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The obligate anaerobe Clostridioides difficile encodes multiple reductases to detoxify molecular oxygen and reactive oxygen species. Caulat and colleagues have characterized the activity and regulation of four such reductases (L. C. Caulat, A. Lotoux, M. C. Martins, N. Kint, et al., mBio 15:e01591-24, 2024, https://doi.org/10.1128/mbio.01591-24). Each proved critical for clostridial survival in a different range of oxygen concentrations; together, they ameliorate a broad range of oxidative stress levels. Moreover, two previously uncharacterized regulators were found to control reductase gene expression in response to oxidative stress. The genetic repressor Rex and the reductase FdpF are both sensitive to the NAP + :NADH ratio, which is affected by a cell's metabolic state as well as redox activity. While oxygen is known to influence the expression of metabolism genes in C. difficile , the mechanisms for cross-talk between the pathways that respond to oxidative and metabolic stress are not well known. The NADH dependence of Rex and FdpF may represent a newly mapped junction between these pathways., Competing Interests: The author declares no conflict of interest.

Conventional plant gene editing requires laborious tissue-culture-mediated transformation, which restricts the range of applicable plant species. In this study, we developed a heritable and tissue-culture-free gene editing method in Nicotiana benthamiana using tobacco ringspot virus (TRSV) as a vector for in planta delivery of Cas9 and single-guide RNA (sgRNA) to shoot apical meristems. Agrobacterium-mediated inoculation of the TRSV vector induced systemic and heritable gene editing in Nicotiana benthamiana PHYTOENE DESATURASE. Transient downregulation of RNA silencing enhanced gene editing efficiency, resulting in an order of magnitude increase (0.8-13.2%) in the frequency of transgenerational gene editing. While the TRSV system had a preference for certain sgRNA sequences, co-inoculation of a TRSV vector carrying only Cas9 and a tobacco rattle virus vector carrying sgRNA successfully introduced systemic mutations with all five tested sgRNAs. Extensively gene-edited lateral shoots occasionally grew from plants inoculated with the virus vectors, the transgenerational gene editing frequency of which ranged up to 100%. This virus-mediated heritable gene editing method makes plant gene editing easy, requiring only the inoculation of non-transgenic plants with a virus vector(s) to obtain gene-edited individuals., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Deoxynivalenol (DON), a type-B trichothecene mycotoxin, is primarily produced by Fusarium species and widely pollutes wheat and other grains. Enzymatic treatment of DON has been widely studied in recent years. Here, we present the biochemical identification of the DON dehydrogenase from Pelagibacterium sp. SCN 63-126 (Pe DDH). After removing the signal peptide, Pe DDH is effectively expressed in its soluble form. Biochemical identification indicates that the optimal temperature and pH of Pe DDH against DON is 35 ℃ and pH 8.5. Furthermore, Pe DDH is activated significantly in the presence of Ca 2+ , Mg 2+ , and Cu 2+ , and alternatively activated by pyrroloquinoline quinone (PQQ), phenazine methosulfate (PMS), and 2, 6-dichlorophenolindophenol (DCPIP). When PQQ, PMS, and DCPIP are combined, Pe DDH (60 µg/mL) effectively degrads DON (150 µM) in just 5 min, suggesting a synergistic effect of three cofactors on DON degradation. All these results suggest a great potential of Pe DDH in the control of DON contamination., Competing Interests: Declarations. Ethical approval: This article does not contain any studies with human participants performed by any of the authors. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In fermented plant foods, phenolic compounds are metabolized by 2-ene reductases, which reduce double bonds adjacent to an aromatic rings in phytochemicals, including hydroxycinnamic acids, isoflavones, and flavones. Only few 2-ene reductases of lactic acid bacteria were characterized, including the hydrocinnamic reductases HcrB and Par1, and the daidzein reductase of Lactococcus lactis. This study aimed to characterize HcrF, a homologue of HcrB, in Limosilactobacillus fermentum. HcrF was purified after cloning in Escherichia coli and purification by affinity chromatography. HcrF was optimally active at 30°C-40°C and pH 7.0 and required both flavin mononucleotide and nicotinamide adenine dinucleotide as co-factors. Ferulic, caffeic, p-coumaric, and sinapic acids but not trans-cinnamic acids were reduced to dihydro derivatives. The maximum reaction velocity Vmax of HcrF was highest for ferulic acid. On a phylogenetic tree of 2-ene reductases, HcrF clustered most closely with the hydroxycinnamic acid reductase HcrB of Lactiplantibacillus plantarum. The hydroxycinnamic acid reductase Par1 of Furfurilactobacillus milii and flavone or isoflavone reductases were only distantly related to HcrF. In summary, current knowledge does not allow to predict the substrate specificity of 2-ene reductases on the basis of the protein sequence; this study adds HcrF to the short list of enzymes with known substrate specificity., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Dihydrochalcones (DHCs) are flavonoids produced as a side branch of the phenylpropanoid pathway. DHCs are found at high concentrations in apples (Malus spp.) but not in pears (Pyrus spp.) or other members of the Rosaceae. Biosynthesis of DHCs in apple has been hypothesized to occur via reduction of p-coumaroyl CoA by a Malus × domestica hydroxycinnamoyl CoA double-bond reductase (MdHCDBR) followed by the action chalcone synthase to produce phloretin or via direct reduction of naringenin chalcone to phloretin via an unknown enzyme. In this study, we report that genetic downregulation of MdHCDBR does not reduce DHC concentrations in apple leaves. We used comparative transcriptome analysis to identify candidate naringenin chalcone reductases (NCRs), designated MdNCR1a-c, expressed in apple leaves but not fruit. These MdNCR1 genes form an expanded gene cluster found exclusively in apple. Transient expression of MdNCR1 genes in Nicotiana benthamiana leaves indicated they produced DHCs at high concentrations in planta. Recombinant MdNCR1 utilized naringenin chalcone to produce phloretin at high efficiency. Downregulation of NCR genes in transgenic apple reduced foliar DHC levels by 85% to 95%. Reducing DHC production redirected flux to the production of flavonol glycosides. In situ localization indicated that NCR proteins were likely found in the vacuolar membrane. Active site analysis of AlphaFold models indicated that MdNCR1a-c share identical substrate binding pockets, but the pockets differ substantially in related weakly active/inactive NCR proteins. Identifying the missing enzyme required for DHC production provides opportunities to manipulate DHC content in apple and other fruits and has other applications, e.g. in biofermentation and biopharming., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Algae such as diatoms and haptophytes have distinct photosynthetic pigments from plants, including a novel set of carotenoids. This includes a primary xanthophyll cycle comprised of diadinoxanthin and its de-epoxidation product diatoxanthin that enables the switch between light harvesting and non-photochemical quenching (NPQ)-mediated dissipation of light energy. The enzyme responsible for the reversal of this cycle was previously unknown. Here, we identified zeaxanthin epoxidase 3 (ZEP3) from Phaeodactylum tricornutum as the candidate diatoxanthin epoxidase. Knocking out the ZEP3 gene caused a loss of rapidly reversible NPQ following saturating light exposure. This correlated with the maintenance of high concentrations of diatoxanthin during recovery in low light. Xanthophyll cycling and NPQ relaxation were restored via complementation of the wild-type ZEP3 gene. The zep3 knockout strains showed reduced photosynthetic rates at higher light fluxes and reduced specific growth rate in variable light regimes, likely due to the mutant strains becoming locked in a light energy dissipation state. We were able to toggle the level of NPQ capacity in a time and dose dependent manner by placing the ZEP3 gene under the control of a β-estradiol inducible promoter. Identification of this gene provides a deeper understanding of the diversification of photosynthetic control in algae compared to plants and suggests a potential target to improve the productivity of industrial-scale cultures., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Background: Deregulation of lipid metabolism is one of the most prominent metabolic features in cancer. The activation of sphingolipid metabolic pathways affects the proliferation, invasion, angiogenesis, chemoresistance, and immune escape of tumors, including colorectal cancer (CRC). Dehydrogenase/reductase member 2 (DHRS2), which belongs to the short-chain dehydrogenase/reductase (SDR) family, has been reported to participate in the regulation of lipid metabolism and impact on cancer progression. Trichothecin (TCN) is a sesquiterpenoid metabolite originating from an endophytic fungus of the herbal plant Maytenus hookeri Loes. Studies have shown that TCN exerts a broad-spectrum antitumor activity., Methods: We evaluated the proliferative ability of CRC cells by CCK8 and colony formation assays. A metabolite profiling using liquid chromatography coupled with mass spectrometry (LC/MS) was adopted to identify the proximal metabolite changes linked to DHRS2 overexpression. RNA stability assay and RNA immunoprecipitation (RIP) experiments were applied to determine the post-transcriptional regulation of SPHK1 expression by DHRS2. We used flow cytometry to detect changes in cell cycle and cell apoptosis of CRC cells in the absence or presence of TCN., Results: We demonstrate that DHRS2 hampers the sphingosine kinases 1 (SPHK1)/sphingosine 1-phosphate (S1P) metabolic pathway to inhibit CRC cell growth. DHRS2 directly binds to SPHK1 mRNA to accelerate its degradation in a post-transcriptionally regulatory manner. Moreover, we illustrate that SPHK1 downregulation induced by DHRS2 contributes to TCN-induced growth inhibition of CRC., Conclusions: The present study provides a mechanistic connection among metabolic enzymes, metabolites, and the malignant progression of CRC. Moreover, TCN could be developed as a potential pharmacological tool against CRC by the induction of DHRS2 and targeting SPHK1/S1P metabolic pathway., Competing Interests: Declaration of competing interest No conflicts of interest, financial or otherwise, are declared by the authors., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Three new strains of dissimilatory perchlorate-reducing bacteria (DPRB), QD19-16, QD1-5, and P3-1, were isolated from an active sludge. Phylogenetic trees based on 16S rRNA genes indicated that QD19-16, QD1-5, and P3-1 belonged to Brucella, Acidovorax, and Citrobacter, respectively, expanding the distribution of DPRB in the Proteobacteria. The three strains were gram-negative and facultative anaerobes with rod-shaped cells without flagella, which were 1.0-1.6 μm long and 0.5-0.6 μm wide. The three DPRB strains utilized similar broad spectrum of electron donors and acceptors and demonstrated a similar capability to reduce perchlorate within 6 days. The enzyme activity of perchlorate reductase in QD19-16 toward chlorate was higher than that toward perchlorate. The high sequence similarity of the perchlorate reductase operon and chlorite dismutase genes in the perchlorate reduction genomic islands (PRI) of the three strains implied that they were monophyletic origin from a common ancestral PRI. Two transposase genes (tnp1 and tnp2) were found in the PRIs of strain QD19-16 and QD1-5, but were absent in the strain P3-1 PRI. The presence of fragments of IR sequences in the P3-1 PRI suggested that P3-1 PRI had previously contained these two tnp genes. Therefore, it is plausible to suggest that a common ancestral PRI transferred across the strains Brucella sp. QD19-16, Acidovorax sp. QD1-5, and Citrobacter sp. P3-1 through horizontal gene transfer, facilitated by transposases. These results provided a direct evidence of horizontal gene transfer of PRI that could jump across phylogenetically unrelated bacteria through transposase. KEY POINTS: • Three new DPRB strains can effectively remove high concentration of perchlorate. • The PRIs of three DPRB strains are acquired from a single ancestral PRI. • PRIs are incorporated into different bacteria genome through HGT by transposase., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Engineering nonribosomal peptide synthetases (NRPSs) has been a "holy grail" in synthetic biology due to their modular nature and limited understanding of catalytic mechanisms. Here, we reported a computational redesign of the "gate-keeper" adenylation domain of the model NRPS-like enzyme carboxylic acid reductases (CARs) by using approximate mechanism-based geometric criteria and the Rosetta energy score. Notably, Mab CAR3 mutants ACA-1 and ACA-4 displayed a remarkable improvement in catalytic efficiency ( k cat / K M ) for 6-aminocaproic acid, up to 101-fold. Furthermore, G418K exhibited an 86-fold enhancement in substrate specificity for adipic acid compared to 6-aminocaproic acid. Our work provides not only promising biocatalysts for nylon monomer biosynthesis but also a strategy for efficient NRPSs engineering.

