Publications by authors named "Yongdong Liu"

64 Publications

Confined Crystallization and Melting Behaviors of 3-Pentadecylphenol in Anodic Alumina Oxide Nanopores.

ACS Omega 2021 Jul 8;6(28):18235-18247. Epub 2021 Jul 8.

State Key Laboratory of High-Efficiency Coal Utilization and Green Chemical Engineering, Ningxia University, 489 Helanshan West Road, Yinchuan 750021, China.

To explore the effects of end groups on the confined crystallization of an alkyl chain, 3-pentadecylphenol (PDP) was infiltrated into the anodic aluminum oxide template (AAO) to investigate the melting and crystallization behaviors of PDP in a nanoconfined environment. Wide-angle X-ray diffraction (WAXD) found that the solid-solid phase transition of PDP occurred under confined conditions, and the absence of the (00L) reflections indicated that the stacking of the end groups of the alkyl chain layered structure was seriously disturbed. Thermal analysis (TG) showed that the thermal stability of the confined samples decreased due to the confinement effect, and the introduction of end groups made the confinement effect more obvious. Differential scanning calorimeter (DSC) results well reflected the space-time equivalence in the PDP crystallization processes, i.e., the solid-solid phase transition can be achieved by reducing the cooling rate or confining PDP in the nanometer space. Compared with C, the introduction of the end groups with a phenol ring led to the disappearance of the solid-solid phase transition of an alkyl chain at high cooling rates. In the confined environment, the introduction of the end groups with a phenol ring caused the melting double peaks of the alkyl chain to become a single melting peak, and it also caused the disappearance of the surface freezing monolayer for alkyl chains. Through the analysis of crystallinity, it was found that AAO-PDP was more sensitive to AAO pore size changes than AAO-C, the of AAO-PDP had a good linear relationship with the pore size , but the of the AAO-C had a nonlinear relationship with the pore size . Attenuated total reflection (ATR)-IR proved that in the confined environment, the order of the alkyl chain decreased and the degree of chain distortion increased.
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http://dx.doi.org/10.1021/acsomega.1c02112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8296606PMC
July 2021

Engineered Human Heavy-Chain Ferritin with Half-Life Extension and Tumor Targeting by PAS and RGDK Peptide Functionalization.

Pharmaceutics 2021 Apr 9;13(4). Epub 2021 Apr 9.

School of Chemical Engineering and Advanced Materials, The University of Adelaide, Adelaide SA5005, Australia.

Ferritin, one of the most investigated protein nanocages, is considered as a promising drug carrier because of its advantageous stability and safety. However, its short half-life and undesirable tumor targeting ability has limited its usage in tumor treatment. In this work, two types of functional peptides, half-life extension peptide PAS, and tumor targeting peptide RGDK (Arg-Gly-Asp-Lys), are inserted to human heavy-chain ferritin (HFn) at C-terminal through flexible linkers with two distinct enzyme cleavable sites. Structural characterizations show both HFn and engineered HFns can assemble into nanoparticles but with different apparent hydrodynamic volumes and molecular weights. RGDK peptide enhanced the internalization efficiency of HFn and showed a significant increase of growth inhibition against 4T1 cell line in vitro. Pharmacokinetic study in vivo demonstrates PAS peptides extended ferritin half-life about 4.9 times in Sprague Dawley rats. RGDK peptides greatly enhanced drug accumulation in the tumor site rather than in other organs in biodistribution analysis. Drug loaded PAS-RGDK functionalized HFns curbed tumor growth with significantly greater efficacies in comparison with drug loaded HFn.
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http://dx.doi.org/10.3390/pharmaceutics13040521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8070472PMC
April 2021

Stent placement combined with intraluminal radiofrequency ablation and hepatic arterial infusion chemotherapy for advanced biliary tract cancers with biliary obstruction: a multicentre, retrospective, controlled study.

Eur Radiol 2021 Aug 13;31(8):5851-5862. Epub 2021 Feb 13.

Department of Interventional Therapy, Cancer Center, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, School of Medicine, South China University of Technology, 106 Zhongshan Second Road, Guangzhou, 510080, Guangdong, China.

Objective: To evaluate the efficacy and safety of stent placement combined with intraluminal radiofrequency ablation (intra-RFA) and hepatic arterial infusion chemotherapy (HAIC) for patients with advanced biliary tract cancers (Ad-BTCs) and biliary obstruction (BO).

Methods: We retrospectively reviewed data for patients with Ad-BTCs and BO who underwent stent placement with or without intra-RFA and HAIC in three centres between November 2013 and November 2018. The stent patency time (SPT), overall survival (OS), and adverse events (AEs) were analysed.

Results: Of the 135 enrolled patients, 64 underwent stent placement combined with intra-RFA and HAIC, while 71 underwent only stent placement. The median SPT was significantly longer in the combination group (8.2 months, 95% confidence interval [CI]: 7.1-9.3) than in the control group (4.3 months, 95% CI: 3.6-5.0; p < 0.001). A similar result was observed for OS (combination: 13.2 months, 95% CI: 11.1-16.5; control: 8.5 months, 95% CI: 7.6-9.6; p < 0.001). The incidence of AEs related to biliary tract operation was not significantly different between the two groups (p > 0.05). The most common AE and serious AE related to HAIC were alanine aminotransferase elevation (24/64; 37.5%) and thrombocytopenia (8/64; 12.5%), respectively. All AEs were tolerable, and there was no death from AEs.

Conclusions: Stent placement combined with intra-RFA and HAIC may be a safe, potential treatment strategy for patients with Ad-BTCs and BO.

Key Points: • Advanced biliary cancers (Ad-BTCs) with biliary obstruction (BO) can rapidly result in liver failure and cachexia with an extremely poor prognosis. • Stent placement combined with intraluminal radiofrequency ablation and hepatic arterial infusion chemotherapy may be safe and effective for patients with Ad-BTCs and BO. • The long-term efficacy and safety of the combined treatment is promising.
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http://dx.doi.org/10.1007/s00330-021-07716-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8270826PMC
August 2021

Adaptation of endothelial cells to shear stress under atheroprone conditions by modulating internalization of vascular endothelial cadherin and vinculin.

Ann Transl Med 2020 Nov;8(21):1423

Department of Cardiology, NHC Key Laboratory of Assisted Circulation, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.

Background: Endothelial cells play a pivotal role in cardiovascular physiology and pathology by providing a barrier to the bloodstream. In the current study, we investigated the phenotype and barrier function of endothelial cells in response to shear stress under pro-atherogenic conditions.

Methods: Endothelial cells were exposed to laminar shear stress (LSS) in a parallel-plate flow chamber containing oxidized low-density lipoprotein (oxLDL) in the perfusion solution, or remained static. We quantified the response of endothelial monolayers to LSS and oxLDL in terms of cell viability, barrier integrity, vascular endothelial cadherin (VE-cadherin) availability, focal adhesion (FA) remodeling, and monocyte-endothelial interactions.

