Glucagon receptor antibody completely suppresses type 1 diabetes phenotype without insulin by disrupting a novel diabetogenic pathway

Insulin monotherapy can neither maintain normoglycemia in type 1 diabetes (T1D) nor prevent the long-term damage indicated by elevated glycation products in blood, such as glycated hemoglobin (HbA1c). Here we find that hyperglycemia, when unaccompanied by an acute increase in insulin, enhances itself by paradoxically stimulating hyperglucagonemia. Raising glucose from 5 to 25 mM without insulin enhanced glucagon secretion ∼two- to fivefold in InR1-G9 α cells and ∼18-fold in perfused pancreata from insulin-deficient rats with T1D. Mice with T1D receiving insulin treatment paradoxically exhibited threefold higher plasma glucagon during hyperglycemic surges than during normoglycemic intervals. Blockade of glucagon action with mAb Ac, a glucagon receptor (GCGR) antagonizing antibody, maintained glucose below 100 mg/dL and HbA1c levels below 4% in insulin-deficient mice with T1D. In rodents with T1D, hyperglycemia stimulates glucagon secretion, up-regulating phosphoenolpyruvate carboxykinase and enhancing hyperglycemia. GCGR antagonism in mice with T1D normalizes glucose and HbA1c, even without insulin.Ninety years of insulin treatment in patients with type 1 diabetes (T1D) have made it clear that insulin alone cannot normalize glucose homeostasis or glycated hemoglobin (HbA1c) levels. Even optimally controlled patients may exhibit postprandial surges of glucose levels to three or four times normal (1, 2), which may explain why HbA1c levels below 6% are so rare in patients with T1D. Current thinking attributes these spikes in peripheral plasma glucose to insufficient uptake of incoming dietary glucose by peripheral target tissues as a result of a lack of insulin. As a consequence, they are often managed by a preprandial bolus of insulin and restriction of dietary carbohydrate. This strategy results in chronic iatrogenic hyperinsulinemia (3) in patients with “well-controlled” T1D and is responsible for a high incidence of hypoglycemic events, which can be life-threatening.In nondiabetic subjects, a glucose load suppresses glucagon levels by stimulating an acute transient rise in paracrine insulin from β-cells juxtaposed to the glucagon-producing α cells (4–6). This glucagon suppression converts the liver from an organ of glucose production to an organ of glucose storage (7). In T1D, paracrine insulin is lacking and is replaced by peripherally injected insulin. The resulting intraislet insulin concentrations are but a small fraction of the paracrine concentrations of undiluted insulin that suppress glucagon in nondiabetic subjects (8, 9). In 1974, it was reported that hyperglycemia paradoxically stimulates glucagon secretion in dogs with chemically induced diabetes (10). More recently, plasma glucagon concentrations were reported to rise, with a tripling of hepatic glucose production, in normal rats continuously infused with glucose at a constant rate (11). Thus, there is evidence that in the absence of adequate insulin, elevated glucose might stimulate glucagon production, which in turn aggravates hyperglycemia. In this setting, the liver would not be reprogrammed to store incoming glucose but, rather, would continue to produce glucose as if it were still in the unfed state (12). This may play a major role in postprandial hyperglycemia (10).Here we find that in T1D, hyperglycemia stimulates, rather than suppresses, glucagon secretion. This suggests that in T1D, a positive hormonal feedback loop enhances hyperglycemia by adding endogenously produced glucose to diet-derived glucose. If this is an important factor in the hyperglycemic surges that plague patients with T1D, then suppressing glucagon secretion or blocking glucagon action should eliminate the surges of hyperglycemia observed in T1D in mice.To measure the normal response of pancreatic islets to elevated glucose, pancreata were isolated from normal mice and perfused with 5 or 25 mM glucose. Glucagon concentrations were measured in the perfusate. Raising the glucose concentration fivefold decreased glucagon concentration in the perfusate approximately sixfold (Fig. 1A). To determine the effect of increased glucose concentration on glucagon secretion without paracrine insulin, we measured glucagon levels in the medium of cultured InR1-G9 α cells in 5, 10, and 25 mM glucose. The rise from 5 to 10 mM glucose caused an approximately threefold increase in glucagon secretion, and the rise from 10 to 25 mM caused another twofold rise (Fig. 1B). Because cultured cells may not reflect the behavior of native α cells in situ in T1D, we isolated pancreata from streptozotocin-induced insulin-deficient T1D rats and perfused them with 5, 10, and 25 mM glucose concentrations. When the perfusate glucose was increased from 5 to 10 mM, glucagon secretion increased fourfold (Fig. 1C). An increase in glucose from 10 to 25 mM increased glucagon secretion another fourfold. Insulin concentrations in all of these perfusates were below the detection limit of a radioimmune assay (RIA) (EMD Millipore). Previously, we reported that the streptozotocin treatment protocol we used resulted in the destruction of 93.4% of β cells (13).Fig. 1.In the absence of paracrine insulin, glucagon secretion increases in response to increases in glucose. (A) An increase from 5 to 25 mM in the glucose perfused into the isolated pancreata of nondiabetic mice causes profound suppression of glucagon secretion. ...The fact that elevations of glucose stimulated glucagon secretion in the absence of an acute paracrine insulin release suggested that in animals with T1D, any rise in glucose would stimulate