Basic Biophysical Properties Research Articles

Estimation of the mass density of biological matter from refractive index measurements

The quantification of physical properties of biological matter gives rise to novel ways of understanding functional mechanisms. One of the basic biophysical properties is the mass density (MD). It affects the dynamics in sub-cellular compartments and plays a major role in defining the opto-acoustical properties of cells and tissues. As such, the MD can be connected to the refractive index (RI) via the well known Lorentz-Lorenz relation, which takes into account the polarizability of matter. However, computing the MD based on RI measurements poses a challenge, as it requires detailed knowledge of the biochemical composition of the sample. Here we propose a methodology on how to account for assumptions about the biochemical composition of the sample and respective RI measurements. To this aim, we employ the Biot mixing rule of RIs alongside the assumption of volume additivity to find an approximate relation of MD and RI. We use Monte-Carlo simulations and Gaussian propagation of uncertainty to obtain approximate analytical solutions for the respective uncertainties of MD and RI. We validate this approach by applying it to a set of well-characterized complex mixtures given by bovine milk and intralipid emulsion and employ it to estimate the MD of living zebrafish (Danio rerio) larvae trunk tissue. Our results illustrate the importance of implementing this methodology not only for MD estimations but for many other related biophysical problems, such as mechanical measurements using Brillouin microscopy and transient optical coherence elastography.

Voltage-gated calcium currents in human dorsal root ganglion neurons.

Voltage-gated calcium channels in sensory neurons underlie processes ranging from neurotransmitter release to gene expression and remain a therapeutic target for the treatment of pain. Yet virtually all we know about voltage-gated calcium channels has been obtained through the study of rodent sensory neurons and heterologously expressed channels. To address this, high voltage-activated (HVA) Ca2+ currents in dissociated human and rat dorsal root ganglion neurons were characterized with whole-cell patch clamp techniques. The HVA currents from both species shared basic biophysical and pharmacological properties. However, HVA currents in human neurons differed from those in the rat in at least 3 potentially important ways: (1) Ca2+ current density was significantly smaller, (2) the proportion of nifedipine-sensitive currents was far greater, and (3) a subpopulation of human neurons displayed relatively large constitutive current inhibition. These results highlight the need to for the study of native proteins in their native environment before initiating costly clinical trials.

Biophysical and synaptic properties of NMDA receptors in the lateral habenula

Excitatory synaptic transmission in the lateral habenula (LHb), an evolutionarily ancient subcortical structure, encodes aversive stimuli and affective states. Habenular glutamatergic synapses contribute to these processes partly through the activation of AMPA receptors. Yet, N-methyl-d-aspartate receptors (NMDARs) are also expressed in the LHb and support the emergence of depressive symptoms. Indeed, local NMDAR blockade in the LHb rescues anhedonia and behavioral despair in rodent models of depression. However, the subunit composition and biophysical properties of habenular NMDARs remain unknown, thereby hindering their study in the context of mental health. Here, we performed electrophysiological recordings and optogenetic-assisted circuit mapping in mice, to study pharmacologically-isolated NMDAR currents in LHb neurons that receive innervation from different brain regions (entopeduncular nucleus, lateral hypothalamic area, bed nucleus of the stria terminalis, or ventral tegmental area). This systematic approach revealed that habenular NMDAR currents are sensitive to TCN and ifenprodil – drugs that specifically inhibit GluN2A- and GluN2B-containing NMDARs, respectively. Whilst these pharmacological effects were consistently observed across inputs, we detected region-specific differences in the current-voltage relationship and decay time of NMDAR currents. Finally, inspired by the firing of LHb neurons in vivo, we designed a burst protocol capable of eliciting calcium-dependent long-term potentiation of habenular NMDAR transmission ex vivo. Altogether, we define basic biophysical and synaptic properties of NMDARs in LHb neurons, opening new avenues for studying their plasticity processes in physiological as well as pathological contexts.

