Ultrafine polysaccharide nanofibrous membranes for water purification

Ultrafine polysaccharide nanofibers (i.e., cellulose and chitin) with 5-10 nm diameters were employed as barrier layers in a new class of thin-film nanofibrous composite (TFNC) membranes for water purification. In addition to concentration, the viscosity of the polysaccharide nanofiber coating suspension was also found to be affected by the pH value and ionic strength. When compared with two commercial UF membranes (PAN10 and PAN400), 10-fold higher permeation flux with above 99.5% rejection ratio were achieved by using ultrafine cellulose nanofibers-based TFNC membranes for ultrafiltration of oil/water emulsions. The very high surface-to-volume ratio and negatively charged surface of cellulose nanofibers, which lead to a high virus adsorption capacity as verified by MS2 bacteriophage testing, offer further opportunities in drinking water applications. The low cost of raw cellulose/chitin materials, the environmentally friendly fabrication process, and the impressive high-flux performance indicate that such ultrafine polysaccharide nanofibers-based TFNC membranes can surpass conventional membrane systems in many different water applications.     

Covalent immobilization of lipase from Candida rugosa onto poly(acrylonitrile-co-2-hydroxyethyl methacrylate) electrospun fibrous membranes for potential bioreactor application

A simple way of fabricating enzymatic membrane reactor with high enzyme loading and activity retention from the conjugation between nanofibrous membrane and lipase was devised. Poly(acrylonitrile-co-2-hydroxyethyl methacrylate) (PANCHEMA) was electrospun into fibrous membrane and used as support for enzyme immobilization. The hydroxyl groups on the fibrous membrane surface were activated with epichlorohydrin, cyanuric chloride or p-benzoquinone, respectively. Lipase from Candida rugosa was covalently immobilized on these fibrous membranes. The resulted bioactive fibrous membranes were examined in catalytic efficiency and activity for hydrolysis. The observed enzyme loading on the fibrous membrane with fiber diameter of 80–150 nm was up to 1.6% (wt/wt), which was as thrice as that on the fibrous membrane with fiber diameter of 800–1000 nm. Activity retention for the immobilized lipase varied between 32.5% and 40.6% with the activation methods of hydroxyl groups. Stabilities of the immobilized lipase were obviously improved. In addition, continuous hydrolysis was carried out with an enzyme-immobilized fibrous membrane bioreactor and a steady hydrolysis conversion (3.6%) was obtained at a 0.23 mL/min flow rate under optimum condition.     

Performance of a novel Viresolve NFR virus filter

Mammalian cell-expressed therapeutic proteins are particularly vulnerable to contamination by endogenous retrovirus-like particles (RVLPs). The Viresolve NFR filter was designed to meet the critical requirement of manufacturing a safe and virus-free therapeutic by retaining RVLPs by a minimum of six log reduction value (LRV). The NFR designation refers to retrovirus removal in a normal flow format. To qualify the product, we tested two model viruses: the 78 nm diameter phi6 bacteriophage and the 80-110 nm diameter Xenotropic Murine Leukemia Virus (X-MuLV). Robust retention was demonstrated over a wide range of process parameters. Viresolve NFR filters also retain other model adventitious viruses including 70-85 nm diameter Reovirus 3 (Reo3), 70-90 nm diameter Adenovirus 2 (Ad2), and 53 nm diameter PR772 by >6 LRV. In addition to these model viruses, the filter retains >7 LRV of both the mycoplasma Acholeplasma laidlawii and the bacterium Brevundimonas diminuta. Protein passage is shown to be consistently high (95-100%) for a variety of therapeutic protein products, including monoclonal antibodies. Characterization of the filter in specific applications is made simple by availability of ultralow surface area (5 cm(2)) disks, which are shown to scale linearly to the manufacturing scale pleated-filters. Viresolve NFR filters provide consistent water permeability performance (34-37 LMH/psi) and show very little plugging for all feedstocks evaluated. The Viresolve NFR filter incorporates Retropore, a unique asymmetric polyethersulfone membrane, the surface of which has been modified to minimize protein binding.     

