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Early-life establishment of tolerance to commensal bacteria at barrier surfaces carries enduring implications for immune health but remains poorly understood. Here, we showed that tolerance in skin was controlled by microbial interaction with a specialized subset of antigen-presenting cells. More particularly, CD301b+ type 2 conventional dendritic cells (DCs) in neonatal skin were specifically capable of uptake and presentation of commensal antigens for the generation of regulatory T (Treg) cells. CD301b+ DC2 were enriched for phagocytosis and maturation programs, while also expressing tolerogenic markers. In both human and murine skin, these signatures were reinforced by microbial uptake. In contrast to their adult counterparts or other early-life DC subsets, neonatal CD301b+ DC2 highly expressed the retinoic-acid-producing enzyme, RALDH2, the deletion of which limited commensal-specific Treg cell generation. Thus, synergistic interactions between bacteria and a specialized DC subset critically support early-life tolerance at the cutaneous interface.

Gases like H2, N2, CO2, and CO are increasingly recognized as critical feedstock in green energy conversion and as sources of nitrogen and carbon for the agricultural and chemical sectors. However, the industrial transformation of N2, CO2, and CO and the production of H2 require significant energy input, which renders processes like steam reforming and the Haber-Bosch reaction economically and environmentally unviable. Nature, on the other hand, performs similar tasks efficiently at ambient temperature and pressure, exploiting gas-processing metalloenzymes (GPMs) that bind low-valent metal cofactors based on iron, nickel, molybdenum, tungsten, and sulfur. Such systems are studied to understand the biocatalytic principles of gas conversion including N2 fixation by nitrogenase and H2 production by hydrogenase as well as CO2 and CO conversion by formate dehydrogenase, carbon monoxide dehydrogenase, and nitrogenase. In this review, we emphasize the importance of the cofactor/protein interface, discussing how second and outer coordination sphere effects determine, modulate, and optimize the catalytic activity of GPMs. These may comprise ionic interactions in the second coordination sphere that shape the electron density distribution across the cofactor, hydrogen bonding changes, and allosteric effects. In the outer coordination sphere, proton transfer and electron transfer are discussed, alongside the role of hydrophobic substrate channels and protein structural changes. Combining the information gained from structural biology, enzyme kinetics, and various spectroscopic techniques, we aim toward a comprehensive understanding of catalysis beyond the first coordination sphere.

Carbon fixation by chemoautotrophic microbes such as homoacetogens had a major impact on the transition from the inorganic to the organic world. Recent reports have shown the presence of genes for key enzymes associated with the Wood-Ljungdahl pathway (WLP) in the phylum Actinobacteria, which adds to the diversity of potential autotrophs. Here, we compiled 42 actinobacterial metagenome-assembled genomes (MAGs) from new and existing metagenomic datasets and propose three novel classes, Ca. Aquicultoria, Ca. Geothermincolia and Ca. Humimicrobiia. Most members of these classes contain genes coding for acetogenesis through the WLP, as well as a variety of hydrogenases (NiFe groups 1a and 3b-3d; FeFe group C; NiFe group 4-related hydrogenases). We show that the three classes acquired the hydrogenases independently, yet the carbon monoxide dehydrogenase/acetyl-CoA synthase complex (CODH/ACS) was apparently present in their last common ancestor and was inherited vertically. Furthermore, the Actinobacteria likely donated genes for CODH/ACS to multiple lineages within Nitrospirae, Deltaproteobacteria (Desulfobacterota), and Thermodesulfobacteria through multiple horizontal gene transfer events. Finally, we show the apparent growth of Ca. Geothermincolia and H2-dependent acetate production in hot spring enrichment cultures with or without the methanogenesis inhibitor 2-bromoethanesulfonate, which is consistent with the proposed homoacetogenic metabolism.

PurposeIn this study we investigate the disease etiology in 12 patients with de novo variants in FAR1 all resulting in an amino acid change at position 480 (p.Arg480Cys/His/Leu).MethodsFollowing next-generation sequencing and clinical phenotyping, functional characterization was performed in patients' fibroblasts using FAR1 enzyme analysis, FAR1 immunoblotting/immunofluorescence, and lipidomics.ResultsAll patients had spastic paraparesis and bilateral congenital/juvenile cataracts, in most combined with speech and gross motor developmental delay and truncal hypotonia. FAR1 deficiency caused by biallelic variants results in defective ether lipid synthesis and plasmalogen deficiency. In contrast, patients' fibroblasts with the de novo FAR1 variants showed elevated plasmalogen levels. Further functional studies in fibroblasts showed that these variants cause a disruption of the plasmalogen-dependent feedback regulation of FAR1 protein levels leading to uncontrolled ether lipid production.ConclusionHeterozygous de novo variants affecting the Arg480 residue of FAR1 lead to an autosomal dominant disorder with a different disease mechanism than that of recessive FAR1 deficiency and a diametrically opposed biochemical phenotype. Our findings show that for patients with spastic paraparesis and bilateral cataracts, FAR1 should be considered as a candidate gene and added to gene panels for hereditary spastic paraplegia, cerebral palsy, and juvenile cataracts.

