Membrane-Induced Technique for the Management of Combined Soft Tissue and Osseous Defects.

The induced membrane technique is a simple, effective, and reproducible treatment method for segmental bone defects. It is a 2-stage approach that requires eventual autologous bone graft to manage the deficit. The first stage requires debridement of all nonviable tissue while preserving a healthy soft tissue envelope. A polymethylmethacrylate is implanted between the osseous segments to maintain length. The osseous defect can be stabilized internally or externally. During the second stage, a vascularized induced membrane is formed and produces multiple growth factors. The induced membrane technique is a valuable option for limb salvage in cases of segmental bone defects.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging.

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism's genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging.

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

Evaluation of physicochemical and biological properties of chitosan/poly (vinyl alcohol) polymer blend membranes and their correlation for Vero cell growth.

The blend membranes with varying weight ratios of chitosan/poly (vinyl alcohol) (CS/PVA) (1:0, 1:1, 1:2.5, 1.5:1, 1.5: 2.5) were prepared using solvent casting method and were evaluated for their potential application in single-use membrane bioreactors (MBRs). The physicochemical properties of the prepared membranes were investigated for chemical interactions (FTIR), surface morphology (SEM), water uptake, protein sorption (qe), ammonia sorption and growth kinetics of Vero cells. CS/PVA blend membrane having weight ratio of 1.5:1 had shown enhanced membrane flexibility, reduced water uptake, less protein sorption and no ammonium sorption compared to CS membrane. This blend membrane also showed comparatively enhanced higher specific growth rate (0.82/day) of Vero cells. Improved physicochemical properties and growth kinetics obtrude CS/PVA (1.5:1) as a potential surface for adhesion and proliferation with possible application in single use membrane bioreactors. Additionally, new insight explaining correlation between water holding (%) of CS/PVA (1.5:1) blend membrane and doubling time (td) of Vero cells is proposed.

Controlled growth of filamentous fatty acid vesicles under flow.

The earliest forms of cellular life would have required a membrane compartment capable of growth and division. Fatty acid vesicles are an attractive model of protocell membranes, as they can grow into filamentous vesicles that readily divide while retaining their contents. In order to study vesicle growth, we have developed a method for immobilizing multilamellar fatty acid vesicles on modified glass surfaces and inducing filamentous membrane growth under flow. Filament formation strictly depended on the presence of freshly neutralized fatty acid micelles in the flow chamber. Using light microscopy, we observed a strong dependence of initial growth velocity on initial vesicle size, suggesting that new fatty acid molecules were incorporated into the membrane over the entire external surface of the vesicle. We examined the influences of flow rate, fatty acid concentration, and salt concentration on filamentous growth and observed drastic shape changes, including membrane pearling, of preexisting membrane tubules in response to osmotic stress. These results illustrate the versatility of flow studies for exploring the process of fatty acid vesicle growth following exposure to free fatty acids.

Development and glycoprotein composition of the perimicrovillar membrane in Triatoma (Meccus) pallidipennis (Hemiptera: Reduviidae).

Hemipterans and thysanopterans (Paneoptera: Condylognatha) differ from other insects by having an intestinal perimicrovillar membrane (PMM) which extends from the base of the microvilli to the intestinal lumen. The development and composition of the PMM in hematophagous Reduviidae depend on factors related to diet. The PMM may also allow the human parasite Trypanosoma cruzi, the etiological agent of human Chagas Disease, to establish and develop in this insect vector. We studied the PMM development in the Mexican vector of Chagas Disease, Triatoma (Meccus) pallidipennis. We describe changes in the midgut epithelial cells of insects in response to starvation, and at different times (10, 15 and 20 days) after bloodfeeding. In starved insects, the midguts showed epithelial cells closely connected to each other but apparently free of PMM with some regions being periodic acid-Schiff (PAS-Schiff) positive. In contrast, the PMM was evident and fully developed in the midgut region of insects 15 days after feeding. After this time, the PMM completely covered the microvilli and reached the midgut lumen. At 15 days following feeding the labeled PAS-Schiff increased in the epithelial apex, suggesting an increase in carbohydrates. Lectins as histochemical reagents show the presence of a variety of glycoconjugates including mannose, glucose, galactosamine, N-acetyl-galactosamine. Also present were N-acetyl-glucosamine and sialic acid which contribute to the successful establishment and replication or T. cruzi in its insect vectors. By means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the formation and structure of the PMM is confirmed at 15 days post feeding. Our results confirmed the importance of the feeding processes in the formation of the PMM and showed the nature of the biochemical composition of the vectors' intestine in this important Mexican vector of Chagas disease.