Stress transfer in cellulose nanowhisker composites--influence of whisker aspect ratio and surface charge

The mechanically induced molecular deformation of cellulose nanowhiskers embedded in subpercolation concentration in an epoxy resin matrix was monitored through Raman spectroscopy. Cellulose nanowhiskers isolated by sulfuric acid hydrolysis from tunicates and by sulfuric acid hydrolysis and hydrochloric acid hydrolysis from cotton were used to study how the aspect ratio (ca. 76 for tunicate and 19 for cotton) and surface charges (38 and 85 mmol SO(4)(-)/kg for sulfuric acid hydrolysis of cotton and tunicate, respectively; no detectable surface charges for hydrochloric acid hydrolysis) originating from the isolation process influence stress transfer in such systems. Atomic force microscopy confirmed that uncharged cellulose nanowhiskers produced by hydrochloric acid hydrolysis have a much higher tendency to aggregate than the charged cotton or tunicate nanowhiskers. Each of these nanowhisker types was incorporated in a concentration of 0.7 vol % in a thermosetting epoxy resin matrix. Mechanically induced shifts of the Raman peak initially located at 1095 cm(-1) were used to express the level of deformation imparted to the nanowhiskers embedded in the resin. Much larger shifts of the diagnostic Raman band were observed for nanocomposites with tunicate nanowhiskers than for the corresponding samples comprising cotton nanowhiskers. In the case of nanocomposites comprising nanowhiskers produced by hydrochloric acid hydrolysis, no significant Raman band shift was observed. These results are indicative of different modes of stress transfer, which in turn appear to originate from the different sample morphologies.     

Optimum Membrane Structures for Growth of Coliform and Fecal Coliform Organisms

The purpose of this study was to determine the optimum membrane filter structure and characteristics for recovery of coliform organisms. Additionally, other factors such as sterilization method and membrane composition were examined. Fecal coliform growth tests with varied samples indicated that the most critical factor in recovery was surface pore morphology and not other factors previously suspected. Fecal coliform counts showed a dramatic increase, with increasing surface opening sizes. Membrane structures with surface openings large enough to surround the entrapped bacteria are required for optimum growth of fecal coliform organisms. Maximum fecal coliform recoveries are obtained using membranes composed of mixed esters of cellulose exhibiting a surface opening diameter of 2.4 μm and a retention pore size of 0.7 μm.     

LBL fabricated biopolymer-layered silicate based nanofibrous mats and their cell compatibility studies

N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC) was synthesized from chitosan (CS). Organic rectorite (OREC) added into cellulose acetate (CA) was used to fabricate electrospun nanofibrous mats with improved thermal properties, as a result of depositing multilayers of the positively charged HTCC-OREC composites and the negatively charged sodium alginate (ALG) via layer-by-layer (LBL) technique. The morphology was affected by the number of deposition bilayers and the component of the outmost layer. Observed from the field emission scanning electron microscopy (FE-SEM) images, the LBL structured nanofibrous mats had much larger fiber sizes than CA-OREC nanofibrous mats. X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) results further confirmed that HTCC-OREC was assembled on nanofibrous mats. Additionally, cell experiments and MTT results demonstrated that OREC had little effect on the cytotoxicity of LBL template, but obviously affected both the cytotoxicity and the cell compatibility of LBL structured mats when OREC was in the deposition films.     

Surface modification of nanofibrous poly(acrylonitrile-co-acrylic acid) membrane with biomacromolecules for lipase immobilization

In this work, poly(acrylonitrile-co-acrylic acid) (PANCAA) was electrospun into nanofibers with a mean diameter of 180 nm. To create a biofriendly microenvironment for enzyme immobilization, collagen or protein hydrolysate from egg skin (ES) was respectively tethered on the prepared nanofibrous membranes in the presence of 1-ethyl-3-(dimethyl-aminopropyl) carbodiamine (EDC)/N-hydroxyl succinimide (NHS). Confocal laser scanning microscopy (CLSM) was used to verify the surface modification and protein density on the nanofibrous membranes. Lipase from Candida rugosa was then immobilized on the protein-modified nanofibrous membranes by covalent binding using glutaraldehyde (GA) as coupling agent, and on the nascent PANCAA nanofibrous membrane using EDC/NHS as coupling agent, respectively. The properties of the immobilized enzyme were assayed. It was found that different pre-tethered biomacromolecules had distinct effects on the immobilized enzyme. The activity retention of the immobilized lipase on ES hydrolysate-modified nanofibrous membrane increased from 15.0% to 20.4% compared with that on the nascent one, while it was enhanced up to more than quadrupled (activity retention of 61.7%) on the collagen-modified nanofibrous membrane. The kinetic parameter, K_m and V_max, were also determined for the free and immobilized lipases. Furthermore, the stabilities of the immobilized lipases were obviously improved compared with the free one.     

