From the Cover: PNAS Plus: Agonist antibody that induces human malignant cells to kill one another

An attractive, but as yet generally unrealized, approach to cancer therapy concerns discovering agents that change the state of differentiation of the cancer cells. Recently, we discovered a phenomenon that we call “receptor pleiotropism” in which agonist antibodies against known receptors induce cell fates that are very different from those induced by the natural agonist to the same receptor. Here, we show that one can take advantage of this phenomenon to convert acute myeloblastic leukemic cells into natural killer cells. Upon induction with the antibody, these leukemic cells enter into a differentiation cascade in which as many as 80% of the starting leukemic cells can be differentiated. The antibody-induced killer cells make large amounts of perforin, IFN-γ, and granzyme B and attack and kill other members of the leukemic cell population. Importantly, induction of killer cells is confined to transformed cells, in that normal bone marrow cells are not induced to form killer cells. Thus, it seems possible to use agonist antibodies to change the differentiation state of cancer cells into those that attack and kill other members of the malignant clone from which they originate.Although adoptive cell transfer has long been an important method in experimental immunology, it only recently has entered clinical practice for the purpose of killing cancer cells (1, 2). In the most popular iteration, T cells harvested from patients are engineered to express single-chain antibodies to tumor antigens on their surface, in a format in which the antibodies are also linked to T-cell receptor (TCR) signal transduction domains. This cellular engineering endows the cells with the ability to bind specifically to tumors and to be activated upon binding. These cells, that now bear chimeric tumor antigen receptors, therefor are referred to as “chimeric antigen receptor T” (CAR-T) cells. At the site of the tumor, the CAR-T cells initiate a cytotoxic cascade that leads to the killing of the malignant cells (1, 2).As an alternative process, one might consider taking advantage of the fact that some cells of the immune system, such as natural killer (NK) cells, already have an innate specificity for cells whose surface is altered because they are malignant or infected (3–5). Indeed, there is a growing consensus that the use of NK cells in immunotherapy is, at present, underappreciated (5). To make the possibility of using NK cells in immunotherapy a reality, it would be helpful if agonists could be discovered that both induce and activate them. In terms of potency, specificity, and half-life, such agonists should go beyond the less specific cytokines such as IL-2 and IL-15 that already are known to activate NK cells but can have profound systemic side effects (5, 6). At a minimum, the induction in vivo of already innately targeted NK cells would reduce the complicated therapeutic work flow inherent in the adoptive transfer process and potentially could give the clinician more control over the therapy.The recent discovery of many agonist antibodies that govern cell fates has opened the way to induce selectively a large variety of specific cells of the immune system from normal or malignant bone marrow (BM) or blood (7–13). Sometimes these agonist antibodies induce cell differentiation along lineages expected from the known function of the receptor to which they bind. In other cases, however, they activate differentiation or transdifferentiation pathways that are different from those expected from the nature of the receptor with which they interact (7). For example, the known function of the granulocyte-colony stimulating factor receptor is to activate a pathway leading to granulocyte formation after binding to its natural agonist, granulocyte-colony stimulating factor. However, when some rare antibodies, which were found by autocrine selection from antibody libraries, bind to this same receptor, neural cells, instead of granulocytes, are formed efficiently (7). We call this phenomenon “receptor pleiotropism,” and we have identified several examples (13). Receptor pleiotropism may relate to the plasticity of fate conversion of hematopoietic cells (14, 15). Here, we report on a rare antibody against the thrombopoietin receptor (TPOR), again obtained by autocrine-based selection, that efficiently transforms malignant acute myeloblastic leukemia (AML) cells into highly activated NK cells. This highly specific antibody has an EC₅₀ of 5 pM (that of TPO is 70 pM) and shows no activity in cells in which the TPOR is knocked out (12). The induced AML cells have extensive filopodia at their surface and express the CD11c dendritic cell marker. These induced cells synthesize large amounts of perforin, granzyme B, and IFN-γ that, as molecules involved in the mechanism of killing, are markers of NK cells. From a therapeutic standpoint, the ability to induce activated NK cells at will is arguably the most important example of receptor pleiotropism observed to date, in that the ability to induce activated NK cells from the readily accessible peripheral blood (PB) and BM cellular compartments may open new routes to cancer therapy that are much simpler than those in use today. We refer to this agonist antibody as “Fratricidin” in keeping with its ability to initiate the process of fratricide in a clone of tumor cells. If other small molecules or proteins with similar effects could be found, they could be referred to as “Fratricidins.”     

Wnt signaling and a Smad pathway blockade direct the differentiation of human pluripotent stem cells to multipotent neural crest cells

Neural crest stem cells can be isolated from differentiated cultures of human pluripotent stem cells, but the process is inefficient and requires cell sorting to obtain a highly enriched population. No specific method for directed differentiation of human pluripotent cells toward neural crest stem cells has yet been reported. This severely restricts the utility of these cells as a model for disease and development and for more applied purposes such as cell therapy and tissue engineering. In this report, we use small-molecule compounds in a single-step method for the efficient generation of self-renewing neural crest-like stem cells in chemically defined media. This approach is accomplished directly from human pluripotent cells without the need for coculture on feeder layers or cell sorting to obtain a highly enriched population. Critical to this approach is the activation of canonical Wnt signaling and concurrent suppression of the Activin A/Nodal pathway. Over 12-14 d, pluripotent cells are efficiently specified along the neuroectoderm lineage toward p75(+) Hnk1(+) Ap2(+) neural crest-like cells with little or no contamination by Pax6(+) neural progenitors. This cell population can be clonally amplified and maintained for >25 passages (>100 d) while retaining the capacity to differentiate into peripheral neurons, smooth muscle cells, and mesenchymal precursor cells. Neural crest-like stem cell-derived mesenchymal precursors have the capacity for differentiation into osteocytes, chondrocytes, and adipocytes. In sum, we have developed methods for the efficient generation of self-renewing neural crest stem cells that greatly enhance their potential utility in disease modeling and regenerative medicine.     

