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Chemistry (Weinheim An Der Bergstrasse,... Jun 2024Rivastigmine is one of the several pharmaceuticals widely prescribed for the treatment of Alzheimer's disease. However, its practical synthesis still faces many issues,...
Rivastigmine is one of the several pharmaceuticals widely prescribed for the treatment of Alzheimer's disease. However, its practical synthesis still faces many issues, such as the involvement of toxic metals and harsh reaction conditions. Herein, we report a chemo-enzymatic synthesis of Rivastigmine. The key chiral intermediate was synthesized by an engineered alcohol dehydrogenase from Lactobacillus brevis (LbADH). A semi-rational approach was employed to improve its catalytic activity and thermal stability. Several LbADH variants were obtained with a remarkable increase in activity and melting temperature. Exploration of the substrate scope of these variants demonstrated improved activities toward various ketones, especially acetophenone analogs. To further recycle and reuse the biocatalyst, one LbADH variant and glucose dehydrogenase were co-immobilized on nanoparticles. By integrating enzymatic and chemical steps, Rivastigmine was successfully synthesized with an overall yield of 66 %. This study offers an efficient chemo-enzymatic route for Rivastigmine and provides several efficient LbADH variants with a broad range of potential applications.
Topics: Rivastigmine; Levilactobacillus brevis; Alcohol Dehydrogenase; Enzymes, Immobilized; Biocatalysis; Acetophenones; Protein Engineering
PubMed: 38568868
DOI: 10.1002/chem.202400454 -
Plant Disease Apr 2024Faba bean () is one of the characteristic economic crops in Qinghai Province of China, which has multiple uses as grain, vegetable, fodder, fertilizer and medicine....
Faba bean () is one of the characteristic economic crops in Qinghai Province of China, which has multiple uses as grain, vegetable, fodder, fertilizer and medicine. Chocolate spot is a critical disease of faba bean in the world, and it is widely spread in all production areas of Qinghai. In August 2021, a severe occurrence of chocolate spot was found in a faba bean field in Xunhua County, Qinghai Province (35°52'N, 102°22'E, alt. 1890m). All plants in the field were affected by this disease. A voucher specimen was deposited in the Herbarium of Plant Pathology, College of Agricultural and Forestry Sciences at Qinghai University under accession No. PY015. The pathogen infected the leaves and stems, causing small irregular red spots to appear, which later coalesce into larger spots and faded green lesions appear around the spots. Diseased leaf pieces 5 mm2 were surface sterilized with 75% ethyl alcohol for 30s, 1.2% NaOCl for 30s, and rinsed three times with sterile water. They were then plated on potato dextrose agar (PDA) at 22℃ for 10 days in the dark. Fungal colonies are initially white, then gray, and have produced spores by 5 days. Conidia are clusters, ellipsoidal or ovoid, 9-14 × 6-9 μm. The conidiophore is straight, terminally enlarged, septate, 300-1500 μm long, 8-13 μm wide. No sclerotia were observed during culture. DNA of the strain PY015 was extracted by CTAB method. Molecular identification was first performed using the universal region of ITS (ITS1/ITS4). The PCR product was sequenced, the sequence was deposited in GenBank under the accession number OR739575. The results showed 100% similarity to spp. (KX301016, MT250940, LC519322) in BLAST search. Molecular characterization was continued using five specific primer pairs: (DNA-dependent RNA polymerase subunit II, RPB2-5F/RPB2-7cR), and (necrosis and ethylene-inducing proteins, NEP1for/ NEP1revB and NEP2forD/NEP2revD), (heat-shock protein 60, HSP60for/HSP60rev), (glyceraldehyde-3-phosphate dehydrogenase, G3PDHfor/G3PDHrev). The sequences of PY015 were deposited in GenBank (accession numbers: OR731179, OR731180, OR731181, OR731182, OR731183), and all five sequences showed 100% similarity to YZU171088 (accession numbers: MH614610 MH614611, MH614612, MH614613 MH614614). A phylogenetic tree based on these five genes was constructed using Mega7.0 (1000 bootstrap replicates, neighbor-joining method), and PY015 was placed in the same clade as YZU171088 with 100% bootstrap values. Morphological and molecular biological results confirmed that isolate PY015 was . To fulfill Koch's postulates, the spore suspension (2 × 105 conidia/ml) was sprayed on healthy faba bean (Yun-122) plants at the 10-leaf stage, while an equal amount of sterile distilled water was applied to controls. After 7 days, the inoculated plants showed symptoms consistent with field infection and was re-isolated using the same protocol, while the control remained asymptomatic. The pathogenicity test was repeated twice. The same isolates were recovered from symptomatic leaves and identified by sequence. was morphologically and molecularly identical to the original isolates, completing Koch's postulates. Currently, , , and are the main pathogens of chocolate spot on faba bean that have been identified and reported nationally and internationally. is a new species discovered from eucalyptus in southern China in 2016, and its current hosts are only eucalyptus and citrus. To our knowledge, the present study is the first report of chocolate spot caused by on faba bean in China.
