Publications by authors named "Qingtao Liu"

55 Publications

Transcriptome analysis reveals new insight of duck Tembusu virus (DTMUV)-infected DF-1 cells.

Res Vet Sci 2021 Jul 29;137:150-158. Epub 2021 Apr 29.

Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Diagnosis, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, Nanjing, 210014, China.

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused huge economic losses to the duck industry in China since 2010. Moreover, the infection has spread rapidly, resulted in a potential public health concern. To improve our understanding of the host cellular responses to virus infection and the pathogenesis of DTMUV infection, we used RNA-Seq to detect the gene changes in DF-1 cells infected and mock-infected with DTMUV. A total of 663 differentially-expressed genes (DEGs) were identified in DTMUV-infected compared with mock-infected DF-1 cells at 24 h post-infection (hpi), among which 590 were up regulated and 73 were down regulated. Gene Ontology analysis indicated that the DEGs were mainly involved in cellular process, immune system processes, metabolic processes, and signal-organism process. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DEGs were mainly involved in several signaling pathways such as Toll-like receptor signaling, Jak-STAT signaling, RIG-I-like receptor signaling and AGE-RAGE signaling pathway. Moreover, some selected DEGs were further confirmed by real-time PCR and the results were consistent with the sequencing data. To our knowledge, this study is the first to analyze the transcriptomic change in DF-1 cells following DTMUV infection. We believe that our research provides useful information in better understanding the host response to DTMUV infection and the inherent mechanism of DTMUV replication and pathogenicity.
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http://dx.doi.org/10.1016/j.rvsc.2021.04.028DOI Listing
July 2021

Highly Accessible Atomically Dispersed Fe-N Sites Electrocatalyst for Proton-Exchange Membrane Fuel Cell.

Adv Sci (Weinh) 2021 Mar 29;8(5):2002249. Epub 2021 Jan 29.

Beijing Advanced Innovation Center for Soft Matter Science and Engineering State Key Laboratory of Organic-Inorganic Composites Beijing University of Chemical Technology Beijing 100029 P. R. China.

Atomically dispersed transition metal-N sites have emerged as a frontier for electrocatalysis because of the maximized atom utilization. However, there is still the problem that the reactant is difficult to reach active sites inside the catalytic layer in the practical proton exchange membrane fuel cell (PEMFC) testing, resulting in the ineffective utilization of the deeply hided active sites. In the device manner, the favorite structure of electrocatalysts for good mass transfer is vital for PEMFC. Herein, a facile one-step approach to synthesize atomically dispersed Fe-N species on hierarchically porous carbon nanostructures as a high-efficient and stable atomically dispersed catalyst for oxygen reduction in acidic media is reported, which is achieved by a predesigned hierarchical covalent organic polymer (COP) with iron anchored. COP materials with well-defined building blocks can stabilize the dopants and provide efficient mass transport. The appropriate hierarchical pore structure is proved to facilitate the mass transport of reactants to the active sites, ensuring the utilization of active sites in devices. Particularly, the structurally optimized HSAC/Fe-3 displays a maximum power density of up to 824 mW cm, higher than other samples with fewer mesopores. Accordingly, this work will offer inspirations for designing efficient atomically dispersed electrocatalyst in PEMFC device.
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http://dx.doi.org/10.1002/advs.202002249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7927611PMC
March 2021

Analysis of the microRNA expression profiles of chicken dendritic cells in response to H9N2 avian influenza virus infection.

Vet Res 2020 Oct 17;51(1):132. Epub 2020 Oct 17.

Key Laboratory of Veterinary Biological Engineering and Technology of Ministry of Agriculture, National Center for Engineering Research of Veterinary Bio-products, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, 50 Zhongling Street, Nanjing, 210014, Jiangsu, China.

MicroRNA (miRNA) plays a key role in virus-host interactions. Here, we employed deep sequencing technology to determine cellular miRNA expression profiles in chicken dendritic cells infected with H9N2 avian influenza virus (AIV). A total of 66 known and 36 novel miRNAs were differently expressed upon H9N2 infection, including 72 up-regulated and 30 down-regulated miRNAs. Functional analysis showed that the predicted targets of these miRNAs were significantly enriched in several pathways including endocytosis, notch, lysosome, p53, RIG-I-like and NOD-like receptor signaling pathways. These data provide valuable information for further investigating the roles of miRNA in AIV pathogenesis and host defense response.
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http://dx.doi.org/10.1186/s13567-020-00856-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568386PMC
October 2020

Peptide inhibitors of tembusu virus infection derived from the envelope protein.

Vet Microbiol 2020 Jun 7;245:108708. Epub 2020 May 7.

