Brahma safeguards canalization of cardiac mesoderm differentiation.

Differentiation proceeds along a continuum of increasingly fate-restricted intermediates, referred to as canalization1,2. Canalization is essential for stabilizing cell fate, but the mechanisms that underlie robust canalization are unclear. Here we show that the BRG1/BRM-associated factor (BAF) chromatin-remodelling complex ATPase gene Brm safeguards cell identity during directed cardiogenesis of mouse embryonic stem cells. Despite the establishment of a well-differentiated precardiac mesoderm, Brm-/- cells predominantly became neural precursors, violating germ layer assignment. Trajectory inference showed a sudden acquisition of a non-mesodermal identity in Brm-/- cells. Mechanistically, the loss of Brm prevented de novo accessibility of primed cardiac enhancers while increasing the expression of neurogenic factor POU3F1, preventing the binding of the neural suppressor REST and shifting the composition of BRG1 complexes. The identity switch caused by the Brm mutation was overcome by increasing BMP4 levels during mesoderm induction. Mathematical modelling supports these observations and demonstrates that Brm deletion affects cell fate trajectory by modifying saddle-node bifurcations2. In the mouse embryo, Brm deletion exacerbated mesoderm-deleted Brg1-mutant phenotypes, severely compromising cardiogenesis, and reveals an in vivo role for Brm. Our results show that Brm is a compensable safeguard of the fidelity of mesoderm chromatin states, and support a model in which developmental canalization is not a rigid irreversible path, but a highly plastic trajectory.

Noise-driven cellular heterogeneity in circadian periodicity.

Nongenetic cellular heterogeneity is associated with aging and disease. However, the origins of cell-to-cell variability are complex and the individual contributions of different factors to total phenotypic variance are still unclear. Here, we took advantage of clear phenotypic heterogeneity of circadian oscillations in clonal cell populations to investigate the underlying mechanisms of cell-to-cell variability. Using a fully automated tracking and analysis pipeline, we examined circadian period length in thousands of single cells and hundreds of clonal cell lines and found that longer circadian period is associated with increased intercellular heterogeneity. Based on our experimental results, we then estimated the contributions of heritable and nonheritable factors to this variation in circadian period length using a variance partitioning model. We found that nonheritable noise predominantly drives intercellular circadian period variation in clonal cell lines, thereby revealing a previously unrecognized link between circadian oscillations and intercellular heterogeneity. Moreover, administration of a noise-enhancing drug reversibly increased both period length and variance. These findings suggest that circadian period may be used as an indicator of cellular noise and drug screening for noise control.

Quantification of pulmonary/systemic shunt ratio by single-acquisition phase-contrast cardiovascular magnetic resonance.

PURPOSE: Phase-contrast cardiovascular magnetic resonance (PC-CMR) quantification of intracardiac shunt (measuring the pulmonary to systemic flow ratio, Qp/Qs) is typically determined by measuring flow through planes perpendicular the pulmonary trunk (PA) and ascending aorta (Ao). This method is subject to error from presence of background velocity offsets and requires two scan acquisitions. We evaluated an alternate PC-CMR technique for quantifying Qp/Qs using a single modified plane that encompasses both the PA and Ao. MATERIAL AND METHODS: In 53 patients evaluated for intracardiac shunting, PC-CMR measurement in the individual Ao and PA planes and also in a single-acquisition plane was obtained and Qp/Qs calculated by each method. Bland-Altman analysis was performed to evaluate the agreement between the two methods. RESULTS: The 95% confidence limits of agreement ranged from -0.52 to +0.34 indicating good agreement between the two methods. There was excellent agreement on the clinically relevant threshold value of Qp/Qs ratio of 1.5 (representing criteria for surgical correction of shunt). CONCLUSIONS: Qp/Qs determined from the single-acquisition approach agrees well with that of the individual PA and Ao method and offers potential improved accuracy (due to background velocity offset).

Perioperative Pain Control in the Ambulatory Setting.

