Lessons from a tarantula: new insights into muscle thick filament and myosin interacting-heads motif structure and function

The tarantula skeletal muscle X-ray diffraction pattern suggested that the myosin heads were helically arranged on the thick filaments. Electron microscopy (EM) of negatively stained relaxed tarantula thick filaments revealed four helices of heads allowing a helical 3D reconstruction. Due to its low resolution (5.0 nm), the unambiguous interpretation of densities of both heads was not possible. A resolution increase up to 2.5 nm, achieved by cryo-EM of frozen-hydrated relaxed thick filaments and an iterative helical real space reconstruction, allowed the resolving of both heads. The two heads, “free” and “blocked”, formed an asymmetric structure named the “interacting-heads motif” (IHM) which explained relaxation by self-inhibition of both heads ATPases. This finding made tarantula an exemplar system for thick filament structure and function studies. Heads were shown to be released and disordered by Ca²⁺-activation through myosin regulatory light chain phosphorylation, leading to EM, small angle X-ray diffraction and scattering, and spectroscopic and biochemical studies of the IHM structure and function. The results from these studies have consequent implications for understanding and explaining myosin super-relaxed state and thick filament activation and regulation. A cooperative phosphorylation mechanism for activation in tarantula skeletal muscle, involving swaying constitutively Ser35 mono-phosphorylated free heads, explains super-relaxation, force potentiation and post-tetanic potentiation through Ser45 mono-phosphorylated blocked heads. Based on this mechanism, we propose a swaying-swinging, tilting crossbridge-sliding filament for tarantula muscle contraction.     

Contrast of nuclei in stratified squamous epithelium in optical coherence tomography images at 800 nm

Imaging nuclei of keratinocytes in the stratified squamous epithelium has been a subject of intense research since nucleus associated cellular atypia is the key criteria for the screening and diagnosis of epithelial cancers and their precursors. However, keratinocyte nuclei have been reported to be either low scattering or high scattering, so that these inconsistent reports might have led to misinterpretations of optical images, and more importantly, hindered the establishment of optical diagnostic criteria. We disclose that they are generally low scattering in the core using Micro‐optical coherence tomography (μOCT) of 1.28‐μm axial resolution in vivo; those previously reported “high scattering” or “bright” signals from nuclei are likely from the nucleocytoplasmic boundary, and the low‐scattering nuclear cores were missed possibly due to insufficient axial resolutions (~4μm). It is further demonstrated that the high scattering signals may be associated with flattening of nuclei and cytoplasmic glycogen accumulation, which are valuable cytologic hallmarks of cell maturation.     

A study of apo- and holo-forms of horse liver alcohol dehydrogenase in solution by diffuse x-ray scattering

Apo- and holo-forms of horse liver alcohol dehydrogenase (LADH) in solution were studied by diffuse x-ray scattering. Experimental scattering curves for apo- and holo-forms coincide both with the curves calculated from the crystal structures of apo- and holo-enzymes, and with each other. Thus the “sliding” of catalytical domains in LADH upon substrate binding, which has been shown by x-ray analysis, cannot be detected by diffuse x-ray scattering. Sensitivity of the scattering curves to the domain displacements of sliding and “locking” types has been investigated. It has been shown that the scattering curves of LADH are rather sensitive to the domain “unlocking.” However, these curves change only slightly upon sliding of domains, including the sliding of domains observed in LADH by x-ray analysis.     

Fluorescence-based real time monitoring and diagnostics of recombinant Pichia pastoris cultivations in a bioreactor

This study describes the application of the multivariate curve resolution (MCR) analysis technique for real-time analysis of culture fluorescence during recombinant Pichia pastoris cultivation in a bioreactor. Fluorescence spectra were acquired with an on-line dual excitation wavelength fluorometer and then used to develop a real time MCR-based bioprocess monitoring and diagnostics tool. Initial bioreactor experiments using two similar recombinant antibody secreting P. pastoris cell lines showed significant differences in protein production. To distinguish between the contributions of operating conditions and the specific cell line's genetic composition to the observed differences in protein production, the bioreactor experiments were repeated and accompanied by real time MCR analysis. The tests demonstrated high sensitivity of MCR-derived “pure concentration” profiles to growth as well as to initial conditions, thus enabling real-time cultivation process trend diagnostics and fault detection. © 2018 Her Majesty the Queen in Right of Canada © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2761, 2019.     

