Topological feature maps with self-organized lateral connections: a population-coded,one-layer model of associative memory

Guided by the neurobiological principles of self-organization and population coding, we develop a simple, neural, one-layer model for auto-association. Its core is a feature map endowed with self-organized lateral connections. Input patterns are coded by small spots of active neurons. The time evolution of neural activity then realizes an auto-association process by a recurrent attractor dynamics. Population coding is preserved due to a balance of diffusive spreading of activity and competitive refocusing. Because of its simplicity, the model allows a thorough qualitative and quantitative understanding. We show that the network is capable of performing a cluster analysis and hierarchical classification of data and, thus, qualifies as a tool for unsupervised statistical data analysis.     

Orientation and lateral mobility of cytochrome c on the surface of ultrathin lipid multilayer films.

We have previously shown that cytochrome c can be electrostatically bound to an ultrathin multilayer film having a negatively charged hydrophilic surface; furthermore, x-ray diffraction and absorption spectroscopy techniques indicated that the cytochrome c was bound to the surface of these ultrathin multilayer films as a molecular monolayer. The ultrathin fatty acid multilayers were formed on alkylated glass, using the Langmuir-Blodgett method. In this study, optical linear dichroism was used to determine the average orientation of the heme group within cytochrome c relative to the multilayer surface plane. The cytochrome c was either electrostatically or covalently bound to the surface of an ultrathin multilayer film. Horse heart cytochrome c was electrostatically bound to the hydrophilic surface of fatty acid multilayer films having an odd number of monolayers. Ultrathin multilayer films having an even number of monolayers would not bind cytochrome c, as expected for such hydrophobic surfaces. Yeast cytochrome c was covalently bound to the surface of a multilayer film having an even number of fatty acid monolayers plus a surface monolayer of thioethyl stearate. After washing extensively with buffer, the multilayer films with either electrostatically or covalently bound cytochrome c were analyzed for bound protein by optical absorption spectroscopy; the orientation of the cytochrome c heme was then investigated via optical linear dichroism. Polarized optical absorption spectra were measured from 450 to 600 nm at angles of 0 degrees, 30 degrees, and 45 degrees between the incident light beam and the normal to the surface plane of the multilayer. The dichroic ratio for the heme alpha-band at 550 nm as a function of incidence angle indicated that the heme of the electrostatically-bound monolayer of cytochrome c lies, on average, nearly parallel to the surface plane of the ultrathin multilayer. Similar results were obtained for the covalently-bound yeast cytochrome c. Furthermore, fluorescence recovery after photobleaching (FRAP) was used to characterize the lateral mobility of the electrostatically bound cytochrome c over the monolayer plane. The optical linear dichroism and these initial FRAP studies have indicated that cytochrome c electrostatically bound to a lipid surface maintains a well-defined orientation relative to the membrane surface while exhibiting measurable, but highly restricted, lateral motion in the plane of the surface.     

Memory Storage Fidelity in the Hippocampal Circuit: The Role of Subregions and Input Statistics

In the last decades a standard model regarding the function of the hippocampus in memory formation has been established and tested computationally. It has been argued that the CA3 region works as an auto-associative memory and that its recurrent fibers are the actual storing place of the memories. Furthermore, to work properly CA3 requires memory patterns that are mutually uncorrelated. It has been suggested that the dentate gyrus orthogonalizes the patterns before storage, a process known as pattern separation. In this study we review the model when random input patterns are presented for storage and investigate whether it is capable of storing patterns of more realistic entorhinal grid cell input. Surprisingly, we find that an auto-associative CA3 net is redundant for random inputs up to moderate noise levels and is only beneficial at high noise levels. When grid cell input is presented, auto-association is even harmful for memory performance at all levels. Furthermore, we find that Hebbian learning in the dentate gyrus does not support its function as a pattern separator. These findings challenge the standard framework and support an alternative view where the simpler EC-CA1-EC network is sufficient for memory storage.     

