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1. chinaXiv:201605.01734 [pdf]

Real-Time Monitoring Surface Chemistry-Dependent In Vivo Behaviors of Protein Nanocages via Encapsulating an NIR-II Ag2S Quantum Dot

Li, Chunyan; Zhang, Yejun; Wang, Qiangbin; Li, Feng; Zhang, Wenjing; Zhang, Xian-En
Subjects: Biology >> Biophysics

Protein nanocages (PNCs) have been recognized as a promising platform for nanomedicine innovation. Real-time in vivo tracking of PNCs can provide critically important information for the development of PNC-based diagnostics and therapeutics. Here we demonstrate a general strategy for monitoring the behaviors of PNCs in vivo by encapsulating a Ag2S quantum dot (QD) with fluorescence in the second near-infrared window (NIR-II, 1000-1700 nm) inside the PNC, using simian virus 40 (SV40) PNC (PNCSV40) as a model. Benefiting from the high spatiotemporal resolution and deep tissue penetration of NIR-II fluorescence imaging, the dynamic distribution of the PNCSV40 in living mice was tracked in real time with high fidelity, and adopting the PEGylation strategy, surface chemistry-dependent in vivo behaviors of PNCSV40 were clearly revealed. This study represents the first evidence of real-time tracking of the intrinsic behaviors of PNCs in vivo without interference in PNC-host interactions by encapsulating nanoprobes inside. The as-described imaging strategy will facilitate the study of interactions between exogenously introduced PNCs and host body and prompt the development of future protein-based drugs, sensors, and high-efficacy targeted delivery systems.

submitted time 2016-05-15 Hits3020Downloads1255 Comment 0

2. chinaXiv:201605.01493 [pdf]

Self-Assembly of Ferritin Nanoparticles into an Enzyme Nanocomposite with Tunable Size for Ultrasensitive Immunoassay

Men, Dong; Zhou, Juan; Zhang, Zhi-Ping; Cui, Zong-Qiang; Deng, Jiao-Yu; Wang, Dian-Bing; Zhang, Xian-En; Men, Dong; Zhang, Ting-Ting; Hou, Li-Wei; Shi, Yuan-Yuan; Zhang, Jin-Li
Subjects: Biology >> Biophysics

The self-assembly of nanoparticles into larger superstructures is a powerful strategy to develop novel functional nanomaterials, as these superstructures display collective properties that are different to those displayed by individual nanoparticles or bulk samples. However, there are increasing bottlenecks in terms of size control and multifunctionalization of nanoparticle assemblies. In this study, we developed a self-assembly strategy for construction of multifunctional nanoparticle assemblies of tunable size, through rational regulation of the number of self-assembling interaction sites on each nanoparticle. As proof-of-principle, a size-controlled enzyme nanocomposite (ENC) was constructed by self-assembly of streptavidin-labeled horseradish peroxidase (SA-HRP) and autobiotinylated ferritin nanoparticles (bFNP). Our ENC integrates a large number of enzyme molecules, together with a streptavidin-coated surface, allowing for a drastic increase in enzymatic signal when the SA is bound to a biotinylated target molecule. As result, a 10000-fold increase in sensitivity over conventional enzyme-linked immunosorbent assays (ELISA) methods was achieved in a cardiac troponin immunoassay. Our method presented here should provide a feasible approach for constructing elaborate multifunctional superstructures of tunable size useful for a broad range of biomedical applications.

submitted time 2016-05-12 Hits1298Downloads758 Comment 0

3. chinaXiv:201605.01459 [pdf]

Structure and function of Mycobacterium smegmatis 7-keto-8-aminopelargonic acid (KAPA) synthase

Fan, Shanghua; Chen, Guanjun; Li, De-Feng; Wang, Da-Cheng; Fleming, Joy; Zhang, Hongtai; Zhou, Ying; Zhang, Xian-En; Bi, Lijun; Li, De-Feng; Wang, Da-Cheng; Fleming, Joy; Zhang, Hongtai; Zhou, Ying; Zhang, Xian-En; Bi, Lijun; Zhou, Lin; Chen, Tao; Zhou, Jie
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

