Zeaxanthin epoxidase 3 Knockout Mutants of the Model Diatom Phaeodactylum tricornutum Enable Commercial Production of the Bioactive Carotenoid Diatoxanthin.

Carotenoids are pigments that have a range of functions in human health. The carotenoid diatoxanthin is suggested to have antioxidant, anti-inflammatory and chemo-preventive properties. Diatoxanthin is only produced by a few groups of microalgae, where it functions in photoprotection. Its large-scale production in microalgae is currently not feasible. In fact, rapid conversion into the inactive pigment diadinoxanthin is triggered when cells are removed from a high-intensity light source, which is the case during large-scale harvesting of microalgae biomass. Zeaxanthin epoxidase (ZEP) 2 and/or ZEP3 have been suggested to be responsible for the back-conversion of high-light accumulated diatoxanthin to diadinoxanthin in low-light in diatoms. Using CRISPR/Cas9 gene editing technology, we knocked out the ZEP2 and ZEP3 genes in the marine diatom Phaeodactylum tricornutum to investigate their role in the diadinoxanthin-diatoxanthin cycle and determine if one of the mutant strains could function as a diatoxanthin production line. Light-shift experiments proved that ZEP3 encodes the enzyme converting diatoxanthin to diadinoxanthin in low light. Loss of ZEP3 caused the high-light-accumulated diatoxanthin to be stable for several hours after the cultures had been returned to low light, suggesting that zep3 mutant strains could be suitable as commercial production lines of diatoxanthin.

Clinical application value of pre-pregnancy carrier screening in Chinese Han childbearing population.

BACKGROUND: To explore the clinical application value of pre-conception expanded carrier screening (PECS) in the Chinese Han ethnicity population of childbearing age. METHODS: The results of genetic testing of infertile parents who underwent PECS in the Reproductive Medicine Center of the Second Affiliated Hospital of Zhengzhou University, China, from September 2019 to December 2021, were retrospectively analyzed. The carrier rate of single gene disease, the detection rate of high-risk parents, and the clinical outcome of high-risk parents were statistically analyzed. RESULTS: A total of 1372 Chinese Han ethnicity patients underwent PECS, among which 458 patients underwent the extended 108-gene test, their overall carrier rate was 31.7%, and the detection rate of high-risk parents was 0.3%. The highest carrier rates were SLC22A (2.4%), ATP7B (2.4%), MMACHC (2.2%), PAH (1.8%), GALC (1.8%), MLC1 (1.3%), UNC13D (1.1%), CAPN3 (1.1%), and PKHD1 (1.1%). There were 488 women with fragile X syndrome-FMR1 gene detection, and 6 patients (1.2%) had FMR1 gene mutation. A total of 426 patients were screened for spinal muscular atrophy-SMN1, and the carrier rate was 3.5%, and the detection rate of parents' co-carrier was 0.5%. CONCLUSION: Monogenic recessive hereditary diseases had a high carrier rate in the population. Pre-pregnancy screening could provide good prenatal and postnatal care guidance for patients and preimplantation genetic testing for monogenic/single gene disorders (PGT-M) and prenatal diagnosis could provide more precise reproductive choices for high-risk parents.

Radical fluorine transfer catalysed by an engineered nonheme iron enzyme.

Nonheme iron enzymes stand out as one of the most versatile biocatalysts for molecular functionalization. They facilitate a wide array of chemical transformations within biological processes, including hydroxylation, chlorination, epimerization, desaturation, cyclization, and more. Beyond their native biological functions, these enzymes possess substantial potential as powerful biocatalytic platforms for achieving abiological metal-catalyzed reactions, owing to their functional and structural diversity and high evolvability. To this end, our group has recently engineered a series of nonheme iron enzymes to employ non-natural radical-relay mechanisms for abiological radical transformations not previously known in biology. Notably, we have demonstrated that a nonheme iron enzyme, (S)-2-hydroxypropylphosphonate epoxidase from Streptomyces viridochromogenes (SvHppE), can be repurposed into an efficient and selective biocatalyst for radical fluorine transfer reactions. This marks the first known instance of a redox enzymatic process for C(sp3)F bond formation. This chapter outlines the detailed experimental protocol for engineering SvHPPE for fluorination reactions. Furthermore, the provided protocol could serve as a general guideline that might facilitate other engineering endeavors targeting nonheme iron enzymes for novel catalytic functions.

