Structural Insights of a cis-Epoxysuccinate Hydrolase Facilitate the Development of Robust Biocatalysts for the Production of l-(+)-Tartrate.

l-(+)-Tartaric acid plays important roles in various industries, including pharmaceuticals, foods, and chemicals. cis-Epoxysuccinate hydrolases (CESHs) are crucial for converting cis-epoxysuccinate to l-(+)-tartrate in the industrial production process. There is, however, a lack of detailed structural and mechanistic information on CESHs, limiting the discovery and engineering of these industrially relevant enzymes. In this study, we report the crystal structures of RoCESH and KoCESH-l-(+)-tartrate complex. These structures reveal the key amino acids of the active pocket and the catalytic triad residues and elucidate a dynamic catalytic process involving conformational changes of the active site. Leveraging the structural insights, we identified a robust BmCESH (550 ± 20 U·mg-1) with sustained catalytic activity even at a 3 M substrate concentration. After six batches of transformation, immobilized cells with overexpressed BmCESH maintained 69% of their initial activity, affording an overall productivity of 200 g/L/h. These results provide valuable insights into the development of high-efficiency CESHs and the optimization of biotransformation processes for industrial uses.

Selection of Peptide-Bismuth Bicycles Using Phage Display.

Cysteine conjugation is widely used to constrain phage displayed peptides for the selection of cyclic peptides against specific targets. In this study, the nontoxic Bi3+ ion was used as a cysteine conjugation reagent to cross-link peptide libraries without compromising phage infectivity. We constructed a randomized 3-cysteine peptide library and cyclized it with Bi3+, followed by a selection against the maltose-binding protein as a model target. Next-generation sequencing of selection samples revealed the enrichment of peptides containing clear consensus sequences. Chemically synthesized linear and Bi3+ cyclized peptides were used for affinity validation. The cyclized peptide showed a hundred-fold better affinity (0.31 ± 0.04 µM) than the linear form (39 ± 6 µM). Overall, our study proved the feasibility of developing Bi3+ constrained bicyclic peptides against a specific target using phage display, which would potentially accelerate the development of new peptide-bismuth bicycles for therapeutic or diagnostic applications.

Effect of scanning speeds on mechanical properties of CoCrNi medium entropy alloy prepared by laser additive manufacturing.

Medium entropy alloy (MEA) is a hot spot in the field of material research in recent years. At present, the most widely used processing method of MEAs is "casting-rolling-heat treatment", and the preparation of CoCrNi MEA by laser additive manufacturing (LAM) is still in primary stage. In this study, CoCrNi MEAs were fabricated with different scanning speeds by laser additive manufacturing, and the influence of scanning speed on its mechanical properties was investigated. The results show that higher scanning speed can significantly improve the mechanical properties of CoCrNi MEA. Compared with the low-speed laser additive manufacturing (LSLAM) MEA, the tensile strength of high-speed laser additive manufacturing (HSLAM) MEA is increased by 5.6 % and the fracture strain is increased by 60 %, which is mainly due to the entanglement of 1/6<112> and 1/2<110> dislocations and the defect structure at the grain boundary. LAM is a promising technology that can promote the development and application of MEAs in industry.

Nomogram development and external validation for predicting overall survival and cancer-specific survival in patients with primary retroperitoneal sarcoma: a retrospective cohort study.

