ptx3a+ fibroblast/epicardial cells provide a transient macrophage niche to promote heart regeneration.

Macrophages conduct critical roles in heart repair, but the niche required to nurture and anchor them is poorly studied. Here, we investigated the macrophage niche in the regenerating heart. We analyzed cell-cell interactions through published single-cell RNA sequencing datasets and identified a strong interaction between fibroblast/epicardial (Fb/Epi) cells and macrophages. We further visualized the association of macrophages with Fb/Epi cells and the blockage of macrophage response without Fb/Epi cells in the regenerating zebrafish heart. Moreover, we found that ptx3a+ epicardial cells associate with reparative macrophages, and their depletion resulted in fewer reparative macrophages. Further, we identified csf1a expression in ptx3a+ cells and determined that pharmacological inhibition of the csf1a pathway or csf1a knockout blocked the reparative macrophage response. Moreover, we found that genetic overexpression of csf1a enhanced the reparative macrophage response with or without heart injury. Altogether, our studies illuminate a cardiac Fb/Epi niche, which mediates a beneficial macrophage response after heart injury.

Neutrophils facilitate the epicardial regenerative response after zebrafish heart injury.

Despite extensive studies on endogenous heart regeneration within the past 20 years, the players involved in initiating early regeneration events are far from clear. Here, we assessed the function of neutrophils, the first-responder cells to tissue damage, during zebrafish heart regeneration. We detected rapid neutrophil mobilization to the injury site after ventricular amputation, peaking at 1-day post-amputation (dpa) and resolving by 3 dpa. Further analyses indicated neutrophil mobilization coincides with peak epicardial cell proliferation, and recruited neutrophils associated with activated, expanding epicardial cells at 1 dpa. Neutrophil depletion inhibited myocardial regeneration and significantly reduced epicardial cell expansion, proliferation, and activation. To explore the molecular mechanism of neutrophils on the epicardial regenerative response, we performed scRNA-seq analysis of 1 dpa neutrophils and identified enrichment of the FGF and MAPK/ERK signaling pathways. Pharmacological inhibition of FGF signaling indicated its' requirement for epicardial expansion, while neutrophil depletion blocked MAPK/ERK signaling activation in epicardial cells. Ligand-receptor analysis indicated the EGF ligand, hbegfa, is released from neutrophils and synergizes with other FGF and MAPK/ERK factors for induction of epicardial regeneration. Altogether, our studies revealed that neutrophils quickly motivate epicardial cells, which later accumulate at the injury site and contribute to heart regeneration.

hapln1a+ cells guide coronary growth during heart morphogenesis and regeneration.

Although several tissues and chemokines orchestrate coronary formation, the guidance cues for coronary growth remain unclear. Here, we profile the juvenile zebrafish epicardium during coronary vascularization and identify hapln1a+ cells enriched with vascular-regulating genes. hapln1a+ cells not only envelop vessels but also form linear structures ahead of coronary sprouts. Live-imaging demonstrates that coronary growth occurs along these pre-formed structures, with depletion of hapln1a+ cells blocking this growth. hapln1a+ cells also pre-lead coronary sprouts during regeneration and hapln1a+ cell loss inhibits revascularization. Further, we identify serpine1 expression in hapln1a+ cells adjacent to coronary sprouts, and serpine1 inhibition blocks vascularization and revascularization. Moreover, we observe the hapln1a substrate, hyaluronan, forming linear structures along and preceding coronary vessels. Depletion of hapln1a+ cells or serpine1 activity inhibition disrupts hyaluronan structure. Our studies reveal that hapln1a+ cells and serpine1 are required for coronary production by establishing a microenvironment to facilitate guided coronary growth.

Bidirectional phonon emission in two-dimensional heterostructures triggered by ultrafast charge transfer.

Photoinduced charge transfer in van der Waals heterostructures occurs on the 100 fs timescale despite weak interlayer coupling and momentum mismatch. However, little is understood about the microscopic mechanism behind this ultrafast process and the role of the lattice in mediating it. Here, using femtosecond electron diffraction, we directly visualize lattice dynamics in photoexcited heterostructures of WSe2/WS2 monolayers. Following the selective excitation of WSe2, we measure the concurrent heating of both WSe2 and WS2 on a picosecond timescale-an observation that is not explained by phonon transport across the interface. Using first-principles calculations, we identify a fast channel involving an electronic state hybridized across the heterostructure, enabling phonon-assisted interlayer transfer of photoexcited electrons. Phonons are emitted in both layers on the femtosecond timescale via this channel, consistent with the simultaneous lattice heating observed experimentally. Taken together, our work indicates strong electron-phonon coupling via layer-hybridized electronic states-a novel route to control energy transport across atomic junctions.

hapln1 Defines an Epicardial Cell Subpopulation Required for Cardiomyocyte Expansion During Heart Morphogenesis and Regeneration.