Tri-enzyme fusion of tryptophan halogenase achieves a concise strategy for coenzyme self-sufficiency and the continuous halogenation of L-tryptophan.

The halogenase-based catalysis is one of the most environmentally friendly methods for the synthesis of halogenated products, among which flavin-dependent halogenases (FDHs) have attracted great interest as one of the most promising biocatalysts due to the remarkable site-selectivity and wide substrate range. However, the complexity of constructing the NAD+-NADH-FAD-FADH2 bicoenzyme cycle system has affected the engineering applications of FDHs. In this work, a coenzyme self-sufficient tri-enzyme fusion was constructed and successfully applied to the continuous halogenation of L-tryptophan. SpFDH was firstly identified derived from Streptomyces pratensis, a highly selective halogenase capable of generating 6-chloro-tryptophan from tryptophan. Then, using gene fusion technology, SpFDH was fused with glucose dehydrogenase (GDH) and flavin reductase (FR) to form a tri-enzyme fusion, which increased the yield by 1.46-fold and making the coenzymes self-sufficient. For more efficient halogenation of L-tryptophan, a continuous halogenation bioprocess of L-tryptophan was developed by immobilizing the tri-enzyme fusion and attaching it to a continuous catalytic device, which resulted in a reaction yield of 97.6% after 12 h reaction. An FDH from S. pratensis was successfully applied in the halogenation and our study provides a concise strategy for the preparation of halogenated tryptophan mediated by multienzyme cascade catalysis.

Identification of novel molecules and pathways associated with fascin actin­bundling protein 1 in laryngeal squamous cell carcinoma through comprehensive transcriptome analysis.

Laryngeal squamous cell carcinoma (LSCC) is a common malignant tumor with a poor prognosis. Fascin actin­bundling protein 1 (FSCN1) has been reported to play a crucial role in the development and progression of LSCC; however, the underlying molecular mechanisms remain unknown. Herein, a whole transcriptome microarray analysis was performed to screen for differentially expressed genes (DEGs) in cells in which FSCN1 was knocked down. A total of 462 up and 601 downregulated mRNA transcripts were identified. Functional annotation analysis revealed that these DEGs were involved in multiple biological functions, such as transcriptional regulation, response to radiation, focal adhesion, extracellular matrix­receptor interaction, steroid biosynthesis and others. Through co­expression and protein­protein interaction analysis, FSCN1 was linked to novel functions, including defense response to virus and steroid biosynthesis. Furthermore, crosstalk analysis with FSCN1­interacting proteins revealed seven DEGs, identified as FSCN1­interacting partners, in LSCC cells, three of which were selected for further validation. Co­immunoprecipitation validation confirmed that FSCN1 interacted with prostaglandin reductase 1 and 24­dehydrocholesterol reductase (DHCR24). Of note, DHCR24 is a key enzyme involved in cholesterol biosynthesis, and its overexpression promotes the proliferation and migration of LSCC cells. These findings suggest that DHCR24 is a novel molecule associated with FSCN1 in LSCC, and that the FSCN1­DHCR24 interaction may promote LSCC progression by regulating cholesterol metabolism­related signaling pathways.

Identification of redox activators for continuous reactivation of glyoxal oxidase from Trametes versicolor in a two-enzyme reaction cascade.

Glyoxal oxidases, belonging to the group of copper radical oxidases (CROs), oxidize aldehydes to carboxylic acids, while reducing O2 to H2O2. Their activity on furan derivatives like 5-hydroxymethylfurfural (HMF) makes these enzymes promising biocatalysts for the environmentally friendly synthesis of the bioplastics precursor 2,5-furandicarboxylic acid (FDCA). However, glyoxal oxidases suffer from inactivation, which requires the identification of suitable redox activators for efficient substrate conversion. Furthermore, only a few glyoxal oxidases have been expressed and characterized so far. Here, we report on a new glyoxal oxidase from Trametes versicolor (TvGLOX) that was expressed at high levels in Pichia pastoris (reclassified as Komagataella phaffii). TvGLOX was found to catalyze the oxidation of aldehyde groups in glyoxylic acid, methyl glyoxal, HMF, 2,5-diformylfuran (DFF) and 5-formyl-2-furancarboxylic acid (FFCA), but barely accepted alcohol groups as in 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), preventing formation of FDCA from HMF. Various redox activators were tested for TvGLOX reactivation during catalyzed reactions. Among them, a combination of horseradish peroxidase and its substrate 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS) most efficiently reactivated TvGLOX. Through continuous reactivation of TvGLOX in a two-enzyme system employing a recombinant Moesziomyces antarcticus aryl-alcohol oxidase (MaAAO) almost complete conversion of 8 mM HMF to FDCA was achieved within 24 h.

