Alcohol Dehydrogenase Research Articles

Solvent isotope and mutagenesis studies on the proton relay system in yeast alcohol dehydrogenase 1

Alcohol dehydrogenase catalyzes the reversible transfer of a hydride directly from an alcohol to the nicotinamide ring of NAD+ to form an aldehyde and NADH, and the proton from the alcohol probably is transferred through a hydrogen-bonded system to the imidazole of His-48. Studies of the pH dependencies, and solvent and substrate isotope effects on the wild-type and the enzyme with His-48 substituted with Gln-48 were used to demonstrate a role for the proton relay system. The H48Q substitution increases affinities for NAD+ and NADH by ∼2-fold, suggesting that the overall protein structure is maintained. In contrast, catalytic efficiencies (V/Km) on ethanol and acetaldehyde and affinity for 2,2,2-trifluoroethanol are decreased by about 10-fold. The pH dependencies for catalytic efficiencies on ethanol and acetaldehyde (log V/Km versus pH), show pK values of about 7.5 for wild-type enzyme, but ethanol oxidation by H48Q ADH is essentially linear over the pH range from 5.5 to 9.2 with a slope of 0.47. Steady-state kinetics and substrate isotope effects suggest that the kinetic mechanism of H48Q ADH has become partly random for oxidation of ethanol. Both wild-type and H48Q ADHs have pH-independent isotope effects for oxidation (V1/Kb) of 1-butanol/1-butanol-d9 of 4, suggesting that hydride transfer is a major rate-limiting step. The pH dependence for butanol oxidation by wild type ADH shows a wavy profile over the pH range from pH 6 to 10, with a ∼2.3-fold larger V1/Kb in D2O than in H2O, an “inverse” isotope effect. The substrate isotope effect of 4 is not altered by the solvent isotope effect, suggesting concerted proton/hydride transfer. The solvent isotope effect can be explained by a ground state with a water bound to the catalytic zinc in the enzyme-NAD+ complex, and a transition state that resembles a complex with NADH and aldehyde. In contrast, the H48Q enzyme has a diminished inverse solvent isotope effect of ∼1.3 and an essentially linear pH dependence with a slope of log V1/Kb against pH of 0.49 for oxidation of 1-butanol, which together are consistent with a transition state where hydroxide ion directly accepts a proton from the 2′-hydroxyl group of the nicotinamide ribose in the proton relay system in the enzyme-NAD+-alcohol complex. The results support a catalytic role for His-48 in the proton relay system.

Biorefinery-centric ethanol and oleochemical production employing Yarrowia lipolytica and Pichia farinosa

The research examined the capabilities of Yarrowia lipolytica (YL) and Pichia farinosa (PF) in converting sugars to ethanol and oleochemicals. Lipid, ethanol, protein yield and gene-expressions were analysed at different substrate concentrations (3 to 30 g/L) with glucose, food waste, and fermentation-effluent. Optimal results were obtained at 20 g/L using both synthetic carbon with 4.6 % of total lipid yield. Lauric and Caprylic acid dominance was noted in total lipid fractions. Protein accumulation (6 g/L) was observed in glucose system (20 g/L) indicating yeast strains potential as single-cell proteins (SCP). Fatty-acid desaturase (FAD12) and alcohol dehydrogenase (ADH) expressions were higher at optimum condition of YL (1.15 × 10−1, 3.8 × 10−2) and PF (5.8 × 10−2, 3.8 × 10−2) respectively. Maximum carbon reduction of 87 % depicted at best condition, aligning with metabolic yield. These findings highlights promising role of yeast as biorefinery biocatalyst.

Molecular and physiological responses to salt stress in salinity-sensitive and tolerant Hibiscus rosa-sinensis cultivars

Ornamental plants are used to decorate urban and peri-urban areas, and during their cultivation or utilisation, they can be exposed to abiotic stress. Salinity is an abiotic stress factor that limits plant growth and reduces the ornamental value of sensitive species. In this study, transcriptomic analysis was conducted to identify genes associated with tolerance or sensitivity to salinity in two hibiscus (Hibiscus rosa-sinensis L.) cultivars, ‘Porto’ and ‘Sunny wind’. The physiological and biochemical parameters of plants exposed to 50, 100, or 200 mM NaCl and water (control) were monitored. Salinity treatments were applied for six weeks. After four weeks, differences between cultivars were clearly evident and ‘Porto’ was more tolerant than ‘Sunny wind’. The tolerant cultivar showed lower electrolyte leakage and ABA concentrations, and higher proline content in the leaves. Accumulation of Na in different organs was lower in the flower organs of ‘Porto’. At the molecular level, several differential expressed genes were observed between the cultivars and flower organs. Among the highly expressed DEGs, coat protein, alcohol dehydrogenase, and AP2/EREBP transcription factor ERF-1. Among the downregulated genes, GH3 and NCED were the most interesting. The differential expression of these genes may explain the salt stress tolerance of ‘Porto’.Graphical

