Symbiotic bacteria confer insecticide resistance by metabolizing buprofezin in the brown planthopper, Nilaparvata lugens (Stål).

Buprofezin, a chitin synthesis inhibitor, is widely used to control several economically important insect crop pests. However, the overuse of buprofezin has led to the evolution of resistance and exposed off-target organisms present in agri-environments to this compound. As many as six different strains of bacteria isolated from these environments have been shown to degrade buprofezin. However, whether insects can acquire these buprofezin-degrading bacteria from soil and enhance their own resistance to buprofezin remains unknown. Here we show that field strains of the brown planthopper, Nilaparvata lugens, have acquired a symbiotic bacteria, occurring naturally in soil and water, that provides them with resistance to buprofezin. We isolated a symbiotic bacterium, Serratia marcescens (Bup_Serratia), from buprofezin-resistant N. lugens and showed it has the capacity to degrade buprofezin. Buprofezin-susceptible N. lugens inoculated with Bup_Serratia became resistant to buprofezin, while antibiotic-treated N. lugens became susceptible to this insecticide, confirming the important role of Bup_Serratia in resistance. Sequencing of the Bup_Serratia genome identified a suite of candidate genes involved in the degradation of buprofezin, that were upregulated upon exposure to buprofezin. Our findings demonstrate that S. marcescens, an opportunistic pathogen of humans, can metabolize the insecticide buprofezin and form a mutualistic relationship with N. lugens to enhance host resistance to buprofezin. These results provide new insight into the mechanisms underlying insecticide resistance and the interactions between bacteria, insects and insecticides in the environment. From an applied perspective they also have implications for the control of highly damaging crop pests.

Phase-Directed Assembly of Triboelectric Nanopaper for Self-Powered Noncontact Sensing.

Noncontact sensing technology serves as a pivotal medium for seamless data acquisition and intelligent perception in the era of the Internet of Things (IoT), bringing innovative interactive experiences to wearable human-machine interaction perception networks. However, the pervasive limitations of current noncontact sensing devices posed by harsh environmental conditions hinder the precision and stability of signals. In this study, the triboelectric nanopaper prepared by a phase-directed assembly strategy is presented, which possesses low charge transfer mobility (1618 cm2 V-1 s-1) and exceptional high-temperature stability. Wearable self-powered noncontact sensors constructed from triboelectric nanopaper operate stably under high temperatures (200 °C). Furthermore, a temperature warning system for workers in hazardous environments is demonstrated, capable of nonintrusively identifying harmful thermal stimuli and detecting motion status. This research not only establishes a technological foundation for accurate and stable noncontact sensing under high temperatures but also promotes the sustainable intelligent development of wearable IoT devices under extreme environments.

High Strength and Toughness Polymeric Triboelectric Materials Enabled by Dense Crystal-Domain Cross-Linking.

Lightweight, easily processed, and durable polymeric materials play a crucial role in wearable sensor devices. However, achieving simultaneously high strength and toughness remains a challenge. This study addresses this by utilizing an ion-specific effect to control crystalline domains, enabling the fabrication of a polymeric triboelectric material with tunable mechanical properties. The dense crystal-domain cross-linking enhances energy dissipation, resulting in a material boasting both high tensile strength (58.0 MPa) and toughness (198.8 MJ m-3), alongside a remarkable 416.7% fracture elongation and 545.0 MPa modulus. Leveraging these properties, the material is successfully integrated into wearable self-powered devices, enabling real-time feedback on human joint movement. This work presents a valuable strategy for overcoming the strength-toughness trade-off in polymeric materials, paving the way for their enhanced applicability and broader use in diverse sensing applications.

Single-cell RNA sequencing reveals the evolution of the immune landscape during perihematomal edema progression after intracerebral hemorrhage.

