Collagen-functionalised electrospun polymer fibers for bioengineering applications

Polymer electrospun fibers are gaining increasing importance in nanobiotechnology, due to their intrinsic three-dimensional topography and biochemical flexibility. Here we present an in-depth study of protein functionalisation for polymethylmethacrylate fibers. We compare different coating approaches for type I collagen, including physisorption and covalent binding methods relying on functional linkers. The biofunctionalised fibers are investigated by scanning electron and confocal laser scanning microscopy, wettability measurements, Fourier-transform infrared spectroscopy, and protein quantification assays. We demonstrate that the largest amount of proteins adsorbed on fibers does not determine the best performance in terms of cell attachment and proliferation in vitro, which is instead related to the type of linking and the relevant role played by adsorption of serum biomolecules on the three-dimensional nanostructures. This study is relevant for designing and engineering novel biomaterials and scaffold architectures based on electrospun nanofibers

Nano-LC–MS/MS for the identification of proteins trapped in sorbent cartridges used for coupled plasma filtration-adsorption treatments of healthy pigs

A dedicated proteomic approach based on nano-Liquid Chromatography coupled with tandem mass spectrometry in ion trap is proposed for the analysis of proteins trapped in sorbent resin cartridges used to remove inflammatory mediators from blood by coupled plasma filtration adsorption (CPFA). The final purpose of the proposed proteomic approach was to obtain a reference map of plasma proteins trapped in CPFA sorbents used for the extracorporeal blood purification of healthy pigs, with the potential impact to design new bio-filters able to control the inflammatory imbalance under pathological conditions, such as severe sepsis. The five main steps of the proteomics analysis, (i) protein extraction from resin cartridges, (ii) two-dimensional gel electrophoresis (2D-PAGE) for protein separation and profiling, (iii) in-gel proteolytic digestion, (iv) tandem mass analysis of peptides resulting from enzymatic cleavage and (v) bioinformatics, for protein identification and post-processing validation of MS/MS data sets, have been carefully evaluated. Prior to electrophoresis, the efficiency of different extraction solutions and procedures to recovery plasma proteins trapped into the sorbents were tested. Then, a rapid one-step procedure for protein extraction was optimized. Protein bands corresponding to the main plasma proteins, namely porcine serum albumin, serotransferrin and immunoglobulins, were identified. In addition, the presence of haptoglobin, hemopexin, α-1 acid glycoprotein and fetuin-A, that are known as acute-phase reaction proteins, was observed, suggesting that CPFA resins led to a non-specifically protein depletion from plasma, rather than targeting specific molecules

Functional Characterization of the OFD1 Protein Reveals a Nuclear Localization and Physical Interaction with Subunits of a Chromatin Remodeling Complex

Oral-facial-digital (OFD) type I syndrome is an X-linked dominant disease (MIM311200) characterized by malformations of oral cavity, face, and digits and by cystic kidneys. We previously identified OFD1, the gene responsible for this disorder, which encodes for a centrosomal protein with an unknown function. We now report that OFD1 localizes both to the primary cilium and to the nucleus. Moreover, we demonstrate that the OFD1 protein is able to self-associate and that this interaction is mediated by its coiled-coil rich region. Interestingly, we identify an OFD1-interacting protein RuvBl1, a protein belonging to the AAA+-family of ATPases, which has been recently associated to cystic kidney in zebrafish and to ciliary assembly and function in Chlamydomonas reinhardtii. We also provide experimental evidence that OFD1, together with RuvBl1, is able to coimmunoprecipitate with subunits of the human TIP60 histone acetyltransferase (HAT) multisubunit complex. On the basis of these results, we hypothesize that OFD1 may be part of a multi-protein complex and could play different biological functions in the centrosome-primary cilium organelles as well as in the nuclear compartment

Bioactive Nanofiber Matrices Functionalized with Fibronectin-Mimetic Peptides Driving the Alignment and Tubular Commitment of Adult Renal Stem Cells

Bioactive, electrospun polymer nanofibers (NFs) are attractive building-block materials for realizing scaffolds for tissue engineering, as these filaments structurally mimic the hierarchical organization of natural extracellular matrix. As presented in this article, demonstrated applications involve the commitment to adipogenic, chondrogenic, osteogenic, neural lineages, and so on, by different types of stem cells obtained from various sources (adult, embryonic, induced pluripotent stem cells, etc.). Since a poor cellular colonization may limit the usefulness of scaffolds for culturing delicate stem cell populations, coupling NFs with cell-adhesive domains is highly desirable. In our work, the surface of either isotropic (randomly orienteor anisotropic (highly aligneelectrospun poly(l-lactic acid) fibrous scaffolds with fibronectin (FN)-mimetic peptides is tailored. The effects of the peptide immobilization on the adhesion and spreading of human, adult renal stem cells are investigated. Functionalized, aligned NFs are suitable scaffolds to promote the confluent growth of stem cells and to enhance their differentiation toward the tubular epithelial lineage. It is anticipated that the functionalization of aligned NFs with FN-mimetic peptides might be a promising method to target a specific response of stem cells in renal tissue engineering. The design and realization of electrospun nanofibers coupled with a fibronectin-mimetic peptide is described. The effect of the scaffolds on the behavior of renal stem cells is analyzed. These bioactive fibers might be useful for the definition of new biomimetic platforms to effectively culture and commit renal stem cells, allowing confluent and functional cell layers to be generated which are analogous to the renal proximal tubule epithelium