College of Animal Science and Technology, Shandong Agricultural University, Tai'an, Shandong province, PR China; Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Tai'an, Shandong, PR China; Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, Tai'an, Shandong, PR China. Electronic address:

The outbreak and spread of Tembusu virus (TMUV) has caused very large losses in the waterfowl-breeding industry since 2010. The viral envelope (E) protein, the principal surface protein of viral particles, plays a vital role in viral entry and fusion. In this study, two peptides derived from domain II (DII) and the stem of the TMUV envelope protein, TP1 and TP2, respectively, were tested for their antiviral activity. TP1 and TP2 inhibited TMUV infection in BHK-21 cells, and their 50% inhibitory concentrations (IC) were 14.19 mg/L and 7.64 mg/L, respectively. Viral inhibition assays in different cell lines of avian origin showed that the inhibitory effects of TP1 and TP2 are not cell type dependent. Moreover, TP2 also exhibited inhibitory activity against Japanese encephalitis virus (JEV) infection. The two peptides inhibited antibody-mediated TMUV infection of duck peripheral blood lymphocytes. Co-immunoprecipitation assays and indirect enzyme-linked immunosorbent assays (ELISAs) indicated that both peptides interact with the surface of the TMUV virion. RNase digestion assays confirmed the release of viral RNA following incubation with TP1, while incubation with TP1 or TP2 interfered with the binding between TMUV and cells. Taken together, these results show that TP1 and TP2 may be developed into antiviral treatments against TMUV infection.
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http://dx.doi.org/10.1016/j.vetmic.2020.108708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7204726PMC
June 2020

Tembusu virus enters BHK-21 cells through a cholesterol-dependent and clathrin-mediated endocytosis pathway.

Microb Pathog 2020 Oct 12;147:104242. Epub 2020 May 12.

Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, Nanjing, China. Electronic address:

Tembusu virus (TMUV) is a newly emerging flavivirus and has caused significant economic loss to the poultry industry in China. To date, the entry of TMUV into host cells remains poorly understood. Here, the mechanism of TMUV entry into BHK-21 cells was investigated. The depletion of cellular cholesterol by methyl-β-cyclodextrin led to a significant decline in the titers and RNA levels of the infectious TMUV. This reduction was restored by supplementation of exogenous cholesterol. Membrane cholesterol depletion mainly blocked viral internalization but not attachment. However, viral infection was unaffected by genistein treatment or caveolin-1 silencing by small interfering RNA. In addition, clathrin-mediated endocytosis might be utilized in TMUV entry given that the viral infection was inhibited by knockdown of clathrin heavy chain and treatment of chlorpromazine (CPZ). Moreover, the number of internalized virus particles decreased under CPZ treatment. Dynasore inhibited TMUV entry suggesting a role for dynamin. Our results reveal that TMUV entry into BHK-21 cells is dependent on cholesterol, clathrin and dynamin but not caveolae.
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http://dx.doi.org/10.1016/j.micpath.2020.104242DOI Listing
October 2020

Global gene expression analysis data of chicken dendritic cells infected with H9N2 avian influenza virus.

Data Brief 2020 Jun 16;30:105430. Epub 2020 Mar 16.

Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, and Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, Nanjing, Jiangsu, China.

This data article reports the global gene expression analysis data of chicken DCs infected with H9N2 avian influenza virus (AIV) compared with mock infection. The differentially expressed genes (DEGs), and the data of GO enrichment analysis and KEGG pathway analysis for DEGs were reported here. In addition, some of these DEGs associated with innate immune response and antigen presentation were also verified by qPCR. The replication of H9N2 AIV in DCs, and the viability kinetic of DCs during H9N2 AIV infection, and the primers for qPCR were also reported in this data article. The data presented here was used on the research article entitled "Transcriptomic profile of chicken bone marrow-derive dendritic cells in response to H9N2 avianinfluenza A virus".
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http://dx.doi.org/10.1016/j.dib.2020.105430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7152653PMC
June 2020

Lipid-based lyotropic liquid crystalline phase transitions as a novel assay platform using birefringence as the visual signal output.

J Mater Chem B 2020 08 30;8(29):6277-6285. Epub 2020 Mar 30.

Drug Delivery, Disposition and Dynamics and ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University Parkville Campus, 381 Royal Parade, Parkville, Victoria 3052, Australia.

Amphiphilic lipids can often form various lipid-based lyotropic liquid crystalline phases when they are exposed to aqueous environments, which includes the inverse hexagonal (H), inverse cubic (V), inverse discontinuous cubic (I) and lamellar (L) phases. Their different interactions with crossed-polarised light make some phases appear bright and some dark, which offers great potential in developing a novel universal assay platform using birefringence as the visual signal output. Here, we have developed a novel strategy for constructing an assay platform using lyotropic liquid crystalline phases as the signal transducer and using changes in their birefringence upon exposure to lipase as the signal output. The hydrolysis of the ester group of glyceryl monooleate (GMO) by lipase induced a phase transition from cubic phase to hexagonal phase and consequently "turned on" the birefringence as the signal output. The change in the intensity of the birefringence depended on both the exposure time and concentration of lipase. The streptavidin-biotin affinity was utilised to demonstrate the potential of the birefringence assay platform, using free biotin as the model analyte, biotinylated lipase and streptavidin-coated magnetic beads in the competitive format. A semi-quantitative assay with a detection limit of 5 μg mL to free biotin as the model analyte was achieved in the visual birefringence mode. This study demonstrated a proof-of-concept lyotropic assay platform with birefringence as the visual signal output that could be deployed as an electronics- and colour-free diagnostic device for a wide range of applications, for example to indicate the presence of toxins in water.
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http://dx.doi.org/10.1039/d0tb00355gDOI Listing
August 2020

Transcriptomic profile of chicken bone marrow-derive dendritic cells in response to H9N2 avian influenza A virus.

Vet Immunol Immunopathol 2020 Feb 9;220:109992. Epub 2019 Dec 9.

Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, and Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, Nanjing, Jiangsu, 50 Zhongling Street, 210014, China. Electronic address:

Avian influenza subtype H9N2 infection is a mild but highly contagious disease that is associated with a decrease in the efficacy of vaccine interventions, and an increase in susceptibility to secondary infections in poultry. However, the immune evasion mechanism of H9N2 avian influenza viruses (AIVs) in chickens is poorly understood. Dendritic cells (DCs) are immune cells of major importance, involved in innate immune responses against viruses, but also in the setting of adaptive immune response due to their high ability to present viral antigen. Therefore, in the present study we used high-throughput RNA-sequencing technology at the transcriptome level to identify the differentially expressed genes (DEGs) between chicken DCs infected with H9N2 virus and mock-infected DCs. We identified 4151 upregulated DEGs and 2138 downregulated DEGs. Further enrichment analysis showed that the upregulated DEGs were enriched in the biological processes mainly involved in signal transduction, transmembrane transport, and innate immune/inflammatory responses. In contrast, the downregulated DEGs were associated with the biological processes mainly including metabolic process, and MHC class I antigen processing and presentation. In addition, 49 of these immune-related DEGs were validated by reverse transcription quantitative PCR (RT-qPCR). Collectively, these data suggest that H9N2 virus infection may enhance the signal transduction, and innate immune responses in chicken DCs, but impair their metabolic functions and antigen-presenting responses, which provide helpful insight into the pathogenesis of H9N2 AIVs in chickens and managing this infection in poultry farms.
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http://dx.doi.org/10.1016/j.vetimm.2019.109992DOI Listing
February 2020

PEGylation and surface functionalization of liposomes containing drug nanocrystals for cell-targeted delivery.

Colloids Surf B Biointerfaces 2019 Oct 15;182:110362. Epub 2019 Jul 15.

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, VIC, 3052, Australia; ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University Parkville Campus, Australia. Electronic address:

Liposomal formulations have important therapeutic applications in anti-cancer treatments but current formulations suffer from serious side effects, high dosage requirements and prolonged treatment. In this study, PEGylated azide-functionalized liposomes containing drug nanocrystals were investigated with the aim of increasing the drug payload and achieving functionalization for targeted delivery. Liposomes were characterized using cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), small and ultra-small angle neutron scattering (SANS/USANS) and small and wide angle X-ray scattering (SAXS/WAXS). Cryo-TEM experiments revealed the dimensions of the nanocrystal-loaded liposomes and the change of shape from spherical to elongated after the formation of nanocrystals. Results from SANS/USANS experiments confirmed the asymmetric particle shape. SAXS/WAXS experiments confirmed that the crystalline drug only occurred in freeze-thawed samples and correlated with a new unidentified polymorphic form of ciprofloxacin. Using a small molecule dye, dibenzocyclooctyne (DBCO)-cy5, specific conjugation between DBCO groups and surface azide groups on the liposomes was confirmed; this indicates the promise of this system for tumour-targeted delivery.
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http://dx.doi.org/10.1016/j.colsurfb.2019.110362DOI Listing
October 2019

The ubiquitin-proteasome system is necessary for the replication of duck Tembusu virus.

Microb Pathog 2019 Jul 1;132:362-368. Epub 2019 May 1.

Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Center for Engineering Research of Veterinary Bio-products, Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, PR China. Electronic address:

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused massive economic losses to the duck industry in China. The cellular factors required for DTMUV replication have been poorly studied. The ubiquitin-proteasome system (UPS), the major intracellular proteolytic pathway, mediates diverse cellular processes, including endocytosis and signal transduction, which may be involved in the entry of virus. In the present study, we explored the interplay between DTMUV replication and the UPS in BHK-21 cells and found that treatment with proteasome inhibitor (MG132 and lactacystin) significantly decreased the DTMUV progency at the early infection stage. We further revealed that inhibition of the UPS mainly occurs on the level of viral protein expression and RNA transcription. In addition, using specific siRNAs targeting ubiquitin reduces the production of viral progeny. In the presence of MG132 the staining for the envelope protein of DTMUV was dramatically reduced in comparison with the untreated control cells. Overall, our observations reveal an important role of the UPS in multiple steps of the DTMUV infection cycle and identify the UPS as a potential drug target to modulate the impact of DTMUV infection.
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http://dx.doi.org/10.1016/j.micpath.2019.04.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7126904PMC
July 2019

[Advances in microbial enzymatic elimination of ethyl carbamate in Chinese rice wine].

Sheng Wu Gong Cheng Xue Bao 2019 Apr;35(4):567-576

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.

Ethyl carbamate (EC), a carcinogenic and teratogenic chemical that is widely distributed in various alcoholic beverages, has attracted much attention. Microbial enzymatic degradation of EC in rice wine is always efficient and attractive. In this review, we summarize the research progress and problems of microbial enzymatic elimination of EC in rice wine from three aspects: the mechanisms of EC formation in rice wine, the research progress of acid urease, and the research progress of urethanase. Then, we propose the corresponding strategies to solve the problems: screening new urethanase with satisfied enzyme properties, food-grade expression and directed evolution of the bifunctional Fe³⁺-dependent acid urease and acid urease used in combination with urethanase to eliminate both urea and EC in rice wine.
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http://dx.doi.org/10.13345/j.cjb.180386DOI Listing
April 2019

Food-grade expression of an iron-containing acid urease in Bacillus subtilis.

