Machine Learning Models Identify Inhibitors of New Delhi Metallo-ß-lactamase.

The worldwide spread of the metallo-ß-lactamases (MBL), especially New Delhi metallo-ß-lactamase-1 (NDM-1), is threatening the efficacy of ß-lactams, which are the most potent and prescribed class of antibiotics in the clinic. Currently, FDA-approved MBL inhibitors are lacking in the clinic even though many strategies have been used in inhibitor development, including quantitative high-throughput screening (qHTS), fragment-based drug discovery (FBDD), and molecular docking. Herein, a machine learning-based prediction tool is described, which was generated using results from HTS of a large chemical library and previously published inhibition data. The prediction tool was then used for virtual screening of the NIH Genesis library, which was subsequently screened using qHTS. A novel MBL inhibitor was identified and shown to lower minimum inhibitory concentrations (MICs) of Meropenem for a panel of E. coli and K. pneumoniae clinical isolates expressing NDM-1. The mechanism of inhibition of this novel scaffold was probed utilizing equilibrium dialyses with metal analyses, native state electrospray ionization mass spectrometry, UV-vis spectrophotometry, and molecular docking. The uncovered inhibitor, compound 72922413, was shown to be 9-hydroxy-3-[(5-hydroxy-1-oxa-9-azaspiro[5.5]undec-9-yl)carbonyl]-4H-pyrido[1,2-a]pyrimidin-4-one.

[The clinical significance of lateral pelvic sentinel lymph node biopsy using indocyanine green fluorescence navigation in laparoscopic lateral pelvic lymph node dissection].

Objectives: This study aims to explore the clinical significance of lateral pelvic sentinel lymph node biopsy (SLNB) using indocyanine green (ICG) fluorescence navigation in laparoscopic lateral pelvic lymph node dissection (LLND) and evaluate the accuracy and feasibility of this technique to predict the status of lateral pelvic lymph nodes (LPLNs). Methods: The clinical and pathological characteristics, surgical outcomes, lymph node findings and perioperative complications of 16 rectal cancer patients who underwent SLNB using ICG fluorescence navigation in laparoscopic LLND in the Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College during April 2017 and October 2022 were retrospectively collected and analyzed. The patients did not receive preoperative neoadjuvant radiotherapy and presented with LPLNs but without LPLN enlargement (MRI showed the maximum short axes of the LPLNs were ≥5 mm and <10 mm at first visit). Results: All 16 patients were successfully performed SLNB using ICG fluorescence navigation in laparoscopic LLND. Three patients underwent bilateral LLND and 13 patients underwent unilateral LLND. The lateral pelvic sentinel lymph nodes (SLNs) were clearly fluorescent before dissection in 14 patients and the detection rate of SLNs for these patients was 87.5%. Lateral pelvic SLN metastasis was diagnosed in 2 patients and negative results were found in 12 patients by frozen pathological examinations. Among the 14 patients in whom lateral pelvic SLNs were detected, the dissected lateral pelvic non-SLNs were all negative. All dissected LPLNs were negative in two patients without fluorescent lateral pelvic SLNs. The specificity, sensitivity, negative predictive value, and accuracy was 85.7%, 100%, 100%, and 100%, respectively. Conclusions: This study indicates that lateral pelvic SLNB using ICG fluorescence navigation shows promise as a safe and feasible procedure with good accuracy. This technique may replace preventive LLND for locally advanced lower rectal cancer.

A spectral CT-based nomogram for predicting the response to induction chemotherapy in nasopharyngeal carcinoma.

AIM: To establish a spectral computed tomography (CT)-based nomogram for predicting the response to induction chemotherapy (ICT) in nasopharyngeal carcinoma (NPC). MATERIALS AND METHODS: Fifty-four patients with NPC who underwent spectral CT examination before ICT were enrolled prospectively. Patients were assigned to response and non-response groups according to response evaluation. The predictive indicators were spectral CT parameters of venous phase, including iodine concentration (IC), normalised IC (NIC), slope of the spectral attenuation curve in Hounsfield units (λHU), effective atomic number (Eff-Z), and water concentration. Multivariate logistic regression was used to construct a predictive model. The receiver operating characteristic (ROC) and calibration curves were used to evaluate the robustness of model, while the bootstrap method was used for internal validation. The Hosmer-Lemeshow test was used to test the goodness of fit of the discriminant model. RESULTS: Multivariate logistic regression analysis showed that NIC, λHU, and Eff-Z were the potential predictors, and the three indicators were further used to establish a predictive model. The nomogram was evaluated to have good predictive performance, the area under the ROC curve was 0.909 (95% confidence interval [CI]: 0.799-0.970), and the model was well calibrated (χ2 = 8.149, p=0.419). CONCLUSIONS: The spectral CT nomogram has potential clinical value in predicting response to ICT in NPC and may help guide individualised treatment decisions.

