Thomas Irving | Illinois Institute of Technology (original) (raw)

Papers by Thomas Irving

Biophysical Journal, 2014

Biophysical Journal, 2013

The asynchronous, indirect flight muscles (IFM) of Drosophila are characterized by a high passive... more The asynchronous, indirect flight muscles (IFM) of Drosophila are characterized by a high passive stiffness and exceptionally fast myosin kinetics, two attributes that enhance power output to sustain flight. Flightin is an IFM-specific, 20kDa myosin rod-binding protein required for normal thick filament stiffness, sarcomere integrity, and flight. Previously, we showed that a COOH-terminal truncation of flightin (fln DeltaC44) decreased myofilament lattice order and myosin kinetics, resulting in lower oscillatory power output and flightlessness. Here, we investigate the function of the flightin N-terminal 62 amino acids by creating transgenic Drosophila (fln DeltaN62) expressing a truncated flightin. fln DeltaN62 flies were flight impaired (flight index: 2.850.1 vs. 4.250.4 for fln DeltaN62 vs. fln þ rescued null control) despite having a normal wing-beat frequency (19554 vs. 19852 Hz for fln þ). Mechanical analysis of skinned IFM fibers showed that the flightin N-terminal truncation reduced passive, active, and rigor stiffness without affecting cross-bridge kinetics (frequency of maximum power: 20557 vs. 21757 Hz for fln þ). fln DeltaN62 fibers produced approximately half the isometric tension (passive: 0.950.1 vs. 1.750.3 kN/m 2 , active: 0.850.1 vs. 1.550.2 kN/m 2 , rigor: 1.150.2 vs. 3.150.4 kN/m 2) and maximum oscillatory power output (38.054.6 vs. 89.559.6 W/m 3) as fln þ fibers. Moreover, about 60% of the fln DeltaN62 fibers tore in rigor, demonstrating mechanical failure near isometric tension values that were sustained by fln þ fibers. Fourier transform analysis of crosssectional electron micrographs revealed that the flightin N-terminal truncation compromised myofilament lattice crystallinity and reduced inter-thick filament spacing by 10% (44.151.3 vs. 49.750.4 nm). These results indicate that the flightin N-terminal region enhances myofilament lattice order and mechanical integrity, which in turn is required for effective force transmission, normal oscillatory power output, and flight.

Methods in Enzymology, 2016

Chromatin accessibility is modulated by structural transitions that provide timely access to the ... more Chromatin accessibility is modulated by structural transitions that provide timely access to the genetic and epigenetic information during many essential nuclear processes. These transitions are orchestrated by regulatory proteins that coordinate intricate structural modifications and signalling pathways. In vitro reconstituted chromatin samples from defined components are instrumental in defining the mechanistic details of such processes. The bottleneck to appropriate in vitro analysis is the production of high quality, and quality-controlled, chromatin substrates. In this chapter we describe methods for in vitro chromatin reconstitution and quality control. We highlight the strengths and weaknesses of various approaches, and emphasize quality control steps that ensure reconstitution of a bona fide homogenous chromatin preparation. This is essential for optimal reproducibility and reliability of ensuing experiments using chromatin substrates.

International Journal of Molecular Sciences, Nov 30, 2019

Many biological processes are triggered or driven by mechanical forces in the cytoskeletal networ... more Many biological processes are triggered or driven by mechanical forces in the cytoskeletal network, but these transducing forces have rarely been assessed. Striated muscle, with its well-organized structure provides an opportunity to assess intracellular forces using small-angle X-ray fiber diffraction. We present a new methodology using Monte Carlo simulations of muscle contraction in an explicit 3D sarcomere lattice to predict the fiber deformations and length changes along thin filaments during contraction. Comparison of predicted diffraction patterns to experimental meridional X-ray reflection profiles allows assessment of the stepwise changes in intermonomer spacings and forces in the myofilaments within living muscle cells. These changes along the filament length reflect the effect of forces from randomly attached crossbridges. This approach enables correlation of the molecular events, such as the current number of attached crossbridges and the distributions of crossbridge forces to macroscopic measurements of force and length changes during muscle contraction. In addition, assessments of fluctuations in local forces in the myofilaments may reveal how variations in the filament forces acting on signaling proteins in the sarcomere M-bands and Z-discs modulate gene expression, protein synthesis and degradation, and as well to mechanisms of adaptation of muscle in response to changes in mechanical loading.

