Collective dynamics in entangled worm and robot blobs

Living systems at all scales aggregate in large numbers for a variety of functions including mating, predation, and survival. The majority of such systems consist of unconnected individuals that collectively flock, school, or swarm. However, some aggregations involve physically entangled individuals, which can confer emergent mechanofunctional material properties to the collective. Here, we study in laboratory experiments and rationalize in theoretical and robophysical models the dynamics of physically entangled and motile self-assemblies of 1-cm-long California blackworms (Lumbriculus variegatus, Annelida: Clitellata: Lumbriculidae). Thousands of individual worms form braids with their long, slender, and flexible bodies to make a three-dimensional, soft, and shape-shifting “blob.” The blob behaves as a living material capable of mitigating damage and assault from environmental stresses through dynamic shape transformations, including minimizing surface area for survival against desiccation and enabling transport (negative thermotaxis) from hazardous environments (like heat). We specifically focus on the locomotion of the blob to understand how an amorphous entangled ball of worms can break symmetry to move across a substrate. We hypothesize that the collective blob displays rudimentary differentiation of function across itself, which when combined with entanglement dynamics facilitates directed persistent blob locomotion. To test this, we develop a robophysical model of the worm blobs, which displays emergent locomotion in the collective without sophisticated control or programming of any individual robot. The emergent dynamics of the living functional blob and robophysical model can inform the design of additional classes of adaptive mechanofunctional living materials and emergent robotics.

Active matter collectives consists of self-propelled individual units (living or artificial) that interact with each other to gain emergent functionality or to achieve common tasks (1–7). In these systems, repeated interactions between the individuals and their environment can produce complex behaviors at the group level (3, 5). Depending on the type of interactions, collectives can display either fluid-like or solid-like properties (2). Fluid-like behavior is typically observed in unconnected individuals that avoid physical contact such as in flocking birds or schooling fish (5, 8–12). On the other hand, solid-like behavior is a consequence of physical contact between individuals such as in ants or bee self-assemblages (13–15). The latter type of entangled active matter aggregates enables the formation of large mechanically functional structures (bivouacs, rafts, bridges, etc.) that enable new functionalities not accessible to the individual as well as enabling survival benefits to the collective, especially in harsh and adverse environmental conditions in which it is impossible for individuals to survive on their own (16–20).In engineered systems, the emergent dynamics of active matter collectives have been explored in particles ranging in size from micrometers (active colloids) to centimeters (robots) (21–25). Specifically, for collective swarm robotics, the majority of past work has focused on mathematical modeling (26–30). These theoretical approaches often fail to adequately capture real-world physical interactions between individual robots, which may critically influence the emergent collective behavior. Experimentally, although swarming systems have been successfully realized to collectively accomplish a common goal (31–33), each individual robot is equipped with costly and sophisticated sensors to leverage some degree of centralized control, which is subject to many limitations including low fault tolerance, scalability problems, and design complexity (31). To overcome these limitations, researchers have proposed decentralized swarms, which eliminates the need for a central control unit, communication between individual agents, and a priori knowledge about the environment (34–36). These decentralized swarm systems have been demonstrated recently using only physical entanglements, either magnetic (35, 37) or geometric (38), that harness physical coupling between simple robots to yield task-oriented collectives capable of emergent functions.In this study, we investigate worm blobs as an example of an entangled active matter where the long flexible bodies of blackworms (Lumbriculus variegatus) form transient links through braiding. The activity of individual worms in a blob enables worms to self-organize and dynamically respond to changing environmental conditions. Depending on the type, history, and gradient of the environmental stimulus (light, temperature, etc.), the blob can respond in a variety of ways. Here, we specifically focus on the evaporation and thermal responses of worm blobs to understand why worms spontaneously aggregate into blobs and how they spontaneously move as whole. By developing robophysical blobs (39, 40) consisting of three-link robots (smarticles) (38), we describe how variation of gaits and mobility of simple individuals in a physically entangled collective can lead to varying levels of locomotive performance, without the need for sophisticated central control.     

Coevolutionary phage training leads to greater bacterial suppression and delays the evolution of phage resistance

The evolution of antibiotic-resistant bacteria threatens to become the leading cause of worldwide mortality. This crisis has renewed interest in the practice of phage therapy. Yet, bacteria’s capacity to evolve resistance may debilitate this therapy as well. To combat the evolution of phage resistance and improve treatment outcomes, many suggest leveraging phages’ ability to counter resistance by evolving phages on target hosts before using them in therapy (phage training). We found that in vitro, λtrn, a phage trained for 28 d, suppressed bacteria ∼1,000-fold for three to eight times longer than its untrained ancestor. Prolonged suppression was due to a delay in the evolution of resistance caused by several factors. Mutations that confer resistance to λtrn are ∼100× less common, and while the target bacterium can evolve complete resistance to the untrained phage in a single step, multiple mutations are required to evolve complete resistance to λtrn. Mutations that confer resistance to λtrn are more costly than mutations for untrained phage resistance. Furthermore, when resistance does evolve, λtrn is better able to suppress these forms of resistance. One way that λtrn improved was through recombination with a gene in a defunct prophage in the host genome, which doubled phage fitness. This transfer of information from the host genome is an unexpected but highly efficient mode of training phage. Lastly, we found that many other independently trained λ phages were able to suppress bacterial populations, supporting the important role training could play during phage therapeutic development.

In 30 y, the World Health Organization predicts that antibiotic-resistant bacteria will kill over 10 million people each year—more deaths than are caused by cancer (1). This health crisis, in part caused by the heavy and often inappropriate way we use antibiotic drugs, has led to the spread of resistance genes through clinical, agricultural, and natural environments and to the emergence of multidrug-resistant (MDR) “superbugs” that are untreatable due to their resistance against all available classes of antibiotics (2–4). As bacteria continue to outpace our discovery and development of new drugs, the evolution of resistance threatens to return us to a preantibiotic era of infectious disease (5, 6).This crisis has renewed interest in the century-old practice of phage therapy: the use of phages, viruses that infect bacteria, to treat bacterial infections (7–11). Recently, phage therapy has shown promise in cases where drugs of last resort fail to treat life-threatening MDR bacterial infections (9–13). However, even in successful cases, the evolution of phage resistance poses a considerable threat to the efficacy of treatment (9, 12, 14). For example, in 2016, at the University of California San Diego, a patient with acute pancreatitis complicated by an MDR Acinetobacter baumannii infection was treated with two four-phage mixtures that suppressed the pathogen in vitro (9). Within 8 d, A. baumannii isolated from the patient was resistant to all eight phages used. Fortunately, the infection resolved following delivery of a ninth phage, and the patient survived. This case is representative of numerous phage therapy studies (9–12, 14). A metanalysis in 2018 reported that phage resistance evolved in 82% of animal gut decolonization studies, 50% of meningitis/sepsis models, and 75% of human clinical cases in which the evolution of resistance was monitored (14). These observations of rapid phage resistance evolution in therapy mirror decades of basic research in the laboratory; mutations that confer resistance to phages are often as common as those for antibiotic resistance (15–17). Furthermore, many of these resistance mutations confer cross-resistance to multiple phages (18).Although resistance to phages is as or more common than to antibiotics, potential advantages of using phages as therapeutics have been proposed time and again (7, 8, 19–22). Notably, unlike antibiotics, phages are biological entities that evolve. By reciprocally adapting to changes in their hosts (coevolution), phages have maintained the ability to infect their hosts for millennia. Many have proposed harnessing this inherent evolutionary potential by preemptively coevolving phages with target bacterial prey (22–24). Proponents of this “phage training” approach suggest that, by experiencing the ways their host can evolve resistance, trained phages will evolve to counter host defenses. Then, trained phages “from the future” can be used to trap the ancestral, uncoevolved bacteria “from their past” that are infecting the patient, making their evolution futile.While the idea of phage training is enticing, it has not yet been adopted for therapy. Contrasting theories of bacteria–phage coevolutionary dynamics make the success of phage training uncertain (12, 24, 25). According to some conceptual models of coevolution (e.g., matching alleles), as phages adapt to their evolving host, they lose the ability to infect past hosts (24). In such cases, phage training would not work because trained phages would lose the ability to infect the original target bacterium. Alternatively, other models of coevolution (e.g., gene for gene and arms race dynamics) argue that as phages adapt to their evolving host, they maintain the ability to infect their original host (24). In this scenario, training would expand phage host range to encompass both original and contemporary bacteria. Regardless of how coevolution affects host range, some opponents of phage training contend that the use of trained phages will apply stronger selection on target bacteria which will accelerate the evolution of resistance and loss of therapeutic efficacy (22).In this study, we conducted a coevolution experiment using Escherichia coli and either untrained or trained phages to evaluate the potential of phage training for therapy. By comparing the population dynamics of coevolving bacteria and phages, we find that trained phages suppress the target bacteria more strongly and for longer than untrained phages. Through post hoc analyses on the bacteria and phages that evolved in our experiment, we identify the factors that allowed trained phages to suppress host populations and delay the evolution of resistance.     