[Application and prospect of cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy in the treatment of peritoneal metastasis from colorectal cancer].

Colorectal cancer is one of the common malignant tumors in China, and its incidence is increasing with years. As the second most common metastatic site of colorectal cancer, peritoneum is difficult to diagnose early and with a poor prognosis. Systemic intravenous chemotherapy was used as the main treatment strategy for peritoneal metastasis in the past, but its systemic toxic and side effects were obvious, and it could not effectively control tumor progression. In recent years, the continuous development of surgical techniques, concepts, and equipment, as well as the introduction of new chemotherapy drugs and targeted drugs have significantly improved the quality of life and prognosis of patients with peritoneal metastasis of colorectal cancer. Cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC) can effectively eradicated the intraperitoneal free cancer cells and subclinical lesions, while reducing systemic side effects of chemotherapy drugs, and achieve the radical cure of the tumor at the macro and micro levels to the greatest extent. It has been used as the first-line treatment program for peritoneal metastasis of colorectal cancer at home and abroad. This article focuses on the analysis and summary of the survival efficacy, prognostic factor analysis, and chemotherapy safety of CRS+ HIPEC in the treatment of colorectal cancer peritoneal metastasis. The existing problems and controversies of HIPEC therapy are discussed simultaneously.

Visualizing the Dynamic Metalation State of New Delhi Metallo-ß-lactamase-1 in Bacteria Using a Reversible Fluorescent Probe.

New Delhi metallo-ß-lactamase (NDM) grants resistance to a broad spectrum of ß-lactam antibiotics, including last-resort carbapenems, and is emerging as a global antibiotic resistance threat. Limited zinc availability adversely impacts the ability of NDM-1 to provide resistance, but a number of clinical variants have emerged that are more resistant to zinc scarcity (e.g., NDM-15). To provide a novel tool to better study metal ion sequestration in host-pathogen interactions, we describe the development of a fluorescent probe that reports on the dynamic metalation state of NDM within Escherichia coli. The thiol-containing probe selectively coordinates the dizinc metal cluster of NDM and results in a 17-fold increase in fluorescence intensity. Reversible binding enables competition and time-dependent studies that reveal fluorescence changes used to detect enzyme localization, substrate and inhibitor engagement, and changes to metalation state through the imaging of live E. coli using confocal microscopy. NDM-1 is shown to be susceptible to demetalation by intracellular and extracellular metal chelators in a live-cell model of zinc dyshomeostasis, whereas the NDM-15 metalation state is shown to be more resistant to zinc flux. The development of this reversible turn-on fluorescent probe for the metalation state of NDM provides a new tool for monitoring the impact of metal ion sequestration by host defense mechanisms and for detecting inhibitor-target engagement during the development of therapeutics to counter this resistance determinant.

Carbapenem Use Is Driving the Evolution of Imipenemase 1 Variants.

Metallo-ß-lactamases (MBLs) are a growing clinical threat because they inactivate nearly all ß-lactam-containing antibiotics, and there are no clinically available inhibitors. A significant number of variants have already emerged for each MBL subfamily. To understand the evolution of imipenemase (IMP) genes (blaIMP) and their clinical impact, 20 clinically derived IMP-1 like variants were obtained using site-directed mutagenesis and expressed in a uniform genetic background in Escherichia coli strain DH10B. Strains of IMP-1-like variants harboring S262G or V67F substitutions exhibited increased resistance toward carbapenems and decreased resistance toward ampicillin. Strains expressing IMP-78 (S262G/V67F) exhibited the largest changes in MIC values compared to IMP-1. In order to understand the molecular mechanisms of increased resistance, biochemical, biophysical, and molecular modeling studies were conducted to compare IMP-1, IMP-6 (S262G), IMP-10 (V67F), and IMP-78 (S262G/V67F). Finally, unlike most New Delhi metallo-ß-lactamase (NDM) and Verona integron-encoded metallo-ß-lactamase (VIM) variants, the IMP-1-like variants do not confer any additional survival advantage if zinc availability is limited. Therefore, the evolution of MBL subfamilies (i.e., IMP-6, -10, and -78) appears to be driven by different selective pressures.

