Biology subjects

Thirumalai, D.

Publications and source records attributed to Thirumalai, D..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

Moderate activity of RNA chaperone maximizes the yield of self-spliced pre-RNA in vivo

CYT-19 is a DEAD-box protein whose ATP-dependent helicase activity facilitates the folding of group I introns in precursor RNA (pre-RNA) of Neurospora crassa. In the process they consume a substantial amount of ATP. While much of the mechanistic insights into CYT-19 activity has been gained through the studies on the folding of Tetrahymena group I intron ribozyme, the more biologically relevant issue, namely the effect of CYT-19 on the self-splicing of pre-RNA, remains largely unexplored. Here, we employ a kinetic network model, based on the generalized iterative annealing mechanism, to investigate the relation between CYT-19 activity, rate of ribozyme folding, and the kinetics of the self-splicing reaction. The network rate parameters are extracted by analyzing the recent biochemical data for CYT-19-facilitated folding of T. ribozyme. We then build extended models to explore the metabolism of pre-RNA. We show that the timescales of chaperone-mediated folding of group I ribozyme and self-splicing reaction compete with each other. As a consequence, in order to maximize the self-splicing yield of group I introns in pre-RNA, the chaperone activity must be sufficiently large to unfold the misfolded structures, but not too large to unfold the native structures prior to the self-splicing event. We discover that despite the promiscuous action on structured RNAs, the helicase activity of CYT-19 on group I ribozyme gives rise to self-splicing yields that are close to the maximum. Significance StatementIn cells, RNA chaperones assist misfolding-prone ribozymes to fold correctly to carry out its biological function. CYT-19 is an ATP-consuming RNA chaperone that accelerates the production of native group I intron ribozyme by partially unfolding the kinetically trapped structures. Using the theoretical framework based on the iterative annealing mechanism, we establish that to maximize the processing of pre-RNA, an optimal balance should exist between the timescales of self-splicing activity and CYT-19-mediated production of the native ribozyme. Remarkably, the activity of CYT-19 has been optimized to unfold the misfolded structures but is not so high that it disrupts the native ribozyme, which ensures that the yield of the self-splicing reaction is maximized in a biologically relevant time scale.

biophysics↗

bioRxiv2022

TMAO Destabilizes RNA Secondary Structurevia Direct Hydrogen Bond Interactions

Trimethylamine N-oxide (TMAO) is an osmolyte that accumulates in cells in response to osmotic stress. TMAO stabilizes proteins by the entropic stabilization mechanism, which pictures TMAO as a nano-crowder that predominantly destabilizes the unfolded state. However, the mechanism of action of TMAO on RNA is much less understood. Here, we use all atom molecular dynamics simulations to investigate how TMAO interacts with a 12-nt RNA hairpin with a high melting temperature, and an 8-nt RNA hairpin, which has a relatively fluid native basin in the absence of TMAO. The use of the two hairpins with different free energy of stabilization allows us to probe the origin of the destabilization effect of TMAO on RNA molecules without the possibility of forming tertiary interactions. We generated multiple trajectories using allatom molecular dynamics (MD) simulations in explicit water by employing AMBER and CHARMM force fields, both in the absence and presence of TMAO. We observed qualitatively similar RNA-TMAO interaction profiles from the simulations using the two force fields. TMAO hydrogen bond interactions are largely depleted around the paired RNA bases and ribose sugars. In contrast, we show that the oxygen atom in TMAO, the hydrogen bond acceptor, preferentially interacts with the hydrogen bond donors in the solvent exposed bases, such as those in the stem-loop, the destabilized base stacks in the unfolded state, especially in the marginally stable 8-nt RNA hairpin. The predicted destabilization mechanism through TMAO-RNA hydrogen bond interactions could be tested using two-dimensional IR spectroscopy. Since TMAO does not significantly interact with the hydroxyl group of the ribose sugars, we predict that similar results must also hold for DNA.

biophysics↗

bioRxiv2022

Structural changes in chromosomes driven by multiple condensin motors during mitosis