PubMed: 38568789
DOI: 10.1094/PDIS-11-23-2477-PDN -
Analytical Chemistry Apr 2024Lipid metabolic alterations are known to play a crucial role in cancer metastasis. As a key hub in lipid metabolism, intracellular neutral lipid accumulation in lipid...
Lipid metabolic alterations are known to play a crucial role in cancer metastasis. As a key hub in lipid metabolism, intracellular neutral lipid accumulation in lipid droplets (LDs) has become a signature of aggressive human cancers. Nevertheless, it remains unclear whether lipid accumulation displays distinctive features in metastatic lesions compared to the primary ones. Here, we integrated multicolor stimulated Raman scattering (SRS) imaging with confocal Raman spectroscopy on the same platform to quantitatively analyze the amount and composition of LDs in intact human thyroid tissues without any processing or labeling. Inspiringly, we found aberrant accumulation of triglycerides (TGs) in lymphatic metastases but not in normal thyroid, primary papillary thyroid carcinoma (PTC), or normal lymph node. In addition, the unsaturation degree of unsaturated TGs was significantly higher in the lymphatic metastases from patients diagnosed with late-stage (T3/T4) PTC compared to those of patients diagnosed with early-stage (T1/T2) PTC. Furthermore, both public sequencing data analysis and our RNA-seq transcriptomic experiment showed significantly higher expression of alcohol dehydrogenase-1B (ADH1B), which is critical to lipid uptake and transport, in lymphatic metastases relative to the primary ones. In summary, these findings unravel the lipid accumulation as a novel marker and therapeutic target for PTC lymphatic metastasis that has a poor response to the regular radioactive iodine therapy.
Topics: Humans; Thyroid Cancer, Papillary; Lymphatic Metastasis; Thyroid Neoplasms; Carcinoma, Papillary; Iodine Radioisotopes; Nonlinear Optical Microscopy; Lipids
PubMed: 38567982
DOI: 10.1021/acs.analchem.4c00306 -
Chembiochem : a European Journal of... Jun 2024Detailed insights into protein structure/function relationships require robust characterization methodologies. Free-solution capillary electrophoresis (CE) is a unique...
Detailed insights into protein structure/function relationships require robust characterization methodologies. Free-solution capillary electrophoresis (CE) is a unique separation technique which is sensitive to the conformation and/or composition of proteins, and therefore provides information on the heterogeneity of these properties. Three unrelated, conformationally/compositionally-altered proteins were separated by CE. An electrophoretic mobility distribution was determined for each protein along with its conformational and/or compositional heterogeneity. The CE results were compared with molar mass distributions obtained from size-exclusion chromatography coupled to light scattering (SEC-MALS). Bovine serum albumin multimers and two monomeric species were separated, highlighting variations in conformational/compositional heterogeneity among the multimers. Analysis of yeast alcohol dehydrogenase resolved two monomeric conformers and various tetrameric species, illustrating the impact of zinc ion removal and disulfide bond reduction on the protein's heterogeneity. The apo (calcium-free) and holo forms of bovine α-lactalbumin were separated and differences in the species' heterogeneity were measured; by contrast, the SEC-MALS profiles were identical. Comparative analysis of these structurally unrelated proteins provided novel insights into the interplay between molar mass and conformational/compositional heterogeneity. Overall, this study expands the utility of CE by demonstrating its capacity to discern protein species and their heterogeneity, properties which are not readily accessible by other analytical techniques.
