Effect of “Xiaoke Tongbi granule” on the proliferation, migration and tubule-forming ability of rat endothelial progenitor cells under high glucose conditions

Purpose: To investigate the effect of Xiaoke Tongbi granule (XTG) on the proliferation, migration and tubule-forming ability of endothelial progenitor cells (EPCs) of rats under high glucose conditions. Methods: Six specific pathogen-free (SPF) and twenty-four healthy rats (mean weight = 200 ± 20 g) were used in this study. Twenty-four (24) healthy rats were treated with graded concentrations of XTG (0.75 – 2.25 g/mL) for 7 days, and were thereafter euthanized to obtain serum which was later used to treat EPCs isolated from bone marrow of SPF rats. The EPCs were seeded in culture plates pre-coated with human fibronectin, and cultured at 37 °C for 72 h in a humidified atmosphere of 5 % CO2 and 95 % air. Cell viability and apoptosis were assessed using 3 (4,5 dimethyl thiazol 2 yl) 2,5 diphenyl 2H tetrazolium bromide (MTT), and flow cytometric assays, respectively. The morphology of isolated EPCs was assessed by immunofluorescence. Results: The isolated EPCs exhibited normal morphology, and were CD34-positive. Proliferation and migration of EPCs, and number of tubular structures formed were significantly suppressed under high glucose conditions, but were significantly and concentration-dependently promoted by XTG treatment (p < 0.05). Treatment with XTG also significantly improved the morphology of isolated EPCs (p < 0.05). Apoptosis was significantly promoted by high glucose conditions, but was significantly and concentration-dependently reduced by XTG treatment (p < 0.05). The incidence of tubule formation in high glucose group was 0.63 %, but was progressively increased from 1.37 to 1.52 % after treatment with graded concentrations of XTG. Conclusion: These results indicate that XTG reverses the effect of high glucose environment on EPC proliferation, migration and tubule-forming ability.

Site-Specific N- and O-Glycosylation Analysis of Human Plasma Fibronectin

Human plasma fibronectin is an adhesive protein that plays a crucial role in wound healing. Many studies had indicated that glycans might mediate the expression and functions of fibronectin, yet a comprehensive understanding of its glycosylation is still missing. Here, we performed a comprehensive N- and O-glycosylation mapping of human plasma fibronectin and quantified the occurrence of each glycoform in a site-specific manner. Intact N-glycopeptides were enriched by zwitterionic hydrophilic interaction chromatography, and N-glycosite sites were localized by the 18O-labeling method. O-glycopeptide enrichment and O-glycosite identification were achieved by an enzyme-assisted site-specific extraction method. An RP–LC–MS/MS system functionalized with collision-induced dissociation and stepped normalized collision energy (sNCE)-HCD tandem mass was applied to analyze the glycoforms of fibronectin. A total of 6 N-glycosites and 53 O-glycosites were identified, which were occupied by 38 N-glycoforms and 16 O-glycoforms, respectively. Furthermore, 77.31% of N-glycans were sialylated, and O-glycosylation was dominated by the sialyl-T antigen. These site-specific glycosylation patterns on human fibronectin can facilitate functional analyses of fibronectin and therapeutics development.

Establishment of an Efficient Primary Culture System for Human Hair Follicle Stem Cells Using the Rho-Associated Protein Kinase Inhibitor Y-27632

BackgroundHair follicle tissue engineering is a promising strategy for treating hair loss. Human hair follicle stem cells (hHFSCs), which play a key role in the hair cycle, have potential applications in regenerative medicine. However, previous studies did not achieve efficient hHFSC expansion in vitro using feeder cells. Therefore, there is a need to develop an efficient primary culture system for the expansion and maintenance of hHFSCs.MethodsThe hHFSCs were obtained by two-step proteolytic digestion combined with microscopy. The cell culture dishes were coated with human fibronectin and inoculated with hHFSCs. The hHFSCs were harvested using a differential enrichment procedure. The effect of Rho-associated protein kinase (ROCK) inhibitor Y-27632, supplemented in keratinocyte serum-free medium (K-SFM), on adhesion, proliferation, and stemness of hHFSCs and the underlying molecular mechanisms were evaluated.ResultsThe hHFSCs cultured in K-SFM, supplemented with Y-27632, exhibited enhanced adhesion and proliferation. Additionally, Y-27632 treatment maintained the stemness of hHFSCs and promoted the ability of hHFSCs to regenerate hair follicles in vivo. However, Y-27632-induced proliferation and stemness in hHFSCs were conditional and reversible. Furthermore, Y-27632 maintained propagation and stemness of hHFSCs through the ERK/MAPK pathway.ConclusionAn efficient short-term culture system for primary hHFSCs was successfully established using human fibronectin and the ROCK inhibitor Y-27632, which promoted the proliferation, maintained the stemness of hHFSCs and promoted the ability to regenerate hair follicles in vivo. The xenofree culturing method used in this study provided a large number of high-quality seed cells, which have applications in hair follicle tissue engineering and stem cell therapy.

