空肠弯曲菌蛋白质组学的研究进展

空肠弯曲菌病在世界各地均有发生,主要导致人和动物的细菌性胃肠炎及急性腹泻等。通过细菌蛋白质组学技术研究该类病原菌应对环境变化,尤其是应对宿主的反应机制,以及重要功能蛋白的亚细胞定位等,能够在蛋白水平深入研究弯曲菌病的致病机理。本文就空肠弯曲菌蛋白组学的研究进展作一综述。     

Synthesis and Biological Evaluation of Dithiobisacetamides as Novel Urease Inhibitors

Thirty-eight disulfides containing N-arylacetamide were designed and synthesized in an effort to develop novel urease inhibitors. Biological evaluation revealed that some of the synthetic compounds exhibited strong inhibitory potency against both cell-free urease and urease in intact cell with low cytotoxicity to mammalian cells even at concentration up to 250 μM. Of note, 2,2′-dithiobis(N-(2-fluorophenyl)acetamide) ( d7 ), 2,2′-dithiobis(N-(3,5-difluorophenyl)acetamide) ( d24 ), and 2,2′-dithiobis(N-(3-fluorophenyl)acetamide) ( d8 ) were here identified as the most active inhibitors with IC₅₀ of 0.074, 0.44, and 0.81 μM, showing 32- to 355-fold higher potency than the positive control acetohydroxamic acid. These disulfides were confirmed to bind urease without covalent modification of the cysteine residue and to inhibit urease reversibly with a mixed inhibition mechanism. They also showed very good anti-Helicobacter pylori activities with d8 showing a comparable potency to the clinical used drug amoxicillin. The impressive in vitro biological profile indicated their immense potential as therapeutic agents to tackle H. pylori caused infections.     

An Updated Model of the Divisome: Regulation of the Septal Peptidoglycan Synthesis Machinery by the Divisome

The synthesis of a peptidoglycan septum is a fundamental part of bacterial fission and is driven by a multiprotein dynamic complex called the divisome. FtsW and FtsI are essential proteins that synthesize the peptidoglycan septum and are controlled by the regulatory FtsBLQ subcomplex and the activator FtsN. However, their mode of regulation has not yet been uncovered in detail. Understanding this process in detail may enable the development of new compounds to combat the rise in antibiotic resistance. In this review, recent data on the regulation of septal peptidoglycan synthesis is summarized and discussed. Based on structural models and the collected data, multiple putative interactions within FtsWI and with regulators are uncovered. This elaborates on and supports an earlier proposed model that describes active and inactive conformations of the septal peptidoglycan synthesis complex that are stabilized by these interactions. Furthermore, a new model on the spatial organization of the newly synthesized peptidoglycan and the synthesis complex is presented. Overall, the updated model proposes a balance between several allosteric interactions that determine the state of septal peptidoglycan synthesis.     

常温酸性除锈液中返诱仰制剂研究

本文研究了常规缓蚀剂对返锈的抑制能力,发现酰胺类化合物(N1/2)具有较高的返锈抑制作用,同时具有与乌洛托品相当的缓蚀性,并且,当二者复合时具有协同效应.     

Discovery of SARS-CoV-2 3CLPro Peptidomimetic Inhibitors through the Catalytic Dyad Histidine-Specific Protein–Ligand Interactions

As the etiological agent for the coronavirus disease 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenges the ongoing efforts of vaccine development and drug design. Due to the accumulating cases of breakthrough infections, there are urgent needs for broad-spectrum antiviral medicines. Here, we designed and examined five new tetrapeptidomimetic anti-SARS-CoV-2 inhibitors targeting the 3C-Like protease (3CL^Pro), which is highly conserved among coronaviruses and essential for viral replications. We significantly improved the efficacy of a ketoamide lead compound based on high-resolution co-crystal structures, all-atom simulations, and binding energy calculations. The inhibitors successfully engaged the catalytic dyad histidine residue (H41) of 3CLPro as designed, and they exhibited nanomolar inhibitory capacity as well as mitigated the viral loads of SARS-CoV-2 in cellular assays. As a widely applicable design principle, our results revealed that the potencies of 3CL^Pro-specific drug candidates were determined by the interplay between 3CL^Pro H41 residue and the peptidomimetic inhibitors.     

MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of Escherichia coli and Pseudomonas aeruginosa

One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans.     