Results: Our results showed that oxLDL stimulation and static conditions synergized to enhance endothelial barrier disruption. Under the same oxLDL challenge, the application of 25 dynes/cm LSS on the endothelial monolayer decreased the passage of fluorescein isothiocyanate (FITC)-dextran by 37.79%, increased transendothelial electrical resistance (TEER) by 24.97% compared with static cells (P<0.05), which was accompanied by reduced intercellular gap formation, relatively solid cell-substrate adhesion. Compared with static cells, endothelial cells exposed to both laminar flow and oxLDL had less small FAs, less monocyte adhesion and transmigration, and alleviated overexpression of VCAM-1 and MCP-1. Meanwhile, the oxLDL-induced internalization of VE-cadherin and vinculin were also attenuated by laminar flow, and this change was more pronounced at LSS of 25 dynes/cm than 5 dynes/cm.

Conclusions: Static conditions favor, whereas physiologically higher levels of LSS ameliorate endothelial barrier disruption under pro-atherogenic stress, which is related to the improved availability of VE-cadherin and vinculin on the cell surface.
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http://dx.doi.org/10.21037/atm-20-3426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7723531PMC
November 2020

Genomic origin and intratumor heterogeneity revealed by sequencing on carcinomatous and sarcomatous components of pulmonary sarcomatoid carcinoma.

Oncogene 2021 01 3;40(4):821-832. Epub 2020 Dec 3.

Department of Medical Oncology, Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou, 510060, Guangdong, P. R. China.

Pulmonary sarcomatoid carcinoma (PSC) contains carcinomatous component (CaC) and sarcomatous component (SaC). Herein, we explored the genomic origin and intratumor heterogeneity (ITH) of PSC. We collected 31 resected PSC tumors and obtained CaC and SaC by laser capture microdissection for next-generation sequencing. The majority of PSCs (97%) had component-shared alterations. Driver mutations in EGFR, KRAS, MET, PIK3CA, and EML4-ALK fusion were mostly component-shared. Twenty-seven (87%) PSCs had component-private alterations. Compared with pure lung adenocarcinoma (LUAD), adenocarcinoma component of PSC showed lower EGFR incidence. Compared with other typical sarcomas, numerous genes of SaC exhibited significant differences. CaC and SaC had equivalent and proportional tumor mutation burden (TMB), as well as PD-L1 level. Compared with LUAD, SaC had significant higher TMB and more patients with high PD-L1 expression (tumor proportion score ≥50%). PSC with lower proportion of component-shared alterations (trunk-ratio) had a prolonged disease-free survival (DFS), regardless of the influence of clinical factors. We conclude that most PSCs originate from a monoclone accompanied by genomic ITH which is a potential independent prognostic factor, and more proportion of PSCs may be beneficial from immune checkpoint inhibitors.
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http://dx.doi.org/10.1038/s41388-020-01573-9DOI Listing
January 2021

Clinical features and factors leading to early recurrence of intussusception after saline reduction.

J Pak Med Assoc 2020 Oct;70(10):1727-1730

Department of Pediatric Surgery, Weifang Peoples Hospital, Weifang City, Shandong Province, China.

Objective: To investigate the clinical characteristics of early recurrent intussusception after ultrasound-guided saline reduction, and to explore the factors leading to early recurrence.

Methods: The retrospective observational case-control study was conducted at Weifang People's Hospital, Shandong, China, and comprised data from January 2015 to December 2017 related to paediatric intussusception patients aged 0-12 years who underwent ultrasound-guided saline enema reduction. The patients were divided into two recurrent and non-recurrent groups. Clinical characteristics of the patients with early recurrence were analysed. Factors compared between the groups were gender, age, onset season, onset-to-treatment time interval, blood in stool, fever, diarrhoea, abdominal pain and vomiting, weight and pathology. Data was analysed using SPSS 22.

Results: Of the 672 subjects, 86(13%) were patient with early recurrence while 586(87%) had no early recurrence and acted as controls. Among the patients, 70(81.4%) were aged 6-36 months. In 52(60.5%) patients, recurrence was once, and in 23(26.7%) twice. There were 141 episodes of intussusception; 24(17%) occurring in <12 hours, 85(60.2%) in 12-24 hours. Also, 5(6%) patients required surgery for reduction. Compared to the controls, the second quarter, heavier body weight and pathology were the factors leading to early recurrence of intussusceptions (p<0.05).

Conclusions: The second quarter, heavier body weight and pathological leading points were found to be factors leading to early recurrent intussusception.
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http://dx.doi.org/10.5455/JPMA.21744DOI Listing
October 2020

In vitro preparation of uniform and nucleic acid free hepatitis B core particles through an optimized disassembly-purification-reassembly process.

Protein Expr Purif 2021 02 6;178:105747. Epub 2020 Sep 6.

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, PR China. Electronic address:

Structure heterogeneity and host nucleic acids contamination are two major problems for virus-like particles (VLPs) produced by various host cells. In this study, an in vitro optimized disassembly-purification-reassembly process was developed to obtain uniform and nucleic acid free hepatitis B core (HBc) based VLPs from E. coli fermentation. The process started with ammonium sulfate precipitation of all heterogeneous HBc structures after cell disintegration. Then, dissolution and disassembly of pellets into basic subunits were carried out under the optimized disassembly condition. All contaminants, including host nucleic acids and proteins, were efficiently removed with affinity chromatography. The purified subunits reassembled into VLPs by final removal of the chaotropic agent. Two uniform and nucleic acid free HBc-based VLPs, truncated HBc and chimeric HBc-MAGE3 I, were successfully prepared. It was found that disassembly degree of HBc-based VLPs had a great influence on the protein yield, nucleic acid removal and reassembly efficiency. 4 M urea was optimal because lower concentration would not disassemble the particles completely while higher concentration would further denature the subunits into disordered aggregate and could not be purified and reassembled efficiently. For removal of strong binding nucleic acids such as in the case of HBc-MAGE3 I, benzonase nuclease was added to the disassembly buffer before affinity purification. Through the optimized downstream process, uniform and nucleic acid free HBc VLPs and HBc-MAGE3 I VLPs were obtained with purities above 90% and yields of 55.2 and 43.0 mg/L, respectively. This study would be a reference for efficient preparation of other VLPs.
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http://dx.doi.org/10.1016/j.pep.2020.105747DOI Listing
February 2021

Critical Role of Intestinal Microbiota in ATF3-Mediated Gut Immune Homeostasis.

J Immunol 2020 08 22;205(3):842-852. Epub 2020 Jun 22.

