Fibrillar Layer Research Articles

Fasciola gigantica: Ultrastructural cytochemistry of the tegumental surface in newly- excysted metacercariae and in vitro-penetrated juvenile flukes informs a concept of parasite defence at the interface with the host

Cytochemical staining techniques were carried out en bloc with in vitro excysted and gut-penetrated Fasciola gigantica larvae in order to visualise the glycocalyx of the tegument, a structure which comprises the parasite component of the host-parasite interface, yet is incompletely preserved by conventional fixation and preparation techniques for electron microscopy. Positive reactivity with ruthenium red and periodic acid-thiocarbohydrazine-osmium (PATCO) techniques revealed that the glycocalyx is polyanionic and carbohydrate-rich throughout its depth. It comprises a trilaminate arrangement, with a thin dense zone and fibrillar layer closely apposed to the outer aspect of the apical plasma membrane, invested by an irregular thick mucopolysaccharide capsule. The latter, not recorded in adult flukes, may represent a specific adaptation to facilitate invasion in the face of host immunity, and may also protect the parasite surface from the action of host- and parasite-derived proteases. Early in the invasion of a naïve host, the glycocalyx may be partly responsible for triggering the responses of innate immunity, while later in infection, or when an anamnestic response is initiated in an immunocompetent host, the antibodies and activated lymphocytes of specific acquired immunity are invoked to interact with the parasite surface. The cytochemical properties of the glycocalyx, together with its potential for dynamic turnover due to exocytosis of the T0 tegumental secretory bodies, are likely to aid neutralisation of potentially damaging immune effectors and ensure their removal from the vicinity of the parasite by sloughing in complex with glycocalyx components.

(Invited) Modeling Morphology Distribution in Injection Molded Polypropylene Parts

The morphology distribution in the final molded object is determined by temperature, pressure and flow, experienced during the process, and their effects on relaxation times, nucleation density, spherulitic growth rate, and the interrelation among these quantities with the deformation rate. The combination of the models developed to describe all these effects becomes a model for predicting the morphology evolution during the injection molding process. This model was implemented in the UNISA code. In this work, samples obtained with a fast evolution of cavity surface temperatures were analyzed and the results were compared with the UNISA code simulations. The fibrillar layer morphology was related to the achievement of critical values of both the molecular stretch and the mechanical work, the latter being performed after the achievement of the critical molecular stretch. The dependence of the morphological layers thicknesses upon the heating conditions was satisfactorily described by the model.

(Invited) Modeling Morphology Distribution in Injection Molded Polypropylene Parts

The UNISA code, a software for the simulation of injection molding developed at the University of Salerno, considers a series of specific models to consider several aspects of the mutual effect of flow and crystallinity: the effects of temperature, pressure and flow on relaxation times, the effect of crystallinity on viscosity, the effect of flow on nucleation density and spherulitic growth rate. Each of the effects mentioned above was experimentally analyzed and described by a model for the iPP grade considered in this work. The combination of all these specific models becomes a model for the morphology evolution during polymer processing. In order to verify the accuracy of the predictions, a series of injection molding tests were obtained applying a system which allows to control the cavity surface temperatures. This technique allows to keep, for assigned time intervals, the cavity surface temperature at intermediate values between injection and cooling channels temperatures. A modulation of the level of the cavity surface temperature and of the time, it was kept active, allowed to control the final sample morphology all the way from the complexity of a standard injection molded part down to a completely (skin and shear layers free) spherulitic structure. The fibrillar layer morphology was related to the achievement of critical values of both the molecular stretch and the mechanical work, the latter being performed after the achievement of the critical molecular stretch. The dependence of the morphological layers (skin, shear, spherulitic layers, the latter including the transition from the shear zone) thicknesses upon the heating conditions was satisfactorily described by the models adopted.