Engineered Protein Scaffolds as Next-Generation Therapeutics

The concept of engineering robust protein scaffolds for novel binding functions emerged 20 years ago, one decade after the advent of recombinant antibody technology. Early examples were the Affibody, Monobody (Adnectin), and Anticalin proteins, which were derived from fragments of streptococcal protein A, from the tenth type III domain of human fibronectin, and from natural lipocalin proteins, respectively. Since then, this concept has expanded considerably, including many other protein templates. In fact, engineered protein scaffolds with useful binding specificities, mostly directed against targets of biomedical relevance, constitute an area of active research today, which has yielded versatile reagents as laboratory tools. However, despite strong interest from basic science, only a handful of those protein scaffolds have undergone biopharmaceutical development up to the clinical stage. This includes the abovementioned pioneering examples as well as designed ankyrin repeat proteins (DARPins). Here we review the current state and clinical validation of these next-generation therapeutics.

De Novo Isolation & Affinity Maturation of yeast-displayed Virion-binding human fibronectin domains by flow cytometric screening against Virions

Background The promise of biopharmaceuticals comprising one or more binding domains motivates the development of novel methods for de novo isolation and affinity maturation of virion-binding domains. Identifying avenues for overcoming the challenges associated with using virions as screening reagents is paramount given the difficulties associated with obtaining high-purity virus-associated proteins that retain the conformation exhibited on the virion surface. Results Fluorescence activated cell sorting (FACS) of 1.5 × 107 clones taken from a naïve yeast surface-displayed human fibronectin domain (Fn3) against whole virions yielded two unique binders to Zika virions. Construction and FACS of site-directed binding loop mutant libraries based on one of these binders yielded multiple progeny clones with enhanced Zika-binding affinities. These affinity-matured clones bound Zika virions with low double- or single-digit nanomolar affinity in ELISA assays, and expressed well as soluble proteins in E. coli shake flask culture, with post-purification yields exceeding 10 mg/L. Conclusions FACS of a yeast-displayed binding domain library is an efficient method for de novo isolation of virion-binding domains. Affinities of isolated virion-binding clones are readily enhanced via FACS screening of mutant progeny libraries. Given that most binding domains are compatible with yeast display, the approach taken in this work may be broadly utilized for generating virion-binding domains against many different viruses for use in passive immunotherapy and the prevention of viral infection.

Characterization of pyruvate dehydrogenase complex E1 alpha and beta subunits ofMycoplasma synoviae

ABSTRACTMycoplasma synoviae(MS) is an important pathogen, causing enormous economic losses to the poultry industry worldwide every year. Therefore, the studies on MS will lay the foundation for diagnosis, prevention and treatment of MS infection. In this study, primers designed based on the sequences of pyruvate dehydrogenase complex (PDC) E1 alpha and beta subunit genes (pdhAandpdhB, respectively) of MS WVU1853 strain in GenBank were used to amplify thepdhAandpdhBgenes of MS WVU1853 strain through PCR. Then the prokaryotic expression vectors pET-pdhA and pET-pdhB were constructed and were expressed inEscherichia coliBL21(DE3) cells. Subsequently, the recombinant proteins rMSPDHA and rMSPDHB were purified and anti-rMSPDHA and anti-rMSPDHB sera were prepared by immunizing rabbits, respectively. Finally, the subcellular localization of PDHA and PDHB in MS, binding activity of rMSPDHA and rMSPDHB to chicken plasminogen (Plg) and human fibronectin (Fn), complement-dependent mycoplasmacidal assays, and adherence and adherence inhibition assays were accomplished. The results showed that PDHA and PDHB were distributed both on the surface membrane and within soluble cytosolic fractions of MS cells. The rMSPDHA and rMSPDHB presented binding activity with chicken Plg and human Fn. The rabbit anti-rMSPDHA and anti-rMSPDHB sera had distinct mycoplasmacidal efficacy in the presence of guinea pig complement, and the adherence of MS to DF-1 cells pretreated with Plg was effectively inhibited by treatment with anti-rMSPDHA or anti-rMSPDHB sera. Hence, the study indicates that the surface-associated MSPDHA and MSPDHB are the adhesion-related factors of MS that contributes to bind to Plg/Fn and adhesion to DF-1 cells.

Biophysical restriction of growth area using a monodispersed gold sphere nanobarrier prolongs the mitotic phase in HeLa cells

Homogeneous 83 nm gold nanospheres with a human fibronectin-coated substrate surrounding the cells induce biophysical cues which result in a delay in the mitotic phase of the cell cycle.