Single channel properties of mitochondrial large conductance potassium channel formed by BK-VEDEC splice variant

The activation of mitochondrial large conductance calcium-activated potassium (mitoBKCa) channels increases cell survival during ischemia/reperfusion injury of cardiac cells. The basic biophysical and pharmacological properties of mitoBKCa correspond to the properties of the BKCa channels from the plasma membrane. It has been suggested that the VEDEC splice variant of the KCNMA1 gene product encoding plasma membrane BKCa is targeted toward mitochondria. However there has been no direct evidence that this protein forms a functional channel in mitochondria. In our study, we used HEK293T cells to express the VEDEC splice variant and observed channel activity in mitochondria using the mitoplast patch-clamp technique. For the first time, we found that transient expression with the VEDEC isoform resulted in channel activity with the conductance of 290 ± 3 pS. The channel was voltage-dependent and activated by calcium ions. Moreover, the activity of the channel was stimulated by the potassium channel opener NS11021 and inhibited by hemin and paxilline, which are known BKCa channel blockers. Immunofluorescence experiments confirmed the partial colocalization of the channel within the mitochondria. From these results, we conclude that the VEDEC isoform of the BKCa channel forms a functional channel in the inner mitochondrial membrane. Additionally, our data show that HEK293T cells are a promising experimental model for expression and electrophysiological studies of mitochondrial potassium channels.

Interplay between Gating and Block of Ligand-Gated Ion Channels

Drugs that inhibit ion channel function by binding in the channel and preventing current flow, known as channel blockers, can be used as powerful tools for analysis of channel properties. Channel blockers are used to probe both the sophisticated structure and basic biophysical properties of ion channels. Gating, the mechanism that controls the opening and closing of ion channels, can be profoundly influenced by channel blocking drugs. Channel block and gating are reciprocally connected; gating controls access of channel blockers to their binding sites, and channel-blocking drugs can have profound and diverse effects on the rates of gating transitions and on the stability of channel open and closed states. This review synthesizes knowledge of the inherent intertwining of block and gating of excitatory ligand-gated ion channels, with a focus on the utility of channel blockers as analytic probes of ionotropic glutamate receptor channel function.

Collagen–iron oxide nanoparticle based ferrogel: large reversible magnetostrains with potential for bioactuation

Smart materials such as stimuli responsive polymeric hydrogels offer unique possibilities for tissue engineering and regenerative medicine. As, however, most synthetic polymer systems and their degradation products lack complete biocompatibility and biodegradability, this study aims to synthesize a highly magnetic responsive hydrogel, based on the abundant natural biopolymer collagen. As the main component of vertebratal extracellular matrix, it reveals excellent biocompatibility. In combination with incorporated magnetic iron oxide nanoparticles, a novel smart nano-bio-ferrogel can be designed. While retaining its basic biophysical properties and interaction with living cells, this collagen-nanoparticle hydrogel can be compressed to 38% of its original size and recovers to 95% in suitable magnetic fields. Besides the phenomenology of this scenario, the underlying physical scenarios are also discussed within the framework of network models. The observed reversible peak strains as large as 150% open up possibilities for the fields of biomedical actuation, soft robotics and beyond.

0093 Topographical Analysis of Sleep Spindles and Their Coordination with Slow Oscillations

Abstract Introduction Brain oscillations found during sleep are hypothesized to mediate sleep-dependent memory consolidation, by coordinating cortical-subcortical activity and enabling synaptic plasticity. In particular, sleep spindles (10-16Hz) density and coordination with slow oscillations (SOs, 0.5–1.5 Hz) have been shown to correlate with memory performance post-sleep. In this study, we characterize how spindles are organized on the electrode manifold, and their relation to SO topography. Methods We conducted a sleep-memory study where subjects learned word-pair associations in the morning and were tested in the evening and the next morning. Polysomnography was collected during the night. We detected sleep spindles at each electrode independently and study their basic biophysical properties (density, amplitude, frequency, duration) in light sleep (stage 2, S2) and deep sleep (slow wave sleep, SWS) separately, including their co-occurrence with SOs. We categorize spindles that are co-detected across electrodes within a short time window and study how properties change across groups. Results We find a gradual increase in average spindle frequency in the frontal-occipital axis, but no bimodality in frequency distribution. Furthermore, spindles paired to SOs (a minority in both stages) are shorter than non-paired spindles. We find that coordination between spindles and SO troughs is frequency but not amplitude selective; and differs between the two sleep stages. In S2, slow spindles precede SOs in frontal electrodes and fast spindles follow SOs in centro-posterior electrodes. Our clusters of spindle topography include a Frontal and a Posterior cluster. These clusters mirror the commonly considered slow-frontal and fast-posterior spindles, but contain less than half of all S2 spindles. Clustering identifies sub-types of spindle-SO complexes whose density is linked to memory performance. Conclusion Our study shows that specific sub-types of sleep oscillations, defined by their topography, support the coordination between spindles and SOs which could mediate cortical-subcortical dialogue during sleep. Support This work was supported by NIH grant (R01 AG046646) to Sara C. Mednick

Stanley Nattel.