The Widened Pipe Model of plant hydraulic evolution

Shaping global water and carbon cycles, plants lift water from roots to leaves through xylem conduits. The importance of xylem water conduction makes it crucial to understand how natural selection deploys conduit diameters within and across plants. Wider conduits transport more water but are likely more vulnerable to conduction-blocking gas embolisms and cost more for a plant to build, a tension necessarily shaping xylem conduit diameters along plant stems. We build on this expectation to present the Widened Pipe Model (WPM) of plant hydraulic evolution, testing it against a global dataset. The WPM predicts that xylem conduits should be narrowest at the stem tips, widening quickly before plateauing toward the stem base. This universal profile emerges from Pareto modeling of a trade-off between just two competing vectors of natural selection: one favoring rapid widening of conduits tip to base, minimizing hydraulic resistance, and another favoring slow widening of conduits, minimizing carbon cost and embolism risk. Our data spanning terrestrial plant orders, life forms, habitats, and sizes conform closely to WPM predictions. The WPM highlights carbon economy as a powerful vector of natural selection shaping plant function. It further implies that factors that cause resistance in plant conductive systems, such as conduit pit membrane resistance, should scale in exact harmony with tip-to-base conduit widening. Furthermore, the WPM implies that alterations in the environments of individual plants should lead to changes in plant height, for example, shedding terminal branches and resprouting at lower height under drier climates, thus achieving narrower and potentially more embolism-resistant conduits.

Water transport through plants is a key driver of the carbon and other biogeochemical cycles (1–3) and is a crucial link in plant adaptation to climate and vegetation response to climate change (4–9). The water conducting cells of plants, xylem conduits, widen with distance from the stem tip, and, therefore, taller plants have wider conduits (6, 10–12). Xylem conduits are of two main types: tracheids, found in most gymnosperms, and vessels, found in most flowering plants. Tracheids have intact cell membranes, so water must flow from cell to cell through these membranes. Vessels are made up of cells aligned vertically end to end, with the cell membranes dissolved between successive members, forming a tube. Whatever their differences in structure, wider conduits are beneficial because they conduct more water. Tip-to-base widening is expected to help maintain conductance per unit leaf area constant as an individual plant grows taller, counterbalancing the resistance that would otherwise accrue with increasing conductive path length the individual grows (2, 13). Wider conduits, however, are more vulnerable to embolisms caused by cold and likely drought (8, 14–18) and cost more in terms of carbon for a plant (ref. 1; cf. ref. 19). Embolisms in the xylem even affect transport of photosynthates in the phloem (8, 20). This means that as trees grow taller, conductance, embolism vulnerability, and carbon costs must interrelate in a delicate evolutionary balance.Because of the importance of this balance in plant hydraulic evolution and in forest reactions to climate change (3, 6, 21–23), an important goal of plant biology is to construct models that predict how and why plants deploy conduit diameters throughout their bodies (1, 2, 17, 24–26). Some models predict that conduits should be of uniform diameter (27, 28), while others predict that they should widen tip to base (1, 2, 13, 24, 29, 30). But even current models include untested assumptions and large numbers of parameters, making it difficult to identify the biological causes of the predictions they make. For example, some invoke Da Vinci’s rule, the largely untested assumption that the summed wood area of the twigs is the same as that at the base (24, 26). Other models depict plant conduits as branching as they do in mammalian circulatory systems, but whether this happens along the entire stem in plants is unclear (30–33). There is an expectation that conduit diameter D should widen with distance from the stem tip L following a power-law (D ∝ L^b), but there is no agreement on the value of b, the conduit widening exponent (1, 2). Furthermore, even though within-individual tip-to-base conduit widening has been confirmed in a handful of species (34–36), and the scaling of conduit diameter with plant size across species is consistent with it (6, 10–12, 34), the expectation that conduits should widen similarly within stems across terrestrial vascular plant lineages and habits has yet to be empirically confirmed. Here we present the Widened Pipe Model (WPM), which correctly predicts the form of tip-to-base conduit widening across the span of plant size, life form, and habitat across the terrestrial plant phylogeny.     

Aneuploidy increases resistance to chemotherapeutics by antagonizing cell division

Aneuploidy, defined as whole chromosome gains and losses, is associated with poor patient prognosis in many cancer types. However, the condition causes cellular stress and cell cycle delays, foremost in G1 and S phase. Here, we investigate how aneuploidy causes both slow proliferation and poor disease outcome. We test the hypothesis that aneuploidy brings about resistance to chemotherapies because of a general feature of the aneuploid condition—G1 delays. We show that single chromosome gains lead to increased resistance to the frontline chemotherapeutics cisplatin and paclitaxel. Furthermore, G1 cell cycle delays are sufficient to increase chemotherapeutic resistance in euploid cells. Mechanistically, G1 delays increase drug resistance to cisplatin and paclitaxel by reducing their ability to damage DNA and microtubules, respectively. Finally, we show that our findings are clinically relevant. Aneuploidy correlates with slowed proliferation and drug resistance in the Cancer Cell Line Encyclopedia (CCLE) dataset. We conclude that a general and seemingly detrimental effect of aneuploidy, slowed proliferation, provides a selective benefit to cancer cells during chemotherapy treatment.

