Pre-existing Fibrils Research Articles

Alzheimer's Disease Immunotherapy: Current Strategies and Future Prospects.

Alzheimer's disease (AD) is an extremely complex and heterogeneous pathology influenced by many factors contributing to its onset and progression, including aging, amyloid-beta (Aβ) plaques, tau fibril accumulation, inflammation, etc. Despite promising advances in drug development, there is no cure for AD. Although there have been substantial advancements in understanding the pathogenesis of AD, there have been over 200 unsuccessful clinical trials in the past decade. In recent years, immunotherapies have been at the forefront of these efforts. Immunotherapy alludes to the immunological field that strives to identify disease treatments via the enhancement, suppression, or induction of immune responses. Interestingly, immunotherapy in AD is a relatively new approach for non-infectious disease. At present, antibody therapy (passive immunotherapy) that targets anti-Aβ aimed to prevent the fibrillization of Aβ peptides and disrupt pre-existing fibrils is a predominant AD immunotherapy due to the continuous failure of active immunotherapy for AD. The most rational and safe strategies will be those targeting the toxic molecule without triggering an abnormal immune response, offering therapeutic advantages, thus making clinical trial design more efficient. This review offers a concise overview of immunotherapeutic strategies, including active and passive immunotherapy for AD. Our review encompasses approved methods and those presently under investigation in clinical trials, while elucidating the recent challenges, complications, successes, and potential treatments. Thus, immunotherapies targeting Aβ throughout the disease progression using a mutant oligomer-Aβ stimulated dendritic cell vaccine may offer a promising therapy in AD.

Cross-Seeding Controls Aβ Fibril Populations and Resulting Functions.

Amyloid peptides nucleate from monomers to aggregate into fibrils through primary nucleation. Pre-existing fibrils can then act as seeds for additional monomers to fibrillize through secondary nucleation. Both nucleation processes occur simultaneously, yielding a distribution of fibril polymorphs that can generate a spectrum of neurodegenerative effects. Understanding the mechanisms driving polymorph structural distribution during both nucleation processes is important for uncovering fibril structure-function relationships, as well as for creating polymorph distributions in vitro that better match fibril structures found in vivo. Here, we explore how cross-seeding wild-type (WT) Aβ1-40 with Aβ1-40 mutants E22G (Arctic) and E22Δ (Osaka), as well as with WT Aβ1-42, affects the distribution of fibril structural polymorphs and how changes in structural distribution impact toxicity. Transmission electron microscopy analysis revealed that fibril seeds derived from mutants of Aβ1-40 imparted their structure to WT Aβ1-40 monomers during secondary nucleation, but WT Aβ1-40 fibril seeds do not affect the structure of fibrils assembled from mutant Aβ1-40 monomers, despite the kinetic data indicating accelerated aggregation when cross-seeding of any combination of mutants. Additionally, WT Aβ1-40 fibrils seeded with mutant fibrils produced similar structural distributions to the mutant seeds with similar cytotoxicity profiles. This indicates that mutant fibril seeds not only impart their structure to growing WT Aβ1-40 aggregates but also impart cytotoxic properties. Our findings establish a relationship between the fibril structure and the phenotype on a polymorph population level and that these properties can be passed on through secondary nucleation to the succeeding generations of fibrils.

Ellagic Acid Modulates Uninduced as well as Mutation and Metal-Induced Aggregation of α-Synuclein: Implications for Parkinson's Disease.

α-Synuclein (αS) is an intrinsically disordered protein whose aggregation and deposition in Lewy bodies is involved in the progression of Parkinson's disease (PD) and other related disorders. The aggregation process of αS is also triggered by mutations like A53T and E46K in the SNCA gene and disruption in metal-ion homeostasis. Currently, there is no obviating therapy available in the market that could effectively prevent the progression of the disease. In this backdrop, there exists an emerging need to consider naturally occurring polyphenols and flavonoids as potential therapeutic agents against PD. In this study, we demonstrate the modulatory effect of ellagic acid (EA) against wild-type as well as mutation and metal-induced aggregation of αS. Thioflavin T (ThT) fluorescence assay suggests that EA acts on the nucleation phase of αS fibrillization, thereby increasing the lag phase from 21.33 ± 3.01 to 48.20 ± 5.05 h and reducing the fibrils growth rate from 4.60 ± 2.06 to 0.890 ± 0.36 h-1. 8-Anilino-1-naphthalene sulfonic acid (ANS), Congo red (CR), and intrinsic fluorescence studies indicate that the interaction of EA with αS facilitates the structural changes in the protein that lead to inhibition of fibril formation. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) images illustrate that the size of fibrils diminishes up to 100 nm in the presence of EA. Dot blot and seeding experiments put forward that EA directs the αS aggregation toward off-pathway fibrillization. Our 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay deciphers the role of EA in minimizing the αS fibril-induced toxicity, thereupon leading to an increase in cell viability. Also, EA attenuates both mutations as well as metal-induced αS fibrillization and disaggregates the preexisting fibrils. Additionally, computational studies elucidate that EA preferentially interacts with the N-terminal and NAC domain of αS. Hence, this work reveals the aggregation inhibition mechanism of EA and provides considerable therapeutic interventions against PD and related disorders.

