[Responses of radial growth of Fraxinus mandshurica from different provenances to climate at Maoershan in Northeast China]. / 东北地区帽儿山种源实验林区不同种源水曲柳径向生长对气候的响应.

To understand the responses of radial growth of Fraxinus mandshurica from different provenances to climatic factors, we used the dendrochronological method to establish the standard chronologies of F. mandshurica from 20 provenances in Maoershan provenance test forest, and analyzed the differences in radial growth and their correlation with climate factors. The results showed that the overall trend of F. mandshurica chronologies from 20 provenances was generally similar. There were differences in growth amplitude, with the average radial growth of F. mandshurica from Dailing, Lushuihe and Sanchazi being the highest. The radial growth of F. mandshurica from 20 provenances was significantly positively correlated with the highest temperature in July and the average temperature in July except for Huinan. The radial growth of F. mandshurica from 14 provenances was significantly positively correlated with the precipitation in August. The radial growth of F. mandshurica was constrained by temperature and precipitation during the growing season. There was difference in radial growth among F. mandshurica from different provenances under drought stress. F. mandshurica from Wangqing, Dailing, and Hailin had stronger resistance to drought, while that from Wandianzi, Zhanhe, and Xinglong had better recovery ability after drought.

Analyzing and engineering of the biosynthetic pathway of mollemycin A for enhancing its production.

Mollemycin A (MOMA) is a unique glyco-hexadepsipeptide-polyketide that was isolated from a Streptomyces sp. derived from the Australian marine environment. MOMA exhibits remarkable inhibitory activity against both drug-sensitive and multidrug-resistant malaria parasites. Optimizing MOMA through structural modifications or product enhancements is necessary for the development of effective analogues. However, modifying MOMA using chemical approaches is challenging, and the production titer of MOMA in the wild-type strain is low. This study identified and characterized the biosynthetic gene cluster of MOMA for the first time, proposed its complex biosynthetic pathway, and achieved an effective two-pronged enhancement of MOMA production. The fermentation medium was optimized to increase the yield of MOMA from 0.9 mg L-1 to 1.3 mg L-1, a 44% boost. Additionally, a synergistic mutant strain was developed by deleting the momB3 gene and overexpressing momB2, resulting in a 2.6-fold increase from 1.3 mg L-1 to 3.4 mg L-1. These findings pave the way for investigating the biosynthetic mechanism of MOMA, creating opportunities to produce a wide range of MOMA analogues, and developing an efficient strain for the sustainable and economical production of MOMA and its analogues.

o-Vanillin binds covalently to MAL/TIRAP Lys-210 but independently inhibits TLR2.

Toll-like receptor (TLR) innate immunity signalling protects against pathogens, but excessive or prolonged signalling contributes to a range of inflammatory conditions. Structural information on the TLR cytoplasmic TIR (Toll/interleukin-1 receptor) domains and the downstream adaptor proteins can help us develop inhibitors targeting this pathway. The small molecule o-vanillin has previously been reported as an inhibitor of TLR2 signalling. To study its mechanism of action, we tested its binding to the TIR domain of the TLR adaptor MAL/TIRAP (MALTIR). We show that o-vanillin binds to MALTIR and inhibits its higher-order assembly in vitro. Using NMR approaches, we show that o-vanillin forms a covalent bond with lysine 210 of MAL. We confirm in mouse and human cells that o-vanillin inhibits TLR2 but not TLR4 signalling, independently of MAL, suggesting it may covalently modify TLR2 signalling complexes directly. Reactive aldehyde-containing small molecules such as o-vanillin may target multiple proteins in the cell.

The interplay of Cxcl10+/Mmp14+ monocytes and Ccl3+ neutrophils proactively mediates silica-induced pulmonary fibrosis.