Archives of Biochemistry and Biophysics, Feb 1, 2015

The flight muscles (DLM 1) of the Hawkmoth, Manduca sexta are synchronous, requiring a neural spi... more The flight muscles (DLM 1) of the Hawkmoth, Manduca sexta are synchronous, requiring a neural spike for each contraction. Stress/strain curves of skinned DLM 1 showed hysteresis indicating the presence of titin-like elastic proteins. Projectin and kettin are titin-like proteins previously identified in Lethocerus and Drosophila flight muscles. Analysis of Manduca muscles with 1% SDS-agarose gels and western blots showed two bands near 1 MDa that cross-reacted with antibodies to Drosophila projectin. Antibodies to Drosophila kettin cross-reacted to bands at ~500 and ~700 kDa, but also to bands at ~1.6 and ~2.1 MDa, that had not been previously observed in insect flight muscles. Mass spectrometry identified the 2.1 MDa protein as a product of the Sallimus (sls) gene. Analysis of the gene sequence showed that all 4 putative Sallimus and kettin isoforms could be explained as products of alternative splicing of the single sls gene. Both projectin and Sallimus isoforms were expressed to higher levels in ventrally located DLM 1 subunits, primarily responsible for active work production, as compared to dorsally located subunits, which may act as damped springs. The different expression levels of the 2 projectin isoforms and 4 Sallimus/kettin isoforms may be adaptations to the specific requirements of individual muscle subunits.

Acta Crystallographica Section A, Aug 8, 1996

The Journal of General Physiology, Feb 21, 2019

bioRxiv (Cold Spring Harbor Laboratory), Apr 12, 2023

Rationale: Mavacamten is a novel, FDA-approved, small molecule therapeutic designed to regulate c... more Rationale: Mavacamten is a novel, FDA-approved, small molecule therapeutic designed to regulate cardiac function at the sarcomere level by selectively but reversibly inhibiting the enzymatic activity of myosin. It shifts myosin towards ordered off states close to the thick filament backbone. Objective: The purpose of this study is to investigate whether mavacamten permanently sequester these myosin heads in the off state(s) and can these heads be recruited in response to physiological stimuli when required to boost cardiac output. Methods and Results: We show that cardiac myosins stabilized in these off state(s) by mavacamten are recruitable by 1) Ca 2+ , 2) increased chronotropy (heart rate), 3) stretch, and 4) β adrenergic (β-AR) stimulation, all known physiological inotropic effectors. At the molecular level, we show that Ca 2+ increases myosin ATPase activity by shifting mavacamten-stabilized myosin heads from the inactive super-relaxed (SRX) state to the active disordered relaxed (DRX) state. At the myofilament level, both Ca 2+ and passive lengthening can shift mavacamtenordered off myosin heads from positions close to the thick filament backbone to disordered on states closer to the thin filaments. In isolated rat cardiomyocytes, increased stimulation rates enhanced shortening fraction in mavacamten-treated cells. This observation was confirmed in vivo in telemetered rats, where left-ventricular dP/dt max, an index of inotropy, increased with heart rate in mavacamten treated animals. Finally, we show that β-AR stimulation in vivo increases left-ventricular function and stroke volume in the setting of mavacamten. Conclusions: Our data demonstrate that the mavacamten-promoted off states of myosin in the thick filament are activable, thus leading to the preservation of the cardiac reserve. These results provide a potential mechanistic explanation, beyond mere LV outflow tract obstruction removal, for the clinical observation of increased peak oxygen uptake (pVO 2) with exercise in HCM patients receiving mavacamten. .