J Biotechnol 2019 Mar 28;293:66-71. Epub 2019 Jan 28.

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu, 214122, China. Electronic address:

Enzymatic degradation of urea, the precursor of carcinogenic compound ethylcarbamate in rice wine, is always attractive. In the present study, we achieved high efficient production of Bacillus paralicheniformis iron-containing urease (Bp_Urease) in B. subtilis with the food-grade expression system. After reassembly of the urease gene cluster with inserting ribosome binding site (RBS), the production was increased from 38 U/L to 187 U/L. After altering the position of ureC and co-expressing the iron transporter encoding gene ureH, the activity was further increased to 1307 U/L. Eventually, the urease production was improved to 21,964 U/L in 3-L fermentor, which is the highest reported value to date. Food-grade production of the iron-containing urease would be favorable to the practical applications in food industries.
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http://dx.doi.org/10.1016/j.jbiotec.2019.01.012DOI Listing
March 2019

The unfolded protein response induced by Tembusu virus infection.

BMC Vet Res 2019 Jan 22;15(1):34. Epub 2019 Jan 22.

Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Jiangsu Province, 210014, People's Republic of China.

Background: Tembusu virus (TMUV), classified in the genus Flavivirus, causes reduced egg production and neurological problems in poultry. Flavivirus replication depends on the host endoplasmic reticulum (ER) and induces ER stress that leads to activation of the cellular unfolded protein response (UPR), an important signalling pathway that regulates many biological functions involved in viral pathogenesis and innate immunity. However, the mechanism of TMUV-induced UPR activation remains unclear.

Results: In this study, we systematically investigated the three UPR pathways in TMUV-infected BHK-21 cells. Our results showed that expression of glucose-related protein 78 (GRP78) and GRP94 was upregulated during the course of TMUV infection. We then demonstrated that TMUV activated the PERK pathway in the early stage of infection, resulting in upregulation of ATF4, GADD34 and CHOP, with CHOP induction leading to caspase-3 activation. We also found the IRE1 pathway to be activated, leading to splicing of X box binding protein 1 (XBP1) mRNA and enhanced expression of p58. Finally, we observed increased expression of ATF6 and activity of ER stress-response elements, suggesting stimulation of the ATF6 pathway. In addition, ATF6 pathway activation correlated with the induction of downstream chaperones calnexin, calreticulin, ERp57 and PDI. UPR activity was also observed by the marked elevation in GRP78 and sXBP1 levels in TMUV-infected DF-1 cells.

Conclusions: This is the first report that TMUV infection-induced ER stress activates three branches of the UPR, and these results lay the foundation for elucidating the pathogenesis of TMUV and understanding the inherent mechanism of TMUV infection as well as the host response.
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http://dx.doi.org/10.1186/s12917-019-1781-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343269PMC
January 2019

Novel self-assembling conjugates as vectors for agrochemical delivery.

J Nanobiotechnology 2018 Nov 21;16(1):94. Epub 2018 Nov 21.

School of Life and Environmental Sciences, Deakin University, Waurn Ponds Campus, Geelong, VIC, 3217, Australia.

Background: Modern agricultural practises rely on surfactant-based spray applications to eliminate weeds in crops. The wide spread and indiscriminate use of surfactants may result in a number of deleterious effects that are not limited to impacts on the crop and surrounding farm eco-system but include effects on human health. To provide a safer alternative to the use of surfactant-based formulations, we have synthesised a novel, self-assembling herbicide conjugate for the delivery of a broad leaf herbicide, picloram.

Results: The synthesized self-assembling amphiphile-picloram (SAP) conjugate has three extending arms: a lipophilic lauryl chain, a hydrophilic polyethylene glycol chain and the amphiphobic agrochemical active picloram. We propose that the SAP conjugate maintains its colloidal stability by quickly transitioning between micellar and inverse micellar phases in hydrophilic and lipophilic environments respectively. The SAP conjugate provides the advantage of a phase structure that enables enhanced interaction with the hydrophobic epicuticular wax surface of the leaf. We have investigated the herbicidal efficiency of the SAP conjugate compared against that of commercial picloram formulations using the model plant Arabidopsis thaliana and found that when tested at agriculturally relevant doses between 0.58 and 11.70 mM a dose-dependent herbicidal effect with comparable kill rates was evident.

Conclusion: Though self-assembling drug carriers are not new to the pharmaceutical industry their use for the delivery of agrochemicals shows great promise but is largely unexplored. We have shown that SAP may be used as an alternative to current surfactant-based agrochemical formulations and has the potential to shift present practises towards a more sustainable approach.
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http://dx.doi.org/10.1186/s12951-018-0423-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6247628PMC
November 2018

Construction of Synthetic Promoters by Assembling the Sigma Factor Binding -35 and -10 Boxes.

Biotechnol J 2019 Jan 7;14(1):e1800298. Epub 2018 Dec 7.

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.