Stanley Nattel.

GPMVs in variable physiological conditions: could they be used for therapy delivery?

BackgroundCell based carriers are increasingly recognized as a good system for cargo delivery to cells. One of the reasons is their biocompatibility and low toxicity compared to artificial systems. Giant plasma membrane vesicles (GPMV) derive from the cell plasma membrane. Thus they offer the closest approximation to it, which makes them good candidates for potential drug delivery systems. To evaluate the applicability of GPMVs as carriers, we analyzed their basic biophysical properties to test their robustness in the face of changeable physiological conditions, as well as their ability to translocate across the membrane into cells.ResultsGPMVs formed from human umbilical vein endothelial cells (HUVEC) sustain a drastic osmotic challenge (50–500 mOsmoL/kg) unlike giant unilamelar vesicles (GUVs). In hyper-osmotic solutions the average volume decreases and membrane invaginations form, while in the hypo-osmolar buffer the volume of GPMVs increases and these changes were not reversible. The membranes of flaccid GPMVs started to wrinkle unevenly giving rise to buds after exposure to lipopolysaccharide (LPS). The shape changes in GUVs are reversible in contrast to GPMVs after LPS removal. GPMVs exposed to fluorescent LPS exhibited a signal that remained visible in some GPMVs even after LPS removal, which was never the case with GUVs. Calcein penetrated both into GUVs and GPMVs, however after the removal from the bulk solution some of the GPMVs still exhibited very bright signal, while in GUVs only a weak fluorescent signal was detected. We could also see that practically all GPMVs incorporated dextran initially, but after the dextran solution was changed with the initial non-fluorescent solution it remained only in 20% of them. The majority of HUVEC cells displayed a fluorescent signal after the incubation with GPMVs that contained fluorescently labeled dextran.ConclusionOur findings indicate that GPMVs behave quite differently from artificially made giant phospholipid vesicles and the changes induced by the different treatments we subjected them to are not reversible. We also demonstrate that different substances can be both loaded into them and delivered into cells, so GPMVs may be of potential use as cargo/therapy delivery systems.

Physiological and pharmacological properties of inhibitory postsynaptic currents mediated by α5β1γ2, α5β2γ2 and α5β3γ2 GABAA receptors

α5-containing GABAARs are potential therapeutic targets for clinical conditions including age-related dementia, stroke, schizophrenia, Down syndrome, anaesthetic-induced amnesia, anxiety and pain. α5-containing GABAARs are expressed in layer 5 cortical neurons and hippocampal pyramidal neurons where they mediate both tonic currents and slow inhibitory postsynaptic currents (IPSCs). A range of drugs has been developed to specifically modulate these receptors. The main α5-containing GABAARs that are likely to exist in vivo are the α5β1γ2, α5β2γ2 and α5β3γ2 isoforms. We currently have few clues as to how these isoforms are distributed between synaptic and extrasynaptic compartments or their relative roles in controlling neuronal excitability. Accordingly, the aim of this study was to define the basic biophysical and pharmacological properties of IPSCs mediated by the three isoforms in a hippocampal neuron-HEK293 cell co-culture assay. The IPSC decay time constants were slow (α5β1γ2L: 45 ms; α5β1γ2L: 80 ms; α5β3γ2L: 184 ms) and were largely dominated by the intrinsic channel deactivation rates. By comparing IPSC rise times, we inferred that α5β1γ2L GABAARs are located postsynaptically whereas the other two are predominantly perisynaptic. α5β3γ2L GABAARs alone mediated tonic currents. We quantified the effects of four α5-specific inverse agonists (TB-21007, MRK-016, α5IA and L-655708) on IPSCs mediated by the three isoforms. All compounds selectively inhibited IPSC amplitudes and accelerated IPSC decay rates, albeit with distinct isoform specificities. MRK-016 also significantly accelerated IPSC rise times. These results provide a reference for future studies seeking to identify and characterize the properties of IPSCs mediated by α5-containing GABAAR isoforms in neurons.