Ruptura prematura de membranas / Premature rupture of membranes

Los nacimientos prematuros son un gran problema en el mundo y la ruptura prematura de membranas contribuyen en cerca de un tercio a esa prematuridad. En Latino América aproximadamente uno de cada 7 nacimientos es prematuro y la ruptura de membranas y trastornos hipertensivos son la principal causa. Una vez confirmada la ruptura de membranas en especial las lejos del término, estamos ante una complicación obstétrica que amerita manejo muy específico y basado en pruebas (evidencia)...

Defects in the outer limiting membrane are associated with rosette development in the Nrl-/- retina.

The neural retinal leucine zipper (Nrl) knockout mouse is a widely used model to study cone photoreceptor development, physiology, and molecular biology in the absence of rods. In the Nrl(-/-) retina, rods are converted into functional cone-like cells. The Nrl(-/-) retina is characterized by large undulations of the outer nuclear layer (ONL) commonly known as rosettes. Here we explore the mechanism of rosette development in the Nrl(-/-) retina. We report that rosettes first appear at postnatal day (P)8, and that the structure of nascent rosettes is morphologically distinct from what is seen in the adult retina. The lumen of these nascent rosettes contains a population of aberrant cells protruding into the subretinal space that induce infolding of the ONL. Morphologically adult rosettes do not contain any cell bodies and are first detected at P15. The cells found in nascent rosettes are photoreceptors in origin but lack inner and outer segments. We show that the adherens junctions between photoreceptors and Müller glia which comprise the retinal outer limiting membrane (OLM) are not uniformly formed in the Nrl(-/-) retina and thus allow protrusion of a population of developing photoreceptors into the subretinal space where their maturation becomes delayed. These data suggest that the rosettes of the Nrl(-/-) retina arise due to defects in the OLM and delayed maturation of a subset of photoreceptors, and that rods may play an important role in the proper formation of the OLM.

Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome.

Sensory and signaling pathways are exquisitely organized in primary cilia. Bardet-Biedl syndrome (BBS) patients have compromised cilia and signaling. BBS proteins form the BBSome, which binds Rabin8, a guanine nucleotide exchange factor (GEF) activating the Rab8 GTPase, required for ciliary assembly. We now describe serum-regulated upstream vesicular transport events leading to centrosomal Rab8 activation and ciliary membrane formation. Using live microscopy imaging, we show that upon serum withdrawal Rab8 is observed to assemble the ciliary membrane in ∼100 min. Rab8-dependent ciliary assembly is initiated by the relocalization of Rabin8 to Rab11-positive vesicles that are transported to the centrosome. After ciliogenesis, Rab8 ciliary transport is strongly reduced, and this reduction appears to be associated with decreased Rabin8 centrosomal accumulation. Rab11-GTP associates with the Rabin8 COOH-terminal region and is required for Rabin8 preciliary membrane trafficking to the centrosome and for ciliogenesis. Using zebrafish as a model organism, we show that Rabin8 and Rab11 are associated with the BBS pathway. Finally, using tandem affinity purification and mass spectrometry, we determined that the transport protein particle (TRAPP) II complex associates with the Rabin8 NH(2)-terminal domain and show that TRAPP II subunits colocalize with centrosomal Rabin8 and are required for Rabin8 preciliary targeting and ciliogenesis.

