The battle between harvest and natural selection creates small and shy fish

Harvest of fish and wildlife, both commercial and recreational, is a selective force that can induce evolutionary changes to life history and behavior. Naturally selective forces may create countering selection pressures. Assessing natural fitness represents a considerable challenge in broadcast spawners. Thus, our understanding about the relative strength of natural and fisheries selection is slim. In the field, we compared the strength and shape of harvest selection to natural selection on body size over four years and behavior over one year in a natural population of a freshwater top predator, the northern pike (Esox lucius). Natural selection was approximated by relative reproductive success via parent–offspring genetic assignments over four years. Harvest selection was measured by comparing individuals susceptible to recreational angling with individuals never captured by this gear type. Individual behavior was measured by high-resolution acoustic telemetry. Harvest and natural size selection operated with equal strength but opposing directions, and harvest size selection was consistently negative in all study years. Harvest selection also had a substantial behavioral component independent of body length, while natural behavioral selection was not documented, suggesting the potential for directional harvest selection favoring inactive, timid fish. Simulations of the outcomes of different fishing regulations showed that traditional minimum size-based harvest limits are unlikely to counteract harvest selection without being completely restrictive. Our study suggests harvest selection may be inevitable and recreational fisheries may thus favor small, inactive, shy, and difficult-to-capture fish. Increasing fractions of shy fish in angling-exploited stocks would have consequences for stock assessment and all fisheries operating with hook and line.

Anticipating and preparing for future evolutionary changes within harvested populations whether by fishing or hunting is critical for sustainable natural resource management and successful conservation of ecosystems (1–6). Harvest-induced evolution is a concern for both commercial and recreational fisheries, and harvest from recreational fisheries now frequently exceeds harvest from commercial fisheries in some marine fish and most inland fish populations (7). Harvesting, firstly, elevates adult mortality which favors the evolution of life history adaptations that maximize current as opposed to future reproduction [i.e., a fast life history characterized by early reproduction at a small size and elevated reproductive effort (1, 2)]. Additionally, harvesting is trait selective. Most individuals in harvested populations are not captured or hunted randomly (8). Instead, a suite of traits elevates the probability of harvest (8–13). In fisheries, vulnerability to harvest and fish body size are positively related across most fishing gears, and the relationship is exacerbated by the widespread use of minimum landing sizes (14, 15). Consequently, the average body size of individuals within fish stocks is commonly observed to decrease (15, 16).Decreasing average body size in fish stocks first results from demographic truncation by direct removal of large individuals within a generation but may also result from evolutionary adaptation to a new fitness landscape (17). Positively size-selective harvesting alters the fitness landscape by favoring early reproduction at smaller sizes, in turn slowing down postmaturation growth due to altered allocation of energy from soma to gonads (2, 18). Additionally, reduced postmaturation growth may arise from evolutionary adaptations in energy acquisition–related behaviors [e.g., evolution of risk-sensitive foraging in response to the selective removal of bold, active, or aggressive behavioral phenotypes (19, 20)]. There is considerable debate whether any observed phenotypic changes, derived from monitoring data from wild fisheries, in life history traits such as maturation timing or growth rate are indeed evolutionary (i.e., genetic) or an effect of phenotypic plasticity (21), and a recent review concluded that no conclusive example for fisheries-induced evolution exists at the scale of wild fisheries (21).Most research on fisheries-induced selection and evolution has been focused on life history traits (2). However, fisheries can also induce adaptive changes in behavior through at least two mechanisms. First, by creating selection pressures that favor fast life histories, fisheries may indirectly alter correlated behavioral traits like aggressive and bold behaviors (22–24). Second, passive gear types such as gill nets, traps, or hooks heavily rely on a behavioral response by individual fish for successful capture (25). Fish that are able to forage more, at the expense of taking more risks, are able to grow faster and may produce more offspring (26–28), but they may also be more vulnerable to capture (10, 27) and mortality by predation (29). Accordingly, models comparing life history outcomes emerging from either purely behavioral to purely size-dependent vulnerability to capture demonstrate that behavioral selection can create the same pressures and ultimately evolutionary outcomes as size-selective capture and, depending on context, either favor bold or shy fish (30, 31). As personality traits are known to have a heritable component (32, 33) and vary consistently among individuals (34, 35), the selective capture of active, aggressive, and bold fish may ultimately promote the emergence of timid populations (10, 19, 27). Independent of life history adaptations, these changes may also disrupt the “pace-of-life” syndrome and the correlation of behavior and life history (24, 36, 37). A widespread increase in timidity implies that fish will become harder to catch (10). If this is the case, challenges in stock assessments will arise as they are built on assumptions of consistent fish availability to sampling gear over time to serve as indices of abundance (19, 38, 39).Our understanding of selective harvest’s impact on phenotypic change has not yet been able to fully explain empirical observations from fisheries in the wild (40, 41). Indeed, the rate and impacts of harvest-induced evolution continues to attract controversy despite more than 20 y of research (2, 21, 41). Models of harvest-induced life history evolution consistently underestimate rates of phenotypic change observed in empirical studies from the wild, while experimental studies in the laboratory tend to overestimate empirical rates of evolution (40–42). The discrepancy between models or laboratory studies and empirical data in the wild may partly result from plastic, rather than evolutionary, impacts on phenotypes collected in the wild (43), from inappropriate assumptions of fitness trade-offs in models (30, 31), from exaggerated fishing mortality induced in selection line experiments (44), or from inadvertent selection on other traits correlated with growth, such as behavioral traits, rather than direct selection on size (30, 31). To understand the potential for harvest-induced evolution, a key first step is to understand the selection pressures induced by exploitation in the wild (42, 45). This is because following the breeder’s equation from quantitative genetics, the selection response in any trait is a product of the selection differentials acting on a trait and the trait’s heritability (46). We focus here on estimating selection acting on adaptive traits in a wild fish population and compare the selection to natural selective forces on the same traits.In particular, the counteracting forces of natural selection must be considered to understand the total selective forces acting on a phenotype (47, 48). However, natural selection has rarely been empirically measured in the context of harvest selection in wild fisheries (45, 47–49). Meta-analyses on selection in the wild indicate that fishing is one of the few anthropogenic selective forces consistently stronger than natural selection (49). Yet, natural selection compared to size-selective fisheries has, so far, only been quantified by fitness proxies such as survival (45), growth rate, or female body size (47, 48), assumed to be positively correlated with lifetime reproductive success (RS) (50). As the RS of fish is challenging to measure in the wild, it is unclear how body size and fitness actually scale (50), and consequently it is largely unclear what natural selection on body size or other traits looks like in exploited stocks. Further, the fitness landscape of behavioral traits has rarely been assessed in the wild, although behavior commonly relates to growth (51), survival (52, 53), and RS (26, 27).Our aim was to quantify the strength and direction of harvest and natural selection in the wild using an experimentally exploited top predatory fish and to improve our understanding of whether a portion of harvest size selection is actually the result of undetected behavioral selection (54, 55). To that end, we investigated the strength and direction of harvest selection on body size and activity in northern pike, Esox lucius, measuring fitness in the context of natural selection as relative reproductive success (RRS) over four years and classification of movement behavior over one year using high-resolution acoustic telemetry (56) covering an entire natural ecosystem. We used hook and line fishing as an example of a widespread fishing gear used by both recreational and commercial fisheries. We predicted that harvest and natural size selection act in opposition in which larger fish would have higher RRS (50) but would also be more likely to be captured by angling (57, 58). Furthermore, we expected that fishing selection on size would be much stronger than natural selection (49). However, we also predicted additional harvest selection on behavior (55) because recreational fishing gear is known to be related to behavioral phenotypes (10, 55, 59–61). Finally, through simulations, we investigated how regulations could alter the relationship between harvest and natural selection and potentially counteract fishing selection considering minimum length limits and harvest slots based on established models (42).     

