Rationalization design, soluble expression and PEG modification of highly active recombinant human-porcine uricase mutant protein.

Uric acid is the end product of purine metabolism in humans due to inactivation of the uricase determined by the mutated uricase gene. Uricase catalyzes the conversion of uric acid into water-soluble allantoin that is easily excreted by the kidneys. Hyperuricemia occurs when the serum concentration of uric acid exceeds its solubility (7 mg/dL). However, modifications to improve the uricase activity is under development for treating the hyperuricemia. Here we designed 7 types of human-porcine chimeric uricase by multiple sequence comparisons and targeted mutagenesis. An optimal human-porcine chimeric uricase mutant (uricase-10) with both high activity (6.33 U/mg) and high homology (91.45 %) was determined by enzyme activity measurement. The engineering uricase was further modified with PEGylation to improve the stability of recombinant protein drugs and reduce immunogenicity, uricase-10 could be more suitable for the treatment of gout and hyperuricemia theoretically.

Association of Maternal Body Mass Index During Early Pregnancy With Offspring Lower Respiratory Infections: A Nationwide Cohort Study.

OBJECTIVE: To assess whether or to what extent maternal obesity during early pregnancy could increase the risk of offspring lower respiratory infections (LRI). STUDY DESIGN: This population-based cohort included 688,457 live singleton births born in Denmark between 2004 and 2016. The exposure was maternal body mass index (BMI) during early pregnancy, and the outcome was LRI in offspring. Cox regression models were used to estimate hazard ratios with their 95% confidence intervals (CI) for the association. We also performed subanalysis stratified by the LRI onset age, number of infection episodes before the age of 3, infection pathogens, infection sites, duration of hospital stay due to LRI and allergic constitution of children. RESULTS: A total of 64,725 LRIs in offspring were identified during follow-up. Maternal overweight (BMI 25.0-29.9 kg/m 2 ), moderate or severe obesity (BMI 30.0-39.9 kg/m 2 ) and very severe obesity (BMI ≥40 kg/m 2 ) were associated with a 7% (95% CI: 5%-9%), 16% (95% CI: 14%-19%) and 21% (95% CI: 13%-28%) increased risk of LRI in offspring, respectively. Higher maternal BMI was positively associated with earlier onset age, more episodes before the age of 3, and longer hospital stay of LRI in offspring. In addition, allergic constitution of offspring significantly enhanced the effect of maternal BMI on offspring LRI (44% increased risk, 95% CI: 5%-97% for very severe obesity). CONCLUSIONS: Maternal BMI during early pregnancy might be a risk factor for offspring LRI, especially in children with allergic constitution.

Discovery and Characteristics of a Novel Antitumor Cyclopeptide Derived from Shark.

Peptides pose a challenge in drug development due to their short half-lives in vivo. In this study, we conducted in vitro degradation experiments on SAIF, which is a shark-derived peptide that we previously studied. The degradation fragments were sequenced and a truncated peptide sequence was identified. The truncated peptide was then cloned and expressed via the E. coli system with traceless cloning to form a novel cyclic peptide in vitro oxidation condition via the formation of a disulfide bond between the N- and C-termini, which was named ctSAIF. ctSAIF exhibited high anti-HCC activity and enhanced enzymatic stability in vitro, and retained antitumor activity and good biocompatibility in systemic circulation in a HCC xenograft model. Our study discovered and characterized a novel shark-derived cyclic peptide with antitumor activity, laying a foundation for its further development as an antitumor drug candidate. The study also provided a new solution for peptide drug development.

The role of flexible bronchoscopy in children with Mycoplasma pneumoniae pneumonia.