Self-contact and instabilities in the anisotropic growth of elastic membranes.

We investigate the morphology of thin discs and rings growing in the circumferential direction. Recent analytical results suggest that this growth produces symmetric excess cones (e cones). We study the stability of such solutions considering self-contact and bending stress. We show that, contrary to what was assumed in previous analytical solutions, beyond a critical growth factor, no symmetric e cone solution is energetically minimal any more. Instead, we obtain skewed e cone solutions having lower energy, characterized by a skewness angle and repetitive spiral winding with increasing growth. These results are generalized to discs with varying thickness and rings with holes of different radii.

The key role of the surface membrane in why gastropod nacre grows in towers.

The nacre of gastropod molluscs is intriguingly stacked in towers. It is covered by a surface membrane, which protects the growing nacre surface from damage when the animal withdraws into its shell. The surface membrane is supplied by vesicles that adhere to it on its mantle side and secretes interlamellar membranes from the nacre side. Nacre tablets rapidly grow in height and later expand sideways; the part of the tablet formed during this initial growth phase is here called the core. During initial growth, the tips of the cores remain permanently submerged within the surface membrane. The interlamellar membranes, which otherwise separate the nacre tablet lamellae, do not extend across cores, which are aligned in stacked tablets forming the tower axis, and thus towers of nacre tablets are continuous along the central axis. We hypothesize that in gastropod nacre growth core formation precedes that of the interlamellar membrane. Once the core is complete, a new interlamellar membrane, which covers the area of the tablet outside the core, detaches from the surface membrane. In this way, the tower-like growth of gastropod nacre becomes comprehensible.

Formation of the arthrodial membrane in the blue crab, Callinectes sapidus.

In this study the pattern of arthrodial membrane deposition in Callinectes sapidus was determined by histological and ultrastructural examination of tissues from the carpus joint of the cheliped collected during premolt, ecdysis, postmolt, and intermolt. Apolysis in the arthrodial membrane occurs at stage D(0) and is synchronous with apolysis of the calcified cuticle. Epicuticle formation begins at early stage D(1) and is completed in late stage D(1). Procuticle deposition starts at D(2) and continues until ecdysis. Numerous cytoplasmic extensions occur throughout the lamellae. Component fibers of the arthrodial membrane are intimately associated with dense plaques on the apical membrane of the underlying hypodermal cells, suggesting a site for fiber polymerization. Deposition of the arthrodial membrane continues after ecdysis, with most of the cuticle thickening occurring during stage C. When stained with PAS and counterstained with hematoxylin, a difference can be discerned between preecdysial and postecdysial procuticle of the arthrodial membrane, a distinction not made in previous studies. The boundary between the arthrodial membrane and calcified cuticle is thicker than either of the two layers and the layers overlap rather than butting up against one another. This pattern suggests that underlying hypodermal cells have to produce multiple types of cuticle over the molt cycle. A summary of the various molting patterns in C. sapidus suggests that the control of these diverse events may prove to be complex.

Cortical neurite outgrowth and growth cone behaviors reveal developmentally regulated cues in spinal cord membranes.