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.     

Global analysis of more than 50,000 SARS-CoV-2 genomes reveals epistasis between eight viral genes

Genome-wide epistasis analysis is a powerful tool to infer gene interactions, which can guide drug and vaccine development and lead to deeper understanding of microbial pathogenesis. We have considered all complete severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes deposited in the Global Initiative on Sharing All Influenza Data (GISAID) repository until four different cutoff dates, and used direct coupling analysis together with an assumption of quasi-linkage equilibrium to infer epistatic contributions to fitness from polymorphic loci. We find eight interactions, of which three are between pairs where one locus lies in gene ORF3a, both loci holding nonsynonymous mutations. We also find interactions between two loci in gene nsp13, both holding nonsynonymous mutations, and four interactions involving one locus holding a synonymous mutation. Altogether, we infer interactions between loci in viral genes ORF3a and nsp2, nsp12, and nsp6, between ORF8 and nsp4, and between loci in genes nsp2, nsp13, and nsp14. The paper opens the prospect to use prominent epistatically linked pairs as a starting point to search for combinatorial weaknesses of recombinant viral pathogens.

The pandemic of the disease COVID-19 has so far led to the confirmed deaths of more than 852,000 people (1) and has hurt millions. As the health crisis has been met by nonpharmacological interventions (2, 3) there has been significant economic disruption in many countries. The search for vaccine or treatment against the new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is therefore a worldwide priority. The Global Initiative on Sharing All Influenza Data (GISAID) repository (4) contains a rapidly increasing collection of SARS-CoV-2 whole-genome sequences, and has already been leveraged to identify mutational hotspots and potential drug targets (5). Coronaviruses, in general, exhibit a large amount of recombination (6–9). The distribution of genotypes in a viral population can therefore be expected to be in the state of quasi-linkage equilibrium (QLE) (10–12), and directly related to epistatic contributions to fitness (13, 14). We have determined a list of the largest such contributions from 51,676 SARS-CoV-2 genomes by a direct coupling analysis (DCA) (15, 16). This family of techniques has earlier been used to infer the fitness landscape of HIV-1 Gag (17, 18) to connect bacterial genotypes and phenotypes through coevolutionary landscapes (19) and to enhance models of amino acid sequence evolution (20). We apply a recent enhancement of this technique to eliminate predictions that can be attributed to phylogenetics (shared inheritance) (21). We find that eight predictions stand out between pairs of polymorphic sites located in genes nsp2 and ORF3a, in genes nsp4 and ORF8, and between genes nsp2, nsp6, nsp12, nsp13, nsp14 and ORF3a. Most of these sites have been documented in the literature when it comes to single-locus variations (22–27). The nsp4–ORF8 pair was additionally found to be strongly correlated, in an early study (28). It does not show prominent correlations today, but is ranked second in our global analysis. The epistasis analysis of this paper brings a different perspective than correlations, and highlights pair-wise associations that have remained stable as orders of more SARS-CoV-2 genomes have been sequenced.     

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.     

Encapsulation of ribozymes inside model protocells leads to faster evolutionary adaptation

Functional biomolecules, such as RNA, encapsulated inside a protocellular membrane are believed to have comprised a very early, critical stage in the evolution of life, since membrane vesicles allow selective permeability and create a unit of selection enabling cooperative phenotypes. The biophysical environment inside a protocell would differ fundamentally from bulk solution due to the microscopic confinement. However, the effect of the encapsulated environment on ribozyme evolution has not been previously studied experimentally. Here, we examine the effect of encapsulation inside model protocells on the self-aminoacylation activity of tens of thousands of RNA sequences using a high-throughput sequencing assay. We find that encapsulation of these ribozymes generally increases their activity, giving encapsulated sequences an advantage over nonencapsulated sequences in an amphiphile-rich environment. In addition, highly active ribozymes benefit disproportionately more from encapsulation. The asymmetry in fitness gain broadens the distribution of fitness in the system. Consistent with Fisher’s fundamental theorem of natural selection, encapsulation therefore leads to faster adaptation when the RNAs are encapsulated inside a protocell during in vitro selection. Thus, protocells would not only provide a compartmentalization function but also promote activity and evolutionary adaptation during the origin of life.