A Criterion for the Formation of Fibrillar Layers in Injection Molded Parts

Abstract It is quite well known that the morphology of an injection molded part made by a semicrystalline polymer presents several layers. In particular spherulitic structures are found in the core region, a layer characterized by highly oriented fibrillar morphology (the shear layer) usually follows and a skin layer is often observed at the sample surface. The thickness of the fibrillar layer deeply influences the mechanical properties of the part. In this work, a criterion to predict the thickness of the fibrillar layer is proposed and verified. The criterion is essentially based on the amount of viscous work done when the molecular stretch is higher than a critical value: the molecular stretch should be above a critical value while a critical amount of viscous work is accumulated. In order to tune the parameters, and to validate the criterion, a well characterized polypropylene was chosen as test material, and four different injection molding conditions were analyzed. The criterion is verified by comparing some experimental results with the prediction of the UNISA code (an injection molding software developed at the University of Salerno), good comparison between software predictions and experimental data confirms the suitability of the criterion.

The propensity of the bacterial rodlin protein RdlB to form amyloid fibrils determines its function in Streptomyces coelicolor

Streptomyces bacteria form reproductive aerial hyphae that are covered with a pattern of pairwise aligned fibrils called rodlets. The presence of the rodlet layer requires two homologous rodlin proteins, RdlA and RdlB, and the functional amyloid chaplin proteins, ChpA-H. In contrast to the redundancy shared among the eight chaplins, both RdlA and RdlB are indispensable for the establishment of this rodlet structure. By using a comprehensive biophysical approach combined with in vivo characterization we found that RdlB, but not RdlA, readily assembles into amyloid fibrils. The marked difference in amyloid propensity between these highly similar proteins could be largely attributed to a difference in amino acid sequence at just three sites. Further, an engineered RdlA protein in which these three key amino acids were replaced with the corresponding residues from RdlB could compensate for loss of RdlB and restore formation of the surface-exposed amyloid layer in bacteria. Our data reveal that RdlB is a new functional amyloid and provide a biophysical basis for the functional differences between the two rodlin proteins. This study enhances our understanding of how rodlin proteins contribute to formation of an outer fibrillar layer during spore morphogenesis in streptomycetes.

Comparative morphological characteristics of three Brettanomyces bruxellensis wine strains in the presence/absence of sulfur dioxide

The red wine spoilage yeast Brettanomyces bruxellensis has been the subject of numerous investigations. Some of these studies focused on spoilage mechanisms, sulfur dioxide tolerance and nutrient requirements. Pseudomycelium formation, although a striking feature of this species, has however been poorly investigated. Furthermore, literature regarding the induction mechanism of pseudomycelium formation in this yeast is limited and lacks clarity, as results published are contradictory. This study elucidates this phenomenon among strains from geographically different areas. Potential environmental cues were investigated, to attain a better understanding of this mechanism and its role as a survival strategy. SO2 was previously reported to induce this morphological change however results obtained in this study did not support this. Nevertheless, the results obtained using scanning and transmission electron microscopy illustrate, for the first time in this yeast, deformity to the cell membrane and alterations to the fibrillar layers in SO2 treated cells. In addition, the SO2 exposed cultures displayed cell size variations, with cells displaying a decrease in length as well as delayed growth, with a prolonged lag phase. Fluorescence microscopy demonstrated a decrease in metabolic activity and the appearance of inclusion body-like structures in the cells following exposure to SO2.

Relaxation of strains in fibrillar porous carbon dioxide saturated media near their critical point

The isothermal relaxation of strains in deformed fibrillar porous layers saturated with subcritical or supercritical carbon dioxide has been studied at different temperatures by full-field speckle correlometry. The relaxation of strains is produced by the porous layer—fluid transition of a system from one equilibrium thermodynamic state to another due to a jump-like change in pressure. It has been established that the relaxation time attains a maximum near a critical point and rapidly decreases with increasing deviation of the system temperature from its critical value. The mechanisms of hydrodynamic relaxation in the density of a fluid layer and viscoelastic relaxation in a porous matrix are discussed. It is shown that the experimentally observed slow relaxation of a system is due to the viscoelastic relaxation of a porous fluid saturated layer at equal fluid densities inside and outside the layer.

Desiccation tolerance in the chlorophyte green alga Ulva compressa: does cell wall architecture contribute to ecological success?

