Extracellular matrix assembly: a multiscale deconstruction.

The biochemical and biophysical properties of the extracellular matrix (ECM) dictate tissue-specific cell behaviour. The molecules that are associated with the ECM of each tissue, including collagens, proteoglycans, laminins and fibronectin, and the manner in which they are assembled determine the structure and the organization of the resultant ECM. The product is a specific ECM signature that is comprised of unique compositional and topographical features that both reflect and facilitate the functional requirements of the tissue.

Mimicking the Natural Basement Membrane for Advanced Tissue Engineering.

Advancements in the field of tissue engineering have led to the elucidation of physical and chemical characteristics of physiological basement membranes (BM) as specialized forms of the extracellular matrix. Efforts to recapitulate the intricate structure and biological composition of the BM have encountered various advancements due to its impact on cell fate, function, and regulation. More attention has been paid to synthesizing biocompatible and biofunctional fibrillar scaffolds that closely mimic the natural BM. Specific modifications in biomimetic BM have paved the way for the development of in vitro models like alveolar-capillary barrier, airway models, skin, blood-brain barrier, kidney barrier, and metastatic models, which can be used for personalized drug screening, understanding physiological and pathological pathways, and tissue implants. In this Review, we focus on the structure, composition, and functions of in vivo BM and the ongoing efforts to mimic it synthetically. Light has been shed on the advantages and limitations of various forms of biomimetic BM scaffolds including porous polymeric membranes, hydrogels, and electrospun membranes This Review further elaborates and justifies the significance of BM mimics in tissue engineering, in particular in the development of in vitro organ model systems.

Basement membrane proteins in various arterial beds from individuals with and without type 2 diabetes mellitus: a proteome study.

BACKGROUND: Basement membrane (BM) accumulation is a hallmark of micro-vessel disease in diabetes mellitus (DM). We previously reported marked upregulation of BM components in internal thoracic arteries (ITAs) from type 2 DM (T2DM) patients by mass spectrometry. Here, we first sought to determine if BM accumulation is a common feature of different arteries in T2DM, and second, to identify other effects of T2DM on the arterial proteome. METHODS: Human arterial samples collected during heart and vascular surgery from well-characterized patients and stored in the Odense Artery Biobank were analysed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We included ascending thoracic aortas (ATA) (n = 10 (type 2 DM, T2DM) and n = 10 (non-DM)); laser capture micro-dissected plaque- and media compartments from carotid plaques (n = 10 (T2DM) and n = 9 (non-DM)); and media- and adventitia compartments from ITAs (n = 9 (T2DM) and n = 7 (non-DM)). RESULTS: We first extended our previous finding of BM accumulation in arteries from T2DM patients, as 7 of 12 pre-defined BM proteins were significantly upregulated in bulk ATAs consisting of > 90% media. Although less pronounced, BM components tended to be upregulated in the media of ITAs from T2DM patients, but not in the neighbouring adventitia. Overall, we did not detect effects on BM proteins in carotid plaques or in the plaque-associated media. Instead, complement factors, an RNA-binding protein and fibrinogens appeared to be regulated in these tissues from T2DM patients. CONCLUSION: Our results suggest that accumulation of BM proteins is a general phenomenon in the medial layer of non-atherosclerotic arteries in patients with T2DM. Moreover, we identify additional T2DM-associated effects on the arterial proteome, which requires validation in future studies.

Optimal Elasticity of Biological Networks.

Reinforced elastic sheets surround us in daily life, from concrete shell buildings to biological structures such as the arthropod exoskeleton or the venation network of dicotyledonous plant leaves. Natural structures are often highly optimized through evolution and natural selection, leading to the biologically and practically relevant problem of understanding and applying the principles of their design. Inspired by the hierarchically organized scaffolding networks found in plant leaves, here we model networks of bending beams that capture the discrete and nonuniform nature of natural materials. Using the principle of maximal rigidity under natural resource constraints, we show that optimal discrete beam networks reproduce the structural features of real leaf venation. Thus, in addition to its ability to efficiently transport water and nutrients, the venation network also optimizes leaf rigidity using the same hierarchical reticulated network topology. We study the phase space of optimal mechanical networks, providing concrete guidelines for the construction of elastic structures. We implement these natural design rules by fabricating efficient, biologically inspired metamaterials.