RNA is believed to have been a central constituent of early life (1–3). In the “RNA world” theory, functional RNAs (e.g., ribozymes) would both perform catalytic functions and store and transfer genetic information in a simple living system (4–6). Encapsulation of ribozymes in cell-like compartments, such as protocells, is thought to be an essential feature for the emergence of early life (7–11). In particular, compartmentalization would retain useful metabolites in the vicinity (12) and prevent a cooperative, self-replicating ribozyme system from collapsing under parasitization by selfish RNAs (13, 14). A major model of protocells is lipid vesicles, which consist of an aqueous interior surrounded by a semipermeable membrane (15, 16). However, while the ultimate advantages of compartmentalization may be clear, how encapsulation and confinement inside protocell vesicles would affect the activity and early evolution of ribozymes is not understood well.Confinement by lipid membranes presents a biophysical environment similar to macromolecular crowding (17). The effect of macromolecular crowding on the activity, function, and specificity of biomolecules (i.e., proteins and nucleic acids) has been examined extensively (18–23) using crowding agents such as dextran, polyethylene glycol, and Ficoll in vitro (24–29). In general, macromolecular crowding agents decrease the accessible volume for biomolecules, leading to the excluded-volume effect, in which the relative stability of compacted and folded structures is increased (30, 31). At the same time, chemical interactions between the crowding agents and the biomolecule can also stabilize or destabilize the folded structure, influencing catalytic activity (24, 32). While chemical interactions depend on the properties of the specific molecules under study, the excluded-volume effect resulting from spatial confinement inside vesicles is expected to be general. The effect of confinement can be studied while controlling for chemical interactions by comparing the encapsulated condition to the nonencapsulated but membrane-exposed condition. This comparison represents the prebiotic scenario in which RNAs would be present in the same milieu as lipids (33) and may become encapsulated or not. In this way, confinement inside vesicles was shown to increase the binding affinity of the malachite green RNA aptamer (34). Interestingly, spatial confinement inside a tetrahedral DNA framework has also been shown to increase thermodynamic stability and binding affinity of aptamers by facilitating folding (35).While these and other case studies (17, 25, 36–43) illustrate mechanisms by which RNA activity might be perturbed inside vesicles, understanding how encapsulation would affect evolution requires a broader scale of information. In particular, detailed knowledge of how encapsulation affects the sequence-activity relationship is required. This information is captured in the “fitness landscape,” or the function of fitness over sequence space, which embodies many important evolutionary features [e.g., fitness maxima, epistasis, and the viability of evolutionary trajectories (44–47)]. In practice, the fitness of a ribozyme can be considered to be its chemical activity for a particular function in the given environment (48–53).In the present work, we investigated how encapsulation inside model protocells would affect the catalytic activity and evolution of self-aminoacylating ribozymes. We studied tens of thousands of RNA sequences derived from five previously selected self-aminoacylating ribozyme families (53). These sequences were encapsulated in a mixed fatty acid/phospholipid vesicle system. Fatty acids mixed with phospholipids (1:1 molar ratio) have been used as model protocell membranes, as the vesicles tolerate Mg²⁺ concentrations needed for ribozyme activity and the membrane allows small, charged molecules to permeate while preserving large polynucleotides in the vesicle interior (54, 55). To study the biophysical effect of confinement rather than chemical interactions with the membrane, RNA activity inside vesicles was compared with RNA activity when exposed to the same vesicles without encapsulation. We show that ribozymes generally exhibit higher catalytic activity inside the vesicles and that more active sequences experience greater benefit. Using in vitro selection, we demonstrate that one of the evolutionary consequences of this trend is that encapsulation inside vesicles causes a greater rate of genotypic change due to natural selection.     

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.     

SLFN11 promotes CDT1 degradation by CUL4 in response to replicative DNA damage,while its absence leads to synthetic lethality with ATR/CHK1 inhibitors

Schlafen-11 (SLFN11) inactivation in ∼50% of cancer cells confers broad chemoresistance. To identify therapeutic targets and underlying molecular mechanisms for overcoming chemoresistance, we performed an unbiased genome-wide RNAi screen in SLFN11-WT and -knockout (KO) cells. We found that inactivation of Ataxia Telangiectasia- and Rad3-related (ATR), CHK1, BRCA2, and RPA1 overcome chemoresistance to camptothecin (CPT) in SLFN11-KO cells. Accordingly, we validate that clinical inhibitors of ATR (M4344 and M6620) and CHK1 (SRA737) resensitize SLFN11-KO cells to topotecan, indotecan, etoposide, cisplatin, and talazoparib. We uncover that ATR inhibition significantly increases mitotic defects along with increased CDT1 phosphorylation, which destabilizes kinetochore-microtubule attachments in SLFN11-KO cells. We also reveal a chemoresistance mechanism by which CDT1 degradation is retarded, eventually inducing replication reactivation under DNA damage in SLFN11-KO cells. In contrast, in SLFN11-expressing cells, SLFN11 promotes the degradation of CDT1 in response to CPT by binding to DDB1 of CUL4^CDT2 E3 ubiquitin ligase associated with replication forks. We show that the C terminus and ATPase domain of SLFN11 are required for DDB1 binding and CDT1 degradation. Furthermore, we identify a therapy-relevant ATPase mutant (E669K) of the SLFN11 gene in human TCGA and show that the mutant contributes to chemoresistance and retarded CDT1 degradation. Taken together, our study reveals new chemotherapeutic insights on how targeting the ATR pathway overcomes chemoresistance of SLFN11-deficient cancers. It also demonstrates that SLFN11 irreversibly arrests replication by degrading CDT1 through the DDB1–CUL4^CDT2 ubiquitin ligase.