Inhibitory effect of naturally occurring Ocimum sanctum extract on α-Synuclein aggregation in aqueous solution

α-Synuclein (α-Syn) has a propensity to form amyloids and is one of the major causes of neurodegenerative disorders like Parkinson’s disease (PD). Despite a lot of research, there is no obviating therapy available for PD and related disorders. Therefore, discovering new inhibitors against α-Syn aggregation is a subject of intense research. Against this backdrop, we have investigated the attenuating effect of naturally occurring Ocimum sanctum (OS) against aggregation in aqueous solution using various biochemical and biophysical studies. In this report, Thioflavin (ThT) and Congo red binding assays have shown the hindering effect of OS extract on β-sheet formation in α-Syn, and eventually leading to inhibition of aggregation. ANS binding assay indicated its modulatory effect on hydrophobic patches of α-Syn protein. SDS-PAGE, turbidity measurement, and light scattering assays indicated the role of OS extract in increasing protein solubility. Morphological studies conducted by TEM and DLS suggested that OS extract prevents the formation of large fibrils. This work also extends to reveal the inhibitory and disaggregation effects of OS extract on A53T mutant induced aggregation and pre-existing fibrils respectively. Also, MTT assay implied the protective effect of OS extract on neuroblastoma cells against fibril induced cytotoxicity. Furthermore, docking studies of α-Syn with different molecules of OS extract navigated for putative interacting amino acid residues. Thus, due to the presence of polyphenolic compounds, OS extract binds with α-Syn and inhibits its aggregation. This study implicates that OS extract and associated molecules may become a potential therapeutic intervention against PD and other aggregation related disorders.

Elongation rate and average length of amyloid fibrils in solution using isotope-labelled small-angle neutron scattering.

We demonstrate a solution method that allows both elongation rate and average fibril length of assembling amyloid fibrils to be estimated. The approach involves acquisition of real-time neutron scattering data during the initial stages of seeded growth, using contrast matched buffer to make the seeds effectively invisible to neutrons. As deuterated monomers add on to the seeds, the labelled growing ends give rise to scattering patterns that we model as cylinders whose increase in length with time gives an elongation rate. In addition, the absolute intensity of the signal can be used to determine the number of growing ends per unit volume, which in turn provides an estimate of seed length. The number of ends did not change significantly during elongation, demonstrating that any spontaneous or secondary nucleation was not significant compared with growth on the ends of pre-existing fibrils, and in addition providing a method of internal validation for the technique. Our experiments on initial growth of alpha synuclein fibrils using 1.2 mg ml−1 seeds in 2.5 mg ml−1 deuterated monomer at room temperature gave an elongation rate of 6.3 ± 0.5 Å min−1, and an average seed length estimate of 4.2 ± 1.3 μm.

The Properties of α-Synuclein Secondary Nuclei Are Dominated by the Solution Conditions Rather than the Seed Fibril Strain.

Amyloid fibrils of α-synuclein (α-syn) are a component of Lewy bodies, the characteristic hallmark of Parkinson's disease. Amyloid fibrils arise through primary nucleation from monomers, which in the case of α-syn is often heterogeneous, followed by the growth of the nuclei by monomer addition. Secondary nucleation corresponds to the formation of new fibrils facilitated by pre-existing fibrils. While it is well-established that the newly added monomer in fibril elongation adopts the conformation of the monomers in the seed ("templating"), it is unclear whether fibrils formed through secondary nucleation of monomers on the surface of seed fibrils copy the structure of the "parent" fibril. Here we show by biochemical and microscopical methods that the secondary nucleation of α-syn, enabled at mildly acidic pH, leads to fibrils that structurally resemble more closely those formed de novo under the same conditions, rather than the seeds if these are formed under different solution conditions. This result has important implications for the mechanistic understanding of the secondary nucleation of amyloid fibrils and its role in the propagation of aggregate pathology in protein misfolding diseases.