Designing Cardiovascular Implants Taking in View the Endothelial Basement Membrane.

Insufficient endothelialization of cardiovascular grafts is a major hurdle in vascular surgery and regenerative medicine, bearing a risk for early graft thrombosis. Neither of the numerous strategies pursued to solve these problems were conclusive. Endothelialization is regulated by the endothelial basement membrane (EBM), a highly specialized part of the vascular extracellular matrix. Thus, a detailed understanding of the structure-function interrelations of the EBM components is fundamental for designing biomimetic materials aiming to mimic EBM functions. In this review, a detailed description of the structure and functions of the EBM are provided, including the luminal and abluminal interactions with adjacent cell types, such as vascular smooth muscle cells. Moreover, in vivo as well as in vitro strategies to build or renew EBM are summarized and critically discussed. The spectrum of methods includes vessel decellularization and implant biofunctionalization strategies as well as tissue engineering-based approaches and bioprinting. Finally, the limitations of these methods are highlighted, and future directions are suggested to help improve future design strategies for EBM-inspired materials in the cardiovascular field.

Two new reliable immunohistochemical methods for simultaneous identification of capillaries, the three types of fibers and basal lamina in human skeletal muscle.

Capillary network of skeletal muscle has a crucial role in oxygen supply and is strongly associated with the phenotype and metabolic profile of muscle fibers. Abundant literature has explored capillarization of skeletal muscle in different populations and in response to different interventions. Capillary and fiber type identification techniques have considerably evolved over the last decades, but to the best of our knowledge, no validated immunohistochemical method has yet been developed to simultaneously identify capillaries (using CD31), the three different muscle fiber types, and basal lamina. Nine human muscle biopsies of vastus lateralis were stained using 5 different methods to test: the reliability of different CD31 antibodies for capillary identification, the reliability between single-section or serial-section methods, and the intra-experimenter reproducibility in visual detection of capillaries. High reliability for the different antibodies directed against capillaries was observed for capillary contacts (CC) measurements (intra-class correlations (ICC) [ICC95%] of 0.89 [0.72; 0.96] for type I fibers, 0.93 [0.81; 0.97] for type IIA fibers, 0.88 [0.71; 0.96] for type IIX fibers, 0.95 [0.86; 0.98] for all fiber types) as well as a high level of similarity between single and serial sections methods. A strong similarity in capillary analysis between the different methods was obtained for each sample measurements. Analysis of Lin's concordance correlation coefficients and Bland and Altman's graphics showed a strong intra-experimenter reproducibility. This article proposes two time- and tissue-sparing immunohistochemical methods to accurately assess a complete fiber typing (type I, IIA, and IIX) along with muscle capillarization on a single muscle section.

Autoantibody Profile of a Cohort of 54 Italian Patients with Linear IgA Bullous Dermatosis: LAD-1 Denoted as a Major Auto-antigen of the Lamina Lucida Subtype.

Linear IgA bullous dermatosis (LABD) is characterized by presence of multiple IgA autoantibodies, and a comparatively lesser number of IgG antibodies, directed against different hemidesmosomal antigens. The main autoantigens are LAD-1, LABD-97, BP180 and BP230, type VII collagen and laminin 332. We retrospectively studied the serology of 54 Italian patients with LABD using enzyme-linked immunosorbent assay (ELISA), immunoblotting assay, and indirect immunofluorescence on monkey oesophagus and salt-split skin. Among these, indirect immunofluorescence of salt-split skin elicits the greatest sensitivity. Sixty-three percent of the sera were observed to be positive, with a lamina lucida pattern observed in 48%, a sub-lamina densa pattern in 2% and a mixed pattern in 13% of the cases. IgA reactivity to LAD-1 on immunoblotting was found in 52% of sera, to BP180-NC16A by ELISA in 32% and to BP230 in 26%. Only 17% of patients possessed circulating IgG autoantibodies. LAD-1 was determined to be a major autoantigen of the lamina lucida subtype. Combined serological assays demonstrated a high sensitivity (82%), suggesting that this approach could support diagnosis when a biopsy is not feasible or direct immunofluorescence results are negative.

