Amine Transaminase Engineering for Spatially Bulky Substrate Acceptance

Amine transaminase (ATA) catalyzing stereoselective amination of prochiral ketones is an attractive alternative to transition metal catalysis. As wild‐type ATAs do not accept sterically hindered ketones, efforts to widen the substrate scope to more challenging targets are of general interest. We recently designed ATAs to accept aromatic and thus planar bulky amines, with a sequence‐based motif that supports the identification of novel enzymes. However, these variants were not active against 2,2‐dimethyl‐1‐phenyl‐propan‐1‐one, which carries a bulky tert‐butyl substituent adjacent to the carbonyl function. Here, we report a solution for this type of substrate. The evolved ATAs perform asymmetric synthesis of the respective R amine with high conversions by using either alanine or isopropylamine as amine donor.     

Targeting of GLUT5 for Transporter-Mediated Drug-Delivery Is Contingent upon Substrate Hydrophilicity

Specific link between high fructose uptake and cancer development and progression highlighted fructose transporters as potential means to achieve GLUT-mediated discrimination between normal and cancer cells. The gained expression of fructose-specific transporter GLUT5 in various cancers offers a possibility for developing cancer-specific imaging and bioactive agents. Herein, we explore the feasibility of delivering a bioactive agent through cancer-relevant fructose-specific transporter GLUT5. We employed specific targeting of GLUT5 by 2,5-anhydro-D-mannitol and investigated several drug conjugates for their ability to induce cancer-specific cytotoxicity. The proof-of-concept analysis was carried out for conjugates of chlorambucil (CLB) in GLUT5-positive breast cancer cells and normal breast cells. The cytotoxicity of conjugates was assessed over 24 h and 48 h, and significant dependence between cancer-selectivity and conjugate size was observed. The differences were found to relate to the loss of GLUT5-mediated uptake upon increased conjugate size and hydrophobicity. The findings provide information on the substrate tolerance of GLUT5 and highlight the importance of maintaining appropriate hydrophilicity for GLUT-mediated delivery.     

Identification and Characterization of Specific Nanobodies against Trop-2 for Tumor Targeting

Trophoblast cell-surface antigen 2 (Trop-2) is a tumor-associated antigen that is connected with the development of various tumors and has been identified as a promising target for tumor immunotherapy. To date, the immunotherapy against Trop-2 mainly relies on the specific targeting by monoclonal antibody in antibody-drug conjugate (ADC). Alternatively, the single domain antibodies of nanobodies (Nbs) possesses unique properties such as smaller size, better tissue penetration, etc., to make them good candidates for tumor targeting. Thus, it was proposed to develop anti-Trop-2 Nbs for tumor targeting in this study. Generally, three consecutive rounds of bio-panning were performed against immobilized recombinant Trop-2, and yielded three Nbs (Nb60, Nb65, and Nb108). The affinity of selected Nbs was determined in the nanomolar range, especially the good properties of Nb60 were verified as a promising candidate for tumor labeling. The binding to native Trop-2 was confirmed by flow cytometry against tumor cells. The inhibitory effects of the selected Nbs on tumor cell proliferation and migration were confirmed by wound healing and Transwell assay. The clear localization of the selected Nbs on the surface of tumor cells verified the potent labeling efficiency. In conclusion, this study provided several Nbs with the potential to be developed as targeting moiety of drug conjugates.     

印制线路板基材用工程塑料的应用进展

综合介绍了聚酰亚胺、聚苯醚及改性聚苯醚、聚芳醚酮、液晶聚合物、芳纶纤维、高性能环氧树脂等工程塑料用于生产印制线路板基材的一些主要特点和发展趋势以及它们的改性体系，指出了各自的不足，并展望了印制线路板基材的可能的发展方向。     

A Simple and Efficient Method for the Substrate Identification of Amino Acid Decarboxylases

Amino acid decarboxylases convert amino acids into different biogenic amines which regulate diverse biological processes. Therefore, identifying the substrates of amino acid decarboxylases is critical for investigating the function of the decarboxylases, especially for the new genes predicted to be amino acid decarboxylases. In the present work, we have established a simple and efficient method to identify the substrates and enzymatic activity of amino acid decarboxylases based on LC-MS methods. We chose GAD65 and AADC as models to validate our method. GAD65 and AADC were expressed in HEK 293T cells and purified through immunoprecipitation. The purified amino acid decarboxylases were subjected to enzymatic reaction with different substrate mixtures in vitro. LC-MS analysis of the reaction mixture identified depleted or accumulated metabolites, which corresponded to candidate enzyme substrates and products, respectively. Our method successfully identified the substrates and products of known amino acid decarboxylases. In summary, our method can efficiently identify the substrates and products of amino acid decarboxylases, which will facilitate future amino acid decarboxylase studies.     