Early observations of cancer noted that cancer cells often possess an abnormal number of chromosomes. About 90% of solid tumors are aneuploid, meaning they harbor a chromosome count that is not a multiple of the haploid complement. Thus, aneuploidy is more common than any individual gene mutations in cancer (1). This abnormal DNA content leads to changes in RNA and protein expression and many phenotypic changes (2–5). Notably, aneuploidy is significantly associated with poor patient prognoses, both in pan-cancer analyses and cancer-type-specific studies (6–13).Despite its prevalence in cancer, recent studies have revealed that aneuploidy causes a wide variety of cellular stresses. As changes in gene copy number generally result in changes in gene expression, stoichiometric imbalances in protein complexes lead to increased protein misfolding and proteotoxic stress due to an increased demand for protein quality-control machinery (14–16). Aneuploidy also alters the metabolic landscape of cells and causes genome instability (5, 17–20). These aneuploidy-associated stresses lead to cell cycle changes and slow proliferation (4, 21). Specifically, aneuploidy causes G1 delays in the yeast, Saccharomyces cerevisiae, and lengthens G1 and S phase in mammalian cells under most circumstances (20, 22, 23).How can a feature of cancer be associated with poor patient prognosis if it slows cancer’s growth? One possibility is that aneuploidy increases a tumor’s resistance to treatment. In cancer, drug resistance has, among other parameters, been associated with cells missegregating chromosomes at a higher rate, a phenomenon known as chromosomal instability (CIN). CIN and aneuploidy exhibit positive feedback, with each driving the other. Colon cancer cell lines with CIN have increased multidrug resistance, and patients with high CIN tumors have a worse prognosis than those with chromosomally stable cancers (24). The connection between CIN and drug resistance has been attributed to increased heterogeneity and, hence, subclones within the tumor that are drug resistant. However, other studies have found that extremely high levels of CIN increase patient survival (25–27). Extreme aneuploidy stress could synergize with the antiproliferative effects of cancer drugs and may prevent cells from “holding onto” a drug-resistant karyotype.Specific aneuploidies also drive drug resistance in human cancer cells. In DLD1, a colon cancer cell line, two trisomic derivatives (trisomy 7 and trisomy 13) grew better than their euploid counterpart when treated with 5-fluorouracil (28). Whether aneuploidy-induced copy number changes of specific genes or some general feature of the aneuploid state drive chemotherapy resistance is currently unknown. Here we investigate this question, focusing on cis-diamminedichloroplatinum(II) (cisplatin) and paclitaxel, two front line chemotherapeutics that are used to treat highly aneuploid cancers such as bladder, ovarian, testicular, breast, and nonsmall cell lung cancer. We show that aneuploidy causes resistance to these chemotherapeutics through a general feature of the aneuploid condition: slowed proliferation. Equalizing growth of aneuploid and euploid control cells eliminates differences in drug resistance between them. Finally, we provide evidence that our findings are clinically relevant. Aneuploidy and chromosomal instability correlate with slowed proliferation and drug resistance in the Cancer Cell Line Encyclopedia (CCLE) dataset. We conclude that proliferative index is a major determinant of chemotherapy efficacy and that aneuploidy’s role in causing chemotherapy resistance is mediated at least in part by its adverse effects on cell proliferation.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:The effect of parathyroid hormone on osteogenesis is mediated partly by osteolectin

We previously described a new osteogenic growth factor, osteolectin/Clec11a, which is required for the maintenance of skeletal bone mass during adulthood. Osteolectin binds to Integrin α11 (Itga11), promoting Wnt pathway activation and osteogenic differentiation by leptin receptor⁺ (LepR⁺) stromal cells in the bone marrow. Parathyroid hormone (PTH) and sclerostin inhibitor (SOSTi) are bone anabolic agents that are administered to patients with osteoporosis. Here we tested whether osteolectin mediates the effects of PTH or SOSTi on bone formation. We discovered that PTH promoted Osteolectin expression by bone marrow stromal cells within hours of administration and that PTH treatment increased serum osteolectin levels in mice and humans. Osteolectin deficiency in mice attenuated Wnt pathway activation by PTH in bone marrow stromal cells and reduced the osteogenic response to PTH in vitro and in vivo. In contrast, SOSTi did not affect serum osteolectin levels and osteolectin was not required for SOSTi-induced bone formation. Combined administration of osteolectin and PTH, but not osteolectin and SOSTi, additively increased bone volume. PTH thus promotes osteolectin expression and osteolectin mediates part of the effect of PTH on bone formation.

The maintenance and repair of the skeleton require the generation of new bone cells throughout adult life. Osteoblasts are relatively short-lived cells that are constantly regenerated, partly by skeletal stem cells within the bone marrow (1). The main source of new osteoblasts in adult bone marrow is leptin receptor-expressing (LepR⁺) stromal cells (2–4). These cells include the multipotent skeletal stem cells that give rise to the fibroblast colony-forming cells (CFU-Fs) in the bone marrow (2), as well as restricted osteogenic progenitors (5) and adipocyte progenitors (6–8). LepR⁺ cells are a major source of osteoblasts for fracture repair (2) and growth factors for hematopoietic stem cell maintenance (9–11).One growth factor synthesized by LepR⁺ cells, as well as osteoblasts and osteocytes, is osteolectin/Clec11a, a secreted glycoprotein of the C-type lectin domain superfamily (5, 12, 13). Osteolectin is an osteogenic factor that promotes the maintenance of the adult skeleton by promoting the differentiation of LepR⁺ cells into osteoblasts. Osteolectin acts by binding to integrin α11β1, which is selectively expressed by LepR⁺ cells and osteoblasts, activating the Wnt pathway (12). Deficiency for either Osteolectin or Itga11 (the gene that encodes integrin α11) reduces osteogenesis during adulthood and causes early-onset osteoporosis in mice (12, 13). Recombinant osteolectin promotes osteogenic differentiation by bone marrow stromal cells in culture and daily injection of mice with osteolectin systemically promotes bone formation.Osteoporosis is a progressive condition characterized by reduced bone mass and increased fracture risk (14). Several factors contribute to osteoporosis development, including aging, estrogen insufficiency, mechanical unloading, and prolonged glucocorticoid use (14). Existing therapies include antiresorptive agents that slow bone loss, such as bisphosphonates (15, 16) and estrogens (17), and anabolic agents that increase bone formation, such as parathyroid hormone (PTH) (18), PTH-related protein (19), and sclerostin inhibitor (SOSTi) (20). While these therapies increase bone mass and reduce fracture risk, they are not a cure.PTH promotes both anabolic and catabolic bone remodeling (21–24). PTH is synthesized by the parathyroid gland and regulates serum calcium levels, partly by regulating bone formation and bone resorption (23–25). PTH1R is a PTH receptor (26, 27) that is strongly expressed by LepR⁺ bone marrow stromal cells (8, 28–30). Recombinant human PTH (Teriparatide; amino acids 1 to 34) and synthetic PTH-related protein (Abaloparatide) are approved by the US Food and Drug Administration (FDA) for the treatment of osteoporosis (19, 31). Daily (intermittent) administration of PTH increases bone mass by promoting the differentiation of osteoblast progenitors, inhibiting osteoblast and osteocyte apoptosis, and reducing sclerostin levels (32–35). PTH promotes osteoblast differentiation by activating Wnt and BMP signaling in bone marrow stromal cells (28, 36, 37), although the mechanisms by which it regulates Wnt pathway activation are complex and uncertain (38).Sclerostin is a secreted glycoprotein that inhibits Wnt pathway activation by binding to LRP5/6, a widely expressed Wnt receptor (7, 8), reducing bone formation (39, 40). Sclerostin is secreted by osteocytes (8, 41), negatively regulating bone formation by inhibiting the differentiation of osteoblasts (41, 42). SOSTi (Romosozumab) is a humanized monoclonal antibody that binds sclerostin, preventing binding to LRP5/6 and increasing Wnt pathway activation and bone formation (43). It is FDA-approved for the treatment of osteoporosis (20, 44) and has activity in rodents in addition to humans (45, 46).The discovery that osteolectin is a bone-forming growth factor raises the question of whether it mediates the effects of PTH or SOSTi on osteogenesis.     