The Dynamics of AR 12700 in Its Early Emerging Phase. I. Interchange Reconnection

Abstract The emergence of active regions (ARs) leads to various dynamic activities. Using high-resolution and long-lasting Hα observations from the New Vacuum Solar Telescope, we report the dynamics of NOAA AR 12700 in its emerging phase on 2018 February 26 in detail. In this AR, constant interchange reconnections (IRs) between emerging fibrils and preexisting ones were detected. Driven by the flux emergence, small-scale fibrils observed in Hα wavelength continuously emerged at the center of the AR and reconnected with the ambient preexisting fibrils, forming new longer fibrils. We investigate three IR scenarios that occurred over two hours. Specially, the third scenario of reconnection resulted in the formation of longer fibrils that show pronounced rotation motion. To derive the evolution of the magnetic structure during the reconnections, we perform nonlinear force-free field extrapolations. The extrapolated three-dimensional magnetic fields clearly depict a set of almost potential emerging loops, two preexisting flux ropes at 03:00 UT before the second reconnection scenario, and a set of newly formed loops with less twist at 03:48 UT after the third reconnection scenario. All of these extrapolated structures are consistent with the fibrils detected at the Hα wavelength. The aforementioned observations and extrapolation results suggest that the constant IRs resulted in the magnetic twist being redistributed from preexisting flux ropes toward the newly formed system with longer magnetic structure and weaker twist.

Observation of Secondary Nucleation on Specific Aβ 42 Fibril Type

Amyloid-β (Aβ) fibril formation is the pivotal phenomenon in Alzheimer's disease (AD). However, the understanding of amyloid fibril mechanism has been hindered by its heterogeneous morphology. Further, recent studies point out the existence of new growth sites on pre-existing fibrils, called secondary nucleation, but there is no report of secondary nucleation for Aβ 42. Here we present a detailed inter-conversion scheme of Aβ42 from the study of Aβ fibrillogenesis by means of electron microscopy and atomic force microscopy. We indentified that the secondary nucleation events occur more favorably on specific fibril type during monomer fibrillization. Further, we proved the stability of the most homogeneous and stable Aβ42 fibril type and analyzed the structural elastic properties by using the statistical theory of semi-flexible polymer. The result suggests that lateral association mechanism gives the most structural stability to certain fibril type with a twisted ribbon structure. The extracted elastic modulus of fibrils is ∼ 1.4 GPa on mica and highly ordered pyrolytic graphite. We suggest that elucidation of secondary nucleation events and elastic modulus value will help progress in our understanding of the aggregation mechanism of Aβ 42 fibrillogenesis in AD.

Template-Assisted Lateral Growth of Amyloid-β42 Fibrils Studied by Differential Labeling with Gold Nanoparticles

Amyloid-β protein (Aβ) aggregation into amyloid fibrils is central to the origin and development of Alzheimer's disease (AD), yet this highly complex process is poorly understood at the molecular level. Extensive studies have shown that Aβ fibril growth occurs through fibril elongation, whereby soluble molecules add to the fibril ends. Nevertheless, fibril morphology strongly depends on aggregation conditions. For example, at high ionic strength, Aβ fibrils laterally associate into bundles. To further study the mechanisms leading to fibril growth, we developed a single-fibril growth assay based on differential labeling of two Aβ42 variants with gold nanoparticles. We used this assay to study Aβ42 fibril growth under different conditions and observed that bundle formation is preceded by lateral interaction of soluble Aβ42 molecules with pre-existing fibrils. Based on this data, we propose template-assisted lateral fibril growth as an additional mechanism to elongation for Aβ42 fibril growth.

