Chitosan fibers: versatile platform for nickel-mediated protein assembly

Fibers are a versatile platform because standard methods are available for the hierarchical assembly of individual fibers into controllable patterns (e.g., fabrics). Here, we report a method to biofunctionalize individual fibers by the reversible binding of proteins, and we suggest the potential of fiber assemblies by generating simple multifiber structures. Specifically, we use chitosan fibers and show that nickel can mediate assembly of histidine-tagged proteins to these fibers. Initial studies with the model His-GFP demonstrate the concept of nickel-mediated protein assembly. Subsequent studies with a His-tagged streptococcal antibody-binding protein (protein G) demonstrate the assembly of antibodies to generate antibody-presenting fibers. Antibody assembly onto the fiber was shown to be controllable, and antigen-binding to these antibody-presenting fibers was measured. Importantly, antibody and antigen were observed to penetrate substantially into the individual fibers (tens of microns) to allow the assembly of pmole levels of protein per cm of fiber length. Finally, antibody-presenting fibers with different specificities were assembled into simple one- and two-dimensional structures, and individual fibers in these fiber assemblies were observed to capture their respective antigens from antigen mixtures. The potential of fiber assemblies for multiplexed analysis is discussed.     

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.     

Stress fibers are generated by two distinct actin assembly mechanisms in motile cells

Stress fibers play a central role in adhesion, motility, and morphogenesis of eukaryotic cells, but the mechanism of how these and other contractile actomyosin structures are generated is not known. By analyzing stress fiber assembly pathways using live cell microscopy, we revealed that these structures are generated by two distinct mechanisms. Dorsal stress fibers, which are connected to the substrate via a focal adhesion at one end, are assembled through formin (mDia1/DRF1)-driven actin polymerization at focal adhesions. In contrast, transverse arcs, which are not directly anchored to substrate, are generated by endwise annealing of myosin bundles and Arp2/3-nucleated actin bundles at the lamella. Remarkably, dorsal stress fibers and transverse arcs can be converted to ventral stress fibers anchored to focal adhesions at both ends. Fluorescence recovery after photobleaching analysis revealed that actin filament cross-linking in stress fibers is highly dynamic, suggesting that the rapid association-dissociation kinetics of cross-linkers may be essential for the formation and contractility of stress fibers. Based on these data, we propose a general model for assembly and maintenance of contractile actin structures in cells.     

Exchangeability of alpha-actinin in living cardiac fibroblasts and muscle cells

We have investigated the exchangeability of alpha-actinin in various structures of cultured chick cardiac fibroblasts and muscle cells using fluorescent analogue cytochemistry in combination with fluorescence recovery after photobleaching. Living cells were microinjected with tetramethylrhodamine-labeled alpha-actinin, which became localized in cellular structures. Small areas of labeled structures were then photobleached with a laser pulse, and the subsequent recovery of fluorescence was monitored with an image intensifier coupled to an image-processing system. In fibroblasts, fluorescence recovery was studied in stress fibers and in adhesion plaques. Bleached spots in adhesion plaques generally attained complete recovery within 20 min; whereas complete recovery in stress fibers occurred within 30 to 60 min. In muscle cells, alpha-actinin became localized in the Z-lines of sarcomeres, in punctate structures, and in apparently continuous bundle-like structures. Fluorescence recovery in Z-lines, punctate structures, and some bundle-like structures was extremely slow. Complete recovery did not occur within the 6- to 7-h observation period. However, some bundle-like structures recovered completely within 60 min, a rate similar to that of stress fibers in fibroblasts. These results indicate that fluorescently labeled alpha-actinin is more stably associated with structures in muscle cells than in fibroblasts. In addition, different structures within the same cell can display different alpha-actinin exchangeabilities which, in muscle cells, could be developmentally related.     