Joint Program in Immunology, Affiliated Guangzhou Women and Children's Medical Center, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510623, China;

Secretory Ig A (sIgA) plays an important role in the maintenance of intestinal homeostasis via cross-talk with gut microbiota. The defects in sIgA production could elicit dysbiosis of commensal microbiota and subsequently facilitate the development of inflammatory bowel disease. Our previous study revealed activating transcription factor 3 (ATF3) as an important regulator of follicular helper T (T) cells in gut. ATF3 deficiency in CD4 T cells impaired the development of gut T cells, and therefore diminished sIgA production, which increased the susceptibility to murine colitis. However, the potential role of microbiota in ATF3-mediated gut homeostasis remains incompletely understood. In this study, we report that both and mice displayed profound dysbiosis of gut microbiota when compared with their littermate controls. The proinflammatory taxa, especially , were more abundant in ATF3-deficient mice when compared with littermate controls. This phenotype was obviously abrogated by adoptive transfer of either T cells or IgA B cells. Importantly, depletion of gut microbiota dramatically alleviated the severity of colitis in mice, whereas transfer of microbiota from mice to wild-type recipients increased their susceptibility to colitis. Collectively, these observations indicate the importance of IgA-microbiota interaction in ATF3-mediated gut homeostasis.
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http://dx.doi.org/10.4049/jimmunol.1901000DOI Listing
August 2020

In vivo toxicity of nitroaromatic compounds to rats: QSTR modelling and interspecies toxicity relationship with mouse.

J Hazard Mater 2020 11 27;399:122981. Epub 2020 May 27.

National Engineering Laboratory for Advanced Municipal Wastewater Treatment and Reuse Technology, Engineering Research Center of Beijing, Beijing University of Technology, Beijing 100124, PR China. Electronic address:

Nitroaromatic compounds (NACs) in the environment can cause serious public health and environmental problems due to their potential toxicity. This study established quantitative structure-toxicity relationship (QSTR) models for the acute oral toxicity of NACs towards rats following the stringent OECD principles for QSTR modelling. All models were assessed by various internationally accepted validation metrics and the OECD criteria. The best QSTR model contains seven simple and interpretable 2D descriptors with defined physicochemical meaning. Mechanistic interpretation indicated that van der Waals surface area, presence of C-F at topological distance 6, heteroatom content and frequency of C-N at topological distance 9 are main factors responsible for the toxicity of NACs. This proposed model was successfully applied to a true external set (295 compounds), and prediction reliability was analysed and discussed. Moreover, the rat-mouse and mouse-rat interspecies quantitative toxicity-toxicity relationship (iQTTR) models were also constructed, validated and employed in toxicity prediction for true external sets consisting of 67 and 265 compounds, respectively. These models showed good external predictivity that can be used to rapidly predict the rat oral acute toxicity of new or untested NACs falling within the applicability domain of the models, thus being beneficial in environmental risk assessment and regulatory purposes.
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http://dx.doi.org/10.1016/j.jhazmat.2020.122981DOI Listing
November 2020

Immunization with a fusion protein vaccine candidate generated from truncated peptides of human enterovirus 71 protects mice from lethal enterovirus 71 infections.

Virol J 2020 04 22;17(1):58. Epub 2020 Apr 22.

NHC Key Laboratory of Human Disease Comparative Medicine, Beijing Key Laboratory for Animal Models of Emerging and Reemerging Infectious Diseases, Beijing Engineering Research Center for Experimental Animal Models of Human Critical Diseases, Institute of Laboratory Animal Science, CAMS&PUMC, Beijing, 100021, People's Republic of China.

Background: Prophylactic vaccines are critical in preventing hand, foot, and mouth disease (HFMD) primarily caused by human enterovirus 71 (EV71) infection. Children aged less than 5 years are especially susceptible to EV71 infections. In addition to the development of vaccines containing the inactivated virus, those containing virus-like particles (VLPs) with repeated antigens also constitute an effective preventive strategy for EV71 infections, with safety and productivity advantages. We previously developed a fusion protein composed with truncated peptides of the EV71 capsid protein, which assembled into spherical particles. This study aimed to assess the immunoprotective effects of this fusion protein as a vaccine candidate in a mouse model of EV71 infection.

Methods: To evaluate the protective effect of fusion protein vaccine candidate, neonatal mice born by immunized female mice, as well as normal neonatal mice immunized twice were infected with EV71 virus. Whereafter, the survival rates, clinical scores and viral loads were measured.

Results: The high dosage and booster immunization helped induce specific serum antibodies with high neutralization titers, which were transferred to neonatal mice, thereby facilitating effective resistance towards EV71 infection. An active immune response was also observed in neonatal mice which generated following immunization.

Conclusions: The present results suggest that this fusion protein is a suitable vaccine candidate in treating EV71 infections.
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http://dx.doi.org/10.1186/s12985-020-01328-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7178760PMC
April 2020

Prediction on the mutagenicity of nitroaromatic compounds using quantum chemistry descriptors based QSAR and machine learning derived classification methods.

Ecotoxicol Environ Saf 2019 Dec 18;186:109822. Epub 2019 Oct 18.

National Engineering Laboratory for Advanced Municipal Wastewater Treatment and Reuse Technology, Engineering Research Center of Beijing, Beijing University of Technology, Beijing, 100124, China. Electronic address:

Nitroaromatic compounds (NACs) are an important type of environmental organic pollutants. However, it is lack of sufficient information relating to their potential adverse effects on human health and the environment due to the limited resources. Thus, using in silico technologies to assess their potential hazardous effects is urgent and promising. In this study, quantitative structure activity relationship (QSAR) and classification models were constructed using a set of NACs based on their mutagenicity against Salmonella typhimurium TA100 strain. For QSAR studies, DRAGON descriptors together with quantum chemistry descriptors were calculated for characterizing the detailed molecular information. Based on genetic algorithm (GA) and multiple linear regression (MLR) analyses, we screened descriptors and developed QSAR models. For classification studies, seven machine learning methods along with six molecular fingerprints were applied to develop qualitative classification models. The goodness of fitting, reliability, robustness and predictive performance of all developed models were measured by rigorous statistical validation criteria, then the best QSAR and classification models were chosen. Moreover, the QSAR models with quantum chemistry descriptors were compared to that without quantum chemistry descriptors and previously reported models. Notably, we also obtained some specific molecular properties or privileged substructures responsible for the high mutagenicity of NACs. Overall, the developed QSAR and classification models can be utilized as potential tools for rapidly predicting the mutagenicity of new or untested NACs for environmental hazard assessment and regulatory purposes, and may provide insights into the in vivo toxicity mechanisms of NACs and related compounds.
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http://dx.doi.org/10.1016/j.ecoenv.2019.109822DOI Listing
December 2019

microRNA-605 directly targets SOX9 to alleviate the aggressive phenotypes of glioblastoma multiforme cell lines by deactivating the PI3K/Akt pathway.

Onco Targets Ther 2019 8;12:5437-5448. Epub 2019 Jul 8.

Department of Pediatrics, Weifang People's Hospital, Weifang, Shandong 261041, People's Republic of China.