Laser-induced Propagation and Destruction of Amyloid β Fibrils

The amyloid deposition of amyloid beta (Abeta) peptides is a critical pathological event in Alzheimer disease (AD). Preventing the formation of amyloid deposits and removing preformed fibrils in tissues are important therapeutic strategies against AD. Previously, we reported the destruction of amyloid fibrils of beta(2)-microglobulin K3 fragments by laser irradiation coupled with the binding of amyloid-specific thioflavin T. Here, we studied the effects of a laser beam on Abeta fibrils. As was the case for K3 fibrils, extensive irradiation destroyed the preformed Abeta fibrils. However, irradiation during spontaneous fibril formation resulted in only the partial destruction of growing fibrils and a subsequent explosive propagation of fibrils. The explosive propagation was caused by an increase in the number of active ends due to breakage. The results not only reveal a case of fragmentation-induced propagation of fibrils but also provide insights into therapeutic strategies for AD.

Mouse model to study human A β2M amyloidosis: generation of a transgenic mouse with excessive expression of human β2-microglobulin

Patients on long-term hemodialysis can develop dialysis-related amyloidosis (DRA) due to deposition of β2-microglobulin (β2m) into amyloid fibrils (Aβ2M). Despite intensive biochemical studies, the pathogenesis of amyloid deposition in DRA patients remains poorly understood. To elucidate the mechanisms that underlie Aβ2M fibril formation in DRA, we generated transgenic mice that overexpress human β2m protein in a mouse β2m gene knockout background (hB2MTg+/+ mB2m+/+). The hB2MTg+/+mB2m−/− mice express a high level of human β2m protein in many tissues as well as a high plasma β2m concentration (192.8 mg/L). This concentration is >100 times higher than that observed in healthy humans and >4 times higher than that detected in patients on dialysis. We examined spontaneous and amyloid fibril-induced amyloid deposition in these mice. Amyloid deposition of β2m protein was not observed in aged or amyloid fibril injected animals. However, mouse senile apolipoprotein A-II amyloidosis (AApoAII) was detected, particularly in the joints of mice that were injected with AApoAII amyloid fibrils.This study demonstrates that this mouse model could be valuable in studying the components and conditions that promote DRA, and indicates that high plasma concentrations of hβ2m as well as seeding with pre-existing amyloid fibrils may not be sufficient to induce Aβ2M.

Templated Lateral Fibril Growth, an Alternative Mechanism for Beta-Amyloid Fibril Formation

One of the major pathological hallmarks of Alzheimer's disease (AD) is the deposition of senile plaques in specific areas of the brain. Amyloid fibrils formed by the amyloid-β peptide (Aβ) are the major components of the senile plaques. Aβ amyloid fibril formation is a complex process that starts with the aggregation of monomeric protein molecules into transient oligomeric species, which grow and reorganize into the characteristic amyloid fibrils. Among the different forms of Aβ, the 42-residue fragment (Aβ42) is the most strongly linked to the ethiology of AD. Although one of the main unresolved issues in the field of Aβ aggregation and its relation to AD concerns the molecular mechanisms leading to cytotoxicity, it is crucial to describe the main players and the pathways involved in this process.In this work we present a single molecule resolution fibril growth assay based on two variants of Aβ42 that can be differentially detected. The presence of a biotin moiety on one of them enables its detection by means of a primary and secondary antibody (the latter conjugated to a gold nanoparticle), and the subsequent visualization by transmission electron microscopy. By mixing non-labeled fibrillar seeds with labeled monomeric Aβ42 we have been able to monitor the process of amyloid fibril formation and fibril growth. The observation of the precise location (with nanometric resolution) of the newly incorporated Aβ42 monomers, has revealed an alternative mechanism for Aβ42 fibril growth based on the lateral association of Aβ42 monomers to pre-existing fibrils, using those as templates for the new born protofilament.

Protein aggregation: more than just fibrils

The aggregation of misfolded proteins into amyloid fibrils, and the importance of this step for various diseases, is well known. However, it is becoming apparent that the fibril is not the only structure that aggregating proteins of widely different types may adopt. Around the isoelectric point, when the net charge is essentially zero, rather monodisperse and quasi-amorphous nanoscale particles form. These particles are found to contain limited runs of beta-sheet structure, but their overall organization is random. These nanoparticles have the potential to be useful for such applications as the slow release of drugs. The amyloid fibrils form away from the isoelectric point, but over certain ranges of, e.g., pH, the fibrils themselves do not exist freely, but form suprafibrillar aggregates termed spherulites. These consist of fibrils radiating from a central nucleus, and form by new species attaching to the ends of growing fibrils, rather than by the aggregation of pre-existing fibrils. Under the polarizing light microscope, they exhibit a Maltese cross shape due to their symmetry. The rate of aggregation is determined by factors involving (at least) protein size, concentration, presence of salt and charge. The occurrence of spherulites, which have been found in vivo as well as in vitro, appears to be generic, although the factors which determine the equilibrium between free fibril and spherulite are not as yet clear.

