SEARCH · Search bioRxiv

Results for “biophysics”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 307 records · Page 17Linked to original sources

bioRxiv2020

Label-free Isolation and Single Cell Biophysical Phenotyping Analysis of Primary Cardiomyocytes using Inertial Microfluidics

To advance our understanding of cardiomyocyte identity and function, we need appropriate tools to isolate pure primary cardiomyocytes. We have developed a label-free method to purify viable cardiomyocytes from mouse neonatal hearts using a simple inertial microfluidics biochip. Cardiomyocytes were sorted from neonatal hearts and isolated to >90% purity and their physico-mechanical properties were evaluated using real time deformability cytometry. Purified cardiomyocytes were viable and retained their identity and function as depicted by expression of cardiac specific markers and contractility. Furthermore, we showed that cardiomyocytes have a distinct physico-mechanical phenotype that could be used as an intrinsic biophysical marker to distinguish these cells from other cell types within the heart. Taken together, this cardiomyocyte isolation and phenotyping method could serve as a valuable tool to progress our understanding of cardiomyocyte identity and function, which will ultimately benefit many diagnostic development and cardiac treatment studies.

bioengineering↗

bioRxiv2020

Spatial information transfer in hippocampal place cells depends on trial-to-trial variability, symmetry of place-field firing and biophysical heterogeneities

The relationship between the feature-tuning curve and information transfer profile of individual neurons provides vital insights about neural encoding. However, the relationship between the spatial tuning curve and spatial information transfer of hippocampal place cells remains unexplored. Here, employing a stochastic search procedure spanning thousands of models, we arrived at 127 conductance-based place-cell models that exhibited signature electrophysiological characteristics and sharp spatial tuning, with parametric values that exhibited neither clustering nor strong pairwise correlations. We introduced trial-to-trial variability in responses and computed model tuning curves and information transfer profiles, using stimulus-specific (SSI) and mutual (MI) information metrics, across locations within the place field. We found spatial information transfer to be heterogeneous across models, but to reduce consistently with increasing degrees of variability. Importantly, whereas reliable low-variability responses implied that maximal information transfer occurred at high-slope regions of the tuning curve, increase in variability resulted in maximal transfer occurring at the peak-firing location in a subset of models. Moreover, experience-dependent asymmetry in place-field firing introduced asymmetries in the information transfer computed through MI, but not SSI, and the impact of activity-dependent variability on information transfer was minimal compared to activity-independent variability. Biophysically, we unveiled a many-to-one relationship between different ion channels and information transfer, and demonstrated critical roles for N-methyl-D-aspartate receptors, transient potassium and dendritic sodium channels in regulating information transfer. Our results emphasize the need to account for trial-to-trial variability, tuning-curve shape and biological heterogeneities while assessing information transfer, and demonstrate ion-channel degeneracy in the regulation of spatial information transfer.

neuroscience↗

bioRxiv2020

Towards Biophysically-Based Neuromorphic Computing at Scale: Markov Abstractions of Electrochemical Reaction-Diffusion in Synaptic Transmission

Progress in computational neuroscience towards understanding brain function is challenged both by the complexity of molecular-scale electrochemical interactions at the level of individual neurons and synapses and the dimensionality of network dynamics across the brain covering a vast range of spatial and temporal scales. Our work abstracts an existing highly detailed, biophysically realistic 3D reaction-diffusion model of a chemical synapse to a compact internal state space representation that maps onto parallel neuromorphic hardware for efficient emulation at a very large scale and offers near-equivalence in input-output dynamics while preserving biologically interpretable tunable parameters.

neuroscience↗

bioRxiv2020

Isolation and biophysical characterization of GSU0105, a triheme c-type cytochrome from Geobacter sulfurreducens