CO dehydrogenase (CODH) from the Gram-negative bacterium Oligotropha carboxidovorans is a complex metalloenzyme from the xanthine oxidase family of molybdenum-containing enzymes, bearing a unique binuclear Mo-S-Cu active site in addition to two [2Fe-2S] clusters (FeSI and FeSII) and one equivalent of FAD. CODH catalyzes the oxidation of CO to CO2 with the concomitant introduction of reducing equivalents into the quinone pool, thus enabling the organism to utilize CO as sole source of both carbon and energy. Using a variety of EPR monitored redox titrations and spectroelectrochemistry, we report the redox potentials of CO dehydrogenase at pH 7.2 namely MoVI/V, MoV/IV, FeSI2+/+, FeSII2+/+, FAD/FADH and FADH/FADH-. These potentials are systematically higher than the corresponding potentials seen for other members of the xanthine oxidase family of Mo enzymes, and are in line with CODH utilising the higher potential quinone pool as an electron acceptor instead of pyridine nucleotides. CODH is also active when immobilised on a modified Au working electrode as demonstrated by cyclic voltammetry in the presence of CO.

BackgroundCancer is frequently associated with tumor-related anemia, and many chemotherapeutic agents impair hematopoiesis, leading to impaired quality of life for affected patients. The use of erythropoiesis-stimulating agents has come under scrutiny after prospective clinical trials using recombinant erythropoietin to correct anemia reported increased incidence of thromboembolic events and cancer-related deaths. Furthermore, previous preclinical reports indicated expansion of the pool of breast cancer-initiating cells when erythropoietin was combined with ionizing radiation.MethodsUsing four established breast cancer cell lines, we test the effects of recombinant human erythropoietin and the number of breast cancer-initiating cells in vitro and in vivo and study if recombinant human erythropoietin promotes the phenotype conversion of non-tumorigenic breast cancer cells into breast cancer-initiating cells. In a prospective study, we evaluate whether elevated endogenous serum erythropoietin levels correlate with increased numbers of tumor-initiating cells in a cohort of breast cancer patients who were scheduled to undergo radiation treatment.ResultsOur results indicate that recombinant erythropoietin increased the number of tumor-initiating cells in established breast cancer lines in vitro. Irradiation of breast cancer xenografts caused a phenotype conversion of non-stem breast cancer cells into induced breast cancer-initiating cells. This effect coincided with re-expression of the pluripotency factors c-Myc, Sox2, and Oct4 and was enhanced by recombinant erythropoietin. Hemoglobin levels were inversely correlated with serum erythropoietin levels, and the latter were correlated with disease stage. However, tumor sections revealed a negative correlation between serum erythropoietin levels and the number of ALDH1A3-positive cells, a marker for breast cancer-initiating cells.ConclusionsWe conclude that physiologically slow-rising serum erythropoietin levels in response to tumor-related or chemotherapy-induced anemia, as opposed to large doses of recombinant erythropoietin, do not increase the pool of breast cancer-initiating cells.

Recent studies have underscored the critical role of retinoic acid (RA) in the development of lineage-committed CD4 and CD8 T cells in vivo. We have shown that under acute graft-versus-host disease (GVHD) inflammatory conditions, RA is upregulated in the intestine and is proinflammatory, as GVHD lethality was attenuated when donor allogeneic T cells selectively expressed a dominant negative RA receptor α that blunted RA signaling. RA can function in an autocrine and paracrine fashion, and as such, the host cell lineage responsible for the production of RA metabolism and the specific RA-metabolizing enzymes that potentiate GVHD severity are unknown. In this study, we demonstrate that enhancing RA degradation in the host and to a lesser extent donor hematopoietic cells by overexpressing the RA-catabolizing enzyme CYP26A1 reduced GVHD. RA production is facilitated by retinaldehyde isoform-2 (RALDH2) preferentially expressed in dendritic cells (DCs). Conditionally deleted RA-synthesizing enzyme RALDH2 in host or to a lesser extent donor DCs reduced GVHD lethality. Improved survival in recipients with RALDH2-deleted DCs was associated with increased T cell death, impaired T effector function, increased regulatory T cell frequency, and augmented coinhibitory molecule expression on donor CD4+ T cells. In contrast, retinaldehydrogenase isoform-1 (RALDH1) is dominantly expressed in intestinal epithelial cells. Unexpectedly, conditional host intestinal epithelial cells RALDH1 deletion failed to reduce GVHD. These data demonstrate the critical role of both donor and especially host RALDH2+ DCs in driving murine GVHD and suggest RALDH2 inhibition or CYP26A1 induction as novel therapeutic strategies to prevent GVHD.