glucagon secretion and give rise to a cycle of self-enhancing hyperglycemia (3, 14). To investigate the possibility of such a diabetogenic pathway, we compared plasma glucagon levels in insulin-treated NOD/ShiLtJ T1D mice during and between hyperglycemic surges (Fig. 1D). The mice were treated with 0.1 U Levemir twice daily, and blood glucose was measured in the morning 17 h after an insulin injection (high blood glucose) and in the afternoon 7 h after an insulin injection (low blood glucose) (Fig. 1E). In mice receiving this insulin regimen, glucagon averaged 138 ± 41 pg/mL and insulin 3.9 ± 1.1 ng/mL in samples in which glucose averaged 500 ± 37 mg/dL This glucagon concentration was significantly higher (P < 0.05) than the mean glucagon level of 55 ± 35 pg/mL, measured in samples from the same mice when their glucose levels averaged 130 ± 71 mg/dL and insulin averaged 14.3 ± 4.5 ng/mL These findings are consistent with a glucagon-mediated contribution to the surges of hyperglycemia.To assess directly the effect on the liver of the hyperglucagonemia accompanying hyperglycemia in the absence of endogenous insulin, we compared activation of key markers of glucagon action in liver. The phosphorylation of cAMP response element binding protein (CREB), a transducer of the glucagon signal, and the expression of a gluconeogenic glucagon target, phosphoenolpyruvate carboxykinase (PEPCK), were measured in T1D and nondiabetic mice. Compared with nondiabetic liver, there was a 3.5-fold elevation in phosphorylated CREB and a 2.5-fold increase in PEPCK expression in T1D livers (Fig. 2 A–C). To demonstrate that these differences were glucagon-mediated, we treated T1D mice with a single injection of 5 mg/kg mAb B (15), a fully human, antiglucagon receptor (GCGR) antibody drug candidate under development by REMD Biotherapeutics, Inc. (15–17). In mice treated with the monoclonal antibody mAb B, daily 10:00 AM blood glucose measurements averaged 85 ± 5 mg/dL and remained normoglycemic for 8 d (Fig. 2D), at which time livers were harvested. In the mAb B-treated T1D livers, phosphorylated CREB protein was reduced to nondiabetic levels and PEPCK protein expression was reduced below that of nondiabetic mice (Fig. 2 A–C). Thus, the activation of hepatic gluconeogenesis in T1D mice was a result of their hyperglucagonemia and disappeared when glucagon actions were blocked.Fig. 2.Glucagon action is chronically high in the livers of diabetic animals. (A) Hepatic markers of glucagon signaling (P-CREB) and of glucagon action (PEPCK) were measured by immunoblotting liver samples from nondiabetic and diabetic mice. (B) The ratio of ...If self-enhancing action of hyperglycemia is mediated by glucose-stimulated increase in glucagon in T1D, it follows that suppressing glucagon secretion should eliminate or reduce the problem. To test this, we placed T1D mice on a low dose of insulin (0.01 U twice daily) and then began continuous s.c. infusions of four peptides known to suppress glucagon directly or indirectly (18–22). A fifth, nonpeptidic suppressor (23), GABA, was given mixed in the chow. Each of the five reagents lowered glucagon levels, and in each case, this was accompanied by reduction of hyperglycemia from >600 mg/dL to 160 ± 75 mg/dL (P < 0.001; Fig. 3A). The average insulin concentrations in these samples were not correlated with either glucagon or glucose concentrations. With the exception of leptin, which reduced food intake by 50% compared with diabetic animals receiving insulin monotherapy, none of these glucagon suppressors caused a significant reduction in food intake.Fig. 3.Agents that suppress glucagon secretion or antagonize GCGR normalize glucagon action in liver and plasma glucose in diabetic mice. (A) Plasma glucose and glucagon levels in diabetic NOD mice (n= 3–10) treated with the agents shown. (B) Blood glucose ...If the glucose-lowering effects of glucagon suppressors resulted entirely from reduced glucagon secretion, rather than from off-target actions, therapy with a GCGR antibody should cause as dramatic an improvement as the glucagon-suppressing agents. Mice with chemically (streptozotocin)-induced T1D and a starting hyperglycemia of ∼325 ± 72 mg/dL were injected i.p. once each week with 7.5 mg/kg anti-GCGR antibody mAb Ac (15) (Fig. 3B), and blood glucose was measured weekly for 12 wk. Blood glucose concentrations returned to normal (∼90 mg/dL) in the mice treated with the antibody 1 wk after a single dose (first time point), and this normalization continued for the duration of treatment. Body weight did not change significantly between the control and antibody-treated groups of mice from the start to finish of the experiment. In the vehicle-treated control mice, blood glucose levels rose to 540 ± 70 mg/dL during the 12-wk study. At the end of this treatment, HbA1c was measured as an indication of chronic hyperglycemia. In the mice treated with mAb Ac, HbA1c levels were normal (4 ± 1%), whereas in the control mice, HbA1c averaged 11 ± 1% (Fig. 3C). GLP-1 averaged 3.64 ± 0.9 pmol/L in the control mice and 3.63 ± 0.52 pmol/L in the mice treated with antibody. By immunohistochemistry, the ratio of insulin-positive cells to glucagon-positive cells in islets observed in sections of pancreas taken from five control mice at the end of the study was 0.15, and using cells from five antibody treated mice, the ratio was 0.16. Because the ratio of β cells to α cells in wild-type mice is ∼6.0 (24), the ratios observed are those expected for severe ablation of β cells by streptozotocin. The fact that the ratio did not change between the two groups of animals indicates that the antibody treatment did not induce α-cell hyperplasia in this experiment.     

