Comparative analysis of optogenetic actuators in cultured astrocytes

Astrocytes modulate synaptic transmission via release of gliotransmitters such as ATP, glutamate, d-serine and l-lactate. One of the main problems when studying the role of astrocytes in vitro and in vivo is the lack of suitable tools for their selective activation. Optogenetic actuators can be used to manipulate astrocytic activity by expression of variants of channelrhodopsin-2 (ChR2) or other optogenetic actuators with the aim to initiate intracellular events such as intracellular Ca²⁺ ([Ca²⁺]_i) and/or cAMP increases. We have developed an array of adenoviral vectors (AVV) with ChR2-like actuators, including an enhanced ChR2 mutant (H134R), and a mutant with improved Ca²⁺ permeability (Ca²⁺ translocating channelrhodopsin, CatCh). We show here that [Ca²⁺]_i elevations evoked by ChR2(H134R) and CatCh in astrocytes are largely due to release of Ca²⁺ from the intracellular stores. The autocrine action of ATP which is released under these conditions and acts on the P2Y receptors also contributes to the [Ca²⁺]_i elevations. We also studied effects evoked using light-sensitive G-protein coupled receptors (opto-adrenoceptors). Activation of optoα1AR (Gq-coupled) and optoβ2AR (G_s-coupled) resulted in astrocytic [Ca²⁺]_i increases which were suppressed by blocking the corresponding intracellular signalling cascade (phospholipase C and adenylate cyclase, respectively). Interestingly, the bulk of [Ca²⁺]_i responses evoked using either optoAR was blocked by an ATP degrading enzyme, apyrase, or a P2Y1 receptor blocker, MRS 2179, indicating that they are to a large extent triggered by the autocrine action of ATP. We conclude that, whilst optimal tools for control of astrocytes are yet to be generated, the currently available optogenetic actuators successfully initiate biologically relevant signalling events in astrocytes.     

Synthesis of soluble coordination polymer nanoparticles using room-temperature ionic liquid

We report a new family of soluble cyano-bridged coordination polymer nanoparticles M₃[Cr(CN)₆]₂/[BMIM][BF₄] (where M²⁺ = Ni, Mn, V^IVO; BMIM = 1-butyl-3-methylimidazolium). These nanoparticles of ca. 6 nm were synthesised in the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate, which acts both as a stabilizing agent and a solvent. The magnetic properties of frozen colloids containing the nanoparticles show that the relaxation of magnetisation is strongly influenced by interparticle interactions leading to the appearance of spin-glass-like dynamics in these systems.     

Immunolocalization of smooth muscle-like cells in the quail ovary.

We localized alpha-smooth muscle actin (alpha-SMA) in the quail ovary, using the peroxidase-anti-peroxidase technique. Special attention was paid to the influence of fixation on the immunoreactivity of the antigen. The immunostaining of alpha-SMA largely depended on the nature of the fixative. The antigen could most successfully be localized in ovaries fixed in Carnoy's fluid. We also localized alpha-SMA and desmin in semi-thin glycol methacrylate sections of the pre-ovulatory follicle, using the immunogold-silver staining method. The sections were pretreated with Lugol's iodine or sodium metaperiodate to enhance the immunoreactivity. alpha-SMA was demonstrated in the cells of the chordae, the tunica albuginea, and the theca externa of each follicle. These structures were inter-connected, forming an ovarian suspensory apparatus. The thecal cells of prelampbrush follicles also expressed alpha-SMA. In the wall of the pre-ovulatory follicle, desmin was found in the cells of the chordae and the tunica albuginea, and in a few cells of the theca externa. In the theca interna, desmin, and sometimes alpha-SMA, was observed in cells adjacent to the endothelium of sinusoids, which are probably pericytes. Our results support the hypothesis that in birds the ovarian follicles possess a thecal contractile system, that is presumably involved in the ovulatory process.     