Degradation and potential metabolism pathway of polystyrene by bacteria from landfill site

Microplastics pollution has garnered significant attention in recent years. The unique cross-linked structure of polystyrene microplastics makes them difficult to biodegrade. In this study, we investigated the microbial community in landfill soil that has the ability to degrade polystyrene, as well as two isolated strains, named Lysinibacillus sp. PS-L and Pseudomonas sp. PS-P. The maximum weight loss of polystyrene film and microplastic in 30 days is 2.25% and 6.99% respectively. The water contact angle of polystyrene film decreased by a maximum of 35.70% during biodegradation. The increase in hydrophilicity is attributed to the oxidation reaction and formation of hydroxyl groups during the degradation of polystyrene. The carbon and oxygen element contents of polystyrene decreased and increased by a maximum of 3.81% and 0.79% respectively. The peak intensity changes at wavelengths of 3285–3648 cm−1 and 1652 cm−1 in Fourier transform infrared spectroscopy confirmed the formation of hydroxyl and carbonyl groups. Furthermore, quantitative PCR revealed the gene expression levels of alkane monooxygenase and alcohol dehydrogenase were upregulated by 8.8-fold and 8.5-fold respectively in PS biodegradation. Additionally, genome annotation of Pseudomonas sp. PS-P identified nine genes associated with polystyrene metabolism. These findings highlight Pseudomonas sp. PS-P as a potential candidate strain for polystyrene degradation enzymes or genes. Thus, they lay the groundwork for understanding the potential metabolic mechanisms and pathways involved in polystyrene degradation.

In situ Generation of Aldehydes for Subsequent Biocatalytic Cascade Reactions in Whole Cells

AbstractAldehydes represent valuable precursors for chemical compounds. However, the incorporation of aldehydes in whole‐cell‐based synthetic enzyme cascades is problematic. Due to their toxicity, they are metabolized to the corresponding alcohols or carboxylic acids by the host enzymatic background. Previous research has shown that these endogenous side reactions can be reversed by alcohol dehydrogenase (AlkJ, Pseudomonas putida) and carboxylic acid reductase (CARNi, Nocardia iowensis) in Escherichia coli. Thereby, the aldehyde is available for further enzymatic conversion to a product of choice. Three different enzymes were incorporated into the concept for the transformation of the aldehyde intermediate: A pyruvate decarboxylase (PDCApE469Q, Acetobacter pasteurianus), ω‐transaminase (ω‐TAVf, Vibrio fluvialis) and imine reductase (IREDCf, Cystobacter ferrugineus). (R)‐Phenylacetylcarbinol and a range of primary and secondary amines were obtained as final products. Either the alcohol, the carboxylic acid or a 50 : 50 mixture could be employed as starting material.

Novel insights into the effects of 5-hydroxymethfurural on genomic instability and phenotypic evolution using a yeast model.

5-Hydroxymethfurural (5-HMF) is naturally found in a variety of foods and beverages and represents a main inhibitor in the lignocellulosic hydrolysates used for fermentation. This study investigated the impact of 5-HMF on the genomic stability and phenotypic plasticity of the yeast Saccharomyces cerevisiae. Using next-generation sequencing technology, we examined the genomic alterations of diploid S. cerevisiae isolates that were subcultured on a medium containing 1.2 g/L 5-HMF. We found that in 5-HMF-treated cells, the rates of chromosome aneuploidy, large deletions/duplications, and loss of heterozygosity were elevated compared with that in untreated cells. 5-HMF exposure had a mild impact on the rate of point mutations but altered the mutation spectrum. Contrary to what was observed in untreated cells, more monosomy than trisomy occurred in 5-HMF-treated cells. The aneuploidy mutant with monosomic chromosome IX was more resistant to 5-HMF than the diploid parent strain because of the enhanced activity of alcohol dehydrogenase. Finally, we found that overexpression of ADH6 and ZWF1 effectively stabilized the yeast genome under 5-HMF stress. Our findings not only elucidated the global effect of 5-HMF on the genomic integrity of yeast but also provided novel insights into how chromosomal instability drives the environmental adaptability of eukaryotic cells.IMPORTANCESingle-cell microorganisms are exposed to a range of stressors in both natural and industrial settings. This study investigated the effects of 5-hydroxymethfurural (5-HMF), a major inhibitor found in baked foods and lignocellulosic hydrolysates, on the chromosomal instability of yeast. We examined the mechanisms leading to the distinct patterns of 5-HMF-induced genomic alterations and discovered that chromosomal loss, typically viewed as detrimental to cell growth under most conditions, can contribute to yeast tolerance to 5-HMF. Our results increased the understanding of how specific stressors stimulate genomic plasticity and environmental adaptation in yeast.