以工程能力培养为导向的"食品工程原理"教学改革探讨

"食品工程原理"是食品科学与工程专业的一门重要基础课程,是培养学生工程能力的重要环节。课程内容与其他多门学科联系紧密、基本理论多、理论性强、教学过程难度较大。本文分析了当前"食品工程原理"课程教学中存在的一些问题,提出了以工程能力培养为导向的理论与实践教学改革举措,并对一线教师自身工程素养的提升途径提出了建议。本文为基于工程能力培养为导向的"食品工程原理"课程改革提供了良好借鉴。     

Mitogen-Activated Protein Kinase and Substrate Identification in Plant Growth and Development

Mitogen-activated protein kinases (MAPKs) form tightly controlled signaling cascades that play essential roles in plant growth, development, and defense response. However, the molecular mechanisms underlying MAPK cascades are still very elusive, largely because of our poor understanding of how they relay the signals. The MAPK cascade is composed of MAPK, MAPKK, and MAPKKK. They transfer signals through the phosphorylation of MAPKKK, MAPKK, and MAPK in turn. MAPKs are organized into a complex network for efficient transmission of specific stimuli. This review summarizes the research progress in recent years on the classification and functions of MAPK cascades under various conditions in plants, especially the research status and general methods available for identifying MAPK substrates, and provides suggestions for future research directions.     

Identification of Salicylates in Willow Bark (Salix Cortex) for Targeting Peripheral Inflammation

Salix cortex-containing medicine is used against pain conditions, fever, headaches, and inflammation, which are partly mediated via arachidonic acid-derived prostaglandins (PGs). We used an activity-guided fractionation strategy, followed by structure elucidation experiments using LC-MS/MS, CD-spectroscopy, and 1D/2D NMR techniques, to identify the compounds relevant for the inhibition of PGE₂ release from activated human peripheral blood mononuclear cells. Subsequent compound purification by means of preparative and semipreparative HPLC revealed 2′-O-acetylsalicortin (1), 3′-O-acetylsalicortin (2), 2′-O-acetylsalicin (3), 2′,6′-O-diacetylsalicortin (4), lasiandrin (5), tremulacin (6), and cinnamrutinose A (7). In contrast to 3 and 7, compounds 1, 2, 4, 5, and 6 showed inhibitory activity against PGE₂ release with different potencies. Polyphenols were not relevant for the bioactivity of the Salix extract but salicylates, which degrade to, e.g., catechol, salicylic acid, salicin, and/or 1-hydroxy-6-oxo-2-cycohexenecarboxylate. Inflammation presents an important therapeutic target for pharmacological interventions; thus, the identification of relevant key drugs in Salix could provide new prospects for the improvement and standardization of existing clinical medicine.     

Identification and Engineering of Aptamers for Theranostic Application in Human Health and Disorders

An aptamer is a short sequence of synthetic oligonucleotides which bind to their cognate target, specifically while maintaining similar or higher sensitivity compared to an antibody. The in-vitro selection of an aptamer, applying a conjoining approach of chemistry and molecular biology, is referred as Systematic Evolution of Ligands by Exponential enrichment (SELEX). These initial products of SELEX are further modified chemically in an attempt to make them stable in biofluid, avoiding nuclease digestion and renal clearance. While the modification is incorporated, enough care should be taken to maintain its sensitivity and specificity. These modifications and several improvisations have widened the window frame of aptamer applications that are currently not only restricted to in-vitro systems, but have also been used in molecular imaging for disease pathology and treatment. In the food industry, it has been used as sensor for detection of different diseases and fungal infections. In this review, we have discussed a brief history of its journey, along with applications where its role as a therapeutic plus diagnostic (theranostic) tool has been demonstrated. We have also highlighted the potential aptamer-mediated strategies for molecular targeting of COVID-19. Finally, the review focused on its future prospective in immunotherapy, as well as in identification of novel biomarkers in stem cells and also in single cell proteomics (scProteomics) to study intra or inter-tumor heterogeneity at the protein level. Small size, chemical synthesis, low batch variation, cost effectiveness, long shelf life and low immunogenicity provide advantages to the aptamer over the antibody. These physical and chemical properties of aptamers render them as a strong biomedical tool for theranostic purposes over the existing ones. The significance of aptamers in human health was the key finding of this review.     