The ancient reductive acetyl-CoA pathway is employed by acetogenic bacteria to form acetate from inorganic energy sources. Since the central pathway does not gain net ATP by substrate-level phosphorylation, chemolithoautotrophic growth relies on the additional formation of ATP via a chemiosmotic mechanism. Genome analyses indicated that some acetogens only have an energy-converting, ion-translocating hydrogenase (Ech) as a potential respiratory enzyme. Although the Ech-encoding genes are widely distributed in nature, the proposed function of Ech as an ion-translocating chemiosmotic coupling site has neither been demonstrated in bacteria nor has it been demonstrated that it can be the only energetic coupling sites in microorganisms that depend on a chemiosmotic mechanism for energy conservation. Here, we show that the Ech complex of the thermophilic acetogenic bacterium Thermoanaerobacter kivui is indeed a respiratory enzyme. Experiments with resting cells prepared from T. kivui cultures grown on carbon monoxide (CO) revealed CO oxidation coupled to H2 formation and the generation of a transmembrane electrochemical ion gradient ([Formula: see text]). Inverted membrane vesicles (IMVs) prepared from CO-grown cells also produced H2 and ATP from CO (via a loosely attached CO dehydrogenase) or a chemical reductant. Finally, we show that Ech activity led to the translocation of both H+ and Na+ across the membrane of the IMVs. The H+ gradient was then used by the ATP synthase for energy conservation. These data demonstrate that the energy-converting hydrogenase in concert with an ATP synthase may be the simplest form of respiration; it combines carbon dioxide fixation with the synthesis of ATP in an ancient pathway.

An overview is provided of the molybdenum- and tungsten-containing enzymes that catalyze the interconversion of formate and CO2 , focusing on common structural and mechanistic themes, as well as a consideration of the manner in which the mature Mo- or W-containing cofactor is inserted into apoprotein.

In a large scale screen for skin, hair, and nail abnormalities in null mice generated by The Jackson Laboratory's KOMP center, homozygous mutant Far2tm2b(KOMP)Wtsi/2J (hereafter referrred to as Far2-/-) mice were found to develop focal areas of alopecia as they aged. As sebocytes matured in wildtype C57BL/NJ mice they became pale with fine, uniformly sized clear lipid containing vacuoles that were released when sebocytes disintegrated in the duct. By contrast, the Far2-/- null mice had sebocytes that were similar within the gland but become brightly eosinophilic when the cells entered the sebaceous gland duct. As sebocytes disintegrated, their contents did not readily dissipate. Scattered throughout the dermis, and often at the dermal hypodermal fat junction, were dystrophic hair follicles or ruptured follicles with a foreign body granulomatous reaction surrounding free hair shafts (trichogranuloma). The Meibomian and clitoral glands (modified sebaceous glands) of Far2-/- mice showed ducts dilated to various degrees that were associated with mild changes in the sebocytes as seen in the truncal skin. Skin surface lipidomic analysis revealed a lower level of wax esters, cholesterol esters, ceramides, and diacylglycerols compared to wildtype control mice. Similar changes were described in a number of other mouse mutations that affected the sebaceous glands resulting in primary cicatricial alopecia.

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Patients with high ALDH1A3-expressing glioblastoma (ALDH1A3 hi 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 ALDH1A3 hi GBM., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Lean body mass, consisting mostly of skeletal muscle, is important for healthy aging. We performed a genome-wide association study for whole body (20 cohorts of European ancestry with n = 38,292) and appendicular (arms and legs) lean body mass (n = 28,330) measured using dual energy X-ray absorptiometry or bioelectrical impedance analysis, adjusted for sex, age, height, and fat mass. Twenty-one single-nucleotide polymorphisms were significantly associated with lean body mass either genome wide (p

Fatty alcohols in the C12-C18 range are used in personal care products, lubricants, and potentially biofuels. These compounds can be produced from the fatty acid pathway by a fatty acid reductase (FAR), yet yields from the preferred industrial host Saccharomyces cerevisiae remain under 2% of the theoretical maximum from glucose. Here we improved titer and yield of fatty alcohols using an approach involving quantitative analysis of protein levels and metabolic flux, engineering enzyme level and localization, pull-push-block engineering of carbon flux, and cofactor balancing. We compared four heterologous FARs, finding highest activity and endoplasmic reticulum localization from a Mus musculus FAR. After screening an additional twenty-one single-gene edits, we identified increasing FAR expression; deleting competing reactions encoded by DGA1, HFD1, and ADH6; overexpressing a mutant acetyl-CoA carboxylase; limiting NADPH and carbon usage by the glutamate dehydrogenase encoded by GDH1; and overexpressing the Δ9-desaturase encoded by OLE1 as successful strategies to improve titer. Our final strain produced 1.2g/L fatty alcohols in shake flasks, and 6.0g/L in fed-batch fermentation, corresponding to ~ 20% of the maximum theoretical yield from glucose, the highest titers and yields reported to date in S. cerevisiae. We further demonstrate high-level production from lignocellulosic feedstocks derived from ionic-liquid treated switchgrass and sorghum, reaching 0.7g/L in shake flasks. Altogether, our work represents progress towards efficient and renewable microbial production of fatty acid-derived products.