Immunoproteomics reveal increased serum IgG3/5 binding to Dermatophagoides and yeast protein antigens in severe equine asthma in a preliminary study.

Severe equine asthma (SEA) is a common, chronic respiratory disease of horses characterized by hyperreactivity to hay dust which has many similarities to severe neutrophilic asthma in humans. SEA-provoking antigens have not been comprehensively characterized, but molds and mites have been suggested as relevant sources. Here, we identified relevant antigen candidates using immunoproteomics with IgG isotype-binding analyses. Proteins from Dermatophagoides pteronyssinus (Der p) were separated by two-dimensional gel electrophoresis followed by immunoblotting (2D immunoblots) resulting in a characteristic pattern of 440 spots. After serum incubation, antibody (Ig)-binding of all Ig (Pan-Ig) and IgG isotypes (type-2-associated IgG3/5, type-1-associated IgG4/7) was quantified per each spot and compared between asthmatic and healthy horses' sera (n=5 per group). Ig binding differences were detected in 30 spots. Pan-Ig binding was higher with asthmatics compared to healthy horses' sera on four spots, and IgG3/5 binding was higher on 18 spots. Small IgG4/7 binding differences were detected on 10 spots with higher binding with asthmatics' sera on four but higher binding with healthy horses' sera on six spots. Proteins from the spots with group differences including mite and yeast proteins were identified by liquid chromatography mass spectrometry. The latter likely originated from the feeding substrate of the Der p culture. Prioritized antigen candidates amongst the proteins identified were Der p 1, Der p 11, group 15 allergens, myosin heavy chain, and uncharacterized Der p proteins. Additionally, yeast enolases, alcohol dehydrogenase (ADH), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase, and heat shock proteins were prioritized. Eleven antigen candidates were tested for confirmation by ELISAs using the respective proteins separately. Differences in asthmatics vs. healthy horses' serum Ig binding to Der p 1, Der p 18, and three yeast enzymes (enolase, ADH, and PGK) confirmed these as promising antigens of immune responses in SEA. Antigens with relevance in SEA were newly identified by immunoproteomics, and yeast antigens were considered for SEA for the first time. Serum IgG3/5 binding to relevant antigens was increased in SEA and is a novel feature that points to increased type-2 responses in SEA but requires confirmation of the corresponding cellular responses.

Using In silico Tools to Analyze the 5ʹ Untranslated Regions of the Alcohol Dehydrogenase Gene from Arabidopsis thaliana and Omega Sequence

Using In silico Tools to Analyze the 5ʹ Untranslated Regions of the Alcohol Dehydrogenase Gene from Arabidopsis thaliana and Omega Sequence

Breath Analysis for Lung Cancer Early Detection-A Clinical Study.

This clinical study presents a comprehensive investigation into the utility of breath analysis as a non-invasive method for the early detection of lung cancer. The study enrolled 14 lung cancer patients, 14 non-lung cancer controls with diverse medical conditions, and 3 tuberculosis (TB) patients for biomarker discovery. Matching criteria including age, gender, smoking history, and comorbidities were strictly followed to ensure reliable comparisons. A systematic breath sampling protocol utilizing a BIO-VOC sampler was employed, followed by VOC analysis using Thermal Desorption-Gas Chromatography-Mass Spectrometry (TD-GC/MS). The resulting VOC profiles were subjected to stringent statistical analysis, including Orthogonal Projections to Latent Structures-Discriminant Analysis (OPLS-DA), Kruskal-Wallis test, and Receiver Operating Characteristic (ROC) analysis. Notably, 13 VOCs exhibited statistically significant differences between lung cancer patients and controls. The combination of eight VOCs (hexanal, heptanal, octanal, benzaldehyde, undecane, phenylacetaldehyde, decanal, and benzoic acid) demonstrated substantial discriminatory power with an area under the curve (AUC) of 0.85, a sensitivity of 82%, and a specificity of 76% in the discovery set. Validation in an independent cohort yielded an AUC of 0.78, a sensitivity of 78%, and a specificity of 64%. Further analysis revealed that elevated aldehyde levels in lung cancer patients' breath could be attributed to overactivated Alcohol Dehydrogenase (ADH) pathways in cancerous tissues. Addressing methodological challenges, this study employed a matching of physiological and pathological confounders, controlled room air samples, and standardized breath sampling techniques. Despite the limitations, this study's findings emphasize the potential of breath analysis as a diagnostic tool for lung cancer and suggest its utility in differentiating tuberculosis from lung cancer. However, further research and validation are warranted for the translation of these findings into clinical practice.