BACKGROUND: Primary retroperitoneal sarcoma (RPS) comprises over 70 histologic subtypes, yet there are limited studies that have developed prognostic nomograms for RPS patients to predict overall survival (OS) and cancer-specific survival (CSS). The objective of this study was to construct prognostic nomograms for predicting OS and CSS in RPS patients. METHODS: We identified a total of 1166 RPS patients from the Surveillance, Epidemiology and End Results (SEER) database, and an additional 261 cases were collected from a tertiary cancer center. The study incorporated various clinicopathological and epidemiologic features as variables, and prediction windows for overall survival (OS) and cancer-specific survival (CSS) were set at 3, 5, and 7 years. Multivariable Cox models were utilized to develop the nomograms, and variable selection was performed using a backward procedure based on the Akaike Information Criterion. To evaluate the performance of the nomograms in terms of calibration and discrimination, we used calibration plots, coherence index, and area under the curve. FINDINGS: The study included 818 patients in the development cohort, 348 patients in the internal validation cohort, and 261 patients in the external validation cohort. The backward procedure selected the following variables: age, French Federation of Cancer Centers Sarcoma Group (FNCLCC) grade, pre-/postoperative chemotherapy, tumor size, primary site surgery, and tumor multifocality. The validation results demonstrated that the nomograms had good calibration and discrimination, with C-indices of 0.76 for OS and 0.81 for CSS. Calibration plots also showed good consistency between the predicted and actual survival rates. Furthermore, the areas under the time-dependent receiver operating characteristic curves for the 3-, 5-, and 7-year OS (0.84, 0.82, and 0.78, respectively) and CSS (0.88, 0.88, and 0.85, respectively) confirmed the accuracy of the nomograms. INTERPRETATION: Our study developed accurate nomograms to predict OS and CSS in patients with RPS. These nomograms have important clinical implications and can assist healthcare providers in making informed decisions regarding patient care and treatment options. They may also aid in patient counseling and stratification in clinical trials.

JMJD2D stabilises and cooperates with HBx protein to promote HBV transcription and replication.

Background & Aims: HBV infection is a global health burden. Covalently closed circular DNA (cccDNA) transcriptional regulation is a major cause of poor cure rates of chronic hepatitis B (CHB) infection. Herein, we evaluated whether targeting host factors to achieve functional silencing of cccDNA may represent a novel strategy for the treatment of HBV infection. Methods: To evaluate the effects of Jumonji C domain-containing (JMJD2) protein subfamily JMJD2A-2D proteins on HBV replication, we used lentivirus-based RNA interference to suppress the expression of isoforms JMJD2A-2D in HBV-infected cells. JMJD2D-knockout mice were generated to obtain an HBV-injected model for in vivo experiments. Co-immunoprecipitation and ubiquitylation assays were used to detect JMJD2D-HBx interactions and HBx stability modulated by JMJD2D. Chromatin immunoprecipitation assays were performed to investigate JMJD2D-cccDNA and HBx-cccDNA interactions. Results: Among the JMJD2 family members, JMJD2D was significantly upregulated in mouse livers and human hepatoma cells. Downregulation of JMJD2D inhibited cccDNA transcription and HBV replication. Molecularly, JMJD2D sustained HBx stability by suppressing the TRIM14-mediated ubiquitin-proteasome degradation pathway and acted as a key co-activator of HBx to augment HBV replication. The JMJD2D-targeting inhibitor, 5C-8-HQ, suppressed cccDNA transcription and HBV replication. Conclusion: Our study clarified the mechanism by which JMJD2D regulates HBV transcription and replication and identified JMJD2D as a potential diagnostic biomarker and promising drug target against CHB, and HBV-associated hepatocarcinoma. Impact and implications: HBV cccDNA is central to persistent infection and is a major obstacle to healing CHB. In this study, using cellular and animal HBV models, JMJD2D was found to stabilise and cooperate with HBx to augment HBV transcription and replication. This study reveals a potential novel translational target for intervention in the treatment of chronic hepatitis B infection.

Hybrid coordination for the fast formation building of multi-small-AUV systems with the on-board cameras and limited communication.

Formation building for multi-small-AUV systems with on-board cameras is crucial under the limited communication underwater environment. A hybrid coordination strategy is proposed for the rapid convergence to a leader-follower pattern. The strategy consists of two parts: a time-optimal local-position-based controller (TOLC) and a distributed asynchronous discrete weighted consensus controller (ADWCC). The TOLC controller is designed to optimize the assignation of AUVs' destinations in the given pattern and guide each AUV to its destination by the shortest feasible distance. The ADWCC controller is developed to direct the AUVs blocked by obstacles to reach their destinations with the information from the perceived neighbors by on-board cameras. The rapidity of the proposed strategy is theoretically discussed. The effectiveness of the proposed algorithm has been verified in the simulation environments in both MATLAB and Blender.