Acta Crystallographica, Jul 29, 2022

For many years, analysis of fiber diffraction patterns from muscle was a rate limiting step in th... more For many years, analysis of fiber diffraction patterns from muscle was a rate limiting step in the preparation of experimental results for publication. Most analysis procedures made extensive use of FIT2D supplemented by locally developed or commercial software tools which were not specifically designed for the task. Analyzing the data from a single experiment could take many weeks or months. To address this bottleneck, we have developed a comprehensive data reduction suite of programs which we call 'MuscleX". The software is open source under a modified BSD license. MacIntosh, Windows and linux installers permit easy deployment. The "Equator' routine can analyze the equatorial patterns from a complete experiment in a few hours without operator intervention, a process that used to take many months. The 'Quadrant-Fold" package averages the four quadrants of a diffraction pattern to improve signal to noise for more detailed analysis. It can also perform global background subtractions using either a "white top hat" real space digital filter algorithm, or a two-dimensional convex hull. It also includes radially symmetric, iterative smoothing and "roving window" background subtraction routines from the now defunct CCP software suite. Quadrant folding may be useful to remove the lines in the patterns created by the dead zones between modules in common pixel array detectors. Other components of the suite include "Projection Traces" and "Diffraction Centroids" that automatically analyze the intensity and spacings of prominent reflections on the meridian and layer lines from the large amount of diffraction images collected in time resolved experiments. Once the programs are set up, they can process an entire directory of data without operator intervention in a few hours. In this presentation we will discuss current capabilities and planned future developments and how the MuscleX package may be useful for all biological fiber diffraction experiments, not just muscle diffraction.

Biophysical Journal, 2014

Mammalian hearts express two myosin heavy chain (MHC) isoforms, which drive contractions with dif... more Mammalian hearts express two myosin heavy chain (MHC) isoforms, which drive contractions with different kinetics and power-generating ability. The expression of the isoform that is associated with more rapid contraction kinetics and greater power output, MHC-a, is down-regulated, with a concurrent increase in the relative amount of the slower isoform, MHC-b, during the progression to experimentally-induced or disease-related heart failure. This change in protein expression has been well studied in right and left ventricles in heart failure models and in humans with failure. Relatively little quantitative data exists regarding MHC isoform expression shifts in human failing atria. We previously reported significant increases in the relative amount of MHC-b in the human failing left atrium. The results of that study suggested that there might be a sex-related difference in the level of MHC-b in the left atrium, but the number of female subjects was insufficient for statistical analysis. The objective of this study was to test whether there is, in fact, a sex-related difference in the level of MHC-b in the right and left atria of humans with cardiomyopathy. The results indicate that significant differences exist in atrial MHC isoform expression between men and women who are in failure. The results unexpectedly also revealed a twofold greater amount of MHC-b in the non-failing left atrium of women, compared to men. The observed sexrelated differences in MHC isoform expression could impact ventricular diastolic filling during normal daily activities, as well as during physiologically stressful events.

Biophysical Journal

Additional information Versions of research works Versions of Record If this version is the versi... more Additional information Versions of research works Versions of Record If this version is the version of record, it is the same as the published version available on the publisher's web site. Cite as the published version.

Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Detai... more Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Detailed mechanism of action of these agents can help predict potential unwanted affects and identify patient populations that can benefit most from them. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. Using porcine cardiac tissue and myofibrils we demonstrate that Danicamtiv increases force and calcium sensitivity via increasing the number of myosin in the “on” state and slowing cross bridge turnover. Our detailed analysis shows that inhibition of ADP release results in decreased cross bridge turnover with cross bridges staying on longer and prolonging myofibril relaxation. Using a mouse model of genetic dilated cardiomyopathy, we demonstrated that Dan...

Biophysical Journal, 2018

Acta Crystallographica Section A, 2020

Nemaline myopathy (NM) is one of the most common congenital non-dystrophic human muscle diseases ... more Nemaline myopathy (NM) is one of the most common congenital non-dystrophic human muscle diseases and is characterized by severe muscle weakness and the presence of nemaline bodies (rods) in skeletal muscle biopsies. In this talk I will highlight two recent X-ray diffraction studies done at the BioCAT Beamline 18ID at the Advanced Photon Source ANL that have yielded new insights into the pathogenic mechanisms in these debilitating diseases. Both of these studies showed that the muscle dysfunction has its basis in changes in the structure of the actin containing thin filaments. One form of nemaline myopathy is caused by mutations in the KBTBD13 (NEM6) gene. The role of KBTBD13 in muscle is unknown. An international team of investigators recently reported In de Winter J et al. 2020 (J Clin Invest. 130(2):754-767 how a combination of transcranial magnetic stimulation-induced muscle relaxation, muscle fiber-and sarcomere-contractility assays, super-resolution microscopy, and low angle Xray diffraction revealed that the impaired muscle relaxation kinetics in NEM6 patients are caused by structural changes in the thin filament that are the fundamental cause of muscle weakness. Other forms of NM are due to mutations in nebulin , a giant protein that winds around the actin filaments in the sarcomeres of skeletal muscle. The authors of a second study in Nature Communications (Lindquist et al., Nat Commun. 2020 Jun 1;11(1):2699.) created a mouse model that mimics the typical nebulin-based NM patient with compound-heterozygous mutations. Functional, structural, and biochemical studies revealed altered thin filament structure, increased myofilament lattice spacing, a reduced myofibrillar fractional area, and reduced force production. In particular, X-ray diffraction studies revealed that the actin filament is twisted with a larger radius, that tropomyosin and troponin behavior is altered, and that the myofilament spacing is increased, again showing that the muscle weakness in nemaline myopathy is caused by changes in thin filament structure. These results will be discussed in the context of technical advances at that BioCAT that enabled these studies along with some future directions.

