Suspicious Ultrasound-Occult Non-Calcified Mammographic Masses, Asymmetries, and Architectural Distortions Are Moderate Probability for Malignancy.

Suspicious non-calcified mammographic findings have not been evaluated with modern mammographic technique, and the purpose of this work is to compare the likelihood of malignancy for those findings. To do this, 5018 consecutive mammographically guided biopsies performed during 2016-2019 at a large metropolitan, community-based hospital system were retrospectively reviewed. In total, 4396 were excluded for targeting calcifications, insufficient follow-up, or missing data. Thirty-seven of 126 masses (29.4%) were malignant, 44 of 194 asymmetries (22.7%) were malignant, and 77 of 302 architectural distortions (AD, 25.5%) were malignant. The combined likelihood of malignancy was 25.4%. Older age was associated with a higher likelihood of malignancy for each imaging finding type (all p ≤ 0.006), and a possible ultrasound correlation was associated with a higher likelihood of malignancy when all findings were considered together (p = 0.012). Two-view asymmetries were more frequently malignant than one-view asymmetries (p = 0.03). There were two false-negative biopsies (98.7% sensitivity and 100% specificity). In conclusion, the 25.4% likelihood of malignancy confirms the recommendation for biopsy of suspicious, ultrasound-occult, mammographic findings. Mammographically guided biopsies were highly sensitive and specific in this study. Older patient age and a possible ultrasound correlation should raise concern given the increased likelihood of malignancy in those scenarios.

Coordinated metabolic responses to cyclophilin D deletion in the developing heart.

In the embryonic heart, the activation of the mitochondrial electron transport chain (ETC) coincides with the closure of the cyclophilin D (CypD) regulated mitochondrial permeability transition pore (mPTP). However, it remains to be established whether the absence of CypD has a regulatory effect on mitochondria during cardiac development. Using a variety of assays to analyze cardiac tissue from wildtype and CypD knockout mice from embryonic day (E)9.5 to adult, we found that mitochondrial structure, function, and metabolism show distinct transitions. Deletion of CypD altered the timing of these transitions as the mPTP was closed at all ages, leading to coupled ETC activity in the early embryo, decreased citrate synthase activity, and an altered metabolome particularly after birth. Our results suggest that manipulating CypD activity may control myocyte proliferation and differentiation and could be a tool to increase ATP production and cardiac function in immature hearts.

Effect of epiretinal electrical stimulation on the glial cells in a rabbit retinal eyecup model.

Introduction: We examined how pulse train electrical stimulation of the inner surface of the rabbit retina effected the resident glial cells. We used a rabbit retinal eyecup preparation model, transparent stimulus electrodes, and optical coherence tomography (OCT). The endfeet of Müller glia processes line the inner limiting membrane (ILM). Methods: To examine how epiretinal electrode stimulation affected the Müller glia, we labeled them post stimulation using antibodies against soluble glutamine synthetase (GS). After 5 min 50 Hz pulse train stimulation 30 µm from the surface, the retina was fixed, immunostained for Müller glia, and examined using confocal microscopic reconstruction. Stimulus pulse charge densities between 133-749 µC/cm2/ph were examined. Results: High charge density stimulation (442-749 µC/cm2/ph) caused significant losses in the GS immunofluorescence of the Müller glia endfeet under the electrode. This loss of immunofluorescence was correlated with stimuli causing ILM detachment when measured using OCT. Müller cells show potassium conductances at rest that are blocked by barium ions. Using 30 msec 20 µA stimulus current pulses across the eyecup, the change in transretinal resistance was examined by adding barium to the Ringer. Barium caused little change in the transretinal resistance, suggesting under low charge density stimulus pulse conditions, the Müller cell radial conductance pathway for these stimulus currents was small. To examine how epiretinal electrode stimulation affected the microglia, we used lectin staining 0-4 h post stimulation. After stimulation at high charge densities 749 µC/cm2/ph, the microglia under the electrode appeared rounded, while the local microglia outside the electrode responded to the stimulated retina by process orientation inwards in a ring by 30 min post stimulation. Discussion: Our study of glial cells in a rabbit eyecup model using transparent electrode imaging suggests that epiretinal electrical stimulation at high pulse charge densities, can injure the Müller and microglia cells lining the inner retinal surface in addition to ganglion cells.