Background: Aberrant microRNA (miRNA) expression has been widely reported to play a crucial role in the progression and development of glioblastoma (GBM). miR-605 has been identified as a tumor-suppressing miRNA in several types of human cancers. Nevertheless, the expression profile and detailed roles of miR-605 in GBM remain unclear and need to be further elucidated.

Materials And Methods: RT-qPCR analysis was utilized for the determination of miR-605 expression in GBM tissues and cell lines. In addition, CCK-8 assay, transwell migration and invasion assays, as well as sub-cutaneous xenograft mouse models were utilized to evaluate the effects of miR-605 upregulation in GBM cells. Notably, the potential mechanisms underlying the activity of miR-605 in the malignant phenotypes of GBM were explored.

Results: We observed that expression of miR-605 was reduced in GBM tissues and cell lines. Decreased miR-605 expression exhibited significant correlation with KPS score. The overall survival rate in GBM patients with low miR-605 expression was lower than that of patients with high miR-605 expression. Increased miR-605 expression suppressed the proliferation, migration, and invasion of U251 and T98 cells. In addition, miR-605 upregulation impaired tumor growth in vivo. Furthermore, SRY-Box 9 (SOX9) was identified as a direct target gene of miR-605 in U251 and T98 cells. SOX9 expression was shown to exhibit an inverse correlation with miR-605 expression in GBM tissues. Moreover, silencing of SOX9 expression mimicked the tumor-suppressing roles of miR-605 in U251 and T98 cells, while SOX9 restoration rescued the suppressive effects of miR-605 overexpression in the same. Notably, miR-605 suppressed the PI3K/Akt pathway in GBM in vitro and in vivo.

Conclusion: These results demonstrated that miR-605 acts as a tumor suppressor in the development of GBM by directly targeting SOX9 and inhibiting the activation of the PI3K/Akt pathway, suggesting its potential role as a therapeutic target for GBM.
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http://dx.doi.org/10.2147/OTT.S213026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6625606PMC
July 2019

Expression, Purification, and Polyethylene Glycol Site-Specific Modification of Recombinant Human Interleukin 24 in Escherichia coli.

Protein J 2019 10;38(5):576-585

College of Life Science and Bioengineering, School of Science, Beijing Jiaotong University, 3 Shangyuancun, Beijing, 100044, People's Republic of China.

Interleukin 24 (IL-24) has a broad spectrum of specific antitumor activities without affecting normal cells. The recombinant human IL-24 (rhIL-24) expressed in E. coli has low biological activity due to lack of necessary glycosylation modification. In this study, based on the modification of the non-glycosylated IL-24 with polyethylene glycol (PEG), we aimed to improve the stability and prolong its half-life in vivo. Firstly, the recombinant plasmid containing the hIL-24 cDNA was prepared by the prokaryotic-expression plasmid pET-28a and transformed into E. coli BL21. After induced by isopropyl β-D-thiogalactoside (IPTG), the target protein rhIL-24 was expressed as insoluble inclusion body, which was solubilized and denatured by 6 M guanidine hydrochloride. The denatured rhIL-24 was diluted to refold in the optimized buffer overnight at the protein concentration of 0.1 mg/mL. The refolded rhIL-24 was mainly in the form of soluble aggregate, but high-purity monomer rhIL-24 was obtained through size exchange chromatography with the addition of SDS in elution buffer. The tertiary structure of rhIL-24 was confirmed by fluorescence spectroscopy. Western blot analysis showed that rhIL-24 could be site-specifically modified by mPEG5000-ALD. Methyl thiazolyl tetrazolium (MTT) assay showed no significant difference between mPEG5000-ALD-rhIL-24 and rhIL-24 in inhibiting the growth of melanoma cell line A375 in vitro. Pharmacokinetic studies showed that PEG modification could significantly improve the stability and prolong the half-life of rhIL-24 from 8.41 to 13.2 h. The data strongly suggested that mPEG-ALD 5000 could site-specifically modify rhIL-24 expressed in E. coli. The PEG modification significantly prolonged the half-life of rhIL-24 without reducing its antitumor activity in vitro.
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http://dx.doi.org/10.1007/s10930-019-09836-5DOI Listing
October 2019

Transcriptional factor ATF3 protects against colitis by regulating follicular helper T cells in Peyer's patches.

Proc Natl Acad Sci U S A 2019 03 12;116(13):6286-6291. Epub 2019 Mar 12.

Joint Program in Immunology, Department of Internal Medicine, Affiliated Guangzhou Women and Children's Medical Center, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510623, China;

Disruption of mucosal immunity plays a critical role in the pathogenesis of inflammatory bowel disease, yet its mechanism remains not fully elucidated. Here, we found that activating transcription factor 3 (ATF3) protects against colitis by regulating follicular helper T (T) cells in the gut. The expression of ATF3 in CD4 T cells was negatively correlated with the severity of ulcerative colitis in clinical patients. Mice with ATF3 deficiency in CD4 T cells ( ) were much more susceptible to dextran sulfate sodium-induced colitis. The frequencies of T cells, not other T cell subsets, were dramatically decreased in Peyer's patches from mice compared with littermate controls. The defective T cells significantly diminished germinal center formation and IgA production in the gut. Importantly, adoptive transfer of T or IgA B cells caused significant remission of colitis in mice, indicating the T-IgA axis mediated the effect of ATF3 on gut homeostasis. Mechanistically, B cell lymphoma 6 was identified as a direct transcriptional target of ATF3 in CD4 T cells. In summary, we demonstrated ATF3 as a regulator of T cells in the gut, which may represent a potential immunotherapeutic target in colitis.
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http://dx.doi.org/10.1073/pnas.1818164116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442638PMC
March 2019

A Novel Hexavalent Capsular Polysaccharide Conjugate Vaccine (GBS6) for the Prevention of Neonatal Group B Streptococcal Infections by Maternal Immunization.

J Infect Dis 2019 06;220(1):105-115

Vaccine Research and Development, Pfizer, Pearl River, New York.

Background: Group B streptococcus (GBS) causes serious diseases in newborn infants, often resulting in lifelong neurologic impairments or death. Prophylactic vaccination of pregnant women prior to delivery could provide comprehensive protection, as early onset and late-onset disease and maternal complications potentially could be addressed.

Methods: Capsular polysaccharide conjugate vaccine GBS6 was designed using surveillance data yielded by whole-genome sequencing of a global collection of recently recovered GBS isolates responsible for invasive neonatal GBS disease. Capsular polysaccharides were isolated, oxidized using sodium periodate, and conjugated to CRM197 by reductive amination in dimethyl sulfoxide. Immune responses in mice and rhesus macaques were measured in a multiplex Luminex immunoglobulin G (IgG) assay and opsonophagocytic activity assays.