A GIM-Based Biomimetic Learning Approach for Motion Generation of a Multi-Joint Robotic Fish

In this paper, we propose a biomimetic learning approach for motion generation of a multi-joint robotic fish. Based on a multi-joint robotic fish model, two basic Carangiform swimming patterns, namely ＂cruise＂ and ＂C sharp turning＂, are extracted as training samples from the observations of real fish swimming. A General Internal Model （GIM）, which is an imitation of Central Pattern Generator （CPG） in nerve systems, is adopted to learn and to regenerate coordinated fish behaviors. By virtue of the universal function approximation ability and the temporal/spatial scalabilities of GIM, the proposed learning approach is able to generate the same or similar fish swimming patterns by tuning two parameters. The learned swimming patterns are implemented on a multi-joint robotic fish in experiments. The experiment results verify the effectiveness of the biomimetic learning approach in generating and modifying locomotion patterns for the robotic fish.     

Multistate Luminescent Solar Concentrator “Smart” Windows

A supertwist liquid crystalline luminescent solar concentrator (LSC) “smart” window is fabricated which can be switched electrically between three states: one designed for increased light absorption and electrical generation (the “dark” state), one for transparency (the “light” state), and one for enhanced haziness (“scattering” state). In the scattering state, the absorption and edge emissions decrease while the face emissions are enhanced. This new LSC “smart” window state can find application as a privacy feature in housing, but could also allow for a new “smart” window application as a diffuse glazing to increase plant growth in horticultural applications.     

An iterative approach from the standpoint of the additive hypothesis to the dendrogram problem posed by molecular data sets

The problem of constructing a dendrogram depicting phylogenetic relationships for a collection of contemporary species is considered. An approach was developed based on the additive hypothesis in which each “length” between two species can be described by the shortest sum of lengths for the individual links on the dendrogram topology which connect the two species. The additive hypothesis holds equally well if the dendro gram is replaced by its corresponding (rootless) network. Network topologies are defined set theoretically in terms of the initial, contemporary species, and a coefficient is defined for each point of any conceivable network. It is proved mathematically that each point of an additive network gives a coefficient value of zero, whereas each point not belonging to an additive network gives a coefficient value greater than zero. This suggests an iterative procedure in which “false” network points are replaced by “true” ones, or more generally in which “very false” network points are replaced by “nearly true” ones. The first procedure follows from the mathematical proof and the second is confirmed by simulation. Since most real data sets are not additive in the strict sense, a real data example was presented in which the iterative procedure produced a plausible network topology.     

The parameters of cardiac-muscle repolarization: The measurement and information value

We performed a comparative analysis of different classes of algorithms for computer-assisted cardiological diagnostics. The concepts of an “internal” and “external” image of myocardial ischemia were formulated. We also discussed the biophysical aspects for the basic indices of spatial heterogeneity of the repolarization process in the myocardium, as well as the problems of measurement of the corresponding parameters. The experimental part was performed on two groups of patients. Both experimental and control groups included two sets of 12-lead electrocardiograms of real patients: “Normal” and “Ischemia” (with lateral localization). The experimental group consisted of 202 and 143 electrocardiograms, while the control group consisted of 200 and 91 electrocardiograms, respectively. The electrocardiograms were verified according to the Minnesota Code criteria.     

Conformations of kinesin: solution vs. crystal structures and interactions with microtubules

Recently, the molecular structures of monomeric and dimeric kinesin constructs in complex with ADP have been determined by X-ray crystallography (Kull et al. 1996; Kozielski et al. 1997 a; Sack et al. 1997). The “motor” or “head” domains have almost identical conformations in the known crystal structures, yet the kinesin dimer is asymmetric: the orientation of the two heads relative to the coiled-coil formed by their neck regions is different. We used small angle solution scattering of kinesin constructs and microtubules decorated with kinesin in order to find out whether these crystal structures are of relevance for kinesin's structure under natural conditions and for its interaction with microtubules. Our preliminary results indicate that the crystal structures of monomeric and dimeric kinesin are similar to their structures in solution, though in solution the center-of-mass distance between the motor domains of the dimer could be slightly greater. The crystal structure of dimeric kinesin can be interpreted as representing two equivalent conformations. Transitions between these or very similar conformational states may occur in solution. Binding of kinesin to microtubules has conformational effects on both, the kinesin and the microtubule. Solution scattering of kinesin decorated microtubules reveals a peak in intensity that is characteristic for the B-surface lattice and that can be used to monitor the axial repeat of the microtubules under various conditions. In decoration experiments, dimeric kinesin dissociates, at least partly, leading to a stoichiometry of 1:1 (one kinesin head per tubulin dimer; Thormählen et al. 1998 a) in contrast to the stoichiometry of 2:1 reported for dimeric ncd. This discrepancy is possibly due to the effect of steric hindrance between kinesin dimers on adjacent binding sites.     