Schlafen-11 (SLFN11) is an emergent restriction factor against genomic instability acting by eliminating cells with replicative damage (1–6) and potentially acting as a tumor suppressor (6, 7). SLFN11-expressing cancer cells are consistently hypersensitive to a broad range of chemotherapeutic drugs targeting DNA replication, including topoisomerase inhibitors, alkylating agents, DNA synthesis, and poly(ADP-ribose) polymerase (PARP) inhibitors compared to SLFN11-deficient cancer cells, which are chemoresistant (1, 2, 4, 8–17). Profiling SLFN11 expression is being explored for patients to predict survival and guide therapeutic choice (8, 13, 18–24).The Cancer Genome Atlas (TCGA) and cancer cell databases demonstrate that SLFN11 mRNA expression is suppressed in a broad fraction of common cancer tissues and in ∼50% of all established cancer cell lines across multiple histologies (1, 2, 5, 8, 13, 25, 26). Silencing of the SLFN11 gene, like known tumor suppressor genes, is under epigenetic mechanisms through hypermethylation of its promoter region and activation of histone deacetylases (HDACs) (21, 23, 25, 26). A recent study in small-cell lung cancer patient-derived xenograft models also showed that SLFN11 gene silencing is caused by local chromatin condensation related to deposition of H3K27me3 in the gene body of SLFN11 by EZH2, a histone methyltransferase (11). Targeting epigenetic regulators is therefore an attractive combination strategy to overcome chemoresistance of SLFN11-deficient cancers (10, 25, 26). An alternative approach is to attack SLFN11-negative cancer cells by targeting the essential pathways that cells use to overcome replicative damage and replication stress. Along these lines, a prior study showed that inhibition of ATR (Ataxia Telangiectasia- and Rad3-related) kinase reverses the resistance of SLFN11-deficient cancer cells to PARP inhibitors (4). However, targeting the ATR pathway in SLFN11-deficient cells has not yet been fully explored.SLFN11 consists of two functional domains: A conserved nuclease motif in its N terminus and an ATPase motif (putative helicase) in its C terminus (2, 6). The N terminus nuclease has been implicated in the selective degradation of type II tRNAs (including those coding for ATR) and its nuclease structure can be derived from crystallographic analysis of SLFN13 whose N terminus domain is conserved with SLFN11 (27, 28). The C terminus is only present in the group III Schlafen family (24, 29). Its potential ATPase activity and relationship to chemosensitivity to DNA-damaging agents (3–5) imply that the ATPase/helicase of SLFN11 is involved specifically in DNA damage response (DDR) to replication stress. Indeed, inactivation of the Walker B motif of SLFN11 by the mutation E669Q suppresses SLFN11-mediated replication block (5, 30). In addition, SLFN11 contains a binding site for the single-stranded DNA binding protein RPA1 (replication protein A1) at its C terminus (3, 31) and is recruited to replication damage sites by RPA (3, 5). The putative ATPase activity of SLFN11 is not required for this recruitment (5) but is required for blocking the replication helicase complex (CMG-CDC45) and inducing chromatin accessibility at replication origins and promoter sites (5, 30). Based on these studies, our current model is that SLFN11 is recruited to “stressed” replication forks by RPA filaments formed on single-stranded DNA (ssDNA), and that the ATPase/helicase activity of SLFN11 is required for blocking replication progression and remodeling chromatin (5, 30). However, underlying mechanisms of how SLFN11 irreversibly blocks replication in DNA damage are still unclear.Increased RPA-coated ssDNA caused by DNA damage and replication fork stalling also triggers ATR kinase activation, promoting subsequent phosphorylation of CHK1, which transiently halts cell cycle progression and enables DNA repair (32). ATR inhibitors are currently in clinical development in combination with DNA replication damaging drugs (33, 34), such as topoisomerase I (TOP1) inhibitors, which are highly synergistic with ATR inhibitors in preclinical models (35). ATR inhibitors not only inhibit DNA repair, but also lead to unscheduled replication origin firing (36), which kills cancer cells (37, 38) by inducing genomic alterations due to faulty replication and mitotic catastrophe (33).The replication licensing factor CDT1 orchestrates the initiation of replication by assembling prereplication complexes (pre-RC) in G1-phase before cells enter S-phase (39). Once replication is started by loading and activation of the MCM helicase, CDT1 is degraded by the ubiquitin proteasomal pathway to prevent additional replication initiation and ensure precise genome duplication and the firing of each origin only once per cell cycle (39, 40). At the end of G2 and during mitosis, CDT1 levels rise again to control kinetochore-microtubule attachment for accurate chromosome segregation (41). Deregulated overexpression of CDT1 results in rereplication, genome instability, and tumorigenesis (42). The cellular CDT1 levels are tightly regulated by the damage-specific DNA binding protein 1 (DDB1)–CUL4^CDT2 E3 ubiquitin ligase complex in G1-phase (43) and in response to DNA damage (44, 45). How CDT1 is recognized by CUL4^CDT2 in response to DNA damage remains incompletely known.In the present study, starting with a human genome-wide RNAi screen, bioinformatics analyses, and mechanistic validations, we explored synthetic lethal interactions that overcome the chemoresistance of SLFN11-deficient cells to the TOP1 inhibitor camptothecin (CPT). The strongest synergistic interaction was between depletion of the ATR/CHK1-mediated DNA damage response pathways and DNA-damaging agents in SLFN11-deficient cells. We validated and expanded our molecular understanding of combinatorial strategies in SLFN11-deficient cells with the ATR (M4344 and M6620) and CHK1 (SRA737) inhibitors in clinical development (33, 46, 47) and found that ATR inhibition leads to CDT1 stabilization and hyperphosphorylation with mitotic catastrophe. Our study also establishes that SLFN11 promotes the degradation of CDT1 by binding to DDB1, an adaptor molecule of the CUL4^CDT2 E3 ubiquitin ligase complex, leading to an irreversible replication block in response to replicative DNA damage.     

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.     