Results: The optimized conjugates were immunogenic, alone and in combination, in mice and rhesus macaques, inducing IgG antibodies that mediated opsonophagocytic killing. Active immunization of murine dams with GBS6 prior to mating resulted in serotype-specific protection of pups from a lethal challenge with GBS. Protection following passive administration of serotype-specific IgG monoclonal antibodies to dams demonstrated conclusively that anticapsular polysaccharide IgG alone is sufficient for protection.

Conclusions: The findings support the ongoing clinical evaluation of maternal GBS6 vaccination as a potential alternative method to prevent GBS disease in infants.
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http://dx.doi.org/10.1093/infdis/jiz062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548902PMC
June 2019

Polymorphonuclear myeloid-derived suppressor cells attenuate allergic airway inflammation by negatively regulating group 2 innate lymphoid cells.

Immunology 2019 04 17;156(4):402-412. Epub 2019 Jan 17.

Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China.

Hyperactivation of the type 2 immune response is the major mechanism of allergic asthma, in which both group 2 innate lymphoid cells (ILC2s) and type 2 helper T (Th2) cells participate. Myeloid-derived suppressor cells (MDSCs) alleviate asthma by suppressing Th2 cells. However, the potential effects of MDSCs on the biological functions of ILC2s remain largely unknown. Here, we examined the roles of MDSCs (MDSCs) in the modulation of ILC2 function. Our results showed that polymorphonuclear (PMN)-MDSCs, but not monocytic (M-) MDSCs, effectively suppressed the cytokine production of ILC2s both in vitro and in vivo, thereby alleviating airway inflammation. Further analyses showed that cyclo-oxygenase-1 may mediate the suppressive effects of PMN-MDSCs on ILC2 responses. Our findings demonstrated that PMN-MDSCs may serve as a potent therapeutic target for the treatment of ILC2-driven allergic asthma.
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http://dx.doi.org/10.1111/imm.13040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418421PMC
April 2019

Risk factors for short-term recurrent intussusception and reduction failure after ultrasound-guided saline enema.

Pediatr Surg Int 2018 Nov 27;34(11):1225-1231. Epub 2018 Aug 27.

Department of Pediatric Surgery, Weifang People's Hospital, Weifang, 261041, Shandong, China.

Objective: To investigate the safety and effectiveness of ultrasound-guided saline enema to treat intussusception and to analyze the risk factors affecting short-term recurrence and reduction failure.

Materials And Methods: We selected patients who had undergone intussusception reduction via ultrasound-guided saline enema from January 2010 to December 2017. The overall success rate, overall pathologic intussusception rate, and pathologic intussusception rate were calculated in each group. All the patients were divided into two groups: the successfully reduced group and the failed reduction group. Then, the successfully reduced patients were divided into two groups: the short-term recurrence group and the short-term non-recurrence group. The differences between each of the two sets of groups were analyzed, and the risk factors affecting short-term recurrence and failure of intussusception were analyzed.

Results: During the 8-year study period, a total of 1793 patients with intussusception were treated with ultrasound-guided saline enema reduction in our hospital. Among these patients, 1743 (97.2%) experienced successful reduction, 29 (1.6%) had pathologic intussusception, and 1 experienced perforation. After applying the univariate analysis and logistic regressive multivariate analysis, we found that age above 2 years and the absence of fever were risk factors for the early recurrence of intussusception. Pathologic intussusception was a risk factor for reduction failure.

Conclusion: The overall success rate of ultrasound-guided saline enemas was 97.2%, and the pathologic intussusception rate was 1.6%. Age above 2 years and the absence of fever were risk factors for short-term recurrence, and pathologic intussusception was a risk factor for the failure of reduction.
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http://dx.doi.org/10.1007/s00383-018-4340-3DOI Listing
November 2018

Suppression of RRM2 inhibits cell proliferation, causes cell cycle arrest and promotes the apoptosis of human neuroblastoma cells and in human neuroblastoma RRM2 is suppressed following chemotherapy.

Oncol Rep 2018 Jul 8;40(1):355-360. Epub 2018 May 8.

Department of Pediatric Surgery, Weifang People's Hospital, Weifang, Shandong 261041, P.R. China.

Ribonucleotide reductase regulatory subunit M2 (RRM2) is a rate‑limiting enzyme for DNA synthesis and repair. RRM2 has vital roles in controlling the progression of cancer. In the present study, we investigated the RRM2 level in neuroblastoma tissues, analyzed its relationship with clinicopathological characteristics of neuroblastoma patients, and explored the effect of RRM2 on the biological functions of neuroblastoma cells. RRM2 levels in 67 pairs of neuroblastoma and matched adjacent non‑cancerous tissues were detected by qRT‑PCR, and its association with patient clinicopathological features was assessed. Using RRM2 siRNA, the role of RRM2 in cell viability was detected by CCK‑8 assay, and the effects on cell cycle distribution and cell apoptosis were detected by flow cytometry. Hoechst 33342 staining was also performed. For RRM2 protein detection in cells and tissues, western blot analyses were employed. Our results revealed that RRM2 expression was significant higher in neuroblastoma tissues than that noted in adjacent non‑cancerous tissues at both the mRNA and protein levels. The increased RRM2 level was significantly associated with clinical stage. RRM2 levels were suppressed in stage III and IV tumors in the chemotherapy subgroup, compared with levels noted in tumors in the preoperative non‑chemotherapy subgroup. RRM2 siRNA significantly inhibited cell viability in the SH‑5Y5Y cells, induced cell arrest in the G0/G1 phase, and enhanced cell apoptosis. Taken together, overexpression of RRM2 is associated with the genesis and progression of neuroblastoma, and may be a potential chemotherapeutic target.
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http://dx.doi.org/10.3892/or.2018.6420DOI Listing
July 2018

Extending Half Life of H-Ferritin Nanoparticle by Fusing Albumin Binding Domain for Doxorubicin Encapsulation.

Biomacromolecules 2018 03 5;19(3):773-781. Epub 2018 Feb 5.

State Key Laboratory of Biochemical Engineering, Institute of Process Engineering , Chinese Academy of Sciences , Beijing 100190 , China.