As a fatal occupational disease with limited therapeutic options, molecular mechanisms underpinning silicosis are still undefined. Herein, single-cell RNA sequencing of the lung tissue of silicosis mice identified two monocyte subsets, which were characterized by Cxcl10 and Mmp14 and enriched in fibrotic mouse lungs. Both Cxcl10+ and Mmp14+ monocyte subsets exhibited activation of inflammatory marker genes and positive regulation of cytokine production. Another fibrosis-unique neutrophil population characterized by Ccl3 appeared to be related to the pro-fibrotic process, specifically the "inflammatory response". Meanwhile, the proportion of monocytes and neutrophils was significantly higher in the serum of silicosis patients and slices of lung tissue from patients with silicosis further validated the over-expression of Cxcl10 and Mmp14 in monocytes, also Ccl3 in neutrophils, respectively. Mechanically, receptor-ligand interaction analysis identified the crosstalk of Cxcl10+/Mmp14+ monocytes with Ccl3+ neutrophils promoting fibrogenesis via coupling of HBEGF-CD44 and CSF1-CSF1R. In vivo, administration of clodronate liposomes, Cxcl10 or Mmp14 siRNA-loaded liposomes, Ccl3 receptor antagonist BX471, CD44 or CSF1R neutralizing antibodies significantly alleviated silica-induced lung fibrosis. Collectively, these results demonstrate that the newly defined Cxcl10+/Mmp14+ monocytes and Ccl3+ neutrophils participate in the silicosis process and highlight anti-receptor-ligand pair treatment as a potentially effective therapeutic strategy in managing silicosis.

Structural Basis of the Bivalency of the TRPV1 Agonist DkTx.

Bivalency is a prevalent natural mechanism to enhance receptor avidity. Various two-domain disulfide-rich peptides exhibiting bivalent action have been identified from animal venoms. A unique characteristic of these peptides is that they induce a pharmacological response different from that provoked by any of the constituent domains. The enhanced potency and avidity of such peptides is therefore a consequence of their domain fusion by a peptide linker. The role of the linker itself, beyond conjugation, remains unclear. Here, we investigate how the linker affects the bivalency of the capsaicin receptor (TRPV1) agonist DkTx. We recombinantly produced isotope labelled DkTx using a protein splicing approach, to solve the high-resolution solution structure of DkTx, revealing residual linker order stabilised by linker-domain interactions leading to biased domain orientations. The significance of this was studied using a combination of mutagenesis, spin relaxation studies and electrophysiology measurements. Our results reveal that disrupting the pre-organisation of the domains of DkTx is accompanied by reductions in potency and onset of avidity. Our findings support a model of pre-configured two-domain binding, in favour of the previously suggested sequential binding model. This highlights the significance of ordered elements in linker design and the natural evolution of these in bivalent toxins.

Associations of combined phenotypic ageing and genetic risk with incidence of chronic respiratory diseases in the UK Biobank: a prospective cohort study.

BACKGROUND: Accelerated biological ageing has been associated with an increased risk of several chronic respiratory diseases. However, the associations between phenotypic age, a new biological age indicator based on clinical chemistry biomarkers, and common chronic respiratory diseases have not been evaluated. METHODS: We analysed data from 308 592 participants at baseline in the UK Biobank. The phenotypic age was calculated from chronological age and nine clinical chemistry biomarkers, including albumin, alkaline phosphatase, creatinine, glucose, C-reactive protein, lymphocyte percent, mean cell volume, red cell distribution width and white blood cell count. Furthermore, phenotypic age acceleration (PhenoAgeAccel) was calculated by regressing phenotypic age on chronological age. The associations of PhenoAgeAccel with incident common chronic respiratory diseases and cross-sectional lung function were investigated. Moreover, we constructed polygenic risk scores and evaluated whether PhenoAgeAccel modified the effect of genetic susceptibility on chronic respiratory diseases and lung function. RESULTS: The results showed significant associations of PhenoAgeAccel with increased risk of idiopathic pulmonary fibrosis (IPF) (hazard ratio (HR) 1.52, 95% CI 1.45-1.59), COPD (HR 1.54, 95% CI 1.51-1.57) and asthma (HR 1.18, 95% CI 1.15-1.20) per 5-year increase and decreased lung function. There was an additive interaction between PhenoAgeAccel and the genetic risk for IPF and COPD. Participants with high genetic risk and who were biologically older had the highest risk of incident IPF (HR 5.24, 95% CI 3.91-7.02), COPD (HR 2.99, 95% CI 2.66-3.36) and asthma (HR 2.07, 95% CI 1.86-2.31). Mediation analysis indicated that PhenoAgeAccel could mediate 10∼20% of the associations between smoking and chronic respiratory diseases, while ∼10% of the associations between particulate matter with aerodynamic diameter <2.5 µm and the disorders were mediated by PhenoAgeAccel. CONCLUSION: PhenoAgeAccel was significantly associated with incident risk of common chronic respiratory diseases and decreased lung function and could serve as a novel clinical biomarker.

