Regenerating animals have the ability to reproduce body parts that were originally made in the embryo and subsequently lost due to injury. Understanding whether regeneration mirrors development is an open question in most regenerative species. Here, we take a transcriptomics approach to examine whether leg regeneration shows similar temporal patterns of gene expression as leg development in the embryo, in the crustacean Parhyale hawaiensis. We find that leg development in the embryo shows stereotypic temporal patterns of gene expression. In contrast, the dynamics of gene expression during leg regeneration show a higher degree of variation related to the physiology of individual animals. A major driver of this variation is the molting cycle. We dissect the transcriptional signals of individual physiology and regeneration to obtain clearer temporal signals marking distinct phases of leg regeneration. Comparing the transcriptional dynamics of development and regeneration we find that, although the two processes use similar sets of genes, the temporal patterns in which these genes are deployed are different and cannot be systematically aligned.

5' AMP-activated protein kinase (AMPK) is a highly conserved serine-threonine kinase that regulates energy expenditure by activating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. Therefore AMPK activators are considered to be drug targets for treatment of metabolic diseases such as diabetes mellitus. To identify novel AMPK activators, we screened xanthene derivatives. We determined that the AMPK activators 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-nitro-phenyl)-thioureido]-ethyl}-amide (Xn) and 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-cyano-phenyl)-thioureido]-ethyl}-amide (Xc) elevated glucose uptake in L6 myotubes by stimulating translocation of glucose transporter type 4 (GLUT4). Treatment with the chemical AMPK inhibitor compound C and infection with dominant-negative AMPKa2-virus inhibited AMPK phosphorylation and glucose uptake in myotubes induced by either Xn or Xc. Of the two major upstream kinases of AMPK, we found that Xn and Xc showed LKB1 dependency by knockdown of STK11, an ortholog of human LKB1. Single intravenous administration of Xn and Xc to high-fat diet-induced diabetic mice stimulated AMPK phosphorylation of skeletal muscle and improved glucose tolerance. Taken together, these results suggest that Xn and Xc regulate glucose homeostasis through LKB1-dependent AMPK activation and that the compounds are potential candidate drugs for the treatment of type 2 diabetes mellitus.

Ultrasound has been used to non-invasively manipulate neuronal functions in humans and other animals. However, this approach is limited as it has been challenging to target specific cells within the brain or body. Here, we identify human Transient Receptor Potential A1 (hsTRPA1) as a candidate that confers ultrasound sensitivity to mammalian cells. Ultrasound-evoked gating of hsTRPA1 specifically requires its N-terminal tip region and cholesterol interactions; and target cells with an intact actin cytoskeleton, revealing elements of the sonogenetic mechanism. Next, we use calcium imaging and electrophysiology to show that hsTRPA1 potentiates ultrasound-evoked responses in primary neurons. Furthermore, unilateral expression of hsTRPA1 in mouse layer V motor cortical neurons leads to c-fos expression and contralateral limb responses in response to ultrasound delivered through an intact skull. Collectively, we demonstrate that hsTRPA1-based sonogenetics can effectively manipulate neurons within the intact mammalian brain, a method that could be used across species.

Epigenetic regulatory mechanisms are divergent across the animal kingdom, yet these mechanisms are not well studied in non-model organisms. Unique features of cephalopods make them attractive for investigating behavioral, sensory, developmental, and regenerative processes, and recent studies have elucidated novel features of genome organization and gene and transposon regulation in these animals. However, it is not known how epigenetics regulates these interesting cephalopod features. We combined bioinformatic and molecular analysis of Octopus bimaculoides to investigate the presence and pattern of DNA methylation and examined the presence of DNA methylation and 3 histone post-translational modifications across tissues of three cephalopod species.

Hydrogen production techniques based on solar-water splitting have emerged as carbon-free energy systems. Many researchers have developed highly efficient thin-film photoelectrochemical (PEC) devices made of low-cost and earth-abundant materials. However, solar water splitting systems suffer from short lifetimes due to catalyst instability that is attributed to both chemical dissolution and mechanical stress produced by hydrogen bubbles. A recent study found that the nanoporous hydrogel could prevent the structural degradation of the PEC devices. In this study, we investigate the protection mechanism of the hydrogel-based overlayer by engineering its porous structure using the cryogelation technique. Tests for cryogel overlayers with varied pore structures, such as disconnected micropores, interconnected micropores, and surface macropores, reveal that the hydrogen gas trapped in the cryogel protector reduce shear stress at the catalyst surface by providing bubble nucleation sites. The cryogelated overlayer effectively preserves the uniformly distributed platinum catalyst particles on the device surface for over 200 h. Our finding can help establish semi-permanent photoelectrochemical devices to realize a carbon-free society.