Our previous studies identified PA5407 in Pseudomonas aeruginosa as a new regulatory protein for bacterial division and named it ZapAL. This protein enhances the assembly of the key bacterial division protein FtsZ and participates in the assembly of the bacterial Z-ring, but its physiological function is not clear. ZapAL is in the same gene cluster as PA5402-5406, and in this study, we found that these genes are involved in the regulation of bacterial growth under nutrient deficiency and high-density conditions. The expression of Aa3 oxidase increases significantly at the end of the stationary phase of bacterial growth under aerobic conditions, and appropriately accelerate energy intake to adapt to the adversity. In our study, we found that the knockout of PA5402-5407 in P. aeruginosa promotes the further expression of Aa3 oxidase and its expression increased more and faster than the wild type, especially under carbon starvation and high-density conditions. This results in a larger bacteria population, but the average length of the bacteria is abnormally reduced. In summary, our study found that the P. aeruginosa gene cluster PA5402-5407 regulated the expression of Aa3 oxidase in the late stationary phase, and these genes balance energy intake, growth and division of bacteria under adverse conditions., Competing Interests: Declarations. Conflict of interest: The authors declare no conflict of interest., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The stereoselectivity of enzymes plays a central role in asymmetric biocatalytic reactions, but there remains a dearth of evolution-driven biochemistry studies investigating the evolutionary trajectory of this vital property. Imine reductases (IREDs) are one such enzyme that possesses excellent stereoselectivity, and stereocomplementary members are pervasive in the family. However, the regulatory mechanism behind stereocomplementarity remains cryptic. Herein, we reconstruct a panel of active ancestral IREDs and trace the evolution of stereoselectivity from ancestors to extant IREDs. Combined with coevolution analysis, we reveal six historical mutations capable of recapitulating stereoselectivity evolution. An investigation of the mechanism with X-ray crystallography shows that they collectively reshape the substrate-binding pocket to regulate stereoselectivity inversion. In addition, we construct an empirical fitness landscape and discover that epistasis is prevalent in stereoselectivity evolution. Our findings emphasize the power of ASR in circumventing the time-consuming large-scale mutagenesis library screening for identifying mutations that change functions and support a Darwinian premise from a molecular perspective that the evolution of biological functions is a stepwise process., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Abnormal melanin synthesis can lead to severe skin problems. This study investigated the anti-melanogenic effects on α-melanocyte stimulating hormone (α-MSH)-induced B16F10 cells using cell-free supernatants of Lactiplantibacillus plantarum WB326 and Levilactobacillus brevis WB2810. Samples were prepared using 1 mg/ml freeze-dried culture supernatant. Cell viability was assessed using B16F10 cells and MTT assay. Tyrosinase inhibition and melanin content decreased in the samples compared to those treated with α-MSH. This effect was also observed when L-DOPA staining was used under a microscope. Moreover, the mRNA expression levels of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 decreased in the sample-treated group. Protein expression of the CREB/MITF/MAPK signaling pathway was also reduced. Using HPLC analysis, lactic and acetic acids were detected in the culture supernatants. Finally, the antioxidant effects of the samples were confirmed by comparison with those of Trolox and arbutin. According to the experimental results, their utilization is possible in the fields of functional materials and cosmetic ingredients.

In this study, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and multicopper oxidase (MCO) of Lactiplantibacillus plantarum W155 with histamine degradation ability were expressed. The mulberry fruit wine (MFW) histamine degradation abilities of GAPDH and MCO were 20.81% and 37.67%, respectively. Compared with the control group, the MFW treated by GAPDH showed higher total phenolic (1.17 g GAE/L) and total flavonoid (0.31 g RE/L) contents, while MFW treated by MCO presented similar total phenolic (1.00 g GAE/L) and total flavonoid (0.29 g RE/L) concentrations. Furthermore, the optimal pH and temperature of GAPDH were 6.0 and 40 °C, respectively, while the optimal pH and temperature of MCO were 3.0 and 50 °C, respectively. Meanwhile, the key action sites for histamine degradation of GAPDH and MCO were minded via homology modeling, molecular docking, and site-directed mutagenesis. Val209 and Ile290 were confirmed as the key action sites for GAPDH, while Qln402 and Leu420 were the pivotal action sites for MCO. Above findings indicated that both GAPDH and MCO of L. plantarum W155 could be used to control the histamine of MFW, and the key action sites of these two enzymes could be used as targets for their subsequent modification.

The litchi genome has five anthocyanidin reductase ( LcANR ) and two leucoanthocyanidin reductase ( LcLAR ) members. The high expression of LcANR1a/2a and LcLAR1/2 is significantly positively correlated with the abundant proanthocyanidins and (-)-epicatechin (EC) in the pericarp, leaf, root, etc. The recombinant LcANR1a/2a converts cyanidin to both EC and (+)-catechin (CT) (EC:CT ≈ 1:1) and converts delphindin to (+)-gallocatechin and (-)-epigallocatechin; the recombinant LcLAR1/2 converts leucocyanidin to CT. The enzymatic kinetics of the four enzymes are presented, with the respective K m of LcLAR1/2 to leucocyanidin, 19 and 34 μM, and the V max , 7 and 5 nmol min -1 mg -1 , which are rarely reported for other plants. Overexpression of LcANR1a/2a and LcLAR1/2 in Arabidopsis ban mutant recovered EC and CT biosynthesis respectively in the seeds; however, the EC-only recovery by LcANR1a/2a is inconsistent with their in vitro activity, indicating that the ANR/LAR function is dependent on characteristic molecular contexts in plants and correlated to the distinct PA profiles in litchi.