PAMAM Dendrimers as Potential Agents against Fibrillation ofα‐Synuclein, a Parkinson's Disease‐Related Protein

The effect of PAMAM dendrimers (generations G3, G4 and G5) on the fibrillation of alpha-synuclein was examined by fluorescence and CD spectroscopy, TEM and SANS. PAMAM dendrimers inhibited fibrillation of alpha-synuclein and this effect increased both with generation number and PAMAM concentration. SANS showed structural changes in the formed aggregates of alpha-synuclein--from cylindrical to dense three-dimensional ones--as the PAMAM concentration increased, on account of the inhibitory effect. PAMAM also effectively promoted the breaking down of pre-existing fibrils of alpha-synuclein. In both processes--that is, inhibition and disassociation of fibrils--PAMAM redirected alpha-synuclein to an amorphous aggregation pathway.

Potential of Fibroblast Cell Therapy for Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin-blistering disorder caused by mutations in the COL7A1 gene that lead to reduced type-VII collagen and defective anchoring fibrils at the dermal-epidermal junction (DEJ). Presently there are no effective treatments for this disorder. Recent mouse studies have shown that intradermal injections of normal human fibroblasts can generate new human type-VII collagen and anchoring fibrils at the DEJ. To assess potential clinical benefits in humans, we gave single intradermal injections of allogeneic fibroblasts to five subjects with RDEB. We noted increased type-VII collagen at the DEJ at 2 weeks and at 3 months following injection and increased anchoring fibrils, although none of these had normal morphology. No adverse effects, clinical or immunopathologic, were noted. We believe the major effect of allogeneic fibroblasts is to increase the recipients' own COL7A1 mRNA levels with greater deposition of mutant type-VII collagen at the DEJ and formation of additional rudimentary anchoring fibrils. Nevertheless, this mutant protein may be partially functional and capable of increasing adhesion at the DEJ. This is the first study demonstrating that intradermal injections of allogeneic fibroblasts have therapeutic potential in human subjects with RDEB.

The toxicity of β-amyloid is attenuated by interaction with its specific human scFv E3 in vitro

β-amyloid (Aβ) has been suggested as a potent neurotoxic agent. The Aβ-targeted immunotherapy aims to clear diffuse amyloid deposits and reverse memory deficits in Alzheimer's disease. We generated a human single chain variable domain antibody fragment (scFv) against Aβ 40, termed E3, by screening a phage antibody library. E3 scFv could recognize Aβ in human cerebral cortex. It was able not only to prevent the aggregation of Aβ but also to disrupt the Aβ preexisting fibrils. Moreover, the Aβ toxicity to SK-N-SH cells was attenuated by addition of E3 scFv. Our results indicate that site-directed human scFv might be a potential therapeutic agent for Alzheimer's disease.

Interaction between A beta(1-42) and A beta(1-40) in Alzheimer's beta-amyloid fibril formation in vitro.

We analyzed the interaction of two kinds of amyloid beta-peptides (A beta), i.e., A beta(1-42) and A beta(1-40), in the kinetics of beta-amyloid fibril (fA beta) formation in vitro, based on a nucleation-dependent polymerization model using fluorescence spectroscopy with thioflavin T. When 25 microM A beta(1-42) was incubated with increasing concentrations of amyloidogenic A beta(1-40), the time to proceed to equilibrium was extended dose-dependently. A similar inhibitory effect was observed when 45 microM A beta(1-40) was incubated with increasing concentrations of A beta(1-42). On the other hand, when 50 microM of nonamyloidogenic A beta(1-40) was incubated with A beta(1-42) at a molar ratio of 10:1 or 5:1, A beta(1-42) initiated fA beta formation from A beta(1-40). The lag time of the reaction shortened in a concentration-dependent manner, with A beta(1-42). We next examined the seeding effect of fA beta formed from A beta(1-42) (fA beta(1-42)) on nonamyloidogenic A beta(1-40). When 50 microM of nonamyloidogenic A beta(1-40) was incubated with 10 or 20 microg/mL (2.2 or 4.4 microM) of fA beta(1-42), the fluorescence showed a sigmoidal increase. The lag time of the reaction was shortened by fA beta(1-42) in a concentration-dependent manner. However, the time to proceed to equilibrium was much longer than when an equal concentration of fA beta formed from A beta(1-40) (fA beta(1-40)) was added to A beta(1-40). The fluorescence increased hyperbolically without a lag phase when 25 microM A beta(1-42) was incubated with 10 or 20 microg/mL (2.3 or 4.6 microM) of fA beta(1-40), and proceeded to equilibrium more rapidly than without fA beta(1-40). An electron microscopic study indicated that the morphology of fA beta formed is governed by the major component of fresh A beta peptides in the reaction mixture, not by the morphology of preexisting fibrils. These results may indicate the central role of A beta(1-42) for fA beta deposition in vivo, among the different coexisting A beta species.