Across the USA and various parts of the world, ambulatory surgery centers have transitioned to accepting patients with advanced ASA statuses, leading to a larger volume and higher complexity of surgeries performed, while still urging for same-day patient discharges. Inadequate postoperative pain management and opioid analgesia side effects, such as sedation, respiratory depression, and postoperative nausea and vomiting, are the most common complications and most common reasons for readmission after ambulatory surgery. The trend to limiting these complications and achieve a more rapid patient discharge currently emphasizes a multifactorial, balanced analgesia strategy. This article reviews the multimodal approach by detailing the important aspects of specific regional nerve blocks, nerve blockade with catheter techniques, acetaminophen, non-selective NSAIDs, Cox-2 inhibitors, membrane stabilizers, and corticosteroids. Pain management in the ambulatory surgery patient will thus be optimized with a thorough preoperative evaluation, recognizing intraoperative events, and implementing multiple analgesic modalities.

Co-transcriptional production of RNA-DNA hybrids for simultaneous release of multiple split functionalities.

Control over the simultaneous delivery of different functionalities and their synchronized intracellular activation can greatly benefit the fields of RNA and DNA biomedical nanotechnologies and allow for the production of nanoparticles and various switching devices with controllable functions. We present a system of multiple split functionalities embedded in the cognate pairs of RNA-DNA hybrids which are programmed to recognize each other, re-associate and form a DNA duplex while also releasing the split RNA fragments which upon association regain their original functions. Simultaneous activation of three different functionalities (RNAi, Förster resonance energy transfer and RNA aptamer) confirmed by multiple in vitro and cell culture experiments prove the concept. To automate the design process, a novel computational tool that differentiates between the thermodynamic stabilities of RNA-RNA, RNA-DNA and DNA-DNA duplexes was developed. Moreover, here we demonstrate that besides being easily produced by annealing synthetic RNAs and DNAs, the individual hybrids carrying longer RNAs can be produced by RNA polymerase II-dependent transcription of single-stranded DNA templates.

Increased ribozyme activity in crowded solutions.

Noncoding RNAs must function in the crowded environment of the cell. Previous small-angle x-ray scattering experiments showed that molecular crowders stabilize the structure of the Azoarcus group I ribozyme, allowing the ribozyme to fold at low physiological Mg(2+) concentrations. Here, we used an RNA cleavage assay to show that the PEG and Ficoll crowder molecules increased the biochemical activity of the ribozyme, whereas sucrose did not. Crowding lowered the Mg(2+) threshold at which activity was detected and increased total RNA cleavage at high Mg(2+) concentrations sufficient to fold the RNA in crowded or dilute solution. After correcting for solution viscosity, the observed reaction rate was proportional to the fraction of active ribozyme. We conclude that molecular crowders stabilize the native ribozyme and favor the active structure relative to compact inactive folding intermediates.

Cell mechanics control rapid transitions between blebs and lamellipodia during migration.

Protrusion formation is an essential step during cell migration. Cells migrating in three-dimensional environments and in vivo can form a wide variety of protrusion types, including actin polymerization-driven lamellipodia, and contractility-driven blebs. The ability to switch between different protrusions has been proposed to facilitate motility in complex environments and to promote cancer dissemination. However, plasticity in protrusion formation has so far mostly been investigated in the context of transitions between amoeboid and mesenchymal migration modes, which involve substantial changes in overall cell morphology. As a result, the minimal requirements of transitions between blebs and lamellipodia, as well as the time scales on which they occur, remain unknown. To address these questions, we investigated protrusion switching during cell migration at the single cell level. Using cells that can be induced to form either blebs or lamellipodia, we systematically assessed the mechanical requirements, as well as the dynamics, of switching between protrusion types. We demonstrate that shifting the balance between actin protrusivity and actomyosin contractility leads to immediate transitions between blebs and lamellipodia in migrating cells. Switching occurred without changes in global cell shape, polarity, or cell adhesion. Furthermore, rapid transitions between blebs and lamellipodia could also be triggered upon changes in substrate adhesion during migration on micropatterned surfaces. Together, our data reveal that the type of protrusion formed by migrating cells can be dynamically controlled independently of overall cell morphology, suggesting that protrusion formation is an autonomous module in the regulatory network that controls the plasticity of cell migration.

Multifunctional RNA nanoparticles.

Our recent advancements in RNA nanotechnology introduced novel nanoscaffolds (nanorings); however, the potential of their use for biomedical applications was never fully revealed. As presented here, besides functionalization with multiple different short interfering RNAs for combinatorial RNA interference (e.g., against multiple HIV-1 genes), nanorings also allow simultaneous embedment of assorted RNA aptamers, fluorescent dyes, proteins, as well as recently developed RNA-DNA hybrids aimed to conditionally activate multiple split functionalities inside cells.