Promoter is one of the key elements in regulating gene expression. Many natural or synthetic promoters have been modulated by their cis- or tans-regulatory elements to confer instant gene expression change in responding to designated stimuli. In addition, bacterial cells also engage different sigma factors to control the gene expression network at different growth phases or in response to the changing environment and external stresses. In this study, a set of promoters that assimilate the endogenous regulation of different sigma factors σ , σ , σ , and σ are synthesized. Promoters are designed to contain two or more kinds of interlocking sigma factor binding sites. The most competitive sigma factors will be automatically selected by the cell to take over the synthetic promoters during the cell growth course. Some of the synthetic promoters exhibit very strong strengths under different conditions, including stationary phase, low temperature, acidic pH, and high osmotic pressure. Comparing to the T7 promoter, synthetic promoter P achieved higher yields of L-asparaginase and acid urease in Escherichia coli. The research not only expands the synthetic biology toolbox but also provide another strategy to design and construct synthetic promoters in prokaryotes.
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http://dx.doi.org/10.1002/biot.201800298DOI Listing
January 2019

Molecular Engineering of Bacillus paralicheniformis Acid Urease To Degrade Urea and Ethyl Carbamate in Model Chinese Rice Wine.

J Agric Food Chem 2018 Dec 29;66(49):13011-13019. Epub 2018 Nov 29.

Bacillus paralicheniformis urease (BpUrease) has been shown to be a promising biocatalyst for degrading the carcinogenic chemical ethyl carbamate (EC or urethane) in rice wine. However, low EC affinity and catalytic efficiency limit the practical application of BpUrease. In this study, we improved the EC degradation capability of BpUrease by site-saturation mutagenesis (SSM). The best variant L253P/L287N showed a 49% increase in EC affinity, 1027% increase in catalytic efficiency ( k/ K), and 583% increase in half-life ( t) at 70 °C. Homology modeling analysis suggest that mutation of Leu253 to Pro increased the BpUrease EC specificity by affecting the interaction between Arg339 with the catalytic residue His323, while Leu287Asn mutation benefits EC specificity and affinity by changing the interaction networks among the residues in the catalytic pocket. Our results show that the L253P/L287N variant efficiently degraded urea and EC in a model rice wine, making it a good candidate for practical application in the food industry.
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http://dx.doi.org/10.1021/acs.jafc.8b04566DOI Listing
December 2018

Identification and immunogenic evaluation of T cell epitopes based on tembusu virus envelope protein in ducks.

Virus Res 2018 09 18;257:74-81. Epub 2018 Sep 18.

Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Jiangsu Province, PR China; Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, Nanjing, Jiangsu Province, PR China. Electronic address:

Newly emerging tembusu virus (TMUV) is a severe threat to poultry industry and causes huge economic losses. Humoral and cell-mediated immunity are both play vital roles in TMUV infection. Up to now, there has been no report on identification of T cell epitopes of the TMUV. In this work, we identified T cell epitopes within TMUV envelope (E) protein using synthesized peptides predicted in silico. A total of ten peptides could stimulate TMUV-specific T cells in murine ELISPOT and duck lymphocyte proliferation assay. Subsequently, DNA vaccine containing these T cell epitopes was constructed (pVAX-T) and the expression of multiepitope protein was confirmed by transfection of BHK-21 cells in vitro. Ducks were administrated intramusclarly to evaluated the immunologic effect of pVAX-T. In ducks immunized with pVAX-T, antibody against TMUV was undetectable, but the expression level of cytokines (IL-2, IL-6, IFN-γ) was upregulated both in peripheral blood lymphocytes and spleen. Furthermore, TMUV challenge revealed that cell-mediated immune response sitmulated by pVAX-T contributed to protection against TMUV infection. The identification of these T cell epitopes will contribute to designing epitope vaccine for preventing infection of TMUV and possibly provide the basis for further studies on cell-mediate immune response activated by TMUV.
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http://dx.doi.org/10.1016/j.virusres.2018.09.008DOI Listing
September 2018

Zigzag carbon as efficient and stable oxygen reduction electrocatalyst for proton exchange membrane fuel cells.

Nat Commun 2018 09 19;9(1):3819. Epub 2018 Sep 19.

School of Materials Science and Engineering, Beihang University, No. 37 Xueyuan Road, Beijing, 100083, China.

Non-precious-metal or metal-free catalysts with stability are desirable but challenging for proton exchange membrane fuel cells. Here we partially unzip a multiwall carbon nanotube to synthesize zigzag-edged graphene nanoribbons with a carbon nanotube backbone for electrocatalysis of oxygen reduction in proton exchange membrane fuel cells. Zigzag carbon exhibits a peak areal power density of 0.161 W cm and a peak mass power density of 520 W g, superior to most non-precious-metal electrocatalysts. Notably, the stability of zigzag carbon is improved in comparison with a representative iron-nitrogen-carbon catalyst in a fuel cell with hydrogen/oxygen gases at 0.5 V. Density functional theory calculation coupled with experimentation reveal that a zigzag carbon atom is the most active site for oxygen reduction among several types of carbon defects on graphene nanoribbons in acid electrolyte. This work demonstrates that zigzag carbon is a promising electrocatalyst for low-cost and durable proton exchange membrane fuel cells.
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http://dx.doi.org/10.1038/s41467-018-06279-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6145918PMC
September 2018

Screening and identification of B-cell epitopes within envelope protein of tembusu virus.

Virol J 2018 09 17;15(1):142. Epub 2018 Sep 17.

Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, 50 Zhongling Street, Nanjing City, Jiangsu Province, 210014, People's Republic of China.