Mutation in BrFLS encoding flavonol synthase induced anthocyanin accumulation in Chinese cabbage.

KEY MESSAGE: BrFLS mutation promoted anthocyanin accumulation in Chinese cabbage, which was verified in four allelic mutants. Chinese cabbage is a major vegetable crop in Eastern Asia. Anthocyanin-rich vibrantly colored varieties are increasingly favored by consumers for their higher nutritional and aesthetic value compared to the typical green varieties of Chinese cabbage. Herein, we identified an anthocyanin accumulation mutant aam1 from a mutant library of EMS-mutagenized Chinese cabbage DH line 'FT', which appeared partial purple on leaves, bolting stems and floral buds. This anthocyanin accumulation trait was genetically controlled by a recessive nuclear gene, and through MutMap mapping and KASP genotyping, BraA10g030950.3C was identified as the candidate causal gene with a G202 to A202 non-synonymous SNP variation in exon 1. Three additional mutants allelic to aam1 were obtained via screening of similar-phenotype mutants from the mutant library, namely aam2/3/4, where the causal SNPs reside in the same gene as aam1, corroborating that the mutation of BraA10g030950.3C caused anthocyanin accumulation. BraA10g030950.3C encodes a flavonol synthase that catalyzes dihydroflavonols substrate into flavonols and is homologous to Arabidopsis FLS1 (AT5G08640), named BrFLS. Compared to wildtype, the expression level of BrFLS was significantly reduced in the mutants, while BrDFR, which is involved in the anthocyanin biosynthesis and competes with FLS for the common substrate dihydroflavonols, was increased. The flavonol synthase activity decreased, and dihydroflavonol 4-reductase activity was elevated. Differentially accumulated flavonoid metabolites were detected between wildtype and aam1, which were enriched primarily in flavonol and anthocyanin pathways. Our results revealed that mutations in the BrFLS gene could contribute to anthocyanin accumulation and provide a new target for Chinese cabbage color modification.

Anaerobic hexadecane degradation by a thermophilic Hadarchaeon from Guaymas Basin.

Hadarchaeota inhabit subsurface and hydrothermally heated environments, but previous to this study, they had not been cultured. Based on metagenome-assembled genomes, most Hadarchaeota are heterotrophs that grow on sugars and amino acids, or oxidize carbon monoxide or reduce nitrite to ammonium. A few other metagenome-assembled genomes encode alkyl-coenzyme M reductases (Acrs), ß-oxidation, and Wood-Ljungdahl pathways, pointing toward multicarbon alkane metabolism. To identify the organisms involved in thermophilic oil degradation, we established anaerobic sulfate-reducing hexadecane-degrading cultures from hydrothermally heated sediments of the Guaymas Basin. Cultures at 70°C were enriched in one Hadarchaeon that we propose as Candidatus Cerberiarchaeum oleivorans. Genomic and chemical analyses indicate that Ca. C. oleivorans uses an Acr to activate hexadecane to hexadecyl-coenzyme M. A ß-oxidation pathway and a tetrahydromethanopterin methyl branch Wood-Ljungdahl (mWL) pathway allow the complete oxidation of hexadecane to CO2. Our results suggest a syntrophic lifestyle with sulfate reducers, as Ca. C. oleivorans lacks a sulfate respiration pathway. Comparative genomics show that Acr, mWL, and ß-oxidation are restricted to one family of Hadarchaeota, which we propose as Ca. Cerberiarchaeaceae. Phylogenetic analyses further indicate that the mWL pathway is basal to all Hadarchaeota. By contrast, the carbon monoxide dehydrogenase/acetyl-coenzyme A synthase complex in Ca. Cerberiarchaeaceae was horizontally acquired from Bathyarchaeia. The Acr and ß-oxidation genes of Ca. Cerberiarchaeaceae are highly similar to those of other alkane-oxidizing archaea such as Ca. Methanoliparia and Ca. Helarchaeales. Our results support the use of Acrs in the degradation of petroleum alkanes and suggest a role of Hadarchaeota in oil-rich environments.