Topics: Electrophoresis, Capillary; Cattle; Animals; Protein Conformation; Alcohol Dehydrogenase; Serum Albumin, Bovine; Lactalbumin
PubMed: 38567504
DOI: 10.1002/cbic.202400108 -
Journal of Hazardous Materials May 2024The study addressed the challenge of treating petroleum industry wastewater with high concentrations of 1,2-dichloroethane (1,2-DCA) ranging from 384 to 1654 mg/L,...
The study addressed the challenge of treating petroleum industry wastewater with high concentrations of 1,2-dichloroethane (1,2-DCA) ranging from 384 to 1654 mg/L, which poses a challenge for bacterial biodegradation and algal photodegradation. To overcome this, a collaborative approach using membrane bioreactors (MBRs) that combine algae and bacteria was employed. This synergistic method effectively mitigated the toxicity of 1,2-DCA and curbed MBR fouling. Two types of MBRs were tested: one (B-MBR) used bacterial cultures and the other (AB-MBR) incorporated a mix of algal and bacterial cultures. The AB-MBR significantly contributed to 1,2-DCA removal, with algae accounting for over 20% and bacteria for approximately 49.5% of the dechlorination process. 1,2-DCA metabolites, including 2-chloroethanol, 2-chloro-acetaldehyde, 2-chloroacetic acid, and acetic acid, were partially consumed as carbon sources by algae. Operational efficiency peaked at a 12-hour hydraulic retention time (HRT) in AB-MBR, enhancing enzyme activities crucial for 1,2-DCA degradation such as dehydrogenase (DH), alcohol dehydrogenase (ADH), and acetaldehyde dehydrogenase (ALDH). The microbial diversity in AB-MBR surpassed that in B-MBR, with a notable increase in Proteobacteria, Bacteroidota, Planctomycetota, and Verrucomicrobiota. Furthermore, AB-MBR showed a significant rise in the dominance of 1,2-DCA-degrading genus such as Pseudomonas and Acinetobacter. Additionally, algal-degrading phyla (e.g., Nematoda, Rotifera, and Streptophyta) were more prevalent in AB-MBR, substantially reducing the issue of membrane fouling.
Topics: Bioreactors; Wastewater; Water Pollutants, Chemical; Ethylene Dichlorides; Membranes, Artificial; Petroleum; Bacteria; Biodegradation, Environmental; Waste Disposal, Fluid
PubMed: 38565016
DOI: 10.1016/j.jhazmat.2024.134125 -
BMC Plant Biology Apr 2024Dendrobium spp. comprise a group of tropical orchids with ornamental and medicinal value. Dendrobium spp. are sensitive to low temperature, and the underlying cold...
BACKGROUND
Dendrobium spp. comprise a group of tropical orchids with ornamental and medicinal value. Dendrobium spp. are sensitive to low temperature, and the underlying cold response regulatory mechanisms in this group are unclear. To understand how these plants respond to cold stress, we compared the transcriptomic responses of the cold-tolerant cultivar 'Hongxing' (HX) and the cold-sensitive cultivar 'Sonia Hiasakul' (SH) to cold stress.
RESULTS
Chemometric results showed that the physiological response of SH in the later stages of cold stress is similar to that of HX throughout the cold treatment. Orthogonal partial least squares discriminant analysis (OPLS-DA) revealed that soluble protein content and peroxidase activity are key physiological parameters for assessing the cold tolerance of these two Dendrobium spp. cultivars. Additionally, weighted gene co-expression network analysis (WGCNA) results showed that many cold response genes and metabolic pathways significantly associated with the physiological indices were enriched in the 12 detected modules. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) enrichment analyses of the 105 hub genes showed that Dendrobium spp. adapt to cold stress by regulating signal transduction, phytohormones, transcription factors, protein translation and modification, functional proteins, biosynthesis and metabolism, cell structure, light, and the circadian clock. Hub genes of the cold stress response network included the remorin gene pp34, the abscisic acid signaling pathway-related genes PROTEIN PHOSPATASE 2 C (PP2C), SNF1-RELATED PROTEIN KINASE 2 (SnRK2), ABRE-BINDING FACTOR 1 (ABF1) and SKI-INTERACTING PROTEIN 17 (SKIP17), the Ca signaling-related GTP diphosphokinase gene CRSH1, the carbohydrate-related gene STARCH SYNTHASE 2 (SS2), the cell wall biosynthesis gene CINNAMYL ALCOHOL DEHYDROGENASE (CAD7), and the endocytosis-related gene VACUOLAR PROTEIN SORTING-ASSOCIATED PROTEIN 52 A (VPS52A).