C-type cytochromes play an important role in respiration of dissimilatory metal-reducing bacteria. They form extended conduits for charge transfer between the cellular metabolism and external electron acceptors such as particles of iron oxide, metal ions, and humic substances. Out of more than a hundred c-type cytochromes in Geobacter sulfurreducens, only a small fraction has been previously characterized. Here we present our results on expression and biophysical characterization of GSU0105, a novel 3-heme cytochrome, important for Fe(III) respiration in G. sulfurreducens. We successfully cloned the gene and achieved ~3 mg/L of culture GSU0105 expression in E.coli. Despite a similar size (71 amino acids) and the same number of c-type hemes to the members of the cytochrome (cyt) c7 family, multiple sequence alignment suggests that GSU0105 does not belong to the cyt c7 family. UV-Vis spectroscopy revealed typical c-type cytochrome spectral features, including a weak iron-sulfur charge transfer band suggesting that at least one heme is ligated with a methionine residue. Far UV circular dichroism studies demonstrate approximately 35% content of -helices and {beta}-sheets, each, as well as thermal aggregation occurring above 60 {degrees}C. A combination of SAXS and analytical size exclusion chromatography data shows that GSU0105 is monomeric in solution. Finally, affinity pull-down assays demonstrate high binding affinity to PpcD and weaker binding to the other members of the cyt c7 family.

biochemistry↗

bioRxiv2020

Cloning, Expression and Biophysical Characterization of a Yeast-expressed Recombinant SARS-CoV-2 Receptor Binding Domain COVID-19 Vaccine Candidate

BackgroundCoronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has now spread worldwide to infect over 110 million people, with approximately 2.5 million reported deaths. A safe and effective vaccine remains urgently needed. MethodWe constructed three variants of the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (residues 331-549) in yeast as follows: (1) a "wild type" RBD (RBD219-WT), (2) a deglycosylated form (RBD219-N1) by deleting the first N-glycosylation site, and (3) a combined deglycosylated and cysteine-mutagenized form (C538A-mutated variant (RBD219-N1C1)). We compared the expression yields, biophysical characteristics, and functionality of the proteins produced from these constructs. Results and conclusionsThese three recombinant RBDs showed similar secondary and tertiary structure thermal stability and had the same affinity to their receptor, angiotensin-converting enzyme 2 (ACE-2), suggesting that the selected deletion or mutations did not cause any significant structural changes or alteration of function. However, RBD219-N1C1 had a higher fermentation yield, was easier to purify, was not hyperglycosylated, and had a lower tendency to form oligomers, and thus was selected for further vaccine development and evaluation. General significanceBy genetic modification, we were able to design a better-controlled and more stable vaccine candidate, which is an essential and important criterion for any process and manufacturing of biologics or drugs for human use.

immunology↗

bioRxiv2020

Mutational and biophysical robustness in a pre-stabilized monobody

The fibronectin type III (FN3) monobody domain is a promising non-antibody scaffold which features a less complex architecture than an antibody while maintaining analogous binding loops. We previously developed FN3Con, a hyper-stable monobody derivative with diagnostic and therapeutic potential. Pre-stabilization of the scaffold mitigates the stability-function trade-off commonly associated with evolving a protein domain towards biological activity. Here, we aimed to examine if the FN3Con monobody could take on antibody-like binding to therapeutic targets, while retaining its extreme stability. We targeted the first of the Adnectin derivative of monobodies to reach clinical trials, which was engineered by directed evolution for binding to the therapeutic target VEGFR2; however, this function was gained at the expense of large losses in thermostability and increased oligomerisation. In order to mitigate these losses, we grafted the binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the pre-stabilized FN3Con scaffold to produce a domain that successfully bound with high affinity to the therapeutic target VEGFR2. This FN3Con-anti-VEGFR2 construct also maintains high thermostability, including remarkable long-term stability, retaining binding activity after 2 years of storage at 36 {degrees}C. Further investigations into buffer excipients doubled the presence of monomeric monobody in accelerated stability trials. These data suggest that loop grafting onto a pre-stabilized scaffold is a viable strategy for the development of monobody domains with desirable biophysical characteristics, and is therefore well-suited to applications such as the evolution of multiple paratopes or shelf-stable diagnostics and therapeutics.

biochemistry↗

bioRxiv2020

Interactions between discrete events and continuous dynamics in the regulation of scallops valve opening: insights from a biophysical model