Identification of MDM2 as a prognostic and immunotherapeutic biomarker in a comprehensive pan-cancer analysis: A promising target for breast cancer, bladder cancer and ovarian cancer immunotherapy.

BACKGROUND: The murine double minute 2 (MDM2) gene is a crucial factor in the development and progression of various cancer types. Multiple rigorous scientific studies have consistently shown its involvement in tumorigenesis and cancer progression in a wide range of cancer types. However, a comprehensive analysis of the role of MDM2 in human cancer has yet to be conducted. METHODS: We used various databases, including TIMER2.0, TCGA, GTEx and STRING, to analyze MDM2 expression and its correlation with clinical outcomes, interacting genes and immune cell infiltration. We also investigated the association of MDM2 with immune checkpoints and performed gene enrichment analysis using DAVID tools. RESULTS: The pan-cancer MDM2 analysis found that MDM2 expression and mutation status were observably different in 25 types of cancer tissue compared with healthy tissues, and prognosis analysis showed that there was a significant correlation between MDM2 expression and patient prognosis. Furthermore, correlation analysis showed that MDM2 expression was correlated with tumor mutational burden, microsatellite instability and drug sensitivity in certain cancer types. We found that there was an association between MDM2 expression and immune cell infiltration across cancer types, and MDM2 inhibitors might enhance the effect of immunotherapy on breast cancer, bladder cancer and ovarian cancer. CONCLUSIONS: The first systematic pan-cancer analysis of MDM2 was conducted, and it demonstrated that MDM2 was a reliable prognostic biomarker and was closely related to cancer immunity, providing a potential immunotherapeutic target for breast cancer, bladder cancer and ovarian cancer.

High performance of visible-light driven hydrogen production over graphdiyne (g-CnH2n-2)/MOF S-scheme heterojunction.

Among carbon allotropes, 2D graphdiyne (GDY) possesses the merits of good ductility, strong conductivity and an adjustable energy band structure. In this study, a GDY/ZnCo-ZIF S-scheme heterojunction photocatalyst has been successfully prepared by a low-temperature mixing method. Using eosin as a photosensitizer and triethanolamine as a solvent, the hydrogen production of the GDY/ZnCo-ZIF-0.9 composite reaches 171.79 µmol, which is 6.67 and 13.5 times that of the GDY and ZnCo-ZIF materials, respectively. The apparent quantum efficiency of the GDY/ZnCo-ZIF-0.9 composite at 470 nm is 2.8%. The improved photocatalytic efficiency may be attributed to the creation of an S-scheme heterojunction structure that enables efficient separation of space charges. In addition, the EY-sensitized GDY/ZnCo-ZIF catalyst endows the GDY with a special structure to provide an abundance of electrons for the ZnCo-ZIF material, thus facilitating the photocatalytic reduction reaction to produce hydrogen. A novel perspective is presented in this study regarding the construction of an S-scheme heterojunction based on graphdiyne for efficient photocatalytic hydrogen generation.

Engineering the Substrate Specificity of a P450 Dimerase Enables the Collective Biosynthesis of Heterodimeric Tryptophan-Containing Diketopiperazines.

Heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are an important class of bioactive secondary metabolites. Biosynthesis offers a practical opportunity to access their bioactive structural diversity, however, it is restricted by the limited substrate scopes of the HTDKPs-forming P450 dimerases. Herein, by genome mining and investigation of the sequence-product relationships, we unveiled three important residues (F387, F388 and E73) in these P450s that are pivotal for selecting different diketopiperazine (DKP) substrates in the upper binding pocket. Engineering these residues in NasF5053 significantly expanded its substrate specificity and enabled the collective biosynthesis, including 12 self-dimerized and at least 81 cross-dimerized HTDKPs. Structural and molecular dynamics analysis of F387G and E73S revealed that they control the substrate specificity via reducing steric hindrance and regulating substrate tunnels, respectively.