Endoscopic closure of a recto-pelvic fistula with a cardiac septal occluder device.

Video 1Endoscopic closure of a recto-pelvic fistula with a cardiac septal occluder device in a patient for whom other surgical and endoscopic interventions had failed.

Breast Imaging Patient Demographics at a Major Comprehensive Cancer Center Before and During the COVID-19 Pandemic.

OBJECTIVE: The coronavirus disease of 2019 (COVID-19) pandemic disproportionately affected certain vulnerable communities. The purpose of our study was to determine how COVID-19 affected the socioeconomic demographics of breast imaging patients at a major comprehensive cancer center. METHODS: This retrospective cohort study compared female patients who underwent screening mammograms, diagnostic mammograms, breast ultrasound, or breast MRI during the following time periods: prepandemic (February 1, 2018, through February 29, 2020), acute pandemic (March 1, 2020, through June 30, 2020), subacute pandemic (August 1, 2020, through December 31, 2020), and chronic pandemic (January 1, 2021, through June 30, 2022). Statistics were performed using the generalized estimating equations approach. RESULTS: A total of 74,398 female patients (mean age, 55.6 ± 12.4 years) underwent 238,776 total breast imaging examinations. For screening mammograms, Hispanics represented 27.1% (9,197 of 33,960) of patients in the prepandemic time period compared with 16.7% (604 of 3,621) in the acute pandemic time period, 18.7% (1,835 of 9,830) in the subacute pandemic time period, and 24.3% (7,492 of 30,869) in the chronic pandemic time period (all P < .0001). Self-pay patients saw similar declines for screening mammograms during the same time periods: 21.7% (7,375 of 33,960), 7.9% (286 of 3,621), 9.5% (933 of 9,830), and 17.4% (5,357 of 30,869), respectively (all P < .0001, compared with the prepandemic time period). Similarly dramatic trends were not observed for race or other imaging examinations. DISCUSSION: At our cancer center, Hispanics and self-pay patients were disproportionately affected by the COVID-19 pandemic. Strategies to improve health inequities are needed.

Effect of direct voltage induction by low-frequency security systems on neurostimulator lead.

Low-frequency (LF) security systems, such as antitheft electronic article surveillance (EAS) gates emit strong magnetic fields that could potentially interfere with neurostimulator operation. Some patients reported pain and shocking sensations near EAS gates, even after they turned off their pulse generator. To investigate the direct voltage induction of EAS systems on neurostimulator leads, we evaluated voltages induced by two EAS systems (14 kHz continuous wave or 58 kHz pulsed) on a 40 cm sacral neurostimulator lead formed in a circular loop attached to a pulse generator that was turned off. The lead and neurostimulator were mounted in a saline-filled rectangular phantom placed within electromagnetic fields emitted by EAS systems. The measured voltage waveforms were applied to computational models of spinal nerve axons to predict whether these voltages may evoke action potentials. Additional in vitro testing was performed on the semicircular lead geometry, to study the effect of lead geometry on EAS induced voltages. While standard neurostimulator testing per ISO 14708-3:2017 recommends electromagnetic compatibility testing with LF magnetic fields for induction of malfunctions of the active electronic circuitry while generating intended stimulating pulses, our results show that close to the EAS antenna frames, the induced voltage on the lead could be strong enough to evoke action potentials, even with the pulse generator turned off. This work suggests that patient reports of pain and shocking sensations when near EAS systems could also be correlated with the direct EAS-induced voltage on neurostimulator lead.

Identification of novel NRF2-dependent genes as regulators of lead and arsenic toxicity in neural progenitor cells.