Patulin (PAT), a toxic secondary metabolite produced mainly by Penicillium species that frequently contaminates fruit and fruit-derived products, poses serious health risks to humans and animals. In the present study, three short-chain dehydrogenases/reductases (SDRs) with PAT-degrading ability, designated Bs SDR1, Bs SDR2, and Bs SDR3, were identified from the genome of Bacillus subtilis 168. Bs SDR1 and Bs SDR2 showed powerful PAT elimination abilities, which can completely convert PAT to nontoxic E-ascladiol. Moreover, Bs SDR1, Bs SDR2, and Bs SDR3 shared the highest sequence identity of 36.03% with the reported PAT-degrading enzymes, indicating that they are novel PAT-degrading enzymes. Bs SDR1, Bs SDR2, and Bs SDR3 exhibited the highest activity against PAT at 40, 40, and 35 °C, respectively. Additionally, Bs SDR1, Bs SDR2, and Bs SDR3 displayed remarkable thermostability, retaining 32.50, 24.63, and 46.74% residual activity, respectively, after incubation at 50 °C for 1 h. Three-dimensional (3D) simulation and site-directed mutagenesis indicated that the catalytic triad formed by the residues (Ser, Tyr, and Lys) was the key for SDR activity, and this conserved catalytic mechanism was followed in the catalytic process of novel PAT-degrading enzymes Bs SDR1, Bs SDR2, and Bs SDR3. More importantly, Bs SDR1, Bs SDR2, and Bs SDR3 can degrade PAT in apple juice at rates of 86.90, 90.17, and 61.57%, respectively. The identification of Bs SDR1, Bs SDR2, and Bs SDR3 enriched the PAT-degrading enzyme libraries, providing promising candidates for PAT decontamination in the food industry.

Pseudomonas aeruginosa PA2196 is a TetR family transcriptional repressor. In this study, the deletion of the PA2196 gene caused increased expression of the downstream gene curA (PA2197), which encodes for a NADPH-dependent curcumin/dihydrocurcumin reductase. The PA2196 gene was then identified as curR, and a DNA footprinting assay showed that CurR directly bound to the curA promoter at an imperfect 15-bp inverted repeat, 5'-TAGTTGA-C-TGGTCTA-3'. A curA promoter-lacZ fusion assay and site-directed mutagenesis further demonstrated that the identified CurR binding site plays a crucial role in curA repression by CurR. curA transcription was inducible by sodium hypochlorite (NaOCl) and N-ethylmaleimide (NEM) but not by hydrogen peroxide, organic hydroperoxide, or curcumin. The oxidation and alkylation of CurR by NaOCl and NEM, respectively, resulted in the inactivation of its DNA-binding activity, which induced curA expression. Under the tested conditions, the deletion of either curR or curA did not affect the survival of P. aeruginosa under NaOCl stress in the absence or presence of curcumin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The increasing use of nanoparticles (NPs) necessitates investigation of their impact on wastewater treatment processes, particularly anammox, a critical biological nitrogen removal pathway. This study explored the effects of short-term exposure to TiO 2 , ZnO, and Ag-NPs on anammox activity in enriched cultures derived from River Nile sediments., Materials and Methods: Anammox bacteria were identified and enriched, with activity confirmed through 16S rRNA and hydrazine oxidoreductase (hzo) gene amplification and sequencing. Activity assays demonstrated efficient ammonium removal by the enriched culture. Subsequently, the impact of different sized and concentrated NPs on anammox activity was assessed., Results: XRD analysis confirmed NP behavior within the microcosms: TiO 2 transformed, ZnO partially dissolved, and Ag remained ionic. hzo gene expression served as a biomarker for anammox bacterial activity. Interestingly, 100 nm TiO 2 -NPs up-regulated hzo expression, potentially indicating a non-inhibitory transformed phase. Conversely, ZnO and Ag-NPs across all sizes and concentrations significantly down-regulated hzo expression, suggesting detrimental effects. Ag-NPs amended microcosms showed a significant reduction (79%) in hzo gene expression and a detrimental effect on bacterial populations. Overall, anammox activity mirrored hzo expression patterns, with TiO 2 (21 and 25 nm, respectively) exhibiting the least inhibition, followed by ZnO and Ag-NPs., Conclusion: This study highlights the differential effects of NPs on anammox, with the order of impact being Ag > ZnO > TiO 2 . These findings provide valuable insights into the potential environmental risks of NPs on anammox-mediated nitrogen cycling in freshwater ecosystems., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not Applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Corpse decomposition affects soil organisms through the formation of "cadaver decomposition islands." Soil diazotrophic microbes possess essential ecological functions on nitrogen input and nutrient cycling in the terrestrial ecosystem. However, our knowledge about how soil diazotrophic communities respond to corpse decomposition is lacking. In this study, we focused on the succession patterns and biological interaction of nitrogen-fixing microorganisms during animal (Ochotona curzoniae) corpse decomposition in terrestrial ecosystems by targeting nifH gene with high-throughput sequencing. Our results revealed that corpse decomposition of pikas reduced the α diversity and significantly impacted the β diversity of diazotrophic community across different decomposition stages. The divergent succession of diazotrophic community occurred under corpse pressure. Furthermore, the relative importance of stochasticity to the community assembly was improved by corpse decomposition, while the importance decreased over decomposition time. Cadaver decay also simplified the diazotrophic networks and weakened the biological interactions among diazotrophic populations. Notably, NH 4 -N was the most important factor affecting diazotrophic community, followed by time and total carbon. This work emphasized that corpse decomposition perhaps influences the process of biological nitrogen fixation by altering soil diazotrophic communities, which is of great significance for understanding the terrestrial ecosystems' nitrogen cycle functions. KEY POINTS: • Corpse decomposition reduced the α diversity of diazotrophic community. • Corpse decomposition improved the stochasticity of diazotrophic community assembly. • Corpse decomposition weakened the interactions among diazotrophic populations., (© 2024. The Author(s).)

Microbial formation and oxidation of volatile alkanes in anoxic environments significantly impacts biogeochemical cycles on Earth. The discovery of archaea oxidizing volatile alkanes via deeply branching methyl-coenzyme M reductase variants, dubbed alkyl-CoM reductases (ACR), prompted the hypothesis of archaea-catalysed alkane formation in nature (alkanogenesis). A combination of metabolic modelling, anaerobic physiology assays, and isotope labeling of Candidatus Syntrophoarchaeum archaea catalyzing the anaerobic oxidation of butane (AOB) show a back flux of CO 2 to butane, demonstrating reversibility of the entire AOB pathway. Back fluxes correlate with thermodynamics and kinetics of the archaeal catabolic system. AOB reversibility supports a biological formation of butane, and generally of higher volatile alkanes, helping to explain the presence of isotopically light alkanes and deeply branching ACR genes in sedimentary basins isolated from gas reservoirs., (© 2024. The Author(s).)

Isoniazid is an important first-line medicine to treat tuberculosis (TB). Isoniazid resistance increases the risk of poor treatment outcomes and development of multidrug resistance, and is driven primarily by mutations involving katG , encoding the prodrug-activating enzyme, rather than its validated target, InhA. The chemical tractability of InhA has fostered efforts to discover direct inhibitors of InhA (DIIs). In this study, we bridge the gap in understanding the potential contribution of DIIs to novel combination regimens and demonstrate a clear distinction of DIIs, like GSK693 and the newly described GSK138, from isoniazid, based on activity against clinical isolates and contribution to novel drug regimens. The results suggest that DIIs, specifically GSK138 and GSK693, could be promising partners in novel drug regimens, including those used against isoniazid-resistant TB, potentially enhancing their efficacy and/or preventing the selection of resistant mutants and supporting the continued exploration of InhA as a promising target for TB drug development., Competing Interests: L.E., J.R.-T., A.G.-P., R.G.D.R., and D.B.-A. are employees of, and shareholders in, GSK. A.M.-L. holds GSK stock and has patents issued during his employment at GSK. J.D.M. and V.S. are employees of GSK. The other authors declare no conflict of interest.

