Bite force is a key metric of organismal performance, and expression of masticatory myosin (MHC-M) is associated with high bite force. However, skeletal muscles are multiscale structures, and it remains unclear how adaptations for force production are integrated across scales. We analyzed myosin isoform composition and physiological cross-sectional area of the jaw muscles and measured their dynamic moment arms ex vivo using XROMM (X-ray Reconstruction Of Moving Morphology) in six rodent species. We found modifications at all scales in hard biters (grey squirrels) to prioritize force production. Related species (chipmunk, woodchuck and red squirrel) showed a mix of adaptations across scales, with different muscle phenotypes producing equivalent bite force outputs. By contrast, rat and guinea pig showed modifications at all scales consistent with reduced force production. Our results suggest that selection for ecologically relevant traits – including MHC-M expression – occurs at multiple organizational scales within the rodent craniofacial system.

Inherited cardiomyopathy is a broad class of heart disease that includes pathological cardiac remodeling such as hypertrophic and dilated cardiomyopathy, affecting 1/250-1/500 people worldwide. In many cases, mutations in proteins that make up the sarcomere, the basic subcellular unit of contraction, alter thin filament regulation and are the root cause of hypertrophic and dilated cardiomyopathy. Initially, compensations can maintain cardiac function, so patients may remain asymptomatic for years before a major cardiac episode. Early therapeutic intervention could rescue the deleterious effects of mutations thereby avoiding pathological remodeling, so prediction of potential outcomes and severity of as yet uncharacterized and known mutants of uncertain significance is critical. To accomplish this goal, we begin with the structure of the thin filament containing actin, tropomyosin, and troponin in its regulatory B- and C-states, incorporate all potential single nucleotide mutations to the tropomyosin sequence (over 1700 unique mutations), and then measure the interaction energy between tropomyosin and actin after energy minimization. Analysis of the database thus generated shows the tropomyosin residues resulting in large changes in tropomyosin-actin interaction, and therefore most likely to be deleterious to function. Some of these mutants have been observed in human patients, whereas others are novel. Global analysis further refines hotspots of mutation-sensitive, coiled-coil tropomyosin residues affecting actin interactions. Altogether, the database will allow research to focus in great depth on key candidates for functional analysis, for instance, by assaying in vitro motility and engineered heart tissue mechanics and assessing outcomes in animal models.

Background Evaluating risk in first-degree relatives of patients diagnosed with hypertrophic cardiomyopathy (HCM) is a critical, ongoing clinical challenge, particularly when genetic testing yields a variant of unknown significance (VUS) in an HCM-related gene. Our goal is to develop a pipeline for efficient and scalable evaluation of VUS pathogenicity. Methods Molecular dynamics simulations were used to screen twenty TPM1 VUS and identify four for further study: A102D, D258E, A239T, and K233N. Each VUS was engineered into an adenoviral TPM1 vector which was in turn applied to human engineered heart tissues (hEHTs) made from iPSC-derived cardiomyocytes. One week after viral exposure, isometric contractile behavior of hEHTs was measured. Results hEHTs expressing A102D were markedly hypercontractile, with a significant increase in contractile force and slowing of relaxation. D258E expression caused a mild phenotype, showing only a mildly increased relaxation rate relative to control. A238T and K233N both caused a dramatic decrease in contractile force. These same four VUS were studied in regulated in vitro motility assays and showed patterns of increased or decreased myofilament activity that were entirely consistent with findings in hEHT. Conclusions A102D displayed a hypercontractile phenotype that resembled that of our benchmark HCM mutation (E62Q), while A238T and K233N produced contractile weakness that closely mimicked that of our benchmark DCM mutation (E54K). D258E had a minimal effect that could be interpreted as mildly hypertrophic. We conclude that hEHTs have the potential to serve as a scalable platform for evaluating pathogenicity of TPM1 VUS. Evaluating risk in first-degree relatives of patients diagnosed with hypertrophic cardiomyopathy (HCM) is a critical, ongoing clinical challenge, particularly when genetic testing yields a variant of unknown significance (VUS) in an HCM-related gene. Our goal is to develop a pipeline for efficient and scalable evaluation of VUS pathogenicity. Molecular dynamics simulations were used to screen twenty TPM1 VUS and identify four for further study: A102D, D258E, A239T, and K233N. Each VUS was engineered into an adenoviral TPM1 vector which was in turn applied to human engineered heart tissues (hEHTs) made from iPSC-derived cardiomyocytes. One week after viral exposure, isometric contractile behavior of hEHTs was measured. hEHTs expressing A102D were markedly hypercontractile, with a significant increase in contractile force and slowing of relaxation. D258E expression caused a mild phenotype, showing only a mildly increased relaxation rate relative to control. A238T and K233N both caused a dramatic decrease in contractile force. These same four VUS were studied in regulated in vitro motility assays and showed patterns of increased or decreased myofilament activity that were entirely consistent with findings in hEHT. A102D displayed a hypercontractile phenotype that resembled that of our benchmark HCM mutation (E62Q), while A238T and K233N produced contractile weakness that closely mimicked that of our benchmark DCM mutation (E54K). D258E had a minimal effect that could be interpreted as mildly hypertrophic. We conclude that hEHTs have the potential to serve as a scalable platform for evaluating pathogenicity of TPM1 VUS.

