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.

[Lysosomal membrane protein Sidt2 knockout induces apoptosis of human hepatocytes in vitro independent of the autophagy-lysosomal pathway].

OBJECTIVE: To explore the regulatory mechanism of human hepatocyte apoptosis induced by lysosomal membrane protein Sidt2 knockout. METHODS: The Sidt2 knockout (Sidt2-/-) cell model was constructed in human hepatocyte HL7702 cells using Crispr-Cas9 technology.The protein levels of Sidt2 and key autophagy proteins LC3-II/I and P62 in the cell model were detected using Western blotting, and the formation of autophagosomes was observed with MDC staining.EdU incorporation assay and flow cytometry were performed to observe the effect of Sidt2 knockout on cell proliferation and apoptosis.The effect of chloroquine at the saturating concentration on autophagic flux, proliferation and apoptosis of Sidt2 knockout cells were observed. RESULTS: Sidt2-/- HL7702 cells were successfully constructed.Sidt2 knockout significantly inhibited the proliferation and increased apoptosis of the cells, causing also increased protein expressions of LC3-II/I and P62(P < 0.05) and increased number of autophagosomes.Autophagy of the cells reached a saturated state following treatment with 50 µmol/L chloroquine, and at this concentration, chloroquine significantly increased the expressions of LC3B and P62 in Sidt2-/- HL7702 cells. CONCLUSION: Sidt2 gene knockout causes dysregulation of the autophagy pathway and induces apoptosis of HL7702 cells, and the latter effect is not mediated by inhibiting the autophagy-lysosomal pathway.

[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.

[Expression of miR-4324 and its targeted gene Talin2 in breast cancer].

OBJECTIVE: To investigate the regulatory effect of miR-4324 on ankyrin 2(Talin2) expression and biological behaviors of breast cancer cells and the clinical implications of changes in miR-4324 and Talin2 expressions in breast cancer. METHODS: In breast cancer and adjacent tissues, the expressions of Talin2 and miR-4324 were examined with immunohistochemistry and qRT-PCR, respectively and the association of Talin2 expression levels with the prognosis and clinicopathological features of breast cancer patients was analyzed.The human breast cancer cell line SKBR-3 was transfected with miR-4324 mimic, miR-4324 inhibitor, si-Talin2, or both miR-4324 inhibitor and si-Talin2, and the changes in biological behaviors of the cells were examined; the cellular expression of Talin2at the mRNA and protein levels were detected with qRT-PCR and Western blotting.Dual luciferase reporter gene assay was used to verify the targeting relationship between miR-4324 and Talin2.The effect of miR-4324-mediated regulation of Talin2 on SKBR-3 cell migration was assessed using Transwell assays. RESULTS: Talin2 expression was significantly higher in breast cancer tissues than in the adjacent tissues, and its expression level was correlated with lymph node metastasis and high HER-2 expression in breast cancer (P < 0.05) but not with the patient's age, clinical stage, histological grade or expressions of estrogen and progesterone receptors (P >0.05).The expression of miR-4324 was significantly reduced in breast cancer tissues as compared with the adjacent tissues (P < 0.01).In SKBR-3 cells, transfection with miR-4324 mimics significantly inhibited proliferation, migration and invasion (P < 0.05) and promoted apoptosis (P < 0.01) of the cells.Dual luciferase reporter gene assay confirmed that cotransfection with miR-4324 mimics significantly reduced luciferase activity of Talin2-3'-UTR WT reporter plasmid (P < 0.05).Transfection of the cells with miR-4324 mimics significantly reduced mRNA and protein expressions of Talin2(P < 0.05).Transwell migration assay showed that the migration ability of SKBR-3 cells was significantly enhanced after transfection with miR-4324 inhibitor (P < 0.01), lowered after transfection with si-Talin2(P < 0.01), and maintained at the intermediate level after co-transfection with miR-4324 inhibitor+si-Talin2 group (P < 0.05). CONCLUSIONS: High expression of Talin2 is associated with lymph node metastasis and HER-2 overexpression in breast cancer patients.Down-regulation of miR-4324 inhibits the proliferation, invasion and migration and induces apoptosis of breast cancer cells, and the inhibitory effect of miR-4324 knockdown on breast cancer cell migration is mediated probably by targeted inhibition of Talin2 expression.

[A fixed cohort study of disability trajectory of the dying elderly in China].