From the Cover: PNAS Plus: Distance from sub-Saharan Africa predicts mutational load in diverse human genomes

The Out-of-Africa (OOA) dispersal ∼50,000 y ago is characterized by a series of founder events as modern humans expanded into multiple continents. Population genetics theory predicts an increase of mutational load in populations undergoing serial founder effects during range expansions. To test this hypothesis, we have sequenced full genomes and high-coverage exomes from seven geographically divergent human populations from Namibia, Congo, Algeria, Pakistan, Cambodia, Siberia, and Mexico. We find that individual genomes vary modestly in the overall number of predicted deleterious alleles. We show via spatially explicit simulations that the observed distribution of deleterious allele frequencies is consistent with the OOA dispersal, particularly under a model where deleterious mutations are recessive. We conclude that there is a strong signal of purifying selection at conserved genomic positions within Africa, but that many predicted deleterious mutations have evolved as if they were neutral during the expansion out of Africa. Under a model where selection is inversely related to dominance, we show that OOA populations are likely to have a higher mutation load due to increased allele frequencies of nearly neutral variants that are recessive or partially recessive.It has long been recognized that a human genome may carry many strongly deleterious mutations; Morton et al. (1) estimated that each human carries on average four or five mutations that would have a “conspicuous effect on fitness” if expressed in a homozygous state. Empirically estimating the deleterious mutation burden is now feasible through next-generation sequencing (NGS) technology, which can assay the complete breadth of variants in a human genome. For example, recent sequencing of over 6,000 exomes revealed that nearly half of all surveyed individuals carried a likely pathogenic allele in a known Mendelian disease gene (i.e., from a disease panel used for newborn screening) (2). Although there is some variation across individuals in the number of deleterious alleles per genome, we still do not know whether there are significant differences in deleterious variation among populations. Human populations vary dramatically in their levels of neutral genetic diversity, which suggests variation in the effective population size, N_e. Theory suggests that the efficacy of natural selection is reduced in populations with lower N_e because they experience greater genetic drift (3, 4). In an idealized population of constant size, the efficacy of purifying selection depends on the relationship between N_e and the selection coefficient s against deleterious mutations. If 4N_es << 1, deleterious alleles evolve as if they were neutral and can, thus, reach appreciable frequencies. This theory raises the question of whether human populations carry differential burdens of deleterious alleles due to differences in demographic history.Several recent papers have tested for differences in the burden of deleterious alleles among populations; these papers have focused on primarily comparing populations of western European and western African ancestry. Despite similar genomic datasets, these papers have reached a variety of contradictory conclusions (4–9). Initially, Lohmueller et al. (10) found that a panel of European Americans carried proportionally more derived, deleterious alleles than a panel of African Americans, potentially as the result of the Out-of-Africa (OOA) bottleneck. More recently, analyses using NGS exome datasets from samples of analogous continental ancestry found small or no differences in the average number of deleterious alleles per genome between African Americans and European Americans—depending on which prediction algorithm was used (11–13). Simulations by Fu et al. (11) found strong bottlenecks with recovery could recapitulate patterns of differences in the number of deleterious alleles between African and non-African populations, supporting Lohmueller et al. (10), but in contrast to work by Simons et al. (12).It is important to note two facts about these contradictory observations. First, these papers tend to use different statistics, which differ in power to detect changes across populations, as well as the impact of recent demographic history (6, 11). Lohmueller et al. (10) compared the relative number of nonsynonymous to synonymous (or “probably damaging” to “benign”) SNPs per population in a sample of n chromosomes, whereas Simons et al. (12) examined the special case of n = 2 chromosomes, namely, the average number of predicted deleterious alleles per genome (i.e., heterozygous + 2 * homozygous derived variants per genome). One way to think about these statistics is that the total number of variants, S, gives equal weight, w = 1, to an SNP regardless of its frequency, p. The average number of deleterious variants statistic gives weights proportional to the expected heterozygous and homozygous frequencies or w = 2p(1 − p) + p² = 2p − p². The average number of deleterious alleles per genome is fairly insensitive to differences in demographic history because heterozygosity is biased toward common variants. In contrast, the proportion of deleterious alleles has greater power to detect the impact of recent demographic history for large n across the populations because it is sensitive to rare variants that tend to be more numerous, younger, and enriched for functionally important mutations (14–16). Second, empirical comparisons between two populations have focused primarily on an additive model for deleterious mutations, even though there is evidence for pathogenic mutations exhibiting a recessive or dominant effect (17, 18), and possibly an inverse relationship between the strength of selection s and the dominance parameter h (19).There remains substantial conceptual and empirical uncertainty surrounding the processes that shape the distribution of deleterious variation across human populations. We aim here to clarify three aspects underlying this controversy: (i) Are there empirical differences in the total number of deleterious alleles among multiple human populations? (ii) Which model of dominance is appropriate for deleterious alleles (i.e., should zygosity be considered in load calculations)? (iii) Are the observed patterns consistent with predictions from models of range expansions accompanied by founder effects? We address these questions with a new genomic dataset of seven globally distributed human populations.     

