The biophysical and compositional properties of human basement membranes.

Basement membranes are among the most widespread, non-cellular functional materials in metazoan organisms. Despite this ubiquity, the links between their compositional and biophysical properties are often difficult to establish due to their thin and delicate nature. In this article, we examine these features on a molecular level by combining results from proteomics, elastic, and nanomechanical analyses across a selection of human basement membranes. Comparing results between these different membranes connects certain compositional attributes to distinct nanomechanical signatures and further demonstrates to what extent water defines these properties. In all, these data underline BMs as stiff yet highly elastic connective tissue layers and highlight how the interplay between composition, mechanics and hydration yields such exceptionally adaptable materials.

Characterization of the dystrophin-associated protein complex by mass spectrometry.

The dystrophin-associated protein complex (DAPC) is a highly organized multiprotein complex that plays a pivotal role in muscle fiber structure integrity and cell signaling. The complex is composed of three distinct interacting subgroups, intracellular peripheral proteins, transmembrane glycoproteins, and extracellular glycoproteins subcomplexes. Dystrophin protein nucleates the DAPC and is important for connecting the intracellular actin cytoskeletal filaments to the sarcolemma glycoprotein complex that is connected to the extracellular matrix via laminin, thus stabilizing the sarcolemma during muscle fiber contraction and relaxation. Genetic mutations that lead to lack of expression or altered expression of any of the DAPC proteins are associated with different types of muscle diseases. Hence characterization of this complex in healthy and dystrophic muscle might bring insights into its role in muscle pathogenesis. This review highlights the role of mass spectrometry in characterizing the DAPC interactome as well as post-translational glycan modifications of some of its components such as α-dystroglycan. Detection and quantification of dystrophin using targeted mass spectrometry are also discussed in the context of healthy versus dystrophic skeletal muscle.

Creation of a decellularized vaginal matrix from healthy human vaginal tissue for potential vagina reconstruction: experimental studies.

BACKGROUND: When a disorder causes the absence of a healthy, full-size vagina, various neovaginal creation methods are available. Sometimes dilation or stretching of the vaginal cavity is sufficient, but intestinal or dermal flap tissue is generally required. However, different inherent tissue properties cause complications. Therefore, a lost body part should be replaced with a similar material. The use of organ-specific acellular vaginal tissue carries great potential, as its similar architecture and matrix composition make it suitable for vaginal regeneration. METHODS: The authors developed an optimized protocol for decellularization of healthy human vaginal tissue. Resected colpectomy tissue from 12 healthy transgender patients was used. Successful decellularization was confirmed by applying acellular criteria from in-vivo remodeling reports. Suitability as a tissue-mimicking scaffold for vaginal reconstruction was determined by visible structural features, biocompatibility during stretching, and the presence of visible collagen, elastin, laminin, and fibronectin. RESULTS: Histological examination confirmed the preservation of structural features, and minimal cellular residue was seen during fluorescence microscopy, DNA and RNA quantification, and fragment length examination. Biomechanical testing showed decreased peak load (55%, P <0.05), strain at rupture (23%, P <0.01), and ultimate tensile stress (55%, P <0.05) after decellularization, while the elastic modulus (68%) did not decrease significantly. Fluorescence microscopy revealed preserved Fibronectin-I/II/III and Laminin-I/II, while Collagen-I and Ficolin-2B were decreased but mostly retained. CONCLUSIONS: The absence of cellular residue, moderately altered biomechanical extracellular matrix properties, and mostly preserved structural proteins appear to make our decellularized human vaginal matrix a suitable tissue-mimicking scaffold for vagina transplantation when tissue survival through vascularization and innervation are accomplished in the future.

New approaches concerning the testis of Astyanax lacustris (Characidae): immunohistochemical studies.

Astyanax lacustris, locally known as lambari-do-rabo-amarelo, is a study model for Neotropical fish. Testis of A. lacustris shows deep morphophysiological changes throughout the annual reproductive cycle. This work analyzed the distribution of claudin-1, actin, and cytokeratin as elements of the cytoskeleton in germinal epithelium and interstitium; the distribution of type I collagen, fibronectin, and laminin as extracellular matrix compounds; and the localization of androgen receptor in the testis of this species. Claudin-1, cytokeratin, and actin were present in the Sertoli cells and modified Sertoli cells, and actin was also detected in peritubular myoid cells. Type I collagen were in the interstitial tissue, laminin in the basement membrane of germinal epithelium and endothelium, but fibronectin was additionally detected in the germinal epithelium compartment. The labeling of androgen receptor was higher in peritubular myoid cells and undifferentiated spermatogonia, and weaker labeling was detected in type B spermatogonia. Therefore, the present work highlights new aspects of the biology of the testis of A. lacustris, and contribute to amplify the understanding of this organ.