Main conclusionDesiccation leads to structural changes of the inner pectic cell wall layers inUlva compressa. This contributes to protection against mechanical damage due to desiccation–rehydration cycles.Ulva compressa, characterized by rbcL phylogeny, is a common species in the Mediterranean Sea. Ulva as an intertidal species tolerates repeated desiccation–rehydration cycles in nature; the physiological and structural basis were investigated under experimental conditions here. Desiccation to 73 % relative water content (RWC) led to a significant decrease of the maximum quantum yield of photosystem II (Fv/Fm) to about half of the initial value. A reduction to 48 or 27 % RWC caused a more drastic effect and thalli were only able to recover fully from desiccation to 73 % RWC. Relative electron transport rates were stimulated at 73 % RWC, but decreased significantly at 48 and 27 % RWC, respectively. Imaging-PAM analysis demonstrated a homogenous desiccation process within individual thallus discs. The different cell wall layers of U. compressa were characterized by standard staining procedures, i.e. calcofluor white and aniline blue for structural components (cellulose, callose), ruthenium red for pectins and toluidine blue for acidic polysaccharides. Already a reduction to 73 % RWC caused severe changes of the cell walls. The inner pectin-rich layers followed the shrinkage process of the cytoplasm, while the outer denser fibrillar layers maintained their shape. In this way, the thalli were not plasmolyzed during water loss, and upon recovery not negatively influenced by any mechanical damage. Transmission electron microscopy corroborated the arrangement of the different layers clearly distinguishable by their texture and electron density. We suggest the flexibility of the pectin-rich cell wall layers as a major contribution to desiccation tolerance in Ulva.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-015-2292-6) contains supplementary material, which is available to authorized users.

Polysaccharides from the green seaweed Codium decorticatum. Structure and cell wall distribution.

The cell wall polysaccharides from Codium decorticatum and their assembly were studied and these results were compared with those obtained previously for this genus. The water soluble polysaccharides are: (i) Pyruvylated and sulfated 3- and 6-linked β-D-galactans with sulfate mainly on C-4 and also on C-6. Pyruvate ketals are linked to O-3 and O-4 of terminal β-D-galactose or O-4 and O-6 of 3-linked β-D-galactose. (ii) Sulfated 3-linked β-L-arabinans substituted on C-2 or C-2 and C-4 predominantly with sulfate, but also with single stubs of arabinose, and (iii) 4-linked β-D-mannans with a low degree of sulfation on C-2. The whole polysaccharide system comprises 6.9% of sulfated polysaccharides and 32.9% of fibrillar polysaccharides, mostly insoluble mannans. By in situ localization it was possible to detect two similar fibrillar layers separated by a zone rich in charged polymers. Besides, arabinogalactan proteins co-localized with the fibrillar components.

Fungal Hydrophobin Proteins Produce Self-Assembling Protein Films with Diverse Structure and Chemical Stability.

Hydrophobins are small proteins secreted by fungi and which spontaneously assemble into amphipathic layers at hydrophilic-hydrophobic interfaces. We have examined the self-assembly of the Class I hydrophobins EAS∆15 and DewA, the Class II hydrophobin NC2 and an engineered chimeric hydrophobin. These Class I hydrophobins form layers composed of laterally associated fibrils with an underlying amyloid structure. These two Class I hydrophobins, despite showing significant conformational differences in solution, self-assemble to form fibrillar layers with very similar structures and require a hydrophilic-hydrophobic interface to trigger self-assembly. Addition of additives that influence surface tension can be used to manipulate the fine structure of the protein films. The Class II hydrophobin NC2 forms a mesh-like protein network and the engineered chimeric hydrophobin displays two multimeric forms, depending on assembly conditions. When formed on a graphite surface, the fibrillar EAS∆15 layers are resistant to alcohol, acid and basic washes. In contrast, the NC2 Class II monolayers are dissociated by alcohol treatment but are relatively stable towards acid and base washes. The engineered chimeric Class I/II hydrophobin shows increased stability towards alcohol and acid and base washes. Self-assembled hydrophobin films may have extensive applications in biotechnology where biocompatible; amphipathic coatings facilitate the functionalization of nanomaterials.