Assessment of the non-woven, goose down and duck down as thermally insulating materials for the clothing protecting against cold

PurposeThe purpose of this paper is to compare morphological and physical features of three kinds of materials intended for the insulating layer in the clothing protecting against cold – high-bulk non-woven, goose down (GD) and duck down (DD).Design/methodology/approachComparison of thermal performance of developed textile systems with the non-woven, GD and DD content was based on basic biophysical properties related to comfort sensations of the user such as thermal resistance, water vapor resistance and air permeability. In this study, light microscopy and scanning electron microscopy methods were employed to visualize the surface and internal structure of non-woven, GD and DD samples.FindingsThe paper indicates the advantages and disadvantages of each of selected insulating material. For the down samples, significantly higher thermal resistance in a dry state than for the non-woven samples can be achieved. Meanwhile, textile systems with the non-woven provide lower value of water vapor resistance. The selected textile systems for the research were characterized by a comparable air permeability.Originality/valueThis paper allows for an evaluation of high-bulk non-woven, DD and GD samples in terms of providing optimal thermal performance in clothing protecting against cold.

Single Molecule Characterisation of the Molecular Motor Myosin Vb

The myosin family of molecular motors comprises several different classes, each driving diverse fundamental processes ranging from muscle contraction, to intracellular trafficking, and cell motility. In particular, myosin V class includes three isoforms among which myosin Va, which has been implicated in several forms of intracellular transport, has been thoroughly investigated during the last 15 years. Molecular and cellular studies elucidated most of the molecular mechanisms at the basis of myosin Va processivity and its regulation by force and calcium. Although myosin Vb is ubiquitously expressed and involved in fundamental cellular processes such as mobilization of recycling endosomes and plasma membrane and receptor recycling, its basic biophysical properties have been poorly investigated and no single molecule study has been reported in the literature so far.Here, we developed single molecule manipulation and imaging methods to characterize the molecular mechanisms at the basis of myosin Vb functioning. Single recombinant myosin Vb motors were labelled with a quantum dot and observed with an in vitro motility assay under fluorescence microscopy. We found that myosin Vb is an efficient processive motor that walks in 36 nm steps at about 700 nm/s velocity, covering a distance of about 1 μm before dissociating from actin. By using ultra-fast force-clamp optical tweezers, we found that both myosin Vb velocity and run length largely decrease under opposing pN range loads and the motor stalls at about 2.5 pN. The observed dependence of myosin Vb processivity on load is accounted for by the ratio between forward and backward steps. We finally found that, similarly to myosin Va, calcium inhibits myosin Vb processivity by inducing calmodulin dissociation, which is partially rescued in the presence of excess calmodulin.

Role of volume-regulated and calcium-activated anion channels in cell volume homeostasis, cancer and drug resistance

Volume-regulated channels for anions (VRAC) / organic osmolytes (VSOAC) play essential roles in cell volume regulation and other cellular functions, e.g. proliferation, cell migration and apoptosis. LRRC8A, which belongs to the leucine rich-repeat containing protein family, was recently shown to be an essential component of both VRAC and VSOAC. Reduced VRAC and VSOAC activities are seen in drug resistant cancer cells. ANO1 is a calcium-activated chloride channel expressed on the plasma membrane of e.g., secretory epithelia. ANO1 is amplified and highly expressed in a large number of carcinomas. The gene, encoding for ANO1, maps to a region on chromosome 11 (11q13) that is frequently amplified in cancer cells. Knockdown of ANO1 impairs cell proliferation and cell migration in several cancer cells. Below we summarize the basic biophysical properties of VRAC, VSOAC and ANO1 and their most important cellular functions as well as their role in cancer and drug resistance.

The identification of a volume-regulated anion channel: an amazing Odyssey.