This study constitutes a first attempt to quantify processes that govern valve gape dynamics in bivalves. We elected to focus on the scallop, Pecten maximus, not only because of its economic importance but also because it has a complex behaviour and high sensitivity to stress, which can be inferred from valve gape dynamics. The adductor muscle is the primary organ implicated in valve movements. Scallops, as other bivalves, move their valves sharply to ensure basic physiological functions or to respond to stressing conditions; these sharp events can be perceived as discrete events within a continuous dynamic. A biophysical model, originally designed for human muscles, was first selected to simulate the adductor muscle contraction, countering the passive valve opening by the umbo ligament. However, to maintain the possibility of rapid valve movements, described as typical of bivalves behaviour, it was necessary to modify the model and propose an original formulation. The resulting hybrid modelling simulates how valve opening tends to converge continuously toward a stable steady-state angle, while being interspersed with discrete, sharp closing events, deviating values from this equilibrium. The parameters of the new model were estimated by optimization using Hall-Effect Sensor valvometry data recorded in controlled conditions. Equilibrium of the continuous regime (when fiber activation equals deactivation) was estimated for a gape angle close to ca. 15 degrees, which is ca. 45% of the maximum opening angle, hence implying a constant effort produced by the adductor muscle. The distribution of time intervals between two successive discrete events did not differ significantly from a random process, but the peak amplitudes deviated from randomness, suggesting they are regulated physiologically. These results suggest that discrete events interact with continuous dynamic regimes, regulating valve opening to minimize physiological efforts and conserve energy. However, because the overall physiological state of the scallop organism conditions the activity of the adductor muscle, a complete understanding of the physiology of bivalves will require linking a more comprehensive model of valve gape dynamics with experimental observations of physiological energy consumption under different conditions.

ecology↗

bioRxiv2021

Sodium channel Nav1.2-L1342P variant displaying complex biophysical properties renders hyperexcitability of cortical neurons derived fromhuman iPSCs

With the wide adoption of whole-exome sequencing in children having seizures, an increasing number of SCN2A variants has been revealed as possible genetic causes of epilepsy. Voltage-gated sodium channel Nav1.2, encoded by gene SCN2A, is strongly expressed in the pyramidal excitatory neurons and supports action potential firing. One recurrent SCN2A variant is L1342P, which was identified in multiple patients with early-onset encephalopathy and intractable seizures. Our biophysical analysis and computational modeling predicted gain-of-function features of this epilepsy-associated Nav1.2 variant. However, the mechanism underlying L1342P mediated seizures and the pharmacogenetics of this variant in human neurons remain unknown. To understand the core phenotypes of the L1342P variant in human neurons, we took advantage of a reference human induced pluripotent stem cell (hiPSC) line, in which L1342P was engineered by CRISPR/Cas9 mediated genome-editing. Using patch-clamping and micro-electrode array (MEA) recording, we found that the cortical neurons derived from hiPSCs carrying heterozygous L1342P variant presented significantly increased intrinsic excitability, higher sodium current density, and enhanced bursting and synchronous network firing, showing clear hyperexcitability phenotypes. Interestingly, the L1342P neuronal culture displayed a degree of resistance to the anti-seizure medication (phenytoin), which likely recapitulated aspects of clinical observation of patients carrying the L1342P variant. In contrast, phrixotoxin-3 (PTx3), a Nav1.2 isoform-specific blocker, was able to potently alleviate spontaneous and chemical-induced hyperexcitability of neurons carrying the L1342P variant. Our results reveal a possible pathogenic underpinning of Nav1.2-L1342P mediated epileptic seizures, and demonstrate the utility of genome-edited hiPSCs as an in vitro platform to advance personalized phenotyping and drug discovery.

physiology↗

bioRxiv2021

Multiparametric Profiling of Single Nanoscale Extracellular Vesicles by Combined Atomic Force and Fluorescence Microscopy: Correlation and Heterogeneity in Their Molecular and Biophysical Features