Process of Making Three-dimensional Microstructures using Vaporization of a Sacrificial Component

Vascular structures in natural systems are able to provide high mass transport through high surface areas and optimized structure. Few synthetic material fabrication techniques are able to mimic the complexity of these structures while maintaining scalability. The Vaporization of a Sacrificial Component (VaSC) process is able to do so. This process uses sacrificial fibers as a template to form hollow, cylindrical microchannels embedded within a matrix. Tin (II) oxalate (SnOx) is embedded within poly(lactic) acid (PLA) fibers which facilitates the use of this process. The SnOx catalyzes the depolymerization of the PLA fibers at lower temperatures. The lactic acid monomers are gaseous at these temperatures and can be removed from the embedded matrix at temperatures that do not damage the matrix. Here we show a method for aligning these fibers using micromachined plates and a tensioning device to create complex patterns of three-dimensionally arrayed microchannels. The process allows the exploration of virtually any arrangement of fiber topologies and structures.     

Autophagic response to strenuous exercise in mouse skeletal muscle fibers

Strenuous physical exercise induces necrosis of skeletal muscle fibers and increases lysosomal enzyme activities in surviving muscle fibers. This study examines the ultrastructural basis of the stimulation of the lysosomal system in mouse vastus medialis muscle during the appearance and repair of exercise-induced (9 h of running) injuries. Necrotic fibers appeared the day after exercise and an inflammatory response with the replacement of necrotic fibers by phagocytes was highest 2-3 days after exertion. Ultrastructural study of surviving muscle fibers revealed numerous autophagic vacuoles, residual bodies, and spheromembranous structures at the periphery of myofibers, especially in fibers adjacent to necrotic fibers. The autophagic response was most prominent between 2 and 7 days after exertion. Autophagic vacuoles with double or single limiting membranes contained mitochondria at various stages of degradation. Vacuolar and multilamellar structures were also observed in regenerating muscle fibers. The structure of injured skeletal muscle fibers returned to normal within 2 weeks. It is proposed that increased autophagic activity could be related to the breakdown of cellular constituents of surviving muscle fibers to provide structural elements for regenerating muscle fibers.     

Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK2 cell

PtK2 cells were grown on gold grids and treated with Triton X-100 in a microtubule stabilizing buffer. The resulting cytoskeletons were fixed with glutaraldehyde and subjected to the indirect immunofluorescence procedure using monospecific tubulin antibodies. Grids were examined first by fluorescence microscopy, and the display of fluorescent cytoplasmic microtubules was recorded. The grids were then stained with uranyl acetate and the display of fibrous structures recorded by electron microscopy. Thus the display of cytoplasmic microtubular structures in the light microscope and the electron microscope can be compared within the same cytoskeleton. The results show a direct correspondence of the fluorescent fibers in the light microscope with uninterrupted fibers of diameter approximately 550 A in the electron microscope. This is the diameter reported for a single microtubule decorated around its circumference by two layers of antibody molecules. Thus under optimal conditions immunofluorescence microscopy can visualize individual microtubules.     

Patterns in the quinary structures of proteins. Plasticity and inequivalence of individual molecules in helical arrays of sickle cell hemoglobin and tubulin.

The four recognized levels of organization of protein structure (primary through quaternary) are extended to add the designation quinary structure for the interactions within helical arrays, such as found for sickle cell hemoglobin fibers or tubulin units in microtubules. For sickle cell hemoglobin the main quinary structure is a 14-filament fiber, with a number of other minor forms also encountered. Degenerate forms of the 14-filament fibers can be characterized that lack specific pairs of filaments; evidence is presented which suggests an overall organization of the 14 filaments in pairs, with particular pairs aligned in an antiparallel orientation. For tubulin, a range of quinary structures can be detected depending on the number of protofilaments and whether adjacent protofilaments composed of alternating alpha- and beta-subunits are aligned with contacts between like or unlike subunits and with parallel or antiparallel polarity. Thus, in contrast to quarternary structure, which generally involves a fixed number of subunits, the quinary structures of proteins can exhibit marked plasticity and inequivalence in the juxtaposition of constituent molecules.     

