Surface modification to improve in vitro attachment and proliferation of human urinary tract cells

To evaluate the attachment and proliferation of cultured human urinary tract cells to culture plates surface-modified by photochemical immobilization of extracellular matrix (ECM) proteins. MATERIALS AND METHODS Human uroepithelial (UEC) and smooth muscle (SMC) cells were harvested from ureter and expanded in culture; 24-well culture plates surface-modified by photochemical covalent immobilization of ECM proteins were then seeded with UEC or SMC. To characterize cellular attachment, cells were incubated on surface-modified plates for 30 and 90 min. For proliferation assays the cells were incubated for 3 12 days. Standard tissue culture plates with no surface modification and sham-modified plates served as controls. Differential attachment and proliferation on the various surfaces were assessed using analysis of variance with Fisher's posthoc test for multiple comparisons. RESULTS Attachment at 30 and 90 min of both UEC and SMC on plates surface-modified with ECM proteins was significantly greater than in control plates. Surface-modification with collagen resulted in significantly greater cellular attachment than with either laminin or fibronectin. UEC proliferation was also significantly greater than in control plates by surface-modification with collagen and fibronectin, but not with laminin. SMC proliferation was significantly better after surface modification than on sham- modified plates, but was no better than standard plates. CONCLUSIONS Covalent photochemical immobilization of ECM proteins to potential growth surfaces enhances the attachment of cultured UEC and SMC and the proliferation of UEC. This technique might be useful in modifying surface properties of synthetic polymer-based materials in a controlled and defined manner, giving them the capacity to promote and sustain the growth of urinary tract cells. This may lead to development of alternative methods of tissue engineering in the urinary tract.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73750/1/j.1464-410X.2003.04418.x.pd

Differential Contribution of Subunit Interfaces to α 9 α 10 Nicotinic Acetylcholine Receptor Function

Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits.Fil: Boffi, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Marcovich, Irina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Gill Thind, JasKiran K.. University College London; Reino UnidoFil: Corradi, Jeremias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Collins, Toby. University College London; Reino UnidoFil: Lipovsek, Maria Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Moglie, Marcelo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Plazas, Paola Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Craig, Patricio Oliver. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Millar, Neil S.. University College London; Reino UnidoFil: Bouzat, Cecilia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

The effect of bacteriochlorophyll derivative WST-D and near infrared light on the molecular and fibrillar architecture of the corneal stroma

A cross-linking technique involving application of Bacteriochlorophyll Derivative WST-11 mixed with dextran (WST-D) to the epithelium-debrided cornea and illumination with Near Infrared (NIR), has been identified as a promising therapy for stiffening pathologically weakened corneas. To investigate its effect on corneal collagen architecture, x-ray scattering and electron microscopy data were collected from paired WST-D/NIR treated and untreated rabbit corneas. The treated eye received 2.5 mg/mL WST-D and was illuminated by a NIR diode laser (755 nm, 10 mW/cm2). An increase in corneal thickness (caused by corneal oedema) occurred at 1-day post-treatment but resolved in the majority of cases within 4 days. The epithelium was fully healed after 6–8 days. X-ray scattering revealed no difference in average collagen interfibrillar spacing, fibril diameter, D-periodicity or intermolecular spacing between treated and untreated specimens. Similarly, electron microscopy images of the anterior and posterior stroma in healed WST-D/NIR corneas and untreated controls revealed no obvious differences in collagen organisation or fibril diameter. As the size and organisation of stromal collagen is closely associated with the optical properties of the cornea, the absence of any large-scale changes following treatment confirms the potential of WST-D/NIR therapy as a means of safely stiffening the cornea

Jute/polypropylene composites: Effect of enzymatic modification on thermo-mechanical and dynamic mechanical properties

In this study, a high-performance composite was prepared from jute fabrics and polypropylene (PP). In order to improve the compatibility of the polar fibers and the non-polar matrix, alkyl gallates with different hydrophobic groups were enzymatically grafted onto jute fabric by laccase to increase the surface hydrophobicity of the fiber. The grafting products were characterized by FTIR. The results of contact angle and wetting time showed that the hydrophobicity of the jute fabrics was improved after the surface modification. The effect of the enzymatic graft modification on the properties of the jute/PP composites was evaluated. Results showed that after the modification, tensile and dynamic mechanical properties of composites improved, and water absorption and thickness swelling clearly decreased. However, tensile properties drastically decreased after a long period of water immersion. The thermal behavior of the composites was evaluated by TGA/DTG. The fiber-matrix morphology in the modified jute/PP composites was confirmed by SEM analysis of the tensile fractured specimens.This work was financially supported by the National Natural Science Foundation of China (51173071), the Program for New Century Excellent Talents in University (NCET-12-0883), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1135) and the Fundamental Research Funds for the Central Universities (JUSRP51312B, JUSRP51505)

Molecular Effects of Doxycycline Treatment on Pterygium as Revealed by Massive Transcriptome Sequencing

Pterygium is a lesion of the eye surface which involves cell proliferation, migration, angiogenesis, fibrosis, and extracellular matrix remodelling. Surgery is the only approved method to treat this disorder, but high recurrence rates are common. Recently, it has been shown in a mouse model that treatment with doxycycline resulted in reduction of the pterygium lesions. Here we study the mechanism(s) of action by which doxycycline achieves these results, using massive sequencing techniques. Surgically removed pterygia from 10 consecutive patients were set in short term culture and exposed to 0 (control), 50, 200, and 500 µg/ml doxycycline for 24 h, their mRNA was purified, reverse transcribed and sequenced through Illumina’s massive sequencing protocols. Acquired data were subjected to quantile normalization and analyzed using cytoscape plugin software to explore the pathways involved. False discovery rate (FDR) methods were used to identify 332 genes which modified their expression in a dose-dependent manner upon exposure to doxycycline. The more represented cellular pathways included all mitochondrial genes, the endoplasmic reticulum stress response, integrins and extracellular matrix components, and growth factors. A high correlation was obtained when comparing ultrasequencing data with qRT-PCR and ELISA results