PURPOSE: To explore the effectiveness of flexible bronchoscopy in pediatric Mycoplasma pneumoniae pneumonia (MPP). METHODS: This retrospective cohort study included children with MPP admitted between 2016 and 2019 in Shanghai. Tracheobronchial manifestations, etiologic findings, therapeutic effect, and health-economic indicators were assessed in bronchoscopy (plus bronchoalveolar lavage (BAL)) and non-bronchoscopy group. We used propensity-score matching and multivariable logistic regression to investigate the effect of bronchoscopy and BAL on disease recovery. RESULTS: In 900 children with MPP, 24/278 (8.6%) of those who underwent bronchoscopy had sputum plugs. Coinfection rate was four-fold enhanced by BAL (19.6% vs. 4.5%, p < 0.01) in patients with severe MPP (SMPP) and nearly doubled (10.8% vs. 5.9%, p = 0.03) in those without SMPP, compared with no BAL. Total of 224 (24.9%) patients had multilobar consolidation; after BAL, a significantly shorter lesion-resolution duration was observed on imaging (OR: 0.2, 95% CI: 0.0-0.7). However, longer fever duration (OR: 2.8, 95% CI: 1.7-4.8), hospital stay (OR: 3.1, 95% CI: 1.9-5.1), and higher costs were found in the bronchoscopy group than in the non-bronchoscopy group. CONCLUSIONS: Through BAL, coinfection may explain one-fifth of causes for SMPP. Bronchoscopy with BAL may increase the detection rate of pathogen and resolve pulmonary lesions in patients with multilobar consolidation. IMPACT: Flexible bronchoscopy with bronchoalveolar lavage is of great assistance in the timely detection of coinfection, sputum plug and inflammatory polyps in children with Mycoplasma pneumoniae pneumonia (MPP), and improves the recovery of lung damage in MPP patients with multilobar consolidation. This study provides new insights into the indications of flexible bronchoscopy for the diagnosis and treatment of pediatric patients with MPP.

Recombinant Expression of Human IL-33 Protein and Its Effect on Skin Wound Healing in Diabetic Mice.

Chronic refractory wounds are one of the complications of diabetes mellitus that require effective therapy. The dermal-wound-healing property of IL-33 in diabetics is little understood. Therefore, this study aimed to express recombinant humanized mature IL-33 (rhmatIL-33) in Escherichia coli BL21 (DE3) and demonstrate its efficacy on dermal wounds in streptozotocin (STZ)-induced diabetic and nondiabetic mice by the dorsal incisional skin wound model. Results revealed that the rhmatIL-33 accelerated the scratch-healing of keratinocytes and fibroblasts at the cellular level. The wounds of diabetic mice (DM) showed more severe ulceration and inflammation than wild-type mice (WT), and the exogenous administration of rhmatIL-33 increased wound healing in both diabetic and wild-type mice. Compared with the up-regulation of endogenous IL-33 mRNA after injury in WT mice, the IL-33 mRNA decreased after injury in DM mice. Exogenous IL-33 administration increased the endogenous IL-33 mRNA in the DM group but decreased the IL-33 mRNA expression level of the WT group, indicating that IL-33 plays a balancing role in wound healing. IL-33 administration also elevated ILC2 cells in the wounds of diabetic and non-diabetic mice and improve the transcript levels of YM1, a marker of M2 macrophages. In conclusion, Hyperglycemia in diabetic mice inhibited the expression of IL-33 in the dermal wound. Exogenous addition of recombinant IL-33 promoted wound healing in diabetic mice by effectively increasing the level of IL-33 in wound tissue, increasing ILC2 cells, and accelerating the transformation of macrophage M1 to M2 phenotype.

Early life environmental antibiotic exposure and preschool allergic diseases: A biomonitoring-based prospective study in eastern China.