Background: Tembusu virus is a newly emerging flavivirus that caused egg-drop syndrome in ducks in China. TMUV envelope protein is a major structural protein locates at the surface of tembusu virus particle. During tembusu virus infection, envelope protein plays a pivotal role in induction of neutralizing antibody. However, B cell epitopes within envelope protein have not been well studied.

Method: A series of 13 peptides derived from E protein of tembusu virus were synthesized and screened by Dot blot with tembusu virus-positive duck serum. Potential B-cell epitopes were respectively fused with GST tag and expressed in E. coli. The immunogenicity and protective efficiency of epitopes were assessed in ducks.

Results: Dot blot assay identified the peptides P21 (amino acids 301-329), P23 (amino acids 369-387), P27 (amino acids 464-471) and P28 (amino acids 482-496) as potential B-cell epitopes within the envelope protein of tembusu virus. Immunization of prokaryotically expressed epitopes elicited specific antibodies in ducks and the specific antibody elicited by P21, P27 and P28 could neutralized tembusu virus. In addition, protective test suggested that P21 and P27 could completely protect immunized ducks from TMUV challenge.

Conclusion: Four potential B cell epiotpes within tembusu virus envelope protein were identified and analyzed in vitro and in vivo. It was demonstrated that two of them (P21 and P27) could elicit neutralizing antibodies in ducks and offer complete protection against tembusu virus challenge. This findings will contribute to the development of epitope vaccine for tembusu virus prevention.
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http://dx.doi.org/10.1186/s12985-018-1052-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6142368PMC
September 2018

A new sRNA-mediated posttranscriptional regulation system for Bacillus subtilis.

Biotechnol Bioeng 2018 12 1;115(12):2986-2995. Epub 2018 Oct 1.

The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.

Many genetic tools for gene regulation have been developed during the past decades. Some of them edit genomic DNA, such as nucleotides deletions and insertions, while the others interfere with the gene transcriptions or messenger RNA translation. Here, we report a posttranscriptional regulation tool which is termed "Modulation via the small RNA (sRNA)-dependent operation system: MS-DOS" by engineering the type I toxin-antitoxin system in Bacillus subtilis. MS-DOS depends simply on insertion of an operation region (OPR; partial toxin-encoding region) downstream of a genomic open reading frame of interest and overexpression of the coupling antitoxin sRNA from a plasmid. Pairing between the OPR and the sRNA will trigger the RNAse degradation of the transcripts of selected genes. MS-DOS allows for the quantitative, specific, and reversible knockdown of single or multiple genomic genes in B. subtilis. We also showed that the truncated antitoxin SR4 with 53 nt length is sufficient to repress gene expression. Superior to other existing RNA based interfering systems, MS-DOS allows simultaneous knockdown of multiple genes with effortless expression of a single antitoxin RNA. This sRNA-guided repression system will further enrich the gene regulation tools and expand the gene regulation flexibility.
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http://dx.doi.org/10.1002/bit.26833DOI Listing
December 2018

Identification of Glucose-Regulated Protein 78 (GRP78) as a Receptor in BHK-21 Cells for Duck Tembusu Virus Infection.

Front Microbiol 2018 9;9:694. Epub 2018 Apr 9.

Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, Nanjing, China.

Since 2010, outbreak and spread of tembusu virus (TMUV) caused huge losses to the breeding industry of waterfowl in several provinces of China. In this study, we identify the glucose-regulated protein 78 (GRP78) as a receptor in BHK-21 cells for duck TMUV infection. Using cell membrane from BHK-21 cells, a TMUV-binding protein of approximately 70 kDa was observed by viral overlay protein binding assay (VOPBA). LC-MS/MS analysis and co-immunoprecipitation identified GRP78 as a protein interacting with TMUV. Antibody against GRP78 inhibited the binding of TMUV to the cell surface of BHK-21 cells. Indirect immunofluorescence studies showed the colocalization of GRP78 with TMUV in virus-infected BHK-21 cells. We found that GRP78 over-expression increased TMUV infection, whereas GRP78 knockdown by using a specific small interfering RNA inhibited TMUV infection in BHK-21 cells. Taken together, our results indicate that GRP78 is a novel host factor involved in TMUV entry.
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http://dx.doi.org/10.3389/fmicb.2018.00694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5903163PMC
April 2018

Identification of determinants that mediate binding between Tembusu virus and the cellular receptor heat shock protein A9.

J Vet Sci 2018 Jul;19(4):528-535

Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, Nanjing 210014, China.

Heat shock protein A9 (HSPA9), a member of the heat shock protein family, is a putative receptor for Tembusu virus (TMUV). By using Western blot and co-immunoprecipitation assays, E protein domains I and II were identified as the functional domains that facilitate HSPA9 binding. Twenty-five overlapping peptides covering domain I and domain II sequences were synthesized and analyzed by using an HSPA9 binding assay. Two peptides showed the capability of binding to HSPA9. Dot blot assay of truncated peptides indicated that amino acid residues 19 to 22 and 245 to 252 of E protein constitute the minimal motifs required for TMUV binding to HSPA9. Importantly, peptides harboring those two minimal motifs could effectively inhibit TMUV infection. Our results provide insight into TMUV-receptor interaction, thereby creating opportunities for elucidating the mechanism of TMUV entry.
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http://dx.doi.org/10.4142/jvs.2018.19.4.528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070589PMC
July 2018

The Solid-Phase Synthesis of an Fe-N-C Electrocatalyst for High-Power Proton-Exchange Membrane Fuel Cells.