Organization of chromatin during spermiogenesis: beaded fibers,partly beaded fibers,and loss of nucleosomal structure

The aggregation of chromatin during spermiogenesis in the house cricket and many other animals is an orderly process involving the formation of a series of long, thick, well defined structures. The differentiation of chromatin preliminary to the development of such unusual structures is given attention here. Examination of nuclei after lysis and spreading indicated that fibers with closely spaced nucleosomes, like the fibers of somatic chromatin, make up the chromatin in all stages of early spermiogenesis and most of middle spermiogenesis. The thick structures of late spermatids cannot be formed by aggregation of fibers of this somatic type, however; just before thick structures form, chromatin fibers lose the nucleosomal structure. During the process, fibers with nucleosomes spaced at irregular intervals and with long stretches of smooth thin fiber are found, as if nucleosomes at one site on a fiber are broken down independently of those at adjacent sites. Since prior studies of cricket proteins have indicated that somatic histones persist during the stages when nucleosome structure disappears, the observations imply that the histones which are organized in nucleosomes during early stages must become incorporated into different kinds of nucleoprotein complexes during succeeding stages of spermiogenesis.     

Control of anti-thrombogenic properties: surface-induced self-assembly of fibrinogen fibers

Wound healing is a complex process initiated by the formation of fibrin fibers and endothelialization. Normally, this process is triggered in a wound by thrombin cleavage of fibrinopeptides on fibrinogen molecules, which allows them to self spontaneously-assemble into large fibers that provide the support structure of the clot and promote healing. We have found that the fibrous structures can also form without thrombin on most polymer or metal surfaces, including those commonly used for stents. We show that the relatively hydrophobic E and D regions of the fibrinogen molecule are adsorbed on these surfaces, exposing the αC domains, which in turn results in the formation of large fiber structures that promote endothelial cell adhesion. We show that the entire process can be suppressed when stents or other substrates are coated with polymers that are functionalized to bind the αC domains, leading to the development of potentially nonthrombogenic implant materials.     

Picrosirius Red Staining of Dental Structures

Histologic sections of dog mandibles and teeth were stained with picrosirius red and Mayer's hematoxylin. Collagenous structures of the mandible stained brilliant red. Dentinal tubules, Sharpey's fibers and other structures not easily seen in sections stained with hematoxylin and eosin alone were seen clearly after this procedure. Under polarized light collagen fibers could be specifically identified and their orientation determined. Picrosirius red-hematoxylin is recommended for examination of normal or pathologic dental specimens.     

A Perspective on the Complexity of Dietary Fiber Structures and Their Potential Effect on the Gut Microbiota

Even though there are many factors that determine the human colon microbiota composition, diet is an important one because most microorganisms in the colon obtain energy for their growth by degrading complex dietary compounds, particularly dietary fibers. While fiber carbohydrates that escape digestion in the upper gastrointestinal tract are recognized to have a range of structures, the vastness in number of chemical structures from the perspective of the bacteria is not well appreciated. In this article, we introduce the concept of “discrete structure” that is defined as a unique chemical structure, often within a fiber molecule, which aligns with encoded gene clusters in bacterial genomes. The multitude of discrete structures originates from the array of different fiber types coupled with structural variations within types due to genotype and growing environment, anatomical parts of the grain or plant, discrete regions within polymers, and size of oligosaccharides and small polysaccharides. These thousands of discrete structures conceivably could be used to favor bacteria in the competitive colon environment. A global framework needs to be developed to better understand how dietary fibers can be used to obtain predicted changes in microbiota composition for improved health. This will require a multi-disciplinary effort that includes biological scientists, clinicians, and carbohydrate specialists.     