Discovery of Covalent Bruton's Tyrosine Kinase Inhibitors with Decreased CYP2C8 Inhibitory Activity

Bruton's tyrosine kinase (BTK) is a member of the Tec kinase family that is expressed in cells of hematopoietic lineage. Evidence has shown that inhibition of BTK has clinical benefit for the treatment of a wide array of autoimmune and inflammatory diseases. Previously we reported the discovery of a novel nicotinamide selectivity pocket (SP) series of potent and selective covalent irreversible BTK inhibitors. The top molecule 1 of that series strongly inhibited CYP2C8 (IC₅₀=100 nM), which was attributed to the bridged linker group. However, our effort on the linker replacement turned out to be fruitless. With the study of the X-ray crystal structure of compound 1 , we envisioned the opportunity of removal of this liability via transposition of the linker moiety in 1 from C6 to C5 position of the pyridine core. With this strategy, our optimization led to the discovery of a novel series, in which the top molecule 18 A displayed reduced CYP inhibitory activity and good potency. To further explore this new series, different warheads besides acrylamide, for example cyanamide, were also tested. However, this effort didn't lead to the discovery of molecules with better potency than 18 A . The loss of potency in those molecules could be related to the reduced reactivity of the warhead or reversible binding mode. Further profiling of 18 A disclosed that it had a strong hERG (human Ether-a-go-go Related Gene) inhibition, which could be related to the phenoxyphenyl group.     

A Review of the Advantages,Disadvantages and Limitations of Chemotaxis Assays for Campylobacter spp.

Reproducible qualitative and quantitative assessment of bacterial chemotactic motility, particularly in response to chemorepellent effectors, is experimentally challenging. Here we compare several established chemotaxis assays currently used to investigate Campylobacter jejuni chemotaxis, with the aim of improving the correlation between different studies and establishing the best practices. We compare the methodologies of capillary, agar, and chamber-based assays, and discuss critical technical points, in terms of reproducibility, accuracy, and the advantages and limitations of each.     

A Quick Route to Multiple Highly Potent SARS-CoV-2 Main Protease Inhibitors**

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2M^Pro) to digest two of its translated long polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replicating in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1M^Pro), we have designed and synthesized a series of SC2M^Pro inhibitors that contain β-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2M^Pro active-site cysteine C145. All inhibitors display high potency with K_i values at or below 100 nM. The most potent compound, MPI3, has as a K_i value of 8.3 nM. Crystallographic analyses of SC2M^Pro bound to seven inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549/ACE2 cells. Two inhibitors, MPI5 and MPI8, completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5–5 μM and A549/ACE2 cells at 0.16–0.31 μM. Their virus inhibition potency is much higher than that of some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2M^Pro inhibitors with ultra-high antiviral potency.     

Crystal Structure of the Recombination Mediator Protein RecO from Campylobacter jejuni and Its Interaction with DNA and a Zinc Ion

Homologous recombination is involved in repairing DNA damage, contributing to maintaining the integrity and stability of viral and cellular genomes. In bacteria, the recombination mediator proteins RecO and RecR are required to load the RecA recombinase on ssDNA for homologous recombination. To structurally and functionally characterize RecO, we determined the crystal structure of RecO from Campylobacter jejuni (cjRecO) at a 1.8 Å resolution and biochemically assessed its capacity to interact with DNA and a metal ion. cjRecO folds into a curved rod-like structure that consists of an N-terminal domain (NTD), C-terminal domain (CTD), and Zn²⁺-binding domain (ZnD). The ZnD at the end of the rod-like structure coordinates three cysteine residues and one histidine residue to accommodate a Zn²⁺ ion. Based on an extensive comparative analysis of RecO structures and sequences, we propose that the Zn²⁺-binding consensus sequence of RecO is CxxC…C/HxxC/H/D. The interaction with Zn²⁺ is indispensable for the protein stability of cjRecO but does not seem to be required for the recombination mediator function. cjRecO also interacts with ssDNA as part of its biological function, potentially using the positively charged patch in the NTD and CTD. However, cjRecO displays a low ssDNA-binding affinity, suggesting that cjRecO requires RecR to efficiently recognize ssDNA for homologous recombination.     

Thiol-ene Reaction as Reversible Covalent Bond for the Design of Shape-Memory Polymers

Besides a stable phase, shape-memory polymers require an additional switchable moiety. In addition to thermal transitions and supramolecular interactions, these units can also be based on covalent bonds. Herein, the use of the reversible thiol-ene reaction as reversible cross-linker for the design of shape-memory polymers is demonstrated. A facile route to polymer networks with a thiol-ene acceptor and a comonomer (butyl methacrylate or 2-ethylhexyl methacrylate) cross-linked by dithiols is introduced. The thermal and mechanical properties of the resulting polymers are characterized in detail. Hereby, the polymers feature excellent shape-memory behavior with fixity and recovery rates above 90%. This study shows that the thiol-ene cross-linker can function as both, the stable and the switchable structural moiety rendering the usage of a covalent cross-linker unnecessary. This partial reversibility can also be proven by temperature-depending Raman spectroscopy.     