Fatty alcohols in the C12-C18 range are used in personal care products, lubricants, and potentially biofuels. These compounds can be produced from the fatty acid pathway by a fatty acid reductase (FAR), yet yields from the preferred industrial host Saccharomyces cerevisiae remain under 2% of the theoretical maximum from glucose. Here we improved titer and yield of fatty alcohols using an approach involving quantitative analysis of protein levels and metabolic flux, engineering enzyme level and localization, pull-push-block engineering of carbon flux, and cofactor balancing. We compared four heterologous FARs, finding highest activity and endoplasmic reticulum localization from a Mus musculus FAR. After screening an additional twenty-one single-gene edits, we identified increasing FAR expression; deleting competing reactions encoded by DGA1, HFD1, and ADH6; overexpressing a mutant acetyl-CoA carboxylase; limiting NADPH and carbon usage by the glutamate dehydrogenase encoded by GDH1; and overexpressing the Δ9-desaturase encoded by OLE1 as successful strategies to improve titer. Our final strain produced 1.2g/L fatty alcohols in shake flasks, and 6.0g/L in fed-batch fermentation, corresponding to ~ 20% of the maximum theoretical yield from glucose, the highest titers and yields reported to date in S. cerevisiae. We further demonstrate high-level production from lignocellulosic feedstocks derived from ionic-liquid treated switchgrass and sorghum, reaching 0.7g/L in shake flasks. Altogether, our work represents progress towards efficient and renewable microbial production of fatty acid-derived products.

Most living organisms developed systems to efficiently time environmental changes. The plant-clock acts in coordination with external signals to generate output responses determining seasonal growth and flowering time. Here, we show that two Arabidopsis thaliana transcription factors, FAR1 RELATED SEQUENCE 7 (FRS7) and FRS12, act as negative regulators of these processes. These proteins accumulate particularly in short-day conditions and interact to form a complex. Loss-of-function of FRS7 and FRS12 results in early flowering plants with overly elongated hypocotyls mainly in short days. We demonstrate by molecular analysis that FRS7 and FRS12 affect these developmental processes in part by binding to the promoters and repressing the expression of GIGANTEA and PHYTOCHROME INTERACTING FACTOR 4 as well as several of their downstream signalling targets. Our data reveal a molecular machinery that controls the photoperiodic regulation of flowering and growth and offer insight into how plants adapt to seasonal changes.

The clustering of neurons sharing similar functional properties and connectivity is a common organizational feature of vertebrate nervous systems. Within motor networks, spinal motor neurons (MNs) segregate into longitudinally arrayed subtypes, establishing a central somatotopic map of peripheral target innervation. MN organization and connectivity relies on Hox transcription factors expressed along the rostrocaudal axis; however, the developmental mechanisms governing the orderly arrangement of MNs are largely unknown. We show that Pbx genes, which encode Hox cofactors, are essential for the segregation and clustering of neurons within motor columns. In the absence of Pbx1 and Pbx3 function, Hox-dependent programs are lost and the remaining MN subtypes are unclustered and disordered. Identification of Pbx gene targets revealed an unexpected and apparently Hox-independent role in defining molecular features of dorsally projecting medial motor column (MMC) neurons. These results indicate Pbx genes act in parallel genetic pathways to orchestrate neuronal subtype differentiation, connectivity, and organization.

We show that Mycobacterium smegmatis mutants disrupted in mscR, coding for a dual function S-nitrosomycothiol reductase and formaldehyde dehydrogenase, and mshC, coding for a mycothiol ligase and lacking mycothiol (MSH), are more susceptible to S-nitrosoglutathione (GSNO) and aldehydes than wild type. MSH is a cofactor for MscR, and both mshC and mscR are induced by GSNO and aldehydes. We also show that a mutant disrupted in egtA, coding for a γ-glutamyl cysteine synthetase and lacking in ergothioneine, is sensitive to nitrosative stress but not to aldehydes. In addition, we find that MSH and S-nitrosomycothiol reductase are required for normal biofilm formation in M. smegmatis, suggesting potential new therapeutic pathways to target to inhibit or disrupt biofilm formation. © 2016 IUBMB Life, 68(8):621-628, 2016.