Engineered Glycosidase for Significantly Improved Production of Naturally Rare Vina-Ginsenoside R7.

Ginsenosides are the main bioactive ingredients in plants of the genus Panax. Vina-ginsenoside R7 (VG-R7) is one of the rare high-value ginsenosides with health benefits. The only reported method for preparing VG-R7 involves inefficient and low-yield isolation from highly valuable natural resources. Notoginsenoside Fc (NG-Fc) isolated in the leaves and stems of Panax notoginseng is a suitable substrate for the preparation of VG-R7 via specific hydrolysis of the outside xylose at the C-20 position. Here, we first screened putative enzymes belonging to the glycoside hydrolase (GH) families 1, 3, and 43 and found that KfGH01 can specifically hydrolyze the ß-d-xylopyranosyl-(1 → 6)-ß-d-glucopyranoside linkage of NG-Fc to form VG-R7. The I248F/Y410R variant of KfGH01 obtained by protein engineering displayed a kcat/KM value (305.3 min-1 mM-1) for the reaction enhanced by approximately 270-fold compared with wild-type KfGH01. A change in the shape of the substrate binding pockets in the mutant allows the substrate to sit closer to the catalytic residues which may explain the enhanced catalytic efficiency of the engineered enzyme. This study identifies the first glycosidase for bioconversion of a ginsenoside with more than four sugar units, and it will inspire efforts to investigate other promising enzymes to obtain valuable natural products.

Comparative Study of Gravimetric Humidity Sensor Platforms Based on CMUT and QCM.

Humidity sensors with comprehensive performance are of great interest for industrial and environmental applications. Most sensors, however, have to compromise on at least one characteristic such as sensitivity, response speed, and linearity. This paper reports a gravimetric humidity sensor based on a capacitive micromachined ultrasonic transducer (CMUT) with exceptional all-around performance, and presents a side-by-side comparative investigation of two types of gravimetric humidity sensors for a better understanding of their characteristics and sensing mechanisms. For these purposes, a circular CMUT and a quartz crystal microbalance (QCM) with a resonance frequency of 10 MHz were designed and fabricated. Poly(vinyl alcohol) (PVA) was employed as the humidity sensing layer for its hydrophilicity and ease of film formation. The electrical properties of the sensors, including the electrical input impedances and quality factors, were characterized by a vector network analyzer. The relative humidity (RH) sensing performance of the sensors was evaluated and compared from RH levels of 11% to 97%. Both sensors exhibited good repeatability and low hysteresis. The unique microscale resonant structure of the CMUT humidity sensor contributed to a high sensitivity of 2.01 kHz/%RH, short response and recovery times of 8 s and 3 s, respectively, and excellent linearity (R2 = 0.973), which were far superior to their QCM counterparts. The underlying mechanism was revealed and discussed.

Herbivore-induced tomato plant volatiles lead to the reduction of insecticides susceptibility in Spodoptera litura.

Herbivore-induced plant volatiles (HIPVs) have been associated with plant-plant-herbivorous-natural enemies communication and an enhanced response to the subsequent attack. Spodoptera litura is a serious cosmopolitan pest that has developed a high level of resistance to many insecticides. However, the underlying molecular and biochemical mechanism by which HIPV priming reduces S. litura larval sensitivity to insecticides remains largely unknown. This study was conducted to explore the potential of volatile from undamaged, or artificially damaged, or S. litura-damaged tomato plants on the susceptibility of S. litura to the insecticides beta-cypermethrin indoxacarb and chlorpyrifos. We found that larvae exposed to volatile from S. litura-damaged or artificially damaged tomato plants were significantly less susceptible to the three insecticides than those exposed to volatile from undamaged tomato plants. Elevated activities of detoxifying enzymes [cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), and esterases (ESTs)], were expressed in S. litura larvae exposed to volatile from S. litura-damaged tomato plants than those exposed to volatile from undamaged tomato plants. Similarly, seven detoxification-related genes [GSTs (SlGSTe1, SlGSTo1, and SlGSTe3) and P450s (CYP6B48, CYP9A40, CYP321A7, and CYP321B1)] in the midgut and fat body of larvae were up-regulated under exposure to volatile from S. litura-damaged tomato plants. Increased volatile organic compounds emissions were detected in the headspace of tomato plants damaged by S. litura compared to the undamaged plants. Collectively, these findings suggest that HIPVs can considerably reduce caterpillar susceptibility to insecticides, possibly through induction-enhanced detoxification mechanisms, and provide valuable information for implementing an effective integrated pest management strategy.

