Efflux of antibacterial compounds is a major mechanism for developing antimicrobial resistance. In the Gram‐positive pathogen Staphylococcus aureus, QacA, a 14 transmembrane helix containing major ...facilitator superfamily antiporter, mediates proton‐coupled efflux of mono and divalent cationic antibacterial compounds. In this study, we report the cryo‐EM structure of QacA, with a single mutation D411N that improves homogeneity and retains efflux activity against divalent cationic compounds like dequalinium and chlorhexidine. The structure of substrate‐free QacA, complexed to two single‐domain camelid antibodies, was elucidated to a resolution of 3.6 Å. The structure displays an outward‐open conformation with an extracellular helical hairpin loop (EL7) between transmembrane helices 13 and 14, which is conserved in a subset of DHA2 transporters. Removal of the EL7 hairpin loop or disrupting the interface formed between EL7 and EL1 compromises efflux activity. Chimeric constructs of QacA with a helical hairpin and EL1 grafted from other DHA2 members, LfrA and SmvA, restore activity in the EL7 deleted QacA revealing the allosteric and vital role of EL7 hairpin in antibacterial efflux in QacA and related members.
Synopsis
Bacterial pathogenicity relies on the export of antibacterial compounds by broad‐specificity transporters; however, the molecular determinants of this resistance remain unclear. Here, structural work describes unique features of the proton‐coupled‐efflux transporter QacA in Staphylococcus aureus that reveal a conserved allosteric gating mechanism, and suggest new therapeutic approaches to combat systemic infection.
Cryo‐EM structure of QacA stabilized in outward‐open state was elucidated in complex with two single‐domain camelid antibodies that served as fiducial markers.
QacA displays a unique extracellular α‐helical hairpin loop (EL7) between transmembrane helices 13 and 14 that is observed in a subset of drug:proton antiporters.
Removal of EL7 leads to loss of efflux activity that can be regained by translocating EL7 regions from related transporters.
Comparison of the outward‐open QacA structure with the inward‐open model reveals the formation of the EL7‐EL1 interface that serves to close the extracellular gate during the efflux cycle.
Outward‐open state cryo‐EM structure of drug:proton antiporter QacA uncovers conserved extracellular α‐helical hairpin loop with allosteric function in antibacterial compound efflux.
Despite the many conveniences of Radio Frequency Identification (RFID) systems, the underlying open architecture for communication between the RFID devices may lead to various security threats. ...Recently, many solutions were proposed to secure RFID systems and many such systems are based on only lightweight primitives, including symmetric encryption, hash functions, and exclusive OR operation. Many solutions based on only lightweight primitives were proved insecure, whereas, due to resource-constrained nature of RFID devices, the public key-based cryptographic solutions are unenviable for RFID systems. Very recently, Gope and Hwang proposed an authentication protocol for RFID systems based on only lightweight primitives and claimed their protocol can withstand all known attacks. However, as per the analysis in this article, their protocol is infeasible and is vulnerable to collision, denial-of-service (DoS), and stolen verifier attacks. This article then presents an improved realistic and lightweight authentication protocol to ensure protection against known attacks. The security of the proposed protocol is formally analyzed using Burrows Abadi-Needham (BAN) logic and under the attack model of automated security verification tool ProVerif. Moreover, the security features are also well analyzed, although informally. The proposed protocol outperforms the competing protocols in terms of security.