Angew Chem Int Ed Engl 2018 01 8;57(5):1204-1208. Epub 2018 Jan 8.

School of Materials Science and Engineering, Beihang University, No. 37 Xueyuan Road, Beijing, 100083, China.

The environmentally friendly synthesis of highly active Fe-N-C electrocatalysts for proton-exchange membrane fuel cells (PEMFCs) is desirable but remains challenging. A simple and scalable method is presented to fabricate Fe -doped ZIF-8, which can be further pyrolyzed into Fe-N-C with 3 wt % of Fe exclusively in Fe-N active moieties. Significantly, this Fe-N-C derived acidic PEMFC exhibits an unprecedented current density of 1.65 A cm at 0.6 V and the highest power density of 1.14 W cm compared with previously reported NPMCs. The excellent PEMFC performance can be attributed to the densely and atomically dispersed Fe-N active moieties on the small and uniform catalyst nanoparticles.
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http://dx.doi.org/10.1002/anie.201709597DOI Listing
January 2018

Clickable Cubosomes for Antibody-Free Drug Targeting and Imaging Applications.

Bioconjug Chem 2018 01 20;29(1):149-157. Epub 2017 Dec 20.

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Science, Monash University , Parkville, VIC 3052, Australia.

The combination of copper-free click chemistry with metabolic labeling offers new opportunities in drug delivery. The objective of this study was to determine whether cubosomes functionalized with azide or dibenzocyclooctyne (DBCO) groups are able to undergo copper-free click chemistry with a strained cyclooctyne or azide, respectively. Phytantriol-based cubosomes were functionalized using phospholipids bearing an azide or DBCO group. The modified cubosome dispersions were characterized using dynamic light scattering, cryo-TEM, and small-angle X-ray scattering. The efficiency of "clickability" was assessed by reacting the cubosomes with a complementary dye and determining bound and unbound dye via size exclusion chromatography. The clickable cubosomes reacted specifically and efficiently with a click-Cy5 dye with minor changes to the size, shape, and structure of the cubosomes. This indicates that cubosomes can retain their unique internal structure while participating in copper-free click chemistry. This proof of concept study paves the way for the use of copper-free click chemistry and metabolic labeling with cubosomes for targeted drug delivery and imaging.
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http://dx.doi.org/10.1021/acs.bioconjchem.7b00659DOI Listing
January 2018

Novel agrochemical conjugates with self-assembling behaviour.

J Colloid Interface Sci 2018 Feb 19;512:369-378. Epub 2017 Oct 19.

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, VIC 3052, Australia; ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, VIC 3052, Australia. Electronic address:

Hypothesis: That conjugation of agrichemicals to pro-assembly hydrophobic moieties will enable enhanced compatibility and loading with host lyotropic liquid crystalline carrier matrix, and potentially self-assemble in their own right in aqueous environments.

Experiments: A series of lipid-like agrochemical-conjugates were synthesized using specific amphiphilic entities conjugated onto the agrochemicals, picloram and 2,4-dichlorophenoxyacetic acid (2,4-D). The self-assembly behaviour and compatibility of the novel entities when incorporated into phytantriol and monoolein-based liquid crystalline systems were examined using small angle X-ray scattering, cryo-TEM and polarized optical microscopy.

Findings: Compared to agrochemical-conjugates with simple alkyl ester groups, the esterification of the agrochemicals with amphiphilic groups such as phytantriol and monoolein led to greater structural compatibility and consequently a greater loading of the agrochemicals in the liquid crystalline systems without destabilizing phase structure. Picloram-monoolein and picloram-monoelaidin can self-assemble to form lamellar structures in water. However, certain agrochemical-conjugates such as picloram-monoelaidin and picloram-PEGn-oleate showed poor compatibility with liquid crystalline systems, resulting in phase separation.
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http://dx.doi.org/10.1016/j.jcis.2017.10.070DOI Listing
February 2018

Construction and Characterization of Broad-Spectrum Promoters for Synthetic Biology.

ACS Synth Biol 2018 01 1;7(1):287-291. Epub 2017 Nov 1.

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University , Wuxi 214122, China.

Characterization of genetic circuits and biosynthetic pathways in different hosts always requires promoter substitution and redesigning. Here, a strong, broad-spectrum promoter, P, for Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae was constructed, and it was incorporated into the minimal E. coli-B. subtilis-S. cerevisiae shuttle plasmid pEBS (5.8 kb). By applying a random mutation strategy, three broad-spectrum promoters P, P, and P, with different strengths were generated and characterized. These broad-spectrum promoters will expand the synthetic biology toolbox for E. coli, B. subtilis, and S. cerevisiae.
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http://dx.doi.org/10.1021/acssynbio.7b00258DOI Listing
January 2018

Recent advances in production of 5-aminolevulinic acid using biological strategies.

World J Microbiol Biotechnol 2017 Oct 16;33(11):200. Epub 2017 Oct 16.

Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.