Lead (Pb) and arsenic (As) are prevalent metal contaminants in the environment. Exposures to these metals are associated with impaired neuronal functions and adverse effects on neurodevelopment in children. However, the molecular mechanisms by which Pb and As impair neuronal functions remain poorly understood. Here, we identified F2RL2, TRIM16L, and PANX2 as novel targets of Nuclear factor erythroid 2-related factor 2 (NRF2)-the master transcriptional factor for the oxidative stress response-that are commonly upregulated with both Pb and As in human neural progenitor cells (NPCs). Using a ChIP (Chromatin immunoprecipitation)-qPCR assay, we showed that NRF2 directly binds to the promoter region of F2RL2, TRIM16L, and PANX2 to regulate expression of these genes. We demonstrated that F2RL2, PANX2, and TRIM16L have differential effects on cell death, proliferation, and differentiation of NPCs in both the presence and absence of metal exposures, highlighting their roles in regulating NPC function. Furthermore, the analyses of the transcriptomic data on NPCs derived from autism spectrum disorder (ASD) patients revealed that dysregulation of F2RL2, TRIM16L, and PANX2 was associated with ASD genetic backgrounds and ASD risk genes. Our findings revealed that Pb and As induce a shared NRF2-dependent transcriptional response in NPCs and identified novel genes regulating NPC function. While further in vivo studies are warranted, this study provides a novel mechanism linking metal exposures to NPC function and identifies potential genes of interest in the context of neurodevelopment.

Hypereosinophilia as a presenting sign of advanced pancreatic adenocarcinoma: a rare, severe presentation.

Pancreatic adenocarcinoma (PA) is the third most lethal malignancy worldwide with only a 7.7% 5-year survival rate. Prognosis is poor with more than 50% of patients presenting with stage IV disease. Despite focused attention on early detection and treatment, pathogenesis and early symptomatology are not well described. In addition to prodromal symptoms, hypereosinophilia has been identified as a marker of malignancy in both PA and other solid tumour and haematological malignancies. Peripheral hypereosinophilia (PH) secondary to solid organ tumours, however, is rare, with only four cases of PA reported to date. We present a case of advanced PA with associated severe PH in a man in his early 50s. Time from diagnosis to death in this patient was only 6 weeks, emphasising the need to consider malignancy in the differential diagnosis for a patient that presents with a severe PH of unknown origin.

Revealing invisible cell phenotypes with conditional generative modeling.

Biological sciences, drug discovery and medicine rely heavily on cell phenotype perturbation and microscope observation. However, most cellular phenotypic changes are subtle and thus hidden from us by natural cell variability: two cells in the same condition already look different. In this study, we show that conditional generative models can be used to transform an image of cells from any one condition to another, thus canceling cell variability. We visually and quantitatively validate that the principle of synthetic cell perturbation works on discernible cases. We then illustrate its effectiveness in displaying otherwise invisible cell phenotypes triggered by blood cells under parasite infection, or by the presence of a disease-causing pathological mutation in differentiated neurons derived from iPSCs, or by low concentration drug treatments. The proposed approach, easy to use and robust, opens the door to more accessible discovery of biological and disease biomarkers.

Stepwise Implementation of 2D Synthesized Screening Mammography and Its Effect on Stereotactic Biopsy of Microcalcifications.

RATIONALE AND OBJECTIVES: Information evaluating the efficacy of 2D synthesized mammography (2Ds) reconstructions in microcalcification detection is limited. This study used stereotactic biopsy data for microcalcifications to evaluate the stepwise implementation of 2Ds in screening mammography. The study aim was to identify whether 2Ds + digital breast tomosynthesis (DBT) is non-inferior to 2D digital mammography (2DM) + 2Ds + DBT, 2DM + DBT, and 2DM in identifying microcalcifications undergoing further diagnostic imaging and stereotactic biopsy. MATERIALS AND METHODS: Retrospective stereotactic biopsy data were extracted following 151,736 screening mammograms of healthy women (average age, 56.3 years; range, 30-89 years), performed between 2012 and 2019. The stereotactic biopsy data were separated into 2DM, 2DM + DBT, 2DM + 2Ds + DBT, and 2Ds + DBT arms and examined using Fisher's exact test to compare the detection rates of all cancers, invasive cancers, DCIS, and ADH between modalities for patients undergoing stereotactic biopsy of microcalcifications. RESULTS: No statistical significance in cancer detection was seen for 2Ds + DBT among those calcifications that underwent stereotactic biopsy when comparing the 2Ds + DBT to 2DM, 2DM + DBT, and 2DM + 2Ds + DBT imaging combinations. CONCLUSION: These data suggest that 2Ds + DBT is non-inferior to 2DM + DBT in detecting microcalcifications that will undergo stereotactic biopsy.

Comparison of prone and upright, stereotactic, and tomosynthesis-guided biopsies with secondary analysis of ultrasound-occult architectural distortions.