The extraordinary diversity of neuron types in the mammalian brain is delineated at the highest resolution by subtle gene expression differences that may require specialized molecular mechanisms to be maintained. Neurons uniquely express the longest genes in the genome and utilize neuron-enriched non-CG DNA methylation (mCA) together with the Rett syndrome protein, MeCP2, to control gene expression, but the function of these unique gene structures and machinery in regulating finely resolved neuron type-specific gene programs has not been explored. Here, we employ epigenomic and spatial transcriptomic analyses to discover a major role for mCA and MeCP2 in maintaining neuron type-specific gene programs at the finest scale of cellular resolution. We uncover differential susceptibility to MeCP2 loss in neuronal populations depending on global mCA levels and dissect methylation patterns and intragenic enhancer repression that drive overlapping and distinct gene regulation between neuron types. Strikingly, we show that mCA and MeCP2 regulate genes that are repeatedly tuned to differentiate neuron types at the highest cellular resolution, including spatially resolved, vision-dependent gene programs in the visual cortex. These repeatedly tuned genes display genomic characteristics, including long length, numerous intragenic enhancers, and enrichment for mCA, that predispose them to regulation by MeCP2. Thus, long gene regulation by the MeCP2 pathway maintains differential gene expression between closely-related neurons to facilitate the exceptional cellular diversity in the complex mammalian brain.

Ambient vibration measurements can detect resonance frequency changes related to rock slope instability damage or boundary condition changes during progressive failure. However, the impact of slope kinematics on resonance changes and the expected form and sensitivity of frequency evolution during destabilization require clarification to improve the implementation of this technique across diverse settings. Since instrumented rock slope failures are rare, numerical modeling is needed to study the anticipated spectral response from in situ monitoring. We used 2D distinct‐element modeling to evaluate the sensitivity and evolution of rock slope resonance behavior for slab toppling, flexural toppling, and planar sliding instabilities during progressive failure. Model simulations revealed that fundamental resonance frequency decreases between 20% and 60% with changes correlated with increasing length of open joints. Changes to higher‐order frequencies associated with landslide sub‐volumes were also detectable for cases with multiple fracture networks. Resonance behavior was most pronounced for failures dominated by steeply dipping open tension cracks, that is, flexural and slab toppling. Additionally, amplification patterns across the slope varied for the flexural toppling and sliding cases, providing potential new information with which to characterize landslide failure mechanisms using ambient vibration array measurements. Our results demonstrate landslide characteristics well‐suited for in situ ambient resonance monitoring and provide new data describing the anticipated changes in resonance frequencies during progressive rock slope failure.

Muscle is highly hierarchically organized, with functions shaped by genetically controlled expression of protein ensembles with different isoform profiles at the sarcomere scale. However, it remains unclear how isoform profiles shape whole muscle performance. We compared two mouse hind limb muscles, the slow, relatively parallel-fibered soleus (SOL) and the faster, more pennate-fibered tibialis anterior (TA), across scales: from gene regulation, isoform expression and translation speed, to force-length-velocity-power for intact muscles. Expression of myosin heavy-chain (MHC) isoforms directly corresponded with contraction velocity. The fast-twitch TA with fast MHC isoforms had faster unloaded velocities (actin sliding velocity, VACTIN; peak fiber velocity, VMAX) than slow-twitch SOL. For SOL, VACTIN was biased towards VACTIN for purely slow MHC I, despite this muscle's even fast and slow MHC isoform composition. Our multi-scale results clearly identified a consistent and significant dampening in fiber shortening velocities for both muscles, underscoring an indirect correlation between VACTIN and fiber VMAX that may be influenced by differences in fiber architecture, along with internal loading due to both passive and active effects. These influences correlate with the increased peak force and power in the slightly more pennate TA, leading to a broader length range of near-optimal force production. Conversely, a greater force-velocity curvature in the near-parallel fibered SOL highlights the fine-tuning by molecular-scale influences including myosin heavy and light chain expression along with whole muscle characteristics. Our results demonstrate that the individual gene, protein, and whole fiber characteristics do not directly reflect overall muscle performance but that intricate fine-tuning across scales shapes specialized muscle function.