The biotin biosynthesis pathway is an attractive target for development of novel drugs against mycobacterial pathogens, however there are as yet no suitable inhibitors that target this pathway in mycobacteria. 7-Keto-8-aminopelargonic acid synthase (KAPA synthase, BioF) is the enzyme which catalyzes the first committed step of the biotin synthesis pathway, but both its structure and function in mycobacteria remain unresolved. Here we present the crystal structure of Mycobacterium smegmatis BioF (MsBioF). The structure reveals an incomplete dimer, and the active site organization is similar to, but distinct from Escherichia coli 8-amino-7-oxononanoate synthase (EcAONS), the E. coli homologue of BioF. To investigate the influence of structural characteristics on the function of MsBioF, we deleted bioF in M. smegmatis and confirmed that BioF is required for growth in the absence of exogenous biotin. Based on structural and mutagenesis studies, we confirmed that pyridoxal 5'-phosphate (PLP) binding site residues His129, Lys235 and His200 are essential for MsBioF activity in vivo and residue Glul 71 plays an important, but not essential role in MsBioF activity. The N-terminus (residues 1-37) is also essential for MsBioF activity in vivo. The structure and function of MsBioF reported here provides further insights for developing new anti-tuberculosis inhibitors aimed at the biotin synthesis pathway. (C) 2014 Elsevier Ltd. All rights reserved.

submitted time 2016-05-12 Hits1300Downloads747 Comment 0

4. chinaXiv:201605.01440 [pdf]

A S-Layer Protein of Bacillus anthracis as a Building Block for Functional Protein Arrays by In Vitro Self-Assembly

Wang, Xu-Ying; Wang, Dian-Bing; Bi, Li-Jun; Zhang, Xian-En; Wang, Xu-Ying; Zhang, Ji-Bin; Zhang, Zhi-Ping; Ding, Wei
Subjects: Biology >> Biophysics

S-layer proteins create a cell-surface layer architecture in both bacteria and archaea. Because S-layer proteins self-assemble into a native-like S-layer crystalline structure in vitro, they are attractive building blocks in nanotechnology. Here, the potential use of the S-layer protein EA1 from Bacillus anthracis in constructing a functional nanostructure is investigated, and apply this nanostructure in a proof-of-principle study for serological diagnosis of anthrax. EA1 is genetically fused with methyl parathion hydrolase (MPH), to degrade methyl parathion and provide a label for signal amplifi cation. EA1 not only serves as a nanocarrier, but also as a specific antigen to capture anthrax-specific antibodies. As results, purified EA1-MPH forms a single layer of crystalline nanostructure through self-assembly. Our chimeric nanocatalyst greatly improves enzymatic stability of MPH. When applied to the detection of anthrax-specific antibodies in serum samples, the detection of our EA1-MPH nanostructure is nearly 300 times more sensitive than that of the unassembled complex. Together, it is shown that it is possible to build a functional and highly sensitive nanosensor based on S-layer protein. In conclusion, our present study should serve as a model for the development of other multifunctional nanomaterials using S-layer proteins.

submitted time 2016-05-12 Hits1204Downloads710 Comment 0

5. chinaXiv:201605.01400 [pdf]

Core component EccB1 of the Mycobacterium tuberculosis type VII secretion system is a periplasmic ATPase

Zhang, Xiao-Li; Zhang, Xian-En; Zhang, Xiao-Li; Li, De-Feng; Fleming, Joy; Wang, Li-Wei; Zhou, Ying; Wang, Da-Cheng; Zhang, Xian-En; Bi, Li-Jun; Zhang, Xiao-Li; Li, De-Feng; Fleming, Joy; Wang, Li-Wei; Zhou, Ying; Wang, Da-Cheng; Zhang, Xian-En; Bi, Li-Jun; Zhang, Xiao-Li
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

