Allometry in limb regeneration and scale-invariant patterning as the basis of normal morphogenesis from different sizes of blastemas.

Axolotl (Ambystoma mexicanum) limb regeneration begins with blastemas of various sizes, in contrast to the limb developmental process. Despite this size variation, normal limb morphology, consistent with a limb stump size, is regenerated. This outcome suggests the existence of underlying scale-invariant mechanisms. To identify such mechanisms, we examined the allometric relationships between blastema size, and Sonic Hedgehog (Shh) and Fibroblast Growth Factor 8 (Fgf8) expression patterns against limb stump size. We found that all factors showed allometric rather than isometric scaling; specifically, their relative sizes decrease with an increase in limb stump size. However, the ratio of Shh/Fgf8 signaling dominant region was nearly constant, independent of blastema/body size. Furthermore, the relative spatial patterns of cell density and proliferation activity and the relative position of first digit formation were scale-invariant in the summed Shh/Fgf8 crosstalk region. This scale-invariant nature may underlie the morphogenesis of normal limbs from different sizes of blastemas.

hoxc12/c13 as key regulators for rebooting the developmental program in Xenopus limb regeneration.

During organ regeneration, after the initial responses to injury, gene expression patterns similar to those in normal development are reestablished during subsequent morphogenesis phases. This supports the idea that regeneration recapitulates development and predicts the existence of genes that reboot the developmental program after the initial responses. However, such rebooting mechanisms are largely unknown. Here, we explore core rebooting factors that operate during Xenopus limb regeneration. Transcriptomic analysis of larval limb blastema reveals that hoxc12/c13 show the highest regeneration specificity in expression. Knocking out each of them through genome editing inhibits cell proliferation and expression of a group of genes that are essential for development, resulting in autopod regeneration failure, while limb development and initial blastema formation are not affected. Furthermore, the induction of hoxc12/c13 expression partially restores froglet regenerative capacity which is normally very limited compared to larval regeneration. Thus, we demonstrate the existence of genes that have a profound impact alone on rebooting of the developmental program in a regeneration-specific manner.

An archetype and scaling of developmental tissue dynamics across species.

Morphometric studies have revealed the existence of simple geometric relationships among various animal shapes. However, we have little knowledge of the mathematical principles behind the morphogenetic dynamics that form the organ/body shapes of different species. Here, we address this issue by focusing on limb morphogenesis in Gallus gallus domesticus (chicken) and Xenopus laevis (African clawed frog). To compare the deformation dynamics between tissues with different sizes/shapes as well as their developmental rates, we introduce a species-specific rescaled spatial coordinate and a common clock necessary for cross-species synchronization of developmental times. We find that tissue dynamics are well conserved across species under this spacetime coordinate system, at least from the early stages of development through the phase when basic digit patterning is established. For this developmental period, we also reveal that the tissue dynamics of both species are mapped with each other through a time-variant linear transformation in real physical space, from which hypotheses on a species-independent archetype of tissue dynamics and morphogenetic scaling are proposed.

Endoscopic Aortic Valve Replacement: Initial Outcomes of Isolated and Concomitant Surgery.

BACKGROUND: The applicability of totally endoscopic surgical aortic valve replacement (AVR) in multivalve operations is unknown. This study describes an approach and perioperative outcomes of totally endoscopic isolated and concomitant AVR using various valve types. METHODS: A total of 216 patients (114 male; mean age, 71.3 ± 11.3 years) underwent totally endoscopic AVR from May 2017 to October 2022 in a tertiary care center. The 3-port technique was used: a 3- to 4-cm main port without rib spreading, a 10-mm 3-dimensional endoscopic port, and a 5-mm left-hand port with femoral cannulations. Sutures were hand tied with a knot pusher. Descriptive analyses compared perioperative outcomes between patients with or without concomitant procedures. RESULTS: Of 216 patients, concomitant surgery was performed in 33 (15.2%) patients. Of the 33, 21 (63.6%) had a concomitant mitral procedure. A stented bioprosthesis was implanted in 165 (76.3%) patients, a mechanical valve in 22 (10.2%) patients, and a rapid deployment or sutureless valve in 29 (13.4%) patients. Median operation time and aortic cross-clamp time were 175 minutes (interquartile range; 150-194 minutes) and 78 minutes (interquartile range; 67-92 minutes) for isolated AVR, respectively. Thirty-day mortality occurred in 1 patient (0.5%). Two patients (0.9%) had conversion to sternotomy. Major neurologic events occurred in 3 patients (1.4%). The major adverse event rate was similar between patients with or without concomitant procedures. CONCLUSIONS: Endoscopic AVR can safely address concomitant valve diseases.