Although the importance of proteins of the biomineral organic matrix and their posttranslational modifications for biomineralization is generally recognized, the number of published matrix proteomes is still small. This is mostly due to the lack of comprehensive sequence databases, usually derived from genomic sequencing projects. However, in-depth mass spectrometry-based proteomic analysis, which critically depends on high-quality sequence databases, is a very fast tool to identify candidates for functional biomineral matrix proteins and their posttranslational modifications. Identification of such candidate proteins is facilitated by at least approximate quantitation of the identified proteins, because the most abundant ones may also be the most interesting candidates for further functional analysis.

Imaging living organisms at high spatial resolution requires effective and innocuous immobilization. Long-term imaging places further demands on sample mounting with minimal perturbation of the organism. Here we present a simple, inexpensive method for rapid encapsulation of small animals of any developmental stage within a photo-crosslinked polyethylene glycol (PEG) hydrogel, gently restricting movement within their confined spaces. Immobilized animals maintain their original morphology in a hydrated environment compatible with chemical treatment, optical stimulation, and light-sheet microscopy. We demonstrate prolonged three-dimensional imaging of neural responses in the nematode Caenorhabditis elegans, recovery of viable organisms after 24 h, and imaging of larger squid hatchlings. We characterize a range of hydrogel and illumination conditions for immobilization quality, and identify paralytic-free conditions suitable for high-resolution single-cell imaging. Overall, PEG hydrogel encapsulation provides fast, versatile, and gentle mounting of small living organisms, from yeast to zebrafish, for continuous observation over hours.

Human and octopus lineages are separated by over 500 million years of evolution [1, 2] and show divergent anatomical patterns of brain organization [3, 4]. Despite these differences, growing evidence suggests that ancient neurotransmitter systems are shared across vertebrate and invertebrate species and in many cases enable overlapping functions [5]. Sociality is widespread across the animal kingdom, with numerous examples in both invertebrate (e.g., bees, ants, termites, and shrimps) and vertebrate (e.g., fishes, birds, rodents, and primates) lineages [6]. Serotonin is an evolutionarily ancient molecule [7] that has been implicated in regulating both invertebrate [8] and vertebrate [9] social behaviors, raising the possibility that this neurotransmitter's prosocial functions may be conserved across evolution. Members of the order Octopoda are predominantly asocial and solitary [10]. Although at this time it is unknown whether serotonergic signaling systems are functionally conserved in octopuses, ethological studies indicate that agonistic behaviors are suspended during mating [11-13], suggesting that neural mechanisms subserving social behaviors exist in octopuses but are suppressed outside the reproductive period. Here we provide evidence that, as in humans, the phenethylamine (+/-)-3,4-methylendioxymethamphetamine (MDMA) enhances acute prosocial behaviors in Octopus bimaculoides. This finding is paralleled by the evolutionary conservation of the serotonin transporter (SERT, encoded by the Slc6A4 gene) binding site of MDMA in the O. bimaculoides genome. Taken together, these data provide evidence that the neural mechanisms subserving social behaviors exist in O. bimaculoides and indicate that the role of serotonergic neurotransmission in regulating social behaviors is evolutionarily conserved.

Collective cell migration plays a critical role in physiological and pathological processes such as development, wound healing, and metastasis. Numerous studies have demonstrated how various types of chemical, mechanical, and electrical cues dictate the collective migratory behaviors of cells. Although an acoustic cue can be advantageous because of its noninvasiveness and biocompatibility, cell migration in response to acoustic stimulation remains poorly understood. In this study, we developed a device that is able to apply surface acoustic waves to a cell culture substrate and investigated the effect of propagating acoustic waves on collective cell migration. The migration distance estimated at various wave intensities revealed that unidirectional cell migration was enhanced at a critical wave intensity and that it was suppressed as the intensity was further increased. The increased migration might be attributable to cell orientation alignment along the direction of the propagating wave, as characterized by nucleus shape. Thicker actin bundles indicative of a high traction force were observed in cells subjected to propagating acoustic waves at the critical intensity. Our device and technique can be useful for regulating cellular functions associated with cell migration.