Mining and tailor-made engineering of a novel keto reductase for asymmetric synthesis of structurally hindered γ- and δ-lactones.

A novel carbonyl reductase from Hyphopichia burtoni (HbKR) was discovered by gene mining. HbKR is a NADPH-dependent dual function enzyme with reduction and oxidation activity belonging to SDR superfamily. HbKR strictly follows Prelog priority in the reduction of long-chain aliphatic keto acids/esters containing remote carbonyl groups, such as 4-oxodecanoic acid and 5-oxodecanoic acid, producing (S)-γ-decalactone and (S)-δ-decalactone in >99 % e.e. Tailor-made engineering of HbKR was conducted to improve its catalytic efficiency. Variant F207A/F86M was obtained with specific activity of 8.37 U/mg toward 5-oxodecanoic acid, which was 9.7-fold of its parent. Employing F207A/F86M, 100 mM 5-oxodecanoic acid could be reduced into optically pure (S)-δ-decalactone. Molecular docking analysis indicates that substitution of aromatic Phe with smaller residues renders sufficient space for accommodating substrates in a more stable conformation. This study offers an efficient biocatalyst for the biosynthesis of (S)-lactones, and provides guidance for engineering carbonyl reductases toward structurally hindered substrates.

Wnt/ß-catenin signalling activates IMPDH2-mediated purine metabolism to facilitate oxaliplatin resistance by inhibiting caspase-dependent apoptosis in colorectal cancer.

BACKGROUND: Oxaliplatin resistance usually leads to therapeutic failure and poor prognosis in colorectal cancer (CRC), while the underlying mechanisms are not yet fully understood. Metabolic reprogramming is strongly linked to drug resistance, however, the role and mechanism of metabolic reprogramming in oxaliplatin resistance remain unclear. Here, we aim to explore the functions and mechanisms of purine metabolism on the oxaliplatin-induced apoptosis of CRC. METHODS: An oxaliplatin-resistant CRC cell line was generated, and untargeted metabolomics analysis was conducted. The inosine 5'-monophosphate dehydrogenase type II (IMPDH2) expression in CRC cell lines was determined by quantitative real-time polymerase chain reaction (qPCR) and western blotting analysis. The effects of IMPDH2 overexpression, knockdown and pharmacological inhibition on oxaliplatin resistance in CRC were assessed by flow cytometry analysis of cell apoptosis in vivo and in vitro. RESULTS: Metabolic analysis revealed that the levels of purine metabolites, especially guanosine monophosphate (GMP), were markedly elevated in oxaliplatin-resistant CRC cells. The accumulation of purine metabolites mainly arose from the upregulation of IMPDH2 expression. Gene set enrichment analysis (GSEA) indicated high IMPDH2 expression in CRC correlates with PURINE_METABOLISM and MULTIPLE-DRUG-RESISTANCE pathways. CRC cells with higher IMPDH2 expression were more resistant to oxaliplatin-induced apoptosis. Overexpression of IMPDH2 in CRC cells resulted in reduced cell death upon treatment with oxaliplatin, whereas knockdown of IMPDH2 led to increased sensitivity to oxaliplatin through influencing the activation of the Caspase 7/8/9 and PARP1 proteins on cell apoptosis. Targeted inhibition of IMPDH2 by mycophenolic acid (MPA) or mycophenolate mofetil (MMF) enhanced cell apoptosis in vitro and decreased in vivo tumour burden when combined with oxaliplatin treatment. Mechanistically, the Wnt/ß-catenin signalling was hyperactivated in oxaliplatin-resistant CRC cells, and a reciprocal positive regulatory mechanism existed between Wnt/ß-catenin and IMPDH2. Blocking the Wnt/ß-catenin pathway could resensitize resistant cells to oxaliplatin, which could be restored by the addition of GMP. CONCLUSIONS: IMPDH2 is a predictive biomarker and therapeutic target for oxaliplatin resistance in CRC.