Drosophila collagens in specialised extracellular matrices.

The basement membrane (BM) constitutes a specialised form of the extracellular matrix (ECM) and plays important roles in many biological processes, such as cell migration, organ and tissue integrity, cell polarity, and the formation of metastases. In metazoans, a canonical BM is formed by only a few conserved structural core proteins: Laminin, Collagen IV, Nidogen and Perlecan. Depending on the tissue's function and mechanical load, additional matrix proteins interact with, or are incorporated into the BM, resulting in tissue-specific mechanical properties, such as higher stiffness or elasticity, or special resistance to mechanical stress or harmful environmental conditions. In flies, the collagen IV-like protein Pericardin forms an integral constituent of matrices around the heart and tension sensors (chordotonal organs) of the peripheral nervous system. The function and integrity of both organ systems strongly relies on the appropriate establishment of a Pericardin (Prc) matrix and the function of its adapter protein-Lonely heart (Loh). In this review, we provide an overview of the four collagens present in flies, and will discuss our recent work on the formation and function of Pericardin-containing matrices, the role of the adapter protein Lonely heart and the necessity of specialised ECM molecules in tissue architecture and function.

Expression of cadherins and some connective tissue components in cow uterus and placenta during pregnancy.

The implantation and placental development processes are regulated with cell adhesion molecules and remodeling of the maternal endometrium's extracellular matrices (ECM) and fetal chorion. This study aimed to investigate the distribution and localization of some classical cadherins (E-, N-, and P-cadherins) and extracellular matrix components collagen type 5α1, fibronectin, and laminin in the cow placentomes during pregnancy using immunohistochemical and Western blotting analyses. The study results confirmed the expression of E- and P-cadherins, collagen type Vα1 (COLVα1), fibronectin, and laminin in the cow placentomes, but not N-cadherin. Throughout the pregnancy, E- and P- cadherins, COLVα1, and laminin were localized in the luminal and glandular epithelium of the inter-caruncular endometrium, caruncular epithelium, and the uninucleate (UNCs) and binucleate trophoblast giant cells (BNCs/TGCs). E- cadherin immunoreactivity in the first pregnancy period was strong in the UNCs while moderate in the BNCs/TGCs. However, it was weak in both trophoblast in the second and third pregnancy periods. In the fetal trophoblasts, P- cadherin and laminin immunostainings were more intense in the BNCs/TGCs than UNCs. The fetal and maternal stromal cells were also positive for P- cadherin, COLVα1, fibronectin, and laminin. The immunostaining intensity of COLVα1 and fibronectin in the stromal extracellular matrix of the placentomes decreased as the pregnancy progressed. The endothelia of fetal and maternal vessels were positive for all proteins. The presence and distinct localization of cadherins and ECM proteins in the cow placentome components support the role of these molecules in regulating placental cell growth, migration, and matrix production during pregnancy.

Effects of ultrasound and stretching on skeletal muscle contusion in rats: immunohistochemistry analysis / Efectos de ultrasonido y estiramiento en la contusión muscular esqueletal en ratas: análisis inmunohistoquímico