Influence of the passenger domain of a model autotransporter on the properties of its translocator domain

Autotransporters are a superfamily of proteins secreted by Gram-negative bacteria including many virulence factors. They are modular proteins composed of an N-terminal signal peptide, a surface-exposed ‘passenger’ domain carrying the activity of the protein, and a C-terminal ‘translocator’ domain composed of an α-helical linker region and a transmembrane β-barrel. The translocator domain plays an essential role for the secretion of the passenger domain across the outer membrane; however, the mechanism of autotransport remains poorly understood. The whooping cough agent Bordetella pertussis produces an autotransporter serine-protease, SphB1, which is involved in the maturation of an adhesin at the bacterial surface. SphB1 also mediates the proteolytic maturation of its own precursor. We used SphB1 as a model autotransporter and performed the first comparisons of the biochemical and biophysical properties of an isolated translocator domain with those of the same domain preceded by the C-terminal moiety of its natural passenger. By using cross-linking and dynamic light scattering, we provide evidence that the passenger domain promotes the auto-association of SphB1, although these interactions appear rather labile. Electrophysiological studies revealed that the passenger domain of the autotransporter appears to maintain the translocator channel in a low-conductance conformation, most likely by stabilizing the α-helix inside the pore. That the passenger may significantly influence AT physicochemical properties is likely to be relevant for the in vivo maturation and stability of AT proteins.     

Influence of the passenger domain of a model autotransporter on the properties of its translocator domain

Autotransporters are a superfamily of proteins secreted by Gram-negative bacteria including many virulence factors. They are modular proteins composed of an N-terminal signal peptide, a surface-exposed 'passenger' domain carrying the activity of the protein, and a C-terminal 'translocator' domain composed of an alpha-helical linker region and a transmembrane beta-barrel. The translocator domain plays an essential role for the secretion of the passenger domain across the outer membrane; however, the mechanism of autotransport remains poorly understood. The whooping cough agent Bordetella pertussis produces an autotransporter serine-protease, SphB1, which is involved in the maturation of an adhesin at the bacterial surface. SphB1 also mediates the proteolytic maturation of its own precursor. We used SphB1 as a model autotransporter and performed the first comparisons of the biochemical and biophysical properties of an isolated translocator domain with those of the same domain preceded by the C-terminal moiety of its natural passenger. By using cross-linking and dynamic light scattering, we provide evidence that the passenger domain promotes the auto-association of SphB1, although these interactions appear rather labile. Electrophysiological studies revealed that the passenger domain of the autotransporter appears to maintain the translocator channel in a low-conductance conformation, most likely by stabilizing the alpha-helix inside the pore. That the passenger may significantly influence AT physicochemical properties is likely to be relevant for the in vivo maturation and stability of AT proteins.     

The location of cytochrome c on the surface of ultrathin lipid multilayer films using x-ray diffraction.

X-ray diffraction and spectroscopic techniques were used to characterize ultrathin fatty acid multilayers having a bound surface layer of cytochrome c. Three to six monolayers of arachidic acid were deposited onto an alkylated glass surface, using the Langmuir-Blodgett method. These fatty acid multilayer films were stored either in a 1 mM NaHCO3 pH 7.5 solution or a buffered 10 microM cytochrome c solution, pH 7.5. After washing extensively with buffer, these multilayer films were assayed for bound cytochrome c by optical spectroscopy. It was found that the cytochrome c bound only to the odd-numbered monolayer films (which have hydrophilic surfaces). The theoretical number of cytochrome c molecules bound to the ultrathin multilayer films having three or five monolayers was calculated as N = 1.2 x 10(13)/cm2 (assuming a hexagonally close-packed monolayer of protein), which would produce an optical density of 0.002 at a wavelength of 550 nm; for a three or five monolayer ultrathin film that was incubated with cytochrome c, OD550 approximately equal to 0.002. The protein was released from the film when treated with greater than 100 mM KCl solution, as would be expected for an electrostatic interaction. Meridional x-ray diffraction data were collected from the arachidic acid films with and without a bound cytochrome c layer. A box refinement technique, previously shown to be effective in deriving the profile structures of nonperiodic ultrathin films, was used to determine the multilayer electron density profiles. The electron density profiles and their autocorrelation functions showed that bound cytochrome c resulted in an additional electron dense feature on the multilayer surface, consistent with a bound cytochrome c monolayer. The position of the bound protein relative to the multilayer surface was independent of the number of fatty acid monolayers in the multilayer. Future studies will use these methods to investigate the structures of membrane protein complexes bound directly to the surface of multilayer films.     