A Lysine-Targeted Affinity Label for Serine-ß-Lactamase Also Covalently Modifies New Delhi Metallo-ß-lactamase-1 (NDM-1).

The divergent sequences, protein structures, and catalytic mechanisms of serine- and metallo-ß-lactamases hamper the development of wide-spectrum ß-lactamase inhibitors that can block both types of enzymes. The O-aryloxycarbonyl hydroxamate inactivators of Enterobacter cloacae P99 class C serine-ß-lactamase are unusual covalent inhibitors in that they target both active-site Ser and Lys residues, resulting in a cross-link consisting of only two atoms. Many clinically relevant metallo-ß-lactamases have an analogous active-site Lys residue used to bind ß-lactam substrates, suggesting a common site to target with covalent inhibitors. Here, we demonstrate that an O-aryloxycarbonyl hydroxamate inactivator of serine-ß-lactamases can also serve as a classical affinity label for New Delhi metallo-ß-lactamase-1 (NDM-1). Rapid dilution assays, site-directed mutagenesis, and global kinetic fitting are used to map covalent modification at Lys211 and determine KI (140 µM) and kinact (0.045 min-1) values. Mass spectrometry of the intact protein and the use of ultraviolet photodissociation for extensive fragmentation confirm stoichiometric covalent labeling that occurs specifically at Lys211. A 2.0 Å resolution X-ray crystal structure of inactivated NDM-1 reveals that the covalent adduct is bound at the substrate-binding site but is not directly coordinated to the active-site zinc cluster. These results indicate that Lys-targeted affinity labels might be a successful strategy for developing compounds that can inactivate both serine- and metallo-ß-lactamases.

Evolution of New Delhi metallo-ß-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity.

Infections by carbapenem-resistant Enterobacteriaceae are difficult to manage owing to broad antibiotic resistance profiles and because of the inability of clinically used ß-lactamase inhibitors to counter the activity of metallo-ß-lactamases often harbored by these pathogens. Of particular importance is New Delhi metallo-ß-lactamase (NDM), which requires a di-nuclear zinc ion cluster for catalytic activity. Here, we compare the structures and functions of clinical NDM variants 1-17. The impact of NDM variants on structure is probed by comparing melting temperature and refolding efficiency and also by spectroscopy (UV-visible, 1H NMR, and EPR) of di-cobalt metalloforms. The impact of NDM variants on function is probed by determining the minimum inhibitory concentrations of various antibiotics, pre-steady-state and steady-state kinetics, inhibitor binding, and zinc dependence of resistance and activity. We observed only minor differences among the fully loaded di-zinc enzymes, but most NDM variants had more distinguishable selective advantages in experiments that mimicked zinc scarcity imposed by typical host defenses. Most NDM variants exhibited improved thermostability (up to ∼10 °C increased Tm ) and improved zinc affinity (up to ∼10-fold decreased Kd, Zn2). We also provide first evidence that some NDM variants have evolved the ability to function as mono-zinc enzymes with high catalytic efficiency (NDM-15, ampicillin: kcat/Km = 5 × 106 m-1 s-1). These findings reveal the molecular mechanisms that NDM variants have evolved to overcome the combined selective pressures of ß-lactam antibiotics and zinc deprivation.

Novel small molecule IL-6 inhibitor suppresses autoreactive Th17 development and promotes Treg development.