Background: Globally, the prevalence of allergic diseases remains high, as does the level of environmental antibiotics. It has been found that clinical antibiotic application may increase preschool allergy risk. However, few biomonitoring studies have been conducted about the association between early life environmental trace dose antibiotic exposure and preschool allergy. Objective: To analyze the association between prenatal environmental antibiotic levels and allergic diseases using logistic regression models. Methods: A total of 743 pregnant women and their offspring from the Shanghai Allergy Birth Cohort completed five years follow-up, and 251 mother-infant pairs were finally included. Maternal urine samples were collected for 15 antibiotic quantitative measurements using liquid chromatography-tandem mass spectrometry. The high-antibiotic group was defined as having at least half of antibiotics exceeding the median concentration. Allergic diseases were assessed by clinicians through clinical history, standardized questionnaires, and annual physical examinations until the age of five. Skin-prick-test (SPT) was performed at 5 years old. Results: The incidence of allergic diseases was generally higher in the high-antibiotic than that in the low-antibiotic group. Compared to the low-comprehensive antibiotic group, children in the high-antibiotic group were weakly associated with allergic diseases but had a 6-fold increased risk of food allergens sensitivity (OR: 7.09, 95% CI: 1.59, 31.74). Association of above-median single prenatal antibiotic concentration exposure and allergic diseases was also observed (azithromycin and asthma, OR: 2.72, 95% CI: 1.15, 6.42; enrofloxacin and wheeze, OR: 2.22, 95% CI: 1.22, 4.05; trimethoprim and atopic dermatitis, OR: 2.00, 95% CI: 1.08, 3.71). Moreover, children with higher prenatal norfloxacin levels were more sensitive to food allergens (OR: 5.52, 95%CI: 1.54, 19.71). Conclusion: Early-life environmental antibiotic exposure may be correlated with an increased risk of asthma, wheeze, atopic dermatitis, and SPT positivity for food allergens in 5-year-old children.

αvß3-targeted sEVs for efficient intracellular delivery of proteins using MFG-E8.

BACKGROUND: Small extracellular vesicles (sEVs) are nanometer-sized membranous particles shed by many types of cells and can transfer a multitude of cargos between cells. Recent studies of sEVs have been focusing on their potential to be novel drug carriers due to natural composition and other promising characteristics. However, there are challenges in sEVs-based drug delivery, one of which is the inefficient loading of drugs into sEVs, especially for large biomolecules. RESULTS: In this study, we proposed a membrane-associated protein, milk fat globule-epidermal growth factor 8 protein (MFG-E8), to produce αvß3-targeted sEVs with high delivery efficiency of interested protein. MFG-E8 is a secreted protein with NH2-terminal epidermal growth factor (EGF)-like domains, containing an Arg-Gly-Asp(RGD) sequence that binds αvß3 and αvß5 integrins, and COOH terminal domains C1 and C2, which can bind to lipid membrane with strong affinity. Firstly, we transiently expressed MFG-E8 in HEK293F cells and found that this protein could be secreted and adhere to the cell membrane. The recombinant MFG-E8 is also found to locate at the outer membrane of sEVs. Then we generated engineered sEVs by expressing high levels of the EGFP fused to MFG-E8 in HEK293F cells and showed that MFG-E8 could increase the delivery efficiency of EGFP into sEVs. Further delivery of Gaussia luciferase (GL) by fusion expression with MFG-E8 in donor cells demonstrated that target proteins fused with MFG-E8 still kept their activity. Finally, we identified the sEVs' target to integrin αvß3 by comparing the transfection efficiency with MFG-E8 loaded sEVs (MFG-E8-sEVs) in αvß3 positive cells and αvß3 negative cells. Analysis showed higher target protein could transfect into αvß3 positive cells with MFG-E8-sEVs than with EGFP loaded sEVs (EGFP-sEVs), meaning the engineered sEVs with MFG-E8 not only could increase the delivery of target protein into sEVs, but also could target the αvß3 positive cells. CONCLUSION: This study suggests that recombinant MFG-E8 is an ideal protein to increasingly deliver the drug into sEVs and give sEVs the ability to target the αvß3 positive cells.

KdPT alleviates imiquimod-induced psoriasis-like skin lesion in mice via inhibiting proliferation and inflammation response.