Biomolecule-based antibacterial coating on a stainless steel surface: multilayer film build-up optimization and stability study

The goal of this paper was to establish the durability profile of antibacterial multilayer thin films under storage and usage conditions. Thin films were built on stainless steel (SS) by means of a layer-by-layer process alternating a negatively charged polyelectrolyte, polyacrylic acid, with a cationic antibacterial peptide, nisin. SS coupons coated with the antibacterial film were challenged under environmental and usage conditions likely to be encountered in real-world applications. The change in antibacterial activity elicited by the challenge was used as an indicator of multilayer film resistance. Antibacterial SS samples could be stored for several weeks at 4°C in ambient air and antibacterial films were resistant to dipping and mild wiping in water and neutral detergent. The multilayer coating showed some weaknesses, however, that need to be addressed.     

Polyelectrolyte multilayer microcapsules: Self-assembly and toward biomedical applications

Over the past few years, many studies have been performed involving the application of the Layer-by-Layer (LbL) deposition of oppositely charged polyelectrolytes onto charged colloidal particles, followed by the dissolution of the templates, ultimately resulting in polyelectrolyte multilayer microcapsules. The ease of preparation of polyelectrolyte multilayer microcapsules afforded by the LbL self-assembly technique, as well as the advantages of accurate control over size, composition, and the thickness of the multilayer shell make these capsules very promising for a number of applications in materials and biomedical science. In this review, we describe the assembly and stimuli-responsive properties (“smart” capsules) of polyelectrolyte multilayer microcapsules, and also discuss the potential of this technique in regard to biomedical applications. In addition, we illustrate two measurement techniques for determining the mechanical properties of polyelectrolyte multilayer microcapsules—(i) osmotic swelling and (ii) AFM compression experiments. These capsules are believed to have great potential for future applications, including biosensors, bioreactors, and carriers for targeted drug delivery.     

Wing colors based on arrangement of the multilayer structure of wing scales in lycaenid butterflies (Insecta: Lepidoptera)

Male wing colors and wing scale morphology were examined for three species of lycaenid butterflies: Chrysozephyrus ataxus, Favonius cognatus and F. jezoensis. Measurement of spectral reflectance on the wing surface with a spectrophotometer revealed species‐specific reflection spectra, with one or two peaks in the ultraviolet and/or green ranges. Observations of wing scales using an optical microscope revealed that light was reflected from the inter‐ridge regions, where transmission electron microscopy revealed a multilayer structure. Based on the multilayer dimensions obtained, three models were devised and compared to explain the measured reflectance spectrum. The results showed that the best fit is a model in which thicknesses of thin films of the multilayer system are not constant and air spaces between cuticle layers are more or less packed with cuticle spacers. This suggests that the specific wing colors of the species examined are produced by the species‐specific arrangement of the multilayer structure of wing scales.     

Raman spectroscopic studies of biological membrane model systems: Dietherlecithins

Raman spectra are obtained for the multilayer dispersion of rac-1,2,dioctadec-9′-cis-enyl-glycero-3-phosphorylcholine (dietherlecithin) in excess water. The CH stretching region was studied as a function of temperature and indicates that the multilayer dispersions undergo a liquid crystal to the gel phase transition at ?21 ± 4°C.     

Polypeptide multilayer film co-delivers oppositely-charged drug molecules in sustained manners

The current state-of-the-art for drug-carrying biomedical devices is mostly limited to those that release a single drug. Yet there are many situations in which more than one therapeutic agent is needed. Also, most polyelectrolyte multilayer films intended for drug delivery are loaded with active molecules only during multilayer film preparation. In this paper, we present the integration of capsules as vehicles within polypeptide multilayer films for sustained release of multiple oppositely charged drug molecules using layer-by-layer nanoassembly technology. Calcium carbonate (CaCO(3)) particles were impregnated with polyelectrolytes, shelled with polyelectrolyte multilayers, and then assembled onto polypeptide multilayer films using glutaraldehyde. Capsule-integrated polypeptide multilayer films were obtained after decomposition of CaCO(3) templates. Two oppositely charged drugs were loaded into capsules within polypeptide multilayer films postpreparation based on electrostatic interactions between the drugs and the polyelectrolytes impregnated within capsules. We determined that the developed innovative capsule-integrated polypeptide multilayer films could be used to load multiple drugs of very different properties (e.g., opposite charges) any time postpreparation (e.g., minutes before surgical implantation inside an operating room), and such capsule-integrated films allowed simultaneous delivery of two oppositely charged drug molecules and a sustained (up to two weeks or longer) and sequential release was achieved.     