Midterm results after seamless patch mitral reconstruction.

Objectives: Some pathologies, including infective endocarditis or sclerotic changes of the mitral leaflet, make the conventional mitral valve repair challenging. Our previously described technique for reconstruction with a seamless pericardial patch makes the repair feasible in some of such difficult pathologies. However, the extent of mitral leaflet segments that could be safely repaired using this technique remains unknown. We investigated the association between the midterm outcome and the extent of mitral leaflet segments replaced by a pericardial patch. Methods: From January 2009 to January 2022, patients who underwent mitral valve repair with the seamless 1-patch reconstruction technique were included. The glutaraldehyde-treated pericardium was trimmed and anchored at the papillary muscle. The edge was sewn to the leaflet and the annulus. Results: A total of 49 patients (aged 60 ± 15 years) underwent mitral valve repair with this technique. The totally endoscopic approach was used in 27 patients (55%). No patient's repair was converted to valve replacement. No operative mortality or disabling stroke was observed during the early postoperative period. In the midterm follow-up, redo surgery was required in 9 patients (18%). Freedom from mitral valve reintervention rates at 1, 5, and 10 years were 84%, 82%, and 82% for all patients, respectively. Freedom from reoperation at 5 years was 100%, 92%, and 46% for commissural lesion, 1- to 2-segment involvement, and 3-segment involvement, respectively. There was a significant difference among the 3 groups with regard to mitral valve reoperation rate (P = .002). Conclusions: Mitral valve seamless patch reconstruction provides excellent midterm results if applied to commissural lesions or lesions involving up to 2 segments.

Engineering multicellular living systems-a Keystone Symposia report.

The ability to engineer complex multicellular systems has enormous potential to inform our understanding of biological processes and disease and alter the drug development process. Engineering living systems to emulate natural processes or to incorporate new functions relies on a detailed understanding of the biochemical, mechanical, and other cues between cells and between cells and their environment that result in the coordinated action of multicellular systems. On April 3-6, 2022, experts in the field met at the Keystone symposium "Engineering Multicellular Living Systems" to discuss recent advances in understanding how cells cooperate within a multicellular system, as well as recent efforts to engineer systems like organ-on-a-chip models, biological robots, and organoids. Given the similarities and common themes, this meeting was held in conjunction with the symposium "Organoids as Tools for Fundamental Discovery and Translation".

Cell disorientation by loss of SHH-dependent mechanosensation causes cyclopia.

The physical causes of organ malformation remain largely unclear in most cases due to a lack of information on tissue/cell dynamics. Here, we address this issue by considering onset of cyclopia in sonic hedgehog (SHH)-inhibited chick embryos. We show that ventral forebrain-specific self-organization ability driven by SHH-dependent polarized patterns in cell shape, phosphorylated myosin localization, and collective cell motion promotes optic vesicle elongation during normal development. Stress loading tests revealed that these polarized dynamics result from mechanical responses. In particular, stress and active tissue deformation satisfy orthogonality, defining an SHH-regulated morphogenetic law. Without SHH signaling, cells cannot detect the direction of stress and move randomly, leading to insufficient optic vesicle elongation and consequently a cyclopia phenotype. Since polarized tissue/cell dynamics are common in organogenesis, cell disorientation caused by loss of mechanosensation could be a pathogenic mechanism for other malformations.

Two types of critical cell density for mechanical elimination of abnormal cell clusters from epithelial tissue.