CYK-1/Formin activation in cortical RhoA signaling centers promotes organismal left–right symmetry breaking

Proper left–right symmetry breaking is essential for animal development, and in many cases, this process is actomyosin-dependent. In Caenorhabditis elegans embryos active torque generation in the actomyosin layer promotes left–right symmetry breaking by driving chiral counterrotating cortical flows. While both Formins and Myosins have been implicated in left–right symmetry breaking and both can rotate actin filaments in vitro, it remains unclear whether active torques in the actomyosin cortex are generated by Formins, Myosins, or both. We combined the strength of C. elegans genetics with quantitative imaging and thin film, chiral active fluid theory to show that, while Non-Muscle Myosin II activity drives cortical actomyosin flows, it is permissive for chiral counterrotation and dispensable for chiral symmetry breaking of cortical flows. Instead, we find that CYK-1/Formin activation in RhoA foci is instructive for chiral counterrotation and promotes in-plane, active torque generation in the actomyosin cortex. Notably, we observe that artificially generated large active RhoA patches undergo rotations with consistent handedness in a CYK-1/Formin–dependent manner. Altogether, we conclude that CYK-1/Formin–dependent active torque generation facilitates chiral symmetry breaking of actomyosin flows and drives organismal left–right symmetry breaking in the nematode worm.

The emergence of left–right asymmetry is essential for normal animal development and, in the majority of animal species, one type of handedness is dominant (1). The actin cytoskeleton plays an instrumental role in establishing the left–right asymmetric body plan of invertebrates like fruit flies (2–6), nematodes (7–11), and pond snails (12–15). Moreover, an increasing number of studies showed that vertebrate left–right patterning also depends on a functional actomyosin cytoskeleton (13, 16–22). Actomyosin-dependent chiral behavior has even been reported in isolated cells (23–28) and such cell-intrinsic chirality has been shown to promote left–right asymmetric morphogenesis of tissues (29, 30), organs (21, 31), and entire embryonic body plans (12, 13, 32, 33). Active force generation in the actin cytoskeleton is responsible for shaping cells and tissues during embryo morphogenesis. Torques are rotational forces with a given handedness and it has been proposed that in plane, active torque generation in the actin cytoskeleton drives chiral morphogenesis (7, 8, 34, 35).What could be the molecular origin of these active torques? The actomyosin cytoskeleton consists of actin filaments, actin-binding proteins, and Myosin motors. Actin filaments are polar polymers with a right-handed helical pitch and are therefore chiral themselves (36, 37). Due to the right-handed pitch of filamentous actin, Myosin motors can rotate actin filaments along their long axis while pulling on them (33, 38–42). Similarly, when physically constrained, members of the Formin family rotate actin filaments along their long axis while elongating them (43). In both cases the handedness of this rotation is determined by the helical nature of the actin polymer. From this it follows that both Formins and Myosins are a potential source of molecular torque generation that could drive cellular and organismal chirality. Indeed, chiral processes across different length scales, and across species, are dependent on Myosins (19), Formins (13–15, 26), or both (7, 8, 21, 44). It is, however, unclear how Formins and Myosins contribute to active torque generation and the emergence chiral processes in developing embryos.In our previous work we showed that the actomyosin cortex of some Caenorhabditis elegans embryonic blastomeres undergoes chiral counterrotations with consistent handedness (7, 35). These chiral actomyosin flows can be recapitulated using active chiral fluid theory that describes the actomyosin layer as a thin-film, active gel that generates active torques (7, 45, 46). Chiral counterrotating cortical flows reorient the cell division axis, which is essential for normal left–right symmetry breaking (7, 47). Moreover, cortical counterrotations with the same handedness have been observed in Xenopus one-cell embryos (32), suggesting that chiral counterrotations are conserved among distant species. Chiral counterrotating actomyosin flow in C. elegans blastomeres is driven by RhoA signaling and is dependent on Non-Muscle Myosin II motor proteins (7). Moreover, the Formin CYK-1 has been implicated in actomyosin flow chirality during early polarization of the zygote as well as during the first cytokinesis (48, 49). Despite having identified a role for Myosins and Formins, the underlying mechanism by which active torques are generated remains elusive.Here we show that the Diaphanous-like Formin, CYK-1/Formin, is a critical determinant for the emergence of actomyosin flow chirality, while Non-Muscle Myosin II (NMY-2) plays a permissive role. Our results show that cortical CYK-1/Formin is recruited by active RhoA signaling foci and promotes active torque generation, which in turn tends to locally rotate the actomyosin cortex clockwise. In the highly connected actomyosin meshwork, a gradient of these active torques drives the emergence of chiral counterrotating cortical flows with uniform handedness, which is essential for proper left–right symmetry breaking. Together, these results provide mechanistic insight into how Formin-dependent torque generation drives cellular and organismal left–right symmetry breaking.     

Synthetic protein conjugate vaccines provide protection against Mycobacterium tuberculosis in mice

The global incidence of tuberculosis remains unacceptably high, with new preventative strategies needed to reduce the burden of disease. We describe here a method for the generation of synthetic self-adjuvanted protein vaccines and demonstrate application in vaccination against Mycobacterium tuberculosis. Two vaccine constructs were designed, consisting of full-length ESAT6 protein fused to the TLR2-targeting adjuvants Pam₂Cys-SK₄ or Pam₃Cys-SK₄. These were produced by chemical synthesis using a peptide ligation strategy. The synthetic self-adjuvanting vaccines generated powerful local CD4⁺ T cell responses against ESAT6 and provided significant protection in the lungs from virulent M. tuberculosis aerosol challenge when administered to the pulmonary mucosa of mice. The flexible synthetic platform we describe, which allows incorporation of adjuvants to multiantigenic vaccines, represents a general approach that can be applied to rapidly assess vaccination strategies in preclinical models for a range of diseases, including against novel pandemic pathogens such as SARS-CoV-2.

Vaccination is the most effective strategy for the prevention of many infectious diseases, but so far, vaccines against the major human pathogen Mycobacterium tuberculosis—the causative agent of tuberculosis (TB)—have shown limited efficacy. TB is the leading cause of death worldwide from a single infectious agent (1), resulting in 10 million new cases and 1.5 million deaths in 2018 alone, with huge socioeconomic costs globally (2). Currently, the only vaccine available for TB is Mycobacterium bovis bacille Calmette–Guérin (BCG), an attenuated Mycobacterium that stimulates immune responses against antigens shared with M. tuberculosis (3). Although BCG prevents severe disseminated forms of TB in infants and children, it fails to provide protection against infectious pulmonary disease in adolescents and adults, and has not prevented the spread of M. tuberculosis among populations (3). In addition, as a live-attenuated vaccine, BCG poses risks to immunocompromised subjects, in particular people living with HIV/AIDS (3). There is therefore an urgent need to develop new types of vaccines that provide safe and more effective protection against TB.Protein-based subunit vaccines are one safe option, but these require adjuvants to activate pattern recognition receptors on antigen-presenting cells (APCs) that stimulate cytokine release and up-regulate cell surface expression of costimulatory molecules essential for the activation of T cells (4, 5). Alternatively, liposomal formulations have also been used to achieve an immunostimulatory effect (6, 7). The adjuvant component of vaccines can either be added as an admixture with the protein, or bound to the antigen to generate a self-adjuvanting vaccine (8, 9). Delivery as a conjugate self-adjuvanting vaccine has the advantage of direct stimulation of the APCs, which take up and process the vaccine antigen for presentation to T cells (5, 10–13). In addition, covalently bound adjuvants enhance uptake of antigens through receptor-mediated phagocytosis (14). In general, self-adjuvanting vaccines have utilized peptide antigens, and these have induced protective immunity in murine models (5, 11, 14). For example, we demonstrated that immunization with a peptide epitope from the M. tuberculosis–derived 6-kDa early secretory antigenic target (ESAT6) protein covalently bound to the palmitoyl (Pam)-containing TLR2/6 heterodimer agonist, Pam₂Cys-SerLys₄ (Pam₂Cys-SK₄), was immunogenic and protective in mice (11). However, because of the diverse human leukocyte antigen (HLA) alleles in humans, TB subunit vaccines must include multiple epitopes in order to stimulate T cell responses in the broad population; this can be achieved through the use of full-length proteins (15).The majority of adjuvants are hydrophobic lipopeptide or glycolipid molecules. A major difficulty in generating self-adjuvanting vaccines is the fusion of an aqueous soluble protein with the hydrophobic adjuvant molecule. To address this, we have developed a robust synthetic platform by which self-adjuvanted protein vaccines can be produced in high purity and without issues associated with solubility during adjuvant conjugation. We opted for a synthetic strategy that harnessed the automation and efficiency of solid-phase peptide and lipopeptide synthesis, combined with efficient peptide ligation technologies to assemble the self-adjuvanted protein vaccines.The route of delivery also influences the efficacy of vaccines. There is growing evidence to support the benefit of mucosal or pulmonary immunization for protection against respiratory pathogens, including M. tuberculosis (16–20), and whole-cell, viral, and peptide conjugate vaccines have been more effective when delivered to the lung (11, 21–23). This approach has been successful with an aerosol measles vaccine (24) and has been recently extended to human clinical trials for TB using aerosolized Modified Vaccinia Ankara-85A (MVA85A) (25), and an adenoviral-vectored vaccine (Ad5Ag85A; identifier: {"type":"clinical-trial","attrs":{"text":"NCT02337270","term_id":"NCT02337270"}}NCT02337270). Vaccination at the pulmonary mucosa generates memory CD4⁺ T cells that are retained in the lungs and provide an early response to M. tuberculosis exposure (11, 26). Inhalable vaccines also provide economic and practical advantages for mass immunization programs, as they can be delivered without the need for needles and trained medical personnel (17). Protein-based subunit vaccines have particular advantages for development as pulmonary vaccines; they remove the risks associated with live vaccines, are appropriate for immunocompromised individuals, and importantly are suitable for repeated use to boost immunity. In this work, we selected ESAT6 (Rv3875) as a vaccine antigen because of its promise in preclinical and clinical studies (7, 27, 28), and fused the protein to Pam₂Cys or Pam₃Cys, adjuvants known to be safe and effective in the lung mucosal environment (11, 29). Mucosal delivery of these self-adjuvanting vaccines to mice led to the induction of substantial Th17-type T cell responses in the lungs and significant protection against experimental M. tuberculosis infection.     