Collagen deposition during wound repair in rat gingiva.

The aim of this study was to examine day-by-day changes at incisional wound edge in rat molar interdental tissue with special reference to early interactions of new and pre-existing collagen fibrils. Twenty rats aged 50 days were divided into experimental groups each comprising two animals. Following incision and post-injury observation periods of 1-10 days, the rats were given an overdose of sodium pentothal and specimens prepared for light and electron microscopy. In 1-3-day post-injury specimens fibrin was replaced by inflammatory cells. Fibroblasts were found along the cut edge in 4-day post-injury specimens. Fibrillogenesis had started at 5 days post-injury. During the period of 5-10 days post-injury increasing amounts of new collagen fibrils were laid down at the wound edge. No granular material was identified at the interface of new and preexisting collagen. In standardized mesiodistal sections, bundles of newly formed fibrils regularly appeared cross-cut in relation to the tangentially cut transseptal fiber system, suggestive of a buccolingual fibril orientation at the wound edge. The ultrastructural observations indicate that during early stages of wound repair, connective tissue continuity is generally established without direct splicing of severed fibers.

Light and electron microscopy of rabbit testes infected with Treponema pallidum (Nichols strain): nature of deposited mucopolysaccharides and localisation of treponemes.

The mucopolysaccharide nature of the material deposited in rabbit testes infected with Treponema pallidum was confirmed by histochemical staining with alcian blue. Differential staining of mucopolysaccharides showed the presence of sulphated mucopolysaccharides as an almost constant feature, whereas in little more than half of the orchitic testes studied variable deposits of hyaluronic acid were seen. The treponemes were almost exclusively present in the areas rich in mucopolysaccharide. A combination staining with the Warthin-Starry method and alcian blue showed treponemes in close association with pre-existing fibrils and cells contained in these fibrils. The latter findings were confirmed by electron microscopy, and the fibroblasts to which treponemes adhered displayed the characteristics of activated cells. The close parallel between the histopathological changes observed here and their descriptions in published reports shows that our specific strain still behaves the same as the original Nichols pathogenic strain of T pallidum.

The microfibrillar structure of the cell walls of the filamentous fungus, Allomyces.

Cell walls of the fungus, Allomyces, were isolated by chemical procedures, using either potassium permanganate oxidation or glacial acetic acid-hydrogen peroxide treatment followed by dilute mineral acid. The structure of the treated walls was investigated by means of electron microscopy and electron diffraction analysis which showed that rhizoidal walls were especially suitable for observation. Chitin microfibrils exist in the extreme tips of rhizoidal walls, and tend to lie in a preferred longitudinal orientation. Older rhizoidal wall segments show a crossed fibrillar structure under a thin layer of short randomly arranged microfibrils. In the possession of systems of crossed fibrils these walls are like the cell walls of certain green algae. Walls of branch rhizoidal filaments were observed in the early stages of development, in which case the observed microfibrillar orientations are such that it is possible to envisage their origin from pre-existing fibrils that have passively reoriented. With respect to the continued growth of the filaments, however, it is difficult to explain the observed microfibrillar arrangements in terms of the "multi-net" theory. Hyphal walls usually show two layers, the outer consisting of microfibrils arranged randomly, and the inner consisting of well oriented microfibrils running parallel with the longitudinal axis of the hypha. The oriented inner layer appears to be similar in structure to the secondary wall of the Phycomyces sporangiophore.