Smooth muscle-like cells in ovaries of the hamster and gerbil

Summary Smooth muscle-like cells are present in thecae externae, corpora lutea, and interstitial tissue of hamsters and gerbils. The smooth muscle-like cells, as examined by electron microscopy, are fusiform with central nuclei; the cytoplasm contains numerous myofilaments, dense bodies, micropinocytotic vesicles, and dense accumulations of glycogen-like particles. In addition to the smooth muscle-like cells, fibroblasts and cells that have characteristics of both fibroblasts and smooth muscle are located in the thecae externae of both species. There is no ultrastructural evidence of innervation in the theca folliculi, corpora lutea, or interstitial tissue of either species.A possible function for the smooth muscle-like cells is discussed.     

Numerical analysis of reflectance spectra

(1) Samples of yeast which varied over an arbitrary 0–100% scale with respect, to cytochrome concentration were prepared. (2) The reflectance spectrum of each sample was scanned. (3) These reflectance spectra were analyzed numerically on a digital computer to determine the bandwidths and intensities as a function of cytochrome concentration. Band shapes using the profiles of Cauchy, Gauss, and Giovanlelli were used. The correlations may be used to estimate cytochrome concentrations with a standard deviation of about 3%.     

Angiotensin II-induced differentiation of adipose tissue-derived mesenchymal stem cells to smooth muscle-like cells

Angiotensin II (Ang II) is involved in the development of cardiovascular disease and vascular remodeling. In this study, we demonstrate that treatment of human adipose tissue-derived mesenchymal stem cells (hADSCs) with Ang II increased the expression of smooth muscle-specific genes, including alpha-smooth muscle actin (alpha-SMA), calponin, h-caldesmon, and smooth muscle myosin heavy chain (SM-MHC), and also elicited the secretion of transforming growth factor-beta1 (TGF-beta1) and delayed phosphorylation of Smad2. The Ang II-induced expression of alpha-SMA and delayed phosphorylation of Smad2 were blocked by pretreatment of the cells with a TGF-beta type I receptor kinase inhibitor, SB-431542, small interference RNA-mediated depletion of endogenous Smad2, and adenoviral expression of Smad7. Furthermore, the Ang II-induced TGF-beta1 secretion, alpha-SMA expression, and delayed phosphorylation of Smad2 in hADSCs were abrogated by the MEK inhibitor U0126, suggesting a pivotal role of MEK/ERK pathway in the Ang II-induced activation of TGF-beta1-Smad2 signaling pathway. The smooth muscle-like cells which were differentiated from hADSCs by Ang II treatment exhibited contraction in response to 60mM KCl. These results suggest that Ang II induces differentiation of hADSCs to contractile smooth muscle-like cells through ERK-dependent activation of the autocrine TGF-beta1-Smad2 crosstalk pathway.     

Numerical analysis of an anastomotic device

The explicit dynamic finite element method was utilized to investigate the deformation behaviour of a woven wire mesh tubular device that is used in a side-to-side anastomotic procedure for achieving gastrointestinal anastomosis. The numerical model was initially verified by comparison to experimental results that were obtained using a specialized testing mechanism. Once validated, the finite element model (FEM) was parameterized to ascertain the influence of several device parameters on its deformation behaviour. The importance of these parameters, as related to its optimal design for use in minimally invasive surgery (MIS), was subsequently ascertained and discussed.     

Biomimetic robotic Venus flytrap (Dionaea muscipula Ellis) made with ionic polymer metal composites

The work described in this paper is a novel design of a robotic Venus flytrap (VFT) (Dionaea muscipula Ellis) by means of ionic polymeric metal composite (IPMC) artificial muscles as distributed nanosensors and nanoactuators. Rapid muscular movements in carnivorous plants, such as VFT, which are triggered by antenna-like sensors (trigger hair), present a golden key to study distributed biomolecular motors. Carnivorous plants, such as VFT, possess built-in intelligence (trigger hairs), as a strategy to capture prey, that can be turned on in a controlled manner. In the case of the VFT, the prey that is lured by the sweet nectar in the VFT pair of jaw-like lobes has to flip and move the trigger hairs, which are colorless, bristle-like and pointed. The dynamically moved trigger hairs then electro-elastically send an electric signal to the internal ions in the lobe to migrate outwardly for the jaw-like lobes to close rapidly to capture the prey. The manner in which the VFT lobes bend inward to capture the prey shows a remarkable similarity with typical IPMCs bending in an electric field. Furthermore, the mechano-electrical sensing characteristics of IPMCs also show a remarkable resemblance to mechano-electrical trigger hairs on the lobes of the VFT. The reader is referred to a number of papers in connection with sensing and actuation of IPMCs in particular. Thus, one can integrate IPMC lobes with a common electrode in the middle of one end of the lobes to act like a spine and use IPMC bristles as trigger finger to sense the intrusion of a fly or insect to send a sensing signal to a solid state relay which then triggers the actuation circuit of the IPMC lobes to rapidly bend toward each other and close. The two lobes, which form the trap, are attached to the midrib common electrode which is conveniently termed the spine. The upper surface of each lobe is dished, and spaced along the free margins of the lobes with some 15-20 prong-like teeth. These are tough and pointed, and are inclined at an inward angle so that when the trap is sprung shut they will interlock. We have been experimenting with the VFT closing of its jaw-like lobes that close in about 0.3 s and have gained a lot of knowledge to report on the ionic and electrical mechanisms involved in the operation of such intelligent distributed biomolecular motors.     