SUMMARY: The aim of this study was to evaluate the effects of stretching and therapeutic ultrasound (TUS) on desmin and laminin contents of rat muscle after contusion. Male Wistar rats (n = 35, 8-9 weeks of age, 271 ± 14g body weight) were divided into five groups: Control group (CG) (n= 03); Injured group (IG) (n= 8); Injured + ultrasound group (IUSG) (n= 8); Injured+stretching group (ISG) (n= 8); Injured +ultrasound + stretching group (IUSSG) (n= 8). The application of ultrasound started 72 hours after the contusion, using the 50 % pulsed mode, 0.5 W/cm2, 5 min, once a day, for five consecutive days. Passive manual stretching was started on the tenth day after injury, with four repetitions of 30 s each and 30 s rest between repetitions, once a day, five times per week, for a total of ten applications. After 22 days, the rats were euthanazied and the gastrocnemius of both limbs removed for desmin and laminin immunohistochemistry morphometric measurement. Analysis was conducted using ANOVA one way post-hoc Tukey to parametric data and Kruskall-Wallis for non-parametric data. The IUSSG animals showed a larger area of desmin than ISG (p<0.05). It was found a decrease in laminin comparing IUSG to IG. However, laminin area was higher in ISG than all groups (p<0.05). UST isolated or in combination with stretching influenced gastrocnemius regeneration in different manners. While stretching applied isolated enhanced gastrocnemius regeneration noticed by the increase in laminin area, in combination with TUS strengthened the muscle healing rising desmin area.

RESUMEN: El objetivo de este estudio fue evaluar los efectos del estiramiento y la ecografía en los contenidos de desmina y laminina del músculo de rata después de la lesión. Ratas Wistar macho (n = 35, 8-9 semanas de edad, 271 ± 14 g de peso corporal) se dividieron en cinco grupos: grupo de control (CG) (n = 03); Grupo lesionado (GL) (n = 8); Lesionado + grupo de ultrasonido (LGU) (n= 8); Lesionado + grupo de estiramiento (LGE) (n = 8); Lesionado + ultrasonido + grupo de estiramiento (LUGE) (n = 8). La aplicación de ultrasonido comenzó 72 horas después de la lesión, usando el modo pulsado al 50 %, 0,5W / cm2, 5 min, una vez al día, durante cinco días consecutivos. El estiramiento manual pasivo se inició el décimo día después de la lesión, con cuatro repeticiones de 30 seg cada una y 30 seg de descanso entre repeticiones, una vez al día, cinco veces por semana, para un total de diez aplicaciones. Las ratas fueron sacrificadas después de 22 días, y se extrajo el músculo gastrocnemio de ambos miembros para la medición morfométrica de desmina y laminina a través de inmunohistoquímica. El análisis se realizó utilizando ANOVA unidireccional Tukey post-hoc para datos paramétricos y Kruskall-Wallis para datos no paramétricos. Los animales LUGE mostraron un área mayor de desmina que LGE (p <0,05). Se encontró una disminución en la laminina comparando LGU con GL. Sin embargo, el área de laminina fue mayor en LGE que en todos los grupos (p <0,05). El tratamiento con ultrasonido aislado o en combinación con estiramiento influyó en la regeneración del músculo gastrocnemio de diferentes maneras. Si bien el estiramiento aplicado, en combinación con tratamiento de ultrasonido, fortaleció el área de desmina, la regeneración del músculo gastrocnemio mejoró por el aumento en el área de laminina aumentando la curación muscular.

Extracellular Matrix Structure and Composition in the Early Four-Chambered Embryonic Heart.

During embryonic development, the heart undergoes complex morphogenesis from a liner tube into the four chambers consisting of ventricles, atria and valves. At the same time, the cardiomyocytes compact into a dense, aligned, and highly vascularized myocardium. The extracellular matrix (ECM) is known to play an important role in this process but understanding of the expression and organization remains incomplete. Here, we performed 3D confocal imaging of ECM in the left ventricle and whole heart of embryonic chick from stages Hamburger-Hamilton 28-35 (days 5-9) as an accessible model of heart formation. First, we observed the formation of a fibronectin-rich, capillary-like networks in the myocardium between day 5 and day 9 of development. Then, we focused on day 5 prior to vascularization to determine the relative expression of fibronectin, laminin, and collagen type IV. Cardiomyocytes were found to uniaxially align prior to vascularization and, while the epicardium contained all ECM components, laminin was reduced, and collagen type IV was largely absent. Quantification of fibronectin revealed highly aligned fibers with a mean diameter of ~500 nm and interfiber spacing of ~3 µm. These structural parameters (volume, spacing, fiber diameter, length, and orientation) provide a quantitative framework to describe the organization of the embryonic ECM.

LAMC1 is a prognostic factor and a potential therapeutic target in endometrial cancer.