Violet light suppresses lens-induced myopia via neuropsin (OPN5) in mice

Myopia has become a major public health concern, particularly across much of Asia. It has been shown in multiple studies that outdoor activity has a protective effect on myopia. Recent reports have shown that short-wavelength visible violet light is the component of sunlight that appears to play an important role in preventing myopia progression in mice, chicks, and humans. The mechanism underlying this effect has not been understood. Here, we show that violet light prevents lens defocus–induced myopia in mice. This violet light effect was dependent on both time of day and retinal expression of the violet light sensitive atypical opsin, neuropsin (OPN5). These findings identify Opn5-expressing retinal ganglion cells as crucial for emmetropization in mice and suggest a strategy for myopia prevention in humans.

Myopia (nearsightedness) in school-age children is generally axial myopia, which is the consequence of elongation of the eyeball along the visual axis. This shape change results in blurred vision but can also lead to severe complications including cataract, retinal detachment, myopic choroidal neovascularization, glaucoma, and even blindness (1–3). Despite the current worldwide pandemic of myopia, the mechanism of myopia onset is still not understood (4–8). One hypothesis that has earned a current consensus is the suggestion that a change in the lighting environment of modern society is the cause of myopia (9, 10). Consistent with this, outdoor activity has a protective effect on myopia development (9, 11, 12), though the main reason for this effect is still under debate (7, 12, 13). One explanation is that bright outdoor light can promote the synthesis and release of dopamine in the eye, a myopia-protective neuromodulator (14–16). Another suggestion is that the distinct wavelength composition of sunlight compared with fluorescent or LED (light-emitting diode) artificial lighting may influence myopia progression (9, 10). Animal studies have shown that different wavelengths of light can affect the development of myopia independent of intensity (17, 18). The effects appear to be distinct in different species: for chicks and guinea pigs, blue light showed a protective effect on experimentally induced myopia, while red light had the opposite effect (18–22). For tree shrews and rhesus monkeys, red light is protective, and blue light causes dysregulation of eye growth (23–25).It has been shown that visible violet light (VL) has a protective effect on myopia development in mice, in chick, and in human (10, 26, 27). According to Commission Internationale de l’Eclairage (International Commission on Illumination), VL has the shortest wavelength of visible light (360 to 400 nm). These wavelengths are abundant in outside sunlight but can only rarely be detected inside buildings. This is because the ultraviolet (UV)-protective coating on windows blocks all light below 400 nm and because almost no VL is emitted by artificial light sources (10). Thus, we hypothesized that the lack of VL in modern society is one reason for the myopia boom (9, 10, 26).In this study, we combine a newly developed lens-induced myopia (LIM) model with genetic manipulations to investigate myopia pathways in mice (28, 29). Our data confirm (10, 26) that visible VL is protective but further show that delivery of VL only in the evening is sufficient for the protective effect. In addition, we show that the protective effect of VL on myopia induction requires OPN5 (neuropsin) within the retina. The absence of retinal Opn5 prevents lens-induced, VL-dependent thickening of the choroid, a response thought to play a key role in adjusting the size of the eyeball in both human and animal myopia models (30–33). This report thus identifies a cell type, the Opn5 retinal ganglion cell (RGC), as playing a key role in emmetropization. The requirement for OPN5 also explains why VL has a protective effect on myopia development.     

Stage-specific overcompensation,the hydra effect,and the failure to eradicate an invasive predator

As biological invasions continue to increase globally, eradication programs have been undertaken at significant cost, often without consideration of relevant ecological theory. Theoretical fisheries models have shown that harvest can actually increase the equilibrium size of a population, and uncontrolled studies and anecdotal reports have documented population increases in response to invasive species removal (akin to fisheries harvest). Both findings may be driven by high levels of juvenile survival associated with low adult abundance, often referred to as overcompensation. Here we show that in a coastal marine ecosystem, an eradication program resulted in stage-specific overcompensation and a 30-fold, single-year increase in the population of an introduced predator. Data collected concurrently from four adjacent regional bays without eradication efforts showed no similar population increase, indicating a local and not a regional increase. Specifically, the eradication program had inadvertently reduced the control of recruitment by adults via cannibalism, thereby facilitating the population explosion. Mesocosm experiments confirmed that adult cannibalism of recruits was size-dependent and could control recruitment. Genomic data show substantial isolation of this population and implicate internal population dynamics for the increase, rather than recruitment from other locations. More broadly, this controlled experimental demonstration of stage-specific overcompensation in an aquatic system provides an important cautionary message for eradication efforts of species with limited connectivity and similar life histories.