Calcium chloride treatment maintains quality and enhances antioxidant capacity of fresh-cut carrots through modulating DcCMLs-DcCAMTA3-mediated phenylpropanoid pathway

Whitening and microbial infection are two major reasons to induce quality deterioration in fresh-cut carrots. Calcium chloride (CaCl2) could enhance the quality attributes in fresh-cut carrots, but the molecular mechanism is still not illuminated. In this study, CaCl2 treatment suppressed the increases of whiteness index and total bacterial count as well as enhanced the antioxidant capacity of fresh-cut carrots. In addition, CaCl2 treatment promoted phenolics accumulation by upregulating the transcripts and enzyme activity of phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H) and 4-coumarate coenzyme A ligase (4CL) in phenylpropanoid pathway. However, this treatment inhibited the increase of enzyme activity and gene expression of cinnamyl alcohol dehydrogenase (CAD), thus suppressing lignin synthesis and whitening process in fresh-cut carrots. Moreover, a calmodulin-binding transcription activator (CAMTA) transcription factor DcCAMTA3, was induced in response to CaCl2 treatment. DcCAMTA3 activated the transcription of DcPAL1, DcC4H and Dc4CL1 genes and repressed the expression of DcCAD1 gene by directly targeting the CG-box. DcCML3/11, a calmodulin-like protein, interacted with DcCAMTA3, and this interaction enhanced DcCAMTA3-mediated transcriptional activation of DcPAL1, DcC4H and Dc4CL1 and repression of DcCAD1. Therefore, CaCl2 treatment is effective in maintaining quality and enhancing antioxidant capacity of fresh-cut carrots by the cooperative action of DcCMLs and DcCAMTA3 in positively regulating phenylpropanoid pathway.

Postharvest caffeic acid dipping enhances disease resistance and storage capacity of ‘Zaosu’ pear fruit via regulating phenylpropane metabolism

Caffeic acid, a naturally occurring and safe phenolic compound, acts as a precursor in lignin synthesis within the phenylpropane metabolism pathway. The aims of this study was to evaluate the impacts of postharvest caffeic acid immersion on Alternaria rot and postharvest quality of ‘Zaosu’ pear fruit, as well as elucidate the underlying mechanisms involved. Results showed that postharvest caffeic acid dipping effectively inhibited spore germination and colony diameter of Alternaria alternata, as well as lesion diameter in A. alternata-inoculated pears. Caffeic acid treatment also delayed the increase of weight loss and decline in flesh firmness, increased total soluble solids content, and reduced surface yellowing of pears. Additionally, caffeic acid treatment enhanced the enzymatic activities and gene transcription levels of phenylalanine ammonialyase, 4-coumarate/coenzyme A ligase, cinnamate 4-hydroxylase, peroxidase, cinnamyl alcohol dehydrogenase, and polyphenol oxidase in pears. Meanwhile, the transcription levels of p-coumarate-3-hydroxylase, caffeic acid O-methyltransferase, cinnamoyl CoA reductase, caffeoyl shikimate esterase, hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase, caffeoyl CoA O-methyltransferase, ferulic acid 5-hydroxylase, and laccase genes were also up-regulated by caffeic acid treatment in pears. Furthermore, caffeic acid dipping enhanced phenylalanine, flavonoids, caffeic acid, total phenolic compounds, and lignin levels in the fruit. Therefore, caffeic acid exhibits promising potential for enhancing resistance against A. alternata and maintaining the storage capacity of pears via inducing phenylpropane metabolism.

Lactic Acid from CO2: A Carbon Capture and Utilization Strategy Based on a Biocatalytic Approach.