5-Aminolevulinic acid (5-ALA) is the precursor for the biosynthesis of tetrapyrrole compounds and has broad applications in the medical and agricultural fields. Because of the disadvantages of chemical synthesis methods, microbial production of 5-ALA has drawn intensive attention and has been regarded as an alternative in the last years, especially with the rapid development of metabolic engineering and synthetic biology. In this mini-review, recent advances on the application and microbial production of 5-ALA using novel biological approaches (such as whole-cell enzymatic-transformation, metabolic pathway engineering and cell-free process) are described and discussed in detail. In addition, the challenges and prospects of synthetic biology are discussed.
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http://dx.doi.org/10.1007/s11274-017-2366-7DOI Listing
October 2017

Nitric oxide-sensing actuators for modulating structure in lipid-based liquid crystalline drug delivery systems.

J Colloid Interface Sci 2017 Dec 24;508:517-524. Epub 2017 Aug 24.

Drug Delivery, Disposition and Dynamics, and ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, VIC 3052, Australia. Electronic address:

Herein we report on the development of a nitric oxide-sensing lipid-based liquid crystalline (LLC) system specifically designed to release encapsulated drugs on exposure to NO through a stimulated phase change. A series of nitric oxide (NO)-sensing lipids compatible with phytantriol and GMO cubic phases were designed and synthesized, and utilized in enabling nitric oxide-sensing LLC systems. The nitric oxide (NO)-sensing lipids react with nitric oxide, resulting in hydrolysis of these lipids and phase transition of the LLC system. Specifically, the N-3-aminopyridinyl myristylamine (NAPyM)+phytantriol mixture formed a lamellar phase in excess aqueous environment. The NAPyM+phytantriol LLC responded to the nitric oxide gas as a chemical stimulus which triggers a phase transition from lamellar phase to inverse cubic and hexagonal phase. The nitric oxide-triggered phase transition of the LLC accelerated the release of encapsulated model drug from the LLC bulk phase, resulting in a 15-fold increase in the diffusion coefficient compared to the starting lamellar structure. The nitric oxide-sensing LLC system has potential application in the development of smart medicines to treat nitric oxide implicated diseases.
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http://dx.doi.org/10.1016/j.jcis.2017.08.079DOI Listing
December 2017

A Bacillus paralicheniformis Iron-Containing Urease Reduces Urea Concentrations in Rice Wine.

Appl Environ Microbiol 2017 09 17;83(17). Epub 2017 Aug 17.

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China, and Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, China

Urease, a nickel-containing metalloenzyme, was the first enzyme to be crystallized and has a prominent position in the history of biochemistry. In the present study, we identified a nickel urease gene cluster, , in ATCC 9945a and characterized it in Enzymatic assays demonstrate that this oxygen-stable urease is also an iron-containing acid urease. Heterologous expression assays of UreH suggest that this accessory protein is involved in the transmembrane transportation of nickel and iron ions. Moreover, this iron-containing acid urease has a potential application in the degradation of urea in rice wine. The present study not only enhances our understanding of the mechanism of activation of urease but also provides insight into the evolution of metalloenzymes. An iron-containing, oxygen-stable acid urease from ATCC 9945a with good enzymatic properties was characterized. This acid urease shows activities toward both urea and ethyl carbamate. After digestion with 6 U/ml urease, approximately 92% of the urea in rice wine was removed, suggesting that this urease has great potential in the food industry.
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http://dx.doi.org/10.1128/AEM.01258-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5561274PMC
September 2017

Two Genetically Similar H9N2 Influenza A Viruses Show Different Pathogenicity in Mice.

Front Microbiol 2016 4;7:1737. Epub 2016 Nov 4.

Key Laboratory of Veterinary Biological Engineering and Technology, National Center for Engineering Research of Veterinary Bio-products, Institute of Veterinary Medicine, Ministry of Agriculture, Jiangsu Academy of Agricultural SciencesNanjing, China; Jiangsu Key Laboratory of Zoonosis, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and ZoonosesYangzhou, China.

H9N2 Avian influenza virus has repeatedly infected humans and other mammals, which highlights the need to determine the pathogenicity and the corresponding mechanism of this virus for mammals. In this study, we found two H9N2 viruses with similar genetic background but with different pathogenicity in mice. The A/duck/Nanjing/06/2003 (NJ06) virus was highly pathogenic for mice, with a 50% mouse lethal dose (MLD) of 10 50% egg infectious dose (EID), whereas the A/duck/Nanjing/01/1999 (NJ01) virus was low pathogenic for mice, with a MLD of >10 EID. Further studies showed that the NJ06 virus grew faster and reached significantly higher titers than NJ01 and . Moreover, the NJ06 virus induced more severe lung lesions, and higher levels of inflammatory cellular infiltration and cytokine response in lungs than NJ01 did. However, only 12 different amino acid residues (HA-K157E, NA-A9T, NA-R435K, PB2-T149P, PB2-K627E, PB1-R187K, PA-L548M, PA-M550L, NP-G127E, NP-P277H, NP-D340N, NS1-D171N) were found between the two viruses, and all these residues except for NA-R435K were located in the known functional regions involved in interaction of viral proteins or between the virus and host factors. Summary, our results suggest that multiple amino acid differences may be responsible for the higher pathogenicity of the NJ06 virus for mice, resulting in lethal infection, enhanced viral replication, severe lung lesions, and excessive inflammatory cellular infiltration and cytokine response in lungs. These observations will be helpful for better understanding the pathogenic potential and the corresponding molecular basis of H9N2 viruses that might pose threats to human health in the future.
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http://dx.doi.org/10.3389/fmicb.2016.01737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5096341PMC
November 2016