干细胞治疗基础与临床——国内外最新动态

近年来,干细胞治疗作为一项治疗人类多种疾病的崭新技术,相关的基础研究与临床试验呈现出快速增长的趋势,该文对胚胎干细胞,成体干细胞及诱导性多能干细胞当前的进展情况进行了综述,干细胞临床治疗拥有广阔的发展前景。     

From the Cover: PNAS Plus: Quantitative optical coherence tomography angiography of vascular abnormalities in the living human eye

Retinal vascular diseases are important causes of vision loss. A detailed evaluation of the vascular abnormalities facilitates diagnosis and treatment in these diseases. Optical coherence tomography (OCT) angiography using the highly efficient split-spectrum amplitude decorrelation angiography algorithm offers an alternative to conventional dye-based retinal angiography. OCT angiography has several advantages, including 3D visualization of retinal and choroidal circulations (including the choriocapillaris) and avoidance of dye injection-related complications. Results from six illustrative cases are reported. In diabetic retinopathy, OCT angiography can detect neovascularization and quantify ischemia. In age-related macular degeneration, choroidal neovascularization can be observed without the obscuration of details caused by dye leakage in conventional angiography. Choriocapillaris dysfunction can be detected in the nonneovascular form of the disease, furthering our understanding of pathogenesis. In choroideremia, OCT''s ability to show choroidal and retinal vascular dysfunction separately may be valuable in predicting progression and assessing treatment response. OCT angiography shows promise as a noninvasive alternative to dye-based angiography for highly detailed, in vivo, 3D, quantitative evaluation of retinal vascular abnormalities.Optical coherence tomography (OCT) has become the most commonly used imaging modality in ophthalmology. It provides cross-sectional and 3D imaging of the retina and optic nerve head with micrometer-scale depth resolution. Structural OCT enhances the clinician’s ability to detect and monitor fluid exudation associated with retinal vascular diseases. Whereas anatomical alterations that impact vision are readily visible, structural OCT has a limited ability to image the retinal or choroidal vasculatures. Furthermore, it is unable to directly detect capillary dropout or pathologic new vessel growth (neovascularization) that are the major vascular changes associated with two of the leading causes of blindness, age-related macular degeneration (AMD) and diabetic retinopathy (1). To visualize these changes, traditional i.v. contrast dye-based angiography techniques are currently used.Fluorescein dye is primarily used to visualize the retinal vasculature. A separate dye, indocyanine green (ICG), is necessary to evaluate the choroidal vasculature. Both fluorescein angiography (FA) and ICG angiography require i.v. injection, which is time consuming, and which can cause nausea, vomiting, and, rarely, anaphylaxis (2). Dye leakage or staining provides information regarding vascular incompetence (e.g., from abnormal capillary growth), but it also obscures the image and blurs the boundaries of neovascularization. Additionally, conventional angiography is 2D, which makes it difficult to distinguish vascular abnormalities within different layers. Therefore, it is desirable to develop a no-injection, dye-free method for 3D visualization of ocular circulation.In recent years, several OCT angiography methods have been developed to detect changes in the OCT signal caused by flowing red blood cells in blood vessels. Initially, Doppler OCT angiography methods were investigated for the visualization and measurement of blood flow (3–8). Because Doppler OCT is only sensitive to motion parallel to the OCT probe beam, it is limited in its ability to image retinal and choroidal circulations, which are predominantly perpendicular to the OCT beam. More recent approaches, based on detecting variation in the speckle pattern over time, are sensitive to both transverse and axial flow. Several types of speckle-based techniques have been described, including amplitude-based (9–11), phase-based (12), or a combination of both amplitude and phase (13) variance methods.We developed an amplitude-based method called split-spectrum amplitude-decorrelation angiography (SSADA). The SSADA algorithm detects motion in the blood vessel lumen by measuring the variation in reflected OCT signal amplitude between consecutive cross-sectional scans. The novelty of SSADA lies in how the OCT signal is processed to enhance flow detection and reject axial bulk motion noise. Compared with the full-spectrum amplitude method, SSADA using fourfold spectral splits improved the signal-to-noise ratio (SNR) by a factor of two, which is equivalent to reducing the scan time by a factor of four (14). More recent SSADA implementations use even more than a fourfold split to further enhance the SNR of flow detection. This highly efficient algorithm generates high-quality angiograms of both the retina and choroid. The angiograms have capillary-level detail and can be obtained with currently available commercial OCT systems.This article uses six illustrative cases and highlights the various types of vascular pathologies that can be detected and measured using SSADA and an OCT angiography system of visualization. Additionally, we describe techniques designed to help clinicians rapidly interpret OCT angiograms and to easily identify pathological vascular features. These techniques include (i) separation of the 3D angiogram into individual vascular beds via segmentation algorithms, (ii) presentation of en face OCT angiograms, analogous to traditional angiography, (iii) creation of cross-sectional structural OCT images with superimposed OCT angiograms to help correlate anatomical alterations with vascular abnormalities, and (iv) quantification of neovascularization and capillary dropout in both the retinal and choroidal circulation.     