醚基衣康酸多元共聚物阻垢剂合成条件研究

以衣康酸、丙烯磺酸钠、α-烯丙基甘油醚、次亚磷酸钠为单体,采用水溶性的自由基反应进行共聚合,制备含羧基、醚基、磺酸基、膦酸基的共聚物。考察了单体配比、引发剂用量、加料方式、反应时间和反应温度等因素对单体聚合和聚合物性能的影响,确定了聚合物阻垢剂的最佳合成条件。     

高效铝换热器酸洗缓蚀剂的研制

以钼酸钠、硫脲、吖啶、六次甲基四胺、硫酸锌、表面活性剂等合成了铝翅片换热器酸洗缓蚀剂,并用腐蚀失重法进行了评价.在8%盐酸介质中,钼酸钠:硫脲:吖啶:六次甲基四胺:硫酸锌:表面活性剂=0.6:1.0:0.2:0.8:0.5:0.05,40℃时,铝缓蚀率可达97%以上.     

Structural analysis of the capsular polysaccharide from Campylobacter jejuni RM1221

The complete genome of Campylobacter jejuni strain RM1221 (Penner serotype HS:53) was reported recently and contains a novel capsular polysaccharide (CPS) biosynthesis locus. Cell-surface carbohydrates such as CPS are known to be important for bacterial survival and often contribute to pathogenesis. In this study, we describe the complete structure of the CPS of C. jejuni RM1221, which was determined by using NMR spectroscopy, MS, and chemical methods. The CPS contains 6-deoxy-D-manno-heptose and D-threo-pent-2-ulose (D-xylulose), two monosaccharides that are rarely found in bacterial polysaccharides. The CPS has a regular structure of a linear main chain of trisaccharide repeating units, composed of two alpha- and one beta-6-deoxy-D-manno-heptopyranose residues, which are linked through a phosphodiester linkage. Branching residues of xylulose are incorporated nonstoichiometrically: each trisaccharide repeating unit of the main chain bears no, one, or two xylulose residues. The xylulose glycosidic linkages are extremely acid labile, and it is not clear how they can be preserved under the acidic conditions of the gastrointestinal tract, where Campylobacter resides during infection. We have also shown that the CPS biosynthesis genes of C. jejuni RM1221 are conserved in other C. jejuni strains of the Penner serotype HS:53, including serotype HS:53 reference strain RM3435.     

红外光谱法测热可逆共价键交联氯醇橡胶的组成

以交联单体双环戊二烯二甲双缩水甘油酯（ＤＧＤＣＡ）的特征峰（１７１２ｃｍ＾－１）和环氧氯丙烷均聚物（ＰＥＣＨ）的特征峰（７５０ｃｍ＾－１）为定量计算的基准峰,测定不同质量比的ＤＧＤＣＡ与ＰＥＣＨ混合物ＩＲ谱图。混合物组成与光密度比值（Ｄ１７１２／Ｄ７５０）关系的回归方程为：（Ｄ１７１２／Ｄ７５０＝０．０２０９＋６．６４ＷＤＧＤＣＷＥＣＨ）,进而建立了通过Ｄ１７１２／Ｄ７５０求取共聚物中ＤＧＤＣＡ结     

Deciphering the Nature of Enzymatic Modifications of Bacterial Cell Walls

The major constituent of bacterial cell walls is peptidoglycan, which, in its crosslinked form, is a polymer of considerable complexity that encases the entire bacterium. A functional cell wall is indispensable for survival of the organism. There are several dozen enzymes that assemble and disassemble the peptidoglycan dynamically within each bacterial generation. Understanding of the nature of these transformations is critical knowledge for these events. Octasaccharide peptidoglycans were prepared and studied with seven recombinant cell‐wall‐active enzymes (SltB1, MltB, RlpA, mutanolysin, AmpDh2, AmpDh3, and PBP5). With the use of highly sensitive mass spectrometry methods, we described the breadth of reactions that these enzymes catalyzed with peptidoglycan and shed light on the nature of the cell wall alteration performed by these enzymes. The enzymes exhibit broadly distinct preferences for their substrate peptidoglycans in the reactions that they catalyze.     

Extraction of Peptidoglycan from L. paracasei subp. Paracasei X12 and Its Preliminary Mechanisms of Inducing Immunogenic Cell Death in HT-29 Cells

L. paracasei subp. paracaseiX12 was previously isolated from a Chinese traditional fermented cheese with anticancer activities and probiotic potential. Herein, the integral peptidoglycan (X12-PG) was extracted by a modified trichloroacetic acid (TCA) method. X12-PG contained the four representative amino acids Asp, Glu, Ala and Lys, and displayed the similar lysozyme sensitivity, UV-visible scanning spectrum and molecular weight as the peptidoglycan standard. X12-PG could induce the production of apoptotic bodies observed by transmission electron microscopy (TEM). X12-PG could significantly induced the translocation of calreticulin (CRT) and the release of high mobility group box 1 protein (HMGB1), the two notable hallmarks of immunogenic cell death (ICD), with the endoplastic reticulum (ER) damaged and subsequently intracellular [Ca²⁺] elevated. Our findings implied that X12-PG could induce the ICD of HT-29 cells through targeting at the ER. The present results may enlighten the prospect of probiotics in the prevention of colon cancer.