Psoriasis is a complex chronic skin inflammatory disease characterized by abnormal proliferation, differentiation of keratinocytes and infiltration of lymphocytes and neutrophils. The tripeptide KdPT, structurally derived from the C-terminal amino acid of alpha-melanocyte-stimulating hormone, has shown a significant anti-inflammatory effect on mild-to-moderate active ulcerative colitis in previous reports. In this research, we investigated whether KdPT could consistently ameliorate disease in a mouse model of imiquimod (IMQ)-induced psoriasis by inhibiting proliferation and inflammation response. We demonstrated that KdPT in vitro significantly inhibited the proliferation of human keratinocytes and endothelial cells, and also downgraded the expression of inflammatory factors in LPS-induced RAW264.7, including IL-6, TNF-α and NO. In vivo, KdPT attenuates the severity of IMQ-induced psoriasis-like phenotype in mice. Such an effect was achieved by downregulating the expression of the inflammatory cytokines interleukin (IL)-6, TNF-α, and the proliferation marker Ki67. These results suggested that KdPT might be useful in the treatment for psoriasis.

Soluble expression of bioactive recombinant porcine-human chimeric uricase mutant employing MBP-SUMO fusion system.

Urate oxidase is a promising biological medicine for hyperuricemia treatment, but immunogenicity obstructs the development of its clinical application. The recombinant porcine-human chimeric uricase mutant named dHU-wPU is a humanized chimeric uricase based on wild porcine uricase (wPU), which can effectively reduce the limitation of potential immunogenicity with a high homology (92.76%) to deduced human uricase (dHU). Unfortunately, the insoluble expression form of dHU-wPU in E. coli increases the difficulty of production. In this study, we described a more convenient method to efficiently obtain recombinant dHU-wPU protein from E. coli. Combination small ubiquitin-related modifier protein (SUMO) and maltose-binding protein (MBP) was employed to achieve the soluble expression of dHU-wPU. MBP-SUMO-dHU-wPU fusion protein was not only overexpressed in a soluble form, but also showed high purification and cleavage efficiency. Subsequently, we optimized the culture conditions of shake flasks and expanded the production of MBP-SUMO-dHU-wPU fusion protein in a 5 L bioreactor. Finally, about 15 mg of recombinant dHU-wPU was obtained from 1 L M9 fermentation culture by using two-step affinity chromatography, with a SDS-PAGE purity over 90%. In vitro activity analysis showed that dHU-wPU had better ability to catalyze uric acid than wPU.

Enhancing hemocompatibility and the performance of Au@silica nanoparticles by coating with cRGD functionalized zein.

Poor safety and effectiveness is an outstanding challenge in the preparation of drug delivery systems (DDS) for cancer treatment. The pursuit of the high curative effect will inevitably increase the risk of adverse side effects. Herein, a bio-safe DDS was constructed by combining the advantages of functional zein and Au doped mesoporous silica nanoparticles (Au@SiO2) to achieve chemo-photothermal therapy. The cRGD functionalized zein (cRGD-Zein) was coated on the surface of Au@SiO2 which effectively avoided premature leakage of paclitaxel and realized sustained drug release. Meanwhile, the high hemolysis rate (107%) of Au@SiO2 had been significantly reduced to 4%. The anti-hemolysis mechanism of functionalized zein was explored to give a deeper understanding of the interaction between nanoparticles and RBCs. The results showed that the functional zein would change the protein conformation during the interaction with Au@SiO2 to protect the RBCs from the damage of Au@SiO2. And the release rate of hemoglobin was limited by the size of RBCs membrane cracks with approximately 40 nm in width and 470 nm in length. The cell cytotoxicity and uptake assays showed that the prepared DDS exhibited low tumour cell viability (35%) and enhanced uptake performance (99.3%). This work suggested that the prepared nanoparticles could serve as a promising carrier to achieve safe and efficacious tumour therapy.

Protein-Protein Interaction Inhibitor of SRPKs Alters the Splicing Isoforms of VEGF and Inhibits Angiogenesis.