OBJECTIVES: Compare prone and upright, stereotactic, and tomosynthesis-guided vacuum-assisted breast biopsies (prone DM-VABB, prone DBT-VABB, upright DM-VABB, and upright DBT-VABB) in a community-practice setting and review outcomes of ultrasound-occult architectural distortions (AD). METHODS: Consecutive biopsies performed at two community-based breast centers from 2016 to 2019 were retrospectively reviewed. Technical details of each procedure and patient outcomes were recorded. Separate analyses were performed for ultrasound-occult ADs. Two sample t-tests and Fisher's exact test facilitated comparisons. RESULTS: A total of 1133 patients underwent 369 prone DM-VABB, 324 prone DBT-VABB, 437 upright DM-VABB, and 123 upright DBT-VABB with 99.2%, 100%, 99.3%, and 99.2% success, respectively (p-values > 0.25). Mean lesion targeting times were greater for prone biopsy (minutes: 6.94 prone DM-VABB, 8.54 prone DBT-VABB, 5.52 upright DM-VABB, and 5.51 upright DBT-VABB; p-values < 0.001), yielding longer total prone procedure times for prone biopsy (p < 0.001). Compared to DM-VABB, DBT-VABB used fewer exposures (p < 0.001) and more commonly targeted AD, asymmetries, or masses (p < 0.001). Malignancy rates were similar between procedures: prone DM-VABB 22.4%, prone DBT-VABB 21.9%, upright DM-VABB 22.8%, and upright DBT-VABB 17.2% (p-values > 0.19). One hundred forty of the 1133 patients underwent 145 biopsies for ultrasound-occult AD (143 DBT-VABB and 2 DM-VABB). Biopsy yielded 27 malignancies and 47 high-risk lesions (74 of 145, 51%). Malignancy rate was 20.7% after surgical upgrade of one benign-discordant and two high-risk lesions. CONCLUSIONS: All biopsy procedure types were extremely successful. The 20.7% malignancy rate for ultrasound-occult AD confirms a management recommendation for tissue diagnosis. Upright biopsy was faster than prone biopsy, and DBT-VABB used fewer exposures than DM-VABB. CLINICAL RELEVANCE: Our results highlight important differences between prone DM-VABB, prone DBT-VABB, upright DM-VABB, and upright DBT-VABB. Moreover, the high likelihood of malignancy for ultrasound-occult AD will provide confidence in recommending tissue diagnosis in lieu of observation or clinical follow-up. KEY POINTS: • Upright and prone stereotactic and tomosynthesis-guided breast biopsies were safe and effective in the community-practice setting. • The malignancy rate for ultrasound-occult architectural distortion of 20.7% confirms the management recommendation for biopsy. • Upright procedures were faster than prone procedures, and tomosynthesis-guided biopsy used fewer exposures than stereotactic biopsy.

Cell painting transfer increases screening hit rate.

Drug discovery uses high throughput screening to identify compounds that interact with a molecular target or that alter a phenotype favorably. The cautious selection of molecules used for such a screening is instrumental and is tightly related to the hit rate. In this work, we wondered if cell painting, a general-purpose image-based assay, could be used as an efficient proxy for compound selection, thus increasing the success rate of a specific assay. To this end, we considered cell painting images with 30,000 molecules treatments, and selected compounds that produced a visual effect close to the positive control of an assay, by using the Frechet Inception Distance. We then compared the hit rates of such a preselection with what was actually obtained in real screening campaigns. As a result, cell painting would have permitted a significant increase in the success rate and, even for one of the assays, would have allowed to reach 80% of the hits with 10 times fewer compounds to test. We conclude that images of a cell painting assay can be directly used for compound selection prior to screening, and we provide a simple quantitative approach in order to do so.

Fast and slow light-induced changes in murine outer retina optical coherence tomography: complementary high spatial resolution functional biomarkers.