From the Cover: PNAS Plus: Thermodynamic origin of surface melting on ice crystals

Since the pioneering prediction of surface melting by Michael Faraday, it has been widely accepted that thin water layers, called quasi-liquid layers (QLLs), homogeneously and completely wet ice surfaces. Contrary to this conventional wisdom, here we both theoretically and experimentally demonstrate that QLLs have more than two wetting states and that there is a first-order wetting transition between them. Furthermore, we find that QLLs are born not only under supersaturated conditions, as recently reported, but also at undersaturation, but QLLs are absent at equilibrium. This means that QLLs are a metastable transient state formed through vapor growth and sublimation of ice, casting a serious doubt on the conventional understanding presupposing the spontaneous formation of QLLs in ice–vapor equilibrium. We propose a simple but general physical model that consistently explains these aspects of surface melting and QLLs. Our model shows that a unique interfacial potential solely controls both the wetting and thermodynamic behavior of QLLs.In general, surfaces and interfaces yield unique phase transitions absent in the bulk (1–5). Surface melting (or premelting) of ice (3, 4) is one typical and classical example that has been known since the first prediction by Michael Faraday in 1842 (6). He hypothesized that thin water layers, now called quasi-liquid layers (QLLs), wet ice crystal surfaces even at a temperature below the melting point. Since then, this phenomenon has attracted considerable attention not only because of its importance in the fundamental understanding of melting (a solid-to-liquid transition) itself but also as a link to a diverse set of natural phenomena in subzero environments: making snowballs, slippage on ice surfaces, frost heave, recrystallization and coarsening of ice grains, morphological change of snow crystals, electrification of thunderclouds, and ozone-depleting reactions (3, 4, 7). Furthermore, it is now recognized that surface melting is not specific to ice but rather is universally seen in a wide range of crystalline surfaces such as metals, semiconductors, ceramics, rare gases, and organic and colloidal systems (8–12). Its underlying physics is therefore also inseparable from material science and technology.Although the origin of surface melting, including the nature of QLLs themselves, is still far from completely understood and a matter of active debate (13–18), it is at least phenomenologically believed that surface melting is driven by the reduction of the surface free energy by the presence of intervening liquid between the solid and gas phases (3, 4, 13, 19). More sophisticated approaches have also been proposed in terms of surface phase transitions (1, 3, 4, 20). In contrast to such theoretical speculations, however, the direct observation and the accurate characterization of QLLs by experiments are still highly challenging because of their thinness, assumed to be less than tens of nanometers (21). Experimental efforts in the past have often been bedeviled by large uncertainties depending on the experimental methods and researchers (see table S1 in ref. 22 for details). Even the first convincing evidence for the existence of surface melting of ice was not provided until 1987 (13, 14), more than one century after Faraday’s suggestion. Thus, the conventional theories, although rigorous themselves, have suffered from the lack of reliably experimental support.Recently, we succeeded in making in situ observations of QLLs on ice surfaces using an advanced optical microscope (laser confocal microscopy combined with differential interference contrast microscopy: LCM-DIM), whose resolution in the height direction reaches the order of an angstrom (22, 23). Surprisingly, this work revealed that, contrary to the common belief that QLLs completely and homogeneously wet ice surfaces, they are spatiotemporally heterogeneous and are absent in the equilibrium conditions (22, 24–26). Furthermore, we have observed that QLLs exhibit more than one wetting morphology: droplet type, thin-layer type, and their coexistence (sunny-side-up type) at supersaturation (22, 24–26). This finding fundamentally requires us to recast the conventional understanding based on spatiotemporally averaged equilibrium theories and experiments (e.g., scattering, spectroscopy, and ellipsometry), because of ignorance of the counterintuitive nature of QLLs.In this paper, we present a simple physical model bridging the gap between the conventional interpretation and the above aspects of surface melting based on in situ observations with our advanced optical microscopy combined with a two-beam interferometer. Here we revisit the thermodynamics of wetting (27). The general nature of surface melting suggests the relevance of the phenomenological approach. Starting from the phenomenological interfacial free energy, we robustly determine a full interfacial potential between ice and vapor in the medium of a QLL, governing both the selection and stability of the wetting states, and the thermodynamic condition for the existence of QLLs. As a theoretical consequence, we extend the concept of surface melting into nonequilibrium regimes, more specifically, supersaturation and undersaturation, which has a significant implication for exploring the possible existence of this phenomenon in a wider range of crystalline surfaces. Our model provides not only a clear-cut answer to the long-standing question of the origin of surface melting of ice but also offers a general insight into the origin of surface melting of other solid–gas interfaces.     

From the Cover: PNAS Plus: Long range order and two-fluid behavior in heavy electron materials

The heavy electron Kondo liquid is an emergent state of condensed matter that displays universal behavior independent of material details. Properties of the heavy electron liquid are best probed by NMR Knight shift measurements, which provide a direct measure of the behavior of the heavy electron liquid that emerges below the Kondo lattice coherence temperature as the lattice of local moments hybridizes with the background conduction electrons. Because the transfer of spectral weight between the localized and itinerant electronic degrees of freedom is gradual, the Kondo liquid typically coexists with the local moment component until the material orders at low temperatures. The two-fluid formula captures this behavior in a broad range of materials in the paramagnetic state. In order to investigate two-fluid behavior and the onset and physical origin of different long range ordered ground states in heavy electron materials, we have extended Knight shift measurements to URu₂Si₂, CeIrIn₅, and CeRhIn₅. In CeRhIn₅ we find that the antiferromagnetic order is preceded by a relocalization of the Kondo liquid, providing independent evidence for a local moment origin of antiferromagnetism. In URu₂Si₂ the hidden order is shown to emerge directly from the Kondo liquid and so is not associated with local moment physics. Our results imply that the nature of the ground state is strongly coupled with the hybridization in the Kondo lattice in agreement with phase diagram proposed by Yang and Pines.     