城市住区绿化生态效益及其可控影响因素的量化分析

基于杭州城西2007年1 m×1 m高分辨率航空遥感图像和夏季30 m×30 m LandsatTM数据,应用GIS和遥感图像解译方法定量计算了代表杭州城西30个典型城市住区总体生态效益的归一化植被指数(NDVI),以及各样本住区6个可控生态效益的影响指标(复层结构高度、软硬比、绿化覆盖率、容积率、绿地面积、建筑密度),并采用多元线性回归和对应分析方法得出6个影响因素对绿化生态效益的贡献度排序,以及提升住区生态效益的措施,分析了城市住区生态效益与典型可控影响因素间的定量关系.结果表明:各影响因素对城市住区生态效益贡献度依次为复层结构高度＞软硬比＞绿化覆盖率＞容积率＞绿地面积＞建筑密度;复层结构高度的贡献率远超过其他因素,建筑密度对住区生态效益的影响极微弱.利用对应分析法所得的各影响因素改进二维图,可方便地用来制定提升住区绿化生态效益的决策方案.     

Classification of complete blood count and haemoglobin typing data by a C4.5 decision tree,a naïve Bayes classifier and a multilayer perceptron for thalassaemia screening

This article presents the classification of blood characteristics by a C4.5 decision tree, a naïve Bayes classifier and a multilayer perceptron for thalassaemia screening. The aim is to classify eighteen classes of thalassaemia abnormality, which have a high prevalence in Thailand, and one control class by inspecting data characterised by a complete blood count (CBC) and haemoglobin typing. Two indices namely a haemoglobin concentration (HB) and a mean corpuscular volume (MCV) are the chosen CBC attributes. On the other hand, known types of haemoglobin from six ranges of retention time identified via high performance liquid chromatography (HPLC) are the chosen haemoglobin typing attributes. The stratified 10-fold cross-validation results indicate that the best classification performance with average accuracy of 93.23% (standard deviation = 1.67%) and 92.60% (standard deviation = 1.75%) is achieved when the naïve Bayes classifier and the multilayer perceptron are respectively applied to samples which have been pre-processed by attribute discretisation. The results also suggest that the HB attribute is redundant. Moreover, the achieved classification performance is significantly higher than that obtained using only haemoglobin typing attributes as classifier inputs. Subsequently, the naïve Bayes classifier and the multilayer perceptron are applied to an additional data set in a clinical trial which respectively results in accuracy of 99.39% and 99.71%. These results suggest that a combination of CBC and haemoglobin typing analysis with a naïve Bayes classifier or a multilayer perceptron is highly suitable for automatic thalassaemia screening.     

Inhibition of nuclease activity in Bacillus subtilis following infection with bacteriophage SP82G.

The Raman Spectra of Blodgett-Langmuir multilayer assemblies made from behenic acid, barium behenate and barium cis-13 erucate are reported. In particular, the peak height intensity ratio of the hydrocarbon chain methylene CH stretch Raman bands, $\text{I}{}_{2890}\text{I}{}_{2850}$, for each multilayer assembly is compared to that of phosphatidylcholine in powders and water dispersions as well as to samples of crystalline hydrocarbon chains. It is found that the fatty acid multilayers are more ordered than the phospholipid samples but less ordered than the crystalline samples. It is suggested that Blodgett-Langmuir multilayer assemblies of lipid might be a useful reference in quantitative studies of packing order in lipid phases.     