Retinoic acid regulation by CYP26 in vertebrate lens regeneration

Xenopus laevis is among the few species that are capable of fully regenerating a lost lens de novo. This occurs upon removal of the lens, when secreted factors from the retina are permitted to reach the cornea epithelium and trigger it to form a new lens. Although many studies have investigated the retinal factors that initiate lens regeneration, relatively little is known about what factors support this process and make the cornea competent to form a lens. We presently investigate the role of Retinoic acid (RA) signaling in lens regeneration in Xenopus. RA is a highly important morphogen during vertebrate development, including the development of various eye tissues, and has been previously implicated in several regenerative processes as well. For instance, Wolffian lens regeneration in the newt requires active RA signaling. In contrast, we provide evidence here that lens regeneration in Xenopus actually depends on the attenuation of RA signaling, which is regulated by the RA-degrading enzyme CYP26. Using RT-PCR we examined the expression of RA synthesis and metabolism related genes within ocular tissues. We found expression of aldh1a1, aldh1a2, and aldh1a3, as well as cyp26a1 and cyp26b1 in both normal and regenerating corneal tissue. On the other hand, cyp26c1 does not appear to be expressed in either control or regenerating corneas, but it is expressed in the lens. Additionally in the lens, we found expression of aldh1a1 and aldh1a2, but not aldh1a3. Using an inhibitor of CYP26, and separately using exogenous retinoids, as well as RA signaling inhibitors, we demonstrate that CYP26 activity is necessary for lens regeneration to occur. We also find using phosphorylated Histone H3 labeling that CYP26 antagonism reduces cell proliferation in the cornea, and using qPCR we find that exogenous retinoids alter the expression of putative corneal stem cell markers. Furthermore, the Xenopus cornea is composed of an outer layer and inner basal epithelium, as well as a deeper fibrillar layer sparsely populated with cells. We employed antibody staining to visualize the localization of CYP26A, CYP26B, and RALDH1 within these corneal layers. Immunohistochemical staining of these enzymes revealed that all 3 proteins are expressed in both the outer and basal layers. CYP26A appears to be unique in also being present in the deeper fibrillar layer, which may contain cornea stem cells. This study reveals a clear molecular difference between newt and Xenopus lens regeneration, and it implicates CYP26 in the latter regenerative process.

Aspergillus fumigatus Hyphal Damage Caused by Noninvasive Radiofrequency Field-Induced Hyperthermia

We studied the effect of noninvasive radiofrequency-induced hyperthermia on the viability of Aspergillus fumigatus hyphae in vitro. Radiofrequency-induced hyperthermia resulted in significant (>70%, P < 0.0001) hyphal damage in a time and thermal dose-dependent fashion as assessed by XTT [(sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl] (1)-2H-tetrazolium inner salt)], DiBAC [bis-(1,3-dibutylbarbituric acid) trimethine oxonol] staining, and transmission electron microscopy. For comparison, water bath hyperthermia was used over the range of 45 to 55°C to study hyphal damage. Radiofrequency-induced hyperthermia resulted in severe damage to the outer fibrillar layer of hyphae at a shorter treatment time compared to water bath hyperthermia. Our preliminary data suggest that radiofrequency-induced hyperthermia might be an additional therapeutic approach to use in the management of mold infections.

Leaf Cuticle Anatomy and the Ultrastructure ofGinkgoites ticoensisArchang. from the Aptian of Patagonia

The leaf cuticle of the Ginkgoites ticoensis Archang. type material from the Aptian Anfiteatro de Tico Formation in Patagonia, Argentina, is fully characterized with additional scanning and transmission electron microscopic observations. Many new anatomical and ultrastructural cuticular features are identified in the four-lobed leaf of G. ticoensis: the leaf shows a hypostomatic and papillate laminae, straight and pitted anticlinal and granulate periclinal walls, actinocytic stomata with between five and seven papillate, striate subsidiary cells, and guard cells with anticlinal smooth walls. The TEM studies on ordinary epidermal cells, papillae, subsidiary cells, and guard cells reveal general ultrastructural features of Ginkgoaceae: an outer polylamellate layer A made with A1 and a granular inner layer A2; A1 with an upper part A1U with continuous and straight translucent lamellae; a lower part A1L with significantly disrupted and waving translucent lamellae; and the fibrillar cuticular layer B1 as the i...