Objective: To analyze regional differences in MMACHC gene variations among patients with cblC-type methylmalonic acidemia (MMA) in China and to explore the relationship between these variations and neonatal screening, biochemical markers and prognosis. Methods: Retrospective case summary. Clinical and laboratory data, including general condition, biochemical markers and genetic analysis, were collected from 1 859 cblC MMA patients from 2005 to 2023. Patients were divided into 7 groups according to their regions: north China, northeast China, east China, central China, south China, southwest China and northwest China. They were also classified into neonatal screening and non-neonatal screening groups. Mann-Whitney U and Kruskal-Wallis tests were used to compare biochemical marker levels. In contrast, the Chi-square test was applied to compare MMACHC gene variant frequencies, neonatal screening proportion, onset age and prognosis between groups. Results: Among 1 859 cases of cblC MMA, 1 019 were male and 840 were female, with a consultation age of 1.0 (0.1, 5.0) month. A total of 1 787 cases carried compound heterozygous or homozygous variants and only 1 variant site was identified in 72 cases. The 10 most frequent variants were c.609G>A (1 238 cases), c.658&#95;660delAAG (343 cases), c.80A>G (284 cases), c.482G>A (239 cases), c.567dupT (191 cases), c.656&#95;658delAGA (131 cases), c.217C>T (109 cases), c.394C>T (105 cases), c.445&#95;446delTG (51 cases) and c.1A>G (50 cases). The frequency of the c.609G>A was the lowest in northwest China (28.8% (44/154), χ ²=-18.42, P <0.05). The frequency of the c.567dupT was the most common in southwest China (25.0% (20/80), χ ²=71.70, P <0.001) and c.656&#95;658delAGA had the highest frequency in northeast China (9.3% (19/205), χ ²=32.08, P <0.001). Non-missense variants (91.2% (62/68), 88.5% (46/52)) and early-onset patients (90.0% (36/40), 94.4% (34/36)) were both more prevalent in southwest and south China ( χ ²=14.95, 31.69, both P <0.05). The proportion of neonatal screening was the lowest in south China (22.2% (8/36), χ ²=98.48, P <0.05), where the mortality rate was the highest (19.1% (4/21), χ ²=38.98, P <0.001). East China exhibited the highest frequency of missense variants (21.5% (339/1 579)), the highest proportion of patients identified through neonatal screening (54.5% (465/853)), and a more significant proportion of patients with good prognosis (36.6% (227/621), χ ²=14.57, 93.49, 38.98, all P <0.05). In addition, the c.482G>A variant was more frequent in patients diagnosed by neonatal screening compared to those diagnosed by other methods (8.3% (132/1 586) vs. 5.9% (122/2 060), χ ²=7.97, P <0.05). Conclusions: The frequency of MMACHC gene variation varies across different regions. The c.609G>A was least frequent in northwest China, c.567dupT was most common in southwest China, and c.656&#95;658delAGA was most prevalent in northeast China. South China had the lowest neonatal screening rate and the highest mortality. At the same time, east China exhibited the highest frequency of missense variants, the highest proportion of patients identified through neonatal screening and the best prognosis. The c.482G>A variant was more frequent in patients diagnosed by neonatal screening compared to those diagnosed by other methods.

Boldenone (BD), a protein anabolic hormone, is commonly used to treat muscle damage, osteoporosis, and off-season muscle building in athletes. Traditional BD synthesis methods rely on chemical processes, which are costly and environmentally impactful. Therefore, developing a more sustainable and economical biosynthetic pathway is crucial for BD production. This study aimed to achieve efficient production of BD. Firstly, the catalytic performance of 17β-hydroxysteroid dehydrogenase and 3-ketosteroid-Δ 1 -dehydrogenase was improved through enzyme engineering, and their expression in the new strain of Mycobacterium neoaurum was enhanced using metabolic engineering. These improvements significantly increased BD production to 4.05 g/L, with a significant decrease in by-product generation. To further increase the yield, a multi-enzyme fusion expression system was constructed, and a key cell wall gene kasB was knocked out, resulting in a spatial-time yield of BD reaching 1.02 g/(L·d). Subsequent optimization of the transformation system further increased the BD production to 5.56 g/L, with a spatiotemporal yield of 1.39 g/(L·d). The green biosynthetic route of phytosterol one-step conversion to BD developed in this study lays the foundation for industrial production., (© 2024 Wiley‐VCH GmbH.)

Hosta is commonly acknowledged as a popular and preferred plant for landscaping and gardening. The 'sunburn' caused by prolonged exposure to strong sunlight is reducing the ornamental values of Hosta plants. However, there is a scarcity of research focusing on the genetic components linked to light-induced harm in Hosta . Here, the violaxanthin de-epoxidase (VDE) homolog from Hosta ventricosa was isolated and functionally identified through conducting HvVDE -overexpression tobacco (Nicotiana tabacum ) lines. The results showed that HvVDE encodes a putative protein comprising 481 amino acids with a molecular weight of 54.304kDa. The phylogenetic analysis found that HvVDE exhibited close similarity to JcVDE. Besides, the expression patterns of HvVDE found that HvVDE was expressed differently across tissues, withexpression induced by high light intensities. And overexpression of HvVDE led to the restoration of non-photochemical quenching in tobacco, suggesting that HvVDE plays a role in dissipating excess light energy as thermal energy in H. ventricosa . These findings underscore the significance of HvVDE in mitigating photoinhibition and enhancing photoprotection mechanisms in H. ventricosa .

Alternative oxidase (AOX) is an enzyme that transfers electrons from reduced quinone directly to oxygen without proton translocation. When AOX from Ciona intestinalis is xenotopically expressed in mice, it can substitute the combined electron-transferring activity of mitochondrial complexes III/IV. Here, we used brain mitochondria from AOX-expressing mice with such a chimeric respiratory chain to study respiratory control bioenergetic mechanisms. AOX expression did not compromise the function of the mammalian respiratory chain at physiological conditions, however the complex IV inhibitor cyanide only partially blocked respiration by AOX-containing mitochondria. The relative fraction of cyanide-insensitive respiration increased at lower temperatures, indicative of a temperature-controlled attenuation of mammalian respiratory enzyme activity. As AOX does not translocate protons, the mitochondrial transmembrane potential in AOX-containing mitochondria was more sensitive to cyanide during succinate oxidation than during malate/pyruvate-supported respiration. High concentrations of cyanide fully collapsed membrane potential during oxidation of either succinate or glycerol 3-phosphate, but not during malate/pyruvate-supported respiration. This confirms AOX's electroneutral redox activity and indicates differences in the proton-translocating capacity of dehydrogenases upstream of the ubiquinone pool. Our respiration data refutes previous proposals for quinone partitioning within the supercomplexes of the respiratory chain, instead supporting the concept of a single homogeneous, freely diffusing quinone pool. Respiration with either succinate or glycerol 3-phosphate promotes reverse electron transfer (RET) towards complex I. AOX expression significantly decreased RET-induced ROS generation, with the effect more pronounced at low temperatures. Inhibitor-sensitivity analysis showed that the AOX-induced decrease in H 2 O 2 release is due to the lower contribution of complex I to net ROS production during RET. Overall, our findings provide new insights into the role of temperature as a mechanism to control respiration and highlight the utility of AOX as a genetic tool to characterize both the distinct pathways of oxygen reduction and the role of redox control in RET., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Enzymatic parameters are classically determined in vitro, under conditions that are far from those encountered in cells, casting doubt on their physiological relevance. We developed a generic approach combining tools from synthetic and systems biology to measure enzymatic parameters in vivo. In the context of a synthetic carotenoid pathway in Saccharomyces cerevisiae, we focused on a phytoene synthase and three phytoene desaturases, which are difficult to study in vitro. We designed, built, and analyzed a collection of yeast strains mimicking substantial variations in substrate concentration by strategically manipulating the expression of geranyl-geranyl pyrophosphate (GGPP) synthase. We successfully determined in vivo Michaelis-Menten parameters (K M , V max , and k cat ) for GGPP-converting phytoene synthase from absolute metabolomics, fluxomics and proteomics data, highlighting differences between in vivo and in vitro parameters. Leveraging the versatility of the same set of strains, we then extracted enzymatic parameters for two of the three phytoene desaturases. Our approach demonstrates the feasibility of assessing enzymatic parameters directly in vivo, providing a novel perspective on the kinetic characteristics of enzymes in real cellular conditions., (© 2024. The Author(s).)