Accurate assessments of the internal structure and boundary conditions of unstable rock slopes are imperative for evaluating landslide hazard scenarios. However, instability characterization at depth remains challenging and is often limited by costly or invasive subsurface investigations. Here, we develop a new approach coupling array‐based ambient vibration modal analysis and numerical modeling to improve structural characterization of rock slope instabilities at depth. We used ambient noise cross‐correlation on 4 hr of seismic data recorded by an array of 30 nodal geophones at a 500‐m‐long toppling rock slab in Utah, USA to identify modal frequencies between 0.8 and 3.5 Hz and derive modal displacements. We show that transverse and longitudinal bending modes span the length of the instability, indicating an interconnected slab. Statistical comparison of field results with outputs from >1,000 finite element models with varying boundary conditions showed that the instability depth varies between 40–70 and 10–20 m in the middle and lateral regions, respectively. Our approach yields new information on the structural conditions of rock cliff and column instabilities at depth, which is not easily obtained by other means but is imperative for change detection monitoring and improved hazard assessments.

Organizations continue to face increased business pressures which force leaders to consider new leadership theories focusing on enabling greater leader and team effectiveness. An increasing diverse ethnic employee constituency is one of these pressures that often result in value clashes. Sephora, an LVMH company, is redesigning its distribution center organizational culture towards shared leadership to respond to these challenges. Our study analyzed 39 managers, working in distribution centers in Salt Lake City and in Las Vegas, supervising employees from 7 different ethnic identities were grouped in 14 teams, using the Competing Values Framework (CVF) in a 360-performance appraisal system, performance metrics and annual performance evaluation data. Our action research used three methods to evaluate effectiveness: annual performance evaluation (rated highly successful), upper and lower group (groups based on organizational hierarchy), and 360 exceeding expectations (peer evaluations above supervisor benchmark). Our findings identify common management behaviors for both effective leaders and effective teams to be Create: Broker (influences, effectively presents ideas), and Team: Culture (builds effective relationships through achieving shared goals). Effective leaders add the Collaborate: Mentor (listening & assisting in meeting team member needs), while the effective teams add the Compete: Producer (results orientation) behavior. Consultants looking to improve organizational effectiveness can use this CVF tool to identify employee strengths and weaknesses. The employee results can then be compared to the profile of an effective leader and thus provide an effective manager behavioral roadmap.

Mutations in DNA methyltransferase 3A (DNMT3A) have been detected in autism and related disorders, but how these mutations disrupt nervous system function is unknown. Here, we define the effects of DNMT3A mutations associated with neurodevelopmental disease. We show that diverse mutations affect different aspects of protein activity but lead to shared deficiencies in neuronal DNA methylation. Heterozygous DNMT3A knockout mice mimicking DNMT3A disruption in disease display growth and behavioral alterations consistent with human phenotypes. Strikingly, in these mice, we detect global disruption of neuron-enriched non-CG DNA methylation, a binding site for the Rett syndrome protein MeCP2. Loss of this methylation leads to enhancer and gene dysregulation that overlaps with models of Rett syndrome and autism. These findings define the effects of DNMT3A haploinsufficiency in the brain and uncover disruption of the non-CG methylation pathway as a convergence point across neurodevelopmental disorders.

Organizational change initiatives are key services which consultants provide to their clients. McKinsey & Company note that nearly 75% of these large scale initiatives fail. This case study identifies and addresses value shortfalls in a manufacturing plant in the southeast. Data collection includes multiple surveys and small group interviews. Analysis uses rigorous coding methods to construct a model of critical organizational values and behaviors essential for leadership effectiveness. We bring “theory to practice” by applying complextiy leadership concepts in our intervention strategy. Our findings are categorized into three parts: identifying critical culture value gaps, applying complexity concepts to a scenario-based training intervention, and identifying intervention outcomes. Outcomes include transformed work environment lead by leaders who respect others, share decision-making, and enable employees to be interdependent. This explanatory case study contributes to research by applying complexity leadership theory to a practical consulting intervention. This work provides a template and process for consultants using complexity leadership to inform their client interventions.

