminal (BET) inhibitors OTX015/birabresib and I-BET-762/molibresib suppressed DTP-related ALDH1A3 expression and preferentially inhibited DTP cell growth. In DTP cells, BRD4, but not BRD2/3, was recruited to the ALDH1A3 promoter and BRD4 knockdown decreased drug-induced ALDH1A3 upregulation. Combination therapy with 5-FU and OTX015 significantly suppressed in vivo tumor growth. These observations suggest that BET inhibitors are efficient DTP cell-targeting agents for gastric cancer treatment. SIGNIFICANCE: Drug resistance hampers the cure of patients with cancer. To prevent stable drug resistance, DTP cancer cells are rational therapeutic targets that emerge during the early phase of chemotherapy. This study proposes that the epigenetic regulation by BET inhibitors may be a rational therapeutic strategy to eliminate DTP cells.

CO Oxidation Mechanism of Silver-Substituted Mo/Cu CO-Dehydrogenase - Analogies and Differences to the Native Enzyme.

The aerobic oxidation of carbon monoxide to carbon dioxide is catalysed by the Mo/Cu-containing CO-dehydrogenase enzyme in the soil bacterium Oligotropha carboxidovorans, enabling the organism to grow on the small gas molecule as carbon and energy source. It was shown experimentally that silver can be substituted for copper in the active site of Mo/Cu CODH to yield a functional enzyme. In this study, we employed QM/MM calculations to investigate whether the reaction mechanism of the silver-substituted enzyme is similar to that of the native enzyme. Our results suggest that the Ag-substituted enzyme can oxidize CO and release CO2 following the same reaction steps as the native enzyme, with a computed rate-limiting step of 10.4 kcal/mol, consistent with experimental findings. Surprisingly, lower activation energies for C-O bond formation have been found in the presence of silver. Furthermore, comparison of rate constants for reduction of copper- and silver-containing enzymes suggests a discrepancy in the transition state stabilization upon silver substitution. We also evaluated the effects that differences in the water-active site interaction may exert on the overall energy profile of catalysis. Finally, the formation of a thiocarbonate intermediate along the catalytic pathway was found to be energetically unfavorable for the Ag-substituted enzyme. This finding aligns with the hypothesis proposed for the wild-type form, suggesting that the creation of such species may not be necessary for the enzymatic catalysis of CO oxidation.

Identification of a cinnamoyl-CoA reductase from Cinnamomum cassia involved in trans-cinnamaldehyde biosynthesis.

MAIN CONCLUSION: The identification of a functional cinnamoyl-CoA reductase enzyme from Cinnamomum cassia involved in trans-cinnamaldehyde biosynthesis offers the potential for enhancing trans-cinnamaldehyde production through genetic engineering. A significant accumulation of trans-cinnamaldehyde has been found in the bark tissues of C. cassia, used in traditional Chinese medicine. trans-Cinnamaldehyde exhibits various pharmacological properties such as anti-inflammatory, analgesic, and protection of the stomach and the digestive tract. However, further elucidation and characterization of the biosynthetic pathway for trans-cinnamaldehyde is required. In this study, we conducted an integrated analysis of trans-cinnamaldehyde accumulation profiles and transcriptomic data from five different C. cassia tissues to identify the genes involved in its biosynthesis. The transcriptome data we obtained included nearly all genes associated with the trans-cinnamaldehyde pathway, with the majority demonstrating high abundance in branch barks and trunk barks. We successfully cloned four C. cassia cinnamoyl-CoA reductases (CcCCRs), a key gene in trans-cinnamaldehyde biosynthesis. We found that the recombinant CcCCR1 protein was the only one that more efficiently converted cinnamoyl-CoA into trans-cinnamaldehyde. CcCCR1 exhibited approximately 14.7-fold higher catalytic efficiency (kcat/Km) compared to the Arabidopsis thaliana cinnamoyl-CoA reductase 1 (AtCCR1); therefore, it can be utilized for engineering higher trans-cinnamaldehyde production as previously reported. Molecular docking studies and mutagenesis experiments also validated the superior catalytic activity of CcCCR1 compared to AtCCR1. These findings provide valuable insights for the functional characterization of enzyme-coding genes and hold potential for future engineering of trans-cinnamaldehyde biosynthetic pathways.

GSNOR negatively regulates the NLRP3 inflammasome via S-nitrosation of MAPK14.