Most amino acid substitutions in a protein either lead to partial loss-of-function or are near neutral. Several studies have shown the existence of second-site mutations that can rescue defects ...caused by diverse loss-of-function mutations. Such global suppressor mutations are key drivers of protein evolution. However, the mechanisms responsible for such suppression remain poorly understood. To address this, we characterized multiple suppressor mutations both in isolation and in combination with inactive mutants. We examined six global suppressors of the bacterial toxin CcdB, the known M182T global suppressor of TEM-1 β-lactamase, the N239Y global suppressor of p53-DBD and three suppressors of the SARS-CoV-2 spike Receptor Binding Domain. When coupled to inactive mutants, they promote increased in-vivo solubilities as well as regain-of-function phenotypes. In the case of CcdB, where novel suppressors were isolated, we determined the crystal structures of three such suppressors to obtain insight into the specific molecular interactions responsible for the observed effects. While most individual suppressors result in small stability enhancements relative to wildtype, which can be combined to yield significant stability increments, thermodynamic stabilisation is neither necessary nor sufficient for suppressor action. Instead, in diverse systems, we observe that individual global suppressors greatly enhance the foldability of buried site mutants, primarily through increase in refolding rate parameters measured in vitro. In the crowded intracellular environment, mutations that slow down folding likely facilitate off-pathway aggregation. We suggest that suppressor mutations that accelerate refolding can counteract this, enhancing the yield of properly folded, functional protein in vivo.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Regulation of biological processes by proteins often involves the formation of transient, multimeric complexes whose characterization is mechanistically important but challenging. The bacterial toxin ...CcdB binds and poisons DNA Gyrase. The corresponding antitoxin CcdA extracts CcdB from its complex with Gyrase through the formation of a transient ternary complex, thus rejuvenating Gyrase. We describe a high throughput methodology called Ter‐Seq to stabilize probable ternary complexes and measure associated kinetics using the CcdA‐CcdB‐GyrA14 ternary complex as a model system. The method involves screening a yeast surface display (YSD) saturation mutagenesis library of one partner (CcdB) for mutants that show enhanced ternary complex formation. We also isolated CcdB mutants that were either resistant or sensitive to rejuvenation, and used surface plasmon resonance (SPR) with purified proteins to validate the kinetics measured using the surface display. Positions, where CcdB mutations lead to slower rejuvenation rates, are largely involved in CcdA‐binding, though there were several notable exceptions suggesting allostery. Mutations at these positions reduce the affinity towards CcdA, thereby slowing down the rejuvenation process. Mutations at GyrA14‐interacting positions significantly enhanced rejuvenation rates, either due to reduced affinity or complete loss of CcdB binding to GyrA14. We examined the effect of different parameters (CcdA affinity, GyrA14 affinity, surface accessibilities, evolutionary conservation) on the rate of rejuvenation. Finally, we further validated the Ter‐Seq results by monitoring the kinetics of ternary complex formation for individual CcdB mutants in solution by fluorescence resonance energy transfer (FRET) studies.
A rare cause of hypokalemia Lee, Rina; Merchant, Ambreen; Ahmed, Shahbaz
Proceedings - Baylor University. Medical Center,
2024, Letnik:
37, Številka:
4
Journal Article
Recenzirano
Hypokalemia is a common electrolyte derangement seen in the inpatient setting, often with multiple plausible explanations. However, for patients with nonobvious causes, obtaining a more thorough ...history, including dietary history, can yield valuable insight and clues to guide clinicians in their evaluation.
Cancer, as a multifactorial disease, damages healthy cells and organs. The major drawback of anticancer drugs is that they have a severe impact on healthy cells due to their toxic effects. ...Encapsulation of anticancer drugs into a nanosized formulation enhances the drug delivery system. Thus, nanoformulation (NF) increases the absorption rate and speeds up the circulation time of conventional water-insoluble drugs. Chlorambucil is a well-studied anticancer drug that controls chronic lymphocytic leukaemia and malignant lymphomas. Chlorambucil, a nitrogen mustard alkylating agent, shows hepatotoxicity, secondary malignancies,
etc.
, in normal cells and tissues. In this study, NFs of chlorambucil were prepared through an ultrasonication technique by varying the surfactant concentration. The characterization studies optimized the size, surface charge, heterogeneity, entrapment efficiency, drug loading capacity,
in vitro
drug release profile, stability, and interaction of the drug and excipient by using dynamic light scattering (DLS), UV-visible spectroscopy, transmission electron microscopy (TEM), Fourier-transform infrared (FTIR) spectroscopy, and nuclear magnetic resonance (NMR) methods. Moreover, MTT assay, cell cycling, and dye staining methods were performed to study the cytotoxicity and apoptotic potential of the NF of chlorambucil in HCT-116 cell lines. The cellular uptake study elucidated the internalization of NF by cancer cells. Microtubule affinity regulating kinase-4 (MARK-4) as a cancer-targeted protein was purified to study the complex formation, conformational changes, dynamics, and stability of proteins upon binding of CH by using multi-spectroscopic and
in silico
methods.
Cancer, as a multifactorial disease, damages healthy cells and organs.