Facilitating the structural characterisation of non-canonical amino acids in biomolecular NMR.

Peptides and proteins containing non-canonical amino acids (ncAAs) are a large and important class of biopolymers. They include non-ribosomally synthesised peptides, post-translationally modified proteins, expressed or synthesised proteins containing unnatural amino acids, and peptides and proteins that are chemically modified. Here, we describe a general procedure for generating atomic descriptions required to incorporate ncAAs within popular NMR structure determination software such as CYANA, CNS, Xplor-NIH and ARIA. This procedure is made publicly available via the existing Automated Topology Builder (ATB) server (https://atb.uq.edu.au, last access: 17 February 2023) with all submitted ncAAs stored in a dedicated database. The described procedure also includes a general method for linking of side chains of amino acids from CYANA templates. To ensure compatibility with other systems, atom names comply with IUPAC guidelines. In addition to describing the workflow, 3D models of complex natural products generated by CYANA are presented, including vancomycin. In order to demonstrate the manner in which the templates for ncAAs generated by the ATB can be used in practice, we use a combination of CYANA and CNS to solve the structure of a synthetic peptide designed to disrupt Alzheimer-related protein-protein interactions. Automating the generation of structural templates for ncAAs will extend the utility of NMR spectroscopy to studies of more complex biomolecules, with applications in the rapidly growing fields of synthetic biology and chemical biology. The procedures we outline can also be used to standardise the creation of structural templates for any amino acid and thus have the potential to impact structural biology more generally.

IL33-mediated NPM1 promotes fibroblast-to-myofibroblast transition via ERK/AP-1 signaling in silica-induced pulmonary fibrosis.

Silicosis is a global occupational pulmonary disease due to the accumulation of silica dust in the lung. Lacking effective clinical drugs makes the treatment of this disease quite challenging in clinics largely because the pathogenic mechanisms remain obscure. Interleukin 33 (IL33), a pleiotropic cytokine, could promote wound healing and tissue repair via the receptor ST2. However, the mechanisms governing the involvement of IL33 in silicosis progression remain to be further explored. Here, we demonstrated that the IL33 levels in the lung sections were significantly overexpressed after bleomycin and silica treatment. Chromatin immunoprecipitation assay, knockdown, and reverse experiments were performed in lung fibroblasts to prove gene interaction following exogenous IL33 treatment or cocultured with silica-treated lung epithelial cells. Mechanistically, we illustrated that silica-stimulated lung epithelial cells secreted IL33 and further promoted the activation, proliferation, and migration of pulmonary fibroblasts by activating the ERK/AP-1/NPM1 signaling pathway in vitro. And more, treatment with NPM1 siRNA-loaded liposomes markedly protected mice from silica-induced pulmonary fibrosis in vivo. In conclusion, the involvement of NPM1 in the progression of silicosis is regulated by the IL33/ERK/AP-1 signaling axis, which is the potential therapeutic target candidate in developing novel antifibrotic strategies for pulmonary fibrosis.