Virus harvesting in perfusion culture: Choosing the right type of hollow fiber membrane

The use of bioreactors coupled to membrane-based perfusion systems enables very high cell and product concentrations in vaccine and viral vector manufacturing. Many virus particles, however, are not stable and either lose their infectivity or physically degrade resulting in significant product losses if not harvested continuously. Even hollow fiber membranes with a nominal pore size of 0.2 µm can retain much smaller virions within a bioreactor. Here, we report on a systematic study to characterize structural and physicochemical membrane properties with respect to filter fouling and harvesting of yellow fever virus (YFV; ~50 nm). In tangential flow filtration perfusion experiments, we observed that YFV retention was only marginally determined by nominal but by effective pore sizes depending on filter fouling. Evaluation of scanning electron microscope images indicated that filter fouling can be reduced significantly by choosing membranes with (i) a flat inner surface (low boundary layer thickness), (ii) a smooth material structure (reduced deposition), (iii) a high porosity (high transmembrane flux), (iv) a distinct pore size distribution (well-defined pore selectivity), and (v) an increased fiber wall thickness (larger effective surface area). Lowest filter fouling was observed with polysulfone (PS) membranes. While the use of a small-pore PS membrane (0.08 µm) allowed to fully retain YFV within the bioreactor, continuous product harvesting was achieved with the large-pore PS membrane (0.34 µm). Due to the low protein rejection of the latter, this membrane type could also be of interest for other applications, that is, recombinant protein production in perfusion cultures.     

Cellulose nanowhiskers extracted from TEMPO-oxidized jute fibers

Cellulose nanowhiskers is a kind of renewable and biocompatible nanomaterials evoke much interest because of its versatility in various applications. Here, for the first time, a novel controllable fabrication of cellulose nanowhiskers from jute fibers with a high yield (over 80%) via a 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)/NaBr/NaClO system selective oxidization combined with mechanical homogenization is reported. The versatile jute cellulose nanowhiskers with ultrathin diameters (3-10nm) and high crystallinity (69.72%), contains C6 carboxylate groups converted from C6 primary hydroxyls, which would be particularly useful for applications in the nanocomposites as reinforcing phase, as well as in tissue engineering, pharmaceutical and optical industries as additives.     

Osmoprotective polymer additives attenuate the membrane pore‐forming activity of antimicrobial peptoids

Antimicrobial peptides (AMPs) are critical components of the innate immune system and exhibit bactericidal activity against a broad spectrum of bacteria. We investigated the use of N‐substituted glycine peptoid oligomers as AMP mimics with potent antimicrobial activity. The antimicrobial mechanism of action varies among different AMPs, but many of these peptides can penetrate bacterial cell membranes, causing cell lysis. We previously hypothesized that amphiphilic cyclic peptoids may act through a similar pore formation mechanism against methicillin‐resistant Staphylococcus aureus (MRSA). Peptoid‐induced membrane disruption is observed by scanning electron microscopy and results in a loss of membrane integrity. We demonstrate that the antimicrobial activity of the peptoids is attenuated with the addition of polyethylene glycol osmoprotectants, signifying protection from a loss of osmotic balance. This decrease in antimicrobial activity is more significant with larger osmoprotectants, indicating that peptoids form pores with initial diameters of ～2.0–3.8 nm. The initial membrane pores formed by cyclic peptoid hexamers are comparable in diameter to those formed by larger and structurally distinct AMPs. After 24 h, the membrane pores expand to >200 nm in diameter. Together, these results indicate that cyclic peptoids exhibit a mechanism of action that includes effects manifested at the cell membrane of MRSA. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 227–236, 2015.     