Identification of bacterial dissimilatory antimonate reductase AnrA: genes and proteins involved in antimonate respiration and resistance in Geobacter sp. strain SVR.

Geobacter sp. strain SVR uses antimonate [Sb(V)] as a terminal electron acceptor for anaerobic respiration. Here, we visualized a possible key enzyme, periplasmic Sb(V) reductase (Anr), via active staining and non-denaturing gel electrophoresis. Liquid chromatography-tandem mass spectrometry analysis revealed that a novel dimethyl sulfoxide (DMSO) reductase family protein, WP_173201954.1, is involved in Anr. This protein was closely related with AnrA, a protein suggested to be the catalytic subunit of a respiratory Sb(V) reductase in Desulfuribacillus stibiiarsenatis. The anr genes of strain SVR (anrXSRBAD) formed an operon-like structure, and their transcription was upregulated under Sb(V)-respiring conditions. The expression of anrA gene was induced by more than 1 µM of antimonite [Sb(III)]; however, arsenite [As(III)] did not induce the expression of anrA gene. Tandem mass tag-based proteomic analysis revealed that, in addition to Anr proteins, proteins in the following categories were upregulated under Sb(V)-respiring conditions: (i) Sb(III) efflux systems such as Ant and Ars; (ii) antioxidizing proteins such as ferritin, rubredoxin, and thioredoxin; (iii) protein quality control systems such as HspA, HslO, and DnaK; and (iv) DNA repair proteins such as UspA and UvrB. These results suggest that strain SVR copes with antimony stress by modulating pleiotropic processes to resist and actively metabolize antimony. To the best of our knowledge, this is the first report to demonstrate the involvement of AnrA in Sb(V) respiration at the protein level. Furthermore, this is the first example to show high expression of the Ant system proteins in the Sb(V)-respiring bacterium.IMPORTANCEAntimony (Sb) exists mainly as antimonite [Sb(III)] or antimonate [Sb(V)] in the environment, and Sb(III) is more toxic than Sb(V). Recently, microbial involvement in Sb redox reactions has received attention. Although more than 90 Sb(III)-oxidizing bacteria have been reported, information on Sb(V)-reducing bacteria is limited. Especially, the enzyme involved in dissimilatory Sb(V) reduction, or Sb(V) respiration, is unclear, despite this pathway being very important for the circulation of Sb in nature. In this study, we demonstrated that the Sb(V) reductase (Anr) of an Sb(V)-respiring bacterium (Geobacter sp. SVR) is a novel member of the dimethyl sulfoxide (DMSO) reductase family. In addition, we found that strain SVR copes with Sb stress by modulating pleiotropic processes, including the Ant and Ars systems, and upregulating the antioxidant and quality control protein levels. Considering the abundance and diversity of putative anr genes in the environment, Anr may play a significant role in global Sb cycling in both marine and terrestrial environments.

Evolutionary Analysis of Six Gene Families Part of the Reactive Oxygen Species (ROS) Gene Network in Three Brassicaceae Species.

Climate change is expected to intensify the occurrence of abiotic stress in plants, such as hypoxia and salt stresses, leading to the production of reactive oxygen species (ROS), which need to be effectively managed by various oxido-reductases encoded by the so-called ROS gene network. Here, we studied six oxido-reductases families in three Brassicaceae species, Arabidopsis thaliana as well as Nasturtium officinale and Eutrema salsugineum, which are adapted to hypoxia and salt stress, respectively. Using available and new genomic data, we performed a phylogenomic analysis and compared RNA-seq data to study genomic and transcriptomic adaptations. This comprehensive approach allowed for the gaining of insights into the impact of the adaptation to saline or hypoxia conditions on genome organization (gene gains and losses) and transcriptional regulation. Notably, the comparison of the N. officinale and E. salsugineum genomes to that of A. thaliana highlighted changes in the distribution of ohnologs and homologs, particularly affecting class III peroxidase genes (CIII Prxs). These changes were specific to each gene, to gene families subjected to duplication events and to each species, suggesting distinct evolutionary responses. The analysis of transcriptomic data has allowed for the identification of genes related to stress responses in A. thaliana, and, conversely, to adaptation in N. officinale and E. salsugineum.