Theoretical population models can produce counterintuitive predictions regarding the consequences of harvest or removal of predatory species. These models show that for simple predator-prey systems, there can be positive population responses to predator mortality resulting from harvest for fisheries or population management, which can create an increased equilibrium level of that predator species (1–5). Among these mortality processes is the “hydra effect,” named after the mythical multi-headed serpent that grew two new heads for each one that was removed (6, 7). This counterintuitive outcome can be driven by a density-dependent process known as overcompensation. The hydra effect typically refers to higher equilibrium or time-averaged densities in response to increased mortality, typically involving consumer populations undergoing population cycles. Population increases in response to mortality can be the result of stage-specific overcompensation, which involves an increase in a specific life history stage or a size class following increased mortality. The first analysis of overcompensatory responses to mortality did not depend on stage specificity and was applied initially to fisheries harvests (1). Subsequent models have included stage specificity and have been applied to a broad range of systems in which species have been harvested for consumption or removed for population control of non-native species (4, 5, 8–15).Theory suggests that overcompensation in response to harvest or removal can occur for a variety of reasons, including 1) reduced competition for resources and increased adult reproduction rates, 2) faster rates of juvenile maturation or greater success in reaching the adult stage, and 3) increased juvenile or adult survival rates (1–7). An increase in reproductive output in response to reduced adult density can be the result of a reduction in resource competition (SI Appendix, Fig. S1).While there is substantial evidence that conditions that could produce density-dependent overcompensation occur frequently, evidence for overcompensation in natural populations is rare. For only a few populations do we have the long-term demographic data collected over a sufficiently long duration and for population densities over a wide enough range to detect this effect. Unfortunately, recent reviews of population increases in response to increased mortality do not include field studies with explicit controls for removals (13–17).There are examples of density-dependent overcompensation from field populations (4, 13–15), as well as a larger number of studies from the laboratory and greenhouse typically involving plant and insect populations (18–22). Among the field examples is a population control program for smallmouth bass in a lake in upstate New York, which paradoxically resulted in greater bass abundance, primarily of juveniles, after 7 y of removal efforts (23, 24). Another field study in the United Kingdom showed that perch populations responded similarly when an unidentified pathogen decimated adults (25). Other programs that attempted to remove invasive fishes, including pikeperch in England (26), brook trout in Idaho (27), and Tilapia in Australia (28), showed similar results. However, although many of these examples involved well-executed studies with substantial field data, none had explicit controls for removal, such as comparable populations without harvest (or disease). Thus, despite the support of current theory in these studies, the contribution of external factors to observed population responses to harvest remains uncertain. To date, we are unaware of any experimental studies with comparable controls in a field population that demonstrates overcompensation in a single species (13–15).     

Conditions and extent of volatile loss from the Moon during formation of the Procellarum basin

Rocks from the lunar interior are depleted in moderately volatile elements (MVEs) compared to terrestrial rocks. Most MVEs are also enriched in their heavier isotopes compared to those in terrestrial rocks. Such elemental depletion and heavy isotope enrichments have been attributed to liquid–vapor exchange and vapor loss from the protolunar disk, incomplete accretion of MVEs during condensation of the Moon, and degassing of MVEs during lunar magma ocean crystallization. New Monte Carlo simulation results suggest that the lunar MVE depletion is consistent with evaporative loss at 1,670 ± 129 K and an oxygen fugacity +2.3 ± 2.1 log units above the fayalite-magnetite-quartz buffer. Here, we propose that these chemical and isotopic features could have resulted from the formation of the putative Procellarum basin early in the Moon’s history, during which nearside magma ocean melts would have been exposed at the surface, allowing equilibration with any primitive atmosphere together with MVE loss and isotopic fractionation.

Returned samples of basaltic rocks from the Moon provided evidence decades ago that the Moon is depleted in volatile elements compared to the Earth (1), with lunar basalt abundances of moderately volatile elements (MVEs) being ∼1/5 that of terrestrial basalt abundances for alkali elements and ∼1/40 for other MVE, such as Zn, Ag, In, and Cd (2). The theme of lunar volatiles thus seemed settled. Yet, the unambiguous detection in 2008 of lunar indigenous hydrogen and other volatile elements, such as F, Cl, and S in pyroclastic glasses (3), heralded a new era of research into lunar volatiles, overturning the decades-old paradigm of a bone-dry Moon (e.g., refs. 4 and 5). Here, we define volatile elements as those with 50% condensation temperatures below these of the major rock-forming elements Fe, Mg, and Si (6). This paradigm shift was accompanied by new measurements of volatile stable isotope compositions (e.g., H, C, N, Cl, K, Cr, Cu, Zn, Ga, Rb, and Sn) in a wealth of bulk lunar samples (7–18) and in the mineral phases and melt inclusions they host (19–28). These studies have shown that the stable isotope compositions of most MVEs (e.g., K, Zn, Ga, and Rb) are enriched in their heavier isotopes with respect to the bulk silicate Earth (BSE) (9, 11, 13–15, 17). Such heavy isotope enrichment is associated with elemental depletion, which has been variously attributed to liquid–vapor exchange and vapor loss from the protolunar disk (17, 18), incomplete accretion of MVEs during condensation of the Moon (13, 29, 30), and degassing of these elements during lunar magma ocean crystallization (9, 11, 14, 15, 25, 31). Almost all these hypotheses have typically assumed that the MVE depletions and associated MVE isotope fractionations are relevant to the whole Moon. However, our lunar sample collections are biased, as all Apollo and Luna returned samples come from the lunar nearside from within or around the anomalous Procellarum KREEP Terrane (PKT) region (e.g., ref. 32), where KREEP stands for enriched in K, REEs, and P. Barnes et al. (26) proposed that the heavy Cl isotope signature measured in KREEP-rich Apollo samples resulted from metal-chloride degassing from late-stage lunar magma ocean melts in response to a large crust-breaching impact event, spatially associated with the PKT region, which facilitated exposure of these late-stage melts to the lunar surface. Here, we further investigate whether a localized impact event could have been responsible for the general MVE depletion and heavy MVE isotope enrichment measured in lunar samples.     

Circular swimming motility and disordered hyperuniform state in an algae system

Active matter comprises individually driven units that convert locally stored energy into mechanical motion. Interactions between driven units lead to a variety of nonequilibrium collective phenomena in active matter. One of such phenomena is anomalously large density fluctuations, which have been observed in both experiments and theories. Here we show that, on the contrary, density fluctuations in active matter can also be greatly suppressed. Our experiments are carried out with marine algae (Effrenium voratum), which swim in circles at the air–liquid interfaces with two different eukaryotic flagella. Cell swimming generates fluid flow that leads to effective repulsions between cells in the far field. The long-range nature of such repulsive interactions suppresses density fluctuations and generates disordered hyperuniform states under a wide range of density conditions. Emergence of hyperuniformity and associated scaling exponent are quantitatively reproduced in a numerical model whose main ingredients are effective hydrodynamic interactions and uncorrelated random cell motion. Our results demonstrate the existence of disordered hyperuniform states in active matter and suggest the possibility of using hydrodynamic flow for self-assembly in active matter.

Active matter exists over a wide range of spatial and temporal scales (1–6) from animal groups (7, 8) to robot swarms (9–11), to cell colonies and tissues (12–16), to cytoskeletal extracts (17–20), to man-made microswimmers (21–25). Constituent particles in active matter systems are driven out of thermal equilibrium at the individual level; they interact to develop a wealth of intriguing collective phenomena, including clustering (13, 22, 24), flocking (11, 26), swarming (12, 13), spontaneous flow (14, 20), and giant density fluctuations (10, 11). Many of these observed phenomena have been successfully described by particle-based or continuum models (1–6), which highlight the important roles of both individual motility and interparticle interactions in determining system dynamics.Current active matter research focuses primarily on linearly swimming particles which have a symmetric body and self-propel along one of the symmetry axes. However, a perfect alignment between the propulsion direction and body axis is rarely found in reality. Deviation from such a perfect alignment leads to a persistent curvature in the microswimmer trajectories; examples of such circle microswimmers include anisotropic artificial micromotors (27, 28), self-propelled nematic droplets (29, 30), magnetotactic bacteria and Janus particles in rotating external fields (31, 32), Janus particle in viscoelastic medium (33), and sperm and bacteria near interfaces (34, 35). Chiral motility of circle microswimmers, as predicted by theoretical and numerical investigations, can lead to a range of interesting collective phenomena in circular microswimmers, including vortex structures (36, 37), localization in traps (38), enhanced flocking (39), and hyperuniform states (40). However, experimental verifications of these predictions are limited (32, 35), a situation mainly due to the scarcity of suitable experimental systems.Here we address this challenge by investigating marine algae Effrenium voratum (41, 42). At air–liquid interfaces, E. voratum cells swim in circles via two eukaryotic flagella: a transverse flagellum encircling the cellular anteroposterior axis and a longitudinal one running posteriorly. Over a wide range of densities, circling E. voratum cells self-organize into disordered hyperuniform states with suppressed density fluctuations at large length scales. Hyperuniformity (43, 44) has been considered as a new form of material order which leads to novel functionalities (45–49); it has been observed in many systems, including avian photoreceptor patterns (50), amorphous ices (51), amorphous silica (52), ultracold atoms (53), soft matter systems (54–61), and stochastic models (62–64). Our work demonstrates the existence of hyperuniformity in active matter and shows that hydrodynamic interactions can be used to construct hyperuniform states.     