BACKGROUND: Certain nonmammalian species such as zebrafish have an elevated capacity for innate heart regeneration. Understanding how heart regeneration occurs in these contexts can help illuminate cellular and molecular events that can be targets for heart failure prevention or treatment. The epicardium, a mesothelial tissue layer that encompasses the heart, is a dynamic structure that is essential for cardiac regeneration in zebrafish. The extent to which different cell subpopulations or states facilitate heart regeneration requires research attention. METHODS: To dissect epicardial cell states and associated proregenerative functions, we performed single-cell RNA sequencing and identified 7 epicardial cell clusters in adult zebrafish, 3 of which displayed enhanced cell numbers during regeneration. We identified paralogs of hapln1 as factors associated with the extracellular matrix and largely expressed in cluster 1. We assessed HAPLN1 expression in published single-cell RNA sequencing data sets from different stages and injury states of murine and human hearts, and we performed molecular genetics to determine the requirements for hapln1-expressing cells and functions of each hapln1 paralog. RESULTS: A particular cluster of epicardial cells had the strongest association with regeneration and was marked by expression of hapln1a and hapln1b. The hapln1 paralogs are expressed in epicardial cells that enclose dedifferentiated and proliferating cardiomyocytes during regeneration. Induced genetic depletion of hapln1-expressing cells or genetic inactivation of hapln1b altered deposition of the key extracellular matrix component hyaluronic acid, disrupted cardiomyocyte proliferation, and inhibited heart regeneration. We also found that hapln1-expressing epicardial cells first emerge at the juvenile stage, when they associate with and are required for focused cardiomyocyte expansion events that direct maturation of the ventricular wall. CONCLUSIONS: Our findings identify a subset of epicardial cells that emerge in postembryonic zebrafish and sponsor regions of active cardiomyogenesis during cardiac growth and regeneration. We provide evidence that, as the heart achieves its mature structure, these cells facilitate hyaluronic acid deposition to support formation of the compact muscle layer of the ventricle. They are also required, along with the function of hapln1b paralog, in the production and organization of hyaluronic acid-containing matrix in cardiac injury sites, enabling normal cardiomyocyte proliferation and muscle regeneration.

Zebrafish heart regeneration after coronary dysfunction-induced cardiac damage.

Over the past 20 years, various zebrafish injury models demonstrated efficient heart regeneration after cardiac tissue loss. However, no established coronary vessel injury methods exist in the zebrafish model, despite coronary endothelial dysfunction occurring in most patients with acute coronary syndrome. This is due to difficulties performing surgery on small coronary vessels and a lack of genetic tools to precisely manipulate coronary cells in zebrafish. We determined that the Notch ligand gene deltaC regulatory sequences drive gene expression in zebrafish coronary endothelial cells, enabling us to overcome these obstacles. We created a deltaC fluorescent reporter line and visualized robust coronary growth during heart development and regeneration. Importantly, this reporter facilitated the visualization of coronary growth without an endocardial background. Moreover, we visualized robust coronary growth on the surface of juvenile hearts and regrowth in the wounded area of adult hearts ex vivo. With this approach, we observed growth inhibition by reported vascular growth antagonists of the VEGF, EGF and Notch signaling pathways. Furthermore, we established a coronary genetic ablation system and observed that severe coronary endothelial cell loss resulted in fish death, whereas fish survived mild coronary cell loss. Coronary cell depletion triggered regenerative responses, which resulted in the restoration of damaged cardiac tissues within several weeks. Overall, our work demonstrated the efficacy of using deltaC regulatory elements for high-resolution visualization of the coronary endothelium; screening small molecules for coronary growth effects; and revealed complete recovery in adult zebrafish after coronary-induced heart damage.

Exchange Bias in a Layered Metal-Organic Topological Spin Glass.

The discovery of conductive and magnetic two-dimensional (2D) materials is critical for the development of next generation spintronics devices. Coordination chemistry in particular represents a highly versatile, though underutilized, route toward the synthesis of such materials with designer lattices. Here, we report the synthesis of a conductive, layered 2D metal-organic kagome lattice, Mn3(C6S6), using mild solution-phase chemistry. Strong geometric spin frustration in this system mediates spin freezing at low temperatures, which results in glassy magnetic dynamics consistent with a rare geometrically frustrated (topological) spin glass. Notably, we show that this geometric frustration engenders a large, tunable exchange bias of 1625 Oe in Mn3(C6S6), providing the first example of exchange bias in a coordination solid or a topological spin glass. Exchange bias is a critical component in a number of spintronics applications, but it is difficult to rationally tune, as it typically arises due to structural disorder. This work outlines a new strategy for engineering exchange bias systems using single-phase, crystalline lattices. More generally, these results demonstrate the potential utility of geometric frustration in the design of new nanoscale spintronic materials.

Manual laryngeal reposturing as a primary approach for mutational falsetto.