Multiple sclerosis (MS) is the leading cause of non-traumatic neurological disability in the United States in young adults, but current treatments are only partially effective, making it necessary to develop new, innovative therapeutic strategies. Myelin-specific interleukin (IL)-17-producing T helper type 17 (Th17) cells are a major subset of CD4 T effector cells (Teff ) that play a critical role in mediating the development and progression of MS and its mouse model, experimental autoimmune encephalomyelitis (EAE), while regulatory T cells (Treg ) CD4 T cells are beneficial for suppressing disease. The IL-6/signal transducer and activator of transcription 3 (STAT-3) signaling pathway is a key regulator of Th17 and Treg cells by promoting Th17 development and suppressing Treg development. Here we show that three novel small molecule IL-6 inhibitors, madindoline-5 (MDL-5), MDL-16 and MDL-101, significantly suppress IL-17 production in myelin-specific CD4 T cells in a dose-dependent manner in vitro. MDL-101 showed superior potency in suppressing IL-17 production compared to MDL-5 and MDL-16. Treatment of myelin-specific CD4 T cells with MDL-101 in vitro reduced their encephalitogenic potential following their subsequent adoptive transfer. Furthermore, MDL-101 significantly suppressed proliferation and IL-17 production of anti-CD3-activated effector/memory CD45RO+ CD4+ human CD4 T cells and promoted human Treg development. Together, these data demonstrate that these novel small molecule IL-6 inhibitors have the potential to shift the Teff  : Treg balance, which may provide a novel therapeutic strategy for ameliorating disease progression in MS.

[Analysis of 17 cases underwent laparoscopic rectal cancer surgery with transanal natural orifice specimen extraction and resection].

Objective: To study the feasibility, safety and short-term efficacy of total laparoscopic rectal cancer surgery with transanal natural orifice specimen extraction and resection. Methods: From May 2014 to March 2016, 17 patients with rectal carcinoma were treated by total laparoscopic rectal cancer surgery with transanal natural orifice specimen extraction and resection. The clinical data of these patients was collected and retrospectively analyzed to assess the impact of the operation on postoperative recovery time and the incidence of complications. Results: All operations had been successfully accomplished without conversion to open surgery or conversional laparoscopic-assisted surgery . The median operative time was 105 minutes. The median blood loss was 35 ml. The median proximal and distal margin of tumor is 16 cm and 3.5 cm. The median number of lymph nodes harvest is 21, and the median first bowl movement is 43 hours. The hospitalization after operation is 8 days. No patient underwent abdomen hemorrhage or anastomotic leakage. Conclusion: Laparoscopic rectal cancer surgery with transanal natural orifice specimen extraction and resection appears to be feasible, safe and with promising efficacy for selected patients.

Structure of a bacterial type IV secretion core complex at subnanometre resolution.

Type IV secretion (T4S) systems are able to transport DNAs and/or proteins through the membranes of bacteria. They form large multiprotein complexes consisting of 12 proteins termed VirB1-11 and VirD4. VirB7, 9 and 10 assemble into a 1.07 MegaDalton membrane-spanning core complex (CC), around which all other components assemble. This complex is made of two parts, the O-layer inserted in the outer membrane and the I-layer inserted in the inner membrane. While the structure of the O-layer has been solved by X-ray crystallography, there is no detailed structural information on the I-layer. Using high-resolution cryo-electron microscopy and molecular modelling combined with biochemical approaches, we determined the I-layer structure and located its various components in the electron density. Our results provide new structural insights on the CC, from which the essential features of T4S system mechanisms can be derived.

[Prognosis factors for the perineal incision complications after abdominoperineal resection for low rectal cancer].

Objective: To evaluate the prognosis factors affecting perineal incision complications after abdominoperineal resection (APR) for the low rectal cancer. Methods: This was a retrospective analysis of 151 consecutive patients with low rectal cancer undergoing APR between January and December 2013 at Department of Colorectal Surgery, Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College. The patients were comprised of 95 males and 56 females. The mean age of the patients was (57.3±10.9) years (ranging from 31 to 79 years). χ(2) test and Logistic regression analysis were used to identify the prognosis factors of perineum incision complications. Results: In all 151 patients, perineal incision complications were confirmed in 31 patients (20.5%), including 8 cases of incision infection, 22 cases of poor healing of perineal wound, and 1 case of incision fistula formation. In univariate analysis, the factors associated with perineal incision complications were American Society of Anesthesiologists grade (χ(2)=7.116, P=0.008), intraoperative blood loss (χ(2)=9.157, P=0.002), while the protective factors associate with perineal incision complications were the intraperitoneal chemotherapy with fluorouracil sustained release (χ(2)=5.020, P=0.025), pelvic restoration (χ(2)=10.158, P=0.001), operation experience (χ(2)=7.334, P=0.007). The gender, age, body mass index, diabetes, preoperative radiochemtherapy, hemoglobin level, albumin level, distance from distal tumor to anal verge, the procedure of APR, operating time, intraoperative blood transfusion, total drainage volume 3 days after operation, tumor differentiation and the postoperative TNM staging were not associated with perineal incision complications (P>0.05). Multivariable logistic regression analysis showed that the intraoperative pelvic restoration (OR=0.200, 95% CI: 0.045 to 0.894, P=0.035) and intraoperative blood loss (OR=2.953, 95% CI: 1.155 to 7.551, P=0.024) were independent prognosis factors of perineum incision complications. Conclusions: For patients with low rectal cancer undergoing APR procedure, pelvic restoration wound be needed. The operation should be performed by experienced doctors, intraoperative blood loss should be reduced when possible.