The EU low-carbon economy aims to reduce the level of CO2 emission in the EU to 80% by 2050. High efforts are required to achieve this goal, where successful CCU (Carbon Capture and Utilization) technologies will have a high impact. Biocatalysts offer a greener alternative to chemical catalysts for the development of CCU strategies since biocatalysis conforms 10 of the 12 principles of green chemistry. In this study, a multienzymatic system, based on alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC), and lactate dehydrogenase (LDH), that converts CO2 and ethanol into lactic acid leading to a 100% atom economy was studied. The system allows cofactor regeneration, thus reducing the process cost. Through reaction media engineering and enzyme ratio study, the performance of the system was able to produce up to 250 μM of lactic acid under the best conditions using 100% CO2, corresponding to the highest concentration of lactic acid obtained up to date using this multienzymatic approach. For the first time, the feasibility of the system to be applied under a real industrial environment has been tested using synthetic gas mimicking real blast furnace off-gases composition from the iron and steel industry. Under these conditions, the system was also capable of producing lactic acid, reaching 62 μM.

Comparative characterization and expression analysis revealed cinnamyl alcohol dehydrogenase genes differentially responding to Colletotrichum fructicola in woodland and cultivated strawberries

BACKGROUND: Strawberry (Fragaria×ananassa) production is hampered by anthracnose caused by Colletotrichum spp. Cinnamyl alcohol dehydrogenase (CAD) catalyzes lignin biosynthesis to fortify plant cell wall upon various stresses, but their involvement in strawberry resistance is elusive. OBJECTIVE: Current work aims to uncover the involvement of CAD genes in both woodland and cultivated strawberry responses to Colletotrichum spp. METHODS: Genome-wide identification of CAD genes was updated in cultivated strawberry. Comparative molecular characterization integrated with pathogenesis phenotyping after C. fructicola inoculation, were followed by RT-qPCR analysis and RNAseq data retrieval. RESULTS: Cultivated strawberry comprises 32 CAD genes of three classes. Thirteen syntenic relationships were observed between cultivated and woodland strawberry, with high homology in protein coding regions while promoters being greatly diversified. F. vesca ‘Hawaii4’ was least susceptible, followed by octoploid cultivar ‘ShenQi’, and ‘Benihoppe’ the most susceptible to C. fructicola. The class I CAD4 and CAD5, preferentially expressed in leaves and crowns, respectively, were up-regulated in susceptible varieties at 72 h post inoculation (hpi). Of class II, CAD8 was up-regulated in three genotypes at distinct timepoints, while CAD12 was quickly down-regulated in ‘ShenQi’ upon inoculation. CAD1 and CAD2 of class III were down-regulated in ‘ShenQi’ and ‘Hawaii4’ at distinct stages. The strong involvement of CAD genes in strawberry response to Colletotrichum spp. sets a basis for future functional study. CONCLUSION: F. vesca is less susceptible to Colletotrichum spp. than cultivated strawberries but the expression of CAD family in F. vesca and F.×ananassa were differentially provoked in timing and magnitude hinting no correlation with strawberry resistance to this pathogen.

An evolved pyrrolysyl-tRNA synthetase with polysubstrate specificity expands the toolbox for engineering enzymes with incorporation of noncanonical amino acids

Aminoacyl-tRNA synthetase (aaRS) is a core component for genetic code expansion (GCE), a powerful technique that enables the incorporation of noncanonical amino acids (ncAAs) into a protein. The aaRS with polyspecificity can be exploited in incorporating additional ncAAs into a protein without the evolution of new, orthogonal aaRS/tRNA pair, which hence provides a useful tool for probing the enzyme mechanism or expanding protein function. A variant (N346A/C348A) of pyrrolysyl-tRNA synthetase from Methanosarcina mazei (MmPylRS) exhibited a wide substrate scope of accepting over 40 phenylalanine derivatives. However, for most of the substrates, the incorporation efficiency was low. Here, a MbPylRS (N311A/C313A) variant was constructed that showed higher ncAA incorporation efficiency than its homologous MmPylRS (N346A/C348A). Next, N-terminal of MbPylRS (N311A/C313A) was engineered by a greedy combination of single variants identified previously, resulting in an IPE (N311A/C313A/V31I/T56P/A100E) variant with significantly improved activity against various ncAAs. Activity of IPE was then tested toward 43 novel ncAAs, and 16 of them were identified to be accepted by the variant. The variant hence could incorporate nearly 60 ncAAs in total into proteins. With the utility of this variant, eight various ncAAs were then incorporated into a lanthanide-dependent alcohol dehydrogenase PedH. Incorporation of phenyllactic acid improved the catalytic efficiency of PedH toward methanol by 1.8-fold, indicating the role of modifying protein main chain in enzyme engineering. Incorporation of O-tert-Butyl-L-tyrosine modified the enantioselectivity of PedH by influencing the interactions between substrate and protein. Enzymatic characterization and molecular dynamics simulations revealed the mechanism of ncAAs affecting PedH catalysis. This study provides a PylRS variant with high activity and substrate promiscuity, which increases the utility of GCE in enzyme mechanism illustration and engineering.Graphical