Octopus laqueus is a small tropical octopus found in Okinawa, Japan and the greater Indo-Pacific. Octopus are often viewed as solitary animals but O. laqueus live in close proximity in the wild, and will potentially encounter one another on a regular basis, raising the possibility of social tolerance. Adopting shared den occupancy in aquaria as a potential measure of social tolerance in O. laqueus, we studied the animals' preference for shared dens over solitude. We characterized dependence of sharing preference on sex, den availability and den occupancy density. We designed two simple social tolerance assays in aquaria with a total of 45 daily measurements: (i) Pots Equal, with equal numbers of octopuses and dens and (ii) Pots Limited, with a 3:1 ratio of octopuses to dens. We found that O. laqueus will socially tolerate other individuals by sharing tanks and dens and with typically no loss to cannibalism or escape. However, animals also exhibit significant levels of social repulsion, and individuals often chose a solitary den when given the option. The patterns of den occupancy are observed to be consistent with a maximum entropy model that balances seeking shelter against avoiding other animals. The model accurately captures and predicts the data and can be generalized to other organisms and their social interactions. Overall, in O. laqueus the preference for a den is stronger than the preference to be solitary. The animals are tolerant of others with a mixture of sizes in the tank and even in a den, a reported first for octopuses outside mating. The relaxed disposition and social tolerance of O. laqueus make it a promising species to work with in the lab to explore social and potentially other behaviors in octopuses.

Epithelial wound healing involves the collective responses of many cells, including those at the wound margin (marginal cells) and those that lack direct contact with the wound (submarginal cells). How these responses are induced and coordinated to produce rapid, efficient wound healing remains poorly understood. Extracellular ATP (eATP) is implicated as a signal in epithelial wound healing in vertebrates. However, the role of eATP in wound healing in vivo and the cellular responses to eATP are unclear. Almost nothing is known about eATP signaling in non-bilaterian metazoans (Cnidaria, Ctenophora, Placozoa, and Porifera). Here, we show that eATP promotes closure of epithelial wounds in vivo in the cnidarian Clytia hemisphaerica (Clytia) indicating that eATP signaling is an evolutionarily ancient strategy in wound healing. Furthermore, eATP increases F-actin accumulation at the edges of submarginal cells. In Clytia, this indicates eATP is involved in coordinating cellular responses during wound healing, acting in part by promoting actin remodeling in cells at a distance from the wound. We also present evidence that eATP activates a cation channel in Clytia epithelial cells. This implies that the eATP signal is transduced through a P2X receptor (P2XR). Phylogenetic analyses identified four Clytia P2XR homologs and revealed two deeply divergent major branches in P2XR evolution, necessitating revision of current models. Interestingly, simple organisms such as cellular slime mold appear exclusively on one branch, bilaterians are found exclusively on the other, and many non-bilaterian metazoans, including Clytia, have P2XR sequences from both branches. Together, these results re-draw the P2XR evolutionary tree, provide new insights into the origin of eATP signaling in wound healing, and demonstrate that the cytoskeleton of submarginal cells is a target of eATP signaling.

The fabrication of functional tissues is essential for clinical applications such as disease treatment and drug discovery. Recent studies have revealed that the mechanical environments of tissues, determined by geometric cell patterns, material composition, or mechanical properties, play critical roles in ensuring proper tissue function. Here, we propose an acoustophoretic technique using surface acoustic waves to fabricate therapeutic vascular tissue containing a three-dimensional collateral distribution of vessels. Co-aligned human umbilical vein endothelial cells and human adipose stem cells that are arranged in a biodegradable catechol-conjugated hyaluronic acid hydrogel exhibit enhanced cell-cell contacts, gene expression, and secretion of angiogenic and anti-inflammatory paracrine factors. The therapeutic effects of the fabricated vessel constructs are demonstrated in experiments using an ischemia mouse model by exhibiting the remarkable recovery of damaged tissue. Our study can be referenced to fabricate various types of artificial tissues that mimic the original functions as well as structures.