Serine-arginine (SR) protein kinases (SRPKs) regulate the functions of the SR-rich splicing factors by phosphorylating multiple serines within their C-terminal arginine-serine-rich domains. Dysregulation of these phosphorylation events has been implicated in many diseases, suggesting SRPKs are potential therapeutic targets. In particular, aberrant SRPK1 expression alters the balances of proangiogenic (VEGF165) and antiangiogenic (VEGF165b) splicing isoforms of the key angiogenesis factor, vascular endothelial growth factor (VEGF), through the phosphorylation of prototypic SR protein SRSF1. Here, we report a protein-protein interaction (PPI) inhibitor of SRPKs, docking blocker of SRPK1 (DBS1), that specifically blocks a conserved substrate docking groove unique to SRPKs. DBS1 is a cell-permeable inhibitor that effectively inhibits the binding and phosphorylation of SRSF1 and subsequently switches VEGF splicing from the proangiogenic to the antiangiogenic isoform. Our findings thus provide a new direction for the development of SRPK inhibitors through targeting a unique PPI site to combat angiogenic diseases.

Capsular Polysaccharide From Bacteroides fragilis Protects Against Ulcerative Colitis in an Undegraded Form.

The prominent human symbiont Bacteroides fragilis protects animals from intestinal diseases, such as ulcerative colitis, and its capsular polysaccharide plays a key role in reducing inflammation. B. fragilis strain ZY-312 was isolated from the feces of a healthy breast-fed infant, and the zwitterionic capsular polysaccharide zwitterionic polysaccharide, TP2, was extracted. In rats with 2,4-dinitrobenzenesulfonic acid (DNBS)-induced enteritis, TP2 at an optimal dose of 2.5 mg/kg could significantly alleviate enteritis and reduced the degree of intestinal adhesions, the intestinal ulcer area, and the incidence of ulcers in rats. To understand the underlying mechanism, TP2 was labeled with Fluorescein isothiocyanate and orally administered at a dose of 2.5 mg/kg in rats. TP2 was mainly distributed in the cecum and colorectum, but it was not detected in the blood and other organs except that a compound with a molecular weight greater than that of TP2-FITC was found in liver tissue. During the absorption, distribution, metabolism, and excretion, TP2 was indigestible. These results were further confirmed by investigation in the simulated gastric, intestinal fluid, and colonic fluid with fecal microbiota in vitro, where TP2 remained unaltered at different time points. Furthermore, flora composition was analyzed in simulated colonic fluid with TP2 added and it was found that TP2 increased the abundance of Faecalibacterium, Enterococcus romboutsia, and Ruminococcaceae, whereas the abundance of the phylum Proteobacteria represented by Sutterella, Desulfovibrio, and Enterobacteriaceae was decreased. However, the amount of short-chain fatty acids in the simulated colonic fluid was not changed by intestinal flora post-TP2 addition. In conclusion, these findings confirmed that TP2, a capsular polysaccharide of B. fragilis, protects against ulcerative colitis in an undegraded form.

Exploration of susceptible genes associated with Henoch-Schönlein purpura by whole exome sequencing.

BACKGROUND: Henoch-Schönlein purpura (HSP) is a systemic small-vessel vasculitis caused by environmental and inherent factors. Although recent research has advanced our understanding of the role of genetic susceptibility in HSP, there are still significant gaps in our knowledge. OBJECTIVES: In this study, we aimed to explore some susceptibility genes likely associated with HSP. MATERIAL AND METHODS: Three DNA samples from a family with HSP were used to perform whole exome sequencing with Illumina Hiseq 2500 high-throughput sequencing. The relevant single nucleotide variants (SNVs) were screened according to specific filter conditions and the screened SNVs were then verified with Sanger sequencing. The Sanger sequencing results were further screened according to available literature. Finally, candidate genes were validated in 92 samples from children with HSP, and also in 1 child with HSP from HSP family, using the polymerase chain reaction technique (PCR). RESULTS: Our analysis revealed that the MIF gene and the MGAT5 gene related to immunity remained after screening. Among the 93 children with HSP, there were 3 patients with MIF mutations and 2 patients with MGAT5 mutations. CONCLUSIONS: Our findings are helpful for providing new methods and ideas for understanding the pathogenesis of HSP by detecting and analyzing gene mutations at the whole-exome level including multi-generation sequencing. MIF and MGAT5 may be new susceptibility loci for HSP, but their roles in the pathogenesis of HSP are worthy of further study.