Silicon-based, high-energy-density electrodes show severe microstructural degradation due to continuous expansion and contraction upon charging and discharging. This mechanical degradation behaviour ...affects the cell’s lifetime by changing the microstructure morphology, altering transport parameters, and active volume losses. Since direct experimental observations of mechanical degradation are challenging, we develop a computer simulation approach that is based on real three-dimensional electrode microstructures. By assuming quasi-static cycling and taking into account the mechanical properties of the electrode’s constituents we calculate the heterogeneous deformation and resulting morphological changes. Additionally, we implement an ageing model that allows us to compute a heterogeneously evolving damage field over multiple cycles. From the damage field, we infer the remaining electrode capacity. Using this technique, an anode blend of graphite particles and silicon carbon composite particles (SiC-C) as well as a cathode consisting of Lithium-Nickel-Manganese-Cobalt Oxide with molar ratio of 8:1:1 (NMC811) are studied. In a two-level homogenization approach, we compute, firstly, the effective mechanical properties of silicon composite particles and, secondly, the whole electrode microstructure. By introducing the damage strain ratio, the degradation evolution of the graphite SiC-C anode blend is studied for up to 95 charge-discharge cycles. With this work, we demonstrate an approach to how mechanical damage of battery electrodes can be treated efficiently. This is the basis for a full coupling to electrochemical simulations.
Dentin hypersensitivity (DH) is one of the most challenging and persistent dental complaints characterized by transient, intense pain triggered by various stimuli. It affects a significant portion of ...the global population, predominantly those aged 20-40. This study aims to evaluate the desensitizing efficacy of seventh-generation dentin bonding agents (Single Bond Universal by 3 M ESPE and Xeno-V + by Dentsply) against a control group using Bifluorid 12 by Voco in mitigating DH within a month of the follow-up period.
This was a single-center, parallel-group, double-blind, controlled randomized clinical trial conducted at Dow University of Health Sciences, Karachi, Pakistan. A total of 105 patients with DH were allocated into three groups for this study. The patients were divided into three groups (Single Bond Universal by 3 M ESPE and Xeno-V + by Dentsply) and the control group containing fluoride varnish (Bifluorid 12 by Voco). Discomfort Interval Scale scores and Schiff Cold Air Sensitivity Scale scores were recorded at baseline, immediately after the intervention, after 01 weeks, and after 01 month.
All the materials demonstrated a statistically significant reduction in discomfort and sensitivity (DIS scores p-value 0.01) immediately after 01 week and over a period of 01 month after treatment compared with the baseline scores before application, with no single material proving superior over the one-month observation period. The study also provided insights into dental hygiene practices, with a significant majority using a toothbrush and sensitivity patterns, with cold stimuli being the most common cause of sensitivity.
The study demonstrates that Single Bond Universal, Xeno V+, and Bifluorid 12 are equally effective in reducing dentin hypersensitivity, with no distinct superiority observed over a one-month period. The findings highlight the potential of fluoride varnishes as a less technique-sensitive and cost-effective option for treating DH, offering valuable insights for future research and clinical practice.
NCT04225247 ( https://clinicaltrials.gov/study/NCT04225247 ), Date of Registration: 13/01/2020. (Retrospectively registered).
For the modern power systems with intensive renewable energy sources integration, the uncertainty analysis of its performance become necessary for desired decision making. An appropriate choice of ...modelling and handling methodology, for uncertain parameters associated with the renewable energy, remains one of major concerns for the power system engineers. This paper presents a stochastic optimization framework for some critical decisions making on reactive power dispatch in two consecutive stages by incorporating both generation outputs of wind farms along with load demand uncertainties. The proposed method is generic for optimal reactive power dispatch in any modern wind-integrated power system. A methodology based on scenario-based analysis is adopted for handling these uncertainties. Moreover, a hybrid fuzzy evolutionary algorithm (HFEA) is applied to obtain high-quality solutions for this complex two-stage stochastic optimization problem. Furthermore, the proposed HFEA-based stochastic optimization approach is tested on IEEE 14-bus power system with two distinct wind farms. The simulation results prove the robustness of HFEA for generating feasible and optimal solutions for all the considered scenarios.
We present a quasi-Yagi antenna mounted on a periodic surface for a wearable UHF RFID reader operating in the UHF RFID frequency band centered at 915 MHz. The periodic surface was co-optimized with ...the antenna to enhance the launching of surface waves to enable the end-fire radiation along the forearm so that a user can identify objects by pointing her/his hand towards them. In addition to the radiation pattern modification, the ground plane of the periodic surface serves the second purpose of isolating the antenna from the human body. We optimized the antenna in a full-wave EM simulator using a simplified cylindrical model of the forearm and in the simulation, it achieved the end-fire directivity of 5.9 dBi along the forearm. In the wireless testing, the quasi-Yagi antenna provided the read range of 3.8 m for a typical UHF RFID tag having 0 dBi gain when the reader's output power was 32 dBm that corresponds with EIRP =0.56 W and SAR =0.191 W/kg in our simulations. Considering both, the RFID emission regulations with EIRP = 3.28 W or 4 W and the SAR limit of 1.6 W/kg averaged over 1 gram of tissue, the read range could be further enhanced for reader units with higher output power.
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