Effects and mechanism of Tripterygium wilfordii on chronic glomerulo nephritis.

The objective of this study was to investigate the clinical effects of Tripterygium wilfordii on chronic glomerulo nephritis (CGN) and its mechanisms. Eighty-two cases of CGN treated in our hospital were randomly divided into observation and control groups. The control group was treated with conventional western medicine, and the observation group was treated with conventional western medicine and orally-administered T. wilfordii pills for three courses of treatment, each consisting of 4 weeks. Changes in serum reatinine, blood urea nitrogen, blood total cholesterol, blood albumin, and 24-h urine protein were observed. The levels of peripheral tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were determined with enzyme-linked immunosorbent assay. The curative effects of both groups were evaluated respectively. Both groups had significantly improved serum creatinine, blood urea nitrogen, blood total cholesterol, blood albumin, and 24-h urine protein (P < 0.05), and the observation group exhibited a more significant improvement (P < 0.05). TNF-α and IL-6 levels in both groups obviously decreased (P < 0.05), and the observation group exhibited remarkable changes (P < 0.05). After treatment, the total efficiency of the observation group was 90.24%, which was significantly higher than the 73.17% of the control group (P < 0.05). In conclusion, T. wilfordii can significantly improve kidney function and clinical symptoms in CGN patients, and the mechanism is possibly related to its inhibition of the secretion of TNF-α and IL-6.

Structural and Biochemical Characterization of AidC, a Quorum-Quenching Lactonase with Atypical Selectivity.

Quorum-quenching catalysts are of interest for potential application as biochemical tools for interrogating interbacterial communication pathways, as antibiofouling agents, and as anti-infective agents in plants and animals. Herein, the structure and function of AidC, an N-acyl-l-homoserine lactone (AHL) lactonase from Chryseobacterium, is characterized. Steady-state kinetics show that zinc-supplemented AidC is the most efficient wild-type quorum-quenching enzymes characterized to date, with a kcat/KM value of approximately 2 × 10(6) M(-1) s(-1) for N-heptanoyl-l-homoserine lactone. The enzyme has stricter substrate selectivity and significantly lower KM values (ca. 50 µM for preferred substrates) compared to those of typical AHL lactonases (ca. >1 mM). X-ray crystal structures of AidC alone and with the product N-hexanoyl-l-homoserine were determined at resolutions of 1.09 and 1.67 Å, respectively. Each structure displays as a dimer, and dimeric oligiomerization was also observed in solution by size-exclusion chromatography coupled with multiangle light scattering. The structures reveal two atypical features as compared to previously characterized AHL lactonases: a "kinked" α-helix that forms part of a closed binding pocket that provides affinity and enforces selectivity for AHL substrates and an active-site His substitution that is usually found in a homologous family of phosphodiesterases. Implications for the catalytic mechanism of AHL lactonases are discussed.

Impact of suppressing retinoic acid-related orphan receptor gamma t (ROR)γt in ameliorating central nervous system autoimmunity.