The next generation light sources such as diffractionlimited storage rings and high repetition ra... more The next generation light sources such as diffractionlimited storage rings and high repetition rate free electron lasers (FELs) will generate x-ray beams with significantly increased peak and average brilliance. These future facilities will require x-ray optical components capable of handling large instantaneous and average power densities while tailoring the properties of the x-ray beams for a variety of scientific experiments. In this paper we report on research and development of a single crystal diamond compound refractive lens. Diamond is the best material for high heat load applications. Moreover single crystal lens preserves coherence of the x-ray beam because scattering from grain boundaries, voids and impurities, typical for current beryllium lenses is minimized. We report the fabrication and performance evaluation of single crystal diamond refractive x-ray lenses with a paraboloid of rotation form factor for focusing x-rays in two dimensions simultaneously. The lenses were...

Structure, 2018

Membrane dynamic processes including vesicle biogenesis depend on Arf guanosine triphosphatase (G... more Membrane dynamic processes including vesicle biogenesis depend on Arf guanosine triphosphatase (GTPase) activation by guanine nucleotide exchange factors (GEFs) containing a catalytic Sec7 domain and a membrane-targeting module such as a pleckstrin homology (PH) domain. The catalytic output of cytohesin family Arf GEFs is controlled by autoinhibitory interactions that impede accessibility of the exchange site in the Sec7 domain. These restraints can be relieved through activator Arf-GTP binding to an allosteric site comprising the PH domain and proximal autoinhibitory elements (Sec7-PH linker and C-terminal helix). Small-angle X-ray scattering and negativestain electron microscopy were used to investigate the structural organization and conformational dynamics of cytohesin-3 (Grp1) in autoinhibited and active states. The results support a model in which hinge dynamics in the autoinhibited state expose the activator site for Arf-GTP binding, while subsequent C-terminal helix unlatching and repositioning unleash conformational entropy in the Sec7-PH linker to drive exposure of the exchange site.

Biophysical Journal, 2017

Biophysical Journal, 2016

Biological systems have long been recognized to be modular. The modular structure of the network ... more Biological systems have long been recognized to be modular. The modular structure of the network is represented by an intrinsic parameter, modularity. Modularity is driven by the fitness of the evolving network. A number of observations show that modularity increases under increased environmental pressure. We developed a quasispecies theory for the dynamics of modularity in populations of complex networks. We show how the steady-state fitness in a randomly changing environment can be computed. We derive a relationship between rate of environmental changes and rate of growth of modularity. This relationship explains how complex networks cope with challenging environment change in order to survive and thrive.

Journal of synchrotron radiation, 2014

The small source sizes of third-generation synchrotron sources are ideal for the production of mi... more The small source sizes of third-generation synchrotron sources are ideal for the production of microbeams for diffraction studies of crystalline and non-crystalline materials. While several such facilities have been available around the world for some time now, few have been optimized for the handling of delicate soft-tissue specimens under cryogenic conditions. Here the development of a new X-ray micro-diffraction instrument at the Biophysics Collaborative Access Team beamline 18-ID at the Advanced Photon Source, and its use with newly developed cryo-diffraction techniques for soft-tissue studies, are described. The combination of the small beam sizes delivered by this instrument, the high delivered flux and successful cryo-freezing of rat-tail tendon has enabled us to record data to better than 4 Å resolution. The ability to quickly raster scan samples in the beam allows selection of ordered regions in fibrous samples for markedly improved data quality. Examples of results of expe...