Adipose tissue is a critical regulator of osteoarthritis

Osteoarthritis (OA), the leading cause of pain and disability worldwide, disproportionally affects individuals with obesity. The mechanisms by which obesity leads to the onset and progression of OA are unclear due to the complex interactions among the metabolic, biomechanical, and inflammatory factors that accompany increased adiposity. We used a murine preclinical model of lipodystrophy (LD) to examine the direct contribution of adipose tissue to OA. Knee joints of LD mice were protected from spontaneous or posttraumatic OA, on either a chow or high-fat diet, despite similar body weight and the presence of systemic inflammation. These findings indicate that adipose tissue itself plays a critical role in the pathophysiology of OA. Susceptibility to posttraumatic OA was reintroduced into LD mice using implantation of a small adipose tissue depot derived from wild-type animals or mouse embryonic fibroblasts that undergo spontaneous adipogenesis, implicating paracrine signaling from fat, rather than body weight, as a mediator of joint degeneration.

Osteoarthritis (OA) is the leading cause of pain and disability worldwide and is associated with increased all-cause mortality and cardiovascular disease (1, 2). OA is strongly associated with obesity, suggesting that either increased biomechanical joint loading or systemic inflammation and metabolic dysfunction related to obesity are responsible for joint degeneration (1, 2). However, increasing evidence is mounting that changes in biomechanical loading due to increased body mass do not account for the severity of obesity-induced knee OA (1–9). These observations suggest that other factors related to the presence of adipose tissue and adipose tissue-derived cytokines—termed adipokines—play critical roles in this process and other musculoskeletal conditions (1, 2, 6, 7, 10). As there are presently no disease-modifying OA drugs available, direct evidence linking adipose tissue and cartilage health could provide important mechanistic insight into the natural history of OA and obesity and therefore guide the development and translation of novel OA therapeutic strategies designed to preserve joint health.The exact contribution of the adipokine-signaling network in OA has been difficult to determine due to the complex interactions among metabolic, biomechanical, and inflammatory factors related to obesity (11). To date, the link between increased adipose tissue mass and OA pathogenesis has largely been correlative (6, 7, 12), and, as such, the direct effect of adipose tissue and the adipokines it releases has been difficult to separate from other factors such as dietary composition or excess body mass in the context of obesity, which is most commonly caused by excessive nutrition (2, 6, 7). In particular, leptin, a proinflammatory adipokine and satiety hormone secreted proportionally to adipose tissue mass is most consistently increased in obesity-induced OA (1), and leptin knockout mice are protected from OA (6, 7). However, it remains to be determined whether leptin directly contributes to OA pathogenesis, independent of its effect on metabolism (and weight). Additional adipokines that have been implicated in the onset and progression of OA include adiponectin, resistin, visfatin, chimerin, and inflammatory cytokines such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α) (13). The infrapatellar fat pad represents a local source of adipokines within the knee joint, but several studies indicate strong correlations with visceral adipose tissue, outside of the joint organ system, with OA severity (14). Furthermore, adipokine receptors are found on almost all cells within the joint and, therefore, could directly contribute to OA pathogenesis through synovitis, cartilage damage, and bone remodeling (13). The role of other adipokines (15) in OA pathogenesis remains to be determined, as it has been difficult to separate and directly test the role of adipokines from other biomechanical, inflammatory, and metabolic factors that contribute to OA pathogenesis.To directly investigate the mechanisms by which adipose tissue affects OA, we used a transgenic mouse with lipodystrophy (LD) that completely lacks adipose tissue and, therefore, adipokine signaling. The LD model system affords the unique opportunity to directly examine the effects of adipose tissue and its secretory factors on musculoskeletal pathology without the confounding effect of diet (16, 17). While LD mice completely lack adipose tissue depots, they demonstrate similar body mass to wild-type (WT) controls on a chow diet (12, 16–19). These characteristics provide a unique model that can be used to eliminate the factor of loading due to body mass on joint damage and, thus, to directly test the effects of fat and factors secreted by fat on musculoskeletal tissues. Of particular interest, LD mice also exhibit several characteristics that have been associated with OA, including sclerotic bone (11, 20), metabolic derangement (3, 5, 7–9, 21, 22), and muscle weakness (2). Despite these OA-predisposing features, LD mice are protected from OA and implantation of adipose tissue back into LD mice restores susceptibility to OA—demonstrating a direct relationship between adipose tissue and cartilage health, independent of the effect of obesity on mechanical joint loading.     

The impact of identity by descent on fitness and disease in dogs

Domestic dogs have experienced population bottlenecks, recent inbreeding, and strong artificial selection. These processes have simplified the genetic architecture of complex traits, allowed deleterious variation to persist, and increased both identity-by-descent (IBD) segments and runs of homozygosity (ROH). As such, dogs provide an excellent model for examining how these evolutionary processes influence disease. We assembled a dataset containing 4,414 breed dogs, 327 village dogs, and 380 wolves genotyped at 117,288 markers and data for clinical and morphological phenotypes. Breed dogs have an enrichment of IBD and ROH, relative to both village dogs and wolves, and we use these patterns to show that breed dogs have experienced differing severities of bottlenecks in their recent past. We then found that ROH burden is associated with phenotypes in breed dogs, such as lymphoma. We next test the prediction that breeds with greater ROH have more disease alleles reported in the Online Mendelian Inheritance in Animals (OMIA). Surprisingly, the number of causal variants identified correlates with the popularity of that breed rather than the ROH or IBD burden, suggesting an ascertainment bias in OMIA. Lastly, we use the distribution of ROH across the genome to identify genes with depletions of ROH as potential hotspots for inbreeding depression and find multiple exons where ROH are never observed. Our results suggest that inbreeding has played a large role in shaping genetic and phenotypic variation in dogs and that future work on understudied breeds may reveal new disease-causing variation.