The volume-regulated anion channel (VRAC) plays a pivotal role in cell volume regulation in essentially all cell types studied. Additionally, VRAC appears to contribute importantly to a wide range of other cellular functions and pathological events, including cell motility, cell proliferation, apoptosis and excitotoxic glutamate release in stroke. Although biophysically, pharmacologically and functionally thoroughly described, VRAC has until very recently remained a genetic orphan. The search for the molecular identity of VRAC has been long and has yielded multiple potential candidates, all of which eventually turned out to have properties not fully compatible with those of VRAC. Recently, two groups have independently identified the protein leucine-rich repeats containing 8A (LRRC8A), belonging to family of proteins (LRRC8A-E) distantly related to pannexins, as the likely pore-forming subunit of VRAC. In this brief review, we summarize the history of the discovery of VRAC, outline its basic biophysical and pharmacological properties, link these to several cellular functions in which VRAC appears to play important roles, and sketch the amazing search for the molecular identity of this channel. Finally, we describe properties of the LRRC8 proteins, highlight some features of the LRRC8A knockout mouse and discuss the impact of the discovery of LRRC8 as VRAC on future research.

Gβγ Subunits Modulation of Kv7.4 Channels Expressed in HEK293 Cells at the Single Channel Level

Recently it was shown that KCNQ4-encoded voltage-dependent potassium channels (Kv7.4) play an important role in the regulation of resting vascular tone and in response to endogenous vasodilators (Hypertension, 59:877–884, 2012). However there is lack of information about Kv7.4 basic biophysical properties on the single channel level and about their regulatory mechanisms. Therefore, using standard cell-attached and inside-out configurations of the patch-clamp technique we investigated the single channel properties of Kv7.4 in HEK293 cells stably expressing the channel. We found that NPo of Kv7.4 was 0.61±0.05 (n=9, mean±SEM) in the cell-attached configuration at −50 mV (voltage applied to the patch pipette), whereas in the inside-out configuration at 0 mV NPo of Kv7.4 was 0.04±0.01 (n=9). Based on the inside-out experiments, single channel conductance of Kv7.4 was calculated as 2.31 pS. Linopirdine (10 µM), the pan-Kv7 blocker, abolished the single channel activity in both cell-attached and inside-out configuration when added to the patch pipette, but did not produce significant changes when applied to the bath solution. Bath application of PI(4,5)P2 (1 µM) to the inside-out patches or application of active Gβγ subunits (50 ng/ml) resulted in significant increase of NPo of Kv7.4 correspondingly to 0.27±0.09 (n=3) or to 0.34±0.06 (n=6) without affecting channel conductance (p<0.001 in both cases, determined by 2-way ANOVA). Moreover, bath application of gallein (100 µM), an inhibitor of Gβγ activity, reduced significantly NPo of Kv7.4 channels to 0.03±0.01 (n=3, p<0.001) in the cell-attached experiments. Apart from revealing the basic biophysical properties of single Kv7.4 channels our results show that, similar to PI(4,5)P2, Gβγ subunits play a critical role in the basal activity of voltage-dependent Kv7.4 channels.Supported by the British Heart Foundation (PG/12/63/29824) and by the Medical Research Council (MR/K019074/1).

DNA nanostructures interacting with lipid bilayer membranes.

CONSPECTUS: DNA has been previously shown to be useful as a material for the fabrication of static nanoscale objects, and also for the realization of dynamic molecular devices and machines. In many cases, nucleic acid assemblies directly mimic biological structures, for example, cytoskeletal filaments, enzyme scaffolds, or molecular motors, and many of the applications envisioned for such structures involve the study or imitation of biological processes, and even the interaction with living cells and organisms. An essential feature of biological systems is their elaborate structural organization and compartmentalization, and this most often involves membranous structures that are formed by dynamic assemblies of lipid molecules. Imitation of or interaction with biological systems using the tools of DNA nanotechnology thus ultimately and necessarily also involves interactions with lipid membrane structures, and thus the creation of DNA-lipid hybrid assemblies. Due to their differing chemical nature, however, highly charged nucleic acids and amphiphilic lipids do not seem the best match for the construction of such systems, and in fact they are rarely found in nature. In recent years, however, a large variety of lipid-interacting DNA conjugates were developed, which are now increasingly being applied also for the realization of DNA nanostructures interacting with lipid bilayer membranes. In this Account, we will present the current state of this emerging class of nanosystems. After a brief overview of the basic biophysical and biochemical properties of lipids and lipid bilayer membranes, we will discuss how DNA molecules can interact with lipid membranes through electrostatic interactions or via covalent modification with hydrophobic moieties. We will then show how such DNA-lipid interactions have been utilized for the realization of DNA nanostructures attached to or embedded within lipid bilayer membranes. Under certain conditions, DNA nanostructures remain mobile on membranes and can dynamically associate into higher order complexes. Hydrophobic modification of DNA nanostructures can further result in intra- or intermolecular aggregation, which can also be utilized as a structural switching mechanism. Appropriate design and chemical modification even allows insertion of DNA nanostructures into lipid bilayer membranes, resulting in artificial ion channel mimics made from DNA. Interactions of DNA nanodevices with living cells also involve interactions with membrane structures. DNA-based nanostructures can be directed to cell surfaces via antibody-antigen interactions, and their cellular uptake can be stimulated by modification with appropriate receptor ligands. In the future, membrane-embedded DNA nanostructures are expected to find application in diverse areas ranging from basic biological research over nanotechnology to synthetic biology.