NMR reveals a novel glutaredoxin-glutaredoxin interaction interface

Glutaredoxins (Grx) represent a large family of glutathione (GSH)-dependent oxidoreductases that catalyse the reduction of disulfides or glutathione mixed disulfide. Grx domains from pathogenic bacteria and plant Grxs have been recently reported to target specific peroxiredoxins (Prxs). The specificity that triggers the interaction between Grx and Prx is poorly understood and is only based on the structure of Haemophilus influenzae Prx-Grx hybrid (hyPrx5). We report here an NMR study of the Populus tremula Grx C4 that targets a P.tremula D-type II Prx. We show that Grx C4 specifically self-associates in a monomer-dimer equilibrium with an apparent K(d) of ca 2.6 mM. Grx C4 homodimer was docked under experimental restraints. The results reveal a novel Grx-Grx interface that is unrelated to the hyPrx5 Grx-Grx dimer interface. Chemical-shift perturbations and 15N spin-relaxation measurements show that the auto-association surface comprises both the active site and the GSH binding site. Reduced GSH is demonstrated to bind reduced Grx with a K(d) of ca 8.6 mM. The potential biological significance of the new Grx-Grx interaction interface is discussed.     

Multilayer rat hepatocyte aggregates formed on expanded polytetrafluoroethylene surface

Feasibility of using a macroporous membrane material, expanded polytetrafluoroethylene (ePTFE), for culturing hepatocytes on its surface was examined. Adult rat hepatocytes were attached to an ePTFE surface and cultured in a hormonally defined medium supplemented with or without fetal calf serum (FCS, 10%) or bovine serum albumin (BSA, 0.03–3%). When cultured in a FCS-suplemented medium, hepatocytes reorganized themselves into multilayer cell aggregates on an ePTFE surface. The morphological characteristics of hepatocytes were influenced by the modification of the ePTFE surface as well as the culture medium. Hepatocytes cultured on a polyvinylalcohol (PVA)-coated ePTFE surface formed many more multilayer cell aggregates than those cultured on an uncoated ePTFE surface. Such highly multilayered hepatocyte aggregates were also noted when the cells were cultivated in a BSA-supplemented medium. On the other hand, when cultured in a FCS- or BSA-free medium, hepatocytes formed cell monolayers on both PVA-coated and uncoated ePTFE surfaces as did the cells on a collagen-coated polystyrene surface. The hepatocytes in the aggregates exhibited high albumin expression capability and low DNA synthesis rate as compared with those in monolayer cultures. The multilayer hepatocyte aggregates, as immobilized on a PVA-coated ePTFE surface in a serum-supplemented medium, are shown to be not only morphologically, but functionally differentiated, and will provide us a model system for the development of a bioreactor using hepatocytes, particularly for a hybrid-type artificial liver. This revised version was published online in June 2006 with corrections to the Cover Date.     

Beyond Langmuir: surface-bound macromolecule condensates

Macromolecule condensates, phase separation, and membraneless compartments have become an important area of cell biology research where new biophysical concepts are emerging. This article discusses the possibility that condensates assemble on multivalent surfaces such as DNA, microtubules, or lipid bilayers by multilayer adsorption. Langmuir isotherm theory conceptualized saturable surface binding and deeply influenced physical biochemistry. Brunauer-Emmett-Teller (BET) theory extended Langmuir’s ideas to multilayer adsorption. A BET-inspired biochemical model predicts that surface-binding proteins with a tendency to self-associate will form multilayered condensates on binding surfaces. These “bound condensates” are expected to assemble well below the saturation concentration for liquid–liquid phase separation, so they can compete subunits away from phase-separated droplets and are thermodynamically pinned to the binding surface. Tau binding to microtubules is an interesting test case. The nonsaturable binding isotherm is reminiscent of BET predictions, but assembly of Tau-rich domains at low concentrations requires a different model. Surface-bound condensates may find multiple biological uses, particularly in situations where it is important that condensate assembly is spatially constrained, such as gene regulation.     