Cardiac muscle contraction occurs when thin filaments slide past thick filaments driven by the action of the molecular motor, myosin.Projecting from thick filaments, myosin heads bind to thin filament actin and undergo ATPase-associated conformational changes to propel the filaments to move past one another.Tropomyosin, a coiled-coil protein that wraps around actin as a continuous cable, regulates this process by sterically controlling myosin's ability to bind to actin.Although several actomyosin structures have been proposed, the structure describing cardiac actin -myosin interactions including tropomyosin has not been solved.Here, we present the structure of the cardiac actomyosin-tropomyosin complex in the nucleotide-free state, solved to a resolution of 4.2 Å using cryo-electron microscopy and helical reconstruction.The atomic model derived from this reconstruction shows a strong, mostly hydrophobic interface between actin and myosin that is consistent with previously determined non-cardiac structures.However, we were able to obtain additional residue-residue discrimination, by combining our cryo-EM structure with results of protein-protein docking methods, revealing novel electrostatic interactions between myosin loop 4 (363-376) and tropomyosin.This hybrid method shows that the tip of myosin loop 4, most notably residue Arg369, forms distinct interactions between successive tropomyosin segments on actin protomers.We propose that these favorable contacts compete with and displace ones normally found between actin and tropomyosin in the absence of myosin, playing a vital role in the transition from the relaxed to contracted states.The impact of these structural studies is twofold: (1) they suggest that the favorable myosin loop 4tropomyosin interaction restructures the thin filament during the actin-myosin ATPase cycle; (2) they lay a foundation for understanding molecular mechanisms by which disease-rendering point mutations perturb normal actin-myosin-tropomyosin binding.

The genomes of mammalian neurons contain uniquely high levels of non-CG DNA methylation that can be bound by the Rett syndrome protein, MeCP2, to regulate gene expression. How patterns of non-CG methylation are established in neurons and the mechanism by which this methylation works with MeCP2 to control gene expression is unclear. Here, we find that genes repressed by MeCP2 are often located within megabase-scale regions of high non-CG methylation that correspond with topologically associating domains of chromatin folding. MeCP2 represses enhancers found in these domains that are enriched for non-CG and CG methylation, with the strongest repression occurring for enhancers located within MeCP2-repressed genes. These alterations in enhancer activity provide a mechanism for how MeCP2 disruption in disease can lead to widespread changes in gene expression. Hence, we find that DNA topology can shape non-CG DNA methylation across the genome to dictate MeCP2-mediated enhancer regulation in the brain.

Mutations in titin are responsible for many cardiac and muscle diseases, yet the underlying mechanisms remain largely unexplained. Numerous studies have established roles for titin in muscle function, and Ca2+-dependent interactions between titin and actin have been suggested to play a role in muscle contraction. The present study used co-sedimentation assays, dynamic force spectroscopy (DFS), and in vitro motility (IVM) assays to determine whether the N2A region of titin, overlooked in previous studies, interacts with actin in the presence of Ca2+. Co-sedimentation demonstrated that N2A – F-actin binding increases with increasing protein and Ca2+ concentration, DFS demonstrated increased rupture forces and decreased koff in the presence of Ca2+, and IVM demonstrated a Ca2+-dependent reduction in motility of F-actin and reconstituted thin filaments in the presence of N2A. These results indicate that Ca2+ increases the strength and stability of N2A – actin interactions, supporting the hypothesis that titin plays a regulatory role in muscle contraction. The results further support a model in which N2A – actin binding in active muscle increases titin stiffness, and that impairment of this mechanism contributes to the phenotype in muscular dystrophy with myositis. Future studies are required to determine whether titin – actin binding occurs in skeletal muscle sarcomeres in vivo.

We conduct a case study that examines consultant efforts to improve the culture of a manufacturing plant in the United States plagued by high turnover rates, low trust in management, limited production levels, and low scores on corporate culture surveys. We collect data through surveys, interviews and observations involving approximately 500 of over 800 employees. We determined there were five primary categories of issues holding significant meaning for participants. These are: 1) lack of awards and recognition, 2) lack of training and development, 3) lack of respect and sense of care, 4) excessive overtime, and 5) adequate inventory. Using complexity leadership to frame the study, we offer initial findings, and describe a two-year period showing significant progress toward a complexity culture of interactive plant members.