Recent technological advances in high-resolution imaging and artificial modulation of genetic functions at different times and regions have enabled direct observations of the formation and elimination of abnormal cell populations. A recent trend in cell competition research is the incorporation of cell mechanics. In different tissues and species, abnormal cells developing in epithelial tissues are mechanically eliminated by cell contraction via actomyosin accumulation at the interface between normal and abnormal cells. This mechanical cell elimination process has attracted attention as a potential universal defense mechanism. Here, we theoretically examined the conditions for mechanical elimination of growing abnormal cell populations. Simulations and mathematical analyses using a vertex dynamics model revealed two types of critical cell density associated with mechanical elimination of abnormal cell clusters. One is a subtype of homeostatic density, in which the frequencies of spontaneous mechanical cell elimination and proliferation are balanced, even if no explicit dependence of proliferation or apoptosis on the cell density is assumed. This density is related to the mechanical stability of a single cell. The other is density related to mechanical stability as a cell population under external pressure. Both density types are determined by tissue mechanical properties. In solid tissues, the former type is reached first as the intensity of interfacial contraction increases, and it functions as a critical density. On the other hand, the latter type becomes critical when tissues are highly fluid. The derived analytical solution explicitly reveals the dependence of critical contractile force and density on different parameters. We also found a negative correlation between the proliferation rate of abnormal cells and the likelihood of the abnormal cell population expanding by escaping elimination. This is counterintuitive because in the context of cell competition, fast-growing cell populations generally win. These findings provide new insight into, and interpretation of, the results from experimental studies.

Light-induced local gene expression in primary chick cell culture system.

The ability to manipulate gene expression at a specific region in a tissue or cell culture system is critical for analysis of target gene function. For chick embryos/cells, several gene introduction/induction methods have been established such as those involving retrovirus, electroporation, sonoporation, and lipofection. However, these methods have limitations in the accurate induction of localized gene expression. Here we demonstrate the effective application of a recently developed light-dependent gene expression induction system (LightOn system) using the Neurospora crassa photoreceptor Vivid fused with a Gal4 DNA binding domain and p65 activation domain (GAVPO) that alters its activity in response to light stimulus in a primary chicken cell culture system. We show that the gene expression level and induction specificity in this system are strongly dependent on the light irradiation conditions. Especially, the irradiation interval is an important parameter for modulating gene expression; for shorter time intervals, higher induction specificity can be achieved. Further, by adjusting light irradiation conditions, the expression level in primary chicken cells can be regulated in a multiple step manner, in contrast to the binary expression seen for gene disruption or introduction (i.e., null or overexpression). This result indicates that the light-dependent expression control method can be a useful technique in chick models to examine how gene function is affected by gradual changes in gene expression levels. We applied this light induction system to regulate Sox9 expression in cultures of chick limb mesenchyme cells and showed that induced SOX9 protein could modulate expression of downstream genes.

Comparison between SBR Compounds Filled with In-Situ and Ex-Situ Silanized Silica.

The main advantages of the use of silica instead of carbon black in rubber compounds are based on the use of a silane coupling agent. The use of a coupling agent to modify the silica surface improves the compatibility between the silica and the rubber. There are two different possibilities of modifying the silica surface by silane: ex-situ and in-situ. The present work studies the differences between these processes and how they affect the in-rubber properties of silica filled SBR compounds.

Dynamic Polymer Networks: A New Avenue towards Sustainable and Advanced Soft Machines.

While the fascinating field of soft machines has grown rapidly over the last two decades, the materials they are constructed from have remained largely unchanged during this time. Parallel activities have led to significant advances in the field of dynamic polymer networks, leading to the design of three-dimensionally cross-linked polymeric materials that are able to adapt and transform through stimuli-induced bond exchange. Recent work has begun to merge these two fields of research by incorporating the stimuli-responsive properties of dynamic polymer networks into soft machine components. These include dielectric elastomers, stretchable electrodes, nanogenerators, and energy storage devices. In this Minireview, we outline recent progress made in this emerging research area and discuss future directions for the field.

A rare case of myocardial calcification secondary to acute myocarditis due to an Escherichia coli infection.