Fast (seconds) and slow (minutes to hours) optical coherence tomography (OCT) responses to light stimulation have been developed to probe outer retinal function with higher spatial resolution than the classical full-field electroretinogram (ERG). However, the relationships between functional information revealed by OCT and ERG are largely unexplored. In this study, we directly compared the fast and slow OCT responses with the ERG. Fast responses [i.e. the optoretinogram (ORG)] are dominated by reflectance changes in the outer segment (OS) and the inner segment ellipsoid zone (ISez). The ORG OS response has faster kinetics and a higher light sensitivity than the ISez response, and both differ significantly with ERG parameters. Sildenafil-inhibition of phototransduction reduced the ORG light sensitivity, suggesting a complete phototransduction pathway is needed for ORG responses. Slower OCT responses were dominated by light-induced changes in the external limiting membrane to retinal pigment epithelium (ELM-RPE) thickness and photoreceptor-tip hyporeflective band (HB) magnitudes, with the biggest changes occurring after prolonged light stimulation. Mice with high (129S6/ev) vs. low (C57BL/6 J) ATP(adenosine triphosphate) synthesis efficiency show similar fast ORG, but dissimilar slow OCT responses. We propose that the ORG reflects passive physiology, such as water movement from photoreceptors, in response to the photocurrent response (measurable by ERG), whereas the slow OCT responses measure mitochondria-driven physiology in the outer retina, such as dark-provoked water removal from the subretinal space.

ATF6 Activation Reduces Amyloidogenic Transthyretin Secretion through Increased Interactions with Endoplasmic Reticulum Proteostasis Factors.

The extracellular aggregation of destabilized transthyretin (TTR) variants is implicated in the onset and pathogenesis of familial TTR-related amyloid diseases. One strategy to reduce the toxic, extracellular aggregation of TTR is to decrease the population of aggregation-prone proteins secreted from mammalian cells. The stress-independent activation of the unfolded protein response (UPR)-associated transcription factor ATF6 preferentially decreases the secretion and subsequent aggregation of destabilized, aggregation-prone TTR variants. However, the mechanism of this reduced secretion was previously undefined. Here, we implement a mass-spectrometry-based interactomics approach to identify endoplasmic reticulum (ER) proteostasis factors involved in ATF6-dependent reductions in destabilized TTR secretion. We show that ATF6 activation reduces amyloidogenic TTR secretion and subsequent aggregation through a mechanism involving ER retention that is mediated by increased interactions with ATF6-regulated ER proteostasis factors including BiP and PDIA4. Intriguingly, the PDIA4-dependent retention of TTR is independent of both the single TTR cysteine residue and the redox activity of PDIA4, indicating that PDIA4 retains destabilized TTR in the ER through a redox-independent mechanism. Our results define a mechanistic basis to explain the ATF6 activation-dependent reduction in destabilized, amyloidogenic TTR secretion that could be therapeutically accessed to improve treatments of TTR-related amyloid diseases.

Neonatal Hyperoxia Activates Activating Transcription Factor 4 to Stimulate Folate Metabolism and Alveolar Epithelial Type 2 Cell Proliferation.

Oxygen supplementation in preterm infants disrupts alveolar epithelial type 2 (AT2) cell proliferation through poorly understood mechanisms. Here, newborn mice are used to understand how hyperoxia stimulates an early aberrant wave of AT2 cell proliferation that occurs between Postnatal Days (PNDs) 0 and 4. RNA-sequencing analysis of AT2 cells isolated from PND4 mice revealed hyperoxia stimulates expression of mitochondrial-specific methylenetetrahydrofolate dehydrogenase 2 and other genes involved in mitochondrial one-carbon coupled folate metabolism and serine synthesis. The same genes are induced when AT2 cells normally proliferate on PND7 and when they proliferate in response to the mitogen fibroblast growth factor 7. However, hyperoxia selectively stimulated their expression via the stress-responsive activating transcription factor 4 (ATF4). Administration of the mitochondrial superoxide scavenger mitoTEMPO during hyperoxia suppressed ATF4 and thus early AT2 cell proliferation, but it had no effect on normative AT2 cell proliferation seen on PND7. Because ATF4 and methylenetetrahydrofolate dehydrogenase are detected in hyperplastic AT2 cells of preterm infant humans and baboons with bronchopulmonary dysplasia, dampening mitochondrial oxidative stress and ATF4 activation may provide new opportunities for controlling excess AT2 cell proliferation in neonatal lung disease.

Does lateral arm technique decrease the rate of clip migration in stereotactic and tomosynthesis-guided biopsies?