BACKGROUND: Perihematomal edema (PHE) after post-intracerebral hemorrhage (ICH) has complex pathophysiological mechanisms that are poorly understood. The complicated immune response in the post-ICH brain constitutes a crucial component of PHE pathophysiology. In this study, we aimed to characterize the transcriptional profiles of immune cell populations in human PHE tissue and explore the microscopic differences between different types of immune cells. METHODS: 9 patients with basal ganglia intracerebral hemorrhage (hematoma volume 50-100 ml) were enrolled in this study. A multi-stage profile was developed, comprising Group1 (n = 3, 0-6 h post-ICH, G1), Group2 (n = 3, 6-24 h post-ICH, G2), and Group3 (n = 3, 24-48 h post-ICH, G3). A minimal quantity of edematous tissue surrounding the hematoma was preserved during hematoma evacuation. Single cell RNA sequencing (scRNA-seq) was used to map immune cell populations within comprehensively resected PHE samples collected from patients at different stages after ICH. RESULTS: We established, for the first time, a comprehensive landscape of diverse immune cell populations in human PHE tissue at a single-cell level. Our study identified 12 microglia subsets and 5 neutrophil subsets in human PHE tissue. What's more, we discovered that the secreted phosphoprotein-1 (SPP1) pathway served as the basis for self-communication between microglia subclusters during the progression of PHE. Additionally, we traced the trajectory branches of different neutrophil subtypes. Finally, we also demonstrated that microglia-produced osteopontin (OPN) could regulate the immune environment in PHE tissue by interacting with CD44-positive cells. CONCLUSIONS: As a result of our research, we have gained valuable insight into the immune-microenvironment within PHE tissue, which could potentially be used to develop novel treatment modalities for ICH.

Self-Assembled Metal-Coordination Nanohelices as Efficient and Robust Chiral Supramolecular Catalysts for Enantioselective Reactions.

The development of chiral nanostructures-based supramolecular catalysts with satisfied enantioselectivity remains a significantly more challenging task. Herein, the synthesis and self-assembly of various amino acid amphiphiles as chiral supramolecular catalysts after metal ion coordination is reported and systematically investigate their enantioselectivity in asymmetric Diels-Alder reactions. In particular, the self-assembly of l/d-phenylglycine-based amphiphiles (l/d-PhgC16) and Cu(II) into chiral supramolecular catalysts in the methanol/water solution mixture is described, which features the interesting M/P nanohelices (diameter ≈8 nm) and mostly well-aligned M/P nanoribbons (NRs). The M/P supramolecular catalysts show both high but inverse enantioselectivity (>90% ee) in Diels-Alder reactions, while their monomeric counterparts display nearly racemic products. Analysis of the catalytic results suggests the outstanding enantioselectivities are closely related to the specific stereochemical microenvironment provided by the arrangement of the amphiphiles in the supramolecular assembly. Based on the experimental evidence of chirality transfer from supramolecular nanohelices to coordinated Cu(II) and substrate aza-chalcone and the molecular dynamics simulations, the enantioselective catalytic mechanisms are proposed. Moreover, the relationships between molecular structures of amino acid amphiphiles (the hydrophilic head group and hydrophobic alkyl chain length) in supramolecular catalysts and enantioselectivity in Diels-Alder reactions are elaborated.

Production of 1,4-Butanediol from Succinic Acid Using Escherichia Coli Whole-Cell Catalysis.

The widespread attention towards 1,4-butanediol (BDO) as a key chemical raw material stems from its potential in producing biodegradable plastics. However, the efficiency of its biosynthesis via current bioprocesses is limited. In this study, a dual-pathway approach for 1,4-BDO production from succinic acid was developed. Specifically, a double-enzyme catalytic pathway involving carboxylic acid reductase and ethanol dehydrogenase was proposed. Optimization of the expression levels of the pathway enzymes led to a significant 318 % increase in 1,4-BDO titer. Additionally, the rate-limiting enzyme MmCAR was engineered to enhance the kcat/KM values by 50 % and increase 1,4-BDO titer by 46.7 %. To address cofactor supply limitations, an NADPH and ATP cycling system was established, resulting in a 48.9 % increase in 1,4-BDO production. Ultimately, after 48 hours, 1,4-BDO titers reached 201 mg/L and 1555 mg/L in shake flask and 5 L fermenter, respectively. This work represents a significant advancement in 1,4-BDO synthesis from succinic acid, with potential applications in the organic chemical and food industries.