Micropatterned multicolor dynamically adhesive substrates to control cell adhesion and multicellular organization.

We present a novel technique to examine cell-cell interactions and directed cell migration using micropatterned substrates of three distinct regions: an adhesive region, a nonadhesive region, and a dynamically adhesive region switched by addition of a soluble factor to the medium. Combining microcontact printing with avidin-biotin capture chemistry, we pattern nonadhesive regions of avidin that become adhesive through the capture of biotinylated fibronectin. Our strategy overcomes several limitations of current two-color dynamically adhesive substrates by incorporating a third, permanently nonadhesive region. Having three spatially and functionally distinct regions allows for the realization of more complex configurations of cellular cocultures as well as intricate interface geometries between two cell populations for diverse heterotypic cell-cell interaction studies. We can now achieve spatial control over the path and direction of migration in addition to temporal control of the onset of migration, enabling studies that better recapitulate coordinated multicellular migration and organization in vitro. We confirm that cellular behavior is unaltered on captured biotinylated fibronectin as compared to printed fibronectin by examining the cells' ability to spread, form adhesions, and migrate. We demonstrate the versatility of this approach in studies of migration and cellular cocultures, and further highlight its utility by probing Notch-Delta juxtacrine signaling at a patterned interface.

Left ventricular torsion shear angle volume analysis in patients with hypertension: a global approach for LV diastolic function.

BACKGROUND: Torsion shear angle φ is an important measure of left ventricular (LV) systolic and diastolic functions. Here we provide a novel index utilizing LV normalized torsion shear angle φ ^ volume V ^ loop to assess LV diastolic functional properties. We defined the area within φ ^ V ^ loop as torsion hysteresis area, and hypothesized that it may be an important global parameter of diastolic function. We evaluated the φ ^ changes to increased V ^ during early diastole - d φ ^ / d V ^ as a potential measure of LV suction. METHODS: Sixty resistant hypertension patients (HTN), forty control volunteers were studied using cardiovascular magnetic resonance with tissue tagging. Volumetric and torsional parameters were evaluated. RESULTS: HTN demonstrated concentric remodeling with preserved ejection fraction. HTN had significantly decreased normalized early filling rate, early diastolic mitral annulus velocity and E/A (1.33 ± 1.13 vs. 2.19 ± 1.07, P < 0.0001) vs. control. Torsion hysteresis area was greater (0.11 ± 0.07 vs. 0.079 ± 0.045, P < 0.001) and peak - d φ ^ / d V ^ at early diastole was higher (10.46 ± 8.51 vs. 6.29 ± 3.85, P = 0.002) than control. Torsion hysteresis area was significantly correlated with E/A (r = -0.23, P = 0.025). Thirteen HTN patients had both E/A ratio < 1.12 (Control mean E/A-1SD) and torsion hysteresis area > 0.12 (Control mean torsion hysteresis area + 1SD). CONCLUSIONS: Torsion hysteresis area and peak early diastolic - d φ ^ / d V ^ were significantly increased in hypertensive concentric remodeling. The φ ^ V ^ loop takes into account the active and passive recoil processes of LV diastolic and systolic phases, therefore provides a new global description of LV diastolic function.

Folding path of P5abc RNA involves direct coupling of secondary and tertiary structures.

Folding mechanisms in which secondary structures are stabilized through the formation of tertiary interactions are well documented in protein folding but challenge the folding hierarchy normally assumed for RNA. However, it is increasingly clear that RNA could fold by a similar mechanism. P5abc, a small independently folding tertiary domain of the Tetrahymena thermophila group I ribozyme, is known to fold by a secondary structure rearrangement involving helix P5c. However, the extent of this rearrangement and the precise stage of folding that triggers it are unknown. We use experiments and simulations to show that the P5c helix switches to the native secondary structure late in the folding pathway and is directly coupled to the formation of tertiary interactions in the A-rich bulge. P5c mutations show that the switch in P5c is not rate-determining and suggest that non-native interactions in P5c aid folding rather than impede it. Our study illustrates that despite significant differences in the building blocks of proteins and RNA, there may be common ways in which they self-assemble.

Paradoxical embolism interrupted.