Control of Lipase Enantioselectivity by Engineering the Substrate Binding Site and Access Channel

Lipase from Burkholderia cepacia (BCL) has proven to be a very useful biocatalyst for the resolution of 2‐substituted racemic acid derivatives, which are important chiral building blocks. Our previous work showed that enantioselectivity of the wild‐type BCL could be improved by chemical engineering of the substrate's molecular structure. From this earlier study, three amino acids (L17, V266 and L287) were proposed as targets for mutagenesis aimed at tailoring enzyme enantioselectivity. In the present work, a small library of 57 BCL single mutants targeted on these three residues was constructed and screened for enantioselectivity towards (R,S)‐2‐chloro ethyl 2‐bromophenylacetate. This led to the fast isolation of three single mutants with a remarkable tenfold enhanced or reversed enantioselectivity. Analysis of substrate docking and access trajectories in the active site was then performed. From this analysis, the construction of 13 double mutants was proposed. Among them, an outstanding improved mutant of BCL was isolated that showed an E value of 178 and a 15‐fold enhanced specific activity compared to the parental enzyme; thus, this study demonstrates the efficiency of the semirational engineering strategy.     

人Makorin环指蛋白1基因shRNA真核表达质粒的构建及鉴定

目的构建人Makorin环指蛋白1(MKRN1)基因shRNA真核表达质粒,并检测其对HEK293细胞MKRN1基因表达的影响。方法设计并合成特异性针对人MKRN1基因的小干扰片段,定向克隆至带有卡那霉素抗性基因和绿色荧光蛋白基因的真核表达载体pGenesil-1中,对重组质粒进行酶切鉴定及DNA序列分析,并用脂质体将其转染到HEK293细胞中,观察其对细胞MKRN1基因mRNA转录和蛋白表达水平的影响。结果经酶切鉴定和序列分析证明,3个人MKRN1基因shRNA真核表达质粒及其阴性对照质粒构建正确,转染HEK293细胞后,3个shRNA真核表达质粒在mRNA水平对细胞MKRN1基因的抑制率分别为52.4%、26.2%和42.9%,在蛋白水平对细胞MKRN1基因的抑制率分别为69.1%、27.9%和50.0%。结论已成功构建了人MKRN1基因的shRNA真核表达质粒,为进一步研究MKRN1基因的功能及其与人端粒酶逆转录酶的关系奠定了基础。     

Exosomes and Microvesicles: Identification and Targeting By Particle Size and Lipid Chemical Probes

Exosomes and microvesicles are two classes of submicroscopic vesicle released by cells into the extracellular space. Collectively referred to as extracellular vesicles, these membrane containers facilitate important cell–cell communication by carrying a diverse array of signaling molecules, including nucleic acids, proteins, and lipids. Recently, the role of extracellular vesicle signaling in cancer progression has become a topic of significant interest. Methods to detect and target exosomes and microvesicles are needed to realize applications of extracellular vesicles as biomarkers and, perhaps, therapeutic targets. Detection of exosomes and microvesicles is a complex problem as they are both submicroscopic and of heterogeneous cellular origins. In this Minireview, we highlight the basic biology of extracellular vesicles, and address available biochemical and biophysical detection methods. Detectible characteristics described here include lipid and protein composition, and physical properties such as the vesicle membrane shape and diffusion coefficient. In particular, we propose that detection of exosome and microvesicle membrane curvature with lipid chemical probes that sense membrane shape is a distinctly promising method for identifying and targeting these vesicles.     

油田地面工程施工管理中存在的问题及针对性处理策略分析

随着经济的发展,能源的消耗也不断增加。粗放型的生产方式使我国能源陷入枯竭状态,其中与石油相关的能源也不例外。石油能源的消耗逐渐增加,提高油田开采及地面管理质量,能够节约石油能源的消耗。同时高质量的油田开采管理,可促进我国石油开采行业的发展,同时以石油为依托的行业能够同时得到完善和发展。     

Structure-Guided Engineering of a Family IV Cold-Adapted Esterase Expands Its Substrate Range

Cold active esterases have gained great interest in several industries. The recently determined structure of a family IV cold active esterase (EstN7) from Bacillus cohnii strain N1 was used to expand its substrate range and to probe its commercially valuable substrates. Database mining suggested that triacetin was a potential commercially valuable substrate for EstN7, which was subsequently proved experimentally with the final product being a single isomeric product, 1,2-glyceryl diacetate. Enzyme kinetics revealed that EstN7’s activity is restricted to C2 and C4 substrates due to a plug at the end of the acyl binding pocket that blocks access to a buried water-filled cavity. Residues M187, N211 and W206 were identified as key plug forming residues. N211A stabilised EstN7 allowing incorporation of the destabilising M187A mutation. The M187A-N211A double mutant had the broadest substrate range, capable of hydrolysing a C8 substrate. W206A did not appear to have any significant effect on substrate range either alone or when combined with the double mutant. Thus, the enzyme kinetics and engineering together with a recently determined structure of EstN7 provide new insights into substrate specificity and the role of acyl binding pocket plug residues in determining family IV esterase stability and substrate range.     