Nanoparticles based on the heavy chain of the human ferritin (HFn) are arousing growing interest in the field of drug delivery due to their exceptional characteristics. However, the unsatisfied plasma half life of HFn substantially limits its application as a delivery platform for antitumor agents. Herein we fused an albumin binding domain (ABD) variant that basically derives from the streptococcal protein G and possesses a long-acting characteristic in serum albumin to the N-terminus of the HFn for the aim of half-life extension. This ABD-HFn construct was highly expressed and fully self-assembled into symmetrical and spherical structure in E. coli bacteria. The purified ABD-HFn showed a similar particle size with wild-type HFn and also exhibited an extremely high binding affinity with human serum albumin. To evaluate the therapeutic potential of this ABD-HFn construct in terms of half-life extension, we encapsulated a model antitumor agent doxorubicin (DOX) into the ABD-HFn. Significantly outstanding loading efficacy of above 60 molecules doxorubicin for each ABD-HFn cage was achieved. The doxorubicin-loaded ABD-HFn nanoparticle was characterized and further compared with the recombinant HFn counterpart. The ABD-HFn/DOX nanoparticle showed dramatically improved stability and comparable cell uptake rate when compared with HFn/DOX counterpart. Pharmacokinetics study in Sprague-Dawley rats showed that ABD-HFn/DOX nanoparticle possessed significantly prolonged plasma half life of ∼17.2 h, exhibiting nearly 19 times longer than that of free doxorubicin and 12 times for HFn/DOX. These optimal results indicated that fusion with ABD will be a promising strategy to extend the half life for protein-based nanoparticles.
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http://dx.doi.org/10.1021/acs.biomac.7b01545DOI Listing
March 2018

O-Acetylation is essential for functional antibody generation against Staphylococcus aureus capsular polysaccharide.

Hum Vaccin Immunother 2018 01 11;14(1):81-84. Epub 2017 Dec 11.

a Pfizer Vaccine Research , Pearl River , NY , USA.

Staphylococcus aureus produces an antiphagocytic polysaccharide capsule to evade neutrophil-mediated killing. Many vaccines against encapsulated bacterial pathogens require generation of functional anti-capsular antibodies to mediate protection against infection and disease. Here it is shown that the generation of such antibody responses to S. aureus in vivo and in vitro requires the presence of O-acetyl modifications on the capsular polysaccharides. O-acetylation of S. aureus capsular polysaccharide therefore should be monitored carefully during vaccine development and production. This finding may provide additional insight into the previous failure of a S. aureus capsular polysaccharide conjugate vaccine.
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http://dx.doi.org/10.1080/21645515.2017.1386360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5791590PMC
January 2018

Novel Bioconjugation Strategy Using Elevated Hydrostatic Pressure: A Case Study for the Site-Specific Attachment of Polyethylene Glycol (PEGylation) of Recombinant Human Ciliary Neurotrophic Factor.

Bioconjug Chem 2017 11 31;28(11):2841-2848. Epub 2017 Oct 31.

State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences , 1 North Second Street, Zhong-Guan Village, Beijing 100190, PR China.

In this paper, we reported a novel strategy for the site-specific attachment of polyethylene glycol (PEGylation) of proteins using elevated hydrostatic pressure. The process was similar to the conventional one except the reactor was under elevated hydrostatic pressure. The model protein was recombinant human ciliary neurotrophic factor (rhCNTF), and the reagent was monomethoxy-polyethylene glycol-maleimide (mPEG-MAL). PEGylation with mPEG (40 kDa)-MAL at pH 7.0 under normal pressure for 5 h achieved a less than 5% yield. In comparison, when the pressure was elevated, the PEGylation yield was increased dramatically, reaching nearly 90% at 250 MPa. Furthermore, the following phenomena were observed: (1) high-hydrostatic-pressure PEGylation (HHPP) could operate at a low reactant ratio of 1:1.2 (rhCNTF to mPEG-MAL), while the conventional process needs a much-higher ratio. (2) Short and long chains of PEG gave a similar yield of 90% in HHPP, while the conventional yield for the short chain of the PEG was higher than that of the long chain. (3) The reaction pH in the range of 7.0 to 8.0 had almost no influence upon the yield of HHPP, while the PEGylation yield was significantly increased by a factor of three from pH 7.0 to 8.0 at normal pressure. Surface accessibility analysis was performed using GRASP2 software, and we found that Cys17 of rhCNTF was located at the concave patches, which may have steric hindrance for the PEG to approach. The speculated benefit of HHPP was the facilitation of target-site exposure, reducing the steric hindrance and making the reaction much easier. Structure and activity analysis demonstrated that the HHPP product was comparable to the PEGylated rhCNTF prepared through a conventional method. Overall, this work demonstrated that HHPP, as we proposed, may have application potentials in various conjugations of biomacromolecules.
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http://dx.doi.org/10.1021/acs.bioconjchem.7b00531DOI Listing
November 2017

Neutrophil killing of in diabetes, obesity and metabolic syndrome: a prospective cellular surveillance study.

Diabetol Metab Syndr 2017 3;9:76. Epub 2017 Oct 3.

Pfizer Vaccine Research and Development, 401 North Middletown Rd, Pearl River, NY 10965 USA.

Background: Obesity, metabolic syndrome (MetS), and diabetes are frequent in surgical populations and can enhance susceptibility to postoperative surgical site infections. Reduced neutrophil function has been linked with diabetes and risk of infection. Therefore, neutrophil function in diabetic and obese subjects (± MetS) was assessed in this prospective serological and cellular surveillance study to determine whether vaccines administered to protect against infections after surgery could be effective in these populations.

Methods: Neutrophil function (chemotaxis, phagocytosis, and opsonophagocytic killing of ) was assessed in subjects classified according to diabetes status, body mass index, and presence/absence of MetS. Neutrophils were characterized within functional subsets by flow cytometry. A serologic assay was used to measure baseline antibody presence to each antigen in SA4Ag: capsular polysaccharide (CP) type 5, CP8, recombinant mutant Clumping factor A (rmClfA), and recombinant Manganese transport protein C (rMntC).

Results: Neutrophil function was similar for comorbid and healthy cohorts, with no significant between-group differences in cell counts, migration, phagocytosis ability, neutrophil subset proportions, and killing ability when neutrophils were isolated 3-6 months apart (Visit 1 [n = 90] and Visit 2 [n = 70]) and assessed. Median pre-existing antibody titers to CP5, CP8, and rmClfA were comparable for all cohorts (insufficient subjects with rMntC titers for determination).

Conclusions: MetS, diabetes, and obesity do not impact in vitro neutrophil function with regard to killing, suggesting that if an effective vaccine is developed it may be effective in individuals with these comorbidities.
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http://dx.doi.org/10.1186/s13098-017-0276-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627489PMC
October 2017

Albumin Binding Domain Fusing R/K-X-X-R/K Sequence for Enhancing Tumor Delivery of Doxorubicin.

Mol Pharm 2017 11 9;14(11):3739-3749. Epub 2017 Oct 9.

State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences , Beijing 100190, China.