Biophysical Journal, 2014

Biophysical Journal, 2013

The asynchronous, indirect flight muscles (IFM) of Drosophila are characterized by a high passive... more The asynchronous, indirect flight muscles (IFM) of Drosophila are characterized by a high passive stiffness and exceptionally fast myosin kinetics, two attributes that enhance power output to sustain flight. Flightin is an IFM-specific, 20kDa myosin rod-binding protein required for normal thick filament stiffness, sarcomere integrity, and flight. Previously, we showed that a COOH-terminal truncation of flightin (fln DeltaC44) decreased myofilament lattice order and myosin kinetics, resulting in lower oscillatory power output and flightlessness. Here, we investigate the function of the flightin N-terminal 62 amino acids by creating transgenic Drosophila (fln DeltaN62) expressing a truncated flightin. fln DeltaN62 flies were flight impaired (flight index: 2.850.1 vs. 4.250.4 for fln DeltaN62 vs. fln þ rescued null control) despite having a normal wing-beat frequency (19554 vs. 19852 Hz for fln þ). Mechanical analysis of skinned IFM fibers showed that the flightin N-terminal truncation reduced passive, active, and rigor stiffness without affecting cross-bridge kinetics (frequency of maximum power: 20557 vs. 21757 Hz for fln þ). fln DeltaN62 fibers produced approximately half the isometric tension (passive: 0.950.1 vs. 1.750.3 kN/m 2 , active: 0.850.1 vs. 1.550.2 kN/m 2 , rigor: 1.150.2 vs. 3.150.4 kN/m 2) and maximum oscillatory power output (38.054.6 vs. 89.559.6 W/m 3) as fln þ fibers. Moreover, about 60% of the fln DeltaN62 fibers tore in rigor, demonstrating mechanical failure near isometric tension values that were sustained by fln þ fibers. Fourier transform analysis of crosssectional electron micrographs revealed that the flightin N-terminal truncation compromised myofilament lattice crystallinity and reduced inter-thick filament spacing by 10% (44.151.3 vs. 49.750.4 nm). These results indicate that the flightin N-terminal region enhances myofilament lattice order and mechanical integrity, which in turn is required for effective force transmission, normal oscillatory power output, and flight.

Methods in Enzymology, 2016

Chromatin accessibility is modulated by structural transitions that provide timely access to the ... more Chromatin accessibility is modulated by structural transitions that provide timely access to the genetic and epigenetic information during many essential nuclear processes. These transitions are orchestrated by regulatory proteins that coordinate intricate structural modifications and signalling pathways. In vitro reconstituted chromatin samples from defined components are instrumental in defining the mechanistic details of such processes. The bottleneck to appropriate in vitro analysis is the production of high quality, and quality-controlled, chromatin substrates. In this chapter we describe methods for in vitro chromatin reconstitution and quality control. We highlight the strengths and weaknesses of various approaches, and emphasize quality control steps that ensure reconstitution of a bona fide homogenous chromatin preparation. This is essential for optimal reproducibility and reliability of ensuing experiments using chromatin substrates.

International Journal of Molecular Sciences, Nov 30, 2019

Many biological processes are triggered or driven by mechanical forces in the cytoskeletal networ... more Many biological processes are triggered or driven by mechanical forces in the cytoskeletal network, but these transducing forces have rarely been assessed. Striated muscle, with its well-organized structure provides an opportunity to assess intracellular forces using small-angle X-ray fiber diffraction. We present a new methodology using Monte Carlo simulations of muscle contraction in an explicit 3D sarcomere lattice to predict the fiber deformations and length changes along thin filaments during contraction. Comparison of predicted diffraction patterns to experimental meridional X-ray reflection profiles allows assessment of the stepwise changes in intermonomer spacings and forces in the myofilaments within living muscle cells. These changes along the filament length reflect the effect of forces from randomly attached crossbridges. This approach enables correlation of the molecular events, such as the current number of attached crossbridges and the distributions of crossbridge forces to macroscopic measurements of force and length changes during muscle contraction. In addition, assessments of fluctuations in local forces in the myofilaments may reveal how variations in the filament forces acting on signaling proteins in the sarcomere M-bands and Z-discs modulate gene expression, protein synthesis and degradation, and as well to mechanisms of adaptation of muscle in response to changes in mechanical loading.