Fabrication and characterization of chlorhexidine gluconate loaded poly(vinyl alcohol)/45S5 nano-bioactive glass nanofibrous membrane for guided tissue regeneration applications

Polymeric barrier membranes are used in periodontal applications to prevent fibroblastic cell migration into the cavities of bone tissue and to properly guide the proliferation of tissues. In this study, the fabrication, characterization, bioactivity, and in vitro biological properties of polyvinyl alcohol-based nanofibrous membranes containing nano-sized 45S5 bioactive glass (BG) loaded with chlorhexidine (CH) gluconate with biocompatible, bioactive, and antibacterial properties for using as dental barrier membranes were investigated. Nanofibrous membranes with an average fiber diameter, pore size, and porosity of 210 nm, 24.73 μm, and 12.42%, respectively, were loaded with 1% and 2% CH, and the release profile was investigated. The presence of BG in the membranes promoted fibroblastic proliferation and the presence of CH provided antibacterial properties. Nanofibrous membranes exhibit a high ability to restrict bacterial growth while fulfilling the necessary conditions for use as a dental barrier thanks to their low swelling rates, significant surface bioactivities, and appropriate degradation levels.     

Development and qualification of a novel virus removal filter for cell culture applications

Commercial bioreactors employing mammalian cell cultures to express biological or pharmaceutical products can become contaminated with adventitious viruses. The high expense of such a contamination can be reduced by passing all gases and fluids feeding the bioreactor through virus inactivation or removal steps, which act as viral barriers around the bioreactor. A novel virus barrier filter has been developed for removing viruses from serum-free cell culture media. This filter removes the 20 nm minute virus of mice by >3 log reduction value (LRV), the 28 nm bacteriophage PhiX174 by >4.5 LRV, the mycoplasma Acholeplasma laidlawii by > or =8.8 LRV, and the bacteria Brevundimonas diminuta by > or =9.2 LRV. Robust removal occurs primarily by size exclusion as demonstrated over a wide range of feedstocks and operating conditions. The filtered media are indistinguishable from unfiltered media in growth of cells to high densities, maintenance of cell viability, and productivity in expressing protein product. Insulin and transferrin show high passage through the filter. The virus barrier filter can be autoclaved. The relatively high membrane permeability enables the use of a moderate filtration area.     

The ultrastructure of Ignicoccus: evidence for a novel outer membrane and for intracellular vesicle budding in an archaeon

A novel genus of hyperthermophilic, strictly chemolithotrophic archaea, Ignicoccus, has been described recently, with (so far) three isolates in pure culture. Cells were prepared for ultrastructural investigation by cultivation in cellulose capillaries and processing by high-pressure freezing, freeze-substitution and embedding in Epon. Cells prepared in accordance with this protocol consistently showed a novel cell envelope structure previously unknown among the Archaea: a cytoplasmic membrane; a periplasmic space with a variable width of 20 to 400 nm, containing membrane-bound vesicles; and an outer sheath, approximately 10 nm wide, resembling the outer membrane of gram-negative bacteria. This sheath contained three types of particles: numerous tightly, irregularly packed single particles, about 8 nm in diameter; pores with a diameter of 24 nm, surrounded by tiny particles, arranged in a ring with a diameter of 130 nm; and clusters of up to eight particles, each particle 12 nm in diameter. Freeze-etched cells exhibited a smooth surface, without a regular pattern, with frequent fracture planes through the outer sheath, indicating the presence of an outer membrane and the absence of an S-layer. The study illustrates the novel complex architecture of the cell envelope of Ignicoccus as well as the importance of elaborate preparation procedures for ultrastructural investigations.     

Electrospun nano-fiber mats containing cationic cellulose derivatives and poly (vinyl alcohol) with antibacterial activity

Nano-fibrous mats have been successfully prepared by electrospinning of the blend solutions of cationic cellulose derivatives (PQ-4) and polyvinyl alcohol (PVA). Effects of the blending ratio and applied voltage on the morphology and diameter of the electrospun nano-fibers were investigated. The average diameter of the PQ-4/PVA blend fibers was in the range of 150–250 nm. The electrospinning process became instable and the fiber diameter distribution broadened with increasing PQ-4 content and applied voltage. The antibacterial activity of electrospun PQ-4/PVA blend mats against Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus indicated potential for biomedical use.     