Objective: To explore the trajectory of disability in the dying elderly in China. Methods: Based on the activity of daily living (ADL) data from the 2002-2018 Chinese Longitudinal Healthy Longevity Survey, the longitudinal item response theory (LIRT) model was fitted with the difficulty threshold parameters to analyze the ADL loss in the elderly in China. Then, a mixed-effects model was fitted to analyze the trajectory of the disability level of the dying elderly. Results: A total of 5 817 old adults who entered the cohort in 2002 were included, in whom 41.81% were males, with a baseline age of (86.80±12.40) years and a follow-up time of 4 (3,8) years. The results of LIRT showed that the lowest difficulty threshold parameter in the basic activity of daily living (BADL) was partially disability on bathing (0.41±0.05), and the highest was entirely disability on indoor movement (6.19±0.16). In comparison, the lowest difficulty threshold parameter in instrumental activity of daily living (IADL) was partially disability on using public transportation (-3.01±0.07), and the highest was entirely disability on visiting neighbors (1.51±0.07). In the trajectory of disability, the average dependency in ADL was lower in dying men than in dying women (P<0.001), in the elderly living alone than in the elderly living with family members (P<0.001) and in the non-illiterate elderly than in the illiterate elderly (P<0.001). The estimated value of both the linear change rate and quadratic coefficient of disability level development with time were 0.231 (P<0.001) and 0.002 (P<0.001). Conclusions: In China, the development of disability in the elderly in China has its characteristics, IADL disability might occurs earlier than BADL. Among the IADL/BADL items, the disability of lower limb-based items is more prone to occur compared with upper limb-based items, and the disability of complex items is more prone to occur compared with simple items, and the growth rate of the disability trajectory also accelerates over time. It is necessary to pay attention to old women, old people living with family members, old people with low education level and old people with poor cognitive function in the disability prevention.

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.

Structural insights into the design of reversible fluorescent probes for metallo-ß-lactamases NDM-1, VIM-2, and IMP-1.

Metallo-ß-lactamases (MBLs) are enzymes that are capable of hydrolyzing most ß-lactam antibiotics and all clinically relevant carbapenems. We developed a library of reversible fluorescent turn-on probes that are designed to directly bind to the dizinc active site of these enzymes and can be used to study their dynamic metalation state and enzyme-inhibitor interactions. Structure-function relationships with regards to inhibitory strength and fluorescence turn-on response were evaluated for three representative MBLs.

Discovery of an Effective Small-Molecule Allosteric Inhibitor of New Delhi Metallo-ß-lactamase (NDM).

To identify novel inhibitors of the carbapenemase New Delhi metallo-ß-lactamase (NDM) as possible therapeutic compounds, we conducted a high-throughput screen of a 43,358-compound library. One of these compounds, a 2-quinazolinone linked through a diacylhydrazine to a phenyl ring (QDP-1) (IC50 = 7.9 ± 0.5 µM), was characterized as a slow-binding reversible inhibitor (Kiapp = 4 ± 2 µM) with a noncompetitive mode of inhibition in which substrate and inhibitor enhance each other's binding affinity. These studies, along with differential scanning fluorimetry, zinc quantitation, and selectivity studies, support an allosteric mechanism of inhibition. Cotreatment with QDP-1 effectively lowers minimum inhibitory concentrations of carbapenems for a panel of resistant Escherichia coli and Klebsiella pneumoniae clinical isolates expressing NDM-1 but not for those expressing only serine carbapenemases. QDP-1 represents a novel allosteric approach for NDM drug development for potential use alone or with other NDM inhibitors to counter carbapenem resistance in enterobacterales.

[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.

[Lysosomal membrane protein Sidt2 deletion impairs autophagy in human hepatocytes].