Titanium Surface Priming with Phase-Transited Lysozyme to Establish a Silver Nanoparticle-Loaded Chitosan/Hyaluronic Acid Antibacterial Multilayer via Layer-by-Layer Self-Assembly

Objectives

The formation of biofilm around implants, which is induced by immediate bacterial colonization after installation, is the primary cause of post-operation infection. Initial surface modification is usually required to incorporate antibacterial agents on titanium (Ti) surfaces to inhibit biofilm formation. However, simple and effective priming methods are still lacking for the development of an initial functional layer as a base for subsequent coatings on titanium surfaces. The purpose of our work was to establish a novel initial layer on Ti surfaces using phase-transited lysozyme (PTL), on which multilayer coatings can incorporate silver nanoparticles (AgNP) using chitosan (CS) and hyaluronic acid (HA) via a layer-by-layer (LbL) self-assembly technique.

Methods

In this study, the surfaces of Ti substrates were primed by dipping into a mixture of lysozyme and tris(2-carboxyethyl)phosphine (TCEP) to obtain PTL-functionalized Ti substrates. The subsequent alternating coatings of HA and chitosan loaded with AgNP onto the precursor layer of PTL were carried out via LbL self-assembly to construct multilayer coatings on Ti substrates.

Results

The results of SEM and XPS indicated that the necklace-like PTL and self-assembled multilayer were successfully immobilized on the Ti substrates. The multilayer coatings loaded with AgNP can kill planktonic and adherent bacteria to 100% during the first 4 days. The antibacterial efficacy of the samples against planktonic and adherent bacteria achieved 65%-90% after 14 days. The sustained release of Ag over 14 days can prevent bacterial invasion until mucosa healing. Although the AgNP-containing structure showed some cytotoxicity, the toxicity can be reduced by controlling the Ag release rate and concentration.

Conclusions

The PTL priming method provides a promising strategy for fabricating long-term antibacterial multilayer coatings on titanium surfaces via the LbL self-assembly technique, which is effective in preventing implant-associated infections in the early stage.     

Multilayer network analyses as a toolkit for measuring social structure

The formalization of multilayer networks allows for new ways to measure sociality in complex social systems,including groups of animals.The same mathematical representation and methods are widely applicable across fields and study systems,and a network can represent drastically different types of data.As such,in order to apply analyses and interpret the results in a meaningful way the researcher must have a deep understanding of what their network is representing and what parts of it are being measured by a given analysis.Multilayer social networks can represent social structure with more detail than is often present in single layer networks,including multiple"types"of individuals,interactions,or relationships,and the extent to which these types are interdependent.Multilayer networks can also encompass a wider range of social scales,which can help overcome complications that are inherent to measuring sociality.In this paper,I dissect multilayer networks into the parts that correspond to different components of social structures.I then discuss common pitfalls to avoid across different stages of multilayer network analyses-some novel and some that always exist in social network analysis but are magnified in multi-layer representations.This paper serves as a primer for building a customized toolkit of multilayer network analyses,to probe components of social structure in animal social systems.     

Incorporation of acylated antibody into planar lipid multilayers: characterization and cell binding

Multiple (up to 14) layers of lipid were deposited onto an alkylated glass surface by dialysis of egg phosphatidylcholine (PC) and deoxycholate mixed micelles in the presence of alkylated glass coverslips. The amount of lipid associated with the coverslips was measured by using radioactive PC. It was found that the number of PC molecules in the multilayer increased with increasing initial lipid concentration in the dialysis mixture. Inclusion of cholesterol resulted in a significant increase in the amount of total lipid deposited in the multilayer. However, the PC/cholesterol ratio was up to 2-fold higher in the multilayers than in the liposomes present in the same dialysis bag. In addition, mouse monoclonal anti-H2Kk antibody which had previously been derivatized with palmitic acid could be readily incorporated into the lipid multilayer during dialysis. Measurements of lateral mobility with the fluorescence recovery after photobleaching technique on fluorescently labeled lipid or antibody in the multilayer showed that the lipid molecules diffused rapidly while the antibodies were essentially immobile. Lymphoma cells such as RDM4 cells expressing surface H2Kk glycoproteins could rapidly bind to the antibody-containing multilayers. The binding was blocked by free antibody or by goat anti-mouse immunoglobulin G, indicating the immunospecificity of the binding. Cell binding to the multilayer also exhibited a threshold dependence on the antibody density of the multilayer. A lower threshold was found for cells expressing a higher surface density of H2Kk. This system may be useful for model studies of cellular recognition.