For the purpose of improving the tumor delivery of doxorubicin (DOX), a kind of peptide-DOXO conjugate was designed and prepared, in which the peptide composed of an albumin-binding domain (ABD) and a tumor-specific internalizing sequence (RGDK or RPARPAR) was conjugated to a (6-maleimidocaproyl) hydrazone derivative of doxorubicin (DOXO-EMCH). The doxorubicin uptake by lung cancer cell line of A549 evidenced that the conjugates are capable of being internalized through a tumor-specific sequence mediated manner, and the intracellular imaging of distribution in A549 cell demonstrated that the conjugated doxorubicin can be delivered to the cell nucleus. The A549 cell cytotoxicity of peptide-DOXO conjugates was presented with IC values and shown in the range of about 9-11 μM. Pharmacokinetics study revealed that both conjugates exhibited nearly 5.5 times longer half-time than DOX, and about 4 times than DOXO-EMCH. The in vivo growth inhibitions of the two peptide-DOXO conjugates on BALB/c nude mice bearing A549 tumor (47.78% for ABD-RGDK-DOXO and 47.09% for ABD-RPARPAR-DOXO) were much stronger than that of doxorubicin and DOXO-EMCH (24.28% and 25.67% respectively) at a doxorubicin equivalent dose. Besides, the in vivo fluorescence imaging study confirmed that the peptide markedly increased the payload accumulation in tumor tissues and indicated that albumin binding domain fusing tumor-specific sequence effectively enhanced the tumor delivery of doxorubicin and thus improved its therapeutic potency.
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http://dx.doi.org/10.1021/acs.molpharmaceut.7b00497DOI Listing
November 2017

Prompt and Robust Humoral Immunity Elicited by a Conjugated Chimeric Malaria Antigen with a Truncated Flagellin.

Bioconjug Chem 2018 03 22;29(3):761-770. Epub 2017 Aug 22.

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering , Chinese Academy of Sciences , Beijing 100190 , PR China.

As one of the pathogen-associated molecular patterns (PAMPs), flagellin is recently utilized as a potent adjuvant for many subunit vaccines. In this study, a truncated flagellin (tFL) with deletion of the hypervariable regions was adopted as a carrier-adjuvant by chemical conjugation with a chimeric malaria antigen M.RCAg-1 (M312) via a heterobifunctional polyethylene glycol (PEG) linker. After booster immunization in mice without any extra adjuvants, the M312-PEG-tFL conjugates elicited M312-specific antibody titers 100-1000 times higher than M312 and 10-100 times higher than the physical mixture of M312 and tFL. The elicited specific antibodies could recognize the native parasites, and the immunofluorescence assay (IFA) titer was 2100 for M312-P5k-tFL, which was about 7 times higher than M312. Furthermore, the IFA titers of the conjugates were comparable to the positive control of complete Freund's adjuvant (CFA). Compared to M312, the M312-PEG-tFL conjugates enhanced the proliferation index, lymphocyte activation, and memory T-cell generation. IgG subclasses of sera and cytokines analysis of splenocytes showed that conjugation with tFL could slightly trigger the Th1 polarization, while the antigen alone predominantly induced a Th2-biased immune response. Furthermore, a more-efficient innate immune response was provoked by the M312-PEG-tFL conjugates, as determined by the detection of antigen-specific TNF-α secretion by splenocytes. Our results indicated that tFL mainly retained the function as an agonist of TLR5. Conjugation of antigen to tFL could induce strong humoral and moderate cellular immune responses. Thus, conjugation of antigen to tFL as a potent carrier-adjuvant is an effective strategy for developing a promising protein-based vaccine.
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http://dx.doi.org/10.1021/acs.bioconjchem.7b00320DOI Listing
March 2018

Purification and characterization of a long-acting ciliary neurotrophic factor via genetically fused with an albumin-binding domain.

Protein Expr Purif 2017 Nov 12;139:14-20. Epub 2017 Jul 12.

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, No.1 Beierjie Street, Zhongguancun, Haidian District, Beijing 100190, PR China.

Ciliary neurotrophic factor (CNTF) is a promising candidate for the treatment of neurodegenerative or metabolic diseases, but suffers rapid clearance in body. Herein we constructed a new long-acting recombinant human CNTF (rhCNTF) by genetic fusion with an albumin-binding domain (ABD) through a flexible peptide linker, hoping to endow the new molecule prolonged serum circulation time by binding with endogenous human serum albumin (HSA) and then utilizing the naturally long-half-life property of HSA. This fused protein rhCNTF-ABD was expressed in Escherichia coli mainly in the soluble form and purified through a two-step chromatography, with purity of 95% and a high yield of 90-100 mg/L culture. The in vitro binding ability of rhCNTF-ABD with HSA was firstly verified by incubation of the two components together followed by HP-SEC analysis. ABD-fused rhCNTF showed similar secondary and tertiary structure as the parent protein. It retained approximately 94.1% of the native bioactivity as demonstrated via CCK-8 cell viability assay analysis. In vivo studies in SD rats were performed and the terminal half-life of 483.89 min for rhCNTF-ABD was determined, which is about 14 folds longer than that of rhCNTF (34.28 min) and comparable with 20 k-40 kDa PEGylated rhCNTFs. The new constructed rhCNTF-ABD represents a potential therapeutic modality, and the proposed strategy may also have useful applications for other long-lasting biopharmaceutics' design.
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http://dx.doi.org/10.1016/j.pep.2017.07.006DOI Listing
November 2017

Development of long-acting ciliary neurotrophic factor by site-specific conjugation with different-sized polyethylene glycols and transferrin.

Int J Pharm 2017 Aug 23;529(1-2):275-284. Epub 2017 Jun 23.

State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China. Electronic address:

To overcome the deficiency of rapid elimination from blood, the truncated human recombinant ciliary neurotrophic factor was formulated by site-specific attachment of different-sized PEG-maleimide or by cross-linking with human transferrin through a hetero-bi-functional PEG linker (NHS-PEG5k-MAL). The PEGylated CNTF was purified by a two-step chromatography procedure and the transferrin coupling CNTF conjugate was separated through an elegant protocol. The conjugation site on CNTF was identified by peptide mapping analysis and validated that the linkage of the conjugates was specifically happened to Cys17 residue. Although both PEGylated and transferrin coupling CNTF demonstrated decreased cell based residual activity, markedly enhanced pharmacokinetic behaviors in normal male Sprague-Dawley rats were observed, especially for the PEG40k-CNTF with approximately 58-times improvement compared with the unmodified counterpart. The evaluation of the in vivo potency of body weight-losing was performed with normal male C57BL6 mice and the results revealed that both PEGylation and transferrin coupling could achieve improved therapeutic benefits relative to that of CNTF. Besides, PEG20k/40k-CNTF demonstrated more effective than transferrin coupling CNTF (Tf-PEG5k-CNTF) despite that the later showed preferable pharmacokinetic profile and cell based residual activity compared with PEG20k-CNTF. Weekly subcutaneous administration of PEG40k-CNTF with 0.5mg/kg and 1.0mg/kg dose resulted in approximately 35% and 50% decrease in food intake during one interval period of injection, indicating that PEG40k-CNTF is the most potential anti-obese agent for therapeutics.
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http://dx.doi.org/10.1016/j.ijpharm.2017.06.074DOI Listing
August 2017

High hydrostatic pressure encapsulation of doxorubicin in ferritin nanocages with enhanced efficiency.