Structures of the “open” and “closed” state of trypanosomal triosephosphate isomerase,as observed in a new crystal form: Implications for the reaction mechanism

The structure of trypanosomal triosephosphate isomerase (TIM)has been solved at a resolution of 2.1Å in a new crystal form grown at pH 8.8 from PEG6000. In this new crystal form (space group C2, cell dimensions 94.8 Å, 48.3 Å, 131.0 Å, 90.0°, 100.3°, 90.0°), TIM is present in a ligand-free state. The asymmetric unit consists of two TIM subunits. Each of these subunits is part of a dimer which is sitting on a crystallographic twofold axis, such that the crystal packing is formed from two TIM dimers in two distinct environments. The two constituent monomers of a given dimer are, therefore, crystallographically equivalent. In the ligand-free state of TIM in this crystal form, the two types of dimer are very similar in structure, with the flexible loops in the “Open” conformation. For one dimer (termed molecule-1), the flexible loop (loop-6) is involved in crystal contacts. Crystals of this type have been used in soaking experiments with 0.4 M ammonium sulphate (studied at 2.4 Å resolution), and with 40 μM phosphoglycolohydroxamate (studied at 2.5 Å resolution). It is found that transfer to 0.4 M ammonuum sulphate (equal to 80 times the Ki of sulphate for TIM), gives rise to significant sulphate binding at the active site of one dimer (termed molecule-2), and less significant binding at the active site of the other. In neither dimer does sulphate induce a “closed” conformation. In a mother liquor containing 40 μM phosphoglycolohydroxamate (equal to 10 times the Ki of phosphoglycolohydroxamate for TIM), an inhibitor molecule binds at the active site of only that dimer of which the flexible loop is free from crystal contacts (molecule-2). In this dimer, it induces a closed conformation. These three structures are compared and discussed with respect to the mode of binding of ligand in the active site as well as with respect to the conformational changes resulting from ligand binding. © 1993 Wiley-Liss, Inc.     

The Crystal Structure of Glutamine-binding Protein fromEscherichia coli

The crystal structure of the glutamine-binding protein (GlnBP) fromEscherichia coliin a ligand-free “open” conformational state has been determined by isomorphous replacement methods and refined to anR-value of 21.4% at 2.3 Å resolution. There are two molecules in the asymmetric unit, related by pseudo 4-fold screw symmetry. The refined model consists of 3587 non-hydrogen atoms from 440 residues (two monomers), and 159 water molecules. The structure has root-mean-square deviations of 0.013 Å from “deal” bond lengths and 1.5° from “ideal” bond angles.The GlnBP molecule has overall dimensions of approximately 60 Å × 40 Å × 35 Å and is made up of two domains (termed large and small), which exhibit a similar supersecondary structure, linked by two antiparallel β-strands. The small domain contains three α-helices and four parallel and one antiparallel β-strands. The large domain is similar to the small domain but contains two additional α-helices and three more short antiparallel β-strands. A comparison of the secondary structural motifs of GlnBP with those of other periplasmic binding proteins is discussed.A model of the “closed form” GlnBP-Gln complex has been proposed based on the crystal structures of the histidine-binding protein-His complex and “open form” GlnBP. This model has been successfully used as a search model in the crystal structure determination of the “closed form” GlnBP-Gln complex by molecular replacement methods. The model agrees remarkably well with the crystal structure of the Gln-GlnBP complex with root-mean-square deviation of 1.29 Å. Our study shows that, at least in our case, it is possible to predict one conformational state of a periplasmic binding protein from another conformational state of the protein. The glutamine-binding pockets of the model and the crystal structure are compared and the modeling technique is described.     