The BHLHE40‒PPM1F‒AMPK pathway regulates energy metabolism and is associated with the aggressiveness of endometrial cancer.

BHLHE40 is a basic helix-loop-helix transcription factor that is involved in multiple cell activities including differentiation, cell cycle, and epithelial-to-mesenchymal transition. While there is growing evidence to support the functions of BHLHE40 in energy metabolism, little is known about the mechanism. In this study, we found that BHLHE40 expression was downregulated in cases of endometrial cancer of higher grade and advanced disease. Knockdown of BHLHE40 in endometrial cancer cells resulted in suppressed oxygen consumption and enhanced extracellular acidification. Suppressed pyruvate dehydrogenase (PDH) activity and enhanced lactated dehydrogenase (LDH) activity were observed in the knockdown cells. Knockdown of BHLHE40 also led to dephosphorylation of AMPKα Thr172 and enhanced phosphorylation of pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1) Ser293 and lactate dehydrogenase A (LDHA) Tyr10. These results suggested that BHLHE40 modulates PDH and LDH activity by regulating the phosphorylation status of PDHA1 and LDHA. We found that BHLHE40 enhanced AMPKα phosphorylation by directly suppressing the transcription of an AMPKα-specific phosphatase, PPM1F. Our immunohistochemical study showed that the expression of BHLHE40, PPM1F, and phosphorylated AMPKα correlated with the prognosis of endometrial cancer patients. Because AMPK is a central regulator of energy metabolism in cancer cells, targeting the BHLHE40‒PPM1F‒AMPK axis may represent a strategy to control cancer development.

In Vitro Comparative Study on Antineoplastic Effects of Pinoresinol and Lariciresinol on Healthy Cells and Breast Cancer-Derived Human Cells.

Background: Herbal medicines are the preferred anticancer agents due to their lower cytotoxic effects on healthy cells. Plant lignans play an important role in treating various diseases, especially cancer. The present study aimed to evaluate the effect of podophyllotoxin, pinoresinol, and lariciresinol on cellular toxicity and inducing apoptosis in fibroblasts, HEK-293, and SkBr3 cell lines. Methods: An in vitro study was conducted from 2017 to 2019 at the Faculty of Biological Sciences, Tarbiat Modares University (Tehran, Iran). The cell lines were treated for 24 and 48 hours with different concentrations of lignans. Cell viability and apoptosis were examined using MTT and flow cytometry, respectively. Expression levels of cell cycle and apoptosis regulator genes were determined using quantitative real-time polymerase chain reaction. Data were analyzed using a two-way analysis of variance followed by Tukey's HSD test. P<0.05 was considered statistically significant. Results: Podophyllotoxin significantly increased apoptosis in fibroblast cells compared to pinoresinol and lariciresinol (P<0.001). The percentage of cell viability of fibroblast cells treated for 48 hours with pinoresinol, lariciresinol, and podophyllotoxin was reduced by 49%, 47%, and 36%, respectively. Treatment with pinoresinol and lariciresinol significantly overexpressed pro-apoptotic genes and underexpressed anti-apoptotic genes in SkBr3 cells (P<0.001). SkBr3 cells treated with lariciresinol significantly reduced gene expression (P<0.001). Conclusion: Pinoresinol and lariciresinol can potentially be used as new therapeutic agents for the treatment of breast cancer.

Genome-Wide Identification, Expression Analysis, and Subcellular Localization of DET2 Gene Family in Populus yunnanensis.

(1) Background: Brassinosteroids (BRs) are important hormones involved in almost all stages of plant growth and development, and sterol dehydrogenase is a key enzyme involved in BRs biosynthesis. However, the sterol dehydrogenase gene family of Populus yunnanensis Dode (P. yunnanensis) has not been studied. (2) Methods: The PyDET2 (DEETIOLATED2) gene family was identified and analyzed. Three genes were screened based on RNA-seq of the stem tips, and the PyDET2e was further investigated via qRT-PCR (quantitative real-time polymerase chain reaction) and subcellular localization. (3) Results: The 14 DET2 family genes in P. yunnanensis were categorized into four groups, and 10 conserved protein motifs were identified. The gene structure, chromosome distribution, collinearity, and codon preference of all PyDET2 genes in the P. yunnanensis genome were analyzed. The codon preference of this family is towards the A/U ending, which is strongly influenced by natural selection. The PyDET2e gene was expressed at a higher level in September than in July, and it was significantly expressed in stems, stem tips, and leaves. The PyDET2e protein was localized in chloroplasts. (4) Conclusions: The PyDET2e plays an important role in the rapid growth period of P. yunnanensis. This systematic analysis provides a basis for the genome-wide identification of genes related to the brassinolide biosynthesis process in P. yunnanensis, and lays a foundation for the study of the rapid growth mechanism of P. yunnanensis.