Stability of single and double layer fibrillar amyloid-β oligomers

Alzheimer's disease (AD) is the most common form of dementia world-wide. The causative agent in this protein misfolding disease is the 39 to 42-residues long amyloid-β (Aβ) peptide, that aggregates into oligomers, filaments, and fibrils found in plaque deposits in the brain of AD patients [1]. Up to now a viable cure for this disease is still not available. One reason for this is Aβ's conformational flexibility and structural heterogeneity in solution paired with its aggregation tendency. This renders the determination and isolation of distinct Aβ structures experimentally challenging. Especially the soluble oligomers, that are thought to be the neurotoxic species in AD, may adopt a plethora of conformations in vivo [1]. It is known from experiment, that there exist toxic fibrillar oligomers, but the details of their topology are not yet known [2]. Thus, we used molecular dynamics simulations to investigate the structural stability of fibrillar single and ouble layer Aβ42 oligomers of different size (4-mer to 48-mer), that we constructed from the experimental structure [3] (cf Figure ​Figure11). Figure 1 Double layer Aβ 48-mer (Met35 in the interface in sticks representation). We found that there is a clear correlation between oligomer size and preference for double layer structure: Large oligomers display an enhanced stability in double layer conformation, whereas small oligomers prefer the single layer structure. On the other hand, large single layer oligomers dissociate into smaller oligomers, while small double layer oligomers collapse or are energetically unfavorable. From our simulations we deduce that the critical number of oligomers to construct a stable Aβ double layer is in the range of 10 to 12. In a more general picture, longitudinal growth along a single layer is limited by the increasing structural instability. Lateral growth, i.e. forming a double layer, creates stable mini-fibrils. These may act as seeds for further stable fibril growth.

Hydrophobins—Unique Fungal Proteins

Microorganisms are often covered by a proteinaceous surface layer that serves as a sieve for external molecular influx, as a shield to protect microbes from external aggression, or as an aid to help microbial dispersion. In bacteria, the latter is called the S-layer, in Actinomycetes, the rod-like fibrillar layer, and in fungi, the rodlet layer [1]. The self-assembly properties and remarkable structural and physicochemical characteristics of hydrophobin proteins underlie the multiple roles played by these unique proteins in fungal biology.

Influence of volcanic gases on the epidermis of Pinus halepensis Mill. in Campi Flegrei, Southern Italy: A possible tool for detecting volcanism in present and past floras

Cuticle micromorphology together with epidermal and epistomatal wax, in both current- and first-year-old needles of conifer Pinus halepensis (Aleppo pine) trees growing under volcanic gas fumigation was analysed in Pisciarelli area, Campi Flegrei, Southern Italy. As a control, current- and first-year-old needles growing far from volcanic gas emission were also sampled. Using a multidisciplinary approach with SEM, TEM and X-ray, volcanic gases were shown to cause degradation on epicuticular and epistomatal waxes. Significant statistical variations of ultrastructural components of the cuticle, with 30 measurements, including total thickness of the cuticle, and details and proportions of all different layers, and use of confidence interval, revealed a high degree of sensitivity of Aleppo pine to this extreme environment. In the present study, non-significant thickness variations of the cell wall plus cuticle among current- and first-year-old needles of both fumigated and non fumigated trees have been found. However, at the ultrastructural level, significant variations in cell wall and total cuticle thickness, especially within the three zones of B1 fibrillar layer, revealed different equilibria for each of the four types of material. Using energy dispersive X-ray microanalysis, no sulphur was found in either cuticle or epidermal cells, but the presence of H2S in the fumarole gas is suspected to cause indirect and/or direct cuticle alterations of wax structure. Ultrastructural characters of plant cuticles related to emission of volcanic gases during the geological past are also discussed. Among these considerations, an identification key enabling distinction between non fumigated and fumigated materials with 9 characters, provides a good tool detecting the influence of volcanism for extant and fossil plants.