From the Cover: PNAS Plus: Prehistoric genomes reveal the genetic foundation and cost of horse domestication

The domestication of the horse ∼5.5 kya and the emergence of mounted riding, chariotry, and cavalry dramatically transformed human civilization. However, the genetics underlying horse domestication are difficult to reconstruct, given the near extinction of wild horses. We therefore sequenced two ancient horse genomes from Taymyr, Russia (at 7.4- and 24.3-fold coverage), both predating the earliest archeological evidence of domestication. We compared these genomes with genomes of domesticated horses and the wild Przewalski’s horse and found genetic structure within Eurasia in the Late Pleistocene, with the ancient population contributing significantly to the genetic variation of domesticated breeds. We furthermore identified a conservative set of 125 potential domestication targets using four complementary scans for genes that have undergone positive selection. One group of genes is involved in muscular and limb development, articular junctions, and the cardiac system, and may represent physiological adaptations to human utilization. A second group consists of genes with cognitive functions, including social behavior, learning capabilities, fear response, and agreeableness, which may have been key for taming horses. We also found that domestication is associated with inbreeding and an excess of deleterious mutations. This genetic load is in line with the “cost of domestication” hypothesis also reported for rice, tomatoes, and dogs, and it is generally attributed to the relaxation of purifying selection resulting from the strong demographic bottlenecks accompanying domestication. Our work demonstrates the power of ancient genomes to reconstruct the complex genetic changes that transformed wild animals into their domesticated forms, and the population context in which this process took place.The domestication of the horse had a far-reaching impact on the sociopolitical and economic trajectories of human societies (1). It not only provided meat and milk (2) but also enabled the development of continent-sized nomadic empires, by transforming warfare and allowing for the rapid spread of goods and information over long distances. However, despite the characterization of the genome of modern horses (3), an understanding of the genetic processes underlying horse domestication is still lacking. In other organisms, such an understanding is usually achieved by comparing the genomes of domesticated species and their wild relatives (4–6), but this approach is not directly applicable to horses. Recent genome-wide analyses have shown that Przewalski’s horse, the last truly wild horse population remaining today, is not the direct ancestor of domesticated horses (7, 8). Instead, it likely represents a sister population that separated from the ancestral population of domesticated horses some 38–72 kya (9). This date significantly predates not only the widely accepted date for the beginning of horse domestication, ca. 5.5 kya (2), but also the earliest potential evidence for horse domestication, ca. 7.5 kya (10). In addition, the current Przewalski’s horse population descends from a captive stock consisting of only 13 founder individuals (7). This severe demographic bottleneck, together with inbreeding resulting from unequal contributions from different captive lineages, likely caused a substantial loss of the diversity once present in Przewalski’s horses. As a result, no modern horse population can fully represent the genetic diversity ancestral to the modern, domesticated gene pool (11–13).Ancient DNA allows tracking of past population histories through time, accessing the gene pools of wild animals predating domestication and exploring genetic variation that has been lost in extant populations. Recovery of ancient DNA, coupled with low-throughput gene candidate analyses, has previously been used to investigate changes in the genetic diversity of horses over time. This approach revealed coat color variation as one early result of domestication, by showing that the selection of multiple alleles driving diverse coloration patterns was already ongoing in the Early Bronze Age (14). It has also revealed a loss of Y-chromosomal haplotypes on both the Przewalski’s and domestic horse lineages (12).Using next-generation sequencing, the complete genome sequence of ancient individuals can now be deciphered (15), with qualities rivaling the qualities of modern genomes (16–18). We characterized complete genome sequences of two ancient horse specimens predating the earliest evidence of horse domestication to reveal the population context in which horse domestication took place. We compared the genomic information of these specimens with genomic information of six domestic horses, representing five breeds, and one Przewalski’s horse (9), ranging from a 7.4- to 32.7-fold average depth of coverage. We also included the domestic donkey as an outgroup, which represents a sister lineage of modern horses and shares a most common recent ancestor with horses 4.0–4.5 Mya ago (9). This comparison enabled us to reconstruct the relationships between wild and domesticated horses, and to explore the genetic mechanisms underlying the behavioral, physiological, and other biological changes that accompanied horse domestication.     

From the Cover: PNAS Plus: Intrathecal gene therapy rescues a model of demyelinating peripheral neuropathy