OBJECTIVE: With the emerging significance of genetic profiles in the management of endometrial cancer, the identification of tumor-driving genes with prognostic value is a pressing need. The LAMC1 gene, encoding the laminin subunit gamma 1 (LAMC1) protein, has been reported to be involved in the progression of various malignant tumors. In this study, we aimed to investigate the role of LAMC1 in endometrial cancer and elucidate the underlying mechanism. METHODS: We evaluated the immunohistochemical expression of LAMC1 in atypical endometrial hyperplasia and endometrial cancer. Within the endometrial cancer cases, we analyzed the association of LAMC1 overexpression with clinicopathological factors and prognosis. Furthermore, to indentify genes influenced by LAMC1 overexpression, we transfected HEC50B and SPAC-S cells with siRNA targeting LAMC1 and conducted microarray gene expression assays. RESULTS: While none of the atypical endometrial hyperplasia specimens exhibited LAMC1 overexpression, endometrial cancer possessed a significantly higher LAMC1 overexpression rate. LAMC1 overexpression was strongly associated with histological type, lymphovascular space invasion, lymph node metastasis, advanced International Federation of Gynecology and Obstetrics stage, and poor overall survival in endometrial cancer. Gene expression microarray analysis identified 8 genes correlated with tumor progression (LZTFL1, TAPT1, SEL1L, PAQR6, NME7, TMEM109, CCDC58, and ANKRD40) that were commonly influenced in HEC50B and SPAC-S by LAMC1 silencing. CONCLUSION: LAMC1 overexpression is a potent biomarker for identifying endometrial cancer patients needing aggressive adjuvant therapy. We elucidated 8 candidate genes that may mediate progression of LAMC1 overexpressing cancer. Further investigation of the underlying mechanism should lead to the discovery of new therapeutic targets.

Agrin plays a major role in the coalescence of the aquaporin-4 clusters induced by gamma-1-containing laminin.

The basement membrane that seperates the endothelial cells and astrocytic endfeet that comprise the blood-brain barrier is rich in collagen, laminin, agrin, and perlecan. Previous studies have demonstrated that the proper recruitment of the water-permeable channel aquaporin-4 (AQP4) to astrocytic endfeet is dependent on interactions between laminin and the receptor dystroglycan. In this study, we conducted a deeper investigation into how the basement membrane might further regulate the expression, localization, and function of AQP4, using primary astrocytes as a model system. We found that treating these cells with laminin causes endogenous agrin to localize to the cell surface, where it co-clusters with ß-dystroglycan (ß-DG). Conversely, agrin sliencing profoundly disrupts ß-DG clustering. As in the case of laminin111, Matrigel™, a complete basement membrane analog, also causes the clustering of AQP4 and ß-DG. This clustering, whether induced by laminin111 or Matrigel™ is inhibited when the astrocytes are first incubated with an antibody against the γ1 subunit of laminin, suggesting that the latter is crucial to the process. Finally, we showed that laminin111 appears to negatively regulate AQP4-mediated water transport in astrocytes, suppressing the cell swelling that occurs following a hypoosmotic challenge. This suppression is abolished if DG expression is silenced, again demonstrating the central role of this receptor in relaying the effects of laminin.

Anti-Wrinkle Benefits of Peptides Complex Stimulating Skin Basement Membrane Proteins Expression.

The dermal-epidermal junction (DEJ) provides a physical and biological interface between the epidermis and the dermis. In addition to providing a structural integrity, the DEJ also acts as a passageway for molecular transport. Based on the recently reported importance of the DEJ in skin aging, novel peptide derivatives have been tested for their effects on basement membrane (BM) protein expressions in cultured human epidermal keratinocytes. As a result, protein expressions of collagen XVII, laminin and nidogen were stimulated by the test peptide and peptides complex. Further ex vivo evaluation using excised human skin, confirmed that the topical application of the peptides complex significantly increased dermal collagen expression, as well as expressions of collagen XVII and laminin. Interestingly, while the origin of the laminin protein is epidermal keratinocytes, the immunohistochemical staining of skin showed that laminin was only detected in the uppermost layer of the dermis, which suggests a tight assembly of laminin protein onto the dermal side of the DEJ. These results suggest that a peptide complex could improve the structural properties of the DEJ through its ability to stimulate BM proteins. In order to evaluate the anti-wrinkle benefits of the peptide complex in vivo, a clinical study was performed on 22 healthy Asian female volunteers older than 40 years. As a result, significant improvements in skin wrinkles for all of the five sites were observed after two weeks, as assessed by skin topographic measurements. Collectively, these results demonstrate the anti-aging efficacy of the peptides complex.