The unique demographic and selective history of dogs has enabled the persistence of deleterious variation, simplified genetic architecture of complex traits, and caused an increase in both runs of homozygosity (ROH) and identity-by-descent (IBD) segments within breeds (1–6). Specifically, the average F_ROH was ∼0.3 in dogs (7), compared to 0.005 in humans, computed from the 1000 Genomes populations (8). The large amount of the genome in ROH in dogs, combined with a wealth of genetic variation and phenotypic data (2, 5, 7, 9–11), allow us to test how ROH and IBD influence complex traits and fitness (Fig. 1). Furthermore, many of the deleterious alleles within dogs likely arose relatively recently within a breed, and dogs tend to share similar disease pathways and genes with humans (4, 12, 13), increasing their relevance for complex traits in humans.Open in a separate windowFig. 1.Potential mechanisms for associations between ROH and phenotypes that depend on recessive mutations. If a recessive deleterious mutation is nonlethal (blue), it may lead to ROH correlating with disease, while lethal (red) recessive mutations will cause a depletion of ROH.Despite IBD segments and ROH being ubiquitous in genomes, the extent to which they affect the architecture of complex traits as well as reproductive fitness has remained elusive. Given that ROH are formed by inheritance of the same ancestral chromosome from both parents, there is a much higher probability of the individual to become homozygous for a deleterious recessive variant (8, 14), leading to a reduction in fitness. This prediction was verified in recent work in nonhuman mammals that has shown that populations suffering from inbreeding depression tend to have an increase in ROH (15, 16). ROH in human populations are enriched for deleterious variants (8, 14, 17). However, the extent to which ROH impact phenotypes remains unclear. For example, several studies have associated an increase in ROH with complex traits in humans (18–23), though some associations remain controversial (24–28). Determining how ROH and IBD influence complex traits and fitness could provide a mechanism for differences in complex-trait architecture across populations that vary in their burden of IBD and ROH.Here, we use IBD segments and ROH from 4,741 breed dogs and village dogs, and 380 wolves to determine the recent demographic history of dogs and wolves and establish a connection between recent inbreeding and deleterious variation associated with both disease and inbreeding depression. This comprehensive dataset contains genotype data from 172 breeds of dog, village dogs from 30 countries, and gray wolves from British Colombia, North America, and Europe. We test for an association with the burden of ROH and case-control status for a variety of complex traits. Remarkably, we also find that the number of disease-associated causal variants identified in a breed is positively correlated with breed popularity rather than burden of IBD or ROH in the genome, suggesting ascertainment biases also exist in databases of dog disease mutations and that many breeds of dog are understudied. Lastly, we identify multiple loci that may be associated with inbreeding depression by examining localized depletions of ROH across dog genomes.     

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.     

Real-time measurements of aminoglycoside effects on protein synthesis in live cells

The spread of antibiotic resistance is turning many of the currently used antibiotics less effective against common infections. To address this public health challenge, it is critical to enhance our understanding of the mechanisms of action of these compounds. Aminoglycoside drugs bind the bacterial ribosome, and decades of results from in vitro biochemical and structural approaches suggest that these drugs disrupt protein synthesis by inhibiting the ribosome’s translocation on the messenger RNA, as well as by inducing miscoding errors. So far, however, we have sparse information about the dynamic effects of these compounds on protein synthesis inside the cell. In the present study, we measured the effect of the aminoglycosides apramycin, gentamicin, and paromomycin on ongoing protein synthesis directly in live Escherichia coli cells by tracking the binding of dye-labeled transfer RNAs to ribosomes. Our results suggest that the drugs slow down translation elongation two- to fourfold in general, and the number of elongation cycles per initiation event seems to decrease to the same extent. Hence, our results imply that none of the drugs used in this study cause severe inhibition of translocation.

Antibiotic resistance has become one of the biggest public health challenges of the 21st century. What used to be easily treatable diseases are becoming deadly as a consequence of commonly used antibiotics increasingly turning ineffective. To aid the development of new strategies to address this challenge, it is necessary to improve our understanding of the mechanism of action of these antibacterial compounds. Many antibiotics currently in use target the bacterial ribosome with high specificity (1). These compounds affect different stages of protein synthesis, depending on their binding sites in the bacterial ribosome or their binding to protein factors involved in protein synthesis.Aminoglycosides are a class of natural and semisynthetic chemical compounds of broad-spectrum therapeutic relevance (2, 3) categorized as critically important by the World Health Organization (4). Aminoglycosides are presently used against multidrug-resistant bacterial infections (5, 6) and, more recently, considered as potential treatments for genetic diseases such as cystic fibrosis and Duchenne muscular dystrophy (3, 7, 8). The clinical relevance of aminoglycosides is only shadowed by side effects such as nephrotoxicity and irreversible ototoxicity (5, 6). A subclass of these molecules has a conserved aminocyclitol, a 2-deoxystreptamine, with linked amino sugar groups at different positions. Structural studies showed that these molecules bind at the major groove of the 16S ribosomal RNA (rRNA) in the A-site in close contact with the decoding center of the bacterial 30S ribosomal subunit (9–12). At the decoding center, the adenines A1492 and A1493 take part in monitoring the correct codon–anticodon interaction (13). Aminoglycoside molecules bound to this site have been suggested to interact with A1492/1493 and restrict their mobility (12, 14), which in turn interferes with the selection of cognate transfer RNA (tRNA) (9, 11, 15–18) as well as with the translocation step (11, 16, 19–22).A secondary binding site for 4,5- and 4,6-substituted aminoglycosides has been identified at H69 in the 50S ribosomal subunit, in close contact with A- and P-site tRNAs (23). Based on crystal structures (23) and in vitro kinetics assays (24), it has been suggested that drugs bound to this secondary binding site affect ribosome recycling and also intersubunit rotation—potentially also affecting translocation.The synergistic effect of aminoglycosides binding to multiple sites in the bacterial ribosome contributes to the misreading of codons and defective translocation, which eventually leads to cell death. The mechanism of action of various aminoglycosides on the ribosome has been characterized using diverse structure biology methods (as reviewed in ref. 25), classical in vitro functional biochemical assays (15, 20, 26), and, more recently, in vitro single-molecule approaches (11, 21, 27). Even though the mechanistic steps are described in detail by these complementary in vitro techniques, the reported effects of these drugs on the kinetics of protein synthesis are significantly different. For example, whereas single-molecule Förster resonance energy transfer (FRET) studies report a four- to sixfold inhibition of messenger RNA (mRNA) movement during translocation (21), stopped-flow experiments report a 160-fold inhibition (20). Recent advances in live-cell single-molecule tracking methods have now opened up the possibility to measure the drug’s effects on protein synthesis kinetics directly in live cells (28, 29).In the present study, we measured the effect of three structurally different aminoglycosides, apramycin, gentamicin, and paromomycin, on the kinetics of translation elongation at a single-ribosome level in live Escherichia coli cells. By tracking single dye-labeled tRNAs and analyzing the diffusion trajectories using a Hidden Markov Model-based (HMM) approach, we measured dwell-times of elongator [Cy5]tRNA^Phe and initiator [Cy5]tRNA^fMet on the ribosome, which suggest an overall slower, but ongoing, protein synthesis in intact cells exposed to the aminoglycosides.     