[Influencing Factors and Prediction Model of Performance of Needle Visualization in Fine Needle Aspiration of Thyroid Nodules].

Objective To investigate the influencing factors and establish a model predicting the performance of needle visualization in fine-needle aspiration (FNA) of thyroid nodules. Methods This study prospectively included 175 patients who underwent FNA of thyroid nodules in the Department of Ultrasound in China-Japan Friendship Hospital and compared the display of the needle tips in the examination of 199 thyroid nodules before and after the application of needle visualization.We recorded the location,the positional relationship with thyroid capsule,ultrasonic characteristics,and the distribution of the soft tissue strip structure at the puncture site of the nodules with unclear needle tips display before using needle visualization.Furthermore,according to the thyroid imaging reporting and data system proposed by the American College of Radiology,we graded the risk of the nodules.Lasso-Logistic regression was employed to screen out the factors influencing the performance of needle visualization and establish a nomogram for prediction. Results The needle tips were not clearly displayed in the examination of 135 (67.8%) and 53 (26.6%) nodules before and after the application of needle visualization,respectively,which showed a significant difference (P<0.001).Based on the positional relationship between the nodule and capsule,anteroposterior/transverse diameter (A/T) ratio,blood supply,and the distribution of subcutaneous strip structure at the puncture site,a nomogram was established to predict the probability of unclear display of the needle tips after application of needle visualization.The C-index of the prediction model was 0.75 (95%CI=0.67-0.84) and the area under the receiver operating characteristic curve was 0.72.The calibration curve confirmed the appreciable reliability of the prediction model,with the C-index of 0.70 in internal validation. Conclusions Needle visualization can improve the display of the needle tip in ultrasound-guided FNA of thyroid nodules.The nomogram established based on ultrasound features such as the positional relationship between the nodule and capsule,A/T ratio,blood supply,and the distribution of subcutaneous strip structure at the puncture site can predict whether needle visualization is suitable for the examination of nodules.

Designing antimicrobial peptides using deep learning and molecular dynamic simulations.

With the emergence of multidrug-resistant bacteria, antimicrobial peptides (AMPs) offer promising options for replacing traditional antibiotics to treat bacterial infections, but discovering and designing AMPs using traditional methods is a time-consuming and costly process. Deep learning has been applied to the de novo design of AMPs and address AMP classification with high efficiency. In this study, several natural language processing models were combined to design and identify AMPs, i.e. sequence generative adversarial nets, bidirectional encoder representations from transformers and multilayer perceptron. Then, six candidate AMPs were screened by AlphaFold2 structure prediction and molecular dynamic simulations. These peptides show low homology with known AMPs and belong to a novel class of AMPs. After initial bioactivity testing, one of the peptides, A-222, showed inhibition against gram-positive and gram-negative bacteria. The structural analysis of this novel peptide A-222 obtained by nuclear magnetic resonance confirmed the presence of an alpha-helix, which was consistent with the results predicted by AlphaFold2. We then performed a structure-activity relationship study to design a new series of peptide analogs and found that the activities of these analogs could be increased by 4-8-fold against Stenotrophomonas maltophilia WH 006 and Pseudomonas aeruginosa PAO1. Overall, deep learning shows great potential in accelerating the discovery of novel AMPs and holds promise as an important tool for developing novel AMPs.

Self-cyclisation as a general and efficient platform for peptide and protein macrocyclisation.

Macrocyclisation of proteins and peptides results in a remarkable increase in structural stability, making cyclic peptides and proteins of great interest in drug discovery-either directly as drug leads or as in the case of cyclised nanodiscs (cNDs), as tools for studies of trans-membrane receptors and membrane-active peptides. Various biological methods have been developed that are capable of yielding head-to-tail macrocyclised products. Recent advances in enzyme-catalysed macrocyclisation include discovery of new enzymes or design of new engineered enzymes. Here, we describe the engineering of a self-cyclising "autocyclase" protein, capable of performing a controllable unimolecular reaction for generation of cyclic biomolecules in high yield. We characterise the self-cyclisation reaction mechanism, and demonstrate how the unimolecular reaction path provides alternative avenues for addressing existing challenges in enzymatic cyclisation. We use the method to produce several notable cyclic peptides and proteins, demonstrating how autocyclases offer a simple, alternative way to access a vast diversity of macrocyclic biomolecules.