Plant root system is significantly influenced by high soil levels of ammonium nitrogen, leading to reduced root elongation and enhanced lateral root branching. In Arabidopsis, these processes have been reported to be mediated by phytohormones and their downstream signaling pathways, while the controlling mechanisms remain elusive in crops. Through a transcriptome analysis of roots subjected to high/low ammonium treatments, we identified a cytokinin oxidase/dehydrogenase encoding gene, CKX3, whose expression is induced by high ammonium. Knocking out CKX3 and its homologue CKX8 results in shorter seminal roots, fewer lateral roots, and reduced sensitivity to high ammonium. Endogenous cytokinin levels are elevated by high ammonium or in ckx3 mutants. Cytokinin application results in shorter seminal roots and fewer lateral roots in wild-type, mimicking the root responses of ckx3 mutants to high ammonium. Furthermore, CKX3 is transcriptionally activated by type-B RR25 and RR26, and ckx3 mutants have reduced auxin content and signaling in roots under low ammonium. This study identified RR25/26-CKX3-cytokinin as a signal module that mediates root responses to external ammonium by modulating of auxin signaling in the root meristem and lateral root primordium. This highlights the critical role of cytokinin metabolism in regulating rice root development in response to ammonium., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Hydrogen sulfide (H 2 S) is an endogenous gaseous signaling molecule, which has been shown to play an important role in plant growth and development by coupling with various phytohormones. However, the relationship between H 2 S and cytokinin (CTK) and the mechanisms by which H 2 S and CTK affect root growth remain poorly understood. Endogenous CTK was analyzed by UHPLC-ESI-MS/MS. Persulfidation of cytokinin oxidase/dehydrogenases (CKXs) was analyzed by mass spectrometry (MS). ckx2/CKX2 wild-type (WT) , OE CKX2 and ckx2/CKX2 Cys(C)62alanine(A) transgenic lines were isolated with the ckx2 background. H 2 S is linked to CTK content by CKX2, which regulates root system architecture (RSA). Persulfidation at cysteine (Cys)62 residue of CKX2 enhances CKX2 activity, resulting in reduced CTK content. We utilized 35S-LCD/oasa1 transgenic lines to investigate the effect of endogenous H 2 S on RSA, indicating that H 2 S reduces the gravitropic set-point angle (GSA), shortens root hairs, and increases the number of lateral roots (LRs). The persulfidation of CKX2 Cys62 changes the elongation of cells on the upper and lower flanks of LR elongation zone, confirming that Cys62 of CKX2 is the specificity target of H 2 S to regulate RSA in vivo. In conclusion, this study demonstrated that H 2 S negatively regulates CTK content and affects RSA by persulfidation of CKX2 Cys62 in Arabidopsis thaliana., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Three strains of Gram-negative bacterium, Rhizobium, were developed by gamma (γ)-irradiation random mutagenesis. The developed strains were evaluated for their augmented features for symbiotic association, nitrogen fixation, and crop yield of three leguminous plants-chickpea, field-pea, and lentil-in agricultural fields of the northern Indian state of Haryana. Crops treated with developed mutants exhibited significant improvement in plant features and the yield of crops when compared to the control-uninoculated crops and crops grown with indigenous or commercial crop-specific strains of Rhizobium. This improvement was attributed to generated mutants, MbPrRz1 (on chickpea), MbPrRz2 (on lentil), and MbPrRz3 (on field-pea). Additionally, the cocultured symbiotic response of MbPrRz1 and MbPrRz2 mutants was found to be more pronounced on all three crops. The statistical analysis using Pearson's correlation coefficients revealed that nodulation and plant biomass were the most related parameters of crop yield. Among the effectiveness of developed mutants, MbPrRz1 yielded the best results for all three tested crops. Moreover, the developed mutants enhanced macro- and micronutrients of the experimental fields when compared with fields harboring the indigenous rhizobial community. These developed mutants were further genetically characterized, predominantly expressing nitrogen fixation marker, nifH, and appeared to belong to Mesorhizobium ciceri (MbPrRz1) and Rhizobium leguminosarum (both MbPrRz2 and MbPrRz3). In summary, this study highlights the potential of developed Rhizobium mutants as effective biofertilizers for sustainable agriculture, showcasing their ability to enhance symbiotic relationships, crop yield, and soil fertility., (© 2024 The Author(s). Journal of Basic Microbiology published by Wiley‐VCH GmbH.)

Recent studies confirm the critical roles of endoplasmic reticulum oxidoreductase 1 alpha (ERO1L) in malignant behavior of various cancers. Nevertheless, what function ERO1L plays in lung adenocarcinoma (LUAD) remains uncovered. The expressions and clinical significance of ERO1L in LUAD were investigated using the TCGA dataset. The ERO1L levels were examined by RT-qPCR. The LUAD cell proliferation was valued using colony formation as well as CCK-8 assays. The invasion and migration abilities of LUAD cells were detected through Transwell in addition to wound healing assays. The effects of ERO1L on LUAD cell apoptosis were determined by flow cytometric analysis. Moreover, we also established mouse xenograft models of LUAD cells to confirm the functions of ERO1L in vivo. The ERO1L levels in tumors were identified by immunohistochemistry. Western blot was used for the detection of the levels of Wnt/βcatenin signaling-related proteins. The TCGA database revealed that ERO1L expressions were higher in LUAD tissues than those in non-cancerous tissues. ERO1L overexpression was related to poorer overall survival of LUAD patients. In addition, ERO1L silence suppresses LUAD cell clone formation, proliferation, migration as well as invasion but induces apoptosis. Moreover, we also verified that ERO1L silence could promote LUAD growth in vivo. Based on the mechanism analysis, ERO1L was confirmed to regulate LUAD development via Wnt/βcatenin cascade signal. ERO1L, the expression of which was increased in LUAD tissues, functioned as an oncogene. ERO1L silence significantly attenuated LUAD tumorigenesis, likely via inhibition of Wnt/βcatenin signaling, indicating that ERO1L could be exploited as a promising biomarker in LUAD treatment.

Changes in the oxidative (redox) environment accompany idiopathic pulmonary fibrosis (IPF). S-glutathionylation of reactive protein cysteines is a post-translational event that transduces oxidant signals into biological responses. We recently demonstrated that increases in S-glutathionylation promote pulmonary fibrosis, which was mitigated by the deglutathionylating enzyme glutaredoxin (GLRX). However, the protein targets of S-glutathionylation that promote fibrogenesis remain unknown. In the present study we addressed whether the extracellular matrix is a target for S-glutathionylation. We discovered increases in COL1A1 (collagen 1A1) S-glutathionylation (COL1A1-SSG) in lung tissues from subjects with IPF compared with control subjects in association with increases in ERO1A (endoplasmic reticulum [ER] oxidoreductin 1) and enhanced oxidation of ER-localized PRDX4 (peroxiredoxin 4), reflecting an increased oxidative environment of the ER. Human lung fibroblasts exposed to TGFB1 (transforming growth factor-β1) show increased secretion of COL1A1-SSG. Pharmacologic inhibition of ERO1A diminished the oxidation of PRDX4, attenuated COL1A1-SSG and total COL1A1 concentrations, and dampened fibroblast activation. Absence of Glrx enhanced COL1A1-SSG and overall COL1A1 secretion and promoted the activation of mechanosensing pathways. Remarkably, COL1A1-SSG resulted in marked resistance to collagenase degradation. Compared with COL1, lung fibroblasts plated on COL1-SSG proliferated more rapidly and increased the expression of genes encoding extracellular matrix crosslinking enzymes and genes linked to mechanosensing pathways. Overall, these findings suggest that glutathione-dependent oxidation of COL1A1 occurs in settings of IPF in association with enhanced ER oxidative stress and may promote fibrotic remodeling because of increased resistance to collagenase-mediated degradation and fibroblast activation.

Background: We recently demonstrated that 48 h exposure of primary human bronchial epithelial (hBE) cells, obtained from both CF (F508del homozygous) and non-CF subjects, to the triple drug combination Elexacaftor/Tezacaftor/Ivacaftor (ETI) results in a CFTR genotype-independent modulation of the de novo synthethic pathway of sphingolipids, with an accumulation of dihydroceramides (dHCer). Since dHCer are converted into ceramides (Cer) by the action of a delta-4 sphingolipid desaturase (DEGS) enzyme, we aimed to better understand this off-target effect of ETI (i.e., not related to CFTR rescue) METHODS: hBE cells, both F508del and wild-type, were cultured to create fully differentiated bronchial epithelia. We analyzed Cer and dHCer using an LC-MS based method previously developed by our lab. DEGS expression levels in differentiated hBE cells lysates were quantified by western blot analysis., Results: We demonstrated that 1) dHCer accumulate in hBE with time following prolonged ETI exposure, that 2) similar inhibition occurs in wild-type primary human hepatocytes and that 3) this does not result in an alteration of DEGS expression. We then proved that 4) ETI is a direct inhibitor of DEGS, that 5) Tezacaftor is the molecule responsible for this effect, that 6) the inhibition is concentration dependent. Finally, after repeated oral administration of ETI to naïve, non-CF, mice, we observed a slight accumulation of dHCer in the brain., Conclusions: We believe that further investigations on Tezacaftor should be envisaged, particularly for the use of ETI during pregnancy, breastfeeding and in the early stages of development. DEGS dysfunction and dHCer accumulation causes impairment in the development of the nervous system, due to a derangement in myelin formation and maintenance., Competing Interests: Declaration of competing interest The Authors declare that no conflict of interest exists., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Cytochrome bds are bacterial terminal oxidases expressed under low oxygen conditions, and they are important for the survival of many pathogens and hence potential drug targets. The largest subunit CydA contains the three redox-active cofactors heme b 558 , heme b 595 and the active site heme d. One suggested proton transfer pathway is found at the interface between the CydA and the other major subunit CydB. Here we have studied the O 2 reduction mechanism in E. coli cyt. bd-I using the flow-flash technique and focused on the mechanism, kinetics and pathway for proton transfer. Our results show that the peroxy (P) to ferryl (F) transition, coupled to the oxidation of the low-spin heme b 558 is pH dependent, with a maximum rate constant (~10 4  s -1 ) that is slowed down at higher pH. We assign this behavior to rate-limitation by internal proton transfer from a titratable residue with pK a  ~ 9.7. Proton uptake from solution occurs with the same P➔F rate constant. Site-directed mutagenesis shows significant effects on catalytic turnover in the CydB variants Asp58 B ➔Asn and Asp105 B ➔Asn variants consistent with them playing a role in proton transfer. Furthermore, in the Asp105 B ➔Asn variant, the reactions up to P formation occur essentially as in the wildtype bd-I, but the P➔F transition is specifically inhibited, supporting a direct and specific role for Asp105 B in the functional proton transfer pathway in bd-I. We further discuss the possible identity of the high pK a proton donor, and the conservation pattern of the Asp-105 B in the cyt. bd superfamily., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