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).     

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.     

Chemokine CCL5 promotes robust optic nerve regeneration and mediates many of the effects of CNTF gene therapy

Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for several ocular diseases and induces optic nerve regeneration in animal models. Paradoxically, however, although CNTF gene therapy promotes extensive regeneration, recombinant CNTF (rCNTF) has little effect. Because intraocular viral vectors induce inflammation, and because CNTF is an immune modulator, we investigated whether CNTF gene therapy acts indirectly through other immune mediators. The beneficial effects of CNTF gene therapy remained unchanged after deleting CNTF receptor alpha (CNTFRα) in retinal ganglion cells (RGCs), the projection neurons of the retina, but were diminished by depleting neutrophils or by genetically suppressing monocyte infiltration. CNTF gene therapy increased expression of C-C motif chemokine ligand 5 (CCL5) in immune cells and retinal glia, and recombinant CCL5 induced extensive axon regeneration. Conversely, CRISPR-mediated knockdown of the cognate receptor (CCR5) in RGCs or treating wild-type mice with a CCR5 antagonist repressed the effects of CNTF gene therapy. Thus, CCL5 is a previously unrecognized, potent activator of optic nerve regeneration and mediates many of the effects of CNTF gene therapy.

Like most pathways in the mature central nervous system (CNS), the optic nerve cannot regenerate once damaged due in part to cell-extrinsic suppressors of axon growth (1, 2) and the low intrinsic growth capacity of adult retinal ganglion cells (RGCs), the projection neurons of the eye (3–5). Consequently, traumatic or ischemic optic nerve injury or degenerative diseases such as glaucoma lead to irreversible visual losses. Experimentally, some degree of regeneration can be induced by intraocular inflammation or growth factors expressed by inflammatory cells (6–10), altering the cell-intrinsic growth potential of RGCs (3–5), enhancing physiological activity (11, 12), chelating free zinc (13, 14), and other manipulations (15–19). However, the extent of regeneration achieved to date remains modest, underlining the need for more effective therapies.Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for glaucoma and other ocular diseases (20–23). Activation of the downstream signal transduction cascade requires CNTF to bind to CNTF receptor-α (CNTFRα) (24), which leads to recruitment of glycoprotein 130 (gp130) and leukemia inhibitory factor receptor-β (LIFRβ) to form a tripartite receptor complex (25). CNTFRα anchors to the plasma membrane through a glycosylphosphatidylinositol linkage (26) and can be released and become soluble through phospholipase C-mediated cleavage (27). CNTF has been reported to activate STAT3 phosphorylation in retinal neurons, including RGCs, and to promote survival, but it is unknown whether these effects are mediated by direct action of CNTF on RGCs via CNTFRα (28). Our previous studies showed that CNTF promotes axon outgrowth from neonate RGCs in culture (29) but fails to do so in cultured mature RGCs (8) or in vivo (6). Although some studies report that recombinant CNTF (rCNTF) can promote optic nerve regeneration (20, 30, 31), others find little or no effect unless SOCS3 (suppressor of cytokine signaling-3), an inhibitor of the Jak-STAT pathway, is deleted in RGCs (5, 6, 32). In contrast, multiple studies show that adeno-associated virus (AAV)-mediated expression of CNTF in RGCs induces strong regeneration (33–40). The basis for the discrepant effects of rCNTF and CNTF gene therapy is unknown but is of considerable interest in view of the many promising clinical and preclinical outcomes obtained with CNTF to date.Because intravitreal virus injections induce inflammation (41), we investigated the possibility that CNTF, a known immune modulator (42–44), might act by elevating expression of other immune-derived factors. We report here that the beneficial effects of CNTF gene therapy in fact require immune system activation and elevation of C-C motif chemokine ligand 5 (CCL5). Depletion of neutrophils, global knockout (KO) or RGC-selective deletion of the CCL5 receptor CCR5, or a CCR5 antagonist all suppress the effects of CNTF gene therapy, whereas recombinant CCL5 (rCCL5) promotes axon regeneration and increases RGC survival. These studies point to CCL5 as a potent monotherapy for optic nerve regeneration and to the possibility that other applications of CNTF and other forms of gene therapy might similarly act indirectly through other factors.     