CONCLUSIONS
The cold-responsive genes and metabolic pathways of Dendrobium spp. revealed in this study provide important insight to enable the genetic enhancement of cold tolerance in Dendrobium spp., and to facilitate cold tolerance breeding in related plants.
Topics: Cold-Shock Response; Dendrobium; Plant Breeding; Gene Expression Profiling; Transcriptome; Stress, Physiological; Gene Expression Regulation, Plant
PubMed: 38561687
DOI: 10.1186/s12870-024-04903-1 -
Reproductive Sciences (Thousand Oaks,... Apr 2024Endometriosis (EMT) -related infertility has been a challenge for clinical research. Many studies have confirmed that abnormal alterations in the immune microenvironment...
Endometriosis (EMT) -related infertility has been a challenge for clinical research. Many studies have confirmed that abnormal alterations in the immune microenvironment and glycolysis are instrumental in causing EMT-related infertility. Recently, our research team identified several key glycolysis-immune-related genes in the endometrial cells of EMT patients. This study aimed to further investigate the expression patterns of pyruvate dehydrogenase kinase 3 (PDK3), glypican-3 (GPC3), and alcohol dehydrogenase 6 (ADH6), which are related to glycolysis and immunity, in the follicular microenvironment of infertile patients with EMT using enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR) assays. According to the results, compared to the patients with tubal factor infertility, the concentrations of PDK3 and GPC3 were considerably increased in the follicular environment of EMT patients, while ADH6 expression was significantly reduced. The number of oocytes retrieved, the transferable embryo rate, and the cumulative clinical pregnancy rate of EMT patients were significantly reduced, and there was a correlation with the level of PDK3, GPC3, and ADH6 in Follicular Fluid (FF). The area under the receiver operating characteristic (ROC) curve for predicting clinical pregnancy in infertile patients with EMT for PDK3, GPC3, ADH6, and their combination was 0.732, 0.705, 0.855, and 0.879, respectively (P < 0.05). In conclusion, our research indicates that glycolysis-immune-related genes may contribute to infertility in EMT patients through immune infiltration, and disruption of mitochondrial and oocyte functions. The combined detection of PDK3, GPC3, and ADH6 in FF helps to predict clinical pregnancy outcomes in infertile patients with EMT.
PubMed: 38561472
DOI: 10.1007/s43032-024-01518-7 -
Angewandte Chemie (International Ed. in... May 2024The design and orderly layered co-immobilization of multiple enzymes on resin particles remain challenging. In this study, the SpyTag/SpyCatcher binding pair was fused...
The design and orderly layered co-immobilization of multiple enzymes on resin particles remain challenging. In this study, the SpyTag/SpyCatcher binding pair was fused to the N-terminus of an alcohol dehydrogenase (ADH) and an aldo-keto reductase (AKR), respectively. A non-canonical amino acid (ncAA), p-azido-L-phenylalanine (p-AzF), as the anchor for covalent bonding enzymes, was genetically inserted into preselected sites in the AKR and ADH. Employing the two bioorthogonal counterparts of SpyTag/SpyCatcher and azide-alkyne cycloaddition for the immobilization of AKR and ADH enabled sequential dual-enzyme coating on porous microspheres. The ordered dual-enzyme reactor was subsequently used to synthesize (S)-1-(2-chlorophenyl)ethanol asymmetrically from the corresponding prochiral ketone, enabling the in situ regeneration of NADPH. The reactor exhibited a high catalytic conversion of 74 % and good reproducibility, retaining 80 % of its initial activity after six cycles. The product had 99.9 % ee, which that was maintained in each cycle. Additionally, the double-layer immobilization method significantly increased the enzyme loading capacity, which was approximately 1.7 times greater than that of traditional single-layer immobilization. More importantly, it simultaneously enabled both the purification and immobilization of multiple enzymes on carriers, thus providing a convenient approach to facilitate cascade biocatalysis.