Electron Microscopic and Immunohistochemical Findings of the Epidermal Basement Membrane in Two Families with Nail-patella Syndrome.

Nail-patella syndrome is an autosomal dominant disorder characterized by nail dysplasia and skeletal anomaly. Some patients have been shown to have ultrastructural abnormalities of the glomerular basement membrane that result in nephrosis. However, little has been reported on the epidermal basement membrane in this condition. This paper reports 2 families with nail-patella syndrome. Direct sequencing analysis of LMX1B revealed that family 1 and family 2 were heterozygous for the mutations c.140-1G>C and c.326+1G>C, respectively. To evaluate the epidermal basement membrane zone, ultrastructural and immunohistochemical analyses were performed using skin specimens obtained from the dorsal thumb. Electron microscopy showed intact hemidesmosomes, lamina lucida, lamina densa, and anchoring fibrils. Immunofluorescence studies with antibodies against components of the epidermal basement membrane zone revealed a normal expression pattern among the components, including type IV collagen. These data suggest that nail dysplasia in patients with nail-patella syndrome is not caused by structural abnormalities of the epidermal basement membrane.

Posterior Vitreous Detachment and the Posterior Hyaloid Membrane.

PURPOSE: Despite posterior vitreous detachment being a common ocular event affecting most individuals in an aging population, there is little consensus regarding its precise anatomic definition. We investigated the morphologic appearance and molecular composition of the posterior hyaloid membrane to determine whether the structure clinically observed enveloping the posterior vitreous surface after posterior vitreous detachment is a true basement membrane and to postulate its origin. Understanding the relationship between the vitreous (in both its attached and detached state) and the internal limiting membrane of the retina is essential to understanding the cause of rhegmatogenous retinal detachment and vitreoretinal interface disorders, as well as potential future prophylactic and treatment strategies. DESIGN: Clinicohistologic correlation study. PARTICIPANTS: Thirty-six human donor globes. METHODS: Vitreous bodies identified to have posterior vitreous detachment were examined with phase-contrast microscopy and confocal microscopy after immunohistochemically staining for collagen IV basement membrane markers, in addition to extracellular proteins that characterize the vitreoretinal junction (fibronectin, laminin) and vitreous gel (opticin) markers. The posterior retina similarly was stained to evaluate the internal limiting membrane. Findings were correlated to the clinical appearance of the posterior hyaloid membrane observed during slit-lamp biomicroscopy after posterior vitreous detachment and compared with previously published studies. MAIN OUTCOME MEASURES: Morphologic appearance and molecular composition of the posterior hyaloid membrane. RESULTS: Phase-contrast microscopy consistently identified a creased and distinct glassy membranous sheet enveloping the posterior vitreous surface, correlating closely with the posterior hyaloid membrane observed during slit-lamp biomicroscopy in patients with posterior vitreous detachment. Immunofluorescent confocal micrographs demonstrated the enveloping membranous structure identified on phase-contrast microscopy to show positive stain results for type IV collagen. Immunofluorescence of the residual intact internal limiting membrane on the retinal surface also showed positive stain results for type IV collagen. CONCLUSIONS: The results of this study provide immunohistochemical evidence that the posterior hyaloid membrane is a true basement membrane enveloping the posterior hyaloid surface. Because this membranous structure is observed only after posterior vitreous detachment, the results of this study indicate that it forms part of the internal limiting membrane when the vitreous is in its attached state.