Efficient production of protocatechuic acid using systems engineering of Escherichia coli.

Protocatechuic acid (3, 4-dihydroxybenzoic acid, PCA) is widely used in the pharmaceuticals, health food, and cosmetics industries owing to its diverse biological activities. However, the inhibition of 3-dehydroshikimate dehydratase (AroZ) by PCA and its toxicity to cells limit the efficient production of PCA in Escherichia coli. In this study, a high-level strain of 3-dehydroshikimate, E. coli DHS01, was developed by blocking the carbon flow from the shikimate-overproducing strain E. coli SA09. Additionally, the PCA biosynthetic pathway was established in DHS01 by introducing the high-activity ApAroZ. Subsequently, the protein structure and catalytic mechanism of 3-dehydroshikimate dehydratase from Acinetobacter pittii PHEA-2 (ApAroZ) were clarified. The variant ApAroZR363A, achieved by modulating the conformational dynamics of ApAroZ, effectively relieved product inhibition. Additionally, the tolerance of the strain E. coli PCA04 to PCA was enhanced by adaptive laboratory evolution, and a biosensor-assisted high-throughput screening method was designed and implemented to expedite the identification of high-performance PCA-producing strains. Finally, in a 5 L bioreactor, the final strain PCA05 achieved the highest PCA titer of 46.65 g/L, a yield of 0.23 g/g, and a productivity of 1.46 g/L/h for PCA synthesis from glucose using normal fed-batch fermentation. The strategies described herein serve as valuable guidelines for the production of other high-value and toxic products.

Serum deprivation protein response intervenes in the proliferation, motility, and extracellular matrix production in keloid fibroblasts by blocking the amplification of TGF-ß1/SMAD signal cascade via ERK1/2.

Keloid formation has been linked to abnormal fibroblast function, such as excessive proliferation and extracellular matrix (ECM) production. Serum deprivation protein response (SDPR) is a crucial regulator of cellular function under diverse pathological conditions, yet its role in keloid formation remains unknown. The current work investigated the function of SDPR in regulating the proliferation, motility, and ECM production of keloid fibroblasts (KFs), as well as to decipher the mechanisms involved. Analysis of RNA sequencing data from the GEO database demonstrated significant down-regulation of SDPR in KF compared to normal fibroblasts (NFs). This down-regulation was also observed in clinical keloid specimens and isolated KFs. Overexpression of SDPR suppressed the proliferation, motility, and ECM production of KFs, while depletion of SDPR exacerbated the enhancing impact of TGF-ß1 on the proliferation, motility, and ECM production of NFs. Mechanistic studies revealed that SDPR overexpression repressed TGF-ß/Smad signal cascade activation in KFs along with decreased levels of phosphorylated Samd2/3, while SDPR depletion exacerbated TGF-ß/Smad activation in TGF-ß1-stimulated NFs. SDPR overexpression also repressed ERK1/2 activation in KFs, while SDPR depletion exacerbated ERK1/2 activation in TGF-ß1-stimulated NFs. Inhibition of ERK1/2 abolished SDPR-depletion-induced TGF-ß1/Smad activation, cell proliferation, motility, and ECM production in NFs. In conclusion, SDPR represses the proliferation, motility, and ECM production in KFs by blocking the TGF-ß1/Smad pathway in an ERK1/2-dependent manner. The findings highlight the role of SDPR in regulating abnormal behaviors of fibroblasts associated with keloid formation and suggest it as a potential target for anti-keloid therapy development.

Systems engineering Escherichia coli for efficient production p-coumaric acid from glucose.