Topics: Enzymes, Immobilized; Alcohol Dehydrogenase; Biocatalysis; Protein Engineering; Aldo-Keto Reductases; Phenylalanine; Azides
PubMed: 38556813
DOI: 10.1002/anie.202403539 -
The Journal of Biological Chemistry May 2024Sterols are ubiquitous membrane constituents that persist to a large extent in the environment due to their water insolubility and chemical inertness. Recently, an...
Sterols are ubiquitous membrane constituents that persist to a large extent in the environment due to their water insolubility and chemical inertness. Recently, an oxygenase-independent sterol degradation pathway was discovered in a cholesterol-grown denitrifying bacterium Sterolibacterium (S.) denitrificans. It achieves hydroxylation of the unactivated primary C26 of the isoprenoid side chain to an allylic alcohol via a phosphorylated intermediate in a four-step ATP-dependent enzyme cascade. However, this pathway is incompatible with the degradation of widely distributed steroids containing a double bond at C22 in the isoprenoid side chain such as the plant sterol stigmasterol. Here, we have enriched a prototypical delta-24 desaturase from S. denitrificans, which catalyzes the electron acceptor-dependent oxidation of the intermediate stigmast-1,4-diene-3-one to a conjugated (22,24)-diene. We suggest an αβ architecture of the 440 kDa enzyme, with each subunit covalently binding an flavin mononucleotide cofactor to a histidyl residue. As isolated, both flavins are present as red semiquinone radicals, which can be reduced by stigmast-1,4-diene-3-one but cannot be oxidized even with strong oxidizing agents. We propose a mechanism involving an allylic radical intermediate in which two flavin semiquinones each abstract one hydrogen atom from the substrate. The conjugated delta-22,24 moiety formed allows for the subsequent hydroxylation of the terminal C26 with water by a heterologously produced molybdenum-dependent steroid C26 dehydrogenase 2. In conclusion, the pathway elucidated for delta-22 steroids achieves oxygen-independent hydroxylation of the isoprenoid side chain by bypassing the ATP-dependent formation of a phosphorylated intermediate.
Topics: Bacterial Proteins; Molybdenum; Stigmasterol; Betaproteobacteria; Fatty Acid Desaturases; Hydroxylation; Flavins
PubMed: 38556086
DOI: 10.1016/j.jbc.2024.107243 -
Biochemical and Biophysical Research... May 2024Hyperuricemia is a chronic metabolic disease caused by purine metabolism disorder. And several gene loci and transporter proteins that associated with uric acid...
Hyperuricemia is a chronic metabolic disease caused by purine metabolism disorder. And several gene loci and transporter proteins that associated with uric acid transport functions have been identified. Retinol Dehydrogenase 12 (RDH12), recognized for its role in safeguarding photoreceptors, and our study investigated the potential impact of Rdh12 mutations on other organs and diseases, particularly hyperuricemia. We assessed Rdh12 mRNA expression levels in various tissues and conducted serum biochemical analyses in Rdh12 mice. Compared with the wild type, significant alterations in serum uric acid levels and kidney-related biochemical indicators have been revealed. Then further analysis, including quantitative RT-PCR of gene expression in the liver and kidney, highlighted variations in the expression levels of specific genes linked to hyperuricemia. And renal histology assessment exposed mild pathological lesions in the kidneys of Rdh12 mice. In summary, our study suggests that Rdh12 mutations impact not only retinal function but also contribute to hyperuricemia and renal disease phenotypes in mice. Our finding implies that individuals with Rdh12 mutations may be prone to hyperuricemia and gout, emphasizing the significance of preventive measures and regular examinations in daily life.
Topics: Mice; Animals; Hyperuricemia; Uric Acid; Alcohol Oxidoreductases; Phenotype
PubMed: 38552555
DOI: 10.1016/j.bbrc.2024.149809