Inherited demyelinating peripheral neuropathies are progressive incurable diseases without effective treatment. To develop a gene therapy approach targeting myelinating Schwann cells that can be translatable, we delivered a lentiviral vector using a single lumbar intrathecal injection and a myelin-specific promoter. The human gene of interest, GJB1, which is mutated in X-linked Charcot–Marie–Tooth Disease (CMT1X), was delivered intrathecally into adult Gjb1-null mice, a genetically authentic model of CMT1X that develops a demyelinating peripheral neuropathy. We obtained widespread, stable, and cell-specific expression of connexin32 in up to 50% of Schwann cells in multiple lumbar spinal roots and peripheral nerves. Behavioral and electrophysiological analysis revealed significantly improved motor performance, quadriceps muscle contractility, and sciatic nerve conduction velocities. Furthermore, treated mice exhibited reduced numbers of demyelinated and remyelinated fibers and fewer inflammatory cells in lumbar motor roots, as well as in the femoral motor and sciatic nerves. This study demonstrates that a single intrathecal lentiviral gene delivery can lead to Schwann cell-specific expression in spinal roots extending to multiple peripheral nerves. This clinically relevant approach improves the phenotype of an inherited neuropathy mouse model and provides proof of principle for treating inherited demyelinating neuropathies.Inherited demyelinating neuropathies result from genetic defects in a variety of genes that are expressed by myelinating Schwann cells (1). These mutations are thought to cause demyelination in a cell-autonomous manner. Recessively inherited disorders cause loss of function, and dominantly inherited disorders cause haplotype insufficiency or toxic gain of function (2, 3). Achieving a therapeutic correction of these genetic defects requires either gene replacement or gene silencing approaches, ideally confined to myelinating Schwann cells (4).Various techniques for gene delivery to peripheral nerves have been attempted, including adenoviral (AV) and adeno-associated viral (AAV) vectors and ubiquitous promoters (5). Intramuscular and direct intraneural injections help restrict expression to Schwann cells, but the duration of expression is typically limited. Lentiviral vectors produce sustained expression and have been injected intraneurally in crushed sciatic nerves to achieve retrograde transport and gene expression in motor neurons (6) and locally to transduce Schwann cells (7). None of the approaches published to date has provided a Schwann cell-specific gene delivery method to achieve widespread and stable expression.We recently reported Schwann cell-specific expression driven by the rat myelin protein zero (Mpz) promoter of a neuropathy gene following intraneural lentiviral vector delivery, alleviating pathological changes in a model of X-linked Charcot–Marie–Tooth disease (CMT1X) (8). Expression was restricted to the injected sciatic nerve, however, thus limiting its usefulness for clinical applications.Here we report a gene delivery approach via a single lumbar intrathecal injection leading to stable Schwann cell gene expression in an unexpectedly widespread distribution—the lumbar spinal roots and along the entire length of the femoral and sciatic nerves. Using this approach to treat a mouse model of CMT1X resulted in significant behavioral, functional, and morphological improvement, providing an important advance toward treating inherited neuropathies.     

From the Cover: PNAS Plus: Whole-brain calcium imaging with cellular resolution in freely behaving Caenorhabditis elegans

The ability to acquire large-scale recordings of neuronal activity in awake and unrestrained animals is needed to provide new insights into how populations of neurons generate animal behavior. We present an instrument capable of recording intracellular calcium transients from the majority of neurons in the head of a freely behaving Caenorhabditis elegans with cellular resolution while simultaneously recording the animal’s position, posture, and locomotion. This instrument provides whole-brain imaging with cellular resolution in an unrestrained and behaving animal. We use spinning-disk confocal microscopy to capture 3D volumetric fluorescent images of neurons expressing the calcium indicator GCaMP6s at 6 head-volumes/s. A suite of three cameras monitor neuronal fluorescence and the animal’s position and orientation. Custom software tracks the 3D position of the animal’s head in real time and two feedback loops adjust a motorized stage and objective to keep the animal’s head within the field of view as the animal roams freely. We observe calcium transients from up to 77 neurons for over 4 min and correlate this activity with the animal’s behavior. We characterize noise in the system due to animal motion and show that, across worms, multiple neurons show significant correlations with modes of behavior corresponding to forward, backward, and turning locomotion.How do patterns of neural activity generate an animal’s behavior? To answer this question, it is important to develop new methods for recording from large populations of neurons in animals as they move and behave freely. The collective activity of many individual neurons appears to be critical for generating behaviors including arm reach in primates (1), song production in zebrafinch (2), the choice between swimming or crawling in leech (3), and decision-making in mice during navigation (4). New methods for recording from larger populations of neurons in unrestrained animals are needed to better understand neural coding of these behaviors and neural control of behavior more generally.Calcium imaging has emerged as a promising technique for recording dynamics from populations of neurons. Calcium-sensitive proteins are used to visualize changes in intracellular calcium levels in neurons in vivo which serve as a proxy for neural activity (5). To resolve the often weak fluorescent signal of an individual neuron in a dense forest of other labeled cells requires a high magnification objective with a large numerical aperture that, consequently, can image only a small field of view. Calcium imaging has traditionally been performed on animals that are stationary from anesthetization or immobilization to avoid imaging artifacts induced by animal motion. As a result, calcium imaging studies have historically focused on small brain regions in immobile animals that exhibit little or no behavior (6).No previous neurophysiological study has attained whole-brain imaging with cellular resolution in a freely behaving unrestrained animal. Previous whole-brain cellular resolution calcium imaging studies of populations of neurons that involve behavior investigate either fictive locomotion (3, 7), or behaviors that can be performed in restrained animals, such as eye movements (8) or navigation of a virtual environment (9). One exception has been the development of fluorescence endoscopy, which allows recording from rodents during unrestrained behavior, although imaging is restricted to even smaller subbrain regions (10).Investigating neural activity in small transparent organisms allows one to move beyond studying subbrain regions to record dynamics from entire brains with cellular resolution. Whole-brain imaging was performed first in larval zebrafish using two-photon microscopy (7). More recently, whole-brain imaging was performed in Caenorhabditis elegans using two-photon (11) and light-field microscopy (12). Animals in these studies were immobilized, anesthetized, or both and thus exhibited no gross behavior.C. elegans’ compact nervous system of only 302 neurons and small size of only 1 mm make it ideally suited for the development of new whole-brain imaging techniques for studying behavior. There is a long and rich history of studying and quantifying the behavior of freely moving C. elegans dating back to the mid-1970s (13, 14). Many of these works involved quantifying animal body posture as the worm moved, for example as in ref. 15. To facilitate higher-throughput recordings of behavior, a number of tracking microscopes (16–18) or multiworm imagers were developed (19, 20) along with sophisticated behavioral analysis software and analytical tools (21, 22). Motivated in part to understand these behaviors, calcium imaging systems were also developed that could probe neural activity in at first partially immobilized (23) and then freely moving animals, beginning with ref. 24 and and then developing rapidly (17, 18, 25–29). One limitation of these freely moving calcium imaging systems is that they are limited to imaging only a very small subset of neurons and lack the ability to distinguish neurons that lie atop one another in the axial direction of the microscope. Despite this limitation, these studies, combined with laser-ablation experiments, have identified a number of neurons that correlate or affect changes in particular behaviors including the AVB neuron pair and VB-type motor neurons for forward locomotion; the AVA, AIB, and AVE neuron pairs and VA-type motor neurons for backward locomotion; and the RIV, RIB, and SMD neurons and the DD-type motor neurons for turning behaviors (17, 18, 25, 26, 28, 30, 31). To move beyond these largely single-cell studies, we sought to record simultaneously from the entire brain of C. elegans with cellular resolution and record its behavior as it moved about unrestrained.     

