MODULATION OF THE NF-KAPPA B SIGNALING PATHWAY BY THE BACTERIAL TYPE III SECRETION SYSTEM EFFECTORS

The type III secretion system (T3SS) is a bacterial injection system expressed by many Gram-negative bacteria. During the last two decades, the repertoire of T3SS effectors has been greatly explored, and several mechanisms of these effectors have been discovered. The identified host targets of T3SS effectors are involved in different biological events including cytoskeleton rearrangement, cellular signaling, transcription and protein degradation. A/E (attaching and effacing) pathogens including EHEC (Enterohaemorrhaigic E. coli), EPEC (Enteropathogenic E. coli) and C. rodentium (Citrobacter rodentium), a pathogen of mice, inhibit NF-κB transcriptional activity by employing unidentified T3SS effectors. However, the identity of these effector(s) was unknown. In this thesis, my goals were to identify T3SS effectors from attaching and effacing (A/E) pathogens responsible for modulating NF-κB activation and reveal the working mechanism of these identified effectors. In the first project, NleH1 and NleH2, which share C-termini similarity with the S. flexneri T3SS effector OspG, were studied. OspG targets ubiquitin-conjugating enzyme UbcH5 to prevent IκBα degredation, which results in the inhibition of NF-κB activation. We discovered that both NleH1 and NleH2 interact with the N-terminus of ribosomal protein S3 (RPS3) after their translocation into host cells. RPS3 is a non-Rel NF-κB subunit which promotes the DNA binding affinity of NF-κB. We found that NleH1, but not NleH2, blocks the nuclear translocation of RPS3 stimulated by TNF and by bacterial infection. By this process, NleH1 selectively attenuates RPS3-mediated NF-κB dependent gene transcription. In addition, we discovered NleH proteins as Ser/Thr kinases and that kinase activity is critical for the effect of NleH1 on RPS3. By collaborating with Dr. Lenardo's group in National Institute of Allergy and Infectious Diseases (NIAID), we discovered that RPS3 is inducibly associated with and phosphorylated by IKKβ at serine 209 (S209) and that NleH1 efficiently blocks this phosphorylation. Moreover, by using a gnotobiotic pig model, we found piglets infected with wild-type E. coli O157:H7 exhibits diffuse and low intensity phospho-RPS3 staining. Surprisingly, although infection by ΔnleH1 EHEC causes mild diarrhea and displays significantly reduced bacterial colonization in piglets, this mutant becomes hypervirulent to the host, as infected piglets die more rapidly and develop systemic intoxication compared to infection with the wild-type strain. Therefore, our data suggests a complex role for NleH1 in mediating bacterial virulence in the host to maximize bacterial survival and growth. In the second project, we identified NleB, another T3SS effector known to target NF-κB activation, as an O-GlcNAc (O-linked N-acetylglucosamine) transferase. We found NleB directly interacts with host glycolytic protein GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) and O-GlcNAcylates GAPDH during infection. GAPDH has many nonglycolytic roles and is involved in a broad range of biological events, such as transcription, cell signaling, membrane integrity and cell survivial. In our study, we demonstrated an essential role for GAPDH in NF-κB activation. We found that GAPDH serves as a co-activator of TRAF2 (TNF receptor-associated factor 2) and promotes TRAF2 polyubiquitination under stress conditions. Targeting the catalytic site C150 of GAPDH by a chemical inhibitor or by site-directed mutagenesis specifically impairs TNF-induced TRAF2 polyubiquitination and NF-κB activation. This function is unrelated to glycolysis, as targeting the rate-limiting glycolytic enzymes does not imipair TRAF2 activation and only leads to a moderate inhibition on NF-κB activation, which is likely due to an unrelated mechanism. Moreover, O-GlcNAcylated GAPDH fails to interact with TRAF2 resulting in the attenuation of TRAF2 polyubiquitination. Eliminating the O-GlcNAc transferase activity of NleB by mutating its catalytic sites or by deleting the nucleotide sugar-binding domain abolishes the effect of NleB on NF-κB activation and reduces bacteria colonization of mice. Taken together, our studies suggest an integral role of the metabolic protein GAPDH in the NF-κB signaling pathway and that the T3SS effector NleB O-GlcNAcylates GAPDH to prevent the participation of GAPDH in NF-κB signaling

Sub-Band Knowledge Distillation Framework for Speech Enhancement

In single-channel speech enhancement, methods based on full-band spectral features have been widely studied. However, only a few methods pay attention to non-full-band spectral features. In this paper, we explore a knowledge distillation framework based on sub-band spectral mapping for single-channel speech enhancement. Specifically, we divide the full frequency band into multiple sub-bands and pre-train an elite-level sub-band enhancement model (teacher model) for each sub-band. These teacher models are dedicated to processing their own sub-bands. Next, under the teacher models' guidance, we train a general sub-band enhancement model (student model) that works for all sub-bands. Without increasing the number of model parameters and computational complexity, the student model's performance is further improved. To evaluate our proposed method, we conducted a large number of experiments on an open-source data set. The final experimental results show that the guidance from the elite-level teacher models dramatically improves the student model's performance, which exceeds the full-band model by employing fewer parameters.Comment: Published in Interspeech 202

Structure of the substrate-engaged SecA-SecY protein translocation machine.