Sucrose density gradient sedimentation of E. coli ribosomes.

The behavior of E. coli ribosomes during sedimentation on sucrose gradients is predicted under a variety of conditions by computer simulations. Since numerous recent kinetic studies indicate equilibration in times short compared to the time of sedimentation, these simulations assume that the system attains local reaction equilibrium at every point in the gradient at all times. For any type of homogeneous equilibrating ribosome population, governed by a single formation constant at one atmosphere pressure for 70S couples, no more than two clearly defined zones will be resolved, although the presence of large dissociating effects due to pressure gradients in high speed experiments will spread the subunit zone. Normally the pattern will consist of a 30S zone and a so-called “70S” zone, which is in reality a mixture of 70S couples and 30S and 50S subunits in local equilibrium. The greater the dissociation into subunits, the more the “70S” zone will be slowed below the nominal rate of 70 Svedberg units. If ribosomes have been collected from the “70S” zone in several successive cycles of purification, the repeated deletion of resolved 30S subunits can result in a preparation with so large a molar excess of 50S subunits that the ensuing sucrose density gradient sedimentation pattern may exhibit a “70S” zone followed by zone of 50S subunits, insteadof a zone of 30S subunits. Our most important conclusion is that whenever a well-resolved 50S zone is present in a sucrose density gradient sedimentation experiment on E. coli ribosomes, in addition to a 30S and a “70S” zone, under conditions where ribosomes and subunits should be in reversible equilibrium, the preparation must be microheterogeneous, containing a mixture of “tight” and “loose” couples. Moreover in such cases the content of large subunits in the 50S zone must be derived entirely from “loose” couples whereas the 30S zone must contain small subunits derived from both “tight” and “loose” couples. Sedimentation patterns predicted for various mixtures of “tight” and “loose” couples display all the major characteristics of published experimental patterns for E. coli ribosomes, including the partial or complete resolution into three zones, depending on rotor velocity and level of Mg²⁺.     

Case study and application of process analytical technology (PAT) towards bioprocessing: Use of tryptophan fluorescence as at‐line tool for making pooling decisions for process chromatography

Process analytical technology (PAT) has been gaining momentum in the biopharmaceutical community due to the potential for continuous real time quality assurance resulting in improved operational control and compliance. Two imperatives for implementing any PAT tool are that “variability is managed by the process” and “product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions.” Recently, we have been examining the feasibility of applying different analytical tools to bioprocessing unit operations. We have previously demonstarted that commercially available online‐high performance liquid chromatography and ultra performance liquid chromatography systems can be used for analysis that can facilitate real‐time decisions for column pooling based on product quality attributes (Rathore et al., 2008 a,b). In this article, we review an at‐line tool that can be used for pooling of process chromatography columns. We have demonstrated that our tryptophan fluorescence method offers a feasible approach and meets the requirements of a PAT application. It is significantly faster than the alternative of fractionation, offline analysis followed by pooling. Although the method as presented here is not an online method, this technique may offer better resolution for certain applications and may be a more optimal approach as it is very conducive to implementation in a manufacturing environment. This technique is also amenable to be used as an online tool via front face fluorescence measurements done concurrently with product concentration determination by UV. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

The effects of peripherally injected serotonin on feeding and locomotor activities in carp Cyprinus carpio L.

The influence of intraperitoneal (IP) and intramuscular (IM) injections of serotonin (5-hydroxytryptamine or 5-HT, 10 μg/g body weight) on a number of parameters of feeding behavior and locomotor activity in carp Cyprinus carpio L. has been investigated. It was shown that exogenous serotonin decreased various parameters of feeding and locomotor activities, and IM injections caused stronger inhibitory effect than IP injections. IP administration of this biogenic amine reduced the food intake in fishes of different age groups, induced an increase of the search reaction time (the latency to leave the starting chamber after its front wall was raised, or latency period for feeding of fish) in carp fingerlings in the experiments with “single” feeding. IM injections significantly lowered food intake of carp fingerlings in 1, 5 and 53 h, two other parameters—during all period of observation. In the experiments with “group” feeding food intake, duration of “group” feeding and total duration of feeding decreased during all period of observation after IM administration and in 1 h after IP injections only. Duration of “single” feeding and locomotor activity were changed less distinctly. The strongest effect of serotonin (up to 100%) was shown for duration of “group” feeding. It was supposed that inhibitory effects of exogenous serotonin on feeding and locomotor activities in carps were caused by its peripheral effects as well as by partial involving of central effect.     