A 41 year-old African-American male presented with syncope preceded by shortness of breath at outside facility and transferred to us for management of extensive pulmonary embolism with unstable vital signs. Electrocardiogram showed sinus tachycardia with S1Q3T3 pattern. A transthoracic echocardiogram revealed a freely mobile strand like mass in the left atrium. A transoesophageal echocardiogram showed a very large freely mobile thrombus extending from a patent foramen ovale in to the left atrium. He underwent emergent surgery for the extraction of clot followed by thromboembolectomy from both pulmonary arteries. He made a remarkable recovery and was discharged after seven days of hospital stay.

Impact of cost on prescribing diabetes medications for older adults with type 2 diabetes in the outpatient setting.

BACKGROUND: Newer diabetes medications have cardiorenal benefits beyond blood sugar lowering that make them a preferred treatment option in many patients. Despite this, studies have shown that prescribing of these medications remains suboptimal with medication costs being hypothesized as a reason for underutilization. OBJECTIVE: To understand clinicians' decision-making processes for prescribing diabetes medications in older adults, focusing on higher cost medications. METHODS: Observations of patient encounters and semi-structured interviews were conducted with clinicians from primary care, endocrinology, and geriatrics to elucidate themes into diabetes medication prescribing. A qualitative descriptive approach was used to analyze the data from interviews using an inductive coding scheme with themes derived from the data. RESULTS: Twenty-one interviews were conducted. Five themes were identified: 1) out-of-pocket costs drive prescribing decisions 2) out-of-pocket costs can be variable due to changing insurance plans or changing coverage 3) clinicians have difficulty with determining patient-specific out-of-pocket costs 4) clinicians manage the tradeoffs existing between cost, efficacy, and safety and 5) clinicians can use cost-modifying strategies such as patient assistance. CONCLUSION: Addressing the challenges that medication costs pose to prescribing evidence-based medications for type 2 diabetes is necessary to optimize diabetes care for older adults.

Accurate Modeling of Ejection Fraction and Stroke Volume With Mobile Phone Auscultation: Prospective Case-Control Study.

BACKGROUND: Heart failure (HF) contributes greatly to morbidity, mortality, and health care costs worldwide. Hospital readmission rates are tracked closely and determine federal reimbursement dollars. No current modality or technology allows for accurate measurement of relevant HF parameters in ambulatory, rural, or underserved settings. This limits the use of telehealth to diagnose or monitor HF in ambulatory patients. OBJECTIVE: This study describes a novel HF diagnostic technology using audio recordings from a standard mobile phone. METHODS: This prospective study of acoustic microphone recordings enrolled convenience samples of patients from 2 different clinical sites in 2 separate areas of the United States. Recordings were obtained at the aortic (second intercostal) site with the patient sitting upright. The team used recordings to create predictive algorithms using physics-based (not neural networks) models. The analysis matched mobile phone acoustic data to ejection fraction (EF) and stroke volume (SV) as evaluated by echocardiograms. Using the physics-based approach to determine features eliminates the need for neural networks and overfitting strategies entirely, potentially offering advantages in data efficiency, model stability, regulatory visibility, and physical insightfulness. RESULTS: Recordings were obtained from 113 participants. No recordings were excluded due to background noise or for any other reason. Participants had diverse racial backgrounds and body surface areas. Reliable echocardiogram data were available for EF from 113 patients and for SV from 65 patients. The mean age of the EF cohort was 66.3 (SD 13.3) years, with female patients comprising 38.3% (43/113) of the group. Using an EF cutoff of ≤40% versus >40%, the model (using 4 features) had an area under the receiver operating curve (AUROC) of 0.955, sensitivity of 0.952, specificity of 0.958, and accuracy of 0.956. The mean age of the SV cohort was 65.5 (SD 12.7) years, with female patients comprising 34% (38/65) of the group. Using a clinically relevant SV cutoff of <50 mL versus >50 mL, the model (using 3 features) had an AUROC of 0.922, sensitivity of 1.000, specificity of 0.844, and accuracy of 0.923. Acoustics frequencies associated with SV were observed to be higher than those associated with EF and, therefore, were less likely to pass through the tissue without distortion. CONCLUSIONS: This work describes the use of mobile phone auscultation recordings obtained with unaltered cellular microphones. The analysis reproduced the estimates of EF and SV with impressive accuracy. This technology will be further developed into a mobile app that could bring screening and monitoring of HF to several clinical settings, such as home or telehealth, rural, remote, and underserved areas across the globe. This would bring high-quality diagnostic methods to patients with HF using equipment they already own and in situations where no other diagnostic and monitoring options exist.

