Repressive Mutations Restore Function-Loss Caused by the Disruption of Trimerization in \u3cem\u3eEscherichia coli\u3c/em\u3e Multidrug Transporter AcrB

AcrAB-TolC and their homologs are major multidrug efflux systems in Gram-negative bacteria. The inner membrane component AcrB functions as a trimer. Replacement of Pro223 by Gly in AcrB decreases the trimer stability and drastically reduces the drug efflux activity. The goal of this study is to identify suppressor mutations that restore function to mutant AcrBP223G and explore the mechanism of function recovery. Two methods were used to introduce random mutations into the plasmid of AcrBP223G. Mutants with elevated drug efflux activity were identified, purified, and characterized to examine their expression level, trimer stability, interaction with AcrA, and substrate binding. Nine single-site repressor mutations were identified, including T199M, D256N, A209V, G257V, M662I, Q737L, D788K, P800S, and E810K. Except for M662I, all other mutations located in the docking region of the periplasmic domain. While three mutations, T199M, A209V, and D256N, significantly increased the trimer stability, none of them restored the trimer affinity to the wild type level. M662, the only site of mutation that located in the porter domain, was involved in substrate binding. Our results suggest that the function loss resulted from compromised AcrB trimerization could be restored through various mechanisms involving the compensation of trimer stability and substrate binding

Repressive Mutations Restore Function-Loss Caused by the Disruption of Trimerization in \u3cem\u3eEscherichia coli\u3c/em\u3e Multidrug Transporter AcrB

AcrAB-TolC and their homologs are major multidrug efflux systems in Gram-negative bacteria. The inner membrane component AcrB functions as a trimer. Replacement of Pro223 by Gly in AcrB decreases the trimer stability and drastically reduces the drug efflux activity. The goal of this study is to identify suppressor mutations that restore function to mutant AcrBP223G and explore the mechanism of function recovery. Two methods were used to introduce random mutations into the plasmid of AcrBP223G. Mutants with elevated drug efflux activity were identified, purified, and characterized to examine their expression level, trimer stability, interaction with AcrA, and substrate binding. Nine single-site repressor mutations were identified, including T199M, D256N, A209V, G257V, M662I, Q737L, D788K, P800S, and E810K. Except for M662I, all other mutations located in the docking region of the periplasmic domain. While three mutations, T199M, A209V, and D256N, significantly increased the trimer stability, none of them restored the trimer affinity to the wild type level. M662, the only site of mutation that located in the porter domain, was involved in substrate binding. Our results suggest that the function loss resulted from compromised AcrB trimerization could be restored through various mechanisms involving the compensation of trimer stability and substrate binding

Functional Relevance of AcrB Trimerization in Pump Assembly and Substrate Binding

AcrB is a multidrug transporter in the inner membrane of Escherichia coli. It is an obligate homotrimer and forms a tripartite efflux complex with AcrA and TolC. AcrB is the engine of the efflux machinery and determines substrate specificity. Active efflux depends on several functional features including proton translocation across the inner membrane through a proton relay pathway in the transmembrane domain of AcrB; substrate binding and migration through the substrate translocation pathway; the interaction of AcrB with AcrA and TolC; and the formation of AcrB homotrimer. Here we investigated two aspects of the inter-correlation between these functional features, the dependence of AcrA-AcrB interaction on AcrB trimerization, and the reliance of substrate binding and penetration on protein-protein interaction. Interaction between AcrA and AcrB was investigated through chemical crosslinking, and a previously established in vivo fluorescent labeling method was used to probe substrate binding. Our data suggested that dissociation of the AcrB trimer drastically decreased its interaction with AcrA. In addition, while substrate binding with AcrB seemed to be irrelevant to the presence or absence of AcrA and TolC, the capability of trimerization and conduction of proton influx did affect substrate binding at selected sites along the substrate translocation pathway in AcrB

Spring-IMU Fusion Based Proprioception for Feedback Control of Soft Manipulators

This paper presents a novel framework to realize proprioception and closed-loop control for soft manipulators. Deformations with large elongation and large bending can be precisely predicted using geometry-based sensor signals obtained from the inductive springs and the inertial measurement units (IMUs) with the help of machine learning techniques. Multiple geometric signals are fused into robust pose estimations, and a data-efficient training process is achieved after applying the strategy of sim-to-real transfer. As a result, we can achieve proprioception that is robust to the variation of external loading and has an average error of 0.7% across the workspace on a pneumatic-driven soft manipulator. The realized proprioception on soft manipulator is then contributed to building a sensor-space based algorithm for closed-loop control. A gradient descent solver is developed to drive the end-effector to achieve the required poses by iteratively computing a sequence of reference sensor signals. A conventional controller is employed in the inner loop of our algorithm to update actuators (i.e., the pressures in chambers) for approaching a reference signal in the sensor-space. The systematic function of closed-loop control has been demonstrated in tasks like path following and pick-and-place under different external loads

Operator radiation dose during trans-hepatic arterial chemoembolization: different patients’ positions via transradial or transfemoral access