Discovery and characterization of CHO host cell protease-induced fragmentation of a recombinant monoclonal antibody during production process development.

Monoclonal antibody (mAb) fragmentation is a widespread issue of protein stability that needs to be carefully monitored for critical mAb quality control during the production process development. This study describes here the discovery and characterization of CHO host cell protease-induced fragmentation of a therapeutic mAb-X in the formulation samples from an early production process. The fragmentation was observed in the sodium dodecyl sulfate capillary electrophoresis (CE-SDS) analysis of mAb-X formulation samples incubated at elevated temperature. Size exclusion liquid chromatography (SEC-HPLC) was used to analyze and collect these cleaved fragments derived from mAb-X. Reversed phase liquid chromatography mass spectrometry (RP-LC-MS) and tandem mass (MS/MS) analysis demonstrated that the fragment was generated mainly due to the hinge region cleavage of mAb-X. The fragmentation rate was characterized in the mAb-X formulation samples at pH from 4.0 to 6.0 using CE-SDS and SDS-PAGE analysis. The percentage of the main fragment increased dramatically from 2.8% to 31.2% as pH decreased from 6.0 to 4.0 at 40 °C for 28 days, which indicated the fragmentation was highly pH-dependent. The SDS-PAGE analysis further verified the pH-dependent property of the framentation of mAb-X. Moreover, the fragmentation was characterized in the presence and absence of pepstatin A, an inhibitor of acidic proteases. Significant inhibition of mAb-X fragmentation was observed with the addition of pepstatin A to mAb-X formulation samples. These results suggested residual acidic host cell protease(s) in the formulation samples from an early production process caused the fragmentation of mAb-X. To prove evidence, we developed an optimized protein A chromatography to enhance the residual host cell protease(s) removal capability of mAb-X purification process and consequently eliminate the above described cleaved fragment of mAb-X, which further supported the hypothesis that the fragmentation of mAb-X was catalyzed by the residual host cell protease(s) in the formulation samples from the early production process. This case study reiterated that residual host cell protease is a critical quality attribute (CQA) that should be carefully controlled and evaluated to guarantee successful manufacture processes for mAb products.

Study of the mechanism for increased protein expression via transcription potency reduction of the selection marker.

Stable transfection of mammalian cells using various expression cassettes for exogenous gene expression has been well established. The impact of critical factors in these cassettes, such as promoter and enhancer elements, on recombinant protein production in mammalian cells has been studied extensively to optimize the expression efficiency. However, few studies on the correlation between the strength of selection marker and the expression of gene of interest (GOI) have been reported. Here we investigated the correlation between the strength of a widely used selection marker, glutamine synthetase (GS) gene, and gene of interest in which the expression of GOI is driven by mouse cytomegalovirus (mCMV) major immediate early (MIE) promoter whereas the expression of GS is controlled by SV40E (Simian vacuolating virus 40E) promoter. We used a green fluorescent protein and the adalimumab antibody (heavy and light chain) as two distinct examples for the gene of interest. We then decreased the expression of GS gene by engineering a specific region of its SV40E promoter in these expression cassettes. By comparing the expression of GS and GOI at transcription and translation level before and after the SV40E promoter was weakened, we found that lower GS expression due to weaker SV40E transcription correlated well with the higher expression of recombinant proteins, mainly by increasing the copy number of GS and GOI integration into host cell genome.

The role of extracellular histones in systemic-onset juvenile idiopathic arthritis.