Mechanical regulation of cell function with geometrically modulated elastomeric substrates.

We report the establishment of a library of micromolded elastomeric micropost arrays to modulate substrate rigidity independently of effects on adhesive and other material surface properties. We demonstrated that micropost rigidity impacts cell morphology, focal adhesions, cytoskeletal contractility and stem cell differentiation. Furthermore, early changes in cytoskeletal contractility predicted later stem cell fate decisions in single cells.

Diagnostic performance of cardiac stress perfusion MRI in the detection of coronary artery disease using fractional flow reserve as the reference standard: a meta-analysis.

OBJECTIVE. This is an analysis of pooled studies for the determination of the test characteristics of stress perfusion cardiac MRI in the diagnosis of flow-limiting obstructive coronary artery disease (CAD) using fractional flow reserve (FFR) at catheter coronary angiography as the reference standard. MATERIALS AND METHODS. Traditionally, planimetric measurement of coronary stenosis at catheter coronary angiography has been considered the reference standard and has been used to verify the diagnostic characteristics of gatekeeper tests. FFR is a physiologic measure of flow limitation and is considered a more authentic reference standard in the diagnosis of CAD. The emergence of a new reference standard questions the true diagnostic accuracy of gatekeeper tests. A systematic literature review was performed for qualifying studies. The DerSimonian-Laird random effects model and a random-effects symmetric summary receiver operating characteristic curve analysis were performed. RESULTS. Twelve studies (761 patients) met the inclusion criteria. Four hundred six stenotic coronary arteries had FFR less than 0.75. Perfusion stress MRI has a sensitivity of 89.1% (95% CI, 84-93%) and specificity of 84.9% (95% CI, 76.6-91.1%) on a patient basis and a sensitivity of 87.7% (95% CI, 84.4-90.6%) and specificity of 88.6% (95% CI, 86.7-90.4%) on a coronary territory basis. CONCLUSION. Stress perfusion MRI remains an accurate test for the detection of flow-limiting stenosis when adjudicated by a physiologic reference standard.

Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid.

Activation of T cells to the capsid of adeno-associated virus (AAV) serotype 2 vectors has been implicated in liver toxicity in a recent human gene therapy trial of hemophilia B. To further investigate this kind of toxicity, we evaluated T-cell responses to AAV capsids after intramuscular injection of vectors into mice and nonhuman primates. High levels of T cells specific to capsids of vectors based on AAV2 and a phylogenetically related AAV variant were detected. Vectors from other AAV clades such as AAV8 (ref. 3), however, did not lead to activation of capsid-specific T cells. Through the generation of AAV2-AAV8 hybrids and the creation of site-directed mutations, we mapped the domain that directs the activation of T cells to the RXXR motif on VP3, which was previously shown to confer binding of the virion to heparan sulfate proteoglycan (HSPG). Evaluation of natural and engineered AAV variants showed direct correlations between heparin binding, uptake into human dendritic cells (DCs) and activation of capsid-specific T cells. The role of heparin binding in the activation of CD8(+) T cells may be useful in modulating the immunogenicity of antigens and improving the safety profile of existing AAV vectors for gene therapy.

Colchicine toxicity precipitated by interaction with sunitinib.