Hyperactivation of the NLRP3 inflammasome has been implicated in the pathogenesis of numerous diseases. However, the precise molecular mechanisms that modulate the transcriptional regulation of NLRP3 remain largely unknown. In this study, we demonstrated that S-nitrosoglutathione reductase (GSNOR) deficiency in macrophages leads to significant increases in the Nlrp3 and Il-1ß expression levels and interleukin-1ß (IL-1ß) secretion in response to NLRP3 inflammasome stimulation. Furthermore, in vivo experiments utilizing Gsnor-/- mice revealed increased disease severity in both lipopolysaccharide (LPS)-induced septic shock and dextran sodium sulfate (DSS)-induced colitis models. Additionally, we showed that both LPS-induced septic shock and DSS-induced colitis were ameliorated in Gsnor-/- Nlrp3-/- double-knockout (DKO) mice. Mechanistically, GSNOR deficiency increases the S-nitrosation of mitogen-activated protein kinase 14 (MAPK14) at the Cys211 residue and augments MAPK14 kinase activity, thereby promoting Nlrp3 and Il-1ß transcription and stimulating NLRP3 inflammasome activity. Our findings suggested that GSNOR is a regulator of the NLRP3 inflammasome and that reducing the level of S-nitrosylated MAPK14 may constitute an effective strategy for alleviating diseases associated with NLRP3-mediated inflammation.

Investigating human-derived lactic acid bacteria for alcohol resistance.

BACKGROUND: Excessive alcohol consumption has been consistently linked to serious adverse health effects, particularly affecting the liver. One natural defense against the detrimental impacts of alcohol is provided by alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), which detoxify harmful alcohol metabolites. Recent studies have shown that certain probiotic strains, notably Lactobacillus spp., possess alcohol resistance and can produce these critical enzymes. Incorporating these probiotics into alcoholic beverages represents a pioneering approach that can potentially mitigate the negative health effects of alcohol while meeting evolving consumer preferences for functional and health-centric products. RESULTS: Five lactic acid bacteria (LAB) isolates were identified: Lactobacillus paracasei Alc1, Lacticaseibacillus rhamnosus AA, Pediococcus acidilactici Alc3, Lactobacillus paracasei Alc4, and Pediococcus acidilactici Alc5. Assessment of their alcohol tolerance, safety, adhesion ability, and immunomodulatory effects identified L. rhamnosus AA as the most promising alcohol-tolerant probiotic strain. This strain also showed high production of ADH and ALDH. Whole genome sequencing analysis revealed that the L. rhamnosus AA genome contained both the adh (encoding for ADH) and the adhE (encoding for ALDH) genes. CONCLUSIONS: L. rhamnosus AA, a novel probiotic candidate, showed notable alcohol resistance and the capability to produce enzymes essential for alcohol metabolism. This strain is a highly promising candidate for integration into commercial alcoholic beverages upon completion of comprehensive safety and functionality evaluations.

[Study based on the acetaldehyde dehydrogenase 2 gene polymorphism and acetaminophen-induced liver injury].

Objective: To explore the association between aldehyde dehydrogenase 2 (ALDH2) gene polymorphisms and abnormal liver function-induced by acetaminophen (APAP) drugs. Methods: An ALDH2 gene knockout mouse model was constructed using CRISPR/Cas9 gene editing technology. The obtained heterozygous mice were mated with opposite sex of heterozygotes. Genomic DNA was extracted from the tail of the offspring mouse. The polymerase chain reaction (PCR) method was used to determine the ALDH2 genotype. APAP was further used to induce acute drug-induced liver injury models in wild-type and ALDH2 knockout mice. Blood and liver tissues of mice were collected for liver function index, HE staining, F4/80 immunohistochemistry, and other detections. The intergroup mean was compared using a one-way ANOVA. The LSD- t test was used for pairwise comparison. Results: ALDH2 knockout mice were bred successfully. The genotyping of the offspring was segregated into the wild-type (ALDH2(+/+)), heterozygous mutant (ALDH2(+/-)), and homozygous mutant (ALDH2(-/-)), respectively. Biochemical and histological results after APAP modeling showed that the level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TBil) was not significantly increased in the blank control group (P < 0.05), while the ALT, AST,ALP, and TBil were all elevated in the APAP experimental group. The levels of ALT (P  = 0.004), AST (P = 0.002), and TBil (P = 0.012) were significantly elevated among the mutant group compared to those in the wild-type group, and the expression levels of these indicators were also significantly elevated among the homozygous mutant group compared to those in the heterozygous mutant group (P = 0.003, 0 and 0.006). In addition, the ALP levels were higher in the heterozygous mutation group than those in the homozygous mutant group (P = 0.085) and wild-type group mice, but the difference was only statistically significant compared to wild-type mice (P = 0.002). HE staining results showed that mice in the APAP experimental group had hepatocyte degeneration, necrosis, and increased inflammatory cell infiltration, which was mostly evident in mutant mice. Simultaneously, the F4/80 immunohistochemical staining results showed that brown granules were visible in the liver tissue of APAP experimental group mice, and its expression levels were significantly enhanced compared to the blank control group. Conclusion: APAP-induced liver function abnormalities were associated with the ALDH2 gene polymorphism. The liver injury symptoms were increased in ALDH2 mutant mice following APAP modeling, and the ALDH2 gene defect may alleviate, to some extent, APAP-induced liver function abnormalities.