Excessive alcohol consumption can have serious negative consequences on health, including addiction, liver damage, and other long-term effects. The causes of hangovers include dehydration, alcohol and alcohol metabolite toxicity, and nutrient deficiency due to absorption disorders. Additionally, alcohol consumption can slow reaction times, making it more difficult to rapidly respond to situations that require quick thinking. Exposure to a large amount of ethanol can also negatively affect a person's righting reflex and balance. In this study, we evaluated the potential of lactic acid bacteria (LAB) to alleviate alcohol-induced effects and behavioral responses. Two LAB strains isolated from kimchi, Levilactobacillus brevis WiKim0168 and Leuconostoc mesenteroides WiKim0172, were selected for their ethanol tolerance and potential to alleviate hangover symptoms. Enzyme activity assays for alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) were then conducted to evaluate the role of these bacteria in alcohol metabolism. Through in vitro and in vivo studies, these strains were assessed for their ability to reduce blood alcohol concentrations and protect against alcohol-induced liver damage. The results indicated that these LAB strains possess significant ethanol tolerance and elevate ADH and ALDH activities. LAB administration remarkably reduced blood alcohol levels in rats after excessive alcohol consumption. Moreover, the LAB strains showed hepatoprotective effects and enhanced behavioral outcomes, highlighting their potential as probiotics for counteracting the adverse effects of alcohol consumption. These findings support the development of functional foods incorporating LAB strains that can mediate behavioral improvements following alcohol intake.

Hepatocellular carcinoma (HCC) is the predominant pathologic type of primary liver cancer. It is a malignant tumor of liver epithelial cells. There are many ways to treat HCC, but the survival rate for HCC patients remains low. Therefore, understanding the underlying mechanisms by which HCC occurs and develops is critical to explore new therapeutic targets. Aldehyde dehydrogenase 2 (ALDH2) is an important player in the redox reaction of ethanol with endogenous aldehyde products released by lipid peroxidation. Increasing evidence suggests that ALDH2 is a crucial regulator of human tumor development, including HCC. Therefore, clarifying the relationship between ALDH2 and HCC is helpful for formulating rational treatment strategies. This review highlights the regulatory roles of ALDH2 in the development of HCC, elucidates the multiple potential mechanisms by which ALDH2 regulates the development of HCC, and summarizes the progress of research on ALDH2 gene polymorphisms and HCC susceptibility. Meanwhile, we envision viable strategies for targeting ALDH2 in the treatment of HCC SIGNIFICANCE STATEMENT: Numerous studies have aimed to explore novel therapeutic targets for HCC, and ALDH2 has been reported to be a critical regulator of HCC progression. This review discusses the functions, molecular mechanisms, and clinical significance of ALDH2 in the development of HCC and examines the prospects of ALDH2-based therapy for HCC., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Objectives: To reveal the effect and mechanism of methyltransferase-like 3 (METTL3) on cancer stem cells (CSCs) of head and neck squamous cell carcinoma (HNSCC)., Materials and Methods: First, we analyzed 14-HNSCC-patients' scRNA-seq dataset and TCGA dataset of HNSCC. Then, Mettl3 knockout or overexpression mice models were studied via tracing and staining technologies. In addition, we took flow cytometry sorting and sphere formation assays to observe tumorigenicity and used cell transfection and western blotting to verify target protein expression levels. Furthermore, methylated RNA immunoprecipitation sequencing (MeRIP-seq) and MeRIP-quantitative real-time PCR (MeRIP-qPCR) were taken to identify the mechanism of Mettl3 regulating Bmi1 + CSCs in HNSCC., Results: Due to SOX4 transcriptional regulation, METTL3 regulated the malignant behavior of BMI1 + HNSCC stem cells through cell division pathway. The progression and malignancy of HNSCC were decreased after Mettl3 knocked-out, while increased after Mettl3 knocked-in in Bmi1 + CSCs in vivo. Knockdown of Mettl3 inhibited stemness properties of CSCs in vitro. Mechanically, Mettl3 mediated the m 6 A modification of ALDH1A3 and ALDH7A1 mRNA in Bmi1 + HNSCC CSCs., Conclusion: Regulated by SOX4, METTL3-mediated ALDH m 6 A methylation regulates the malignant behavior of BMI1 + HNSCC CSCs through cell division pathway., (© 2023 Wiley Periodicals LLC.)