Multiple sclerosis (MS) is an immune-mediated chronic central nervous system (CNS) disease affecting more than 400 000 people in the United States. Myelin-reactive CD4 T cells play critical roles in the formation of acute inflammatory lesions and disease progression in MS and experimental autoimmune encephalomyelitis (EAE), a well-defined mouse model for MS. Current MS therapies are only partially effective, making it necessary to develop more effective therapies that specifically target pathogenic myelin-specific CD4 T cells for MS treatment. While suppressing T-bet, the key transcription factor in T helper type 1 (Th1) cells, has been demonstrated to be beneficial in prevention and treatment of EAE, the therapeutic potential of retinoic acid-related orphan receptor gamma t (ROR)γt, the key transcription factor for Th17 cells, has not been well-characterized. In this study, we characterized the correlation between RORγt expression and other factors affecting T cell encephalitogenicity and evaluated the therapeutic potential of targeting RORγt by siRNA inhibition of RORγt. Our data showed that RORγt expression correlates with interleukin (IL)-17 production, but not with the encephalitogenicity of myelin-specific CD4 T cells. IL-23, a cytokine that enhances encephalitogenicity, does not enhance RORγt expression significantly. Additionally, granulocyte-macrophage colony-stimulating factor (GM-CSF) levels, which correlate with the encephalitogenicity of different myelin-specific CD4 T cell populations, do not correlate with RORγt. More importantly, inhibiting RORγt expression in myelin-specific CD4 T cells with an siRNA does not reduce disease severity significantly in adoptively transferred EAE. Thus, RORγt is unlikely to be a more effective therapeutic target for ameliorating pathogenicity of encephalitogenic CD4 T cells.

Structure of the VirB4 ATPase, alone and bound to the core complex of a type IV secretion system.

Type IV secretion (T4S) systems mediate the transfer of proteins and DNA across the cell envelope of bacteria. These systems play important roles in bacterial pathogenesis and in horizontal transfer of antibiotic resistance. The VirB4 ATPase of the T4S system is essential for both the assembly of the system and substrate transfer. In this article, we present the crystal structure of the C-terminal domain of Thermoanaerobacter pseudethanolicus VirB4. This structure is strikingly similar to that of another T4S ATPase, VirD4, a protein that shares only 12% sequence identity with VirB4. The VirB4 domain purifies as a monomer, but the full-length protein is observed in a monomer-dimer equilibrium, even in the presence of nucleotides and DNAs. We also report the negative stain electron microscopy structure of the core complex of the T4S system of the Escherichia coli pKM101 plasmid, with VirB4 bound. In this structure, VirB4 is also monomeric and bound through its N-terminal domain to the core's VirB9 protein. Remarkably, VirB4 is observed bound to the side of the complex where it is ideally placed to play its known regulatory role in substrate transfer.

Covalent inhibition of New Delhi metallo-ß-lactamase-1 (NDM-1) by cefaclor.

Covalent irreversible inhibitors can successfully treat antibiotic-resistant infections by targeting serine ß-lactamases. However, this strategy is useless for New Delhi metallo-ß-lactamase (NDM), which uses a non-covalent catalytic mechanism and lacks an active-site serine. Here, NDM-1 was irreversibly inactivated by three ß-lactam substrates: cephalothin, moxalactam, and cefaclor, albeit at supratherapeutic doses (e.g., cefaclor KI =2.3 ± 0.1 mM; k(inact) =0.024 ± 0.001 min(-1)). Inactivation by cephalothin and moxalactam was mediated through Cys208. Inactivation by cefaclor proceeded through multiple pathways, in part mediated by Lys211. Use of a cefaclor metabolite enabled mass spectrometric identification of a +346.0735 Da covalent adduct on Lys211, and an inactivation mechanism is proposed. Lys211 was identified as a promising "handhold" for developing covalent NDM-1 inhibitors and serves as a conceptual example for creating covalent inhibitors for enzymes with non-covalent mechanisms.

A phenylalanine clamp controls substrate specificity in the quorum-quenching metallo-γ-lactonase from Bacillus thuringiensis.

Autoinducer inactivator A (AiiA) is a metal-dependent N-acyl homoserine lactone hydrolase that displays broad substrate specificity but shows a preference for substrates with long N-acyl substitutions. Previously, crystal structures of AiiA in complex with the ring-opened product N-hexanoyl-l-homoserine revealed binding interactions near the metal center but did not identify a binding pocket for the N-acyl chains of longer substrates. Here we report the crystal structure of an AiiA mutant, F107W, determined in the presence and absence of N-decanoyl-l-homoserine. F107 is located in a hydrophobic cavity adjacent to the previously identified ligand binding pocket, and the F107W mutation results in the formation of an unexpected interaction with the ring-opened product. Notably, the structure reveals a previously unidentified hydrophobic binding pocket for the substrate's N-acyl chain. Two aromatic residues, F64 and F68, form a hydrophobic clamp, centered around the seventh carbon in the product-bound structure's decanoyl chain, making an interaction that would also be available for longer substrates, but not for shorter substrates. Steady-state kinetics using substrates of various lengths with AiiA bearing mutations at the hydrophobic clamp, including insertion of a redox-sensitive cysteine pair, confirms the importance of this hydrophobic feature for substrate preference. Identifying the specificity determinants of AiiA will aid the development of more selective quorum-quenching enzymes as tools and as potential therapeutics.

