RESUMO
Metallo-ß-lactamases (MßLs) hydrolyze and inactivate ß-lactam antibiotics, are a pivotal mechanism conferring resistance against bacterial infections. SMB-1, a novel B3 subclass of MßLs from Serratia marcescens could deactivate almost all ß-lactam antibiotics including ampicillin (AMP), which has posed a serious threat to public health. To illuminate the mechanism of recognition and interaction between SMB-1 and AMP, various fluorescence spectroscopy techniques and molecular dynamics simulation were employed. The results of quenching spectroscopy unraveled that AMP could make SMB-1 fluorescence quenching that mechanism was the static quenching; the synchronous and three-dimensional fluorescence spectra validated that the microenvironment and conformation of SMB-1 were altered after interaction with AMP. The molecular dynamics results demonstrated that the whole AMP enters the binding pocket of SMB-1, even though with a relatively bulky R1 side chain. Loop1 and loop2 in SMB-1 undergo significant fluctuations, and α2 (71-73) and local α5 (186-188) were turned into random coils, promoting zinc ion exposure consistent with circular dichroism spectroscopy results. The binding between them was driven by a combination of enthalpy and entropy changes, which was dominated by electrostatic force in agreement with the fluorescence observations. The present study brings structural insights and solid foundations for the design of new substrates for ß-lactamases and the development of effective antibiotics that are resistant to superbugs.
Assuntos
Ampicilina , Simulação de Dinâmica Molecular , Serratia marcescens , Espectrometria de Fluorescência , beta-Lactamases , beta-Lactamases/química , beta-Lactamases/metabolismo , Ampicilina/química , Ampicilina/metabolismo , Ampicilina/farmacologia , Serratia marcescens/enzimologia , Ligação Proteica , Sítios de Ligação , Antibacterianos/química , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismoRESUMO
Given the significant clinical potential of human plasminogen Kringle 5 on tumours, it is crucial to seek its receptors for a thorough comprehension of its physiological functions and mechanism. Eleven candidates have been screened out in our previous works. In the present work, we further inquired whether the candidate, von Willebrand factor type A domain 1 in coagulation factor C homology protein (abbr. vWA1), was a potential receptor of Kringle 5, and investigated their binding mechanism by bio-specific experiments, frontal affinity analysis (FA), and molecular dynamic simulation (MDS). After the potential was validated by bio-specific experiments, the FA results stated that vWA1 exhibited a strong interaction towards Kringle 5 in the proportion of 1:1 with the binding constant of 4.18 × 104 L/mol. The MDS results showed that the binding was mainly driven by electrostatic and Van der Waals forces and occurred spontaneously, during which vWA1 and Kringle 5 mutually fit each other by conformational changing into more flexible and suitable structures including fluctuations for five loops and partial transformation into a random coil for α6-helix in vWA1. Moreover, lysine binding site Leu71-Tyr74 was speculated responsible for Kringle 5 in binding and Tyr72 to be the key amino acid residue. In short, this work not only confirmed vWA1 as a potential Kringle 5 receptor but also provided valuable information on the detailed binding, facilitating the application development of Kringle 5 in regulating immune or inhibiting tumour migration through vWA1.
Assuntos
Proteínas da Matriz Extracelular , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Fragmentos de Peptídeos , Plasminogênio , Ligação Proteica , Conformação ProteicaRESUMO
The evolution of metallo-beta-lactamase CphA in discontinuous gradient concentration of imipenem was investigated in this work. The results suggested that single-base mutations K218R, K249T, K249M, Q253H, and a frameshift mutation M1 were observed. Compared with wild type, the minimum inhibitory concentration (MICs) of K249T, K249M, and M1 increased by at least 128 times and that of K218R increased by 64 times. And the catalytic efficiency increased by 312% and 653%, respectively. It is speculated from the details of the structural changes revealed by molecular dynamics simulations that the carbon skeleton migration caused by the outward motion of the loop 3 in the mutant may have significantly increased the cavity volume of the binding pocket, which is more conducive to the entry and expulsion of imipenem and its hydrolytic product. And the conformational change of the TDRAGGN (71-77) is located at the bottom of the binding pocket from order α-helix to disorder random coil enabled the binding pocket to be more conducive to accommodate and hold the imipenem respectively. All these indicated that during the repeated drug resistance, the wild-type achieved gene mutations and conformational change and evolved to the mutant enzymes with a more delicate structure and stronger hydrolysis ability. KEY POINTS: ⢠The mutation and evolution of CphA under the selective pressure of imipenem. ⢠The CphA evolved to the mutants with stronger hydrolysis capacity. ⢠A novel pathway for the resistance of super bacteria.