We created a computational framework that describes the loop extrusion (LE) by multiple condensin I and II motors in order to investigate the changes in chromosome organization during mitosis. The theory accurately reproduces the experimentally measured contact probability profiles for the mitotic chromosomes in HeLa and DT40 cells. The rate of loop extrusion is smaller at the start of mitosis and increases as the cells approach the metaphase. The mean loop size generated by condensin II is about six times larger than the ones created by condensin I. The loops, which overlap with each other, are stapled to a central dynamically changing helical scaffold formed by the motors during the LE process. The structures of the mitotic chromosomes, using a data-driven method that uses the Hi-C contact map as input, are best described as random helix perversion (RHP) in which the handedness changes randomly along the scaffold. The extent of propagation in the RHP structures is less in HeLa cells than in the DT40 chromosomes.

biophysics↗

bioRxiv2022

Energy landscapes of Aβ monomers are sculpted in accordance with Ostwald's rule of stages

The transition from a disordered to an assembly-competent and sparsely populated monomeric state (N*) in amyloidogenic sequences is a crucial event in the aggregation cascade. Using a well-calibrated model for Intrinsically Disordered Proteins (IDPs), we show that the N* states, which bear considerable resemblance to distinct polymorphic fibril structures found in experiments, not only appear as excitations on the monomer free energy landscapes of A{beta}40 and A{beta}42 but also initiate the aggregation cascade. Interestingly, for A{beta}42, the transitions to the different N* states are in accord with Ostwalds rule of stages, with the least stable structures forming ahead of thermodynamically favored structures, which appear only on longer time-scales. Despite having similar topographies, the A{beta}40 and A{beta}42 monomer landscapes exhibit different extent of ruggedness, particularly in the vicinity of N* states, which we show have profound implications in dictating the intramolecular diffusion rates, and subsequent self-assembly into higher order structures. The network of connected kinetic states, which for A{beta}42 is considerably more complex than for A{beta}40, shows that the most favored dimerization routes proceed via the N* states. Direct transition between the disordered ground states within the monomer and dimer basins is less likely. The Ostwalds rule of stages holds widely, qualitatively explaining the unusual features in other fibril forming IDPs, such as Fused in Sarcoma (FUS). Similarly, the N* theory accounts for dimer formation in small disordered polyglutamine peptides, implicated in the Huntington disease. Graphical TOC Entry O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biophysics↗

bioRxiv2022

Molecular Mechanisms of Steric Pressure Generation and Membrane Remodeling by Intrinsically Disordered Proteins

Cellular membranes, which are densely crowded by proteins, take on an elaborate array of highly curved shapes. Steric pressure generated by protein crowding plays a significant role in shaping membrane surfaces. It is increasingly clear that many proteins involved in membrane remodeling contain substantial regions of intrinsic disorder. These domains have large hydrodynamic radii, suggesting that they may contribute significantly to steric congestion on membrane surfaces. However, it has been unclear to what extent they are capable of generating steric pressure, owing to their conformational flexibility. To address this gap, we use a recently developed sensor based on Forster resonance energy transfer to measure steric pressure generated at membrane surfaces by the intrinsically disordered domain of the endocytic protein, AP180. We find that disordered domains generate substantial steric pressure that arises from both entropic and electrostatic components. Interestingly, this steric pressure is largely invariant with the molecular weight of the disordered domain, provided that coverage of the membrane surface is held constant. Moreover, equivalent levels of steric pressure result in equivalent degrees of membrane remodeling, regardless of protein molecular weight. This result, which is consistent with classical polymer scaling relationships for semi-dilute solutions, helps to explain the molecular and physical origins of steric pressure generation by intrinsically disordered domains. From a physiological perspective, these findings suggest that a broad range of membrane-associated disordered domains are likely to play a significant and previously unknown role in controlling membrane shape. SignificanceWith nearly half their surfaces covered by proteins, biological membranes are highly crowded. Rapid diffusion and collision of membrane-bound proteins generates substantial steric pressure that is capable of shaping membrane surfaces. Many proteins involved in membrane remodeling, are intrinsically disordered. Having large hydrodynamic radii, disordered domains could contribute substantially to membrane crowding. However, it is unclear to what extent they are capable of generating steric pressure, owing to their conformational flexibility. Toward resolving this uncertainty, we have measured steric pressure at membrane surfaces during dynamic membrane remodeling events. Our data indicate that disordered domains generate significant steric pressure through entropic and electrostatic mechanisms, suggesting that they may constitute a critical, yet previously neglected class of membrane remodeling proteins.