An altered zinc-binding site confers resistance to a covalent inactivator of New Delhi metallo-beta-lactamase-1 (NDM-1) discovered by high-throughput screening.

Due to the global threat of antibiotic resistance mediated by New Delhi metallo-beta-lactamase-1 (NDM-1) and the lack of structurally diverse inhibitors reported for this enzyme, we developed screening and counter-screening assays for manual and automated formats. The manual assay is a trans-well absorbance-based endpoint assay in 96-well plates and has a Z' factor of 0.8. The automated assay is an epi-absorbance endpoint assay in 384-well plates, has a Z' factor of ≥0.8, good signal/baseline ratios (>3.8), and is likely scalable for high-throughput screening (HTS). A TEM-1-based counter-screen is also presented to eliminate false positives due to assay interference or off-target activities. A pilot screen of a pharmacologically characterized compound library identified two thiol-modifying compounds as authentic NDM-1 inhibitors: p-chloromecuribenzoate (p-CMB) and nitroprusside. Recombinant NDM-1 has one Cys residue that serves as a conserved active-site primary zinc ligand and is selectively modified by p-CMB as confirmed by LC-MS/MS. However a C208D mutation results in an enzyme that maintains almost full lactamase activity, yet is completely resistant to the inhibitor. These results predict that covalent targeting of the conserved active-site Cys residue may have drawbacks as a drug design strategy.

[M2 macrophage-derived exosomal lncRNA NR_028113.1 promotes macrophage polarization possibly by activating the JAK2/STAT3 signaling pathway].

OBJECTIVE: To explore the effect of M2 macrophage-derived exosomal lncRNA NR_028113.1 on macrophage polarization and its possible mechanism. METHODS: Bone marrow-derived macrophages (BMDMs) from BALB/c mice were isolated and cultured in vitro. After IL-4 treatment to induce M2 macrophage polarization, exosomes (M2-exo) were extracted from the supernatant of M2 macrophages and identified. The expression of lncRNA in M2-exo was detected by qRT-PCR. BMDMs were co-cultured with M2-exo (100 µg/mL) or PBS for 48 h, and the changes in cellular expression levels of Arg1, YM-1, FIZZ1, iNOS and TNF-α were detected using qRT-PCR and Western blotting. The percentage of CD206+ cells was analyzed using flow cytometry, and the phosphorylation levels of JAK2/STAT3 proteins were detected using Western blotting. A lncRNA smart silencer was designed to specifically inhibit the expression of lncRNA NR_028113.1 in the M2 macrophages, from which exosomes were extracted and co-cultured with BMDMs for 48 h. The mRNA expression levels of Arg1, YM-1, FIZZ1, iNOS and TNF-α, CD206+ cell percentage and the phosphorylation levels of JAK2/STAT3 proteins were detected using qRT-PCR, flow cytometry and Western blotting. RESULTS: LncRNA NR_028113.1 was highly expressed in the exosomes of M2 macrophages (P < 0.05). Co-culture with M2-exo significantly increased mRNA expressions of M2 macrophage marker genes Arg1, YM-1 and FIZZ1 (P < 0.05), lowered the expressions of iNOS and TNF-α (P < 0.05), and increased CD206+ cell percentage and JAK2/STAT3 protein phosphorylation level in BMDMs (P < 0.05). After inhibiting the expression of lncRNA NR_028113.1 in M2 macrophages, the extracted M2-exo caused significant down-regulation of the mRNA expressions of Arg1, YM-1 and FIZZ1 and up-regulation of iNOS and TNF-α mRNA (P < 0.05), resulting also in signi-ficantly reduced CD206+ cell percentage and lowered phosphorylation levels of JAK2/STAT3 proteins in co-cultured BMDM (P < 0.05). CONCLUSIONS: M2 macrophage-derived exosomal lncRNA NR_028113.1 can significantly promote M2 polarization of macrophages possibly by activating the JAK2/STAT3 signaling pathway.