Clinical and electroencephalogram characteristics of methylmalonic acidemia with MMACHC and MUT gene mutations.

OBJECTIVE: This study investigated the clinical, imaging, and electroencephalogram (EEG) characteristics of methylmalonic acidemia (MMA) with nervous system damage as the primary manifestation. METHODS: From January 2017 to November 2022, patients with nervous system injury as the main clinical manifestation, diagnosed with methylmalonic acidemia by metabolic and genetic testing, were enrolled and analyzed. Their clinical, imaging, and electroencephalogram data were analyzed. RESULTS: A total of 18 patients were enrolled, including 15 males and 3 females. The clinical symptoms were convulsions, poor feeding, growth retardation, disorder of consciousness, developmental delay, hypotonia, and blood system changes. There were 6 cases (33%) of hydrocephalus, 9 (50%) of extracerebral space widened, 5 (27%) of corpus callosum thinning, 3 (17%) of ventricular dilation, 3 (17%) of abnormal signals in the brain parenchyma (frontal lobe, basal ganglia region, and brain stem), and 3 (17%) of abnormal signals in the lateral paraventricular. In addition, there were 3 cases (17%) of cerebral white matter atrophy and 1 (5%) of cytotoxic edema in the basal ganglia and cerebral peduncle. EEG data displayed 2 cases (11%) of hypsarrhythmia, 3 (17%) of voltage reduction, 12(67%) of abnormal discharge, 13 (72%) of abnormal sleep physiological waves or abnormal sleep structure, 1 (5%) of immature (delayed) EEG development, and 8 (44%) of slow background. There were 2 cases (11%) of spasms, 1 (5%) of atonic seizures, and 1 (5%) of myoclonic seizures. There were 16 patients (89%) with hyperhomocysteinemia. During follow-up, 1 patient was lost to follow-up, and 1 died. In total, 87.5% (14/16) of the children had varying developmental delays. EEG was re-examined in 11 cases, of which 8 were normal, and 3 were abnormal. Treatments included intramuscular injections of vitamin B12, L-carnitine, betaine, folic acid, and oral antiepileptic therapy. Acute treatment included anti-infective, blood transfusion, fluid replacement, and correcting acidosis. The other treatments included low-protein diets and special formula milk powder. CONCLUSION: Methylmalonic acidemia can affect the central nervous system, leading to structural changes or abnormal signals on brain MRI. Metabolic screening and genetic testing help clarify the diagnosis. EEG can reflect changes in brain waves during the acute phase.

Dynamic nitrogen fixation in an aerobic endophyte of Populus.

Biological nitrogen fixation by microbial diazotrophs can contribute significantly to nitrogen availability in non-nodulating plant species. In this study of molecular mechanisms and gene expression relating to biological nitrogen fixation, the aerobic nitrogen-fixing endophyte Burkholderia vietnamiensis, strain WPB, isolated from Populus trichocarpa served as a model for endophyte-poplar interactions. Nitrogen-fixing activity was observed to be dynamic on nitrogen-free medium with a subset of colonies growing to form robust, raised globular like structures. Secondary ion mass spectrometry (NanoSIMS) confirmed that N-fixation was uneven within the population. A fluorescent transcriptional reporter (GFP) revealed that the nitrogenase subunit nifH is not uniformly expressed across genetically identical colonies of WPB and that only ~11% of the population was actively expressing the nifH gene. Higher nifH gene expression was observed in clustered cells through monitoring individual bacterial cells using single-molecule fluorescence in situ hybridization. Through 15N2 enrichment, we identified key nitrogenous metabolites and proteins synthesized by WPB and employed targeted metabolomics in active and inactive populations. We cocultivated WPB Pnif-GFP with poplar within a RhizoChip, a synthetic soil habitat, which enabled direct imaging of microbial nifH expression within root epidermal cells. We observed that nifH expression is localized to the root elongation zone where the strain forms a unique physical interaction with the root cells. This work employed comprehensive experimentation to identify novel mechanisms regulating both biological nitrogen fixation and beneficial plant-endophyte interactions.