biophysics↗

bioRxiv2022

Transcription-induced active forces suppress chromatin motion by inducing a transient disorder-to-order transition

The organization of interphase chromosomes in a number of species is starting to emerge thanks to advances in a variety of experimental techniques. However, much less is known about the dynamics, especially in the functional states of chromatin. Some experiments have shown that the mobility of individual loci in human interphase chromosome decreases during transcription, and increases upon inhibiting transcription. This is a counter-intuitive finding because it is thought that the active mechanical force (F) on the order of ten pico-newtons, generated by RNA polymerase II (RNAPII) that is pre-sumably transmitted to the gene-rich region of the chromatin, would render it more open, thus enhancing the mobility. Inspired by these observations, we developed a minimal active copolymer model for interphase chromosomes to investigate how F affects the dynamical properties of chromatin. The movements of the loci in the gene-rich region are suppressed in an intermediate range of F, and are enhanced at small F values, which has also been observed in experiments. In the intermediate F, the bond length between consecutive loci increases, becoming commensurate with the distance at the minimum of the attractive interaction between non-bonded loci. This results in a transient disorder-to-order transition, leading to the decreased mobility during transcription. Strikingly, the F -dependent change in the locus dynamics preserves the organization of the chromosome at F = 0. Transient ordering of the loci, which is not found in the polymers with random epigenetic profiles, in the gene-rich region might be a plausible mechanism for nucleating a dynamic network involving transcription factors, RNAPII, and chromatin. Significance StatementIn order to explain a physically counter-intuitive experimental finding that chromatin mobility is reduced during transcription, we introduced a polymer model for interphase chromosome that accounts for RNA polymerase (RNAP) induced active force. The simulations show that, over a range of active forces, the mobility of the gene-rich loci is suppressed. Outside this range, chromosomes are compact and exhibit glass-like dynamics. Our study, which accounts for the experimental observations, leads to a novel and testable mechanism of how transcription could shape the coexistence of fluid- and solid-like properties within chromosomes.

biophysics↗

bioRxiv2022

A method to predict 3D structural ensembles of chromatins from pairwise distances: Applications to Interphase Chromosomes and Structural Variants

The principles that govern the organization of genomes, which are needed for a deeper understanding of how chromosomes are packaged and function in eukaryotic cells, could be deciphered if the three dimensional (3D) structures are known. Recently, single-cell imaging experiments have determined the 3D coordinates of a number of loci in a chromosome. Here, we introduce a computational method (Distance Matrix to Ensemble of Structures, DIMES), based on the maximum entropy principle, with experimental pair-wise distances between loci as constraints, to generate a unique ensemble of 3D chromatin structures. Using the ensemble of structures, we quantitatively account for the distribution of pair-wise distances, three-body co-localization and higher-order interactions. We demonstrate that the DIMES method can be applied to both small length-scale and chromosome-scale imaging data to quantify the extent of heterogeneity and fluctuations in the shapes on various length scales. We develop a perturbation method that is used in conjunction with DIMES to predict the changes in 3D structures from structural variations. Our method also reveals quantitative differences between the 3D structures inferred from Hi-C and the ones measured in imaging experiments. Finally, the physical interpretation of the parameters extracted from DIMES provides insights into the origin of phase separation between euchromatin and heterochromatin domains.

biophysics↗

bioRxiv2022

Universality of distribution of tumor mutation burden - a biomarker for the tumor evolution and disease risk

Cancers, resulting in uncontrolled cell proliferation, are driven by accumulation of somatic mutations. Genome-wide sequencing has produced a catalogue of millions of somatic mutations, which contain the evolutionary history of the cancers. However, the connection between the mutation accumulation and disease development and risks is poorly understood. Here, we analyzed more than 1,200,000 mutations from 5,000 cancer patients with whole-exome sequencing, and discovered two novel signatures for 16 cancer types in The Cancer Genome Atlas (TCGA) database. A clock-like mutational process, a strong correlation between Tumor Mutation Burden (TMB) and the Patient Age at Diagnosis (PAD), is observed for cancers with low TMB (mean value less than 3 mutations per million base pairs) but is absent in cancers with high TMB. We also validate this finding using whole-genome sequencing data from more than 2,000 patients for 24 cancer types. Surprisingly, we discovered that the distribution of TMB are universal. At low TMB it exhibits a Gaussian distribution and transitions to a power law at hight TMB. The differences in cancer risk between the sexes are also mainly driven by the disparity in mutation burden. The TMB variations, imprinted at the chromosome level, also reflect accumulation of mutation clusters within small chromosome segments in high TMB cancers. By analyzing the characteristics of mutations based on multi-region sequencing, we found that a combination of TMB and intratumor heterogeneity could be a potential biomarker for predicting the patient survival and response to treatment.