Effect of changes of pHi on intracellular calcium in a smooth muscle-like cell line

The effect of changes of pH_i on Ca_i were studied using fluorescent dyes in cells of the cultured smooth muscle-like line, BC3H-1. Resting Ca_i in these cells was 182 ± 12 nM (n = 74) at pH_o of 7.4. Upon exposure to NH₄Cl, which rapidly alkalinized cells, a transient increase of Cai to 349 ± 55 nM (n = 29) was observed. The peak of the transient occurred within 30 s of exposure to NH₄Cl and returned to baseline within 1 minute. Two other procedures which resulted in rapid cellular alkalinization also caused a transient rise in Ca_i: exposure to and then removal of CO₂ (Ca_i increased from 182 ± 22 to 248 ± 28 nM; n = 8); and exposure to and then removal of Na propionate (Ca_i increased from 242 ± 32 to 456 ± 71 nM; n = 9). The NH4Cl-induced Ca_i transient was eliminated by exposure to 0.2 mM TMB8 and to Ca-free solutions, but not by exposure to 0.5 mM LaCl₃. Sustained changes of pH_i can be induced by varying pH_o. When pH_o was lowered to 6.9, Ca_i fell by 49 ± 11 nM but increased by 203 ± 51 nM (n = 6) when pH_o was raised to 7.9. These data indicate that rapid alkalinization of BC3H-1 cells results in a rapid transient rise of Ca_i. This transient is most likely due to the release of Ca from intracellular stores but may also involve an increase of Ca influx. Steady state values of Cai are positively correlated with steady state pH_i. These data may have implications for the contractile state of smooth muscle during periods of acid/base disturbances and relate to the role of elevated pH_i in cells from hypertensive animals.     

Transdifferentiation of preadipose cells into smooth muscle-like cells: role of aortic carboxypeptidase-like protein

Adipocyte differentiation involves dramatic cell shape alterations that are accompanied by changes in the expression of cytoskeletal and extracellular matrix (ECM) proteins. Aortic carboxypeptidase-like protein (ACLP) is a secreted protein associated with the extracellular matrix whose expression is induced during smooth muscle (SM) differentiation. We analyzed the expression of ACLP gene during adipocyte differentiation of 3T3-F442A, 3T3-L1, and Ob1771 preadipocytes. Our results show that ACLP mRNA and protein are expressed in growing cells and after commitment. Thereafter, their expression levels decrease, as opposed to that of aP2 and PPARgamma2. Consistent with these observations, ACLP mRNA is expressed in the stromal-vascular fraction of adipose tissue but not in the adipocyte fraction. Overexpression of ACLP in 3T3-F442A preadipocytes inhibits adipocyte differentiation at both morphological and molecular level. However, ACLP overexpression promotes transdifferentiation of preadipocytes into smooth muscle-like cells, which express specific markers such as SM22alpha, SM alpha-actin, SM-MHC, and caldesmon. These findings demonstrate that overexpression of a single extracellular matrix protein is sufficient to induce transdifferentiation and that ACLP may modulate the commitment of mesodermal cells into different lineages depending upon its pattern of expression.     