P-coumaric acid (p-CA), a pant metabolite with antioxidant and anti-inflammatory activity, is extensively utilized in biomedicine, food, and cosmetics industry. In this study, a synthetic pathway (PAL) for p-CA was designed, integrating three enzymes (AtPAL2, AtC4H, AtATR2) into a higher l-phenylalanine-producing strain Escherichia coli PHE05. However, the lower soluble expression and activity of AtC4H in the PAL pathway was a bottleneck for increasing p-CA titers. To overcome this limitation, the soluble expression of AtC4H was enhanced through N-terminal modifications. And an optimal mutant, AtC4HL373T/G211H, which exhibited a 4.3-fold higher kcat/Km value compared to the wild type, was developed. In addition, metabolic engineering strategies were employed to increase the intracellular NADPH pool. Overexpression of ppnk in engineered E. coli PHCA20 led to a 13.9-folds, 1.3-folds, and 29.1% in NADPH content, the NADPH/NADP+ ratio and p-CA titer, respectively. These optimizations significantly enhance p-CA production, in a 5-L fermenter using fed-batch fermentation, the p-CA titer, yield and productivity of engineered strain E. coli PHCA20 were 3.09 g/L, 20.01 mg/g glucose, and 49.05 mg/L/h, respectively. The results presented here provide a novel way to efficiently produce the plant metabolites using an industrial strain.

Chiral Supramolecular Self-Assembly Catalysts with Enhanced Metal Ion Interaction for Higher Enantioselectivity.

Understanding the interaction between metal ions as catalytic centers and supramolecular scaffolds as chiral substrates plays an important role in developing chiral supramolecular catalysts with high enantioselectivity. Herein, we found that compared with l-norleucine chiral amphiphile (l-NorC16), l-methionine chiral amphiphile (l-MetC16) with the only heteroatom of S site difference in the hydrophilic group can form a similar supramolecular chiral nanoribbon (NR) with the bilayer structure through the self-assembly approach; yet, the interaction between the Cu(II) ion catalytic centers and supramolecular scaffolds is reinforced, favoring the chirality transfer and therefore enhancing their catalytic enantioselectivity of Diels-Alder reaction from 23% [l-NorC16-NR-Cu(II)] to 78% [l-MetC16-NR-Cu(II)]. Our work demonstrates a new strategy from the perspective of strengthening the metal ion-supramolecular scaffold interaction for the preparation of chiral supramolecular catalysts with good catalytic enantioselectivity.

Trajectory-dependent threshold effects of proton stopping power in LiF nanosheets.

We conducted a study on the trajectory-dependent threshold effects of proton stopping power in LiF nanosheets using time-dependent density functional theory non-adiabatically coupled to the molecular dynamics. This study covered protons with initial velocities in the range of 0.1-1.0 a.u., offering a vast amount of detailed information on the electronic structure during the stopping process with superior spatial and temporal resolution. Our results show that the impact parameters of incident protons play a crucial role in determining the threshold behavior of proton stopping power in LiF nanosheets. Most importantly, we found that close collisions do not exhibit a discernible threshold. In addition, the research results also revealed the time dependence of the number of electrons occupying the atomic orbitals of F and Li as protons pass through the nanosheets.

Shortening Oral Simethicone-to-Colonoscopy Interval Increases Bowel Preparation Quality.

BACKGROUND Simethicone can improve bowel preparation quality, but the optimal timing of oral simethicone before colonoscopy has not been determined. This study aimed to explore the effect of the time interval between oral simethicone and the start of colonoscopy (S-C) on bowel preparation quality. MATERIAL AND METHODS A total of 364 patients undergoing colonoscopy at our department from August 1, 2021 to November 30, 2021 were included in the training cohort, and 420 consecutive patients from December 15, 2021 to January 31, 2022 comprised the validation cohort. They were classified into short and long S-C groups according to the median S-C. Bowel preparation quality evaluated by the Boston Bowel Preparation Scale was compared between the 2 groups. Logistic regression analyses were performed to explore the correlation between S-C and bowel preparation quality, and we explored the effect of run-way time and time of starting colonoscopy on bowel preparation quality. RESULTS In the training cohort, 182 and 182 patients were classified into the short and long S-C groups, respectively; in the validation cohort, 210 and 210 patients were classified into the 2 groups, respectively. In the 2 cohorts, the short S-C group had a significantly higher rate of adequate/excellent bowel preparation than the long S-C group. Logistic regression analyses showed that shorter S-C, shorter run-way time, and colonoscopy in the morning were all correlated with adequate/excellent bowel preparation. CONCLUSIONS Bowel preparation quality may be affected by S-C, run-way time, and time of starting colonoscopy. S-C shortening should be given equal importance as run-way time shortening.