Circulating Vascular Basement Membrane Fragments are Associated with the Diameter of the Abdominal Aorta and Their Expression Pattern is Altered in AAA Tissue.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is characterised by enhanced proteolytic activity, and extracellular matrix (ECM) remodelling in the vascular wall. Type IV and XVIII collagen/endostatin are structural proteins in vascular basement membrane (VBM), a specialised ECM structure. Here the association between plasma levels of these collagens with the aortic diameter and expansion rate is studied, and their expression in aortic tissue characterised. METHODS: This was a retrospective population based cohort study. Type IV and XVIII collagen/endostatin were analysed in plasma by ELISA assay in 615 men, divided into three groups based on the aortic diameter: 1) normal aorta ≤ 25 mm, 2) sub-aneurysmal aorta (SAA) 26-29 mm, and 3) AAA ≥ 30 mm. Follow up data were available for 159 men. The association between collagen levels and aortic diameter at baseline, and with the expansion rate at follow up were analysed in ordinal logistic regression and linear regression models, controlling for common confounding factors. Tissue expression of the collagens was analysed in normal aorta (n = 6) and AAA (n = 6) by immunofluorescence. RESULTS: Plasma levels of type XVIII collagen/endostatin (136 ng/mL [SD 29] in individuals with a normal aorta diameter, 154 ng/ml [SD 45] in SAA, and 162 ng/ml [SD 46] in AAA; p = .001) and type IV collagen (105 ng/mL [SD 42] normal aorta, 124 ng/ml [SD 46] SAA, and 127 ng/ml [SD 47] AAA; p = .037) were associated with a larger aortic diameter. A significant association was found between the baseline levels of type XVIII/endostatin and the aortic expansion rate (p = .035), but in the multivariable model, only the initial aortic diameter remained significantly associated with expansion (p = .005). Altered expression patterns of both collagens were observed in AAA tissue. CONCLUSION: Plasma levels of circulating type IV and XVIII collagen/endostatin increase with AAA diameter. The expression pattern of VBM proteins is altered in the aneurysm wall.

Supportive properties of basement membrane layer of human amniotic membrane enable development of tissue engineering applications.

Human amniotic membrane (HAM) has been widely used as a natural scaffold in tissue engineering due to many of its unique biological properties such as providing growth factors, cytokines and tissue inhibitors of metalloproteinases. This study aimed at finding the most suitable and supportive layer of HAM as a delivery system for autologous or allogeneic cell transplantation. Three different layers of HAM were examined including basement membrane, epithelial and stromal layers. In order to prepare the basement membrane, de-epithelialization was performed using 0.5 M NaOH and its efficiency was investigated by histological stainings, DNA quantification, biomechanical testing and electron microscopy. Adipose-derived stromal cells (ASCs) and a human immortalized keratinocyte cell line (HaCaT) were seeded on the three different layers of HAM and cultured for 3 weeks. The potential of the three different layers of HAM to support the attachment and viability of cells were then monitored by histology, electron microscopy and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Moreover, mechanical strengths of the basement membrane were assessed before and after cell culture. The results indicated that the integrity of extra cellular matrix (ECM) components was preserved after de-epithelialization and resulted in producing an intact basement amniotic membrane (BAM). Moreover, all three layers of HAM could support the attachment and proliferation of cells with no visible cytotoxic effects. However, the growth and viability of both cell types on the BAM were significantly higher than the other two layers. We conclude that growth stimulating effectors of BAM and its increased mechanical strength after culturing of ASCs, besides lack of immunogenicity make it an ideal model for delivering allogeneic cells and tissue engineering applications.

Basement Membrane Mimics of Biofunctionalized Nanofibers for a Bipolar-Cultured Human Primary Alveolar-Capillary Barrier Model.