OBJECTIVE: To study the effect of lysosomal membrane protein Sidt2 deletion on autophagy in human hepatocytes. METHODS: Crispr-Cas9 technology was used to construct a human hepatocyte (HL7702) model of Sidt2 knockout (Sidt2-/-), and the expression levels of the key autophagy proteins LC3II/I, P62 and autophagy-related proteins Atg5, Atg7, and Atg12 were detected.The co-localization of LC3B and P62 in the cells were analyzed with immunofluorescence assay to assess the identification and storage of P62 cargo proteins by the autophagosomes and the degradation of the autophagolysosomes.The co-localization of LC3B and LAMP1 was also determined with immunofluorescence assay to detect the fusion of the autophagosomes with the lysosomes, and LysoTracker was used to trace the acidic lysosomes. RESULTS: We successfully constructed a HL7702 cell model of Sidt2+/+ and Sidt2-/-, and compared with Sidt2+/+ cells, the Sidt2-/- cell model showed significantly increased expressions of LC3-II/I and P62 (P < 0.01).Immunofluorescence assay showed a significant increase of LC3B and P62 expressions (P < 0.001) and obviously lowered expressions of Atg5, Atg7, and Atg12 in Sidt2-/- cells (P < 0.05).The co-localization of LC3B and P62 and that of LC3B and LAMP1 were both reduced and the number of acidic lysosomes was significantly lowered in Sidt2-/- cells (P < 0.05). CONCLUSION: Sidt2 gene deletion disturbs the recognition and sequestration of P62 cargo protein by autophagosomes in human hepatocytes.At the same time, the decreased number of acidic lysosomes and the dysfunction of autophagosome and lysosome fusion cause the block of the autophagy-lysosome pathway, leading eventually to LC3B and P62 accumulation and impaired autophagy in the hepatocytes.

Cyclobutanone Inhibitor of Cobalt-Functionalized Metallo-γ-Lactonase AiiA with Cyclobutanone Ring Opening in the Active Site.

An α-amido cyclobutanone possessing a C10 hydrocarbon tail was designed as a potential transition-state mimetic for the quorum-quenching metallo-γ-lactonase autoinducer inactivator A (AiiA) with the support of in-house modeling techniques and found to be a competitive inhibitor of dicobalt(II) AiiA with an inhibition constant of K i = 0.007 ± 0.002 mM. The catalytic mechanism of AiiA was further explored using our product-based transition-state modeling (PBTSM) computational approach, providing substrate-intermediate models arising during enzyme turnover and further insight into substrate-enzyme interactions governing native substrate catalysis. These interactions were targeted in the docking of cyclobutanone hydrates into the active site of AiiA. The X-ray crystal structure of dicobalt(II) AiiA cocrystallized with this cyclobutanone inhibitor unexpectedly revealed an N-(2-oxocyclobutyl)decanamide ring-opened acyclic product bound to the enzyme active site (PDB 7L5F). The C10 alkyl chain and its interaction with the hydrophobic phenylalanine clamp region of AiiA adjacent to the active site enabled atomic placement of the ligand atoms, including the C10 alkyl chain. A mechanistic hypothesis for the ring opening is proposed involving a radical-mediated process.

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.

Effects of CAR structure and culture conditions on memory CAR-T cells.

Adoptive CAR T cell therapy (chimeric antigen receptor T-Cell) has received increasing attention in recent years; however, its efficacy is undesirable and differs from person to person. Understanding how to overcome this obstacle is important to improve therapy. Infusion of poorly differentiated CAR-CD62L+ T cells, such as T memory stem cell populations, leads to enhanced T cell implantation, expansion, and persistence, which ultimately leads to more stable tumour regression. Here, we reviewed emerging findings demonstrating that CAR structure and cell culture conditions can influence CAR T cell differentiation and antitumour efficacy.

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.

[Analysis of risk factors of severe postoperative complications in elderly patients with colorectal cancer aged over 80 years].