Being a key player in intercellular communications, nanoscale extracellular vesicles (EVs) offer unique opportunities for both diagnostics and therapeutics. However, their cellular origin and functional identity remain elusive due to the high heterogeneity in their molecular and physical features. Here, for the first time, multiple EV parameters involving membrane protein composition, size and mechanical properties on single small EVs (sEVs) are simultaneously studied by combined fluorescence and atomic force microscopy. Furthermore, their correlation and heterogeneity in different cellular sources are investigated. The study, performed on sEVs derived from Human Embryonic Kidney 293, Cord Blood Mesenchymal Stromal and Human Acute Monocytic Leukemia cell lines, identifies both common and cell line-specific sEV subpopulations bearing distinct distributions of the common tetraspanins (CD9, CD63 and CD81) and biophysical properties. Although the tetraspanin abundances of individual sEVs are independent of their sizes, the expression levels of CD9 and CD63 are strongly correlated. A sEV population co-expressing all the three tetraspanins in relatively high abundance, however, having on average diameters <100 nm and relatively low Young moduli, is also found in all cell lines. Such a multiparametric approach is expected to provide new insights regarding EV biology and functions, potentially deciphering unsolved questions in this field.

bioengineering↗

bioRxiv2021

Ultrafast large-scale simulations of biophysically realistic neurons using deep learning

Understanding the activity of the mammalian brain requires an integrative knowledge of circuits at distinct scales, ranging from ion channel gating to circuit connectomics. To understand how multiple parameters contribute synergistically to circuit behavior, neuronal computational models are regularly employed. However, traditional models containing anatomically and biophysically realistic neurons are computationally demanding even when scaled to model local circuits. To overcome this limitation, we trained several artificial neural net (ANN) architectures to model the activity of realistic, multicompartmental neurons. We identified a single ANN that accurately predicted both subthreshold and action potential firing and correctly generalized its responses to previously unobserved synaptic input. When scaled, processing times were orders of magnitude faster compared with traditional approaches, allowing for rapid parameter-space mapping in a circuit model of Rett syndrome. Thus, we present a novel ANN approach that allows for rapid, detailed network experiments using inexpensive, readily available computational resources.

neuroscience↗

bioRxiv2021

Biophysical properties governing septin assembly

Septin filaments build structures such as rings, lattices and gauzes that serve as platforms for localizing signaling and organizing cell membranes. How cells control the geometry of septin assemblies in poorly understood. We show here that septins are isodesmic polymers, in contrast to cooperative polymerization exhibited by F-actin and microtubules. We constructed a physical model to analyze and interpret how septin assemblies change in the presence of regulators in yeast extracts. Notably filaments differ in length and curvature in yeast extract compared to pure protein indicating cellular regulators modulate intrinsic biophysical features. Combining analysis of extracts from regulatory mutants with simulations, we found increased filament flexibility and reduced filament fragmentation promote assembly of septin rings, whereas reduced flexibility in crowded environments promotes local filament alignment. This work demonstrates how tuning of intrinsic features of septin filament assembly by regulatory proteins yields a diverse array of structures observed in cells.

cell biology↗

bioRxiv2021

Genetic, Genomic and Biophysical Case Study: Familial 46, XY Sex Reversal due to a Novel Inherited Mutation in Human Testis-Determining Factor SRY

ObjectiveTo describe the clinical, histopathological and molecular features of a novel inherited SRY allele (p.Met64Val; consensus box position 9) observed within an extensive pedigree: two 46, XY sisters with primary amenorrhea (16 and 14 years of age; probands P1 and P2), their normal father and brother, and an affected paternal XY grandaunt. Design, Setting, Participants and Outcome MeasurementsFollowing DNA sequencing to identify the SRY mutation, hormonal studies of the probands and histopathological examination of their gonads were performed. Functional consequences of p.Met64Val (and other mutations at this site) were also investigated. ResultsBreast development in P1 and P2 was Tanner II and IV, respectively. Mullerian structures and gonads resembling ovaries were found in each sister. Histopathology revealed gonadal dysgenesis, gonadablastoma and dysgerminoma. AMH/MIS, P450 SCC, and P450 aromatase were expressed in gonadoblastoma tissues. Genomic sequencing revealed no candidate mutations in other genes related to sexual differentiation. Variant p.Met64Val impaired Sox9 transcriptional activation associated with attenuated occupancy of the testis-specific enhancers Enh13 and TESCO. Biophysical studies indicated that the mutant HMG box retains specific DNA binding and DNA bending but with accelerated rate of protein-DNA dissociation. ConclusionThe partial biological activity of p.Met64Val SRY, maintained at the threshold of SRY function, rationalizes opposing paternal and proband phenotypes (the "the father-daughter paradox"). Steroidal biosynthesis by gonadoblastoma may delay genetic diagnosis and recognition of gonadal tumors. Quantitative assessment of inherited SRY alleles highlights the tenuous transcriptional threshold of developmental decision-making in the bipotential gonadal ridge.