Osteoclasts, the exclusive bone resorptive cells, are indispensable for bone remodeling. Hence, understanding novel signaling modulators regulating osteoclastogenesis is clinically important. Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) is a master transcription factor in osteoclastogenesis, and binding of NF-κB p65 subunit to NFATc1 promoter is required for its expression. It is well-established that DNA binding activity of p65 can be regulated by various post-translational modifications, including S-nitrosation. Recent studies have demonstrated that S-nitrosoglutathione reductase (GSNOR)-mediated protein denitrosation participated in cell fate commitment by regulating gene transcription. However, the role of GSNOR in osteoclastogenesis remains unexplored and enigmatic. Here, we investigated the effect of GSNOR-mediated denitrosation of p65 on osteoclastogenesis. Our results revealed that GSNOR was up-regulated during osteoclastogenesis in vitro. Moreover, GSNOR inhibition with a chemical inhibitor impaired osteoclast differentiation, podosome belt formation, and bone resorption activity. Furthermore, GSNOR inhibition enhanced the S-nitrosation level of p65, precluded the binding of p65 to NFATc1 promoter, and suppressed NFATc1 expression. In addition, mouse model of lipopolysaccharides (LPS)-induced calvarial osteolysis was employed to evaluate the therapeutic effect of GSNOR inhibitor in vivo. Our results indicated that GSNOR inhibitor treatment alleviated the inflammatory bone loss by impairing osteoclast formation in mice. Taken together, these data have shown that GSNOR activity is required for osteoclastogenesis by facilitating binding of p65 to NFATc1 promoter via promoting p65 denitrosation, suggesting that GSNOR may be a potential therapeutic target in the treatment of osteolytic diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Vanillin dehydrogenase (VDH) has recently come forward as an important enzyme for the commercial production of vanillic acid from vanillin in a one-step enzymatic process. However, VDH with high alkaline tolerance and efficiency is desirable to meet the biorefinery requirements. In this study, computationally guided site-directed mutagenesis was performed by increasing the positive and negative charges on the surface and near the active site of the VDH from the alkaliphilic marine bacterium Bacillus ligniniphilus L1, respectively. In total, 20 residues including 15 from surface amino acids and 5 near active sites were selected based on computational analysis and were subjected to site-directed mutations. The optimum pH of the two screened mutants including I132R, and T235E from surface residue and near active site mutant was shifted to 9, and 8.6, with a 2.82- and 2.95-fold increase in their activity compared to wild enzyme at pH 9, respectively. A double mutant containing both these mutations i.e., I132R/T235E was produced which showed a shift in optimum pH of VDH from 7.4 to 9, with an increase of 74.91 % in enzyme activity. Therefore, the double mutant of VDH from the L1 strain (I132R/T235E) produced in this study represents a potential candidate for industrial applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cyanobacteria possess the unique capacity to naturally produce hydrocarbons from fatty acids. Hydrocarbon compositions of thirty-two strains of cyanobacteria were characterized to reveal novel structural features and insights into hydrocarbon biosynthesis in cyanobacteria. This investigation revealed new double bond (2- and 3-heptadecene) and methyl group positions (3-, 4- and 5-methylheptadecane) for a variety of strains. Additionally, results from this study and literature reports indicate that hydrocarbon production is a universal phenomenon in cyanobacteria. All cyanobacteria possess the capacity to produce hydrocarbons from fatty acids yet not all accomplish this through the same metabolic pathway. One pathway comprises a two-step conversion of fatty acids first to fatty aldehydes and then alkanes that involves a fatty acyl ACP reductase (FAAR) and aldehyde deformylating oxygenase (ADO). The second involves a polyketide synthase (PKS) pathway that first elongates the acyl chain followed by decarboxylation to produce a terminal alkene (olefin synthase, OLS). Sixty-one strains possessing the FAAR/ADO pathway and twelve strains possessing the OLS pathway were newly identified through bioinformatic analyses. Strains possessing the OLS pathway formed a cohesive phylogenetic clade with the exception of three Moorea strains and Leptolyngbya sp. PCC 6406 which may have acquired the OLS pathway via horizontal gene transfer. Hydrocarbon pathways were identified in one-hundred-forty-two strains of cyanobacteria over a broad phylogenetic range and there were no instances where both the FAAR/ADO and the OLS pathways were found together in the same genome, suggesting an unknown selective pressure maintains one or the other pathway, but not both.

The reaction of the air-tolerant CO dehydrogenase from Oligotropha carboxidovorans with H2 has been examined. Like the Ni-Fe CO dehydrogenase, the enzyme can be reduced by H2 with a limiting rate constant of 5.3 s(-1) and a dissociation constant Kd of 525 μM; both kred and kred/Kd, reflecting the breakdown of the Michaelis complex and the reaction of free enzyme with free substrate in the low [S] regime, respectively, are largely pH-independent. During the reaction with H2, a new EPR signal arising from the Mo/Cu-containing active site of the enzyme is observed which is distinct from the signal seen when the enzyme is reduced by CO, with greater g anisotropy and larger hyperfine coupling to the active site (63,65)Cu. The signal also exhibits hyperfine coupling to at least two solvent-exchangeable protons of bound substrate that are rapidly exchanged with solvent. Proton coupling is also evident in the EPR signal seen with the dithionite-reduced native enzyme, and this coupling is lost in the presence of bicarbonate. We attribute the coupled protons in the dithionite-reduced enzyme to coordinated water at the copper site in the native enzyme and conclude that bicarbonate is able to displace this water from the copper coordination sphere. On the basis of our results, a mechanism for H2 oxidation is proposed which involves initial binding of H2 to the copper of the binuclear center, displacing the bound water, followed by sequential deprotonation through a copper-hydride intermediate to reduce the binuclear center.