Objective: To investigate the risk factors of severe postoperative complications in elderly patients with colorectal cancer aged over 80 years old. Methods: A retrospective case-control study was conducted to collect and analyze the clinicopathological data of patients (≥80 years old) who underwent radical colorectal cancer surgery at department of Colorectal Surgery, Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College from January 2010 to December 2018. A total of 269 patients were included in the study, including 160 males and 109 females. The average age was 83 (80-94) years. Among them, the pathological TNM stage was 16 in stage I, 76 in stage II, 167 in stage III, and 10 in stage IV. According to Clavien-Dindo classification, the postoperative complications of grade III and above were defined as serious complications. To analyze the relationship between the patient's clinical data, such as general information, the surgeon's experience (whether to complete more than 500 radical colorectal cancer surgery), intraoperative conditions and the occurrence of severe complications. Univariate analysis was conducted with the chi-squared test. Multivariate logistic regression analysis was used for statistically significant variables in univariate analysis. Results: Of the 269 patients, 34 (12.6%) had severe complications after surgery. The incidence of postoperative complications ranged from high to low, respectively, for pulmonary infection (8/269,3.0%), intestinal obstruction (8/269, 3.0%) and anastomotic leakage (7/269, 2.6%). One patient died of pulmonary embolism and one patient died of multiple organ failure, with a perioperative mortality rate of 0.7% (2/269). On univariate analysis, the occurrence of severe postoperative complications was associated with age (χ(2)=8.181, P=0.004), American society of anesthesiologists grade (χ(2)=7.945, P=0.005), preoperative albumin level (χ(2)=9.088, P=0.003), operation experience (χ(2)=9.395, P=0.002). Multivariable logistic regression analysis showed that age ≥85 years old (OR=4.415, 95% CI: 1.702-11.453, P=0.080), preoperative albumin <35 g/L (OR=2.544, 95%CI: 1.083-5.974, P=0.032), and less-experieced group (OR=2.475, 95% CI:1.082-5.661, P=0.032) was independent risk factor for severe postoperative complications. The incidence of serious postoperative complications was similar in patients undergoing laparoscopy and laparotomy [10.1% (17/169) vs. 17.0% (17/100), χ(2)=2.741, P=0.098]. Conclusion: Adequate preoperative evaluation, appropriate perioperative nutritional support and experienced specialists are the key factors to ensure the successful perioperative period of elderly patients with colorectal cancer aged over 80 years old. In addition, more attention should be paid to the elderly patients aged ≥85 years.

[Characteristics and prognosis of ear fullness feeling in patients with all-frequency sudden deafness].

Objective: The characteristics and prognosis of ear fullness feeling in patients with all-frequency sudden deafness were explored. Method: 104 patients (104 ears) with unilateral all-frequency sudden deafness were collected in study from June 2015 to March 2019, including 50 males and 54 females, the mean age ranged from 23-65 years, and the disease duration ranged from 1-9 days. Of those, 56 patients accompanied with the feeling of ear fullness (FEF) were enrolled into'the ear fullness group', and 48 patients without FEF were included in'the without ear fullness group'. Patients' treatment strategy followed the Chinese Medical Association Guidelines for the diagnosis and treatment of sudden deafness (2015). Moreover, VAS scale scores and subjective grading of FEF were acquired in patients with FEF. We analyzed the clinical characteristics and prognosis of FEF with SPSS 23.0 software. Results: There were no differences between the two groups in terms of age, gender, duration of disease, the side of deaf ear, degree of hearing loss, and auditory brainstem evoked potential results (age,t=1.566; gender,χ(2)=0.001; duration, t=0.057; side,χ(2)=0.033; degree of hearing loss Z=-0.180; ABR,χ(2)=0.001;all P>0.05) . There was a positive correlation between the subjective grading of FEF and the degree of hearing loss in patients with FEF (r=0.599, P<0.001) . The total rate of hearing improvement following one month of treatments in patients with FEF vs with no FEF was 35/56(62.5%) vs 28/48(58.3%) (Z=-0.641, P=0.521). After one month of treatment, the total effective rate of FEF was 94.6%(53/56), and the improvement of FEF had nothing to improvement of hearing (r=0.040, P=0.769) . Conclusions: The degree of hearing loss is positively correlated with the degree of FEF in patients with all-frequency sudden deafness. Hearing recovery is not related to FEF. The recovery effect of FEF is good, and has no correlation with hearing recovery.

Iminodiacetic Acid as a Novel Metal-Binding Pharmacophore for New Delhi Metallo-ß-lactamase Inhibitor Development.

The fungal natural product aspergillomarasmine A (AMA) has been identified as a noncompetitive inhibitor of New Delhi metallo-ß-lactamase-1 (NDM-1) that inhibits by removing ZnII from the active-site. The nonselective metal-chelating properties and difficult synthesis and derivatization of AMA have hindered the development of this scaffold into a potent and selective inhibitor of NDM-1. Iminodiacetic acid (IDA) has been identified as the metal-binding pharmacophore (MBP) core of AMA that can be leveraged for inhibitor development. Herein, we report the use of IDA for fragment-based drug discovery (FBDD) of NDM-1 inhibitors. IDA (IC50 =120 µM) was developed into inhibitor 23 f (IC50 =8.6 µM, Ki =2.6 µM), which formed a ternary complex with NDM-1, as evidenced by protein thermal-shift and native-state electrospray ionization mass spectrometry (ESI-MS) experiments. Combining mechanistic analysis with inhibitor derivatization, the use of IDA as an alternative AMA scaffold for NDM-1 inhibitor development is detailed.