Numerical analysis of sexual dimorphism inSaguinus dentition

Among New World monkeys, more or less sexual dimorphism exists in the dentition, especially in the Cebidae. On the other hand, the Callitrichidae includingSaguinus are said to be characterized by a broad lack of sexual dimorphism with the exception of the reproductive organs. In the present article, sexual dimorphism in the dentition of someSaguinus species was reconfirmed using univariate and multivariate analytical methods. The results of the analysis were as follows: (1) there is no sexual dimorphism in the canine tooth size, except for the upper canine ofS. geoffroyi and lower canine ofS. mystax; (2) the overall tooth size difference between males and females is slight or none inS. geoffroyi, S. leucopus, andS. fuscicollis, relatively small inS. oedipus andS. mystax, and rather larger inS. midas; (3) an overall difference in shape factor between both sexes exists in all species ofSaguinus to a greater or lesser extent; (4) although only slight sexual dimorphism is recognized in the canine tooth itself, sexual dimorphism does exist in some adjacent teeth of the canine in a few species; and (5) there are some interspecific differences in the magnitude of the sexual dimorphism of theSaguinus dentition and these differences are more evident in species inhabiting the peripheral regions of the distribution areas of this genus. Taking all the evidence obtained into account, the sexual dimorphism in theSaguinus dentition must be re-investigated in comparison with other genera of the Callitrichidae.     

Numerical analysis of allozyme variation inCucurbita pepo

Cucurbita pepo, represented by cultivated forms of squash, pumpkins, and ornamental gourds, is a morphologically diverse species. Although several classifications of the cultivars have been proposed, none has been fully accepted. In an attempt to clarify the systematics of theC. pepo cultivars, allozyme variation was assayed. Twelve loci representing 6 enzyme systems (GOT, IDH, MDH, PER, PGI, and PGM) were screened. Seven of the loci were polymorphic. Electrophoretic data were collected for 50 accessions representing 14 commercial cultivars and a landrace from Mexico. Statistical analyses of the allozyme data revealed a biochemical basis for characterizing cultivars that agrees with morphology. Principal component analysis and cluster analysis revealed a major subdivision within the species above the level of cultivars. Genetic identities among groups of cultivars were much lower than those usually found among conspecific populations for outcrossing plants, possibly reflecting the influences of populational subdivision, drift, and selection particular toC. pepo.     

Numerical analysis of Leptospira DNA-restriction endonuclease patterns

Abstract Fifteen test strains, representing 5 leptospire serogroups were studied by DNA-restriction endonuclease ( Eco RI) analysis. The resulting patterns for each strain were scanned by a densitometer and the absorbance values were recorded along the pattern at regular intervals. The absorbance profiles were compared using the correlation coefficient and the similarity matrix was clustered by the UPGMA algorithm. The results were displayed as a simplified dendrogram. The numerical analysis of DNA-restriction endonuclease patterns can be used successfully in the identification of Leptospira strains.     

Statistical analysis of intrahelical ionic interactions in alpha-helices and coiled coils

There are many controversies concerning whether ionic interactions in alpha-helices and coiled coils actually contribute to the stabilisation and formation of these structures. Here we used a statistical approach to probe this question. We extracted unique alpha-helical and coiled coil structures from the protein database and analysed the ionic interactions between positively and negatively charged residues. The ionic interactions were categorized according to the type, spacing and order of the residues involved. Separate datasets were produced depending on the number of alpha-helices in the coiled coils and the mutual orientation of the helices. We compared the frequency of residue configurations able to form ionic interactions with their probability to form the interaction. We found a correlation between the two variables in alpha-helices, antiparallel two-stranded coiled coils and parallel two-stranded coiled coils. This indicates that some ionic interactions are indeed important for the formation and stabilisation of alpha-helices and coiled coils. We concluded that the configurations, which have simultaneously a large probability to form the ionic interaction and a frequent occurrence, are those, which have the most stabilising effect. These are the 4RE, 3ER and 4ER interactions.     