Myocardial calcification secondary to acute myocarditis is a rare but possibly life-threatening complication. We report a 43-year-old woman with minimal change nephrotic syndrome who developed sepsis caused by Escherichia coli. We simultaneously detected the complication of acute myocarditis in the patient. Although echocardiography showed hypokinesis of the apical segment when acute myocarditis was diagnosed, no sign of myocardial calcification was observed. After two weeks, a CT showed myocardial calcification in the same area. Although myocardial calcification was still observed 12 months later, the patient's cardiac function had improved.

A developmental checkpoint directs metabolic remodelling as a strategy against starvation in Drosophila.

Steroid hormones are crucial regulators of life-stage transitions during development in animals. However, the molecular mechanisms by which developmental transition through these stages is coupled with optimal metabolic homeostasis remains poorly understood. Here, we demonstrate through mathematical modelling and experimental validation that ecdysteroid-induced metabolic remodelling from resource consumption to conservation can be a successful life-history strategy to maximize fitness in Drosophila larvae in a fluctuating environment. Specifically, the ecdysteroid-inducible protein ImpL2 protects against hydrolysis of circulating trehalose following pupal commitment in larvae. Stored glycogen and triglycerides in the fat body are also conserved, even under fasting conditions. Moreover, pupal commitment dictates reduced energy expenditure upon starvation to maintain available resources, thus negotiating trade-offs in resource allocation at the physiological and behavioural levels. The optimal stage-specific metabolic shift elucidated by our predictive and empirical approaches reveals that Drosophila has developed a highly controlled system for ensuring robust development that may be conserved among higher-order organisms in response to intrinsic and extrinsic cues.

Quantitative Analysis of 3D Tissue Deformation Reveals Key Cellular Mechanism Associated with Initial Heart Looping.

Despite extensive study, the morphogenetic mechanisms of heart looping remain controversial because of a lack of information concerning precise tissue-level deformation and the quantitative relationship between tissue and cellular dynamics; this lack of information causes difficulties in evaluating previously proposed models. To overcome these limitations, we perform four-dimensional (4D) high-resolution imaging to reconstruct a tissue deformation map, which reveals that, at the tissue scale, initial heart looping is achieved by left-right (LR) asymmetry in the direction of deformation within the myocardial tube. We further identify F-actin-dependent directional cell rearrangement in the right myocardium as a major contributor to LR asymmetric tissue deformation. Our findings demonstrate that heart looping involves dynamic and intrinsic cellular behaviors within the tubular tissue and provide a significantly different viewpoint from current models that are based on LR asymmetry of growth and/or stress at the tube boundaries. Finally, we propose a minimally sufficient model for initial heart looping that is also supported by mechanical simulations.

Critical contractility and cell size for mechanical cell elimination from epithelial tissue.

A defense mechanism in epithelial tissue can mechanically eliminate abnormal cells by contracting the cell boundary or area via actomyosin activity. From numerical simulations of the vertex dynamics model and approximate analytical solutions based on deviations from the ground state, here we derived general conditions for mechanical cell elimination (MCE) occurring via cell contraction. In particular, we found that MCE is realized by saddle-node bifurcation in a wide parameter range, and that the size of the eliminated cell is almost constant at the bifurcation point, suggesting the existence of an intrinsic threshold cell area for MCE.

Variant PRC1 competes with retinoic acid-related signals to repress Meis2 in the mouse distal forelimb bud.

Suppression of Meis genes in the distal limb bud is required for proximal-distal (PD) specification of the forelimb. Polycomb group (PcG) factors play a role in downregulation of retinoic acid (RA)-related signals in the distal forelimb bud, causing Meis repression. It is, however, not known whether downregulation of RA-related signals and PcG-mediated proximal gene repression are functionally linked. Here, we reveal that PcG factors and RA-related signals antagonize each other to polarize Meis2 expression along the PD axis in mouse. Supported by mathematical modeling and simulation, we propose that PcG factors are required to adjust the threshold for RA-related signaling to regulate Meis2 expression. Finally, we show that a variant Polycomb repressive complex 1 (PRC1), incorporating PCGF3 and PCGF5, represses Meis2 expression in the distal limb bud. Taken together, we reveal a previously unknown link between PcG proteins and downregulation of RA-related signals to mediate the phase transition of Meis2 transcriptional status during forelimb patterning.