PURPOSEThis study aimed to compare the radiation dose received by the operator among different patients’ positions via transradial access (TRA) or transfemoral access (TFA) during transarterial chemoembolization (TACE) for hepatocellular carcinoma (HCC).METHODSA total of 120 patients with HCC undergoing TACE for the first time between January and November 2019 were randomized into 4 groups with 30 patients in each group. In group A, patients were placed in the foot-first position with the left upper arm abducted, and TACE was performed via the left radial artery. In group B, patients were placed in the conventional headfirst position with the left hand placed at the left groin, and TACE was performed via the left radial artery. In group C, patients were placed in the conventional head-first position, and TACE was performed via the right radial artery. In group D, patients were placed in the conventional head-first position, and TACE was performed via the right femoral artery. Before each procedure, thermoluminescent dosimeters were taped at 7 different body parts of the operator and the radiation dose was measured and collected after the procedure. The normalized radiation dose was also calculated. Procedural parameters included radiation dose, fluoroscopy time (FT), dose–area product (DAP), and air kerma (AK) were recorded. Patients’ demographics, tumor baseline characteristics, radiation dose, and procedural parameters were compared between groups.RESULTSNo significant differences were found in patients’ demographics, tumor baseline characteristics, as well as in total FT, DAP, and AK. However, significant differences were found in the total radiation dose received by the operator and the doses on the pelvic cavity and the right wrist (P < .05). In group C, the radiation doses received on the pelvic cavity, the right wrist, and the total radiation doses were relatively higher. Significant differences were also found in the normalized radiation doses received by the operator on the thyroid, chest, left wrist, right wrist, and pelvic cavity, and the total normalized doses (all P < .05). Similarly, the radiation doses received by the operator at the aforementioned parts in group C were higher, while those in group A were lower.CONCLUSIONNo statistically significant differences were observed in the FT, DAP, and AK in TACE via TRA when patients were placed in different positions. However, TACE via the left TRA, with patients in the feet-first position, reduced the radiation dose received by the operator, thereby reducing the radiation risk

On-Chip Super-Resolution Imaging with Fluorescent Polymer Films

Wide field of view (FOV), label-free super-resolution imaging is demonstrated using a specially designed waveguide chip that can illuminate a sample with multi-colour evanescent waves travelling along different directions. The method is enabled by a polymer fluorescent film which emits over a broad wavelength range. Its polygonal geometry ensures coverage over all illumination directions, enabling high fidelity image reconstruction whilst minimizing distortion and image blurring. By frequency shifting and iterative stitching of different spatial frequencies in Fourier space, the reconstruction of two dimensional samples is achieved without distortion over wide FOVs. The fabrication process is facile and compatible with conventional semiconductor-fabrication methods. The super-resolution chip (SRC) can thus be produced with high yield, offer opportunities for potential conjunction of super-resolution techniques integrated optical circuits or for the development of single-use diagnostic kits

Speech enhancement under low SNR conditions via noise estimation using sparse and low-rank NMF with Kullback–Leibler divergence

© 2015 IEEE. A key stage in speech enhancement is noise estimation which usually requires prior models for speech or noise or both. However, prior models can sometimes be difficult to obtain. In this paper, without any prior knowledge of speech and noise, sparse and low-rank nonnegative matrix factorization (NMF) with Kullback-Leibler divergence is proposed to noise and speech estimation by decomposing the input noisy magnitude spectrogram into a low-rank noise part and a sparse speech-like part. This initial unsupervised speech-noise estimation allows us to set a subsequent regularized version of NMF or convolutional NMF to reconstruct the noise and speech spectrogram, either by estimating a speech dictionary on the fly (categorized as unsupervised approaches) or by using a pre-trained speech dictionary on utterances with disjoint speakers (categorized as semi-supervised approaches). Information fusion was investigated by taking the geometric mean of the outputs from multiple enhancement algorithms. The performance of the algorithms were evaluated on five metrics (PESQ, SDR, SNR, STOI, and OVERALL) by making experiments on TIMIT with 15 noise types. The geometric means of the proposed unsupervised approaches outperformed spectral subtraction (SS), minimum mean square estimation (MMSE) under low input SNR conditions. All the proposed semi-supervised approaches showed superiority over SS and MMSE and also obtained better performance than the state-of-the-art algorithms which utilized a prior noise or speech dictionary under low SNR conditions.Sun M., Li Y., Gemmeke J.F., Zhang X., ''Speech enhancement under low SNR conditions via noise estimation using sparse and low-rank NMF with Kullback–Leibler divergence'', IEEE/ACM transactions on audio, speech, and language processing, vol. 23, no. 7, pp. 1233-1242, July 2015.status: publishe

Targeted inhibition of human hematological cancers in vivo by doxorubicin encapsulated in smart lipoic acid-crosslinked hyaluronic acid nanoparticles

The chemotherapy of hematological cancers is challenged by its poor selectivity that leads to low therapeutic efficacy and pronounced adverse effects. Here, we report that doxorubicin encapsulated in lipoic acid-crosslinked hyaluronic acid nanoparticles (LACHA-DOX) mediate highly efficacious and targeted inhibition of human hematological cancers including LP-1 human multiple myeloma (MM) and AML-2 human acute myeloid leukemia xenografted in nude mice. LACHA-DOX had a size of ca. 183 nm and a DOX loading content of ca. 12.0 wt.%. MTT and flow cytometry assays showed that LACHA-DOX possessed a high targetability and antitumor activity toward CD44 receptor overexpressing LP-1 human MM cells and AML-2 human acute myeloid leukemia cells. The in vivo and ex vivo images revealed that LACHA-DOX achieved a significantly enhanced accumulation in LP-1 and AML-2 tumor xenografts. Notably, LACHA-DOX effectively suppressed LP-1 as well as AML-2 tumor growth and drastically increased mice survival rate as compared to control groups receiving free DOX or PBS. Histological analyses exhibited that LACHA-DOX caused little damage to the major organs like liver and heart. This study provides a proof-of-concept that lipoic acid-crosslinked hyaluronic acid nanoparticulate drugs may offer a more safe and effective treatment modality for CD44 positive hematological malignancies