A broadband optical fiber transmission-based time domain measurement system for nanosecond-level transient electric field.

A novel nanosecond transient electric field (E-field) measurement system is developed in this paper to measure the E-field pulse caused by the operation of the high-voltage switch (switching E-field pulse) in the substation. An electrically small rod antenna is used as the receiving antenna and is matched by the operational amplifier with high input impedance to achieve broadband frequency response and stable working performance. A broadband analog optical fiber transmission system is further designed based on the high-frequency circuit model of the electronic components. Unlike the traditional frequency domain E-field measurement methods, the developed measurement system can directly output the time domain waveform of the switching E-field pulse. It also has the advantages of adjustable sensitivity, portability, and anti-electromagnetic interference. The calibrated measurement bandwidth ranges from 200 Hz to 680 MHz. Furthermore, the switching E-field pulse in an ultra-high voltage substation is measured and analyzed to verify the effectiveness of the fabricated measurement system.

Generation of a 100-billion cyclic peptide phage display library having a high skeletal diversity.

Phage display is a powerful technique routinely used for the generation of peptide- or protein-based ligands. The success of phage display selections critically depends on the size and structural diversity of the libraries, but the generation of large libraries remains challenging. In this work, we have succeeded in developing a phage display library comprising around 100 billion different (bi)cyclic peptides and thus more structures than any previously reported cyclic peptide phage display library. Building such a high diversity was achieved by combining a recently reported library cloning technique, based on whole plasmid PCR, with a small plasmid that facilitated bacterial transformation. The library cloned is based on 273 different peptide backbones and thus has a large skeletal diversity. Panning of the peptide repertoire against the important thrombosis target coagulation factor XI enriched high-affinity peptides with long consensus sequences that can only be found if the library diversity is large.

Towards the Development of Orally Available Peptide Therapeutics.

Peptides have a number of attractive properties that make them an interesting modality for drug development, including their ability to bind challenging targets, their high target specificity, and their non-toxic metabolic products. However, a major limitation of peptides as drugs is their typically poor oral availability, hindering their convenient and flexible application as pills. Of the more than 60 approved peptide drugs, the large majority is not orally applicable. The oral delivery of peptides is hampered by their metabolic instability and/or limited intestinal uptake. In this article, we review the barriers peptides need to overcome after their oral administration to reach disease targets, we highlight two recent successes of pharma companies in developing orally applicable peptide drugs, and we discuss efforts of our laboratory towards the generation of bioavailable cyclic peptides.

A minimalistic cyclic ice-binding peptide from phage display.

Developing molecules that emulate the properties of naturally occurring ice-binding proteins (IBPs) is a daunting challenge. Rather than relying on the (limited) existing structure-property relationships that have been established for IBPs, here we report the use of phage display for the identification of short peptide mimics of IBPs. To this end, an ice-affinity selection protocol is developed, which enables the selection of a cyclic ice-binding peptide containing just 14 amino acids. Mutational analysis identifies three residues, Asp8, Thr10 and Thr14, which are found to be essential for ice binding. Molecular dynamics simulations reveal that the side chain of Thr10 hydrophobically binds to ice revealing a potential mechanism. To demonstrate the biotechnological potential of this peptide, it is expressed as a fusion ('Ice-Tag') with mCherry and used to purify proteins directly from cell lysate.

Development of Selective FXIa Inhibitors Based on Cyclic Peptides and Their Application for Safe Anticoagulation.