Ultrastructure and development during meiosis and the tetrad period of sporogenesis in the leptosporangiate fern Alsophila setosa (Cyatheaceae) compared with corresponding stages in Psilotum nudum (Psilotaceae)

The pre-meiotic, meiotic and tetrad stages of development in microsporangia of Alsophila setosa were studied with particular emphasis on the early establishment of patterning in the microspore wall and the subsequent development of the sporoderm. The data obtained were compared with corresponding ontogenetic stages of Psilotum nudum. Tapetal behaviour was also examined. During the tetrad period, only one layer, a thin undulating sheet, appeared alongside the plasma membrane of the tetraspores, and this was evidently formed on a pre-patterned structure – a fibrillar layer, corresponding to a kind of primexine matrix. The early free microspores had a wavy plasma membrane with a parallel, sinusoidal, thin initial sporoderm layer. The proximal apertural fold was observed to be an extended outgrowth of this initial spore envelope. Sporoderm ontogeny during the tetrad period in Alsophila and Psilotum show some common points, but also fundamental differences, mainly in the relative timing of events: in Alsophila the end of the tetrad period is the starting point for exospore development, whereas in Psilotum the exospore is already complete at this stage. Considerable differences were also observed in the tapetum of the two species.

Culture of human corneal endothelial cells isolated from corneas with Fuchs endothelial corneal dystrophy

The purpose of this study was to assess the feasibility of initiating primary cultures of corneal endothelial cells from patients suffering from Fuchs endothelial corneal dystrophy (FECD; MIM# 1036800). We also evaluated which conditions yielded the best results for culture. Twenty-nine patients undergoing Descemet stripping automated endothelial keratoplasty consented to the use of their excised Descemet's membrane for this study. Out of the 29 specimens, 18 successfully initiated a culture. Cell morphology varied between endothelial (rounded, slightly elongated cells, n = 12) and fibroblastic-like (thin and very elongated cells, n = 6). These differences in cell morphology were also observed with the normal human corneal endothelial cell cultures. The cultures that initially presented an endothelial morphology maintained their shape in subcultures. Clusterin expression was similar in FECD and normal endothelial cells. Transmission electron microscopy of FECD Descemet's membranes showed a high degree of various abnormalities generally found in this disease, such as a thickened Descemet's membrane, presence of a posterior banded layer, presence of a fibrillar layer and striated bodies of various sizes and periodicities. Patient's age was predictive of culture success, all younger FECD donors generating cultures of endothelial morphology. The absence of a fibrillar layer was also a factor associated with greater success. Culture success was not dependent on specimen size, specimen pigmentation, or patient's preoperative central corneal thickness. In conclusion, this paper shows for the first time that central Descemet's membranes of patients suffering from FECD possess proliferative endothelial cells that can be isolated and cultured without viral transduction, opening the way for new in vitro studies of this disease.

Morphology, developmental ultrastructure and ultracytochemistry of staminal hairs in Bulbine inflata (Asphodelaceae) in relation to function

Results of light and electron microscopy and preliminary ultracytochemical studies of the staminal hairs of Bulbine inflata at different stages of development are reported here. The staminal filaments are covered with yellow, unicellular, linear, erecto-patent hairs. These staminal hairs arise directly as single cell outgrowths from epidermal cells of the filament. The surface of each hair is patterned with helical wall thickenings in an anticlockwise direction. This wall is covered by a thick folded cuticle, and formed of a loosely fibrillar cellulose layer. The hair cell possesses a cytoplasm rich in organelles. Especially ribosomes are abundant. Plastids contain large starch grains and peripheral lipid droplets. The smooth endoplasmic reticulum cisternae (SER) encircle the plastids and mitochondria; it is extended in the cytoplasm along the hair length. These hairs have functions in flower pollination attracting pollinators visually, secreting specific substances, providing increased surface area, protecting the filaments and being involved in their movement and vibration.

The Structure of E. coli IgG-Binding Protein D Suggests a General Model for Bending and Binding in Trimeric Autotransporter Adhesins

The Escherichia coli Ig-binding (Eib) proteins are trimeric autotransporter adhesins (TAAs) and receptors for IgG Fc. We present the structure of a large fragment of the passenger domain of EibD, the first TAA structure to have both a YadA-like head domain and the entire coiled-coil stalk. The stalk begins as a right-handed superhelix, but switches handedness halfway down. An unexpected β-minidomain joins the two and inserts a ∼120° rotation such that there is no net twist between the beginning and end of the stalk. This may be important in folding and autotransport. The surprisingly large cavities we found in EibD and other TAAs may explain how TAAs bend to bind their ligands. We identified how IgA and IgG bind and modeled the EibD-IgG Fc complex. We further show that EibD promotes autoagglutination and biofilm formation and forms a fibrillar layer covering the cell surface making zipper-like contacts between cells.