Picrosirius red staining of dental structures

Histologic sections of dog mandibles and teeth were stained with picrosirius red and Mayer's hematoxylin. Collagenous structures of the mandible stained brilliant red. Dentinal tubules, Sharpey's fibers and other structures not easily seen in sections stained with hematoxylin and eosin alone were seen clearly after this procedure. Under polarized light collagen fibers could be specifically identified and their orientation determined. Picrosirius red-hematoxylin is recommended for examination of normal or pathologic dental specimens.     

Transversal Stiffness and Young's Modulus of Single Fibers from Rat Soleus Muscle Probed by Atomic Force Microscopy

The structural integrity of striated muscle is determined by extra-sarcomere cytoskeleton that includes structures that connect the Z-disks and M-bands of a sarcomere to sarcomeres of neighbor myofibrils or to sarcolemma. Mechanical properties of these structures are not well characterized. The surface structure and transversal stiffness of single fibers from soleus muscle of the rat were studied with atomic force microscopy in liquid. We identified surface regions that correspond to projections of the Z-disks, M-bands, and structures between them. Transversal stiffness of the fibers was measured in each of these three regions. The stiffness was higher in the Z-disk regions, minimal between the Z-disks and the M-bands, and intermediate in the M-band regions. The stiffness increased twofold when relaxed fibers were maximally activated with calcium and threefold when they were transferred to rigor (ATP-free) solution. Transversal stiffness of fibers heavily treated with Triton X-100 was about twice higher than that of the permeabilized ones, however, its regional difference and the dependence on physiological state of the fiber remained the same. The data may be useful for understanding mechanics of muscle fibers when it is subjected to both axial and transversal strain and stress.     

Spatial arrangement of white muscle fibers and myoseptal tendons in fishes

We describe the arrangement of white muscle fibers and tendinous myoseptal structures and the relation of these structures to each other in order to provide an anatomical framework for discussions and experimental research on fish swimming mechanics. For the three major craniate groups, the petromyzontids, myxinids and gnathostomes, we identify three conditions that differ remarkably. Myxinids are characterized by asymmetrical myosepta with long cones. Within a single myoseptum these are connected by collagenous fibers that are almost oriented longitudinally. Distinct tendons are absent in myxinid myosepta. Petromyzontid myosepta lack cones and distinct myoseptal tendons, whereas gnathostomes bear cones and distinct tendinous structures: the lateral band, epineural (epipleural) tendon and myhabdoid tendon. Myoseptal fibers of petromyzontids and myoseptal tendons of gnathostome myosepta are firmly anchored in the skin. Myxinids lack firm myoseptal-skin-connections. Their muscular arrangement is neither comparable to that of petromyzontids nor to that of gnathostomes. The latter two bear archlike arrangements of muscle fibers spanning several segments that are hypothesized to play a role during bending. In gnathostomes, archlike helical muscle fiber arrangements (HMFAs) are present that span the length of several body segments and are multiply intersected by myosepta. Hence, a series of tendinous lateral bands of myosepta is embedded in HMFAs. The posterodorsally oriented HMFAs are underlain by posteroventrally oriented crossing muscle fibers (CMFs). Bending may be generated by contraction of the muscle fibers belonging to an HMFA and the simultaneous counteraction of CMFs. Moving caudally, this anterior muscle fiber arrangement gradually changes, eventually becoming the posterior muscle fiber arrangement. This pattern suggests that the function of the myomeres will also change. Three additional putative roles of myoseptal tendons can be deduced from their relations to white muscle fibers in gnathostomes (and in part in petromyzontids): (1) Posterior transmission of anteriorly generated muscular forces via lateral bands and/or myorhabdoid tendons. These tendons are more robust posteriorly. Anterior and posterior cones appear to play an important role in force transmission. (2) Pulling on collagen fibers of the skin via lateral bands and myorhabdoid tendons, suggesting a transmission of muscular forces that puts the skin into tension. (3) Resisting radial expansion of contracting muscle fibers by epineural (epipleural) tendons. By the latter two mechanisms modulation of body stiffness is likely to be achieved.     