Collagen-functionalised electrospun polymer fibers for bioengineering applications

Polymer electrospun fibers are gaining increasing importance in nanobiotechnology, due to their intrinsic three-dimensional topography and biochemical flexibility. Here we present an in-depth study of protein functionalisation for polymethylmethacrylate fibers. We compare different coating approaches for type I collagen, including physisorption and covalent binding methods relying on functional linkers. The biofunctionalised fibers are investigated by scanning electron and confocal laser scanning microscopy, wettability measurements, Fourier-transform infrared spectroscopy, and protein quantification assays. We demonstrate that the largest amount of proteins adsorbed on fibers does not determine the best performance in terms of cell attachment and proliferation in vitro, which is instead related to the type of linking and the relevant role played by adsorption of serum biomolecules on the three-dimensional nanostructures. This study is relevant for designing and engineering novel biomaterials and scaffold architectures based on electrospun nanofibers

Aligned Nanofiber Topographies Enhance the Differentiation of Adult Renal Stem Cells into Glomerular Podocytes

The use of adult stem/progenitor cells is a challenge in current research about kidney functional regeneration. In this framework, material-induced stem cell differentiation can become a new paradigm to promote advances in therapies. Here, the effects of both isotropic and anisotropic fibrous topographies on the podocyte differentiation of adult renal stem cells (RSCs) from human donors are investigated. The proliferation rate of RSCs is analyzed, immunofluorescence and genetic analyses of specific podocyte markers (Wilms’ tumor 1 gene, nephrin, and podocin) are performed to assess the differentiation level on the scaffolds. The studied markers are over-expressed in RSCs cultured on aligned fibers compared to cells cultured on either protein-functionalized films or randomly oriented fibers. In addition, RSCs cultured on aligned fibers are found to differentiate toward podocyte precursors even in basal medium conditions, thus highlighting scaffold-induced commitment without exogenous chemicals or cellular reprogramming. Aligned polymer fiber scaffolds which provide instructive cues for RSC differentiation might lead to new biomimetic systems for renal stem cell engineering

Establishment and characterization of a highly immunogenic human renal carcinoma cell line

Renal cell carcinoma (RCC) is the most common kidney cancer, and accounts for ~3% of all adult malignancies. RCC has proven refractory to conventional treatment modalities but appears to be the only histological form that shows any consistent response to immunotherapeutic approaches. The development of a clinically effective vaccine remains a major strategic target for devising active specific immunotherapy in RCC. We aimed to identify a highly immunogenic antigenic format for immunotherapeutic approaches, so as to boost immune responses in RCC patients. We established and cloned an immunogenic cell line, RCC85#21 named Elthem, which was derived from a non-aggressive and non-metastatic clear cell carcinoma. The cell line characterization was performed by genomics (real-time PCR, genome instability), proteomics (two dimensional electrophoresis, mass spectro-metry) and immunological analysis (mixed lymphocytes tumor cell cultures). Real-time PCR confirmed the RCC85#21 cell expression of tumor antigens and cytokine genes. No difference in microsatellite instability (MSI) in RCC85#21 cell line was found as compared to control, loss of heterozygosity was observed in the RCC85#21 clone, but not in the renal cancer cell lines from which it was generated. The image analysis of RCC85#21 by two-dimensional gels showed 700±26 spots and 119 spots were identified by mass spectrometry analysis. RCC85#21 promoted a significant RCC-specific T cells activation by exhibiting a cytotoxic phenotype after mixed lymphocyte and tumor cell cultures. CD8+ T cells isolated from RCC patients displayed an elevated reactivity against RCC85#21 and efficiently lysed the RCC85#21 clone. The RCC85#21 immunogenic cell line will be suitable for immune stimulation. The identification of novel tumor associated antigens will allow the evaluation of the immune response in vitro and, subsequently, in vivo paving the way for new immunotherapeutic strategies in the RCC setting

Semaphorin 3F expression is reduced in pregnancy complicated by preeclampsia. An observational clinical study

Background and objective Preeclampsia is a systemic disorder, affecting 2-10% of pregnancies, characterized by a deregulated pro-and anti-angiogenic balance. Semaphorin 3F is an angiogenesis inhibitor. We aimed to investigate whether semaphorin 3F expression is modulated in preeclampsia. Design, setting, participants, and measurements We performed two observational single center cohort studies between March 2013 and August 2014. In the first we enrolled 110 consecutive women, undergoing an elective cesarean section; in the second we included 150 consecutive women undergoing amniocentesis for routine clinical indications at 16-18 week of gestation. Semaphorin 3F concentration was evaluated in maternal peripheral blood, venous umbilical blood and amniotic fluid, along with its placenta protein expression at the time of delivery in the first study group and in the amniotic fluid at 16-18 weeks of gestation in the second study group. Results In the first study 19 patients presented at delivery with preeclampsia. Semaphorin 3F placenta tissue expression was significantly reduced in preeclampsia. In addition, semaphorin 3F level at delivery was significantly lower in serum, amniotic fluid and venous umbilical blood of preeclamptic patients compared with normal pregnant women. In the prospective cohort study 14 women developed preeclampsia. In this setting, semaphorin 3F amniotic level at 16-18 weeks of gestation was reduced in women who subsequently developed preeclampsia compared to women with a normal pregnancy. ROC curve analysis showed that semaphorin 3F amniotic levels could identify women at higher risk of preeclampsia. Conclusions Semaphorin 3F might represent a predictive biomarker of preeclampsia