OBJECTIVES/HYPOTHESIS: Mutational falsetto is a functional voice disorder characterized by failure of the male high-pitched preadolescent voice to transition to the lower pitch of adolescence and adulthood. Few objective outcomes data exist regarding the effectiveness of voice therapy for this poorly understood disorder. This study examined the immediate effects of a single therapy session using manual laryngeal reposturing as a primary approach in the management of mutational falsetto. STUDY DESIGN: Retrospective case series, pre-/posttreatment. METHODS: Manual circumlaryngeal techniques, including digital laryngeal reposturing maneuvers, were used as the primary approach to treat 12 consecutive males with mutational falsetto (mean age 17.5 yrs., range 14-25 years). Pre- and posttreatment audiorecordings of connected speech and sustained vowel samples were submitted to auditory-perceptual and acoustic analysis to assess the effects of a single voice therapy session. Acoustic estimates of severity in both connected speech and sustained vowel productions were computed using the Cepstral Spectral Index of Dysphonia (CSID), a multivariate dysphonia summary tool that incorporates cepstral and spectral measures. RESULTS: Pre- and posttreatment comparisons confirmed a significant change in the direction of normal vocal function within a single treatment session for both listener- and CSID-based estimates of dysphonia severity. CONCLUSION: Behavioral therapy using manual laryngeal reposturing by an experienced voice clinician is an effective and efficient primary approach for mutational falsetto. LEVEL OF EVIDENCE: 4. Laryngoscope, 127:645-650, 2017.

Toward validation of the cepstral spectral index of dysphonia (CSID) as an objective treatment outcomes measure.

OBJECTIVES: To examine the validity of the Cepstral Spectral Index of Dysphonia (CSID) as an objective treatment outcomes measure of dysphonia severity. METHOD: Pre- and posttreatment samples of sustained vowel and connected speech productions were elicited from 112 patients across six diagnostic categories: unilateral vocal fold paralysis, adductor spasmodic dysphonia, primary muscle tension dysphonia, benign vocal fold lesions, presbylaryngis, and mutational falsetto. Listener ratings of severity in connected speech were compared with a three-factor CSID model consisting of the cepstral peak prominence (CPP), the low-to-high spectral energy ratio, and its standard deviation. Two additional variables, the CPP standard deviation and gender, were included in the five-factor CSID model to estimate severity of vowels. RESULTS: CSID-estimated severity for sustained vowels and connected speech was strongly associated with listener ratings pretreatment, posttreatment, and change observed pre- to posttreatment. Spectrum effects were examined, and severity of dysphonia did not influence the relationship between listener perceived severity and CSID-estimated severity. CONCLUSIONS: The results confirm a robust relationship between listener perceived and CSID-derived dysphonia severity estimates in sustained vowels and connected speech across diverse diagnoses and severity levels and support the clinical utility of the CSID as an objective treatment outcomes measure.

Innate immune responses of airway epithelium to house dust mite are mediated through beta-glucan-dependent pathways.

BACKGROUND: House dust mite (HDM) induces allergic asthma in sensitized individuals, although the mechanisms by which HDM is sensed and recognized by the airway mucosa, leading to dendritic cell (DC) recruitment, activation, and subsequent T(H)2-mediated responses, are unknown. OBJECTIVE: We sought to define the pathways by which HDM activates respiratory epithelium to induce allergic airway responses. METHODS: Using a human airway epithelial cell line (16HBE14o-), we studied secretion of the DC chemokine CCL20 after exposure to HDM or other allergens, investigated components of the HDM responsible for the induction of chemokine release, and examined activation of signaling pathways. Central findings were also confirmed in primary human bronchial cells. RESULTS: We demonstrate that exposure of airway epithelium to HDM results in specific and rapid secretion of CCL20, a chemokine attractant for immature DCs. The induction of CCL20 secretion is dose and time dependent and quite specific to HDM because other allergens, such as ragweed pollen and cockroach antigen, fail to significantly induce CCL20 secretion. Induction of CCL20 secretion is not protease or Toll-like receptor 2/4 dependent but, interestingly, relies on beta-glucan moieties within the HDM extract, as evidenced by the ability of other beta-glucans to competitively inhibit its secretion and by the fact that disruption of these structures by treatment of HDM with beta-glucanase significantly reduces subsequent chemokine secretion. CONCLUSION: Taken together, our results describe a novel mechanism for specific pattern recognition of HDM-derived beta-glucan moieties, which initiates allergic airway inflammation and, through recruitment of DCs, might link innate pattern recognition at the airway surface with adaptive immune responses.

Trisomy for the Down syndrome 'critical region' is necessary but not sufficient for brain phenotypes of trisomic mice.

Trisomic Ts65Dn mice show direct parallels with many phenotypes of Down syndrome (DS), including effects on the structure of cerebellum and hippocampus. A small segment of Hsa21 known as the 'DS critical region' (DSCR) has been held to contain a gene or genes sufficient to cause impairment in learning and memory tasks involving the hippocampus. To test this hypothesis, we developed Ts1Rhr and Ms1Rhr mouse models that are, respectively, trisomic and monosomic for this region. Here, we show that trisomy for the DSCR alone is not sufficient to produce the structural and functional features of hippocampal impairment that are seen in the Ts65Dn mouse and DS. However, when the critical region is returned to normal dosage in trisomic Ms1Rhr/Ts65Dn mice, performance in the Morris water maze is identical to euploid, demonstrating that this region is necessary for the phenotype. Thus, although the prediction of the critical region hypothesis was disproved, novel gene dosage effects were identified, which help to define how trisomy for this segment of the chromosome contributes to phenotypes of DS.