The Sec61/SecY channel allows the translocation of many proteins across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. In bacteria, most secretory proteins are transported post-translationally through the SecY channel by the SecA ATPase. How a polypeptide is moved through the SecA-SecY complex is poorly understood, as structural information is lacking. Here, we report an electron cryo-microscopy (cryo-EM) structure of a translocating SecA-SecY complex in a lipid environment. The translocating polypeptide chain can be traced through both SecA and SecY. In the captured transition state of ATP hydrolysis, SecAs two-helix finger is close to the polypeptide, while SecAs clamp interacts with the polypeptide in a sequence-independent manner by inducing a short β-strand. Taking into account previous biochemical and biophysical data, our structure is consistent with a model in which the two-helix finger and clamp cooperate during the ATPase cycle to move a polypeptide through the channel

Receiver Function Imaging of Mantle Transition Zone Discontinuities Beneath the Tanzania Craton and Adjacent Segments of the East African Rift System

The mantle transition zone (MTZ) discontinuities beneath the Tanzania Craton and the Eastern and Western Branches of the East African Rift System are imaged by stacking over 7,100 receiver functions. The mean thickness of the MTZ beneath the Western Branch and Tanzania Craton is about 252 km, which is comparable to the global average and is inconsistent with the existence of present-day thermal upwelling originating from the lower mantle. In contrast, beneath the Eastern Branch, an up to 30 km thinning of the MTZ is observed and is attributable to upwelling of higher temperature materials from either the upper MTZ or the lower mantle. The observations are in agreement with the hypothesis that rifting in Africa is primarily driven by gradients of gravitational potential energy and lateral variations of basal traction force along zones of significant changes of lithospheric thickness such as the edges of the Tanzania Craton

Characterization of anti-leukemia components from Indigo naturalis using comprehensive two-dimensional K562/cell membrane chromatography and in silico target identification.

Traditional Chinese Medicine (TCM) has been developed for thousands of years and has formed an integrated theoretical system based on a large amount of clinical practice. However, essential ingredients in TCM herbs have not been fully identified, and their precise mechanisms and targets are not elucidated. In this study, a new strategy combining comprehensive two-dimensional K562/cell membrane chromatographic system and in silico target identification was established to characterize active components from Indigo naturalis, a famous TCM herb that has been widely used for the treatment of leukemia in China, and their targets. Three active components, indirubin, tryptanthrin and isorhamnetin, were successfully characterized and their anti-leukemia effects were validated by cell viability and cell apoptosis assays. Isorhamnetin, with undefined cancer related targets, was selected for in silico target identification. Proto-oncogene tyrosine-protein kinase (Src) was identified as its membrane target and the dissociation constant (Kd) between Src and isorhamnetin was 3.81 μM. Furthermore, anti-leukemia effects of isorhamnetin were mediated by Src through inducing G2/M cell cycle arrest. The results demonstrated that the integrated strategy could efficiently characterize active components in TCM and their targets, which may bring a new light for a better understanding of the complex mechanism of herbal medicines

Coupling fault transfer characteristics of fixed-axis gear crack and planetary gear missing tooth

The test signal of multistage gear transmission system is complex. When coupling faults occur in the system, it is often difficult to accurately extract all kinds of fault features. In the past studies, we found that the fault signals have modulation and coupling effects. The failure of a gear is often reflected in the power spectrum in the form of associated changes in the meshing frequency of other stages. The separation of these features will facilitate the analysis of the coupling effects of multiple faults. The transfer characteristic method provides an effective method for decomposing such signal features. In this paper, through the analysis of transfer characteristics, the coupling fault transfer characteristics and the associated influence relationship of fixed-axis gear crack and planetary gear missing tooth are revealed. The contact force signals of each pair of gears with the coupling fault are obtained by dynamic simulation. The test signals of the fixed-axis gearbox and planetary gearbox are obtained under normal and coupling fault states. According to the method of system identification, the corresponding transfer function model of each path is established. The transfer process is revealed by the analysis of transfer characteristics. The association between two kinds of faults is found, which does not exist in a single fault and is difficult to obtain by signal analysis. This study reveals the association and vibration mechanism of the coupled fault, which provides a theoretical basis for fault diagnosis of multistage gears

Low-Cost Smart Antenna Using Active Frequency Selective Surfaces

Smart antenna is a key technology for advanced wireless systems and one of the most important features of smart antenna is electronically beam scanning or switching. It is highly desirable to reduce the mass, power consumption and cost of smart antennas, as the traditional phased array is always associated with high cost due to the use of many T/R modules and complicated beamforming network (BFN). This paper presents the University of Kent's recent research progress in the field of low-cost smart antenna design using active frequency selective surfaces (AFSS). Firstly, this paper presents a brief review of AFSS based beam-reconfigurable antenna including several recent designs reported by the authors' group. Then, a new high-gain AFSS antenna design with some preliminary results will be presented. This is design achieves higher gain than the reported AFSS antennas. A detailed list of references is given at the end of this paper