Assuntos
Imipenem , beta-Lactamases , Bactérias/metabolismo , Imipenem/química , Imipenem/metabolismo , Imipenem/farmacologia , Testes de Sensibilidade Microbiana , Mutação , beta-Lactamases/metabolismoRESUMO
Metallo-ß-lactamases (MßLs) are the primary mechanism of resistance to carbapenem antibiotics. To elucidate how MßLs have evolved with the introduction and use of antibiotics, the mutation and evolution of SMB-1 from Serratia marcescens were investigated in microbial evolution plates containing discontinuous meropenem (MEM) concentration gradients. The results revealed 2-point mutations, A242G and S257R; 1 double-site mutation, C240G/E258G; and 3 frameshift mutations, M5, M12, and M13, which are all missense mutations situated at the C-terminus. Compared with that of the wild-type (WT), the minimum inhibitory concentrations (MICs) of MEM for A242G, C240G/E258G, M5, M12, and M13 increased at least 120-fold, and that of S257R increased 8-fold. The catalytic efficiency kcat/Km increased by 365% and 647%, respectively. Concerning the structural changes, the structure at the active site changed from an ordered structure to an unordered conformation. Simultaneously, the flexibility of loop 1 was enhanced. These changes increased the volume of the active site cavity; thus, this was more conducive to exposing the Zn2+ site, facilitating substrate binding and conversion to products. In A242G, structural changes in Gly-242 can be transmitted to the active region via a network of interactions between the side chains of Gly-242 and the amino acid side chains near the active pocket. Together, these results pointed to the process of persistent drug tolerance and resistance, the SMB-1 enzyme evolved into a more exquisite structure with increased flexibility and stability, and stronger hydrolysis activity via genetic mutations and structural changes.
Assuntos
Antibacterianos , beta-Lactamases , Meropeném , beta-Lactamases/metabolismo , Antibacterianos/química , Carbapenêmicos/química , Serratia marcescens/genética , Serratia marcescens/metabolismoRESUMO
An efficient fluorescence reversible regulation system with HEX-OND was developed. Then the application potential was explored in probing Hg(II) & Cysteine (Cys) in real samples and the thermodynamic mechanism was further investigated by precise theory analysis combining multiple spectroscopic methods. The results showed that only mere disturbances were observed among 15 and 11 kinds of other substances for the optimal system in detecting Hg(II) & Cys, respectively; The linear ranges of quantification were identified as 1.0 â¼ 14.0 and 2.0 â¼ 20.0 (×10-8 mol/L) with LODs of 8.75 and 14.09 (×10-9 mol/L) for Hg(II) and Cys, respectively; no significant deviations were found in the quantification results of Hg(II) in three traditional Chinese herbs and Cys in two samples between the well-understood methods with ours respectively, showing excellent selectivity, sensitivity, and tremendous application feasibility. The detailed mechanism was further verified as that the introduced Hg(II) forced HEX-OND to transform into the Hairpin structure with the apparent equilibrium association constant of 6.02 ± 0.62 × 1010 L/mol in the bimolecular ratio, leading to the equimolar quencher, consecutive two guanine bases ((G)2), approaching and spontaneously static-quenching the reporter HEX (hexachlorofluorescein) (equilibrium constant, 8.75 ± 1.97 × 107 L/mol) in the Photo-induced Electron Transfer (PET) way that was driven by the Electrostatic Interaction. The additional Cys destructed the equimolar Hairpin structure with the apparent equilibrium constant of 8.87 ± 2.47 × 105 L/mol through breaking one of the formed T-Hg(II)-T mismatches by association with the involved Hg(II), occasioning (G)2 apart from HEX and consequently the fluorescence recovery.
Assuntos
Cisteína , Mercúrio , Cisteína/análise , Oligonucleotídeos/química , Mercúrio/análise , Limite de Detecção , Espectrometria de Fluorescência/métodos , Corantes Fluorescentes/químicaRESUMO
The robot task sequencing problem and trajectory planning problem are two important issues in the robotic optimization domain and are solved sequentially in two separate levels in traditional studies. This paradigm disregards the potential synergistic impact between the two problems, resulting in a local optimum solution. To address this problem, this paper formulates a co-optimization model that integrates the task sequencing problem and trajectory planning problem into a holistic problem, abbreviated as the robot TSTP problem. To solve the TSTP problem, we model the optimization process as a Markov decision process and propose a deep reinforcement learning (DRL)-based method to facilitate problem solving. To validate the proposed approach, multiple test cases are used to verify the feasibility of the TSTP model and the solving capability of the DRL method. The real-world experimental results demonstrate that the DRL method can achieve a 30.54% energy savings compared to the traditional evolution algorithm, and the computational time required by the proposed DRL method is much shorter than those of the evolutionary algorithms. In addition, when adopting the TSTP model, a 18.22% energy reduction can be achieved compared to using the sequential optimization model.