In vitro reconstruction of an alveolar barrier for modeling normal lung functions and pathological events serve as reproducible, high-throughput pharmaceutical platforms for drug discovery, diagnosis, and regenerative medicine. Despite much effort, the reconstruction of organ-level alveolar barrier functions has failed due to the lack of structural similarity to the natural basement membrane, functionalization with specific ligands for alveolar cell function, the use of primary cells and biodegradability. Here we report a bipolar cultured alveolar-capillary barrier model of human primary cells supported by a basement membrane mimics of fully synthetic bifunctional nanofibers. One-step electrospinning process using a bioresorbable polyester and multifunctional star-shaped polyethylene glycols (sPEG) enables the fabrication of an ultrathin nanofiber mesh with interconnected pores. The nanofiber mesh possessed mechanical stability against cyclic expansion as seen in the lung in vivo. The sPEGs as an additive provide biofunctionality to fibers through the conjugation of peptide to the nanofibers and hydrophilization to prevent unspecific protein adsorption. Biofunctionalized nanofiber meshes facilitated bipolar cultivation of endothelial and epithelial cells with fundamental alveolar functionality and showed higher permeability for molecules compared to microporous films. This nanofiber mesh for a bipolar cultured barrier have the potential to promote growth of an organ-level barrier model for modeling pathological conditions and evaluating drug efficacy, environmental pollutants, and nanotoxicology.

Multiple and repeated sampling increases the sensitivity of direct immunofluorescence testing for the diagnosis of mucous membrane pemphigoid.

BACKGROUND: Mucous membrane pemphigoid (MMP) is an autoimmune disease characterized by the predominant blistering of mucosal surfaces and the linear deposition of complement, IgG, or IgA along the basement membrane detected by direct immunofluorescence (DIF) test. OBJECTIVE: To assess the impact of multiple and repeated DIF sampling on establishing the diagnosis of MMP. METHODS: We reviewed the results of DIF studies in 136 nonlesional biopsies from 78 patients who were immunologically confirmed to have MMP. RESULTS: Thirty-six of 52 patients (69%) who underwent only 1 biopsy at the first workup were positive. In 13 cases, the initial single biopsy was negative, and later biopsies were positive. Twenty-two of 26 patients (85%) who underwent multiple biopsies at the initial workup showed ≥1 positive DIF test result. Simultaneously obtained biopsies yielded discordant positive and negative findings in 11 patients. Overall, 74 of 78 patients (95%) had ≥1 positive result by DIF test. In the remaining 4 cases, the diagnosis was confirmed by the detection of circulating autoantibodies against BP180. LIMITATIONS: This is a retrospective, single-center study. CONCLUSION: Our data demonstrate that multiple and repeated biopsies increase the sensitivity of the DIF test for MMP diagnosis. Negative DIF test findings in cases clinically suggestive of MMP should prompt repeat biopsies.

Matrigel basement membrane matrix induces eccrine sweat gland cells to reconstitute sweat gland-like structures in nude mice.

BACKGROUND: Severe burn results in irreversible damage to eccrine sweat glands, for which no effective treatment is available. Interaction between the extracellular matrix and epithelial cells is critical for proper three-dimensional organization and function of the epithelium. METHODS: Matrigel-embedded eccrine sweat gland cells were subcutaneously implanted into the inguinal regions of nude mice. Two weeks later, the Matrigel plugs were removed and evaluated for series of detection items. RESULTS: Sweat gland cells developed into sweat gland-like structures in the Matrigel plugs based on: (1) de novo formation of tubular-like structures with one or more hollow lumens, (2) expression of epithelial and sweat gland markers (pancytokeratin, CK5/7/14/19, α-SMA and CEA), (3) basement membrane formation, (4) myoepithelial cells presenting in and encompassing the tubular-like structures, (5) cellular polarization, evident by the expression of tight junction proteins (claudin-1 and ZO-2), anchoring junctions (desmoglein-1 and -2 and E-cadherin) and CEA in the luminal membrane, (6) expression of proteins related to sweat secretion and absorption (Na(+)-K(+)-ATPase α/ß, Na(+)-K(+)-2Cl-cotranspoter 1, Na(+)/H(+) exchanger 1, aquaporin-5, epithelial sodium channel, cystic fibrosis transmembrane conductance regulator, potassium channel and vacuolar-type H+-ATPase), and (7) about 20% of the tubular-like structures are de novo coils and 80% are de novo ducts. CONCLUSIONS: This study provides not only an excellent model to study eccrine sweat gland development, cytodifferentiation and reconstitution, but also an in vivo model for regeneration of eccrine sweat glands.