A protein-expressing citrus tristeza virus-based vector construct, pT36CA-V1.3, obtained from a California isolate of the T36 strain (T36CA), was retooled into a virus-induced gene silencing system intended for use with studies of California citrus. Virus-induced gene silencing constructs engineered with a truncated Citrus macrophylla PHYTOENE DESATURASE ( CmPDS ) gene sequence in the sense or antisense orientation worked equally well to silence the endogenous CmPDS gene. In a parallel effort to optimize vector performance, two nonsynonymous nucleotides in open reading frame 1a of pT36CA-V1.3 were replaced with those conserved in the reference sequences from the T36CA cDNA library. The resulting viruses, T36CA-V1.4 (with one amino acid modification: D760N) and T36CA-V1.5 (with two amino acid modifications: D760N and P1174L), along with T36CA-V1.3, were individually propagated in Nicotiana benthamiana and C. macrophylla plants. Enzyme-linked immunosorbent assay (ELISA) measurements of extracts of the newly emerged leaves suggested that all three viruses accumulated to similar levels in N. benthamiana plants at 5 weeks postinoculation. ELISA values of T36CA-V1.4- and -V1.5-infected C. macrophylla samples were significantly higher than that of T36CA-V1.3-infected samples within an 8- to 12-month postinoculation window, suggesting a higher accumulation of T36CA-V1.4 and -V1.5 than T36CA-V1.3. However, at 36 months postinoculation, the ELISA values suggested that all three viruses accumulated to similar levels. When C. macrophylla plants infected with each of the three viruses were grafted to commercial citrus varieties, a limited number of receptor plants became infected, demonstrating a weak but nonetheless (the first) successful delivery of T36CA to California-grown commercial citrus., Competing Interests: The author(s) declare no conflict of interest.

CRISPR-dead Cas9 interference (CRISPRi) has become a valuable tool for precise gene regulation. In this study, CRISPRi was designed to target the inhA gene of Mycobacterium smegmatis (Msm), a gene necessary for mycolic acid synthesis. Our findings revealed that sgRNA2 induced with 100 ng/ml aTc achieved over 90% downregulation of inhA gene expression and inhibited bacterial viability by approximately 1,000-fold. Furthermore, CRISPRi enhanced the susceptibility of M. smegmatis to isoniazid and rifampicin, which are both 50% and 90% lower than those of the wild-type strain or other strains, respectively. This study highlights the ability of CRISPRi to silence the inhA gene, which impacts bacterial viability and drug susceptibility. The findings provide valuable insights into the utility of CRISPRi as an alternative tool for gene regulation. CRISPRi might be further assessed for its synergistic effect with current anti-tuberculosis drugs and its possible implications for combating mycobacterial infections, especially drug-resistant tuberculosis., (© 2024. The Author(s).)

Manganese is an essential trace element for humans, animals, and plants, but excessive amounts of manganese can cause serious harm to organisms. The biological manganese oxidation process mainly oxidizes Mn(II) through the secretion of unique manganese oxidase by manganese-oxidizing bacteria. The T1 Cu site of multicopper oxidase is the main site for substrate oxidation, and its role is to transfer electrons to TNC, where dioxygen reduction occurs. In this study, methionine (Met) No. 444 interacting with the T1Cu-coordinating amino acid in the multicopper oxidase CopA from Brevibacillus panacihumi MK-8 was mutated to phenylalanine (Phe) and leucine (Leu) by the enzyme. Based on the analysis of enzymatic properties and the structural model, the mutant protein M444F with 4.58 times the catalytic efficiency of the original protein CopA and the mutant protein M444L with 1.67 times the catalytic efficiency of the original protein CopA were obtained. The study showed that the manganese removal rate of the manganese-oxidizing engineered bacterium Rosetta-pET-copA M444L cultured for 7 days was 88.87%, which was 10.77% higher than that of the original engineered bacterium. Overall, this study provides a possibility for the application of genetic engineering in the field of biological manganese removal., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Copper homeostasis mechanisms are critical for bacterial resistance to copper-induced stress. The Escherichia coli multicopper oxidase copper efflux oxidase (CueO) is part of the copper detoxification system in aerobic conditions. CueO contains a methionine-rich (Met-rich) domain believed to interact with copper, but its exact function and the importance of related copper-binding sites remain unclear. This study investigates these open questions by employing a multimodal and multiscale approach. Through the design of various E. coli CueO (EcCueO) variants with altered copper-coordinating residues and domain deletions, we employ biological, biochemical, and physico-chemical approaches to unravel in vitro CueO catalytic properties and in vivo copper resistance. Strong correlation between the different methods enables evaluation of EcCueO variants' activity as a function of Cu+ availability. Our findings demonstrate the Met-rich domain is not essential for cuprous oxidation, but it facilitates Cu+ recruitment from strongly chelated forms, acting as transient copper binding domain thanks to multiple methionines. They also indicate that the Cu6/7 copper-binding sites previously observed within the Met-rich domain play a negligible role. Meanwhile, Cu5, located at the interface with the Met-rich domain, emerges as the primary and sole substrate-binding active site for cuprous oxidation. The Cu5 coordination sphere strongly affects the enzyme activity and the in vivo copper resistance. This study provides insights into the nuanced role of CueO Met-rich domain, enabling the functions of copper-binding sites and the entire domain itself to be decoupled. This paves the way for a deeper understanding of Met-rich domains in the context of bacterial copper homeostasis., Competing Interests: Competing interests statement:The authors declare no competing interest.

Electron transfer flavoprotein (ETF) plays an important function in fatty acid beta oxidation and the amino acid metabolic pathway. It can provide pathogenicity to some opportunistic fungi via modulating cellular metabolite composition. Arthrobotrys oligospora is a typical invasion fungus to nematodes. Its ETF characterization is still unknown. Here, we showed that the mutations of A. oligospora ETF ( Aoetfα and Aoetfβ ) and its dehydrogenase ( Aoetfdh ) led to severe defects in mitochondrial integrity and blocked fatty acid metabolism. The pathogenicity-associated trap structures were completely suppressed when exposed to nematode-derived ascarosides and nutrition signals, including ammonia and urea. Compared to the wild-type strain, the nematode predatory activity was significantly reduced and delayed. But surprisingly, the rich nutrition could restore the massive trap and robust predatory activity in the mutant Aoetfβ beyond all induction cues. Moreover, the deletion of Aoetfβ has led to the accumulation of butyrate-like smell, which has a strong attraction to Caenorhabditis elegans nematodes. Ultimately, ETF and its dehydrogenase play a crucial role in nematode-trapping fungi, highlighting mitochondrial metabolite fluctuations that are connected to pathogenesis and further regulating the interactions between fungi and nematodes.

Axially chiral biaryl compounds are ubiquitous scaffolds in natural products, bioactive molecules, chiral ligands and catalysts, but biocatalytic methods for their asymmetric synthesis are limited. Herein, we report a highly efficient biocatalytic route for the atroposelective synthesis of biaryls by dynamic kinetic resolution (DKR). This DKR approach features a transient six-membered aza-acetal-bridge-promoted racemization followed by an imine reductase (IRED)-catalyzed stereoselective reduction to construct the axial chirality under ambient conditions. Directed evolution of an IRED from Streptomyces sp. GF3546 provided a variant (S-IRED-Ss-M11) capable of catalyzing the DKR process to access a variety of biaryl aminoalcohols in high yields and excellent enantioselectivities (up to 98 % yield and >99 : 1 enantiomeric ratio). Molecular dynamics simulation studies on the S-IRED-Ss-M11 variant revealed the origin of its improved activity and atroposelectivity. By exploiting the substrate promiscuity of IREDs and the power of directed evolution, our work further extends the biocatalysts' toolbox to construct challenging axially chiral molecules., (© 2024 Wiley-VCH GmbH.)