Analysis of protein chlorination by mass spectrometry.

Chlorination of tyrosine is a commonly known effect/consequence of myeloperoxidase activity at sites of inflammation, and detection of 3-chlorotyrosine has been used as biomarker for inflammatory diseases. However, few studies have addressed site specific chlorination in proteins, and no methods for large scale chloroproteomics studies have yet been published. In this study, we present an optimized mass spectrometry based protocol to identify and quantify chlorinated peptides from single proteins modified by HOCl (100 and 500 µM, within estimated pathophysiological levels), at a high level of sensitivity and accuracy. Particular emphasis was placed on 1) sensitive and precise detection of modification sites, 2) the avoidance of loss or artefactual creation of modifications, 3) accurate quantification of peptide abundance and reduction of missing values problem, 4) monitoring the dynamics of modification in samples exposed to different oxidant concentrations and 5) development of guidelines for verification of chlorination sites assignment. A combination of an optimised sample preparation protocol, and improved data analysis approaches have allowed identification of 33 and 15 chlorination sites in laminin and fibronectin, respectively, reported in previous manuscripts [1,2]. The method was subsequently tested on murine basement membrane extract, which contains high levels of laminin in a complex mixture. Here, 10 of the major chlorination sites in laminin were recapitulated, highlighting the utility of the method in detecting damage in complex samples.

Alteration of cystic airway mesenchyme in congenital pulmonary airway malformation.

Congenital pulmonary airway malformation (CPAM) is the most common congenital lesion detected in the neonatal lung, which may lead to respiratory distress, infection, and pneumothorax. CPAM is thought to result from abnormal branching morphogenesis during fetal lung development, arising from different locations within the developing respiratory tract. However, the pathogenic mechanisms are unknown, and previous studies have focused on abnormalities in airway epithelial cells. We have analyzed 13 excised lung specimens from infants (age < 1 year) with a confirmed diagnosis of type 2 CPAM, which is supposed to be derived from abnormal growth of intrapulmonary distal airways. By examining the mesenchymal components including smooth muscle cells, laminin, and elastin in airway and cystic walls using immunofluorescence staining, we found that the thickness and area of the smooth muscle layer underlining the airway cysts in these CPAM tissue sections were significantly decreased compared with those in bronchiolar walls of normal controls. Extracellular elastin fibers were also visually reduced or absent in airway cystic walls. In particular, a layer of elastin fibers seen in normal lung between airway epithelia and underlying smooth muscle cells was missing in type 2 CPAM samples. Thus, our data demonstrate for the first time that airway cystic lesions in type 2 CPAM occur not only in airway epithelial cells, but also in adjacent mesenchymal tissues, including airway smooth muscle cells and their extracellular protein products. This provides a new direction to study the molecular and cellular mechanisms of CPAM pathogenesis in human.

Differential Distribution of Laminin N-Terminus α31 Across the Ocular Surface: Implications for Corneal Wound Repair.

Purpose: Laminin N-terminus (LaNt) α31 is a relatively unstudied protein derived from the laminin α3 gene but structurally similar to netrins. LaNt α31 has, to date, been investigated only in two-dimensional (2D) keratinocyte culture where it influences cell migration and adhesion, processes integral to wound repair. Here we investigated LaNt α31 distribution in ocular surface epithelium, during limbal stem cell activation, and corneal wound healing. Methods: Human, mouse, and pig eyes, ex vivo limbal explant cultures, and alkali burn wounds were processed for immunohistochemistry with antibodies against LaNt α31 along with progenitor cell-associated proteins. LaNt α31 expression was induced via adenoviral transduction into primary epithelial cells isolated from limbal explants, and cell spreading and migration were analyzed using live imaging. Results: LaNt α31 localized to the basal layer of the conjunctival, limbal, and corneal epithelial cells. However, staining was nonuniform with apparent subpopulation enrichment, and some suprabasal reactivity was also noted. This LaNt α31 distribution largely matched that of keratin 15, epidermal growth factor receptor, and transformation-related protein 63α (p63α), and displayed similar increases in expression in activated limbal explants. During active alkali burn wound repair, LaNt α31 displayed increased expression in limbal regions and loss of basal restriction within the cornea. Distribution returned to predominately basal cell restricted once the wounded epithelium matured. Cultured corneal epithelial cells expressing LaNt α31 displayed increased 2D area and reduced migration, suggesting a functional link between this protein and key wound repair activities. Conclusions: These data place LaNt α31 in position to influence laminin-dependent processes including wound repair and stem cell activation.