BACKGROUND: To explore the effects of extracellular histones released by activated neutrophils on systemic-onset juvenile idiopathic arthritis (SoJIA), and to study the change of serum histone level between the active and remissive stage of SoJIA, then to clarify the role of serum histone in the pathogenesis of SoJIA. METHODS: Twenty-six patients with SoJIA were recruited, and clinical informations were collected, and the serum histone was detected by ELISA. While neutrophils from normal children were incubated with the serum from the patients with SoJIA, also including incubated with FeCL3 and histone, the extracellular histone was detected, respectively; heparin was added to the above-mentioned groups to observe the changes of extracellular histone levels. The proportions of neutrophils, which released NETs, were calculated by confocal microscope. RESULTS: The levels of serum histones in active SoJIA group (0.90 ± 0.90) were significantly higher than in remissive SoJIA group (0.17 ± 0.10) (P = 0.0009), and also higher than in control group (0.14 ± 0.09) (P = 0.246). Histone affects on clinical manifestations (including fever, rash, joint pain, liver and spleen enlargement, and serositis), except for joint pain. The proportions of neutrophils releasing NETs, that neutrophils were incubated with the serum from active SoJIA group, were 31.93% significantly higher than 12.32% from remissive SoJIA group (P < 0.0001). The proportions of neutrophils releasing NETs, that neutrophils were incubated with different concentration FeCl3 or with different concentration histones respectively, were positively correlated with the concentration of incubation; while heparins were added, NETs from neutrophils could be reduced effectively. CONCLUSIONS: The level of serum histone is positively correlated with the activity of SoJIA. Serum histone may be from NETs, which were released by activated neutrophils. Free iron can activate neutrophils to produce NETs, which may release histones, and histones can further promote NETs to be released, that results in a positive feedback loop of histones, and that may be one of the pathogenesis of acute SoJIA or MAS secondary to SoJIA. Histones maybe play one of important roles in the pathogenesis of SoJIA. Heparin can act on histones to prevent histone-induced inflammation. TRIAL REGISTRATION: ChiCTR-OOC-15006228. Registered 9 April 2015, http://www.chictr.org.cn/showproj.aspx?proj=10752.

The Effect of Culture Temperature on the Aggregation of Recombinant TNFR-Fc is Regulated by the PERK-eIF2a Pathway in CHO Cells.

BACKGROUND: Recombinant human Tumor necrosis factor receptor Ⅱ-Fc (TNFR-Fc) is a therapeutic protein which is expressed in Chinese Hamster Ovary (CHO) cells. The desired TNFRFc is a dimeric form, however, polymeric TNFR-Fc aggregation often occurs during cell culture which needs to be removed to minimize the potential risk of immunogenicity to patients. Lowering the culture temperature is useful to improve the production of many recombinant proteins in CHO cells. OBJECTIVE: The effect of different culture temperatures (37°C and 31°C, or a shift from 37°C to 31°C on the 3rd day) on the aggregation of recombinant TNFR-Fc were investigated. METHODS: Recombinant cells were cultivated at three different temperatures, namely consistent 37°C, 31°C and temperature shifted from 37°C to 31°C on the 3rd day. Intracellular and extracellular TNFR-Fc were quantified by ELISA. Bioactivity of TNFR-Fc was measured by WST-8. UPR sensors such as IRE1, PERK, ATF6, and BiP tested by Q-PCR and WB. The fusion protein TNFRFc was purified by affinity column Protein A. Size-Exclusion Chromatography (SEC) was used to determine the amount of dimeric and multimeric TNFR-Fc. RESULTS: Culture at 31°C could improve the bioactivity, assembly and secretion of TNFR-Fc, while protein aggregation decreased. The temperature shift led to a high TNFR-Fc titer, decreased aggregation, and increased anti-TNF-α activity. Surprisingly, PERK mRNA and protein levels were higher at 31°C than at 37°C, and phosphorylation of eIF2a increased. PERK inhibition and overexpression experiments confirmed its regulatory role in protein aggregation and the overall reduced protein production at low temperatures (31°C). CONCLUSION: Culture temperature affects TNFR-Fc folding and secretion. Protein aggregation was partly reduced in a PERK-dependent way at 31°C.

Hsp90 inhibitor induces KG-1a cell differentiation and apoptosis via Akt/NF-κB signaling.