Coagulation factor XI (FXI) has emerged as a promising target for the development of safer anticoagulation drugs that limit the risk of severe and life-threatening bleeding. Herein, we report the first cyclic peptide-based FXI inhibitor that selectively and potently inhibits activated FXI (FXIa) in human and animal blood. The cyclic peptide inhibitor (Ki = 2.8 ± 0.5 nM) achieved anticoagulation effects that are comparable to that of the gold standard heparin applied at a therapeutic dose (0.3-0.7 IU/mL in plasma) but with a substantially broader estimated therapeutic range. We extended the plasma half-life of the peptide via PEGylation and demonstrated effective FXIa inhibition over extended periods in vivo. We validated the anticoagulant effects of the PEGylated inhibitor in an ex vivo hemodialysis model with human blood. Our work shows that FXI can be selectively targeted with peptides and provides a promising candidate for the development of a safe anticoagulation therapy.

Identification of the potential roles of ring finger protein 8 in TP53-mutant breast cancer.

Breast cancer is one of the malignant tumors with the highest mortality rate. With the development of precise treatment technology for cancer, numerous molecular targets have been identified and applied in the treatment of diseases. The present study investigated the potential role of ring finger protein 8 (RNF8) in TP53-mutant breast cancer and explored its possible mechanisms of action through a combination of bioinformatics techniques and cell biology. The results revealed that significantly different genes were expressed in RNF8-knockout mice sequencing data compared with in the control group in the presence of TP53 mutations. Downregulated genes were significantly enriched in several pathways of cell proliferation and apoptosis regulation, development and transcription regulation, while upregulated genes were mainly enriched in immune response-associated signaling pathways. Therefore, the consensus genes of the major signaling pathways were further analyzed, revealing that among patients with TP53 wild-type breast cancer, the prognosis of patients with low expression levels of fibroblast growth factor receptor 1, LIM homeobox 2 and EPH receptor B2 was improved compared with that of patients with high expression levels, while among patients with TP53-mutant breast cancer, there was no significant difference in survival status. In addition, among patients with TP53-mutant breast cancer, the prognosis of patients with high BR serine/threonine kinase 1 expression was significantly improved compared with that in patients with low expression. Finally, cell biology experiments demonstrated that in TP53-mutant breast cancer cells (HCC1937), inhibition of RNF8 significantly inhibited the proliferation of TP53-mutant HCC1937 cells and promoted their apoptosis. The present findings may enrich the understanding of the role of RNF8 and indicated that RNF8 may be used as a potential molecular target in TP53-mutant breast cancer, which may lead to the development of clinical treatment strategies.

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Some fungi engage symbiosis with bacteria, which can effectively promote the metabolism and growth of fungi. The diversity and community structure of bacteria can reflect the growth and substrate utilization of fungi. In this study, we analyzed the effects of different sawdust dosages on the community structure and diversity of bacteria associated with the hyphae of Pleurotus eryngii using high-throughput sequencing technology (HST) based on PCR-amplified 16S rRNA V3-V4 fragments. The results showed that the high-quality sequences from five groups of mycelia samples were clustered into 25 phyla, 52 classes, 114 orders, 199 families, and 406 genera. Proteobacteria (35.0%-85.9%) and Firmicutes (6.5%-38.4%) were the most abundant bacterial phyla, while Acinetobacter (14.8%-71.6%) and Pseudomonas (1.7%-22.3%) were the dominant symbiotic genera. Compared with the mycelia grown on the complete culture medium, sawdust addition could increase the diversity of bacteria coexisting with P. eryngii mycelia, and change the community structure of 10 dominant phyla and 9 dominant genera. P. eryngii cultivated in substrate containing 5 g sawdust had the fastest mycelium growth rate, thick mycelia, and neatly edges. Furthermore, judging from the abundance and diversity, Pseudomonas and Lactobacillus became the dominant genera, which were positively correlated with the mycelia growth vigour. Sawdust, as an important carbon source, could affect the growth and development of P. eryngii and the community structure and diversity of bacteria coexisting with mycelia. This study would lay a theoretical foundation for exploring the molecular mechanism of sawdust and mycelium symbiosis affecting the growth and development of P. eryngii.