From the Cover: Neural interfacing architecture enables enhanced motor control and residual limb functionality postamputation

Despite advancements in prosthetic technologies, patients with amputation today suffer great diminution in mobility and quality of life. We have developed a modified below-knee amputation (BKA) procedure that incorporates agonist–antagonist myoneural interfaces (AMIs), which surgically preserve and couple agonist–antagonist muscle pairs for the subtalar and ankle joints. AMIs are designed to restore physiological neuromuscular dynamics, enable bidirectional neural signaling, and offer greater neuroprosthetic controllability compared to traditional amputation techniques. In this prospective, nonrandomized, unmasked study design, 15 subjects with AMI below-knee amputation (AB) were matched with 7 subjects who underwent a traditional below-knee amputation (TB). AB subjects demonstrated significantly greater control of their residual limb musculature, production of more differentiable efferent control signals, and greater precision of movement compared to TB subjects (P < 0.008). This may be due to the presence of greater proprioceptive inputs facilitated by the significantly higher fascicle strains resulting from coordinated muscle excursion in AB subjects (P < 0.05). AB subjects reported significantly greater phantom range of motion postamputation (AB: 12.47 ± 2.41, TB: 10.14 ± 1.45 degrees) when compared to TB subjects (P < 0.05). Furthermore, AB subjects also reported less pain (12.25 ± 5.37) than TB subjects (17.29 ± 10.22) and a significant reduction when compared to their preoperative baseline (P < 0.05). Compared with traditional amputation, the construction of AMIs during amputation confers the benefits of enhanced physiological neuromuscular dynamics, proprioception, and phantom limb perception. Subjects’ activation of the AMIs produces more differentiable electromyography (EMG) for myoelectric prosthesis control and demonstrates more positive clinical outcomes.

The standard-of-care surgical approach to amputation has not seen considerable innovation since its conception in the mid-1800s (1), despite significant progress in biomechatronics and advanced reconstructive techniques. The typical amputation procedure neglects neurological substrates and disrupts key neuromuscular relationships responsible for bidirectional (efferent, afferent) signaling. The deafferentation of lower motor neurons triggers central reorganization of motor circuits, which negatively impacts motor imagery and motor coordination (2, 3) and results in significant neuroma pain (4–6), phantom pain (7), and maladaptive or diminishing phantom sensation (8–10). Phantom sensation, the perception of one’s phantom limb while at rest and in motion, is an important component of motor imagery utilized in the preparation of motor control commands and can be a source of chronic irritation, if unpleasant. The haphazard arrangement and myodesis of residual musculature further constrain the ability for individual muscles to dynamically excurse, causing cocontraction of muscles and changing gait patterns (11–14). Together, these peripheral and central modifications result in the poor production of efferent signals for direct myoelectric control (15, 16). To compensate for these shortcomings, considerable effort has been spent on developing and deploying pattern-recognition–based myoelectric control strategies (17–19). However, even with these advanced myoelectric devices and controllers, end users find their operation cumbersome and time consuming (20). Motor control is also challenged by the lack of proprioceptive sensory feedback from prostheses (12, 21–23). Together, the limitations of the current amputation approaches significantly lower the quality of life for persons with amputation (24–27).In recognition of these shortcomings, surgical researchers have recently begun to explore new strategies to modify standard amputation procedures. Targeted muscle reinnervation (TMR) (28, 29) and regenerative peripheral nerve interfaces (RPNIs) (30, 31) represent approaches that are designed to provide greater efferent motor signals for myoelectric control and mitigate neuroma pain. However, neither approach offers muscle–tendon afferent proprioceptive signaling, which is physiologically mediated by agonist–antagonist muscle dynamics and critical for trajectory planning, fine motor control, and reflexes (32, 33).The agonist–antagonist myoneural interface (AMI) is a more recent surgical approach and neural interfacing strategy designed to augment volitional motor control and restore muscle–tendon proprioception (11, 12, 34–37) by surgically coapting agonist and antagonist muscles to restore natural physiological muscle pairing and dynamics. When the agonist muscle contracts, the antagonist muscle stretches (or vice versa), giving rise to musculotendinous afferent feedback from muscle spindle fibers (length and velocity) and Golgi tendon organs (force). For each joint in a bionic limb, one AMI is surgically constructed in the residuum. Functional electrical stimulation (FES) applied to the antagonist muscle of the AMI can provide force or position feedback onto the agonist (or vice versa) from a bionic prosthesis to inform the user of prosthetic torque or position, respectively (11). In Clites et al. (11), an early human subject with an AMI amputation demonstrated dynamic muscle excursions, individualized contraction of each AMI muscle, and graded proprioceptive muscle–tendon feedback in response to muscle activation. This subject additionally demonstrated greater control of joint position, impedance, and FES-based torque feedback from a bionic prosthesis when compared to subjects with a traditional amputation. This pilot study demonstrated the potential of the AMI and paved the way for further implementation and investigation of the physiological properties, phantom limb perceptions, pain, and motor control of the AMI neuromuscular constructs.In this study, we characterize the physiological outcomes of subjects with an AMI below-knee amputation (AB) (n = 15) and compare them against those of matched control subjects with a traditional below-knee amputation (TB) (n = 7). Given the emphasis placed on the reconstruction of peripheral neuromusculature with AMIs, we hypothesize that the AB cohort will experience an enhancement in phantom sensation and range of motion (ROM) percepts compared with the TB cohort. As a result of the dynamically coupled agonist–antagonist muscles comprising the AMI constructs, we also hypothesize that the AB cohort will demonstrate greater muscle excursion and fascicle strains compared to the TB population. With these improvements in residual limb muscle dynamics, motor capabilities, and perception, we further anticipate that AB subjects will demonstrate greater accuracy and precision of performance on ankle and subtalar intended movements compared to matched TB participants. These hypotheses are evaluated through a combination of electromyography (EMG), goniometry, ultrasonography, and surveys.     

Improved bounds on entropy production in living systems

Living systems maintain or increase local order by working against the second law of thermodynamics. Thermodynamic consistency is restored as they consume free energy, thereby increasing the net entropy of their environment. Recently introduced estimators for the entropy production rate have provided major insights into the efficiency of important cellular processes. In experiments, however, many degrees of freedom typically remain hidden to the observer, and, in these cases, existing methods are not optimal. Here, by reformulating the problem within an optimization framework, we are able to infer improved bounds on the rate of entropy production from partial measurements of biological systems. Our approach yields provably optimal estimates given certain measurable transition statistics. In contrast to prevailing methods, the improved estimator reveals nonzero entropy production rates even when nonequilibrium processes appear time symmetric and therefore may pretend to obey detailed balance. We demonstrate the broad applicability of this framework by providing improved bounds on the energy consumption rates in a diverse range of biological systems including bacterial flagella motors, growing microtubules, and calcium oscillations within human embryonic kidney cells.