Heat-shock protein 90 (Hsp 90) acts as a molecular chaperone that maintains protein stability and regulates cell proliferation, survival, differentiation and apoptosis. The present study investigated the effect of Hsp90 inhibition on human acute myeloid leukemia (AML) cells using the novel small-molecule inhibitor SNX-2112. We found that SNX-2112 more potently inhibited KG-1a cell growth than the classical Hsp90 inhibitor 17-(2-dimethylaminoethyl)amino­17-demethoxygeldanamycin as determined by CCK-8 assay. Flow cytometry was used to examine the cell cycle, differentiation, and apoptosis, and western blotting and qRT-PCR were used to analyze the underlying mechanism. The results revealed that low concentrations of SNX-2112 arrested the cells in the G2/M phase and induced their differentiation and apoptosis, possibly by suppressing Akt and inhibitor of κB kinase, a component of the nuclear factor (NF)-κB signaling pathway. We also found that SNX-2112 increased the expression of the differentiation transcription factors PU.1 and CCAAT­enhancer-binding protein-α. Thus, SNX-2112 induced KG-1a cell differentiation, cell cycle arrest and apoptosis via modulation of Akt and NF-κB signaling, suggesting that it is a promising therapeutic agent for the treatment of AML.

Facile encapsulation of hydroxycamptothecin nanocrystals into zein-based nanocomplexes for active targeting in drug delivery and cell imaging.

Nano-drug delivery systems that integrate inorganic and organic or even bioactive components into a single nanoscale platform are playing a hugely important role in cancer treatment. In this article, the fabrication of a versatile nanocarrier based on self-assembled structures of gold nanoparticles (AuNPs)-zein is reported, which displays high drug-loading efficiency for needle-shaped hydroxycamptothecin (HCPT) nanocrystals. The surface modification with folate-conjugated polydopamine (PFA) renders them stable and also facilitates their selective cellular internalization and enhancement of endocytosis. The release of payloads from nanocomplexes (NCs) was shown to be limited at physiological pH (17.1±2.8%) but significantly elevated at endosomal/lysosomal pH (58.4±3.0%) and at enzymatic environment (81.4±4.2%). Compared to free HCPT and its non-targeting equivalent, HCPT@AuNPs-Zein-PFA exerted a superior tumor suppression capacity as well as low side effects due to its active and passive targeting delivery both in vitro and in vivo. These results suggest that the NCs with well-defined core@shell nanostructures encapsulated with HCPT nanocrystals hold great promise to improve cancer therapy with high efficiency in the clinic. STATEMENT OF SIGNIFICANCE: A novel nanocomplex with HCPT nanocrystals encapsulated was designed to achieve selective cellular uptake by endocytosis, acid responsive release in the tumor microenvironment and excellent tumor suppression without toxicity. This nanocomplex with conjugation of folate was stable in the bloodstream, with minimal drug release in extracellular conditions, leading to prolonged blood circulation and high accumulation in tumor tissues. The entrapment of a nanocrystal drug into nanomaterials might be capable of delivering drugs in a predictable and controllable manner.

[Association of Cosmc gene mutation with susceptibility to Henoch-Schönlein purpura in children].

OBJECTIVE: To investigate the presence of Cosmc gene mutation in children with Henoch-Schönlein purpura (HSP) and the association between Cosmc gene mutation and the susceptibility to HSP. MESULTS: Eighty-four children who were diagnosed with HSP between March 2014 and December 2015 were selected as the HSP group. Fifty-eight healthy volunteers matched for age and sex were enrolled as the control group. Fasting venous blood (5 mL) from the two groups was collected in EDTA anticoagulated tubes, followed by the isolation of peripheral blood mononuclear cells (PBMCs) through density gradient centrifugation. Genomic DNA was extracted from PBMCs according to the manufacturer's protocol, and the whole exon region of Cosmc gene was amplified by touch-down polymerase chain reaction (touch-down PCR). The PCR products were identified by 1% agarose gel and sequenced in order to further examine the association between Cosmc gene mutation and the susceptibility to HSP. RESULTS: Sequencing results showed two mutations (c.393T>A and c.72A>G) of Cosmc gene in children with HSP. There were no significant differences in the genotype and allele frequencies at the two loci between the HSP and control groups, and this distribution was not associated with sex. CONCLUSIONS: The mutations c.393T>A and c.72A>G in the exon region of Cosmc gene in children with HSP are not associated with the onset of HSP.