Physical Biology in Cancer. 2. The physical biology of circulating tumor cells

The identification, isolation, and characterization of circulating tumor cells (CTCs) promises to enhance our understanding of the evolution of cancer in humans. CTCs provide a window into the hematogenous, or "fluid phase," of cancer, underlying the metastatic transition in which a locally contained tumor spreads to other locations in the body through the bloodstream. With the development of sensitive and specific CTC identification and isolation methodologies, the role of CTCs in clinical diagnostics, disease surveillance, and the physical basis of metastasis continues to be established. This review focuses on the quantification of the basic biophysical properties of CTCs and the use of these metrics to understand the hematogenous dissemination of these enigmatic cells.

Biological Diversity Mapping Comes of Age

Over the past 60 years, Earth observing has evolved from aerial photographic studies to high-tech airborne 3-D imaging and global satellite-based monitoring. These technological advances have been driven by an increasing call for quantitative and information-rich data on changes in Earth properties and processes. Within the biospheric remote sensing arena, focus has mostly been placed on changes in land cover and land use, ecological disturbance including fire, and basic biophysical properties such as vegetation light absorption and greenness. In recent years, however, interest has rapidly increased in the area of biological diversity monitoring. This special issue of Remote Sensing [1] captures some of the latest thinking on how both traditional and newer mapping technologies can contribute to biodiversity monitoring and analysis. [...]

Chemical composition of human and canine fascia lata.

The fascial system is an integral part of the musculoskeletal system. It is a three-dimensional network of connective tissue spreading ubiquitously throughout the body, surrounding muscles, bones, internal organs, nerves, vessels, and other structures. The basic biophysical properties of the fascial system are determined by its structure and chemical composition. This study aimed to determine the elemental composition of pathologically unchanged fascia lata of the thigh, collected during autopsies on humans and dogs. The wide spectrum of elements analysed included both macro and micro elements. The analyses were conducted using scanning electron microscopy with X-ray microanalysis (SEM-EDS). Concentrations of the following macro and micro elements were determined: C, N, O, Na, Mg, Al, Si, P, S, Cl, K, Ca, Ti, Fe Co, Ni, Cu, and Zn. The obtained results showed significant differences between human and canine fascia lata regarding the content of most of the examined elements (p < 0.05), except for N. These data may in future provide a starting point for the establishment of reference values for the content of various elements in normal fascial tissue and may also serve to verify the usefulness of experimental animal material as a substitute for human tissue.

The expanding view of protein–protein interactions: complexes involving intrinsically disordered proteins

A frequently neglected aspect of protein–protein interactions is flexibility. Small-scale fluctuations are present even in globular proteins, and alternative conformations can have a significant influence on the binding process. However, flexibility becomes highly prominent in complexes involving intrinsically disordered proteins. The importance of disordered regions in protein interactions has been recognized only relatively recently. In this survey we examine the basic properties of the complexes of disordered and ordered proteins from three different directions. The comparison of the interface properties shows that although disordered proteins can also adopt well-defined conformations in their bound form, their inherently dynamic nature is cast into their complexes. Furthermore, an overview of prediction methods indicates that disordered proteins as well as their binding regions can be recognized from the amino acid sequence by capturing the basic biophysical properties of these segments. Finally, we propose the generalization of the ‘energy landscape model’ for the description of complex formation that can help to put the various types of protein associations on a common ground.