Pathogenic mycobacteria transport virulence factors across their complex cell wall via a type VII secretion system (T7SS)/early secreted antigenic target-6 of kDa secretion system (ESX). ESX conserved component (Ecc) B, a core component of the T7SS architecture, is predicted to be a membrane bound protein, but little is known about its structure and function. Here, we characterize EccB1, showing that it is an ATPase with no sequence or structural homology to other ATPases located in the cell envelope of Mycobacterium tuberculosis H37Rv. We obtained the crystal structure of an EccB1-DN72 truncated transmembrane helix and performed modeling and ATP docking studies, showing that EccB1 likely exists as a hexamer. Sequence alignment and ATPase activity determination of EccB1 homologues indicated the presence of 3 conserved motifs in the N- and C-terminals of EccB1-DN72 that assemble together between 2 membrane proximal domains of the EccB1-DN72 monomer. Models of the EccB1 hexamer show that 2 of the conserved motifs are involved in ATPase activity and form an ATP binding pocket located on the surface of 2 adjacent molecules. Our results suggest that EccB may act as the energy provider in the transport of T7SS virulence factors and may be involved in the formation of a channel across the mycomembrane.

submitted time 2016-05-12 Hits1481Downloads913 Comment 0

6. chinaXiv:201605.01364 [pdf]

Novel near-infrared BiFC systems from a bacterial phytochrome for imaging protein interactions and drug evaluation under physiological conditions

Chen, Minghai; Li, Wei; Zhang, Zhiping; Liu, Sanying; Zhang, Xiaowei; Zhang, Xian-En; Cui, Zongqiang; Zhang, Xian-En; Chen, Minghai; Liu, Sanying
Subjects: Biology >> Biophysics >> Imaging Medicine and Biomedical Engineering

Monitoring protein protein interactions (PPIs) in live subjects is critical for understanding these fundamental biological processes. Bimolecular fluorescence complementation (BiFC) provides a good technique for imaging PPIs; however, a BiFC system with a long wavelength remains to be pursued for in vivo imaging. Here, we conducted systematic screening of split reporters from a bacterial phytochrome-based, near-infrared fluorescent protein (iRFP). Several new near-infrared phytochrome BiFC systems were built based on selected split sites including the amino acids residues 97/98, 99/100,122/123, and 123/124. These new near-infrared BiFC systems from a bacterial phytochrome were verified as powerful tools for imaging PPIs under physiological conditions in live cells and in live mice. The interaction between HIV-1 integrase (IN) and cellular cofactor protein Lens epithelium-derived growth factor (LEDGF/p75) was visualized in live cells using the newly constructed iRFP BiFC system because of its important roles in HIV-1 integration and replication. Because the HIV IN-LEDGF/p75 interaction is an attractive anti-HIV target, drug evaluation assays to inhibit the HIV IN-LEDGF/p75 interaction were also performed using the newly constructed BiFC system. The results showed that compound 6 and carbidopa inhibit the HIV IN-LEDGF/p75 interaction in a dose-dependent manner under physiological conditions in the BiFC assays. This study provides novel near-infrared BiFC systems for imaging protein interactions under physiological conditions and provides guidance for splitting other bacterial phytochrome-like proteins to construct BiFC systems. The study also provides a new method for drug evaluation in live cells based on iRFP BiFC systems and supplies some new information regarding candidate drugs for anti-HIV therapies. (C) 2015 Elsevier Ltd. All rights reserved.

submitted time 2016-05-12 Hits2340Downloads1310 Comment 0

7. chinaXiv:201605.01262 [pdf]

Construction of a chimeric lysin Ply187N-V12C with extended lytic activity against staphylococci and streptococci

Dong, Qiuhua; Dong, Qiuhua; Wang, Jing; Yang, Hang; Wei, Cuihua; Yu, Junping; Zhang, Yun; Huang, Yanling; Wei, Hongping; Zhang, Xian-En
Subjects: Biology >> Biophysics