Genetic lineage tracing of targeted cell populations during enthesis healing.

Rotator cuff supraspinatus tendon injuries are clinically challenging due to the high rates of failure after surgical repair. One key limitation to functional healing is the failure to regenerate the enthesis transition between tendon and bone, which heals by disorganized scar formation. Using two models of supraspinatus tendon injury in mouse (partial tear and full detachment/repair), the purpose of the study was to determine functional gait outcomes and identify the origin of the cells that mediate healing. Consistent with previous reports, enthesis injuries did not regenerate; partial tear resulted in a localized scar defect adjacent to intact enthesis, while full detachment with repair resulted in full disruption of enthesis alignment and massive scar formation between tendon and enthesis fibrocartilage. Although gait after partial tear injury was largely normal, gait was permanently impaired after full detachment/repair. Genetic lineage tracing of intrinsic tendon and cartilage/fibrocartilage cells (ScxCreERT2 and Sox9CreERT2 , respectively), myofibroblasts (αSMACreERT2 ), and Wnt-responsive stem cells (Axin2CreERT2 ) failed to identify scar-forming cells in partial tear injury. Unmineralized enthesis fibrocartilage was strongly labeled by Sox9CreERT2 while Axin2CrERT2 labeled a subset of tendon cells away from the skeletal insertion site. In contrast to the partial tear model, Axin2CreERT2 labeling showed considerable contribution of Axin2lin cells to the scar after full detachment/repair. Clinical Significance: Clinically relevant models of rotator cuff tendon injuries in mouse enable the use of genetic tools; lineage tracing suggests that distinct mechanisms of healing are activated with full detachment/repair injuries versus partial tear. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:3275-3284, 2018.

Re-investigating the Basement Membrane Zone of Psoriatic Epidermal Lesions: Is Laminin-511 a New Player in Psoriasis Pathogenesis?

Psoriasis is a complex chronic inflammatory skin disease characterized by epidermal thickening on the basis of increased keratinocyte proliferation and insufficient apoptosis. Laminins are important components of the basement membrane (BM) and impact on epidermal keratinocyte growth/apoptosis. Although several laminins are involved in the pathogenesis of psoriasis, it is still controversial about the expression patterns of laminin isoforms and which laminins are important in the development of psoriasis. Because laminin-511 and -332 are key BM components in human skin, and laminin-511 stimulates human hair follicle growth, we asked whether the BM zone in psoriasis shows any laminin-related abnormalities. This showed that the BM expression of laminin-511 and -332 was significantly increased within the skin lesion of psoriasis. Immunofluorescence microscopy revealed that laminin-511, -332, and collagen type IV proteins were also significantly increased in psoriasis-like skin lesions of Imiquimod-treated mice. Transmission electron microscopy showed a few gaps of lamina densa, and its thickness was significantly increased. Finally, laminin-511 treatment significantly stimulated the proliferation and inhibited apoptosis of HaCaT cells, while laminin-α5 chain gene knockdown decreased proliferation and induced apoptosis. These phenomenological observations raise the question of whether laminin-511-controlled keratinocyte growth/death may be a previously overlooked player in the pathogenesis of psoriatic epidermal lesions.

Exocellular extract of Fusarium oxysporum, fungus free, is able to permeate and act selectively in skin.