RESUMO
All-trans retinoic acid (ATRA)-induced differentiation of acute promyelocytic leukemia (APL) toward granulocytes may trigger APL differentiation syndrome (DS), but there is less knowledge about the mechano-chemical regulation mechanism of APL DS under the mechano-microenvironment. We found that ATRA-induced changes in proliferation, morphology, and adhesive molecule expression levels were either dose or stimulus time dependent. An optimal ATRA stimulus condition for differentiating HL60 cells toward neutrophils consisted of 1 × 10-6 M dose and 120 h of stimulus time. Under wall shear stresses, catch-slip bond transition governs P-selectin-mediated rolling for neutrophils and untreated or ATRA-treated (1 × 10-6 M, 120 h) HL60 cells. The ATRA stimuli slowed down the rolling of HL60 cells on immobilized P-selectin no matter whether ICAM-1 was engaged. The ß2 integrin near the PSGL-1/P-selectin axis would be activated within sub-seconds for each cell group mentioned above, thus contributing to slow rolling. A faster ß2 integrin activation rate and the higher expression levels of PSGL-1 and LFA-1 were assigned to induce the over-enhancement of ATRA-treated HL60 adhesion in flow, causing APL DS development. These findings provided an insight into the mechanical-chemical regulation for APL DS development via ATRA treatment of leukemia and a novel therapeutic strategy for APL DS through targeting the relevant adhesion molecules.
Assuntos
Leucemia Promielocítica Aguda , Selectina-P , Humanos , Células HL-60 , Antígenos CD18 , Tretinoína/farmacologia , Tretinoína/uso terapêutico , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/metabolismoRESUMO
One of the resistance mechanisms of superbugs is to hydrolyze antibiotics by producing metallo-ß-lactamases (MßLs). To verify how MßLs evolved to increase in activity in response to various ß-lactam antibiotics, the mutation and evolution of CphA from Aeromonas hydrophila (Zn2+-dependent MßL) was investigated in a medium with a continuous biapenem (BIA) concentration gradient. The results showed that a single-base mutation M1 and two frameshift mutations M3 and M4 were observed. Furthermore, a nonsense mutation M2 was observed. Compared with wild-type (WT), the minimum inhibitory concentrations (MICs) of the M3 and M4 increased by more than 128 times, and the catalytic efficiency of BIA by the M3 and M4 increased by 752% and 376% respectively. In the mutants, the carbon skeleton migration caused by the outward motion of the loop3 near the entrance of the binding pocket increased the cavity volume of the binding pocket and was more conducive to the entry and expulsion of BIA and its hydrolytic product in the binding pocket. The conformational change effect originated from mutations is transmitted to the binding pocket through the interactions between the side chain amino acid residues of the C-terminal and those of the loop3, thus affecting the binding and hydrolysis capability of the mutants to BIA in the binding pocket. All these indicated that during the repeated drug-endurance and -resistance, the CphA completed its mutation and conformational change and evolved to the mutants with a more delicate structure and stronger hydrolysis ability by a genetic mutation.
Assuntos
Tienamicinas , beta-Lactamases , Antibacterianos , Proteínas de Bactérias/química , Mutação , Tienamicinas/química , Tienamicinas/metabolismo , beta-Lactamases/metabolismoRESUMO
The recognition and interaction of FEZ-1 from Legionella (FEZ-1) with penicillin V(PV) and cefoxitin(CFX) were investigated using fluorescence spectra in combination with molecular dynamics simulation (MD). The results revealed that the CFX bind with FEZ-1 in stronger interaction and induced larger conformational change than PV, despite all being forced by the electrostatic interaction and along with the changing in an environment of amino acid residues as well as the polypeptide skeleton inside the FEZ-1. Moreover, only the loop1, loop2, and N-terminal were observed locating near the binding pocket of FEZ-1, consisting of a flexible "gate-like" zone with better adaptability that controlled the entrance of antibiotic into the pocket by allowing the newly introduced antibiotic to match the pocket better through the conformational changes of these three substructures in the binding procedure. The current study may provide some valuable information on the antibiotic hydrolytic process by metallo-beta-lactamase and thus the references for the development of new antibiotics for super bacteria.