Matriglycan: a novel polysaccharide that links dystroglycan to the basement membrane.

Associations between cells and the basement membrane are critical for a variety of biological events including cell proliferation, cell migration, cell differentiation and the maintenance of tissue integrity. Dystroglycan is a highly glycosylated basement membrane receptor, and is involved in physiological processes that maintain integrity of the skeletal muscle, as well as development and function of the central nervous system. Aberrant O-glycosylation of the α subunit of this protein, and a concomitant loss of dystroglycan's ability to function as a receptor for extracellular matrix (ECM) ligands that bear laminin globular (LG) domains, occurs in several congenital/limb-girdle muscular dystrophies (also referred to as dystroglycanopathies). Recent genetic studies revealed that mutations in DAG1 (which encodes dystroglycan) and at least 17 other genes disrupt the ECM receptor function of dystroglycan and cause disease. Here, we summarize recent advances in our understanding of the enzymatic functions of two of these disease genes: the like-glycosyltransferase (LARGE) and protein O-mannose kinase (POMK, previously referred to as SGK196). In addition, we discuss the structure of the glycan that directly binds the ECM ligands and the mechanisms by which this functional motif is linked to dystroglycan. In light of the fact that dystroglycan functions as a matrix receptor and the polysaccharide synthesized by LARGE is the binding motif for matrix proteins, we propose to name this novel polysaccharide structure matriglycan.

A synthetic nanofibrillar matrix promotes in vitro hepatic differentiation of embryonic stem cells and induced pluripotent stem cells.

Embryonic stem (ES) cells recapitulate normal developmental processes and serve as an attractive source for routine access to a large number of cells for research and therapies. We previously reported that ES cells cultured on M15 cells, or a synthesized basement membrane (sBM) substratum, efficiently differentiated into an endodermal fate and subsequently adopted fates of various digestive organs, such as the pancreas and liver. Here, we established a novel hepatic differentiation procedure using the synthetic nanofiber (sNF) as a cell culture scaffold. We first compared endoderm induction and hepatic differentiation between murine ES cells grown on sNF and several other substrata. The functional assays for hepatocytes reveal that the ES cells grown on sNF were directed into hepatic differentiation. To clarify the mechanisms for the promotion of ES cell differentiation in the sNF system, we focused on the function of Rac1, which is a Rho family member protein known to regulate the actin cytoskeleton. We observed the activation of Rac1 in undifferentiated and differentiated ES cells cultured on sNF plates, but not in those cultured on normal plastic plates. We also show that inhibition of Rac1 blocked the potentiating effects of sNF on endoderm and hepatic differentiation throughout the whole differentiation stages. Taken together, our results suggest that morphological changes result in cellular differentiation controlled by Rac1 activation, and that motility is not only the consequence, but is also able to trigger differentiation. In conclusion, we believe that sNF is a promising material that might contribute to tissue engineering and drug delivery.

Method for perfusion decellularization of porcine whole liver and kidney for use as a scaffold for clinical-scale bioengineering engrafts.