We report here an ENDOR study of an S = 1/2 intermediate state trapped during reduction of the binuclear Mo/Cu enzyme CO dehydrogenase by CO. ENDOR spectra of this state confirm that the (63,65)Cu nuclei exhibits strong and almost entirely isotropic coupling to the unpaired electron, show that this coupling atypically has a positive sign, aiso = +148 MHz, and indicate an apparently undetectably small quadrupolar coupling. When the intermediate is generated using (13)CO, coupling to the (13)C is observed, with aiso = +17.3 MHz. A comparison with the couplings seen in related, structurally assigned Mo(V) species from xanthine oxidase, in conjunction with complementary computational studies, leads us to conclude that the intermediate contains a partially reduced Mo(V)/Cu(I) center with CO bound at the copper. Our results provide strong experimental support for a reaction mechanism that proceeds from a comparable complex of CO with fully oxidized Mo(VI)/Cu(I) enzyme.

The major active retinoid, all-trans retinoic acid, has long been recognized as critical for the development of several organs, including the eye. Mutations in STRA6, the gene encoding the cellular receptor for vitamin A, in patients with Matthew-Wood syndrome and anophthalmia/microphthalmia (A/M), have previously demonstrated the importance of retinol metabolism in human eye disease. We used homozygosity mapping combined with next-generation sequencing to interrogate patients with anophthalmia and microphthalmia for new causative genes. We used whole-exome and whole-genome sequencing to study a family with two affected brothers with bilateral A/M and a simplex case with bilateral anophthalmia and hypoplasia of the optic nerve and optic chiasm. Analysis of novel sequence variants revealed homozygosity for two nonsense mutations in ALDH1A3, c.568A>G, predicting p.Lys190*, in the familial cases, and c.1165A>T, predicting p.Lys389*, in the simplex case. Both mutations predict nonsense-mediated decay and complete loss of function. We performed antisense morpholino (MO) studies in Danio rerio to characterize the developmental effects of loss of Aldh1a3 function. MO-injected larvae showed a significant reduction in eye size, and aberrant axonal projections to the tectum were noted. We conclude that ALDH1A3 loss of function causes anophthalmia and aberrant eye development in humans and in animal model systems.

Hepatocellular carcinoma (HCC) is one of the most common and deadly human cancers and it remains poorly managed. Human HCC development is often associated both with elevated expression of inducible nitric oxide synthase (iNOS) and with genetic deletion of the major denitrosylase S-nitrosoglutathione reductase (GSNOR/ADH5). However, their causal involvement in human HCC is not established. In mice, GSNOR deficiency causes S-nitrosylation and depletion of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (AGT) and increases rates of both spontaneous and DEN carcinogen-induced HCC. Here, we report that administration of 1400W, a potent and highly selective inhibitor of iNOS, blocked AGT depletion and rescued the repair of mutagenic O6-ethyldeoxyguanosines following DEN challenge in livers of GSNOR-deficient (GSNOR(-/-)) mice. Notably, short-term iNOS inhibition following DEN treatment had little effect on carcinogenesis in wild-type mice, but was sufficient to reduce HCC multiplicity, maximal size, and burden in GSNOR(-/-) mice to levels comparable with wild-type controls. Furthermore, increased HCC susceptibility in GSNOR(-/-) mice was not associated with an increase in interleukin 6, tumor necrosis factor-α, oxidative stress, or hepatocellular proliferation. These results suggested that GSNOR deficiency linked to defective DNA damage repair likely acts at the tumor initiation stage to promote HCC carcinogenesis. Together, our findings provide the first proof of principle that HCC development in the context of uncontrolled nitrosative stress can be blocked by pharmacologic inhibition of iNOS, possibly providing an effective therapy for patients with HCC.

Nitric oxide (NO) exerts a wide range of cellular effects in the cardiovascular system. NO is short lived, but S-nitrosoglutathione (GSNO) functions as a stable intracellular bioavailable NO pool. Accordingly, increased levels can facilitate NO-mediated processes, and conversely, catabolism of GSNO by the regulatory enzyme GSNO reductase (GSNOR) can impair these processes. Because dysregulated GSNOR can interfere with processes relevant to cardiovascular health, it follows that inhibition of GSNOR may be beneficial. However, the effect of GSNOR inhibition on vascular activity is unknown. To study the effects of GSNOR inhibition on endothelial function, we treated rats with a small-molecule inhibitor of GSNOR (N6338) that has vasodilatory effects on isolated aortic rings and assessed effects on arterial flow-mediated dilation (FMD), an NO-dependent process. GSNOR inhibition with a single intravenous dose of N6338 preserved FMD (15.3 ± 5.4 vs. 14.2 ± 6.3%, P = nonsignificant) under partial NO synthase inhibition that normally reduces FMD by roughly 50% (14.1 ± 2.9 vs. 7.6 ± 4.4%, P < 0.05). In hypertensive rats, daily oral administration of N6338 for 14 days reduced blood pressure (170.0 ± 5.3/122.7 ± 6.4 vs. 203.8 ± 1.9/143.7 ± 7.5 mmHg for vehicle, P < 0.001) and vascular resistance index (1.5 ± 0.4 vs. 3.2 ± 1.0 mmHg · min · l(-1) for vehicle, P < 0.001), and restored FMD from an initially impaired state (7.4 ± 1.7%, day 0) to a level (13.0 ± 3.1%, day 14, P < 0.001) similar to that observed in normotensive rats. N6338 also reversed the pathological kidney changes exhibited by the hypertensive rats. GSNOR inhibition preserves FMD under conditions of impaired NO production and protects against both microvascular and conduit artery dysfunction in a model of hypertension.