ARMH3-mediated recruitment of PI4KB directs Golgi-to-endosome trafficking and activation of the antiviral effector STING.

The cGAS-STING pathway mediates cytoplasmic DNA-triggered innate immunity. STING activation is initiated by cyclic-GMP-AMP (cGAMP)-induced translocation from the endoplasmic reticulum and sulfated glycosaminoglycans-induced polymerization at the Golgi. Here, we examine the mechanisms underlying STING transport and activation beyond the Golgi. A genome-wide CRISPR-Cas9 screen identified Armadillo-like helical domain-containing protein 3 (ARMH3) as critical for STING activation. Upon cGAMP-triggered translocation, ARMH3 interacted with STING at the Golgi and recruited phosphatidylinositol 4-kinase beta (PI4KB) to synthesize PI4P, which directed STING Golgi-to-endosome trafficking via PI4P-binding proteins AP-1 and GGA2. Disrupting PI4P-dependent lipid transport through RNAi of other PI4P-binding proteins impaired STING activation. Consistently, disturbed lipid composition inhibited STING activation, whereas aberrantly elevated cellular PI4P led to cGAS-independent STING activation. Armh3fl/fllLyzCre/Cre mice were susceptible to DNA virus challenge in vivo. Thus, ARMH3 bridges STING and PIK4B to generate PI4P for STING transportation and activation, an interaction conserved in all eukaryotes.

Comprehensive Peptide Cyclization Examination Yields Optimized APP Scaffolds with Improved Affinity toward Mint2.

Peptides targeting disease-relevant protein-protein interactions are an attractive class of therapeutics covering the otherwise undruggable space between small molecules and therapeutic proteins. However, peptides generally suffer from poor metabolic stability and low membrane permeability. Hence, peptide cyclization has become a valuable approach to develop linear peptide motifs into metabolically stable and potentially cell-permeable cyclic leads. Furthermore, cyclization of side chains, also known as "stapling", can stabilize particular secondary peptide structures. Here, we demonstrate that a comprehensive examination of cyclization strategies in terms of position, chemistry, and length is a prerequisite for the selection of optimal cyclic peptide scaffolds. Our systematic approach identifies cyclic APP dodecamer peptides targeting the phosphotyrosine binding domain of Mint2 with substantially improved affinity. We show that especially all-hydrocarbon stapling provides improved metabolic stability, a significantly stabilized secondary structure and membrane permeability.

A bivalent remipede toxin promotes calcium release via ryanodine receptor activation.

Multivalent ligands of ion channels have proven to be both very rare and highly valuable in yielding unique insights into channel structure and pharmacology. Here, we describe a bivalent peptide from the venom of Xibalbanus tulumensis, a troglobitic arthropod from the enigmatic class Remipedia, that causes persistent calcium release by activation of ion channels involved in muscle contraction. The high-resolution solution structure of φ-Xibalbin3-Xt3a reveals a tandem repeat arrangement of inhibitor-cysteine knot (ICK) domains previously only found in spider venoms. The individual repeats of Xt3a share sequence similarity with a family of scorpion toxins that target ryanodine receptors (RyR). Single-channel electrophysiology and quantification of released Ca2+ stores within skinned muscle fibers confirm Xt3a as a bivalent RyR modulator. Our results reveal convergent evolution of RyR targeting toxins in remipede and scorpion venoms, while the tandem-ICK repeat architecture is an evolutionary innovation that is convergent with toxins from spider venoms.

Crystal structure determination of the armadillo repeat domain of Drosophila SARM1 using MIRAS phasing.