化工设计中安全危险识别及控制

经济的快速发展,使得化工行业变成我国工业行业组成的重要部分,然而,由于化工行业的原料以及内容具备一定的危险性,所以,化工设计中安全危险识别及控制就显得尤为重要。     

Rational Engineering of Hydratase from Lactobacillus acidophilus Reveals Critical Residues Directing Substrate Specificity and Regioselectivity

Enzymatic conversion of fatty acids (FAs) by fatty acid hydratases (FAHs) presents a green and efficient route for high-value hydroxy fatty acid (HFA) production. However, limited diversity was achieved among HFAs, to date, with respect to chain length and hydroxy position. In this study, two highly similar FAHs from Lactobacillus acidophilus were compared: FA-HY2 has a narrow substrate scope and strict regioselectivity, whereas FA-HY1 utilizes longer chain substrates and hydrates various double-bond positions. It is revealed that three active-site residues play a remarkable role in directing substrate specificity and regioselectivity of hydration. If these residues on FA-HY2 are mutated to the corresponding ones in FA-HY1, a significant expansion of substrate scope and a distinct enhancement in hydration of double bonds towards the ω-end of FAs is observed. A three-residue mutant of FA-HY2 (TM-FA-HY2) displayed an impressive reversal of regioselectivity towards linoleic acid, shifting the ratio of the HFA regioisomers (10-OH/13-OH) from 99:1 to 12:88. Notable changes in regioselectivity were also observed for arachidonic acid and for C₁₈ polyunsaturated fatty acid substrates. In addition, TM-FA-HY2 converted eicosapentaenoic acid into its 12-hydroxy product with high conversion at the preparative scale. Furthermore, it is demonstrated that microalgae are a source of diverse FAs for HFA production. This study paves the way for tailor-made FAH design to enable the production of diverse HFAs for various applications from the polymer industry to medical fields.     

CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering**

CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure–function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.     

Expanding Substrate Promiscuity by Engineering a Novel Adenylating‐Methylating NRPS Bifunctional Enzyme

Nonribosomal peptides synthetases (NRPSs), which are multifunctional mega‐enzymes producing many biologically active metabolites, are ideal targets for enzyme engineering. NRPS adenylation domains play a critical role in selecting/activating the amino acids to be transferred to downstream NRPS domains in the biosynthesis of natural products. Both monofunctional and bifunctional A domains interrupted with an auxiliary domain are found in nature. Here, we show that a bifunctional interrupted A domain can be uninterrupted by deleting its methyltransferase auxiliary domain portion to make an active monofunctional enzyme. We also demonstrate that a portion of an auxiliary domain with almost no sequence identity to the original auxiliary domain can be insert into naturally interrupted A domain to develop a new active bifunctional A domain with increased substrate profile. This work shows promise for the creation of new interrupted A domains in engineered NRPS enzymes.     

Identification of B-Cell Epitopes for Eliciting Neutralizing Antibodies against the SARS-CoV-2 Spike Protein through Bioinformatics and Monoclonal Antibody Targeting

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global public health crisis. Effective COVID-19 vaccines developed by Pfizer-BioNTech, Moderna, and Astra Zeneca have made significant impacts in controlling the COVID-19 burden, especially in reducing the transmission of SARS-CoV-2 and hospitalization incidences. In view of the emergence of new SARS-CoV-2 variants, vaccines developed against the Wuhan strain were less effective against the variants. Neutralizing antibodies produced by B cells are a critical component of adaptive immunity, particularly in neutralizing viruses by blocking virus attachment and entry into cells. Therefore, the identification of protective linear B-cell epitopes can guide epitope-based peptide designs. This study reviews the identification of SARS-CoV-2 B-cell epitopes within the spike, membrane and nucleocapsid proteins that can be incorporated as potent B-cell epitopes into peptide vaccine constructs. The bioinformatic approach offers a new in silico strategy for the mapping and identification of potential B-cell epitopes and, upon in vivo validation, would be useful for the rapid development of effective multi-epitope-based vaccines. Potent B-cell epitopes were identified from the analysis of three-dimensional structures of monoclonal antibodies in a complex with SARS-CoV-2 from literature mining. This review provides significant insights into the elicitation of potential neutralizing antibodies by potent B-cell epitopes, which could advance the development of multi-epitope peptide vaccines against SARS-CoV-2.