Archives of Biochemistry and Biophysics, Feb 1, 2015

The flight muscles (DLM 1) of the Hawkmoth, Manduca sexta are synchronous, requiring a neural spi... more The flight muscles (DLM 1) of the Hawkmoth, Manduca sexta are synchronous, requiring a neural spike for each contraction. Stress/strain curves of skinned DLM 1 showed hysteresis indicating the presence of titin-like elastic proteins. Projectin and kettin are titin-like proteins previously identified in Lethocerus and Drosophila flight muscles. Analysis of Manduca muscles with 1% SDS-agarose gels and western blots showed two bands near 1 MDa that cross-reacted with antibodies to Drosophila projectin. Antibodies to Drosophila kettin cross-reacted to bands at ~500 and ~700 kDa, but also to bands at ~1.6 and ~2.1 MDa, that had not been previously observed in insect flight muscles. Mass spectrometry identified the 2.1 MDa protein as a product of the Sallimus (sls) gene. Analysis of the gene sequence showed that all 4 putative Sallimus and kettin isoforms could be explained as products of alternative splicing of the single sls gene. Both projectin and Sallimus isoforms were expressed to higher levels in ventrally located DLM 1 subunits, primarily responsible for active work production, as compared to dorsally located subunits, which may act as damped springs. The different expression levels of the 2 projectin isoforms and 4 Sallimus/kettin isoforms may be adaptations to the specific requirements of individual muscle subunits.

Acta Crystallographica Section A, Aug 8, 1996

The Journal of General Physiology, Feb 21, 2019

bioRxiv (Cold Spring Harbor Laboratory), Apr 12, 2023

Rationale: Mavacamten is a novel, FDA-approved, small molecule therapeutic designed to regulate c... more Rationale: Mavacamten is a novel, FDA-approved, small molecule therapeutic designed to regulate cardiac function at the sarcomere level by selectively but reversibly inhibiting the enzymatic activity of myosin. It shifts myosin towards ordered off states close to the thick filament backbone. Objective: The purpose of this study is to investigate whether mavacamten permanently sequester these myosin heads in the off state(s) and can these heads be recruited in response to physiological stimuli when required to boost cardiac output. Methods and Results: We show that cardiac myosins stabilized in these off state(s) by mavacamten are recruitable by 1) Ca 2+ , 2) increased chronotropy (heart rate), 3) stretch, and 4) β adrenergic (β-AR) stimulation, all known physiological inotropic effectors. At the molecular level, we show that Ca 2+ increases myosin ATPase activity by shifting mavacamten-stabilized myosin heads from the inactive super-relaxed (SRX) state to the active disordered relaxed (DRX) state. At the myofilament level, both Ca 2+ and passive lengthening can shift mavacamtenordered off myosin heads from positions close to the thick filament backbone to disordered on states closer to the thin filaments. In isolated rat cardiomyocytes, increased stimulation rates enhanced shortening fraction in mavacamten-treated cells. This observation was confirmed in vivo in telemetered rats, where left-ventricular dP/dt max, an index of inotropy, increased with heart rate in mavacamten treated animals. Finally, we show that β-AR stimulation in vivo increases left-ventricular function and stroke volume in the setting of mavacamten. Conclusions: Our data demonstrate that the mavacamten-promoted off states of myosin in the thick filament are activable, thus leading to the preservation of the cardiac reserve. These results provide a potential mechanistic explanation, beyond mere LV outflow tract obstruction removal, for the clinical observation of increased peak oxygen uptake (pVO 2) with exercise in HCM patients receiving mavacamten. .