The crystal structure determination of the armadillo repeat motif (ARM) domain of Drosophila SARM1 (dSARM1ARM) is described, which required the combination of a number of sources of phase information in order to obtain interpretable electron-density maps. SARM1 is a central executioner of programmed axon degeneration, a common feature of the early phase of many neurodegenerative diseases. SARM1 is held in the inactive state in healthy axons by its N-terminal auto-inhibitory ARM domain, and is activated to cleave NAD upon injury, triggering subsequent axon degeneration. To characterize the molecular mechanism of SARM1 activation, it was sought to determine the crystal structure of the SARM1 ARM domain. Here, the recombinant production and crystallization of dSARM1ARM is described, as well as the unconventional process used for structure determination. Crystals were obtained in the presence of NMN, a precursor of NAD and a potential activator of SARM1, only after in situ proteolysis of the N-terminal 63 residues. After molecular-replacement attempts failed, the crystal structure of dSARM1ARM was determined at 1.65 Šresolution using the MIRAS phasing technique with autoSHARP, combining data from native, selenomethionine-labelled and bromide-soaked crystals. The structure will further the understanding of SARM1 regulation.

Smart Self-Cleaning Membrane via the Blending of an Upper Critical Solution Temperature Diblock Copolymer with PVDF.

Poly(2,2,2-trifluoroethyl methacrylate)-b-poly(imidazoled glycidyl methacrylate-co-diethylene glycol methyl ether methacrylate) (PTFEMA-b-P(iGMA-co-MEO2MA)) containing an upper critical solution temperature (UCST) polymer chain was prepared and blended with poly(vinylidene fluoride) (PVDF) to produce a thermoresponsive membrane with smart self-cleaning performance. The successful preparation of the membrane was demonstrated by attenuated total reflection-Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, and scanning electron microscopy characterization. The membrane shows UCST performance, and its flux changes with the filtrate temperature as the UCST polymer chain stretches out and contracts in response to various temperatures. In addition, the UCST polymer chain can disrupt the foulant and push it away from the membrane when the temperature is above the UCST and thus enables membranes to exhibit a smart self-cleaning behavior. To the best of our knowledge, this work is the first report of a smart self-cleaning membrane based on the blending of a diblock copolymer containing a UCST polymer chain with PVDF.

Structural basis for the binding of the cancer targeting scorpion toxin, ClTx, to the vascular endothelia growth factor receptor neuropilin-1.

Chlorotoxin (ClTx) is a 36-residue disulfide-rich peptide isolated from the venom of the scorpion Leiurus quinquestriatus. This peptide has been shown to selectively bind to brain tumours (gliomas), however, with conflicting reports regarding its direct cellular target. Recently, the vascular endothelial growth factor receptor, neuropilin-1 (NRP1) has emerged as a potential target of the peptide. Here, we sought to characterize the details of the binding of ClTx to the b1-domain of NRP1 (NRP1-b1) using solution state nuclear magnetic resonance (NMR) spectroscopy. The 3D structure of the isotope labelled peptide was solved using multidimensional heteronuclear NMR spectroscopy to produce a well-resolved structural ensemble. The structure points to three putative protein-protein interaction interfaces, two basic patches (R14/K15/K23 and R25/K27/R36) and a hydrophobic patch (F6/T7/T8/H10). The NRP1-b1 binding interface of ClTx was elucidated using 15N chemical shift mapping and included the R25/K27/R36 region of the peptide. The thermodynamics of binding was determined using isothermal titration calorimetry (ITC). In both NMR and ITC measurements, the binding was shown to be competitive with a known NRP1-b1 inhibitor. Finally, combining all of this data we generate a model of the ClTx:NRP1-b1 complex. The data shows that the peptide binds to the same region of NRP1 that is used by the SARS-CoV-2 virus for cell entry, however, via a non-canonical binding mode. Our results provide evidence for a non-standard NRP1 binding motif, while also providing a basis for further engineering of ClTx to generate peptides with improved NRP1 binding for future biomedical applications.