Rotifers are common prey of predatory zooplankton and have evolved a suite of defensive and escape strategies to avoid being consumed. Species of the genus Hexarthra are extraordinary in bearing six highly setose, arm-like appendages that function in saltational jumps through the water column to escape predation. To date, there are no observations on the structure of these escape organs despite their exceptionality within Rotifera. Here, we apply transmission electron microscopy (TEM) to study the ultrastructure of the arm-like appendages, their setae, and their muscle supply. TEM reveals that the arms are hollow extensions of the trunk integument with a similar ultrastructure. The integument is entirely syncytial. The syncytium is bordered apically by a double-layered plasma membrane beneath which is a layered cytoplasm: The top layer is a thin and fibrous intracytoplasmic lamina and the bottom layer is an electron lucent region containing cellular organelles bounded by a basal plasma membrane and thin basal lamina. Arm spines are hollow evaginations of the integument with no special ultrastructure. The arms terminate in primary setae that give rise to secondary setae, all of which possess an ultrastructure similar to the arms. There are two types of primary setae: unarticulated setae that are direct extensions of the arm integument, and articulated setae that fit into a ball-and-socket-type joint in the arm. Neither type is innervated nor supplied with muscles. The skeletal muscles in the trunk and arms are all cross-striated with distinct sarcomeres. All muscles are richly supplied with glycogen granules and mitochondria. A complex sarcotubular system supplies the myofibrils and is proximal to dense regions of glycogen, suggesting a glycolytic pathway for fast ATP production and the rapid release and reuptake of Ca2+ for muscle contraction.

Missense mutations to human alpha-tropomyosin (TPM1) have been implicated in the development of various cardiomyopathies, including hypertrophic (HCM), dilated (DCM), and more recently left ventricular noncompaction (LVNC). TPM1 variant E192K has been found in both HCM and LVNC patients, though its functional and molecular consequences remain largely uncharacterized. E192K substitutes an opposite charge residue on the outer surface to tropomyosin (TM) coiled coil, which may lead to altered interaction of TM with the surface of actin. Molecular dynamics simulations suggest that E192K substantially increases the flexibility of the TM chain compared to wild-type tropomyosin with a dynamic persistence length in E192K less than half that of wild-type tropomyosin. Preliminary modeling of thin filament activation under conditions of decreased stiffness suggests that it may lead to an altered twitch phenotype, with both increased systolic and diastolic force production. In order to study the consequences of the sarcomeric mutation TM E192K in the context of human cardiac muscle, we generated induced pluripotent stem cell cardiomyocytes (iPSC-CMs) from a patient with the mutation E192K who had family history of HCM and manifested severe hypertrophy. Engineered heart tissues (EHTs) were created by seeding IPSC-CMs onto decellularized laser-cut porcine myocardium and cultured under standard conditions. Preliminary contractile characterization after just two weeks of culture revealed that the peak tension (PT) generated by the mutant EHTs were several times higher than non-mutant control EHTs and that the time to peak (TTP) and time from peak to 50% relaxation (RT50) were increased compared to non-mutant control EHTs. Ongoing work will develop a comprehensive functional phenotype of E192K including contractile mechanics, diastolic tension, passive mechanics, and calcium handling in intact human EHT, allowing us to fully dissect the role of this rare but clinically-relevant TM mutation that may have broader implications for other thin filament mutations involved in genetic cardiomyopathies.

Coiled-coil tropomyosin binds super-helically along F-actin. Together with the troponin complex, tropomyosin inhibits myosin-binding. McKillop and Geeves (1993) introduced a three-state model for thin filament regulation with tropomyosin occupying three positions on the actin filament; blocked, closed and open. In the absence of myosin, tropomyosin primarily occupies the closed and blocked-states with the blocked-state being stabilized by Ca2+-free troponin. The probability of tropomyosin occupying any one of these two states is dependent on factors including cytosolic Ca2+-concentration (altering troponin conformation), mechanical strain, ionic strength, pH, and mutations that alter tropomyosin affinity for actin. Recently, we examined the impact of cardiomyopathy-linked tropomyosin mutations on the energy landscape of the tropomyosin/actin interaction. Our results demonstrated that mutations associated with hypertrophic cardiomyopathy showed reduced interaction-energy with actin resulting in a destabilization of the blocked-state. Using an in vitro motility assay here, we tested the ability of the mutant tropomyosins to regulate acto-myosin interactions, as well as the impact of strong binding cross-bridges on reconstituted thin filaments containing E40K and E62Q mutations in human alpha-tropomyosin (which lead to dilated (DCM) and hypertrophic (HCM) cardiomyopathies, respectively). Regulated thin filament velocity measurements showed an increase in low-Ca2+ contractility of E62Q reconstituted thin filaments when compared to wild-type and E40K reconstituted thin filaments. The ability of myosin to activate reconstituted mutant thin filaments at low-Ca2+ was not significantly different between the three tropomyosins, indicating that the enhanced motile fraction observed with E62Q is likely due to a destabilization of the blocked-state rather than alterations in myosin-dependent activation. The motility data is consistent with molecular dynamics simulations of the mutant tropomyosins in the presence of actin which show significant, yet subtle and complex alterations to the electrostatic interactions between tropomyosin and actin.