cancer biology↗

bioRxiv2022

Statistical Mechanical theory for spatio-temporal evolution of Intra-tumor heterogeneity in cancers: Analysis of Multiregion sequencing data

Variations in characteristics from one region (sub-population) to another is commonly observed in complex systems, such as glasses and a collection of cells. Such variations are manifestations of heterogeneity, whose spatial and temporal behavior is hard to describe theoretically. In the context of cancer, intra-tumor heterogeneity (ITH), characterized by cells with genetic and phenotypic variability that co-exist within a single tumor, is often the cause of ineffective therapy and recurrence of cancer. Next-generation sequencing, obtained by sampling multiple regions of a single tumor (multi-region sequencing, M-Seq), has vividly demonstrated the pervasive nature of ITH, raising the need for a theory that accounts for evolution of tumor heterogeneity. Here, we develop a statistical mechanical theory to quantify ITH, using the Hamming distance, between genetic mutations in distinct regions within a single tumor. An analytic expression for ITH, expressed in terms of cell division probability () and mutation probability (p), is validated using cellular-automaton type simulations. Application of the theory successfully captures ITH extracted from M-seq data in patients with exogenous cancers (melanoma and lung). The theory, based on punctuated evolution at the early stages of the tumor followed by neutral evolution, is accurate provided the spatial variation in the tumor mutation burden is not large. We show that there are substantial variations in ITH in distinct regions of a single solid tumor, which supports the notion that distinct subclones could co-exist. The simulations show that there are substantial variations in the sub-populations, with the ITH increasing as the distance between the regions increases. The analytical and simulation framework developed here could be used in the quantitative analyses of the experimental (M-Seq) data. More broadly, our theory is likely to be useful in analyzing dynamic heterogeneity in complex systems such as super-cooled liquids.

biophysics↗

bioRxiv2022

Tracer particles sense local stresses in an evolving multicellular spheroid without affecting the anomalous dynamics of the cancer cells

Measurements of local stresses on the cancer cells (CCs), inferred by embedding inert compressible tracer particles (TPs) in a growing multicellular spheroid (MCS), show that pressure decreases monotonically as the distance from the core of the MCS increases. How faithfully do the TPs report the local stresses in the CCs is an important question because pressure buildup in the MCS is dynamically generated due to CC division, which implies that the CC dynamics should be minimally altered by the TPs. Here using theory and simulations, we show that although the TP dynamics is unusual, exhibiting sub-diffusive behavior on times less than the CC division times and hyper-diffusive dynamics on in the long-time limit, they do not affect the long-time CC dynamics or the local CC stress distributions. The CC pressure profile within the MCS, which decays from a high value at the core to the periphery, is almost identical with and without the TPs. That the TPs have insignificant effect on the local stresses in the MCS implies that they are reliable reporters of the CC microenvironment.

biophysics↗

bioRxiv2022

Differences in Ion-RNA binding modes due to charge density variations explain the stability of RNA in monovalent salts

The stability of RNA increases as the charge density of the alkali metal cations increases. The molecular mechanism for the stability does not exist. To fill this gap, we performed all-atom molecular dynamics (MD) pulling simulations to dissect the microscopic origin of this phenomenon. We first established that the free energy landscape obtained in the simulations is in excellent agreement with the single-molecule optical tweezer experiments. The origin of the stronger stability in Na+ compared to K+ is found to be due to the differences in the charge-density related binding modes. The smaller hydrated Na+ ion preferentially binds to the highly charged phosphates. In contrast, the larger K+ ions interact with the major grooves. As a result, the electrostatic repulsion between the phosphate groups is reduced more effectively by Na+ ions. Because the proposed mechanism is generic, we predict that the same conclusions are valid for divalent alkaline earth metal cations.