Acta Crystallographica, Jul 29, 2022

For many years, analysis of fiber diffraction patterns from muscle was a rate limiting step in th... more For many years, analysis of fiber diffraction patterns from muscle was a rate limiting step in the preparation of experimental results for publication. Most analysis procedures made extensive use of FIT2D supplemented by locally developed or commercial software tools which were not specifically designed for the task. Analyzing the data from a single experiment could take many weeks or months. To address this bottleneck, we have developed a comprehensive data reduction suite of programs which we call 'MuscleX". The software is open source under a modified BSD license. MacIntosh, Windows and linux installers permit easy deployment. The "Equator' routine can analyze the equatorial patterns from a complete experiment in a few hours without operator intervention, a process that used to take many months. The 'Quadrant-Fold" package averages the four quadrants of a diffraction pattern to improve signal to noise for more detailed analysis. It can also perform global background subtractions using either a "white top hat" real space digital filter algorithm, or a two-dimensional convex hull. It also includes radially symmetric, iterative smoothing and "roving window" background subtraction routines from the now defunct CCP software suite. Quadrant folding may be useful to remove the lines in the patterns created by the dead zones between modules in common pixel array detectors. Other components of the suite include "Projection Traces" and "Diffraction Centroids" that automatically analyze the intensity and spacings of prominent reflections on the meridian and layer lines from the large amount of diffraction images collected in time resolved experiments. Once the programs are set up, they can process an entire directory of data without operator intervention in a few hours. In this presentation we will discuss current capabilities and planned future developments and how the MuscleX package may be useful for all biological fiber diffraction experiments, not just muscle diffraction.

Biophysical Journal, 2014

Mammalian hearts express two myosin heavy chain (MHC) isoforms, which drive contractions with dif... more Mammalian hearts express two myosin heavy chain (MHC) isoforms, which drive contractions with different kinetics and power-generating ability. The expression of the isoform that is associated with more rapid contraction kinetics and greater power output, MHC-a, is down-regulated, with a concurrent increase in the relative amount of the slower isoform, MHC-b, during the progression to experimentally-induced or disease-related heart failure. This change in protein expression has been well studied in right and left ventricles in heart failure models and in humans with failure. Relatively little quantitative data exists regarding MHC isoform expression shifts in human failing atria. We previously reported significant increases in the relative amount of MHC-b in the human failing left atrium. The results of that study suggested that there might be a sex-related difference in the level of MHC-b in the left atrium, but the number of female subjects was insufficient for statistical analysis. The objective of this study was to test whether there is, in fact, a sex-related difference in the level of MHC-b in the right and left atria of humans with cardiomyopathy. The results indicate that significant differences exist in atrial MHC isoform expression between men and women who are in failure. The results unexpectedly also revealed a twofold greater amount of MHC-b in the non-failing left atrium of women, compared to men. The observed sexrelated differences in MHC isoform expression could impact ventricular diastolic filling during normal daily activities, as well as during physiologically stressful events.

Biophysical Journal

Additional information Versions of research works Versions of Record If this version is the versi... more Additional information Versions of research works Versions of Record If this version is the version of record, it is the same as the published version available on the publisher's web site. Cite as the published version.

Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Detai... more Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Detailed mechanism of action of these agents can help predict potential unwanted affects and identify patient populations that can benefit most from them. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. Using porcine cardiac tissue and myofibrils we demonstrate that Danicamtiv increases force and calcium sensitivity via increasing the number of myosin in the “on” state and slowing cross bridge turnover. Our detailed analysis shows that inhibition of ADP release results in decreased cross bridge turnover with cross bridges staying on longer and prolonging myofibril relaxation. Using a mouse model of genetic dilated cardiomyopathy, we demonstrated that Dan...

Biophysical Journal, 2018

Acta Crystallographica Section A, 2020

Nemaline myopathy (NM) is one of the most common congenital non-dystrophic human muscle diseases ... more Nemaline myopathy (NM) is one of the most common congenital non-dystrophic human muscle diseases and is characterized by severe muscle weakness and the presence of nemaline bodies (rods) in skeletal muscle biopsies. In this talk I will highlight two recent X-ray diffraction studies done at the BioCAT Beamline 18ID at the Advanced Photon Source ANL that have yielded new insights into the pathogenic mechanisms in these debilitating diseases. Both of these studies showed that the muscle dysfunction has its basis in changes in the structure of the actin containing thin filaments. One form of nemaline myopathy is caused by mutations in the KBTBD13 (NEM6) gene. The role of KBTBD13 in muscle is unknown. An international team of investigators recently reported In de Winter J et al. 2020 (J Clin Invest. 130(2):754-767 how a combination of transcranial magnetic stimulation-induced muscle relaxation, muscle fiber-and sarcomere-contractility assays, super-resolution microscopy, and low angle Xray diffraction revealed that the impaired muscle relaxation kinetics in NEM6 patients are caused by structural changes in the thin filament that are the fundamental cause of muscle weakness. Other forms of NM are due to mutations in nebulin , a giant protein that winds around the actin filaments in the sarcomeres of skeletal muscle. The authors of a second study in Nature Communications (Lindquist et al., Nat Commun. 2020 Jun 1;11(1):2699.) created a mouse model that mimics the typical nebulin-based NM patient with compound-heterozygous mutations. Functional, structural, and biochemical studies revealed altered thin filament structure, increased myofilament lattice spacing, a reduced myofibrillar fractional area, and reduced force production. In particular, X-ray diffraction studies revealed that the actin filament is twisted with a larger radius, that tropomyosin and troponin behavior is altered, and that the myofilament spacing is increased, again showing that the muscle weakness in nemaline myopathy is caused by changes in thin filament structure. These results will be discussed in the context of technical advances at that BioCAT that enabled these studies along with some future directions.