An investigation of the structure of Alfalfa mosaic virus by small-angle neutron scattering

Small-angle neutron scattering experiments have been performed on the tubular bottom component of Alfalfa mosaic virus (AMV) and the “30 S” particle (a quasispherical reassembled AMV coat protein particle) with the aim of determining the internal structure of the virus. Scattering curves were obtained out to a resolution of $\text{1}\text{50}\text{A}\text{?}{}^{\mathrm{?1}}$ at a number of H₂O/²H₂O ratios and were analysed using a model fitting technique. This involves calculating the scattering intensity due to a parameterised distribution of scattering density representing the particle and comparing this to the experimental data after taking into account the effect of instrumental smearing. The use of the contrast variation method enables the internal consistency of the model to be well tested.Three models are used in an attempt to explain the scattering curve of the 30 S particle. A single homogeneous shell is shown to be inadequate and two other models introducing the presumed T = 1 icosahedral symmetry of the particle are presented and discussed. The most satisfactory of these consists of 60 spherical monomers of radius 19 Å symmetrically placed in pairs about the 2-fold icosahedral positions.The analysis of the bottom component data has yielded a low resolution model for the virus, which is shown to be consistent with its composition as given by earlier physico-chemical measurements. In the model the RNA is uniformly packed throughout the interior of the capsid (which is cylindrical with hemispherical ends) out to a radius of about 65 Å and with a packing fraction of 20%. Within the limitations of an homogeneous shell model, the protein capsid has an outer radius of 94 Å and thickness of 23 Å, but arguments are presented based on the marked lattice structure of the cylindrical capsid and the analysis of the scattering data of the 30 S particle, that this model underestimates the thickness of the protein shell and that it in fact makes contact with the RNA at about 65 Å.     

Development of a room laser based real-time alignment monitoring system using an array of photodiodes

PurposeTo develop a real-time alignment monitoring system (RAMS) to compensate for the limitations of the conventional room-laser-based alignment system. To verify the feasibility of the RAMS, reproducibility and accuracy tests were conducted.MethodsRAMS was composed of a room laser sensing array (RLSA), an electric circuit, an analog-to-digital converter (ADC), and a control PC. The RLSA was designed to arrange photodiodes in a pattern that results in the RAMS having a resolution of 1 mm. The photodiodes were used for quantitative assessment of the alignment condition. To verify the usability of the developed system, we conducted tests of temporal reproducibility, repeatability, and accuracy.ResultsThe results of the temporal reproducibility test suggested that the signal of the RAMS was stable with respect to time. Further, the repeatability test resulted in a maximum coefficient of variance of 1.14%, suggesting that the signal of the RAMS was stable over repeated set-ups. The accuracy test confirmed that the “on” and “off” signals could be distinguished by signal intensity, considering that the “off” signal was below 75% of the “on” signal in every case. In addition, we confirmed that the system can detect 1 mm of movement by monitoring the pattern of the “on” and “off” signals.ConclusionWe developed a room laser based alignment monitoring system. The feasibility test verified that the system is capable of quantitative alignment monitoring in real time. We expect that the RAMS can propose the potential of the room laser based alignment monitoring method.     

Mitochondrial groups of germinal cells of teleost Cyprinidae. II. High resolution autoradiographic study of the incorporation of 3H phenylalanine and 3H uridine

A high resolution autoradiographic study of the incorporation of tritiated uridine and amino acids by the mitochondrial groups, which are typical of most of germ cells at the beginning of gametogenesis, has been made on tench spermatocytes. By this technique, we confirm the partially proteinacious composition of the intermitochondrial “cement”; and for the first time, the presence of RNA in the “cement” is demonstrated by autoradiography. Moreover, a study of the kinetics of the incorporation of both precursors makes likely the hypothesis that at least a part of the “cement” derived from nucleocytoplasmic transfer. Since the biogenesis of mitochondria results from the complementary functioning of the two protein synthesic systems, the cytoplasmic one, which is preponderant, and the mitochondrial one, the mitochondrial groups seem to be the direct visualization of the contribution of the nuclear genome to the edification of new mitochondria.