Economic bioconversion of plant cell wall hydrolysates into fuels and chemicals has been hampered mainly due to the inability of microorganisms to efficiently co-ferment pentose and hexose sugars, especially glucose and xylose, which are the most abundant sugars in cellulosic hydrolysates. Saccharomyces cerevisiae cannot metabolize xylose due to a lack of xylose-metabolizing enzymes. We developed a rapid and efficient xylose-fermenting S. cerevisiae through rational and inverse metabolic engineering strategies, comprising the optimization of a heterologous xylose-assimilating pathway and evolutionary engineering. Strong and balanced expression levels of the XYL1, XYL2, and XYL3 genes constituting the xylose-assimilating pathway increased ethanol yields and the xylose consumption rates from a mixture of glucose and xylose with little xylitol accumulation. The engineered strain, however, still exhibited a long lag time when metabolizing xylose above 10 g/l as a sole carbon source, defined here as xylose toxicity. Through serial-subcultures on xylose, we isolated evolved strains which exhibited a shorter lag time and improved xylose-fermenting capabilities than the parental strain. Genome sequencing of the evolved strains revealed that mutations in PHO13 causing loss of the Pho13p function are associated with the improved phenotypes of the evolved strains. Crude extracts of a PHO13-overexpressing strain showed a higher phosphatase activity on xylulose-5-phosphate (X-5-P), suggesting that the dephosphorylation of X-5-P by Pho13p might generate a futile cycle with xylulokinase overexpression. While xylose consumption rates by the evolved strains improved substantially as compared to the parental strain, xylose metabolism was interrupted by accumulated acetate. Deletion of ALD6 coding for acetaldehyde dehydrogenase not only prevented acetate accumulation, but also enabled complete and efficient fermentation of xylose as well as a mixture of glucose and xylose by the evolved strain. These findings provide direct guidance for developing industrial strains to produce cellulosic fuels and chemicals.

Main Conclusion: Overexpression of OsDJ-1C in rice improves root architecture, photosynthesis, yield and abiotic stress tolerance through modulating methylglyoxal levels, antioxidant defense, and redox homeostasis. Exposure to abiotic stresses leads to elevated methylglyoxal (MG) levels in plants, impacting seed germination and root growth. In response, the activation of NADPH-dependent aldo-keto reductase and glutathione (GSH)-dependent glyoxalase enzymes helps to regulate MG levels and reduce its toxic effects. However, detoxification may not be carried out effectively due to the limitation of GSH and NADPH in plants under stress. Recently, a novel enzyme called glyoxalase III (GLY III) has been discovered which can detoxify MG in a single step without needing GSH. To understand the physiological importance of this pathway in rice, we overexpressed the gene encoding GLYIII enzyme (OsDJ-1C) in rice. It was observed that OsDJ-1C overexpression in rice regulated MG levels under stress conditions thus, linked well with plants' abiotic stress tolerance potential. The OsDJ-1C overexpression lines displayed better root architecture, improved photosynthesis, and reduced yield penalty compared to the WT plants under salinity, and drought stress conditions. These plants demonstrated an improved GSH/GSSG ratio, reduced level of reactive oxygen species, increased antioxidant capacity, and higher anti-glycation activity thereby indicating that the GLYIII mediated MG detoxification plays a significant role in plants' ability to reduce the impact of abiotic stress. Furthermore, these findings imply the potential of OsDJ-1C in crop improvement programs., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (© 2024. The Author(s).)

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.

Objective: This research aimed to explore a diagnostic method based on serum ALDH3B1 and to evaluate the clinical diagnostic efficacy in gastric cancer (GC) by comparing it with the traditional GC diagnostic method, the carcinoembryonic protein (CEA) assay. Methods: Serum samples were collected from 70 healthy volunteers and various patients (GC: 76, benign gastric lesions: 20, postoperative: 37, recurrence: 56). The diagnostic efficacy of serum ALDH3B1 , CEA and the co-diagnosis were evaluated by receiver operating characteristic curve. ALDH3B1 protein levels were evaluated by western blot. Results: The co-diagnosis of ALDH3B1 and CEA had the highest diagnostic efficacy (area under the curve = 0.841). Conclusion: Serum ALDH3B1 may be used as an auxiliary diagnostic biomarker for GC, and its co-diagnosis with CEA can improve diagnostic efficacy.