The next generation light sources such as diffractionlimited storage rings and high repetition ra... more The next generation light sources such as diffractionlimited storage rings and high repetition rate free electron lasers (FELs) will generate x-ray beams with significantly increased peak and average brilliance. These future facilities will require x-ray optical components capable of handling large instantaneous and average power densities while tailoring the properties of the x-ray beams for a variety of scientific experiments. In this paper we report on research and development of a single crystal diamond compound refractive lens. Diamond is the best material for high heat load applications. Moreover single crystal lens preserves coherence of the x-ray beam because scattering from grain boundaries, voids and impurities, typical for current beryllium lenses is minimized. We report the fabrication and performance evaluation of single crystal diamond refractive x-ray lenses with a paraboloid of rotation form factor for focusing x-rays in two dimensions simultaneously. The lenses were...

Structure, 2018

Membrane dynamic processes including vesicle biogenesis depend on Arf guanosine triphosphatase (G... more Membrane dynamic processes including vesicle biogenesis depend on Arf guanosine triphosphatase (GTPase) activation by guanine nucleotide exchange factors (GEFs) containing a catalytic Sec7 domain and a membrane-targeting module such as a pleckstrin homology (PH) domain. The catalytic output of cytohesin family Arf GEFs is controlled by autoinhibitory interactions that impede accessibility of the exchange site in the Sec7 domain. These restraints can be relieved through activator Arf-GTP binding to an allosteric site comprising the PH domain and proximal autoinhibitory elements (Sec7-PH linker and C-terminal helix). Small-angle X-ray scattering and negativestain electron microscopy were used to investigate the structural organization and conformational dynamics of cytohesin-3 (Grp1) in autoinhibited and active states. The results support a model in which hinge dynamics in the autoinhibited state expose the activator site for Arf-GTP binding, while subsequent C-terminal helix unlatching and repositioning unleash conformational entropy in the Sec7-PH linker to drive exposure of the exchange site.

Biophysical Journal, 2017

Biophysical Journal, 2016

Biological systems have long been recognized to be modular. The modular structure of the network ... more Biological systems have long been recognized to be modular. The modular structure of the network is represented by an intrinsic parameter, modularity. Modularity is driven by the fitness of the evolving network. A number of observations show that modularity increases under increased environmental pressure. We developed a quasispecies theory for the dynamics of modularity in populations of complex networks. We show how the steady-state fitness in a randomly changing environment can be computed. We derive a relationship between rate of environmental changes and rate of growth of modularity. This relationship explains how complex networks cope with challenging environment change in order to survive and thrive.

Journal of synchrotron radiation, 2014

The small source sizes of third-generation synchrotron sources are ideal for the production of mi... more The small source sizes of third-generation synchrotron sources are ideal for the production of microbeams for diffraction studies of crystalline and non-crystalline materials. While several such facilities have been available around the world for some time now, few have been optimized for the handling of delicate soft-tissue specimens under cryogenic conditions. Here the development of a new X-ray micro-diffraction instrument at the Biophysics Collaborative Access Team beamline 18-ID at the Advanced Photon Source, and its use with newly developed cryo-diffraction techniques for soft-tissue studies, are described. The combination of the small beam sizes delivered by this instrument, the high delivered flux and successful cryo-freezing of rat-tail tendon has enabled us to record data to better than 4 Å resolution. The ability to quickly raster scan samples in the beam allows selection of ordered regions in fibrous samples for markedly improved data quality. Examples of results of expe...