WHAT IS KNOWN AND OBJECTIVE: Colchicine is an anti-inflammatory agent used primarily in treatment of gout and familial Mediterranean fever. Toxicity is uncommon, and depends on dose, hepatic or renal impairment, co-administration with P-glycoprotein or CYP3A4 inhibitors and route of administration. In patients taking p-glycoprotein inhibitors, maximum recommended dose is 0·3 mg per day. In renal or hepatic impairment, recommendation is to avoid concomitant administration of p-glycoprotein inhibitors and colchicine. CASE SUMMARY: We present an 82 year old patient, with a history of gout, chronic kidney disease and recurrent renal cell carcinoma who was admitted with features of colchicine toxicity after taking a cumulative dose of 41·4 mg over ten days, and taking sunitinib 50 mg daily from day seven of his high dose colchicine regimen. Symptoms started after commencing his cycle of sunitinib, which he had taken in 14 day cycles for many years. He developed severe diarrhea, normal anion gap metabolic acidosis, fever, pneumonia, white cell abnormalities including 30% bands and toxic granulation with Dohle bodies. Red cell abnormalities included anemia, burr cells and acanthocytosis. He also developed acute cardiovascular collapse with hypotension and acute systolic heart failure. Cardiac catheterization showed previously known coronary artery disease, with no significant progression to explain degree of cardiovascular collapse. WHAT IS NEW AND CONCLUSION: P-glycoprotein inhibition by sunitinib has been demonstrated. Interaction with colchicine metabolism precipitated colchicine toxicity in this case. Knowledge of p-glycoprotein and its role in drug interactions and potential drug toxicity may not be widespread among clinicians. We report the first case of colchicine toxicity precipitated by interaction with a tyrosine kinase inhibitor.

Sonographic Assessment of Hand Injuries: Diagnostic Accuracy and Review of Pathology.

Background: The high soft-tissue contrast of magnetic resonance imaging (MRI) makes it useful for evaluation of hand injuries, but its limitations include cost, imaging artifacts, and patient claustrophobia. Ultrasound is readily available, fast, noninvasive, and radiation free, but its utility for the evaluation of hand soft-tissue injury and pathology is less well known. Purpose: We sought to examine the accuracy of ultrasound for the evaluation of hand injury at a single institution. Methods: We queried a radiology information system for ultrasound cases between 2014 and 2020 at a tertiary care institution using the keyword "hand" and injury terms. We performed a retrospective chart review of cases found according to the type of injury detected on ultrasound. To evaluate the diagnostic accuracy of ultrasound in hand injury and pathology, we recorded postimaging clinical diagnoses and surgical findings. Results: We found 154 patients who underwent ultrasound for hand injuries and had confirmed surgical diagnosis and/or robust clinical follow-up. Tendon injury was the most commonly diagnosed condition on ultrasound (70/154); others detected were retained foreign body (31), mass (21), ligamentous injury (9), pulley injury (8), nerve injury (11), and traumatic arthropathy (4). Ultrasound correctly characterized hand injury in 150/154 cases (97.4%) based on surgical and/or clinical follow-up. Ultrasound failed to diagnose 3 cases of partial tendon tear and 1 case of digital nerve injury. Conclusion: In this retrospective, single-institution review, ultrasound was found to be highly accurate in the detection of soft tissue hand injury and pathology, demonstrating a high concordance rate with surgical and clinical findings. Further study is warranted.

Decoupling diffusional from dimensional control of signaling in 3D culture reveals a role for myosin in tubulogenesis.

We present a novel microfabricated platform to culture cells within arrays of micrometer-scale three-dimensional (3D) extracellular matrix scaffolds (microgels). These microscale cultures eliminate diffusion barriers that are intrinsic to traditional 3D culture systems (macrogels) and enable uniform cytokine stimulation of the entire culture population, as well as allow immunolabeling, imaging and population-based biochemical assays across the relatively coplanar microgels. Examining early signaling associated with hepatocyte growth factor (HGF)-mediated scattering and tubulogenesis of MDCK cells revealed that 3D culture modulates cellular responses both through dimensionality and altered stimulation rates. Comparing responses in 2D culture, microgels and macrogels demonstrated that HGF-induced ERK signaling was driven by the dynamics of stimulation and not by whether cells were in a 2D or 3D environment, and that this ERK signaling was equally important for HGF-induced cell scattering on 2D substrates and tubulogenesis in 3D. By contrast, we discovered a specific HGF-induced increase in myosin expression leading to sustained downregulation of myosin activity that occurred only within 3D contexts and was required for 3D tubulogenesis but not 2D scattering. Interestingly, although absent in cells on collagen-coated plates, downregulation of myosin activity also occurred for cells on collagen gels, but was transient and mediated by a combination of myosin dephosphorylation and enhanced myosin expression. Furthermore, upregulating myosin activity via siRNA targeted to a myosin phosphatase did not attenuate scattering in 2D but did inhibit tubulogenesis in 3D. Together, these results demonstrate that cellular responses to soluble cues in 3D culture are regulated by both rates of stimulation and by matrix dimensionality, and highlight the importance of decoupling these effects to identify early signals relevant to cellular function in 3D environments.