biophysics↗

bioRxiv2022

Mechanical feedback controls the emergence of dynamical memory in growing tissue monolayers

The growth of a tissue, which depends on cell-cell interactions and biologically relevant process such as cell division and apoptosis, is regulated by a mechanical feedback mechanism. We account for these effects in a minimal two-dimensional model in order to investigate the consequences of mechanical feedback, which is controlled by a critical pressure, pc. A cell can only grow and divide if the pressure it experiences, due to interaction with its neighbors, is less than pc. Because temperature is an irrelevant variable in the model, the cell dynamics is driven by self-generated active forces (SGAFs) that are created by cell division. It is shown that even in the absence of intercellular interactions, cells undergo diffusive behavior. The SGAF-driven diffusion is indistinguishable from the well-known dynamics of a free Brownian particle at a fixed finite temperature. When intercellular interactions are taken into account, we find persistent temporal correlations in the force-force autocorrelation function (FAF) that extends over timescale of several cell division times. The time-dependence of the FAF reveals memory effects, which increases as pc increases. The observed non-Markovian effects emerge due to the interplay of cell division and mechanical feedback, and is inherently a non-equilibrium phenomenon.

biophysics↗

bioRxiv2022

Cooperativity and folding kinetics in a multi-domain protein with interwoven chain topology

Although a large percentage of eukaryotic proteomes consist of proteins with multiple domains, not much is known about their assembly mechanism, especially those with complicated native state architectures. Some have complex topology in which the structural elements along the sequence are interwoven in such a manner that the domains cannot be separated by cutting at any location along the sequence. We refer to such proteins as Multiply connected Multidomain Proteins (MMPs). The phoshotransferase enzyme Adenylate Kinase (ADK) with three domains (NMP, LID, and CORE), the subject of this study, is an example of MMP. We devised a coarse-grained model to simulate ADK folding initiated by changing either the temperature or guanidinium chloride (GdmCl) concentration. The simulations reproduce the experimentally measured melting temperatures that are associated with two equilibrium transitions, FRET efficiency as a function of GdmCl concentration, and the global folding times nearly quantitatively. Although the NMP domain orders independently, cooperative interactions between the LID and the CORE domains are required for complete assembly of the enzyme. The kinetic simulations show that on the collapse time scale, but less than the global folding time, multiple interconnected metastable states are populated, attesting to the folding heterogeneity. The network connectivity between distinct states shows that the CORE domain folds only after the NMP and LID domains are formed, reflecting the interwoven nature of the chain topology. We propose that the rules for MMP folding must also hold for the folding of RNA enzymes.

biophysics↗

bioRxiv2021

Information flow, Gating, and Energetics in dimeric molecular motors

Molecular motors belonging to the kinesin and myosin super family hydrolyze ATP by cycling through a sequence of chemical states. These cytoplasmic motors are dimers made up of two linked identical monomeric globular proteins. Fueled by the free energy generated by ATP hydrolysis, the motors walk on polar tracks (microtubule or filamentous actin) processively, which means that only one head detaches and executes a mechanical step while the other stays bound to the track. Thus, the one motor head must regulate chemical state of the other, referred to as "gating", a concept that is not fully understood. Inspired by experiments, showing that only a fraction of the energy from ATP hydrolysis is used to advance the kinesin motors against load, we demonstrate that additional energy is used for coordinating the chemical cycles of the two heads in the dimer - a feature that characterizes gating. To this end, we develop a general framework based on information theory and stochastic thermodynamics, and establish that gating could be quantified in terms of information flow between the motor heads. Applications of the theory to kinesin-1 and Myosin V show that information flow occurs, with positive cooperativity, at external resistive loads that are less than a critical value, Fc. When force exceeds Fc, effective information flow ceases. Interestingly, Fc, which is independent of the input energy generated through ATP hydrolysis, coincides with force at which the probability of backward steps starts to increase. Our findings suggest that transport efficiency is optimal only at forces less than Fc, which implies that these motors must operate at low loads under in vivo conditions.

biophysics↗

bioRxiv2021

Adhesion strength between cells regulate non-monotonic growth by a biomechanical feedback mechanism