developmental biology↗

bioRxiv2021

Changes of the biophysical properties of voltage-gated Na+ currents during maturation of the sodium-taste cells in rat fungiform papillae

Taste cells are a heterogeneous population of sensory receptors that undergoes a continuous turnover. Different chemo-sensitive cell lines rely on action potentials to release the neurotransmitter onto nerve endings. The electrical excitability is due to the presence of a tetrodotoxin-sensitive, voltage-gated sodium current (INa) similar to that found in neurons. Since the biophysical properties of INa change during neuronal development, we wondered whether the same also occurred in taste cells. Here, we used the patch-clamp recording technique to study INa in sodium sensing cells of rat fungiform papillae. We identified these cells by exploiting the known blocking effect of amiloride on ENaC, the sodium (salt) receptor. Then, based on the amplitude of INa and a morphological analysis, we subdivided sodium cells into two broad developmental groups, namely immature and mature cells. We found that: the voltage dependence of activation and inactivation changed in the transition from immature to mature state (depolarizing shift); the membrane capacitance significantly decreased in mature cells, enhancing the density of INa; a persistent sodium current, absent in immature cells, appeared in mature cells. mRNA expression analysis of the -subunits of voltage-gated sodium channels in fungiform taste buds supported the electrophysiological data. As a whole, our findings provide evidence for a noticeable change in membrane excitability during development, which is consistent with the key role played by electrical signaling in the release of neurotransmitter by mature sodium cells. Key Points SummaryO_LITaste cells are sensory receptors that undergo continuous turnover while they detect food chemicals and communicate with afferent nerve fibers. C_LIO_LIThe voltage-gated sodium current (INa) is a key ion current for generating action potentials in fully differentiated and chemo-sensitive taste cells, which use electrical signaling to release neurotransmitters. C_LIO_LIHere we report that in rat taste cells involved in salt detection, the properties of INa, such as voltage dependence of activation and inactivation, undergo significant changes during the transition from immature to mature state. C_LIO_LIOur results help understand how taste cells gain electrical excitability during turnover, a property critical to operate as chemical detectors that relay sensory information to nerve fibers. C_LI

neuroscience↗

bioRxiv2021

Composition and Biophysical Properties of the Sorting Platform Pods in the Shigella Type III Secretion System

Shigella flexneri, causative agent of bacillary dysentery (shigellosis), uses a type III secretion system (T3SS) as its primary virulence factor. The T3SS injectisome delivers effector proteins into host cells to promote entry and create an important intracellular niche. The injectisomes cytoplasmic sorting platform (SP) is a critical assembly that contributes to substrate selection and energizing secretion. The SP consists of oligomeric Spa33 "pods" that associate with the basal body via MxiK and connect to the Spa47 ATPase via MxiN. The pods contain heterotrimers of Spa33 with one full-length copy associated with two copies of a C-terminal domain (Spa33C). The structure of Spa33C is known, but the precise makeup and structure of the pods in situ remains elusive. We show here that recombinant wild-type Spa33 can be prepared as a heterotrimer that forms distinct stable complexes with MxiK and MxiN. In two-hybrid analyses, association of the Spa33 complex with these proteins occurs via the full-length Spa33 component. Furthermore, these complexes each have distinct biophysical properties. Based on these properties, new high-resolution cryo-electron tomography data and architectural similarities between the Spa33 and flagellar FliM-FliN complexes, we provide a preliminary model of the Spa33 heterotrimers within the SP pods. From these findings and evolving models of SP interfaces and dynamics in the Yersinia and Salmonella T3SS, we suggest a model for SP function in which two distinct complexes come together within the context of the SP to contribute to form the complete pod structures during the recruitment of T3SS secretion substrates.

microbiology↗

bioRxiv2021

Multispecies phase diagram reveals biophysical principles of bacterial biofilm architectures