J Biotechnol 2017 Jul 4;254:34-42. Epub 2017 Jun 4.

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, No.1 Beierjie Street, Zhongguancun, Haidian District, Beijing 100190, PR China; Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing 210023, PR China. Electronic address:

Human ferritin (HFn) nanocaging is becoming an appealing platform for anticancer drugs delivery. However, protein aggregation always occurs during the encapsulation process, resulting in low production efficiency. A new approach using high hydrostatic pressure (HHP) was explored in this study to overcome the problem of loading doxorubicin (DOX) in HFn. At the pressure of 500MPa and pH 5.5, DOX molecules were found to be encapsulated into HFn. Meanwhile, combining it with an additive of 20mM arginine completely inhibited precipitation and aggregation, resulting in highly monodispersed nanoparticles with almost 100% protein recovery. Furthermore, stepwise decompression and incubation of the complex in atmospheric pressure at pH 7.4 for another period could further increase the DOX encapsulation ratio. The HFn-DOX nanoparticles (NPs) showed similar morphology and structural features to the hollow cage and no notable drug leakage occurred for HFn-DOX NPs when stored at 4°C and pH 7.4 for two weeks. HFn-DOX NPs prepared through HHP also showed significant cytotoxicity in vitro and higher antitumor bioactivity in vivo than naked DOX. Moreover, This HHP encapsulation strategy could economize on DOX that was greatly wasted during the conventional preparation process simply through a desalting column. These results indicated that HHP could offer a feasible approach with high efficiency for the production of HFn-DOX NPs.
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http://dx.doi.org/10.1016/j.jbiotec.2017.05.025DOI Listing
July 2017

High hydrostatic pressure enables almost 100% refolding of recombinant human ciliary neurotrophic factor from inclusion bodies at high concentration.

Protein Expr Purif 2017 05 18;133:152-159. Epub 2017 Mar 18.

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, No.1 Beierjie Street, Zhongguancun, Haidian District, Beijing 100190, PR China. Electronic address:

Protein refolding from inclusion bodies (IBs) often encounters a problem of low recovery at high protein concentration. In this study, we demonstrated that high hydrostatic pressure (HHP) could simultaneously achieve high refolding concentration and high refolding yield for IBs of recombinant human ciliary neurotrophic factor (rhCNTF), a potential therapeutic for neurodegenerative diseases. The use of dilution refolding obtained 18% recovery at 3 mg/mL, even in the presence of 4 M urea. In contrast, HHP refolding could efficiently increase the recovery up to almost 100% even at 4 mg/mL. It was found that in the dilution, hydrophobic aggregates were the off-path products and their amount increased with the protein concentration. However, HHP could effectively minimize the formation of hydrophobic aggregates, leading to almost complete conversion of the rhCNTF IBs to the correct configuration. The stable operation range of concentration is 0.5-4.0 mg/mL, in which the refolding yield was almost 100%. Compared with the literatures where HHP failed to increase the refolding yield beyond 90%, the reason could be attributed to the structural difference that rhCNTF has no disulfide bond and is a monomeric protein. After purification by one-step of anionic chromatography, the purity of rhCNTF reached 95% with total process recovery of 54.1%. The purified rhCNTF showed similar structure and in vitro bioactivity to the native species. The whole process featured integration of solubilization/refolding, a high refolding yield of 100%, a high concentration of 4 mg/mL, and a simple chromatography to ensure a high productivity.
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http://dx.doi.org/10.1016/j.pep.2017.03.014DOI Listing
May 2017

Inhibition of eukaryotic initiation factor 4E phosphorylation by cercosporamide selectively suppresses angiogenesis, growth and survival of human hepatocellular carcinoma.

Biomed Pharmacother 2016 Dec 20;84:237-243. Epub 2016 Sep 20.

Hepatobiliary and Vascular Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning, PR China. Electronic address:

Mnk kinase is required for the phosphorylation and activation of the eukaryotic initiation factor 4E (eIF4E), which regulates translation of proteins involve in important aspects of hepatocellular carcinoma (HCC). Here we investigated whether an antifungal agent, cercosporamide, which had been recently identified as a potent Mnk inhibitor, is active against HCC and angiogenesis. We showed that cercosporamide significantly inhibited growth and induced caspase-dependent apoptosis on numerous HCC cell lines, while sparing normal liver cells. In addition, cercosporamide impaired HCC angiogenesis via inhibiting HCC-endothelial cells (HCC-EC) capillary network formation, migration, proliferation and survival. Importantly, cercosporamide sensitized HCC cells to cisplatin in in vitro cell culture and in vivo HCC xenograft mouse model. Cercosporamide blocked the phosphorylation of eIF4E but not Erk or p38 in a dose- and time-dependent manner in HCC and HCC-EC cells, suggesting that suppression of eIF4E phosphorylation was the result of inhibition of Mnk but not Mnk upstream pathways. Overexpression of constitutively active eIF4E (S209D) but not the nonphosphorylatable eIF4E (S209A) abolished the inhibitory effects of cercosporamide in HepG2 cells. Altogether, our work demonstrates that cercosporamide acts as a Mnk inhibitor through blockage of eIF4E phosphorylation and selectively exhibits anti-HCC activities. Our work suggests that targeting MNK-eIF4E pathway represents a therapeutic strategy to overcome chemo-resistance for HCC treatment.
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http://dx.doi.org/10.1016/j.biopha.2016.09.038DOI Listing
December 2016

Development of a Biomimetic Chondroitin Sulfate-modified Hydrogel to Enhance the Metastasis of Tumor Cells.

Sci Rep 2016 07 19;6:29858. Epub 2016 Jul 19.

Department of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China.

Tumor metastasis with resistance to anticancer therapies is the main cause of death in cancer patients. It is necessary to develop reliable tumor metastasis models that can closely recapitulate the pathophysiological features of the native tumor tissue. In this study, chondroitin sulfate (CS)-modified alginate hydrogel beads (ALG-CS) are developed to mimic the in vivo tumor microenvironment with an abnormally increased expression of CS for the promotion of tumor cell metastasis. The modification mechanism of CS on alginate hydrogel is due to the cross-linking between CS and alginate molecules via coordination of calcium ions, which enables ALG-CS to possess significantly different physical characteristics than the traditional alginate beads (ALG). And quantum chemistry calculations show that in addition to the traditional egg-box structure, novel asymmetric egg-box-like structures based on the interaction between these two kinds of polymers are also formed within ALG-CS. Moreover, tumor cell metastasis is significantly enhanced in ALG-CS compared with that in ALG, as confirmed by the increased expression of MMP genes and proteins and greater in vitro invasion ability. Therefore, ALG-CS could be a convenient and effective 3D biomimetic scaffold that would be used to construct standardized tumor metastasis models for tumor research and anticancer drug screening.
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http://dx.doi.org/10.1038/srep29858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4949442PMC
July 2016
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