Comparative transcript abundance of gibberellin oxidases genes in two barley (Hordeum vulgare) genotypes with contrasting lodging resistance under different regimes of water deficit.

Barley (Hordeum vulgare ) is the world's fourth most important cereal crop, and is particularly well adapted to harsh environments. However, lodging is a major productivity constraint causing 13-65% yield losses. Gibberellic acid (GA) homeostatic genes such as HvGA20ox, HvGA3ox and HvGA2ox are responsible for changes in plant phenotype for height and internodal length that contribute towards lodging resistance. This study explored the expression of different HvGAox transcripts in two contrasting barley genotypes (5-GSBON-18, lodging resistant; and 5-GSBON-70, lodging sensitive), which were sown both under controlled (hydroponic, completely randomised factorial design) and field conditions (split-plot, completely randomised block design) with two irrigation treatments (normal with three irrigation events; and water deficit with one irrigation event). In the hydroponic experiment, expression analysis was performed on seedlings at 0, ¾, 1½, 3 and 6h after application of treatment. In the field experiment, leaf, shoot nodes and internodes were sampled. Downregulation of HvGA20ox.1 transcript and 2-fold upregulation of HvGA2ox.2 transcript were observed in 5-GSBON-18 under water deficit conditions. This genotype also showed a significant reduction in plant height (18-20%), lodging (<10%), and increased grain yield (15-18%) under stress. Utilisation of these transcripts in barley breeding has the potential to reduce plant height, lodging and increased grain yield.

Rapid reaction studies on the chemistry of flavin oxidation in urocanate reductase.

Urocanate reductase (UrdA) is a bacterial flavin-dependent enzyme that reduces urocanate to imidazole propionate, enabling bacteria to use urocanate as an alternative respiratory electron acceptor. Elevated serum levels of imidazole propionate are associated with the development of type 2 diabetes, and, since UrdA is only present in humans in gut bacteria, this enzyme has emerged as a significant factor linking the health of the gut microbiome and insulin resistance. Here, we investigated the chemistry of flavin oxidation by urocanate in the isolated FAD domain of UrdA (UrdA') using anaerobic stopped-flow experiments. This analysis unveiled the presence of a charge-transfer complex between reduced FAD and urocanate that forms within the dead time of the stopped-flow instrument (∼1 ms), with flavin oxidation subsequently occurring with a rate constant of ∼60 s-1. The pH dependence of the reaction and analysis of an Arg411Ala mutant of UrdA' are consistent with Arg411 playing a crucial role in catalysis by serving as the active site acid that protonates urocanate during hydride transfer from reduced FAD. Mutational analysis of urocanate-binding residues suggests that the twisted conformation of urocanate imposed by the active site of UrdA' facilitates urocanate reduction. Overall, this study provides valuable insight into the mechanism of urocanate reduction by UrdA.

Proximity to Photosystem II is necessary for activation of Plastid Terminal Oxidase (PTOX) for photoprotection.

The Plastid Terminal Oxidase (PTOX) is a chloroplast localized plastoquinone oxygen oxidoreductase suggested to have the potential to act as a photoprotective safety valve for photosynthesis. However, PTOX overexpression in plants has been unsuccessful at inducing photoprotection, and the factors that control its activity remain elusive. Here, we show that significant PTOX activity is induced in response to high light in the model species Eutrema salsugineum and Arabidopsis thaliana. This activation correlates with structural reorganization of the thylakoid membrane. Over-expression of PTOX in mutants of Arabidopsis thaliana perturbed in thylakoid stacking also results in such activity, in contrast to wild type plants with normal granal structure. Further, PTOX activation protects against photoinhibition of Photosystem II and reduces reactive oxygen production under stress conditions. We conclude that structural re-arrangements of the thylakoid membranes, bringing Photosystem II and PTOX into proximity, are both required and sufficient for PTOX to act as a Photosystem II sink and play a role in photoprotection.