Bacterial biofilms are among the most abundant multicellular structures on Earth and play essential roles in a wide range of ecological, medical, and industrial processes. However, general principles that govern the emergence of biofilm architecture across different species remain unknown. Here, we combine experiments, simulations and statistical analysis to identify shared biophysical mechanisms that determine biofilm architecture development at the single-cell level, for the species Vibrio cholerae, Escherichia coli, Salmonella enterica, and Pseudomonas aeruginosa. Our data-driven analysis reveals that despite the many molecular differences between these species, the biofilm architecture differences can be described by only two control parameters: cellular aspect ratio and cell density. Further experiments using single-species mutants for which the cell aspect ratio and the cell density are systematically varied, and mechanistic simulations, show that tuning these two control parameters reproduces biofilm architectures of different species. Altogether, our results show that early-stage biofilm architecture is determined by mechanical cell-cell interactions which are conserved across different species and, therefore, provide a unifying understanding of biofilm architecture development.

microbiology↗

bioRxiv2021

A Biophysical Counting Mechanism for Keeping Time

The ability to estimate and produce appropriately timed responses is central to many behaviors including speaking, dancing, and playing a musical instrument. A classical framework for estimating or producing a time interval is the pacemaker-accumulator model in which pulses of a pacemaker are counted and compared to a stored representation. However, the neural mechanisms for how these pulses are counted remains an open question. The presence of noise and stochasticity further complicate the picture. We present a biophysical model of how to keep count of a pacemaker in the presence of various forms of stochasticity using a system of bistable Wilson-Cowan units asymmetrically connected in a one-dimensional array; all units receive the same input pulses from a central clock but only one unit is active at any point in time. With each pulse from the clock, the position of the activated unit changes thereby encoding the total number of pulses emitted by the clock. This neural architecture maps the counting problem into the spatial domain, which in turn translates count to a time estimate. We further extend the model to a hierarchical structure to be able to robustly achieve higher counts.

neuroscience↗

bioRxiv2021

Highly similar sequence and structure yet different biophysical behaviour: A computational study of two triosephosphate isomerases

Homodimeric triosephosphate isomerases (TIM) from Trypanosoma cruzi (TcTIM) and Trypanosoma brucei (TbTIM) have a markedly similar amino acid sequences and three-dimensional structures. However, several of their biophysical parameters, such as their susceptibility to sulfhydryl agents and their reactivation speed after being denatured, have significant differences. The causes of these differences were explored with microsecond-scale molecular dynamics (MD) simulations of three different TIM proteins: TcTIM, TbTIM and a chimeric protein, Mut1. We examined their electrostatic interactions and explored the impact of simulation length on them. The same salt bridge between catalytic residues Lys 14 and Glu 98 was observed in all three proteins but key differences were found in other interactions that the catalytic amino acids form. In particular, a cation-{pi} interaction between catalytic amino acids Lys 14 and His 96, and both a salt bridge and a hydrogen bond between catalytic Glu168 and residue Arg100, were only observed in TcTIM. Furthermore, although TcTIM forms less hydrogen bonds than TbTIM and Mut1, its hydrogen bond network spans almost the entire protein, connecting the residues in both monomers. This work provides new insight on the mechanisms that give rise to the different behaviour of these proteins. The results also show the importance of long simulations.

biochemistry↗

bioRxiv2021

Biophysical Kv channel alterations dampen excitability of cortical PV interneurons and contribute to network hyperexcitability in early Alzheimer's

In Alzheimers disease (AD), a multitude of genetic risk factors and early biomarkers are known. Nevertheless, the causal factors responsible for initiating cognitive decline in AD remain controversial. Toxic plaques and tangles correlate with progressive neuropathology, yet disruptions in circuit activity emerge before their deposition in AD models and patients. Parvalbumin (PV) interneurons are potential candidates for dysregulating cortical excitability, as they display altered AP firing before neighboring excitatory neurons in prodromal AD. Here we report a novel mechanism responsible for PV hypoexcitability in young adult familial AD mice. We found that biophysical modulation of K+ channels, but not changes in mRNA expression, are responsible for dampened excitability. These K+ conductances could efficiently regulate near-threshold AP firing, resulting in gamma-frequency specific network hyperexcitability. Our findings suggest that posttranslational modulation of ion channels can reshape cortical network activity prior to changes in their gene expression in early AD.

neuroscience↗