Thermodynamic laws place fundamental limits on the efficiency and fitness of living systems (1, 2). To maintain cellular order and perform essential biological functions such as sensing (3–6), signaling (7), replication (8, 9) or locomotion (10), organisms consume energy and dissipate heat. In doing so, they increase the entropy of their environment (2), in agreement with the second law of thermodynamics (11). Obtaining reliable estimates for the energy consumption and entropy production in living matter holds the key to understanding the physical boundaries (12–14) that constrain the range of theoretically and practically possible biological processes (3). Recent experimental (6, 15, 16) and theoretical (17–20) advances in the imaging and modeling of cellular and subcellular dynamics have provided groundbreaking insights into the thermodynamic efficiency of molecular motors (14, 21), biochemical signaling (16, 22, 23) and reaction (24) networks, and replication (9) and adaption (25) phenomena. Despite such major progress, however, it is also known that the currently available entropy production estimators (26, 27) can fail under experimentally relevant conditions, especially when only a small set of observables is experimentally accessible or nonequilibrium transport currents (28–30) vanish.To help overcome these limitations, we introduce here a generic optimization framework that can produce significantly improved bounds on the entropy production in living systems. We will prove that these bounds are optimal given certain measurable statistics. From a practical perspective, our method requires observations of only a few coarse-grained state variables of an otherwise hidden Markovian network. We demonstrate the practical usefulness by determining improved entropy production bounds for bacterial flagella motors (10, 31), growing microtubules (32, 33), and calcium oscillations (7, 34) in human embryonic kidney cells.Generally, entropy production rates can be estimated from the time series of stochastic observables (35). Thermal equilibrium systems obey the principle of detailed balance, which means that every forward trajectory is as likely to be observed as its time reversed counterpart, neutralizing the arrow of time (36). By contrast, living organisms operate far from equilibrium, which means that the balance between forward and reversed trajectories is broken and net fluxes may arise (1, 37–39). When all microscopic details of a nonequilibrium system are known, one can measure the rate of entropy production by comparing the likelihoods of forward and reversed trajectories in sufficiently large data samples (35, 36). However, in most if not all biophysical experiments, many degrees of freedom remain hidden to the observer, demanding methods (28, 40, 41) that do not require complete knowledge of the system. A powerful alternative is provided by thermodynamic uncertainty relations (TURs), which use the mean and variance of steady-state currents to bound entropy production rates (18, 19, 26, 42–48). Although highly useful when currents can be measured (44–47), or when the system can be externally manipulated (40, 49), these methods give, by construction, trivial zero bounds for current-free nonequilibrium systems, such as driven one-dimensional (1D) nonperiodic oscillators. In the absence of currents, potential asymmetries in the forward and reverse trajectories can still be exploited to bound the entropy production rate (29, 30, 50), but to our knowledge no existing method is capable of producing nonzero bounds when forward and reverse trajectories are statistically identical. Moreover, even though previous bounds can become tight in some cases (51), optimal entropy production estimators for nonequilibrium systems are in general unknown.To obtain bounds that are provably optimal under reasonable conditions on the available data, we reformulate the problem here within an optimization framework. Formally, we consider any steady-state Markovian dynamics for which only coarse-grained variables are observable, where these observables may appear non-Markovian. We then search over all possible underlying Markovian systems to identify the one which minimizes the entropy production rate while obeying the observed statistics. More specifically, our algorithmic implementation leverages information about successive transitions, allowing us to discover nonzero bounds on entropy production even when the coarse-grained statistics appear time symmetric. We demonstrate this for both synthetic test data and experimental data (52) for flagella motors. Subsequently, we consider the entropy production of microtubules (33), which slowly grow before rapidly shrinking in steady state, to show how refined coarse graining in space and time leads to improved bounds. The final application to calcium oscillations in human embryonic kidney cells (34) illustrates how external stimulation with drugs can increase entropy production.     

Conditional destabilization of the TPLATE complex impairs endocytic internalization

In plants, endocytosis is essential for many developmental and physiological processes, including regulation of growth and development, hormone perception, nutrient uptake, and defense against pathogens. Our toolbox to modulate this process is, however, rather limited. Here, we report a conditional tool to impair endocytosis. We generated a partially functional TPLATE allele by substituting the most conserved domain of the TPLATE subunit of the endocytic TPLATE complex (TPC). This substitution destabilizes TPC and dampens the efficiency of endocytosis. Short-term heat treatment increases TPC destabilization and reversibly delocalizes TPLATE from the plasma membrane to aggregates in the cytoplasm. This blocks FM uptake and causes accumulation of various known endocytic cargoes at the plasma membrane. Short-term heat treatment therefore transforms the partially functional TPLATE allele into an effective conditional tool to impair endocytosis. Next to their role in endocytosis, several TPC subunits are also implicated in actin-regulated autophagosomal degradation. Inactivating TPC via the WDX mutation, however, does not impair autophagy, thus enabling specific and reversible modulation of endocytosis in planta.

Endocytosis is an evolutionarily conserved eukaryotic pathway by which extracellular material and plasma membrane (PM) components are internalized via vesicles (1, 2). Clathrin-mediated endocytosis (CME), relying on the scaffolding protein clathrin, is the most prominent and the most studied endocytic pathway (3–5). As clathrin does not interact directly with the PM, nor does it recognize cargoes, adaptor proteins are required to act as essential links between the clathrin coat and the PM (6). In plant cells, material selected for CME is recognized by two adaptor complexes, the adaptor complex 2 (AP-2) and the TPLATE complex (TPC) (7–9). In contrast to TPC, single subunit mutants of AP-2 are viable (7, 8, 10–13) and AP-2 recruitment and dynamics appear to rely on TPC function (8, 14).TPC represents an ancestral adaptor complex, which is however absent in present-day metazoans and yeasts. It was experimentally identified as an octameric complex in Arabidopsis and as a hexametric complex in Dictyostelium (8, 15). Plants, however, are the only eukaryotic supergroup identified so far where TPC is essential for life (8, 15), as knockout or severe knockdown of single subunits of TPC in Arabidopsis leads to pollen or seedling lethality, respectively (8, 13). Two TPC subunits, AtEH1/Pan1 and AtEH2/Pan1, were not associated with the other TPC core components when the complex was forced into the cytoplasm by truncating the TML subunit and did not copurify with the other TSET components in Dictyostelium. This indicates that they may be auxiliary components to the core TPC (8, 15). These AtEH/Pan1 proteins were recently identified as important players in actin-regulated autophagy in plants. AtEH/Pan1 proteins recruit several components of the endocytic machinery to the autophagosomes, and are degraded together with them under stress conditions (16). However, whether this pathway serves to degrade specific cargoes or to regulate the endocytic machinery itself (17), and whether the whole TPC is required for this degradation pathway, remains unclear.Genetic and chemical tools to manipulate endocytosis have been extensively investigated via interfering with the functions of endocytic players, such as clathrin (18–22), adaptor proteins (7, 10–12, 14, 23–25), and dynamin-related proteins (26–30). The chemical inhibitors originally used to affect CME in plants have recently been described to possess undesirable side effects (31) or to affect proteins that are not only specific for endocytosis: for example, clathrin itself, as it is also involved in TGN trafficking (19, 22). The same is true for several genetic tools currently available to affect CME in plants (18, 21, 22, 30). Manipulation of TPC, functioning exclusively at the PM, represents a very good candidate to affect CME more specifically. So far however, there are no chemical tools to target TPC functions or dominant-negative mutants available. Inducible silencing works, but causes seedling lethality and takes several days to become effective (8). The only tools to manipulate TPC function in viable plants consist of knock-down mutants with very mild reduction of expression and consequently similar mild effects on CME (8, 14, 16, 32).