We probe the interplay between intercellular interactions and pressure fluctuations associated with single cells in regulating cell proliferation using simulations of a minimal model for three-dimensional multicellular spheroid (MCS) growth. The emergent spatial variations in the cell division rate, that depends on the location of the cells within the MCS, is regulated by intercellular adhesion strength (fad). This in turn results in non-monotonic proliferation of cells in the MCS with varying adhesion strength, which accords well with experimental results. A biomechanical feedback mechanism coupling the fad and cell-dependent pressure fluctuations relative to a threshold value (pc) determines the onset of a dormant phase, and explains the non-monotonic proliferation response. Increasing fad from low values enhances cell proliferation because pressure on individual cells is smaller compared to pc. In contrast, at high fad, cells readily become dormant and cannot rearrange effectively, leading to arrested cell proliferation. Our work, which shows that proliferation is regulated by pressure-adhesion feedback loop, may be a general feature of tumor growth.

biophysics↗

bioRxiv2021

Plus and minus ends of microtubule respond asymmetrically to kinesin binding by a long range directionally driven allosteric mechanism

Many members in the kinesin superfamily walk predominantly towards the plus end of the micro-tubule (MT) in a hand-over-hand manner. Despite great progress in elucidating the mechanism of stepping kinetics, the origin of stepping directionality is not fully understood. To provide quantitative insights into this important issue, we represent the structures of conventional kinesin (Kin1), MT, and the Kin1-MT complex using the elastic network model, and calculate the residue-dependent responses to a local perturbation in these constructs. Fluctuations in the residues in the {beta} domain of the /{beta}-tubulin are distinct from the domain. Surprisingly, the Kin1-induced asymmetry, which is more pronounced in /{beta}-tubulin in the plus end of MT than in the minus end, propagates spatially across multiple /{beta}-tubulin dimers. Kin1 binding expands the MT lattice by mechanical stresses, resulting in a transition in the cleft of /{beta} tubulin dimer between a closed (CC for closed cleft) state (not poised for Kin1 to bind) to an open (OC for open cleft) binding competent state. The long-range asymmetric responses in the MT, leading to the creation of OC states with high probability in several /{beta} dimers on the plus end of the bound Kin1, is needed for the motor to take multiple steps towards the plus end of the MT. Reciprocally, kinesin binding to the MT stiffens the residues in the MT binding region, induces correlations between switches I and II in the motor, and enhances fluctuations in ADP and the residues in the binding pocket. Our findings explain both the directionality of stepping and MT effects on a key step in the catalytic cycle of Kin1.

biophysics↗

bioRxiv2021

Temperature and Guanidine Hydrochloride effects on the folding thermodynamics of WW domain and variants

We used simulations based on an all atom Go model to calculate the folding temperatures (Tfs) and free energies ({Delta}Gs) of two variants of the WW domain, which is a small all {beta}-sheet protein. The results, without adjusting any parameter, are in good agreement with experiments, thus validating the simulations. We then used the Molecular Transfer Model to predict the changes in the {Delta}G and Tfs as guanidine hydrochloride concentration is varied. The predictions can be readily tested in experiments.

biophysics↗

bioRxiv2021

Sequence determines the switch in the fibril forming regions in the low complexity FUS protein and its variants

Residues spanning distinct regions of the low-complexity domain of the RNA-binding protein, Fused in Sarcoma (FUS-LC), form fibril structures with different core morphologies. NMR experiments show that the 214 residue FUS-LC forms a fibril with an S-bend (core-1, residues 39-95), while the rest of the protein is disordered. In contrast, the fibrils of the C-terminal variant (FUS-LC-C; residues 111-214) has a U-bend topology (core-2, residues 112-150). Absence of the U-bend in FUS-LC implies that the two fibril cores do not coexist. Computer simulations show that these perplexing findings could be understood in terms of the population of sparsely-populated fibril-like excited states in the monomer. The propensity to form core-1 is higher compared to core-2. We predict that core-2 forms only in truncated variants that do not contain the core-1 sequence. At the monomer level, sequence-dependent enthalpic effects determine the relative stabilities of the core-1 and core-2 topologies. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/451535v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@f81959org.highwire.dtl.DTLVardef@4859f3org.highwire.dtl.DTLVardef@96aecorg.highwire.dtl.DTLVardef@4ab94e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