Morphometric staging of organ development based on cross sectional images.

An objective, continuous, and robust method for staging developing embryos or organs is essential for providing a common measure of time when studying quantitative/systems developmental biology. However, classical methods based on factors such as somite number or qualitative visual attributes are discrete and/or ambiguous due to observers' subjectivity. Thus, an alternative staging method based on an explicit and continuous description of developmental states over time, such as anatomy/morphology, is needed. Here, we briefly propose a novel staging method as a natural extension of classical staging based on cross sectional images of organs, which are more accessible than full 3D structures. The contours are represented as 2D closed curves and quantified using elliptic Fourier descriptors. Treating the ambiguity in classical staging as a statistical model, the relationship between the novel morphometric staging and classical staging can be determined. This method was validated by applying it to two different sets of data: chick forebrain and Xenopus hindlimb development. Using this method, it is also possible to reconstruct the time evolution of the average morphology, which would be useful for quantitatively comparing morphologies between embryos or between normal and abnormal conditions.

Dynamics of angiogenesis in ischemic areas of the infarcted heart.

Cardiomyocytes are susceptible to apoptosis caused by hypoxia during the acute and subacute phases of myocardial infarction (MI). Angiogenesis can reduce MI-induced damage by mitigating hypoxia. It has been speculated that the ischemic border zone is a unique area rescued by angiogenic therapy. However, the mechanism and timing for new vessel formation in the mammalian heart following hypoxia are unclear. Identifying targets that benefit from angiogenesis treatment is indispensable for the development of revolutionary therapies. Here, we describe a novel circulatory system wherein new vessels develop from the endocardium of the left ventricle to perfuse the hypoxic area and salvage damaged cardiomyocytes at 3-14 days after MI by activating vascular endothelial growth factor signaling. Moreover, enhanced angiogenesis increased cardiomyocyte survival along the endocardium in the ischemic zone and suppressed ventricular remodeling in infarcted hearts. In contrast, cardiomyocytes in the border zone's hypoxic area underwent apoptosis within 12 h of MI, and the border area that was amenable to treatment disappeared. These data indicate that the non-perfused area along the endocardium is a site of active angiogenesis and a promising target for MI treatment.

Possible roles of mechanical cell elimination intrinsic to growing tissues from the perspective of tissue growth efficiency and homeostasis.

Cell competition is a phenomenon originally described as the competition between cell populations with different genetic backgrounds; losing cells with lower fitness are eliminated. With the progress in identification of related molecules, some reports described the relevance of cell mechanics during elimination. Furthermore, recent live imaging studies have shown that even in tissues composed of genetically identical cells, a non-negligible number of cells are eliminated during growth. Thus, mechanical cell elimination (MCE) as a consequence of mechanical cellular interactions is an unavoidable event in growing tissues and a commonly observed phenomenon. Here, we studied MCE in a genetically-homogeneous tissue from the perspective of tissue growth efficiency and homeostasis. First, we propose two quantitative measures, cell and tissue fitness, to evaluate cellular competitiveness and tissue growth efficiency, respectively. By mechanical tissue simulation in a pure population where all cells have the same mechanical traits, we clarified the dependence of cell elimination rate or cell fitness on different mechanical/growth parameters. In particular, we found that geometrical (specifically, cell size) and mechanical (stress magnitude) heterogeneities are common determinants of the elimination rate. Based on these results, we propose possible mechanical feedback mechanisms that could improve tissue growth efficiency and density/stress homeostasis. Moreover, when cells with different mechanical traits are mixed (e.g., in the presence of phenotypic variation), we show that MCE could drive a drastic shift in cell trait distribution, thereby improving tissue growth efficiency through the selection of cellular traits, i.e. intra-tissue "evolution". Along with the improvement of growth efficiency, cell density, stress state, and phenotype (mechanical traits) were also shown to be homogenized through growth. More theoretically, we propose a mathematical model that approximates cell competition dynamics, by which the time evolution of tissue fitness and cellular trait distribution can be predicted without directly simulating a cell-based mechanical model.