Effect of Body Mass Index on Cecal Intubation Time During Unsedated Colonoscopy: Variation Across the Learning Stages of an Endoscopist.

BACKGROUND Body mass index (BMI) and endoscopists' experiences can be associated with cecal intubation time (CIT), but such associations are controversial. This study aimed to clarify the association between BMI and CIT during unsedated colonoscopy at 3 learning stages of a single endoscopist. MATERIAL AND METHODS A total of 1500 consecutive patients undergoing unsedated colonoscopy by 1 endoscopist at our department from December 11, 2020, to August 21, 2022, were reviewed. They were divided into 3 learning stages according to the number of colonoscopies performed by 1 endoscopist, including intermediate (501-1000 colonoscopies), experienced (1001-1500 colonoscopies), and senior stages (1501-2000 colonoscopies). Variables that significantly correlated with CIT were identified by Spearman rank correlation analyses and then included in multiple linear regression analysis. RESULTS Overall, 1233 patients were included. Among them, 392, 420, and 421 patients were divided into intermediate, experienced, and senior stages, respectively. Median CIT was 7.83, 6.38, and 5.58 min at intermediate, experienced, and senior stages, respectively (P.

Cholecystokinin-like peptide mediates satiety by inhibiting sugar attraction.

Feeding is essential for animal survival and reproduction and is regulated by both internal states and external stimuli. However, little is known about how internal states influence the perception of external sensory cues that regulate feeding behavior. Here, we investigated the neuronal and molecular mechanisms behind nutritional state-mediated regulation of gustatory perception in control of feeding behavior in the brown planthopper and Drosophila. We found that feeding increases the expression of the cholecystokinin-like peptide, sulfakinin (SK), and the activity of a set of SK-expressing neurons. Starvation elevates the transcription of the sugar receptor Gr64f and SK negatively regulates the expression of Gr64f in both insects. Interestingly, we found that one of the two known SK receptors, CCKLR-17D3, is expressed by some of Gr64f-expressing neurons in the proboscis and proleg tarsi. Thus, we have identified SK as a neuropeptide signal in a neuronal circuitry that responds to food intake, and regulates feeding behavior by diminishing gustatory receptor gene expression and activity of sweet sensing GRNs. Our findings demonstrate one nutritional state-dependent pathway that modulates sweet perception and thereby feeding behavior, but our experiments cannot exclude further parallel pathways. Importantly, we show that the underlying mechanisms are conserved in the two distantly related insect species.

The G932C mutation of chitin synthase 1 gene (CHS1) mediates buprofezin resistance as confirmed by CRISPR/Cas9-mediated knock-in approach in the brown planthopper, Nilaparvata lugens.

The brown planthopper (Nilaparvata lugens) is a major destructive rice pest in Asia. High levels of insecticide resistance have been frequently reported, and the G932C mutation in the chitin synthase 1 (CHS1) gene has been found to mediate buprofezin resistance. However, there has been no direct evidence to confirm the functional significance of the single G932C substitution mutation leading to buprofezin resistance in N. lugens. Here, we successfully constructed a knock-in homozygous strain (Nl-G932C) of N. lugens using CRISPR/Cas9 coupled with homology-directed repair (HDR). Compared with the background strain susceptible to buprofezin (Nl-SS), the knock-in strain (Nl-G932C) showed a 94.9-fold resistance to buprofezin. Furthermore, resistant strains (Nl-932C) isolated from the field exhibited a 2078.8-fold resistance to buprofezin, indicating that there are other mechanisms contributing to buprofezin resistance in the field. Inheritance analysis showed that the resistance trait is incomplete dominance. In addition, the Nl-G932C strain had a relative fitness of 0.33 with a substantially decreased survival rate, emergence rate, and fecundity. This study provided in vivo functional evidence for the causality of G932C substitution mutation of CHS1 with buprofezin resistance and valuable information for facilitating the development of resistance management strategies in N. lugens. This is the first example of using CRISPR/Cas9 gene-editing technology in a hemipteran insect to directly confirm the role of a candidate target site mutation in insecticide resistance.