Elusive structural changes of New Delhi metallo-ß-lactamase revealed by ultraviolet photodissociation mass spectrometry.

We use mass spectrometry (MS), under denaturing and non-denaturing solution conditions, along with ultraviolet photodissociation (UVPD) to characterize structural variations in New Delhi metallo-ß-lactamase (NDM) upon perturbation by ligands or mutation. Mapping changes in the abundances and distributions of fragment ions enables sensitive detection of structural alterations throughout the protein. Binding of three covalent inhibitors was characterized: a pentafluorphenyl ester, an O-aryloxycarbonyl hydroxamate, and ebselen. The first two inhibitors modify Lys211 and maintain dizinc binding, although the pentafluorophenyl ester is not selective (Lys214 and Lys216 are also modified). Ebselen reacts with the sole Cys (Cys208) and ejects Zn2 from the active site. For each inhibitor, native UVPD-MS enabled simultaneous detection of the closing of a substrate-binding beta-hairpin loop, identification of covalently-modified residue(s), reporting of the metalation state of the enzyme, and in the case of ebselen, observation of the induction of partial disorder in the C-terminus of the protein. Owing to the ability of native UVPD-MS to track structural changes and metalation state with high sensitivity, we further used this method to evaluate the impact of mutations found in NDM clinical variants. Changes introduced by NDM-4 (M154L) and NDM-6 (A233V) are revealed to propagate through separate networks of interactions to direct zinc ligands, and the combination of these two mutations in NDM-15 (M154L, A233V) results in additive as well as additional structural changes. Insight from UVPD-MS helps to elucidate how distant mutations impact zinc affinity in the evolution of this antibiotic resistance determinant. UVPD-MS is a powerful tool capable of simultaneous reporting of ligand binding, conformational changes and metalation state of NDM, revealing structural aspects of ligand recognition and clinical variants that have proven difficult to probe.

A Single Salt Bridge in VIM-20 Increases Protein Stability and Antibiotic Resistance under Low-Zinc Conditions.

To understand the evolution of Verona integron-encoded metallo-ß-lactamase (VIM) genes (blaVIM) and their clinical impact, microbiological, biochemical, and structural studies were conducted. Forty-five clinically derived VIM variants engineered in a uniform background and expressed in Escherichia coli afforded increased resistance toward all tested antibiotics; the variants belonging to the VIM-1-like and VIM-4-like families exhibited higher MICs toward five out of six antibiotics than did variants belonging to the widely distributed and clinically important VIM-2-like family. Generally, maximal MIC increases were observed when cephalothin and imipenem were tested. Additionally, MIC determinations under conditions with low zinc availability suggested that some VIM variants are also evolving to overcome zinc deprivation. The most profound increase in resistance was observed in VIM-2-like variants (e.g., VIM-20 H229R) at low zinc availability. Biochemical analyses reveal that VIM-2 and VIM-20 exhibited similar metal binding properties and steady-state kinetic parameters under the conditions tested. Crystal structures of VIM-20 in the reduced and oxidized forms at 1.25 Å and 1.37 Å resolution, respectively, show that Arg229 forms an additional salt bridge with Glu171. Differential scanning fluorimetry of purified proteins and immunoblots of periplasmic extracts revealed that this difference increases thermostability and resistance to proteolytic degradation when zinc availability is low. Therefore, zinc scarcity appears to be a selective pressure driving the evolution of multiple metallo-ß-lactamase families, although compensating mutations use different mechanisms to enhance resistance.IMPORTANCE Antibiotic resistance is a growing clinical threat. One of the most serious areas of concern is the ability of some bacteria to degrade carbapenems, drugs that are often reserved as last-resort antibiotics. Resistance to carbapenems can be conferred by a large group of related enzymes called metallo-ß-lactamases that rely on zinc ions for function and for overall stability. Here, we studied an extensive panel of 45 different metallo-ß-lactamases from a subfamily called VIM to discover what changes are emerging as resistance evolves in clinical settings. Enhanced resistance to some antibiotics was observed. We also found that at least one VIM variant developed a new ability to remain more stable under conditions where zinc availability is limited, and we determined the origin of this stability in atomic detail. These results suggest that zinc scarcity helps drive the evolution of this resistance determinant.