The skin is an important gateway for Fusarium infection in humans. Our hypothesis is that metabolites produced by Fusarium oxysporum should change the barrier structure to permeate the skin. Male Wistar rats received a topical application of a solution (0.05 mg/mL) of Fusarium metabolites. The animals were euthanized 3, 6, 12, 24 h after and the skin was processed for immunostaining by laminin and E-cadherin to investigate whether the Fusarium metabolites can break the barrier of healthy skin. Other techniques were employed: H&E to study the morphology; metalloproteinase-9 (MMP-9), TUNEL, and PCNA immunostaining to evaluate the inflammation, cell death, and proliferation, respectively. There was an inflammatory response mainly centered in the dermis. Qualitatively, the skin of the experimental group showed reduced E-cadherin and laminin immunostaining at 3, 12, and 24 h. Higher intensity staining by TUNEL at 3 h, and PCNA at 6, 12, and 24 h. There was intense MMP-9 activity at 6, 12, and 24 h. None of analyses revealed any changes in the epidermis. It was concluded that the fraction was able to permeate the skin and act selectively in dermis, inducing inflammatory response, increasing MMP-9 immunostaining, inducing apoptosis, and reducing E-cadherin and laminin immunostaining.

Orienting proteins by nanostructured surfaces: evidence of a curvature-driven geometrical resonance.

Experimental and theoretical reports have shown that nanostructured surfaces have a dramatic effect on the amount of protein adsorbed and the conformational state and, in turn, on the performances of the related devices in tissue engineering strategies. Here we report an innovative method to prepare silica-based nanostructured surfaces with a reproducible, well-defined local curvature, consisting of ordered hexagonally packed arrays of curved hemispheres, from nanoparticles of different diameters (respectively 147 nm, 235 nm and 403 nm). The nanostructured surfaces have been made chemically homogeneous by partially embedding silica nanoparticles in poly(hydroxymethylsiloxane) films, further modified by means of UV-O3 treatments. This paper has been focused on the experimental and theoretical study of laminin, taken as a model protein, to study the nanocurvature effects on the protein configuration at nanostructured surfaces. A simple model, based on the interplay of electrostatic interactions between the charged terminal domains of laminin and the nanocurved charged surfaces, closely reproduces the experimental findings. In particular, the model suggests that nanocurvature drives the orientation of rigid proteins by means of a "geometrical resonance" effect, involving the matching of dimensions, charge distribution and spatial arrangement of both adsorbed molecules and adsorbent nanostructures. Overall, the results pave the way to unravel the nanostructured surface effects on the intra- and inter-molecular organization processes of proteins.

Isolation and analysis of laminins.

Laminins are large glycoproteins forming structural and signaling networks with two major physiological roles: one role crucial for the formation and stability of basement membranes and the other role, as crucial as the first, in cell anchorage and signaling. Laminins come in several flavors as 16 different isoforms are known, each with both common and unique functions. Here the most current techniques for purification and identification of laminins in tissues and cultivated cells as well as for testing the cell adhesion-promoting activity of laminins will be described.

Ultrasensitive electrochemiluminescence biosensor for detection of laminin based on DNA dendrimer-carried luminophore and DNA nanomachine-mediated target recycling amplification.

Herein, a novel electrochemiluminescence (ECL) biosensor was proposed for ultrasensitive detection of laminin (LN), in which DNA dendrimer (D) as a promising nanocarrier for luminophore and DNA nanomachine as tactic for target recycling. The DNA dendrimer was synthesized by hybridization between sense and its antisense Y-shaped DNAs which were formed via reaction between single-stranded DNA (ssDNA) with a thiol group at the 5'-end and a synthesized trimeric cross-linker of tris(2-maleimidoethyl)amine. This dendrimer provided abundant double-stranded DNA (dsDNA) to achieve high loading efficiency for ECL luminophore. Simultaneously, N-(aminobutyl)-N-(ethylisoluminol) (ABEI) was conjugated with doxorubicin (Dox, a intercalator of dsDNA) to form the ECL indicator (Dox-ABEI) which could effectively intercalate DNA dendrimer to form the ECL probe (D-Dox-ABEI). Subsequently, a DNA nanomachine activated by target protein was involved to obtain numerous output ssDNA (S2) which was amplified by exonuclease III-assisted recycle and immobilized on ssDNA (S1)-modified sensing electrode surface via complementation. Thereby, abundant D-Dox-ABEI probes were captured by S2 to construct the biosensor for target protein detection. The proposed ECL biosensor realized the ultrasensitive detection of LN with a linear range from 0.1pg/mL to 100ng/mL and a low detection limit of 0.0661pg/mL. Impressively, the application of this ECL biosensor would provide the great potential for analysis of other proteins, revealing a new avenue for early diagnosis and the prognosis evaluation of various diseases.