Supramolecular structure characterization of cellulose II nanowhiskers produced by acid hydrolysis of cellulose I substrates

Cellulose II nanowhiskers (CNW-II) were produced by treatment of microcrystalline cellulose with sulfuric acid by both controlling the amount of H(2)SO(4) introduced and the time of addition during the hydrolysis process. The crystalline structure was confirmed by both XRD and (13)C CP-MAS NMR spectroscopy. When observed between crossed polarizers, the cellulose II suspension displayed flow birefringence and was stable for several months. The CNW-II nanowhiskers were significantly smaller than the cellulose I nanowhiskers (CNW-I) and had a rounded shape at the tip. The CNW-II average length and height were estimated by AFM to be 153 ± 66 and 4.2 ± 1.5 nm, respectively. An average width of 6.3 ± 1.7 nm was found by TEM, suggesting a ribbon-shape morphology for these whiskers. The average dimensions of the CNW-II elementary crystallites were estimated from the XRD data, using Scherrer's equation. A tentative cross-sectional geometry consistent with both XRD and NMR data was then proposed and compared with the geometry of the CNW-I nanowhiskers.     

Immobilization of Candida rugosa lipase on electrospun cellulose nanofiber membrane

A biocatalyst with high activity retention of lipase was fabricated by the covalent immobilization of Candida rugosa lipase on a cellulose nanofiber membrane. This nanofiber membrane was composed of nonwoven fibers with 200 nm nominal fiber diameter. It was prepared by electrospinning of cellulose acetate (CA) and then modified with alkaline hydrolysis to convert the nanofiber surface into regenerated cellulose (RC). The nanofiber membrane was further oxidized by NaIO₄. Aldehyde groups were simultaneously generated on the nanofiber surface for coupling with lipase. Response surface methodology (RSM) was applied to model and optimize the modification conditions, namely NaIO₄ content (2–10 mg/mL), reaction time (2–10 h), reaction temperature (25–35 °C) and reaction pH (5.5–6.5). Well-correlating models were established for the residual activity of the immobilized enzyme (R² = 0.9228 and 0.8950). We found an enzymatic activity of 29.6 U/g of the biocatalyst was obtained with optimum operational conditions. The immobilized lipase exhibited significantly higher thermal stability and durability than equivalent free enzyme.     

Nuclear surface complex as observed with the high resolution scanning electron microscope. Visualization of the membrane surfaces of the neclear envelope and the nuclear cortex from Xenopus laevis oocytes

The nuclear envelope and associated structures from Xenopus laevis oocytes (stage VI) have been examined with the high resolution scanning electron microscope (SEM). The features of the inner and outer surfaces of the nuclear surface complex were revealed by manual isolation , whereas the membranes facing the perinuclear space (the space between the inner and outer nuclear membranes) were observed by fracturing the nuclear envelope in this plane and splaying the corresponding regions apart. Pore complexes were observed on all four membrane surfaces of this double-membraned structure. The densely packed pore complexes (55/micron2) are often clustered into triplets with shared walls (outer diameter = 90 nm; inner diameter = 25 nm; wall thickness = aproximately 30 nm), and project aproximately 20 nm above each membrane except where they are flush with the innermost surface. The pore complex appears to be an aggregate of four 30-nm subunits. The nuclear cortex, a fibrous layer (300 nm thickness) associated with the inner surface of the nuclear envelope, has been revealed by rapid fixation. This cortical layer is interrupted by funnel-shaped intranuclear channels (120-640 nm diam) which narrow towards the pore complexes. Chains of particles, arranged in spirals, are inserted into these intranuclear channels. The fibers associated with the innermost face of the nuclear envelope can be extraced with 0.6 MKI to reveal the pore complexes. A model of the nuclear surface complex, compiled from the visualization of all the membrane faces and the nuclear cortex, demonstrates relations between the intranuclear channels (3.2/micron2) and the numerous pore complexes, and the possibility of their role in nucleocytoplasmic interactions.     