From the Cover: Evidence from South Africa for a protracted end-Permian extinction on land

Earth’s largest biotic crisis occurred during the Permo–Triassic Transition (PTT). On land, this event witnessed a turnover from synapsid- to archosauromorph-dominated assemblages and a restructuring of terrestrial ecosystems. However, understanding extinction patterns has been limited by a lack of high-precision fossil occurrence data to resolve events on submillion-year timescales. We analyzed a unique database of 588 fossil tetrapod specimens from South Africa’s Karoo Basin, spanning ∼4 My, and 13 stratigraphic bin intervals averaging 300,000 y each. Using sample-standardized methods, we characterized faunal assemblage dynamics during the PTT. High regional extinction rates occurred through a protracted interval of ∼1 Ma, initially co-occurring with low origination rates. This resulted in declining diversity up to the acme of extinction near the Daptocephalus–Lystrosaurus declivis Assemblage Zone boundary. Regional origination rates increased abruptly above this boundary, co-occurring with high extinction rates to drive rapid turnover and an assemblage of short-lived species symptomatic of ecosystem instability. The “disaster taxon” Lystrosaurus shows a long-term trend of increasing abundance initiated in the latest Permian. Lystrosaurus comprised 54% of all specimens by the onset of mass extinction and 70% in the extinction aftermath. This early Lystrosaurus abundance suggests its expansion was facilitated by environmental changes rather than by ecological opportunity following the extinctions of other species as commonly assumed for disaster taxa. Our findings conservatively place the Karoo extinction interval closer in time, but not coeval with, the more rapid marine event and reveal key differences between the PTT extinctions on land and in the oceans.

Mass extinctions are major perturbations of the biosphere resulting from a wide range of different causes including glaciations and sea level fall (1), large igneous provinces (2), and bolide impacts (3, 4). These events caused permanent changes to Earth’s ecosystems, altering the evolutionary trajectory of life (5). However, links between the broad causal factors of mass extinctions and the biological and ecological disturbances that lead to species extinctions have been difficult to characterize. This is because ecological disturbances unfold on timescales much shorter than the typical resolution of paleontological studies (6), particularly in the terrestrial record (6–8). Coarse-resolution studies have demonstrated key mass extinction phenomena including high extinction rates and lineage turnover (7, 9), changes in species richness (10), ecosystem instability (11), and the occurrence of disaster taxa (12). However, finer time resolutions are central to determining the association and relative timings of these effects, their potential causal factors, and their interrelationships. Achieving these goals represents a key advance in understanding the ecological mechanisms of mass extinctions.The end-Permian mass extinction (ca. 251.9 Ma) was Earth’s largest biotic crisis as measured by taxon last occurrences (13–15). Large outpourings from Siberian Trap volcanism (2) are the likely trigger of calamitous climatic changes, including a runaway greenhouse effect and ocean acidification, which had profound consequences for life on land and in the oceans (16–18). An estimated 81% of marine species (19) and 89% of tetrapod genera became extinct as established Permian ecosystems gave way to those of the Triassic. In the ocean, this included the complete extinction of reef-forming tabulate and rugose corals (20, 21) and significant losses in previously diverse ammonoid, brachiopod, and crinoid families (22). On land, many nonmammalian synapsids became extinct (16), and the glossopterid-dominated floras of Gondwana also disappeared (23). Stratigraphic sequences document a global “coral gap” and “coal gap” (24, 25), suggesting reef and forest ecosystems were rare or absent for up to 5 My after the event (26). Continuous fossil-bearing deposits documenting patterns of turnover across the Permian–Triassic transition (PTT) on land (27) and in the oceans (28) are geographically widespread (29, 30), including marine and continental successions that are known from China (31, 32) and India (33). Continental successions are known from Russia (34), Australia (35), Antarctica (36), and South Africa’s Karoo Basin (Fig. 1 and 37–40), the latter providing arguably the most densely sampled and taxonomically scrutinized (41–43) continental record of the PTT. The main extinction has been proposed to occur at the boundary between two biostratigraphic zones with distinctive faunal assemblages, the Daptocephalus and Lystrosaurus declivis assemblage zones (Fig. 1), which marks the traditional placement of the Permian–Triassic geologic boundary [(37) but see ref. 44]. Considerable research has attempted to understand the anatomy of the PTT in South Africa (38, 39, 45–52) and to place it in the context of biodiversity changes across southern Gondwana (53, 54) and globally (29, 31, 32, 44, 47, 55).Open in a separate windowFig. 1.Map of South Africa depicting the distribution of the four tetrapod fossil assemblage zones (Cistecephalus, Daptocephalus, Lystrosaurus declivis, Cynognathus) and our two study sites where fossils were collected in this study (sites A and B). Regional lithostratigraphy and biostratigraphy within the study interval are shown alongside isotope dilution–thermal ionization mass spectrometry dates retrieved by Rubidge et al., Botha et al., and Gastaldo et al. (37, 44, 80). The traditional (dashed red line) and associated PTB hypotheses for the Karoo Basin (37, 44) are also shown. Although traditionally associated with the PTB, the Daptocephalus–Lystrosaurus declivis Assemblage Zone boundary is defined by first appearances of co-occurring tetrapod assemblages, so its position relative to the three PTB hypotheses is unchanged. The Ripplemead member (*) has yet to be formalized by the South African Committee for Stratigraphy.Decades of research have demonstrated the richness of South Africa’s Karoo Basin fossil record, resulting in hundreds of stratigraphically well-documented tetrapod fossils across the PTT (37, 39, 56). This wealth of data has been used qualitatively to identify three extinction phases and an apparent early postextinction recovery phase (39, 45, 51). Furthermore, studies of Karoo community structure and function have elucidated the potential role of the extinction and subsequent recovery in breaking the incumbency of previously dominant clades, including synapsids (11, 57). Nevertheless, understanding patterns of faunal turnover and recovery during the PTT has been limited by the scarcity of quantitative investigations. Previous quantitative studies used coarsely sampled data (i.e., assemblage zone scale, 2 to 3 Ma time intervals) to identify low species richness immediately after the main extinction, potentially associated with multiple “boom and bust” cycles of primary productivity based on δ¹³C variation during the first 5 My of the Triassic (41, 58). However, many details of faunal dynamics in this interval remain unknown. Here, we investigate the dynamics of this major tetrapod extinction at an unprecedented time resolution (on the order of hundreds of thousands of years), using sample-standardized methods to quantify multiple aspects of regional change across the Cistecephalus, Daptocephalus, and Lystrosaurus declivis assemblage zones.