The role of the Bemisia tabaci and Trialeurodes vaporariorum cytochrome-P450 clade CYP6DPx in resistance to nicotine and neonicotinoids.

The alkaloid, nicotine, produced by tobacco and other Solanaceae as an anti-herbivore defence chemical is one of the most toxic natural insecticides in nature. However, some insects, such as the whitefly species, Trialeurodes vaporariorum and Bemisia tabaci show strong tolerance to this allelochemical and can utilise tobacco as a host. Here, we used biological, molecular and functional approaches to investigate the role of cytochrome P450 enzymes in nicotine tolerance in T. vaporariorum and B. tabaci. Insecticide bioassays revealed that feeding on tobacco resulted in strong induced tolerance to nicotine in both species. Transcriptome profiling of both species reared on tobacco and bean hosts revealed profound differences in the transcriptional response these host plants. Interrogation of the expression of P450 genes in the host-adapted lines revealed that P450 genes belonging to the CYP6DP subfamily are strongly upregulated in lines reared on tobacco. Functional characterisation of these P450s revealed that CYP6DP1 and CYP6DP2 of T. vaporariorum and CYP6DP3 of B. tabaci confer resistance to nicotine in vivo. These three genes, in addition to the B. tabaci P450 CYP6DP5, were also found to confer resistance to the neonicotinoid imidacloprid. Our data provide new insight into the molecular basis of nicotine resistance in insects and illustrates how divergence in the evolution of P450 genes in this subfamily in whiteflies may have impacted the extent to which different species can tolerate a potent natural insecticide.

Identification and Expression Analysis of Putative Sugar Transporter Gene Family during Bulb Formation in Lilies.

Sugar transporters play important roles in plant growth and development, flowering and fruiting, as well as responses to adverse abiotic and biotic environmental conditions. Lilies (Lilium spp.) are some of the most representative ornamental bulbous flowers. Sugar metabolism is critical for bulb formation in lilies; therefore, clarifying the amount and expression pattern of sugar transporters is essential for further analyzing their roles in bulb formation. In this study, based on the transcriptome data of the Lilium Oriental hybrid 'Sorbonne' and Lilium × formolongi, a total of 69 and 41 sugar transporters were identified in 'Sorbonne' and Lilium × formolongi, respectively, by performing bioinformatics analysis. Through phylogenetic analysis, monosaccharide transporters (MSTs) can be divided into seven subfamilies, sucrose transporters (SUTs) can be divided into three subgroups, and sugars will eventually be exported transporters (SWEETs) can be divided into four clades. According to an analysis of conserved motifs, 20, 14, and 12 conserved motifs were predicted in MSTs, SUTs, and SWEETs, respectively. A conserved domain analysis showed that MSTs and SUTs contained a single domain, whereas most of the SWEETs harbored two MtN3/saliva domains, also known as a PQ-loop repeat. The LohINT1, which was predicted to have a smaller number of transmembrane structural domains, was cloned and analyzed for subcellular localization. It was found that the LohINT1 protein is mainly localized in the cell membrane. In addition, the expression analysis indicated that 22 LohMSTs, 1 LohSUTs, and 5 LohSWEETs were upregulated in 'Sorbonne' 1 day after scale detachment treatment, suggesting that they may regulate the initiation of the bulblet. A total of 10 LflMSTs, 1 LflSUTs, and 6 LflSWEETs were upregulated 4~6 months after sowing, which corresponds to the juvenile-to-adult transition phase of Lilium × formolongi, suggesting that they may also play a role in the accompanying bulb swelling process. Combined with quantitative real-time PCR (qRT-PCR) analysis, LohSTP8 and LohSTP12 were significantly overexpressed during the extremely early stage of bulblet initiation, and LflERD6.3 was significantly overexpressed during the growth of the underground bulblet, suggesting that they may be key sugar transporters in the formation of lily bulbs, which needs further functional verification.