From the Cover: PNAS Plus: Functional screen identifies kinases driving prostate cancer visceral and bone metastasis

Mutationally activated kinases play an important role in the progression and metastasis of many cancers. Despite numerous oncogenic alterations implicated in metastatic prostate cancer, mutations of kinases are rare. Several lines of evidence suggest that nonmutated kinases and their pathways are involved in prostate cancer progression, but few kinases have been mechanistically linked to metastasis. Using a mass spectrometry-based phosphoproteomics dataset in concert with gene expression analysis, we selected over 100 kinases potentially implicated in human metastatic prostate cancer for functional evaluation. A primary in vivo screen based on overexpression of candidate kinases in murine prostate cells identified 20 wild-type kinases that promote metastasis. We queried these 20 kinases in a secondary in vivo screen using human prostate cells. Strikingly, all three RAF family members, MERTK, and NTRK2 drove the formation of bone and visceral metastasis confirmed by positron-emission tomography combined with computed tomography imaging and histology. Immunohistochemistry of tissue microarrays indicated that these kinases are highly expressed in human metastatic castration-resistant prostate cancer tissues. Our functional studies reveal the strong capability of select wild-type protein kinases to drive critical steps of the metastatic cascade, and implicate these kinases in possible therapeutic intervention.Metastatic prostate cancer is responsible for the deaths of ∼30,000 men in the United States each year (1, 2). Ninety percent of patients develop bone metastases, and other major sites of metastases include lymph nodes, liver, adrenal glands, and lung (3). First-line treatments for metastatic disease are androgen deprivation therapies that block androgen synthesis or signaling through the androgen receptor (AR) (2). Inevitably, metastatic prostate cancer becomes resistant to androgen blockade. Second-line treatments such as chemotherapy (docetaxel, cabazitaxel) and radiation only extend survival 2–4 mo (4, 5).Identifying new therapeutic targets for metastatic prostate cancer has proven difficult. Exome and whole-genome sequencing of human metastatic prostate cancer tissues have found frequent mutations and/or chromosomal aberrations in numerous genes, including AR, TP53, PTEN, BRCA2, and MYC (6–11). The precise functional contribution of these genes to prostate cancer metastasis remains unknown. Genomic and phosphoproteomic analyses have also revealed that metastatic prostate cancer is molecularly heterogeneous, which has complicated the search for common therapeutic targets (12). Few murine models of prostate cancer develop metastases. Mice having prostate-specific homozygous deletions in SMAD4 and PTEN or expression of mutant KRAS develop metastases in visceral organs but rarely in bone (13–15).Targeting genetically altered constitutively active protein kinases such as BCR-ABL in chronic myelogenous leukemia and BRAF^V600E in melanoma has led to dramatic clinical responses (16). Although numerous oncogenic alterations have been identified in prostate cancer, DNA amplifications, translocations, or other mutations resulting in constitutive activity of kinases are rare (6, 9, 17). Genome sequencing of metastatic prostate cancer tissues from >150 patients found translocations involving the kinases BRAF and CRAF in <1% of patients (8, 18). Although uncommon, these genomic aberrations cause enhanced BRAF and CRAF kinase activity and suggest that kinase-driven pathways can be crucial in prostate cancer. Multiple lines of evidence indicate that nonmutated kinases may contribute to prostate cancer progression, castration resistance, and metastasis. SRC kinase synergizes with AR to drive the progression of early-stage prostatic intraepithelial neoplasia to advanced adenocarcinoma (19). SRC, BMX, and TNK2 kinases promote castration resistance by phosphorylating and stabilizing AR (20–22). Moreover, FGFR1, AKT1, and EGFR kinases activate pathways in prostate cancer cells to drive epithelial-to-mesenchymal transition and angiogenesis, both of which are key steps in metastasis (23–25). Despite the strong evidence implicating kinases in advanced prostate cancer, a systematic analysis of the functional role of kinases in prostate cancer metastasis has been lacking.Metastasis of epithelial-derived cancers encompasses a complex cascade of steps, including (i) migration and invasion through surrounding stroma/basement membrane, (ii) intravasation and survival in circulation/lymphatics, (iii) extravasation through the vasculature, and (iv) survival and growth at a secondary site (26). With the exception of genetically engineered mouse models, no single experimental assay can model all steps of the metastatic cascade. As a result, most screens for genes involved in metastasis have focused on testing one step of the cascade. The migration/invasion step of metastasis is commonly interrogated in vitro by determining the ability of cells to invade through small pores in a membrane (27–29). Genes that function in other steps, or those dependent on the in vivo microenvironment to promote metastasis, are likely to be overlooked in these screens.Multiple groups have performed in vivo screens for regulators of metastasis by manipulating cell lines in vitro with shRNA libraries or using genome editing techniques, and injecting cells either subcutaneously or into the tail vein of mice (30, 31). These methods are advantageous, because they interrogate multiple steps of the metastatic cascade (survival in circulation, extravasation, and colonization and growth at a secondary site) in a physiologically relevant environment. However, the majority of in vivo screens conducted so far have been based on loss-of-function genetics. These screens are limited to inhibiting the function of proteins expressed by a particular cell line. Using a gain-of-function in vivo screen, we sought to identify kinases that activate pathways leading to prostate cancer metastasis.     