Arsenic is recognized as a hazardous environmental toxicant strongly associated with neurological damage, but the mechanism is ambiguous. Neuronal cell death is one of the mechanisms of arsenic-induced neurological injury. Ferroptosis is involved in the pathophysiological process of many neurological diseases, however, the role and regulatory mechanism of ferroptosis in nerve injury under arsenic exposure remains uncovered. Our findings confirmed the role of ferroptosis in arsenic-induced learning and memory disorder and revealed miR-21 played a regulatory role in neuronal ferroptosis. Further study discovered that miR-21 regulated neuronal ferroptosis by targeting at FTH1, a finding which has not been documented before. We also found an extra increase of ferroptosis in neuronal cells conditionally cultured by medium collected from arsenic-exposed microglial cells when compared with neuronal cells directly exposed to the same dose of arsenic. Moreover, microglia-derived exosomes removal or miR-21 knockdown in microglia inhibited neuronal ferroptosis, suggesting the role of intercellular communication in the promotion of neuronal ferroptosis. In summary, our findings highlighted the regulatory role of miR-21 in ferroptosis and the contribution of microglia-derived miR-21 in exosomes to arsenic-induced neuronal ferroptosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Since the discovery of complete ammonia oxidizers (comammox) within the genus Nitrospira , their distribution and abundance across habitats have been intensively studied to better understand their ecological significance. Many primers targeting their ammonia monooxygenase subunit A gene ( amoA ) have been designed to detect and quantify comammox bacteria and to describe their community structure. We identified 38 published primers, but only few had high coverage and specificity for all known comammox Nitrospira or one of the two described subclades. For each target group, we comprehensively evaluated selected primer pairs using in silico analyses, endpoint PCRs, qPCRs, and amplicon sequencing on samples from various environments. Endpoint PCRs and qPCRs showed that the most commonly used primer pairs (comaA-244F/659R, comaB-244F/659R, and Ntsp-amoA162F/359R) produced several bands, which likely inflated quantifications via qPCR. In contrast, the recently published primer combinations CA377F/C576R, CB377F/C576R, and CA-CB377F/C576R resulted mostly in a single band. Furthermore, amplicon sequencing demonstrated that these primer combinations also captured the highest richness of comammox Nitrospira . Taken together, our results indicate that few existing comammox amoA primer combinations have both high specificity and coverage and that the choice of these high-specificity and high-coverage primer pairs substantially impacts the accurate detection, quantification, and community description of comammox bacteria. We, therefore, recommend using the CA377F/C576R, CB377F/C576R, and CA-CB377F/C576R primer pairs.IMPORTANCEBacteria that can fully convert ammonia via nitrite to nitrate, the complete ammonia oxidizers (comammox), were recently discovered and are found in many natural and engineered environments. PCR-based tools to study their abundance and diversity were rapidly developed, resulting in a plethora of primers available, many of which are widely used. The presence of comammox bacteria in an environment can, however, only be correctly determined if the used primers detect all members of this group while not detecting any other guilds. This study assesses the coverage and specificity of existing primers targeting comammox bacteria using both computational and standard molecular techniques, revealing large differences in their performance. The uniform usage of well-performing primers across studies could aid in generating comparable and generalizable data to better understand the importance of comammox bacteria in the environment., Competing Interests: The authors declare no conflict of interest.

Background: Tuberculosis (TB) is an airborne disease caused by Mycobacterium tuberculosis (M. tuberculosis). The world is currently facing challenges due to the spread of anti-tuberculosis drug-resistant of M. tuberculosis. Isoniazid-resistant (INH), is one of the first-line anti-tuberculosis agents that has a high resistance case. This study used Multiplex allele-specific Polymerase Chain Reaction (MAS-PCR) to detect the most common mutations associated with isoniazid resistance on inhA, katG, and ahpC gene., Methods: This study used samples from clinical isolates of M. tuberculosis which had been tested for their antibiotic sensitivity of first-line anti-tuberculosis drugs. The DNA extraction process was carried out using the boiling method and then amplified with specific primers for inhA, katG, and ahpC genes using the MAS-PCR method. The results are then read on the electrophoretic gel with an interpretation of the mutation gene when the target gene DNA bands were absent according to the allele-specific fragments target., Results: A total of 200 isolates were tested in this study consisting of isoniazid-resistant and susceptible with the largest distribution of Multi-Drug Resistant (MDR) isolates with a total of 146 isolates (73%). The most significant gene mutation was on the ahpC gene in 61 isolates (30,5%) and the combination mutation of the katG + ahpC gene in 52 isolates (26%) with sensitivity and specificity of the test reaching 87% and 42% for the detection of INH-resistant., Conclusion: Mutation on the ahpC gene has the highest percentage in this study. AhpC gene can be considered one of the essential genes to be tested for the cause of isoniazid-resistant. Using MAS-PCR for detecting gene mutation in isoniazid-resistant was simple and easy, it has the potential to be widely used as a rapid screening molecular test., (Copyright © 2023. Published by Elsevier B.V.)

Dehydroascorbate reductase (DHAR), an indispensable enzyme in the production of ascorbic acid (AsA) in plants, is vital for plant tolerance to various stresses. However, there is limited research on the stress tolerance functions of DHAR genes in sweet potato (Ipomoea batatas [L.] Lam). In this study, the full-length IbDHAR1 gene was cloned from the leaves of sweet potato cultivar Xu 18. The IbDHAR1 protein is speculated to be located in both the cytoplasm and the nucleus. As revealed by qRT-PCR, the relative expression level of IbDHAR1 in the proximal storage roots was much greater than in the other tissues, and could be upregulated by high-temperature, salinity, drought, and abscisic acid (ABA) stress. The results of pot experiments indicated that under high salinity and drought stress conditions, transgenic Arabidopsis and sweet potato plants exhibited decreases in H 2 O 2 and MDA levels. Conversely, the levels of antioxidant enzymes APX, SOD, POD, and ACT, and the content of DHAR increased. Additionally, the ratio of AsA/DHA was greater in transgenic lines than in the wild type. The results showed that overexpression of IbDHAR1 intensified the ascorbic acid-glutathione cycle (AsA-GSH) and promoted the activity of the related antioxidant enzyme systems to improve plant stress tolerance and productivity., Competing Interests: Declarations. Conflict of interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Cobalamin C is the most common inborn error of intracellular cobalamin metabolism caused by biallelic pathogenic variants in the MMACHC gene, leading to impaired conversion of dietary vitamin B12 into its two metabolically active forms, methylcobalamin and adenosylcobalamin. Biochemical hallmarks are elevated plasma total homocysteine (HCYs) and low methionine accompanied by methylmalonic aciduria. This study aimed to evaluate the clinical, biochemical, and molecular analysis of Pakistani patients with CblC defect., Methods: Medical charts, urine organic acid (UOA) chromatograms, plasma amino acid levels, plasma tHcy and MMACHC gene results of patients presenting at the Biochemical Genetics Clinic, AKUH from 2013-2021 were reviewed. Details were collected on a pre-structured questionnaire. SPSS 22 was used for data analysis., Results: CblC was found in 33 cases (Male:Female 19:14). The median age of symptoms onset and diagnosis were 300 (IQR:135-1800) and 1380 (IQR: 240-2730) days. The most common clinical features were cognitive impairment ( n  = 29), seizures ( n  = 23), motor developmental delay ( n  = 20), hypotonia ( n  = 17), and sparse/hypopigmented scalp hair ( n  = 16). The MMACHC gene sequencing revealed homozygous pathogenic variant c.394C > T, (p.Arg132*) in 32 patients, whereas c.609G > A, (p.TRP203*) in one patient whose ancestors had settled in Pakistan from China decades ago. The median age of treatment initiation was 1530 (IQR: 240-2790). The median pre-treatment HCYs levels were 134 (IQR:87.2-155.5) compared to post-treatment levels of 33.3 (IQR: 27.3-44.95) umol/L., Conclusions: Thirty-three cases of CblC defect from a single center underscores a significant number of the disorder within Pakistan. Late diagnosis emphasizes the need for increased clinical awareness and adequate diagnostic facilities.