BACKGROUND: Mammography-guided vacuum-assisted biopsies (MGVAB) can be done with stereotaxis or digital breast tomosynthesis guidance. Both methods can be performed with a conventional (CBA) or a lateral arm biopsy approach (LABA). Marker clip migration is relatively frequent in MGVAB (up to 44%), which in cases requiring surgery carries a risk of positive margins and re-excision. We aimed to compare the rates of clip migration and hematoma formation between the CBA and LABA techniques of prone MGVAB. Our HIPAA compliant retrospective study included all consecutive prone MGVAB performed in a single institution over a 20-month period. The LABA approach was used with DBT guidance; CBA utilized DBT or stereotactic guidance. The tissue sampling techniques were otherwise identical. RESULTS: After exclusion, 389 biopsies on 356 patients were analyzed. LABA was done in 97 (25%), and CBA in 292 (75%) cases. There was no statistical difference in clip migration rate with either 1 cm or 2 cm distance cut-off [15% for CBA and 10% for LABA for 1 cm threshold (p = 0.31); 5.8% or CBA and 3.1% or LABA for 2 cm threshold (p = 0.43)]. There was no difference in the rate of hematoma formation (57.5% in CDB and 50.5% in LABA, p = 0.24). The rates of technical failure were similar for both techniques (1.7% for CBA and 3% for LABA) with a combined failure rate of 1%. CONCLUSIONS: LABA and CBA had no statistical difference in clip migration or hematoma formation rates. Both techniques had similar success rates and may be helpful in different clinical situations.

A Deep Learning Approach to Re-create Raw Full-Field Digital Mammograms for Breast Density and Texture Analysis.

PURPOSE: To develop a computational approach to re-create rarely stored for-processing (raw) digital mammograms from routinely stored for-presentation (processed) mammograms. MATERIALS AND METHODS: In this retrospective study, pairs of raw and processed mammograms collected in 884 women (mean age, 57 years ± 10 [standard deviation]; 3713 mammograms) from October 5, 2017, to August 1, 2018, were examined. Mammograms were split 3088 for training and 625 for testing. A deep learning approach based on a U-Net convolutional network and kernel regression was developed to estimate the raw images. The estimated raw images were compared with the originals by four image error and similarity metrics, breast density calculations, and 29 widely used texture features. RESULTS: In the testing dataset, the estimated raw images had small normalized mean absolute error (0.022 ± 0.015), scaled mean absolute error (0.134 ± 0.078) and mean absolute percentage error (0.115 ± 0.059), and a high structural similarity index (0.986 ± 0.007) for the breast portion compared with the original raw images. The estimated and original raw images had a strong correlation in breast density percentage (Pearson r = 0.946) and a strong agreement in breast density grade (Cohen κ = 0.875). The estimated images had satisfactory correlations with the originals in 23 texture features (Pearson r ≥ 0.503 or Spearman ρ ≥ 0.705) and were well complemented by processed images for the other six features. CONCLUSION: This deep learning approach performed well in re-creating raw mammograms with strong agreement in four image evaluation metrics, breast density, and the majority of 29 widely used texture features.Keywords: Mammography, Breast, Supervised Learning, Convolutional Neural Network (CNN), Deep learning algorithms, Machine Learning AlgorithmsSee also the commentary by Chan in this issue.Supplemental material is available for this article.©RSNA, 2021.

Coordination of endothelial cell positioning and fate specification by the epicardium.

The organization of an integrated coronary vasculature requires the specification of immature endothelial cells (ECs) into arterial and venous fates based on their localization within the heart. It remains unclear how spatial information controls EC identity and behavior. Here we use single-cell RNA sequencing at key developmental timepoints to interrogate cellular contributions to coronary vessel patterning and maturation. We perform transcriptional profiling to define a heterogenous population of epicardium-derived cells (EPDCs) that express unique chemokine signatures. We identify a population of Slit2+ EPDCs that emerge following epithelial-to-mesenchymal transition (EMT), which we term vascular guidepost cells. We show that the expression of guidepost-derived chemokines such as Slit2 are induced in epicardial cells undergoing EMT, while mesothelium-derived chemokines are silenced. We demonstrate that epicardium-specific deletion of myocardin-related transcription factors in mouse embryos disrupts the expression of key guidance cues and alters EPDC-EC signaling, leading to the persistence of an immature angiogenic EC identity and inappropriate accumulation of ECs on the epicardial surface. Our study suggests that EC pathfinding and fate specification is controlled by a common mechanism and guided by paracrine signaling from EPDCs linking epicardial EMT to EC localization and fate specification in the developing heart.