Innervation and acetylcholine sensitivity of skeletal muscle cells differentiated in vitro from chick embryo

Trypsin-dissociated myoblasts from leg muscle of 12-day chick embryos have been cultured in monolayers. After four days the muscle cultures have been confronted with fragments of the spinal cord of six-day chick embryos. Electrophysiological and morphological analysis demonstrate that characteristic neuromuscular transmission can develop in these cultures. Electrical stimulation of the cord fragment evokes contractions of innervated muscle fibers, from which end plate potentials and miniature end plate potentials with average frequency around one per second or more can be recorded. D-tubocurarine (1 μg/ml) suppresses reversibly these synaptic potentials. Non-innervated muscle fibers are sensitive to acetylcholine over all their surface, while innervated muscle fibers are sensitive at the regions where structures suggestive of motor end plate (“bulb-type”) are found. We can conclude that neuromuscular connections developed in vitro in our experiments are functional in respect of transmission of impulses but also in respect of neurotrophic influences for restriction of chemosensitivity.     

Transmission of 308 nm excimer laser radiation for ophthalmic microsurgery--medical, technical and safety aspects

A surgical system using 308 nm excimer laser radiation transmitted by quartz fibers is described. In-vitro surgery has been performed on the cornea, in the anterior chamber angle, on the lens and in the vitreous with a special application tip for each procedure. Possible damage of ocular structures by 308 nm radiation is discussed. In-vitro experiments showed that UV exposure of intraocular structures sensitive to 308 nm radiation can be reduced by appropriate design of the application tips and by the application of UV absorbing drugs.     

Polymerization of deoxy-sickle cell hemoglobin in high-phosphate buffer

Deoxy-sicklecell hemoglobin (HbS) polymerizes in 0.05 M phosphate buffer to form long helical fibers. The reaction typically occurs when the concentration of HbS is about 165 mg/ml. Polymerization produces a variety of polymorphic forms. The structure of the fibers can be probed by using site-directed mutants to examine the effect of altering the residues involved in intermolecular interactions. Polymerization can also be induced in the presence of 1.5 M phosphate buffer. Under these conditions polymerization occurs at much lower concentrations (ca. 5 mg/ml), which is advantageous when site-directed mutants are being used because only small quantities of the mutants are available. We have characterized the structure of HbS polymers formed in 1.5 M phosphate to determine how their structures are related to the polymers formed under more physiological conditions. Under both sets of conditions fibers are the first species to form. At pHs between 6.7 and 7.3 fibers initially form bundles and then crystals. At lower pHs fibers form macrofibers and then crystals. Fourier transforms of micrographs of the polymers formed in 1.5 M phosphate display the 32- and 64-A(-1) periodicity characteristic of fibers formed in 0.05 M phosphate buffer. The 64-A(-1) layer line is less prominent in Fourier transforms of negatively stained fibers formed in 1.5 M phosphate possibly because salt interferes with staining of the fibers. However, micrographs and Fourier transforms of frozen hydrated fibers formed in high and low phosphate display the same periodicities. Under both sets of reaction conditions HbS polymers form crystals with the same unit cell parameters as Wishner-Love crystals (a = 64 A, b = 185 A, c = 53 A). Some of the polymerization intermediates were examined in the frozen-hydrated state in order to determine whether their structures were significantly perturbed by negative staining. We have also carried out reconstructions of the frozen-hydrated fibers in high and low phosphate to compare their molecular coordinates. The helical projection of the reconstructions in low phosphate shows the expected 14-strand structure. In high phosphate the 14-strand fibers are also formed and their molecular coordinates are the same (within experimental error) as those of fibers formed in 0.05 M phosphate. In addition, the reconstructions of high-phosphate fibers reveal a new minor variant of fiber containing 10 strands. The polymerization products in 1.5 M phosphate buffer were generally indistinguishable from those formed in 0.05 M phosphate buffer. Micrographs of frozen hydrated specimens have facilitated the interpretation of previously published micrographs using negative staining.