A polysaccharide from Alhagi honey protects the intestinal barrier and regulates the Nrf2/HO-1-TLR4/MAPK signaling pathway to treat alcoholic liver disease in mice

Ethnopharmacological relevanceAccording to the theory of traditional Chinese medicine, the main factors related to alcoholic liver disease (ALD) are qi stagnation and blood stasis of the five viscera. Previously, we showed that the bioactive components of Alhagi honey have various pharmacological effects in treating liver diseases, but the influence of Alhagi honey on ALD (and its mechanism of action) is not known. Aim of the studyTo determine the efficacy of the main active component of Alhagi honey, the polysaccharide AHPN80, in ALD and to explore the potential mechanism of action. Materials and methodsAHPN80 was isolated from dried Alhagi honey and identified by transmission electron microscopy, Fourier-transform infrared spectroscopy, and gas chromatography. Venous blood, liver tissue, and colon tissue were collected in a mouse model of alcohol-induced acute liver injury. Histology, staining (Oil Red O, Alcian Blue–Periodic Acid Schiff) and measurement of reactive oxygen species (ROS) levels were used to detect histopathologic and lipid-accumulation changes in the liver and colon. Lipopolysaccharide (LPS) levels and the content of proinflammatory cytokines in serum were measured by enzyme-linked immunosorbent assays. Commercial kits were employed to detect biochemistry parameters in serum and the liver. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining kit was used to identify hepatocyte apoptosis. Expression of tight junction-associated proteins in colon tissues and nuclear factor erythroid 2-related factor 2/heme oxygenase-1/toll-like receptor-4/mitogen-activated protein kinase (Nrf2/HO-1/TLR4/MAPK) pathway-related proteins in liver tissues and HepG2 cells were analyzed by immunofluorescence or western blotting. ResultsIn a mouse model of alcohol-induced acute liver injury, AHPN80 therapy: significantly improved liver parameters (cytochrome P450 2E1, alcohol dehydrogenase, aldehyde dehydrogenase, superoxide dismutase, malondialdehyde, glutathione peroxidase, catalase, total cholesterol, triglycerides, alanine transaminase, aspartate transaminase); reduced serum levels of LPS, interleukin (IL)-1β, IL-6, and tumor necrosis faction-α; increased levels of IL-10 and interferon-gamma. AHPN80 reduced ALD-induced lipid accumulation and ROS production, improved alcohol-induced inflammatory damage to hepatocytes, and inhibited hepatocyte apoptosis. Immunofluorescence staining and western blotting suggested that AHPN80 might eliminate hepatic oxidative stress by activating the Nrf2/HO-1 signaling pathway, repair the intestinal barrier, inhibit the LPS/TLR4/MAPK signaling pathway, and reduce liver inflammation. ConclusionsAHPN80 may activate the Nrf2/HO-1 pathway to eliminate oxidative stress, protect the intestinal barrier, and regulate the TLR4/MAPK pathway to treat ALD in mice. AHPN80 could be a functional food and natural medicine to prevent ALD and its complications.

Melatonin enhances waterlogging tolerance of field-grown cotton through quiescence adaptation and compensatory growth strategies