SARM1 is a metabolic sensor activated by an increased NMN/NAD+ ratio to trigger axon degeneration.

Axon degeneration is a central pathological feature of many neurodegenerative diseases. Sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1) is a nicotinamide adenine dinucleotide (NAD+)-cleaving enzyme whose activation triggers axon destruction. Loss of the biosynthetic enzyme NMNAT2, which converts nicotinamide mononucleotide (NMN) to NAD+, activates SARM1 via an unknown mechanism. Using structural, biochemical, biophysical, and cellular assays, we demonstrate that SARM1 is activated by an increase in the ratio of NMN to NAD+ and show that both metabolites compete for binding to the auto-inhibitory N-terminal armadillo repeat (ARM) domain of SARM1. We report structures of the SARM1 ARM domain bound to NMN and of the homo-octameric SARM1 complex in the absence of ligands. We show that NMN influences the structure of SARM1 and demonstrate via mutagenesis that NMN binding is required for injury-induced SARM1 activation and axon destruction. Hence, SARM1 is a metabolic sensor responding to an increased NMN/NAD+ ratio by cleaving residual NAD+, thereby inducing feedforward metabolic catastrophe and axonal demise.

Preparation of Multipurpose Polyvinylidene Fluoride Membranes via a Spray-Coating Strategy Using Waterborne Polymers.

As reported herein, the waterborne polymers poly(glycidyl methacrylate-co-poly(ethylene glycol) methyl ether methacrylate) P(GMA-co-mPEGMA) and polyethyleneimine (PEI) were used to prepare multipurpose polyvinylidene fluoride (PVDF) membranes via a direct spray-coating method. P(GMA-co-mPEGMA) and PEI were alternately sprayed onto the PVDF membrane to yield stable cross-linked copolymer coatings. The successful coating of polymers onto the membrane surface was verified by scanning electron microscopy, attenuated total reflectance-Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy characterization. The coated membrane exhibited oil rejection rates that exceeded 99.0% for oil water mixture separation and 98.0% for oil/water emulsion separation. The flux recovery ratio reached 96.7% after bovine serum albumin filtration and washing with water. The removal efficiencies of the coated membrane M3 for Congo red, methyl orange, methylene blue, and crystal violet, Pb(II), Cu(II), and Cd(II) were 82.4, 83.9, 6.3, 26.8, 90.6, 91.3, and 86.2%, respectively. Thus, it can be used for the removal of dyes and heavy metal ions from wastewater. The antibacterial activities of the coated membranes were also confirmed by the inhibition zone tests and confocal laser scanning microscopy analysis. In addition, the cross-linking strategy provides the coated membranes with excellent durability and repeatability. More importantly, the use of water as the solvent can ensure that the application of these membrane coatings proceeds via a very safe and environmentally friendly coating process.

Molecular basis of regio- and stereo-specificity in biosynthesis of bacterial heterodimeric diketopiperazines.

Bacterial heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are a growing family of bioactive natural products. They are challenging to prepare by chemical routes due to the polycyclic and densely functionalized backbone. Through functional characterization and investigation, we herein identify a family of three related HTDKP-forming cytochrome P450s (NasbB, NasS1868 and NasF5053) and reveal four critical residues (Qln65, Ala86, Ser284 and Val288) that control their regio- and stereo-selectivity to generate diverse dimeric DKP frameworks. Engineering these residues can alter the specificities of the enzymes to produce diverse frameworks. Determining the crystal structures (1.70-1.47 Å) of NasF5053 (ligand-free and substrate-bound NasF5053 and its Q65I-A86G and S284A-V288A mutants) and molecular dynamics simulation finally elucidate the specificity-conferring mechanism of these residues. Our results provide a clear molecular and mechanistic basis into this family of HTDKP-forming P450s, laying a solid foundation for rapid access to the molecular diversity of HTDKP frameworks through rational engineering of the P450s.