Developing chimeric lysins with a wide lytic spectrum would be important for treating some infections caused by multiple pathogenic bacteria. In the present work, a novel chimeric lysin (Ply187N-V12C) was constructed by fusing the catalytic domain (Ply187N) of the bacteriophage lysin Ply187 with the cell binding domain (146-314aa, V12C) of the lysin PlyV12. The results showed that the chimeric lysin Ply187N-V12C had not only lytic activity similar to Ply187N against staphylococcal strains but also extended its lytic activity to streptococci and enterococci, such as Streptococcusdysgalactiae, Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecium and Enterococcus faecalis, which Ply187N could not lyse. Our work demonstrated that generating novel chimeric lysins with an extended lytic spectrum was feasible through fusing a catalytic domain with a cell-binding domain from lysins with lytic spectra across multiple genera.

submitted time 2016-05-11 Hits1034Downloads608 Comment 0

8. chinaXiv:201605.01261 [pdf]

Detection of Bacillus anthracis spores by super-paramagnetic lateral-flow immunoassays based on 'Road Closure'

Wang, Dian-Bing; Fleming, Joy; Bi, Li-Jun; Zhang, Xian-En; Tian, Bo; Zhang, Zhi-Ping; Wang, Xu-Ying; Tian, Bo; Yang, Rui-Fu
Subjects: Biology >> Biophysics

Detection of Bacillus anthracis in the field, whether as a natural infection or as a biothreat remains challenging. Here we have developed a new lateral-flow immunochromatographic assay (LFIA) for B. anthracis spore detection based on the fact that conjugates of B. anthracis spores and super-paramagnetic particles labeled with antibodies will block the pores of chromatographic strips and form retention lines on the strips, instead of the conventionally reported test lines and control lines in classic LFIA. As a result, this new LFIA can simultaneously realize optical, magnetic and naked-eye detection by analyzing signals from the retention lines. As few as 500-700 pure B. anthracis spores can be recognized with CV values less than 8.31% within 5 min of chromatography and a total time of 20 min. For powdery sample tests, this LFIA can endure interference from 25% (w/v) milk, 10% (w/v) baking soda and 10% (w/v) starch without any sample pre-treatment, and has a corresponding detection limit of 6 x 10(4) spores/g milk powder, 2 x 10(5) spores/g starch and 5 x 10(5) spores/g baking soda. Compared with existing methods, this new approach is very competitive in terms of sensitivity, specificity, cost and ease of operation. This proof-of-concept study can also be extended for detection of many other large-sized analytes. (C) 2014 The Authors. Published by Elsevier B.V.

submitted time 2016-05-11 Hits1220Downloads671 Comment 0

9. chinaXiv:201605.00720 [pdf]

The beta(2) clamp in the Mycobacterium tuberculosis DNA polymerase III alpha beta(2)epsilon replicase promotes polymerization and reduces exonuclease activity

Gu, Shoujin; Li, Wenjuan; Zhang, Hongtai; Fleming, Joy; Wei, Wenjing; Hou, Jian; Zhou, Ying; Lin, Shiqiang; Zhang, Xian-En; Bi, Lijun; Gu, Shoujin; Li, Wenjuan; Zhang, Hongtai; Fleming, Joy; Wei, Wenjing; Hou, Jian; Zhou, Ying; Lin, Shiqiang; Zhang, Xian-En; Bi, Lijun
Subjects: Biology >> Biophysics

DNA polymerase III (DNA pol III) is a multi-subunit replication machine responsible for the accurate and rapid replication of bacterial genomes, however, how it functions in Mycobacterium tuberculosis (Mtb) requires further investigation. We have reconstituted the leading-strand replication process of the Mtb DNA pol III holoenzyme in vitro, and investigated the physical and functional relationships between its key components. We verify the presence of an alpha beta(2)epsilon polymerase-clamp-exonuclease replicase complex by biochemical methods and protein-protein interaction assays in vitro and in vivo and confirm that, in addition to the polymerase activity of its a subunit, Mtb DNA pol III has two potential proofreading subunits; the alpha and epsilon subunits. During DNA replication, the presence of the beta(2) clamp strongly promotes the polymerization of the alpha beta(2)epsilon replicase and reduces its exonuclease activity. Our work provides a foundation for further research on the mechanism by which the replication machinery switches between replication and proofreading and provides an experimental platform for the selection of antimicrobials targeting DNA replication in Mtb.

submitted time 2016-05-05 Hits1321Downloads722 Comment 0

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