Virus filtration of high‐concentration monoclonal antibody solutions

The ability to process high‐concentration monoclonal antibody solutions (> 10 g/L) through small‐pore membranes typically used for virus removal can improve current antibody purification processes by eliminating the need for feed stream dilution, and by reducing filter area, cycle‐time, and costs. In this work, we present the screening of virus filters of varying configurations and materials of construction using MAb solutions with a concentration range of 4–20 g/L. For our MAbs of interest—two different humanized IgG1s—flux decay was not observed up to a filter loading of 200 L/m² with a regenerated cellulose hollow fiber virus removal filter. In contrast, PVDF and PES flat sheet disc membranes were plugged by solutions of these same MAbs with concentrations >4 g/L well before 50 L/m². These results were obtained with purified feed streams containing <2% aggregates, as measured by size exclusion chromatography, where the majority of the aggregate likely was composed of dimers. Differences in filtration flux performance between the two MAbs under similar operating conditions indicate the sensitivity of the system to small differences in protein structure, presumably due to the impact of these differences on nonspecific interactions between the protein and the membrane; these differences cannot be anticipated based on protein pI alone. Virus clearance data with two model viruses (XMuLV and MMV) confirm the ability of hollow fiber membranes with 19 ± 2 nm pore size to achieve at least 3–4 LRV, independent of MAb concentration, over the range examined. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Assembly of Cellulose Synthesizing Complexes on the Plasma Membrane of Boodlea coacta

The assembly of cellulose synthesizing complexes (terminal complexes,TCs) on the plasma membrane of Boodlea coacta was investigatedduring the formation of both the matrix-rich layer (MRL) andfibril-rich layers (FRLs) of cell walls. The TCs appeared tobe located mostly within the outer leaflet of the plasma membrane,and were observed as elliptical protrusions consisting of manyparticles of about 9 nm in diameter. Their length varied from100 to 500 nm (average, 220 nm) during MRL formation and from100 to 860 nm (average, 360 nm) during FRL formation. A correlationwas found between the length of TCs and the microfibril widthin both MRL and FRL. On the E-face of the plasma membrane, numerous round protrusions(30–130 nm in diameter), consisting of many particles,8–10 nm in diameter, were also present. Their densitywas greater during FRL formation than during MRL formation.Some of these structures larger than 100 nm were associatedwith microfibril impressions and some appeared to be bound tothe TCs. These protrusions increased in number with Calcofluortreatment but decreased in number when the dye was removed fromthe culture medium. Thus, the TCs may be assembled from massesof particles aggregated on the outer surface of the plasma membrane,and may grow longer by incorporation of these masses. The appearanceof the longer TCs during FRL formation is probably due to thegreater density of these masses. (Received May 1, 1985; Accepted August 16, 1985)     

High resolution analysis of the formation of cellulose synthesizing complexes inVaucheria hamata

Summary Ultrastructure and assembly of cellulose terminal synthesizing complexes (terminal complexes, TCs) in the algaVaucheria hamata (Waltz) were investigated by high resolution analytical techniques for freeze-fracture replication.Vaucheria TCs consist of many diagonal rows of subunits located on the inner leaflet of the plasma membrane. Each row contains about 10–18 subunits. The subunits themselves are rectangular, approx. 7×3.5 nm, and each has a single elliptical hole which may be the site of a single glucan chain polymerization. The subunits are connected with extremely small filaments (0.3–0.5 nm). Connections are more extensive in a direction parallel to the subunit rows and less extensive perpendicular to them. Nascent TC subunits are found to be packed within globules (15–20 nm in diameter) which are larger than typical intramembranous particles (IMPS are 10–11 nm in diameter) distributed in the plasma membrane. The subunits in the globule, which may be a zymogenic precursor of the TC, are generally exhibited in the form of doublets. Approximately 6 doublets are connected to a center core with small filaments. The globules are inserted into the plasma membrane together with IMPS by the fusion of cytoplasmic (Golgi derived) vesicles. Two or three globules attach to each other, unfold, and expand to form the first subunit rows of the TC on the inner leaflet of the plasma membrane. More globules attach to the structure and unfold until the nascent TC consists of a few rows of subunits. These rows are arranged almost parallel to each other. Two formation centers of subunits appear at both ends of an elongating TC. New subunits carried by the globules are added at each of these centers to create new rows until the elongating TC structure is completed. On the basis of this study, a model of TC assembly and early initiation of microfibril formation inVaucheria is proposed.Abbreviations IMPS intramembranous particles - MF microfibril - TC terminal complex