ContextWaterlogging stress inhibits plant growth and development, resulting in significant yield losses. Melatonin, a naturally occurring molecule in plants, has attracted attention in recent years for its potential role in aiding plants in response to abiotic stresses. However, it remains unclear whether melatonin can enhance cotton’s tolerance to waterlogging stress. MethodsTo fill this gap, field-grown cotton plants at early flowering were subjected to a 10-day waterlogging stress and were foliar sprayed with melatonin during waterlogging. The effects of melatonin on the endogenous melatonin concentration, plant biomass, and related physiological and molecular events, as well as the seedcotton yield and yield components, were evaluated over two consecutive years. ResultsIn waterlogged cotton without melatonin treatment (W), melatonin concentration increased by 10.2%, but it increased by 26.9% with melatonin treatment (W+M), compared to the non-waterlogged cotton (NW). Under waterlogging without the application of melatonin, there was a reduction of 27.1% in biological yield and 46.6% in seedcotton yield. However, these reductions were reduced to 21.8% and 39.0%, respectively, following melatonin application. Quantitative real-time PCR analysis revealed that melatonin up-regulated the melatonin biosynthesis genes GhSNAT1 and GhCOMT, as well as the gibberellin biosynthetic gene GhGA20ox1 by 2.1, 3.6 and 1.8 times, respectively, compared to non-waterlogged cotton. However, melatonin down-regulated the ABA synthesis gene GhNCED2, the H2O2 generation related gene GhRBOHC, and the glycolysis and fermentation gene GhADH2 by 61.9%, 65.0% and 40.0%, respectively. Additionally, melatonin increased the leaf chlorophyll content, photosynthetic rate, levels of auxin (IAA) and gibberellic acid (GA), as well as the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT). Conversely, melatonin reduced contents of hydrogen peroxide (H2O2), malonaldehyde (MDA) and ABA, as well as the activities of alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC), compared to waterlogging without application of melatonin. ConclusionsThese findings demonstrate that foliar application of melatonin effectively improved the waterlogging tolerance of field-grown cotton, leading to a reduction in yield loss by 16.5% under waterlogged conditions. This outcome was achieved by a series of physiological, biological and molecular adjustments involved in quiescence adaption and compensatory growth strategies. Therefore, melatonin plays a crucial role in enhancing waterlogging tolerance and mitigating yield loss in waterlogged cotton through these strategies.

Metabolic engineering of Escherichia coli for efficient production of 1,4-butanediol from crude glycerol

1,4-butanediol (1,4-BDO) is a significant non-natural chemical with diverse applications in various fields, particularly in the production of biodegradable plastic products as an essential monomer. The conversion of starch-based sugars using microbial cell factories provides a green alternative for the production of 1,4-BDO, but was limited by the low efficiency and high production cost. In this work, the classic CoA-dependent 1,4-BDO synthesis pathway was exogenously introduced into Escherichia coli, but the titer of 1,4-BDO was only 0.72 g/L using glucose as the substrate. The poorly expressed enzymes and the ineffective distribution of central carbon flux were considered the main limiting factors. Combined genetic manipulation of the synthesis pathway and bioprocess engineering, glycerol was proven to be a more favorable feedstock for 1,4-BDO synthesis, and a novel pathway functioned by carboxylic acid reductase could achieve one-step catalysis from 4-hydroxybutyric acid to 4-hydroxybutyraldehyde, which avoided the rate-limiting steps involved by hydroxybutyrate CoA transferase (Cat2) and alcohol dehydrogenase (Bld). The titer of 1,4-BDO reached 1.31 g/L and 1.07 g/L in the novel pathway using pure glycerol and the industrial by-product crude glycerol as the substrate, respectively. The fed-batch fermentation was finally conducted, and the titer of 1,4-BDO was increased to 5.75 g/L with a yield of 143.75 mg/g glycerol. Our work removed the bottleneck of the 1,4-BDO synthesis pathway by introducing carboxylic acid reductase and effectively converted crude glycerol to 1,4-BDO, which enabled us to build a sustainable microbial cell factory to cost-efficiently produce 1,4-BDO as well as other biochemicals from crude glycerol.

Combined metabolome and transcriptome analysis reveals a critical role of lignin biosynthesis and lignification in stem-like pneumatophore development of the mangrove Avicennia marina.

Transcriptional and metabolic regulation of lignin biosynthesis and lignification plays crucial roles in Avicennia marina pneumatophore development, facilitating its adaptation to coastal habitats. Avicennia marina is a pioneer mangrove species in coastal wetland. To cope with the periodic intertidal flooding and hypoxia environment, this species has developed a complex and extensive root system, with its most unique feature being a pneumatophore with a distinct above- and below-ground morphology and vascular structure. However, the characteristics of pneumatophore lignification remain unknown. Studies comparing the anatomy among above-ground pneumatophore, below-ground pneumatophore, and feeding root have suggested that vascular structure development in the pneumatophore is more like the development of a stem than of a root. Metabolome and transcriptome analysis illustrated that the accumulation of syringyl (S) and guaiacyl (G) units in the pneumatophore plays a critical role in lignification of the stem-like structure. Fourteen differentially accumulated metabolites (DAMs) and 10 differentially expressed genes involved in the lignin biosynthesis pathway were targeted. To identify genes significantly associated with lignification, we analyzed the correlation between 14 genes and 8 metabolites and further built a co-expression network between 10 transcription factors (TFs), including 5 for each of MYB and NAC, and 23 enzyme-coding genes involved in lignin biosynthesis. 4-Coumarate-CoA ligase, shikimate/quinate hydroxycinnamoyl transferase, cinnamyl alcohol dehydrogenase, caffeic acid 3-O-methyltransferase, phenylalanine ammonia-lyase, and peroxidase were identified to be strongly correlated with these TFs. Finally, we examined 9 key candidate genes through quantitative real-time PCR to validate the reliability of transcriptome data. Together, our metabolome and transcriptome findings reveal that lignin biosynthesis and lignification regulate pneumatophore development in the mangrove species A. marina and facilitate its adaptation to coastal habitats.