Assuntos
Cefoxitina , Legionella , Penicilina V , beta-Lactamases , Cefoxitina/farmacologia , Legionella/metabolismo , Simulação de Dinâmica Molecular , Penicilina V/farmacologia , beta-Lactamases/metabolismoRESUMO
BACKGROUND: Droplet digital polymerase chain reaction (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, a ddPCR assay for the epidermal growth factor receptor (EGFR) p.Thr790Met (T790M) mutation suitable for clinical use remains to be established with analytical and clinical validations. OBJECTIVE: We aimed to develop and validate a new ddPCR assay to quantify the T790M mutation in plasma for monitoring and predicting the progression of advanced non-small-cell lung cancer (NSCLC). METHODS: Specificity of the ddPCR assay was evaluated with genomic DNA samples from healthy individuals. The inter- and intraday variations of the assay were evaluated using mixtures of plasmid DNA containing wild-type EGFR and T790M mutation sequences. We assessed the clinical utility of the T790M assay in a multicenter prospective study in patients with advanced NSCLC receiving tyrosine kinase inhibitor (TKI) treatment by analyzing longitudinal plasma DNA samples. RESULTS: We set the criteria for a positive call when the following conditions were satisfied: (1) T790M mutation frequency > 0.098% (3 standard deviations above the background signal); (2) at least two positive droplets in duplicate ddPCR reactions. Among the 62 patients with advanced NSCLC exhibiting resistance to TKI treatment, 15 had one or more serial plasma samples that tested positive for T790M. T790M mutation was detected in the plasma as early as 205 days (median 95 days) before disease progression, determined by imaging analysis. Plasma T790M concentrations also correlated with intervention after disease progression. CONCLUSIONS: We developed a ddPCR assay to quantify the T790M mutation in plasma. Quantification of longitudinal plasma T790M mutation may allow noninvasive assessment of drug resistance and guide follow-up treatment in TKI-treated patients with NSCLC. TRIAL REGISTRATION: Clinical Trials.gov identifier: NCT02804100.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/enzimologia , Neoplasias Pulmonares/enzimologia , Mutação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/genética , Estudos de Casos e Controles , DNA/sangue , DNA/genética , Progressão da Doença , Receptores ErbB/sangue , Receptores ErbB/genética , Feminino , Humanos , Estudos Longitudinais , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Seleção de Pacientes , Estudos ProspectivosRESUMO
Nano erythrocyte ghosts have recently been used as drug carriers of water-soluble APIs due to inherit biological characteristics of good compatibility, low toxicity, and small side-effect. In this study, we developed a novel drug delivery system based on nano erythrocyte ghosts (STS-Nano-RBCs) to transport Sodium Tanshinone IIA sulfonate (STS) for intravenous use in rat. STS-Nano-RBCs were prepared by hypotonic lysis and by extrusion methods, and its biological properties were investigated compared with STS injection. The results revealed that STS-Nano-RBCs have narrow particle size distribution, good drug loading efficiency, and good stability within 21 days. Compared with STS injection, STS-Nano-RBCs extended the drug release time in vitro and in vivo with better repairing effect on oxidative stress-impaired endothelial cells. These results suggest that the nano erythrocyte ghosts system could be used to deliver STS.
RESUMO
Phosphatase and tensin homolog (PTEN) is a potent tumor suppressor which regulates various cellular functions. The aim of the present study was to analyze the function of PTEN gene expression in squamous cell carcinoma (SCC) cells. This gene exhibits a unique function in cell migration and proliferation during the early stages of embryonic development. However, its role as a tumor suppressor gene in tongue squamous carcinoma cells remains unclear. In the present study, an SCC-4 cell line stably expressing PTEN was established and the effects of PTEN gene expression on SCC-4 cell proliferation, invasion and apoptosis were investigated. PTEN expression was found to induce apoptosis in SCC-4 cells, possibly via negative regulation of the phosphatidylinositide 3-kinase/Akt signaling pathway and increased expression of Bcl-2-interacting mediator of cell death. In addition, PTEN was found to control the epithelial-mesenchymal transition in SCC cells, thereby reducing their invasive ability. Furthermore, Transwell assay revealed that the expression of E-cadherin was increased, while the expression of vimentin and SNAIL was decreased. This study has provided an important insight into the mechanisms by which PTEN mediates the progression and early metastasis of tongue carcinoma.