Corticospinal axon outgrowth in vivo and the ability to sprout or regenerate after injury decline with age. This developmental decline in growth potential has been correlated with an increase in inhibitory myelin-associated proteins in older spinal cord. However, previous results have shown that sprouting of corticospinal fibers after contralateral lesions begins to diminish prior to myelination, suggesting that a decrease in growth promoting and/or an increase in inhibitory molecules in spinal gray matter may also regulate corticospinal axon outgrowth. To address this possibility, we carried out in vitro experiments to measure neurite outgrowth from explants of 1-day-old hamster forelimb sensorimotor cortex that were plated onto membrane carpets or membrane stripe assays prepared from white or gray matter of 1-to 22-day-old cervical spinal cord. On uniform carpets and in the stripe assays cortical neurites grew robustly on young but not older membranes from both white and gray matter. Mixtures of membranes from 1- and 15-day spinal cord inhibited neurite outgrowth, suggesting that the presence of inhibitory molecules in the 15-day cord overwhelmed permissive or growth promoting molecules in membranes from 1-day cord. Video microscopic observations of growth cone behaviors on membrane stripe assays transferred to glass coverslips supported this view. Cortical growth cones repeatedly collapsed at borders between permissive substrates (laminin or young membrane stripes) and nonpermissive substrates (older membrane stripes). Growth cones either turned away from the older membranes or reduced their growth rates. These results suggest that molecules in both the gray and white matter of the developing spinal cord can inhibit cortical neurite outgrowth.

Molecular automata assembly: principles and simulation of bacterial membrane construction.

The motivation to understand the basic rules and principles governing molecular self-assembly may be relevant to explain in the context of molecular biology the self-organization and biological functions exhibited within cells. This paper presents a molecular automata model to simulate molecular self-assembly introducing the concept of molecular programming to simulate the biological function or operation performed by an assembled molecular state machine. The method is illustrated modelling Escherichia coli membrane construction including the assembly and operation of ATP synthase as well as the assembly of the bacterial flagellar motor. Flagellar motor operation was simulated using a different approach based on state machine definition used in virtual reality systems. The proposed methodology provides a modelling framework for simulation of biological functions performed by cellular components and other biological systems suitable to be modelled as molecular state machines.

Conjuntivite membranosa lenhosa / Lignous membranous conjunctivitis

A conjuntivite membranosa lenhosa é considerada uma patologia de etiologia desconhecida, cuja principal manifestaçäo clínica é a presença de membranas fortemente aderidas ao epitélio. Apresenta uma evoluçäo insidiosa, necessitando de tratamento adequado por um largo período de tempo. Procurou-se relatar nesse artigo um caso típico dessa rara patologia

Ontogeny of membrane and soluble amino-oligopeptidases in rat intestine.

Intestinal amino-oligopeptidase (AOP) plays an essential role in protein digestion. To characterize its postnatal development, we measured AOP activity in intestinal membrane and cytosolic fractions in suckling and weaned rats, compared the subunit structures of the membrane and soluble enzymes, and assessed the biochemical relationship of these peptidases. At weaning, jejunal membrane AOP activity doubled while soluble AOP activity in the ileum fell abruptly. The maturational increase in the molecular mass of ileal membrane AOP was due to alterations in the N-linked glycosylation of this protein. Ileal membrane and soluble AOP exhibited similar substrate affinities, pH optima, inhibition characteristics, and antigenic epitopes. However, soluble AOP was 25-35 kDa smaller than the membrane enzyme. Peak incorporation of [35S]methionine into ileal brush-border AOP preceded maximal radioactivity in soluble AOP, suggesting that the membrane peptidase is a precursor of the soluble enzyme. We conclude that membrane and soluble AOP are closely related proteins with distinct developmental profiles and that the soluble peptidase may be derived from endocytosis of the membrane enzyme.

Copper deficiency in the laying hen.

Copper deficiency in the laying hen resulted in anemia and the production of eggs which were abnormal in size and shape. Many of the eggs had shells which were wrinkled and rough in texture. There was also an increase in the number of shell-less eggs. Examination of malformed egg shells using the scanning electron microscope revealed ultrastructural changes in the mammillary layer of the shell. The effect of copper deficiency on shell formation was attributed to the shell membranes which were altered in color, appearance, and physical consistency. Amino acid analysis of the membranes indicated that the membranes from copper deficient hens were characterized by an increase in lysine content. This suggests that copper is necessary for the formation of lysine derived cross-links in a manner similar to that which occurs in connective tissue. The exact nature of these cross-links is unknown at this time.