BACKGROUND: Whole-organ engineering provides a new alternative source of donor organs for xenotransplantation. Utilization of decellularized whole-organ scaffolds, which can be created by detergent perfusion, is a strategy for tissue engineering. In this article, our aim is to scale up the decellularization process to human-sized liver and kidney to generate a decellularized matrix with optimal and stable characteristics on a clinically relevant scale. METHODS: Whole porcine liver and kidney were decellularized by perfusion using different detergents (1% SDS, 1% Triton X-100, 1% peracetic acid (PAA), and 1% NaDOC) via the portal vein and renal artery of the liver and kidney, respectively. After rinsing with PBS to remove the detergents, the obtained liver and kidney extracellular matrix (ECM) were processed for histology, residual cellular content analysis, and ECM components evaluation to investigate decellularization efficiency, xenoantigens removal, and ECM preservation. RESULTS: The resulting liver and kidney scaffolds in the SDS-treated group showed the most efficient clearance of cellular components and xenoantigens, including DNA and protein, and preservation of the extracellular matrix composition. In comparison, cell debris was observed in the other decellularized groups that were generated using Triton X-100, PAA, and NaDOC. Special staining and immunochemistry of the porcine liver and kidney ECMs further confirmed the disrupted three-dimension ultrastructure of the ECM in the Triton X-100 and NaDOC groups. Additionally, Triton X-100 effectively eliminated the residual SDS in the SDS-treated group, which ensured the scaffolds were not cytotoxic to cells. Thus, we have developed an optimal method that can be scaled up for use with other solid whole organs. CONCLUSIONS: Our SDS-perfusion protocol can be used for porcine liver and kidney decellularization to obtain organ scaffolds cleared of cellular material, xenoimmunogens, and preserved vital ECM components.

Intracellular immunohistochemical detection of tetrodotoxin in Pleurobranchaea maculata (Gastropoda) and Stylochoplana sp. (Turbellaria).

Tetrodotoxin (TTX), is a potent neurotoxin targeting sodium channels that has been identified in multiple marine and terrestrial organisms. It was recently detected in the Opisthobranch Pleurobranchaea maculata and a Platyhelminthes Stylochoplana sp. from New Zealand. Knowledge on the distribution of TTX within these organisms is important to assist in elucidating the origin and ecological role of this toxin. Intracellular micro-distribution of TTX was investigated using a monoclonal antibody-based immunoenzymatic technique. Tetrodotoxin was strongly localized in neutral mucin cells and the basement membrane of the mantle, the oocytes and follicles of the gonad tissue, and in the digestive tissue of P. maculata. The ova and pharynx were the only two structures to contain TTX in Stylochoplana sp. Using liquid chromatography-mass spectrometry, TTX was identified in the larvae and eggs, but not the gelatinous egg cases of P. maculata. Tetrodotoxin was present in egg masses of Stylochoplana sp. These data suggest that TTX has a defensive function in adult P. maculata, who then invest this in their progeny for protection. Localization in the digestive tissue of P. maculata potentially indicates a dietary source of TTX. Stylochoplana sp. may use TTX in prey capture and for the protection of offspring.

Generalized linear IgA dermatosis with palmar involvement.

Linear IgA bullous dermatosis (LABD) is a sub-epidermal blistering disorder characterized by deposition of IgA along the basement membrane zone (BMZ) as detected by immunofluorescence microscopy. The diagnosis is made by clinicopathologic correlation with immunofluorescence confirmation. Differentiation from other bullous dermatoses is important because therapeutic measures differ. Prompt initiation of the appropriate therapies can have a major impact on outcomes. We present three cases with prominent palmar involvement to alert the clinician of this potential physical exam finding and to consider LABD in the right context.

Developments in purification methods for obtaining and evaluation of collagen derived endogenous angioinhibitors.

Collagen constitutes one of the vital components of the basement membrane scaffolds. Non-collagenous domains (NC1) derived from collagens exhibit potent anti-angiogenic properties, thus attaining significance in regulation of angiogenesis promoted diseases. Individual NC1 domains essential for anti-angiogenic evaluations are generally obtained through purification of individual non-collagenous domains, which have undergone steady developments for enhancing the yields, purpose of biological evaluations and solubility based on the nature of different NC1 domains. This review focuses on the method developments in obtaining biologically active NC1 domains and for specific evaluations in different scenarios.