Effects of Hovenia dulcis fruit and peduncle extract on alcohol metabolism

Ethnopharmacological relevanceThe dried fruit and peduncle of Hovenia dulcis Thunberg (Rhamnaceae) (HD) has been used as a folk medicine to treat liver disease, detoxify alcoholism, and prevent and cure hangovers. Aim of the studyWe investigated the pharmacology of HD on the kinetics of EtOH and on the enzymes related to alcohol metabolism to seek the scientific evidence of HD to prevent hangover, the effectiveness as a folk medicine. Materials and methodsEtOH was orally administered 30 min after oral administration of HD boiling water extract in rats. Then, the profiles of blood EtOH concentrations were measured. Mice were reared with food containing powdered HD for 7 days, and the activities of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) in liver were measured. Hepa1c1c7 cells were cultured with the medium containing HD extract, and the activities of ADH and ALDH were measured. ResultsHD extract reduced the blood EtOH concentrations in rats and induced the activities of ADH and ALDH and mRNA and protein expressions of ADH1B, ALDH1A1, and ALDH2 in the liver of mice and Hepa1c1c7 cells. Dihydromyricetin, one of the ingredients of HD, significantly induced the activities of ADH and ALDH in Hepa1c1c7 cells, however, the fractions containing hydrophilic organic compounds with small molecular weight contributed the most of the activities of HD extract. ConclusionsWe clarified the experimental pharmacological evidences of HD as a folk medicine to detoxify alcoholism and prevent hangovers.

Implementation of mandatory blood alcohol screening in trauma patients: A retrospective study from a tertiary trauma center in a Middle Eastern country

BackgroundAlcohol consumption is a significant risk factor for several types of injuries and trauma recidivism and remains an important public health concern worldwide. We aimed to describe the implementation of mandatory alcohol screening and the AUDIT tool among trauma patients admitted to a level 1 trauma center in a country with a partial ban on alcohol consumption. MethodsThis was a retrospective analysis of trauma patients (>12 years old) who required hospital admission and underwent blood alcohol concentration (BAC) screening between 2014 and 2019. This was achieved via an enzymatic method using alcohol dehydrogenase for ethanol detection in the plasma and serum samples. Trauma patients with a BAC <2.2 mmol/L were referred to as “negative”, and BAC >2.2 mmol/L was referred to as “BAC positive”. A comparative analysis was performed between the two BAC groups. Alcohol Screening, Brief Intervention, Referral for Treatment [ASBIRT] program, and AUDIT were applied. ResultsA total of 7326 BAC screening tests were performed on 7284 patients during the study period. With slight variation over the years, the compliance rate was 77% (70.4%–85.3%), and the test positivity rate was 10% (8.6%–12.5%). There were 42 repeated admissions, of which seven patients were BAC positive at every admission. Young age and non-Arab patients were more likely to test positive, and the main mechanism of injury (MOI) was road traffic-related trauma (p < 0.05). Assault and self-inflicted injuries were significantly higher in BAC-positive patients than in BAC-negative patients (18% vs. 4% and 2.7% vs. 1.3%, respectively; p = 0.001). The injury severity score (ISS) and mortality rate were comparable between the study groups. Patients with a positive BAC were significantly more likely to undergo pan-CT scan in the emergency department, intubation, and exploratory laparotomy than those with a negative BAC. In patients who sustained injuries due to assault, all-terrain vehicles, or motorcycle crashes, there was a significant association between the positivity of BAC tests and the patient's ISS. ConclusionDespite improvements in BAC screening in trauma admissions over the years, almost 20% of cases were missed. Although the mortality rates were comparable, alcohol consumption burdens resources in terms of excess imaging, intubation, open abdominal surgery, and possible disability. Further studies are needed to understand the key obstacles and challenges to achieving optimum compliance for screening in trauma settings.