From the Cover: PNAS Plus: The K+ channel KIR2.1 functions in tandem with proton influx to mediate sour taste transduction

Sour taste is detected by a subset of taste cells on the tongue and palate epithelium that respond to acids with trains of action potentials. Entry of protons through a Zn²⁺-sensitive proton conductance that is specific to sour taste cells has been shown to be the initial event in sour taste transduction. Whether this conductance acts in concert with other channels sensitive to changes in intracellular pH, however, is not known. Here, we show that intracellular acidification generates excitatory responses in sour taste cells, which can be attributed to block of a resting K⁺ current. We identify K_IR2.1 as the acid-sensitive K⁺ channel in sour taste cells using pharmacological and RNA expression profiling and confirm its contribution to sour taste with tissue-specific knockout of the Kcnj2 gene. Surprisingly, acid sensitivity is not conferred on sour taste cells by the specific expression of K_ir2.1, but by the relatively small magnitude of the current, which makes the cells exquisitely sensitive to changes in intracellular pH. Consistent with a role of the K⁺ current in amplifying the sensory response, entry of protons through the Zn²⁺-sensitive conductance produces a transient block of the K_IR2.1 current. The identification in sour taste cells of an acid-sensitive K⁺ channel suggests a mechanism for amplification of sour taste and may explain why weak acids that produce intracellular acidification, such as acetic acid, taste more sour than strong acids.Sour taste is mediated by a subset of taste cells on the tongue and palate epithelium that respond to acids with trains of action potentials and transmitter release (1–3). Both strong acids, such as hydrochloric acid, and weak acids, such as acetic or citric acid, produce a sour sensation in humans and evoke sensory responses in nerve recordings in a variety of model organisms, including rat, mouse, and hamster (4–7). A number of molecules have been proposed to transduce sour taste, most recently the ion channel PKD2L1/PKD1L3 (8–12), but their role in taste transduction remains unclear as subsequent studies using knockout mouse strains have failed to identify significant effects on sour taste (13–15). Nonetheless, the Pkd2l1 gene serves as a useful marker for sour taste cells (also designated type III cells), which account for ∼10% of the ∼50–100 taste cells found in each taste bud (1, 9, 11, 16, 17). Previously, using a Pkd2l1-YFP mouse, we showed that sour cells express a unique Zn²⁺-sensitive proton conductance, of unknown identity, that is likely to mediate the initial event in taste transduction (16). Whether this conductance acts “alone” or in concert with other channels sensitive to changes in intracellular pH is not known. In this report, we provide evidence for a second component of the transduction cascade: a resting K⁺ current, mediated by K_IR2.1 channels, which have an unexpected sensitivity to intracellular pH.Several pieces of evidence argue for a second component of taste transduction, sensitive to intracellular acidification. First, it was demonstrated nearly a century ago (18) that weak acids, which can penetrate the cell membrane and acidify the cell cytosol, taste more sour than strong acids, at the same pH. Mirroring this effect, it is well established that the gustatory nerve response is greater when the tongue is stimulated with weak acids than with strong acids at the same pH, and varies both as a factor of pH and of the concentration of the undissociated acid (5, 19). Similarly, calcium responses from sour-sensitive cells in slice recording can be evoked with weak acids at a higher pH compared with strong acids (20). Moreover, we previously reported that action potentials can be elicited in sour taste cells in response to extracellular pH of 6.5–6.7, where the current carried by protons (2–3 pA) is unlikely to be sufficient to depolarize the cell (21). All of these phenomena can be explained if intracellular acidification increases membrane excitability of sour taste cells. Indeed, it has been proposed that two-pore domain K⁺ channels, several of which are expressed at high levels in sour taste cells, could serve as sensors of intracellular pH (22, 23). However, to date, there is no direct evidence showing that sour taste cells are activated by intracellular acidification, and the molecular mechanisms by which intracellular acidification could excite sour taste cells remain largely unexplored.Here, using genetically identified sour taste cells, we show that intracellular acidification, in the absence of extracellular acidification, is sufficient to produce robust trains of action potentials in sour taste cells but not in taste cells that detect bitter, sweet, and umami. Intracellular acidification blocks an inwardly rectifying K⁺ current in sour taste cells, which we identify as K_IR2.1 based on expression profiling and pharmacological analysis, and confirm using a newly developed, cell-type–specific K_IR2.1-knockout model. We propose that block of the resting K⁺ current in sour taste cells contributes to the taste of weak acids and provides a mechanism for amplification of the sensory response.