Rheological investigation on dynamic and structure of separated phases in polymer mixture–ionic surfactant ternary system

Ternary systems composed of 0.30% sodium carboxymethyl cellulose (NaCMC), 0.70% hydroxypropylmethyl cellulose (HPMC), and sodium dodecylsulfate (SDS) of various concentrations (0.00–2.00%), were investigated by oscillatory and flow shear rheometry. The investigation on binary mixtures of HPMC/SDS (0.7% HPMC, 0.00–2.00% SDS) and HPMC/NaCMC (1.00% polymer mixtures with HPMC/NaCMC mass ratio ranging from 1/0 to 0/1) was also carried out. In the examined systems, various interactions between HPMC–NaCMC, HPMC–SDS and NaCMC–(HPMC–SDS) take place. The interactions may bring about phase separation in the ternary system into HPMC–SDS complex rich phase (coacervate), and NaCMC rich phase (supernatant). The possibility of employing the interactions in the ternary system to vary structural properties of the coacervate, in terms of viscoelasticity and shear rate influence, was investigated. Rheological properties of the ternary system, as well as of the separated coacervate and the supernatant were tested. Influence of shear rate on the interactions taking place in both the binary and the ternary mixtures was examined. It was found that the applied shear rate can influence the interactions. Molecular mechanisms determining the rheological properties of the coacervate were suggested. It was found that complex intermolecular interactions in the ternary system can be employed to attain control over the structural properties of the coacervate phase.     

Numerical analysis of telluric nonfermenting Gram-negative bacteria]

A numerical analysis was carried out from a set of 165 telluric Gram-negative bacterial strains. The results allowed to join up 130 of them divided into eight phenons. Two of these phenons represent on their own 70% of the classified strains. The first of these phenons (52 strains) can be assimilated to the genus Pseudomonas in the fluorescent group; the second one (41 strains) offers some analogies with the Acinetobacter. A representative strain type of the latter phenon was retained for later taxonomic comparisons.     

Numerical analysis of random drift in a cline

The equilibrium state of a diffusion model for random genetic drift in a cline is analyzed numerically. The monoecious organism occupies an unbounded linear habitat with constant, uniform population density. Migration is homogeneous, symmetric and independent of genotype. A single diallelic locus with a step environment is investigated in the absence of dominance and mutation. The flattening of the expected cline due to random drift is very slight in natural populations. The ratio of the variance of either gene frequency to the product of the expected gene frequencies decreases monotonically to a nonzero constant. The correlation between the gene frequencies at two points decreases monotonically to zero as the separation is increased with the average position fixed; the decrease is asymptotically exponential. The correlation decreases monotonically to a positive constant depending on the separation as the average position increasingly deviates from the center of the cline with the separation fixed. The correlation also decreases monotonically to zero if one of the points is fixed and the other is moved outward in the habitat, the ultimate decrease again being exponential. Some asymptotic formulae are derived analytically.——The loss of an allele favored in an environmental pocket is investigated by simulating a chain of demes exchanging migrants, the other assumptions being the same as above. For most natural populations, provided the allele would be maintained in the population deterministically, this process is too slow to have evolutionary importance.     

Numerical analysis of thermal structure in heated room

1. 1. Two-dimensional numerical analysis is carried out on the thermal structure in an empty heated room of actual size under a steady state.

2. 2. Three types of heater, floor heating, side-wall heating and hot-air heating were used.

3. 3. The thermal structure in a room formed by each heating type is investigated in consideration of the energy transfer through the window of the room.

4. 4. The results indicate that floor heating is most suitable in a room without window from a viewpoint of the thermal comfort.

5. 5. In a windowed room, it is desirable that a heater should be located by the side of the window to prevent the cold draft.

In vitro ovulation of trout oocytes : Effect of prostaglandins on smooth muscle-like cells of the theca

Ovulation (active expulsion of oocyte from the mature follicle) of trout follicles matured can be induced by adding PGF_2α at doses of 1 and 5 μg/ml. PGE₂ is ineffective.The induction of ovulation by PGF_2α is inhibited in a calcium free medium or by inhibitors of calcium influx, particularly by Mn⁺⁺ and La⁺⁺⁺, suggesting that ovulation process implies active contraction of the smooth muscle cells of the theca.A significant but partial inhibition is also observed with cytochalasin B (1 and 5 μg/ml) demonstrating that contraction of other cell types than muscle, containing actin-like filaments, may also participate in the process.