Background: Body-color polymorphisms in insects are often explained by environmental selective advantages. Differential fitness related to body coloration has been demonstrated in Myzus persicae (Sulzer): performance of the red morph is in general better than that of the green morph on tobacco plants. However, the molecular mechanism involved is largely unclear., Results: Here we showed that the red morph of M. persicae had higher expression of a carotenoid desaturase CarD763 in the whole body, salivary gland and saliva relative to the green morph. Also, 18% individuals displayed faded red body color 5 days post dsCarD763 treatment. Furthermore, knockdown of CarD763 in the red morph significantly prolonged the time needed to locate phloem and shortened the duration of phloem feeding. Honeydew production and survival rate decreased as well. In contrast, overexpression of CarD763 in tobacco leaves facilitated aphid feeding, enhanced honeydew production and improved the survival rate of aphids. Compared with those fed by dsGFP aphids, plants infested by dsCarD763-treated aphids had higher ROS accumulation, lower lycopene content and photosynthetic rate, and maximum photon quantum yield. The reverse was true when plants overexpressed CarD763., Conclusion: These findings demonstrated that CarD763, a red morph-specific salivary protein, could enhance aphid feeding and early colonization by promoting plant photosynthesis. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Cytokinin oxidase/dehydrogenase (CKX) regulates cytokinin levels in plants which are vital for plant growth and development. However, there is a paucity of evidence regarding their role in controlling embryo/seed development in pigeonpea. This comprehensive study provides information on the identification and characterization of CKX genes in pigeonpea. A genome-wide analysis identified 18 CKX genes, each with distinct structure, expression patterns, and possible diverse functions. Domain analysis revealed the presence of the sequences including FAD and CK-Binding domain, and subcellular localization analysis showed that almost 50 % of them reside within the nucleus. They were observed to be located unevenly on chromosome numbers 2, 4, 6, 7, and 11 with a majority of them present on the scaffolds. The 8 homologous pairs and various orthologous gene pairs provided further insights into their evolution pattern. Further, SNP/Indels variation in CKX genes and haplotype groups among contrasting genotypes for SNPP (seed number per pod) were analyzed. Spatiotemporal expression analysis revealed the significant expression pattern of CcCKX15, CcCKX17, and CcCKX2 in genotypes carrying low SNPP reiterating their possible role as negative regulators. These genes can be potential targets to undertake seed and biomass improvement in pigeonpea., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Iron (Fe) toxicity is a major abiotic stress in lowland rice production. Breeding tolerant varieties has proven challenging due to the complex genetic architecture of Fe toxicity tolerance and the strong genotype-by-environment interactions. Additionally, conventional methods for phenotyping visible stress symptoms are often inaccurate, inconsistent, and lack reproducibility. In our previous work, we identified that ascorbate redox regulation, mediated by the activities of dehydroascorbate reductase (DHAR) and ascorbate oxidase (AO), contributed to high tolerance in an indica rice genotype across various environments. To explore whether this mechanism is common among other rice genotypes, we selected ten genotypes with contrasting stress symptoms under Fe-toxic conditions to examine the roles of DHAR and AO in regulating Fe toxicity tolerance. Additionally, we aimed to develop objective and accurate image-based phenotyping methods to replace the traditional leaf bronzing scoring method. Among the ten genotypes we tested, we found significant positive correlations between DHAR activity and stress symptoms in plants grown under both Fe toxicity and control conditions, suggesting a general link between ascorbate redox regulation and Fe toxicity tolerance. Using RGB signals from leaf images of plants exposed to 1000 mg/L Fe 2+ , we evaluated 36 different color indices to quantify stress symptoms. We identified the normalized green‒red difference index as most significant in quantifying stress symptoms under Fe toxicity conditions. Our findings suggest that DHAR activity could be potentially employed as a biomarker in the screening of rice germplasms and breeding tolerant cultivars to Fe toxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Masson SAS.)

The development of point-of-care testing (POCT) for circulating tumor DNA (ctDNA) is meaningful for the non-invasive cancers screening and diagnosis, particularly in resource-limited settings. The microfluidic paper-based analytical device (μPAD) provides an ideal platform, its application in ctDNA assays remains underexplored. In this work, a multifunctional μPAD was manufactured, which can enhance the efficiency and reduce the cost of ctDNA sensing. Additionally, a smartphone-based application analysis was fabricated for convenient, portable detection and colorimetric signal readout. Moreover, the novel oxidase-like MnB 2 nanozyme was introduced in the sandwiches sensing strategy, utilizing its catalytic properties to effectively generate a colorimetric signal. The use of MnB 2 nanozyme in sensing application is relatively novel, and its catalytic performance and mechanism was thoroughly evaluated via experiment and density functional theory (DFT) calculations. After optimizing the detection conditions, the proposed biosensor exhibited satisfactory results. Furthermore, the method was successfully used to detect ctDNA in tumor cell lysates and peripheral blood samples from tumor-bearing mice. The results were consistent with standard qPCR method, affirming the reliability of our POCT analysis device in ctDNA detection. Thus, this work not only provides a paper-based POCT device and intelligent analysis tool for portable cancers diagnosis, but it also paves a new application path for MnB 2 nanozyme in the sensing filed., Competing Interests: Declaration of Competing interest The author(s) declared no potential conflicts of interest with respect to the research, author-ship, and/or publication of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Millettia pinnata is an important medicinal plant that has been used as a treatment of various diseases due to presence of wide range of pharmacological properties. The plant contains quercetin, kaempferol, karanjin, pongaglabrone, kanjone, kanugin, gammatin, pongaglabol, and other bioflavonoids. Kaempferol is a natural flavonol that shows many pharmacological properties including anti-inflammatory, antioxidant, anticancer, and antidiabetic activities etc. The enzyme flavonol synthase (FLS, EC 1.14.20.6) catalyses the conversion of dihydroflavonols to flavonols, i.e. biosynthesis of kaempferol from dihydrokaempferol. The current work examined the binding affinity-based approach to improve the enzyme catalytic activity using computational methods. Sequential site-directed mutagenesis was used to create four mutants with the goal to increase hydrogen bonds and further improving the ligand (dihydrokaempferol) binding efficiency. Simulations were done to monitor the stability of the mutants followed by molecular docking to confirm interactions with ligand. For structure validation, various dynamic analysis like RMSD, RMSF, ROG, SASA, H-bond, PCA, DCCM, and FEL were performed, which predicts the stability of wild-type (WT) proteins and mutants. The Mutant&#95;2 and Mutant&#95;3 showed maximum H-bonding and better stability than other mutants and WT that proved higher affinity suggesting improved catalysis. Mutant&#95;2 and Mutant&#95;3 exhibited binding affinities of -7.6 and -8.2 kcal/mol, respectively for the ligand. The outcome of present study will provide significant improvement in synthesis of kaempferol and other plant-based flavonoids.Communicated by Ramaswamy H. Sarma.

Chlorophyll degradation is a characteristic process of leaf senescence. Two mutant lines, which showed green leaves and seeds during senescence, were identified by screening a Tnt-1 retrotransposon-tagged population of Medicago truncatula. Genetic and molecular analyses indicated that the mutated gene is NON-YELLOW COLORING 1 (MtNYC1) in M. truncatula. MtNYC1 encoded a chlorophyll b reductase, characterized by three transmembrane domains and a catalytic site (Y***K). Our investigation further identified three splicing variants of MtNYC1, encoding a full-length protein (MtNYC1A) and two truncated proteins (MtNYC1B, MtNYC1C). Genetic evidence indicated that the catalytic site and the third transmembrane domain were critical domains for chlorophyll b reductase. The coordinated action of three splicing variants plays a pivotal role in the degradation of chlorophyll during the senescence of leaves. This discovery provides precise target sites for the development of stay-green legume cultivars., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aflatoxin B1 (AFB1) produced by some Aspergillus species belongs to the most dangerous contaminants of animal feeds. Development of safe and cost efficient decontamination methods saving feed quality and nutritional value are of paramount importance. The use of recombinant AFB1-detoxifying microbial enzymes represents a promising biotechnological approach meeting the aforementioned requirements. In this study, three AFB1-degrading oxidases (AFOs) from edible basidiomycetes Cantharellus cibarius , Lentinula edodes and Pleurotus eryngii as well as AFO from Armillaria tabescens were expressed in E. coli Rosetta (DE3) and purified by immobilized metal-chelate chromatography. The stabilizing effect of the addition of glycerol and β-mercaptoethanol during protein extraction is shown. The catalytic constants of the recombinant AFOs (rAFOs) and other characteristics, which might be important for their practical application (and optimal temperature and pH, thermolability, regulation of the activity by metal ions and chelating agents, storage stability) were investigated. Among the obtained enzymes, rAFO from P. eryngii (Pe-AFO), which was characterized by the highest specific activity, thermostability and pH stability (especially at acidic pH range), the lowest K m , and relative resistance to the inhibition by phytate, showed the best AFB1-degrading efficacy. However, Pe-AFO and all other rAFOs significantly decreased the target activity during heating above 45 °C, storage frozen or lyophilization.