Design and Catalytic Asymmetric Synthesis of Furan-Indole Compounds Bearing both Axial and Central Chirality.

In the chemistry community, catalytic asymmetric synthesis of furan-based compounds bearing both axial and central chirality has proven to be a significant but challenging issue owing to the importance and difficulty in constructing such frameworks. In this work, we have realized the first catalytic asymmetric synthesis of five-five-membered furan-based compounds bearing both axial and central chirality via organocatalytic asymmetric (2+4) annulation of achiral furan-indoles with 2,3-indolyldimethanols with uncommon regioselectivity. By this strategy, furan-indole compounds bearing both axial and central chirality were synthesized in high yields with excellent regio-, diastereo-, and enantioselectivities. Moreover, theoretical calculations were conducted to provide an in-depth understanding of the reaction pathway, activation mode, and the origin of the selectivity.

Rational Design of the Spatial Effect in a Fe(II)/α-Ketoglutarate-Dependent Dioxygenase Reverses the Regioselectivity of C(sp3)-H Bond Hydroxylation in Aliphatic Amino Acids.

The hydroxylation of remote C(sp3)-H bonds in aliphatic amino acids yields crucial precursors for the synthesis of high-value compounds. However, accurate regulation of the regioselectivity of remote C(sp3)-H bonds hydroxylation in aliphatic amino acids continues to be a common challenge in chemosynthesis and biosynthesis. In this study, the Fe(II)/α-ketoglutarate-dependent dioxygenase from Bacillus subtilis (BlAH) was mined and found to catalyze hydroxylation at the γ and δ sites of aliphatic amino acids. Crystal structure analysis, molecular dynamics simulations, and quantum chemical calculations revealed that regioselectivity was regulated by the spatial effect of BlAH. Based on these results, the spatial effect of BlAH was reconstructed to stabilize the transition state at the δ site of aliphatic amino acids, thereby successfully reversing the γ site regioselectivity to the δ site. For example, the regioselectivity of L-Homoleucine (5 a) was reversed from the γ site (1 : 12) to the δ site (>99 : 1). The present study not only expands the toolbox of biocatalysts for the regioselective functionalization of remote C(sp3)-H bonds, but also provides a theoretical guidance for the precision-driven modification of similarly remote C(sp3)-H bonds in complex molecules.

Comparative transcriptome and metabolome analyses identified the mode of sucrose degradation as a metabolic marker for early vegetative propagation in bulbs of Lycoris.

Vegetative propagation (VP) is an important practice for production in many horticultural plants. Sugar supply constitutes the basis of VP in bulb flowers, but the underlying molecular basis remains elusive. By performing a combined sequencing technologies coupled with ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry approach for metabolic analyses, we compared two Lycoris species with contrasting regeneration rates: high-regeneration Lycoris sprengeri and low-regeneration Lycoris aurea. A comprehensive multi-omics analyses identified both expected processes involving carbohydrate metabolism and transcription factor networks, as well as the metabolic characteristics for each developmental stage. A higher abundance of the differentially expressed genes including those encoding ethylene responsive factors was detected at bulblet initiation stage compared to the late stage of bulblet development. High hexose-to-sucrose ratio correlated to bulblet formation across all the species examined, indicating its role in the VP process in Lycoris bulb. Importantly, a clear difference between cell wall invertase (CWIN)-catalyzed sucrose unloading in high-regeneration species and the sucrose synthase-catalyzed pathway in low-regeneration species was observed at the bulblet initiation stage, which was supported by findings from carboxyfluorescein tracing and quantitative real-time PCR analyses. Collectively, the findings indicate a sugar-mediated model of the regulation of VP in which high CWIN expression or activity may promote bulblet initiation via enhancing apoplasmic unloading of sucrose or sugar signals, whereas the subsequent high ratio of hexose-to-sucrose likely supports cell division characterized in the next phase of bulblet formation.