Early procurement of scarlet macaws and the emergence of social complexity in Chaco Canyon,NM

High-precision accelerator mass spectrometer (AMS) ¹⁴C dates of scarlet macaw (Ara macao) skeletal remains provide the first direct evidence from Chaco Canyon in northwestern New Mexico that these Neotropical birds were procured from Mesoamerica by Pueblo people as early as ∼A.D. 900–975. Chaco was a prominent prehistoric Pueblo center with a dense concentration of multistoried great houses constructed from the 9th through early 12th centuries. At the best known great house of Pueblo Bonito, unusual burial crypts and significant quantities of exotic and symbolically important materials, including scarlet macaws, turquoise, marine shell, and cacao, suggest societal complexity unprecedented elsewhere in the Puebloan world. Scarlet macaws are known markers of social and political status among the Pueblos. New AMS ¹⁴C-dated scarlet macaw remains from Pueblo Bonito demonstrate that these birds were acquired persistently from Mesoamerica between A.D. 900 and 1150. Most of the macaws date before the hypothesized apogeal Chacoan period (A.D. 1040–1110) to which they are commonly attributed. The 10th century acquisition of these birds is consistent with the hypothesis that more formalized status hierarchies developed with significant connections to Mesoamerica before the post-A.D. 1040 architectural florescence in Chaco Canyon.Archaeologists have known for more than a century that the prehispanic Pueblo people of the American Southwest (hereafter SW) acquired goods from Mesoamerica. Such items included marine shell from the Gulf of California, raw copper and crafted copper bells from West Mexico (1–3), cacao from the Neotropics (4), and tropical birds such as scarlet and military macaws whose feathers were important in ritual (5, 6).Scarlet macaws (Ara macao; Fig. 1A) are the most exotic birds recovered by archaeologists excavating settlements in the SW from southern Utah and Colorado to northern Mexico. Along with cacao, shell, and copper, these birds signal the type of long-distance transport of goods often argued to have been an important dimension of emergent sociopolitical complexity in prehistoric societies. Like cacao, to which access was probably restricted to high-status individuals or groups (7), the acquisition and control of scarlet macaws was likely the province of social and religious elites.Open in a separate windowFig. 1.(A) Scarlet macaws, (B) the historic range of scarlet macaws, (C) locations of sites cited in text or with larger concentrations of macaws in the SW, and (D) a prehistoric feather bundle (H/13338) from Allen Canyon in Grand Gulch.The skeletal remains of more than 400 scarlet macaws have been recovered from the SW, with most concentrated in Chaco Canyon (n = 35 macaws), the Mimbres region of southwestern New Mexico (n ≥ 10), the Mogollon Rim area of east-central Arizona (n ≥ 27), Wupatki in north-central Arizona (n = 22), and Paquimé (n = 322) in northern Mexico (5, 6) (Fig. 1C). The range of scarlet macaws is restricted to humid forests in tropical America (8, 9), primarily the Gulf Coast region of Mexico, Central America, and northern sections of South America (Fig. 1B). The northernmost populations (Mexico to Nicaragua) have been recognized as a distinct subspecies, Ara macao cyanoptera, and inhabit more open habitats, including deciduous, mixed pine–broadleaf, and gallery forests (10).The acquisition of scarlet macaws likely required lengthy trips between the northern SW and the lowland tropical forests of Mesoamerica (11). These birds historically lived as far north as the Gulf Coast state of Tamaulipas near Tampico, ∼1,800 km from Chaco, until the late 19th century (12). The northern limit of this species along the Pacific coast was southern Oaxaca, even more distant (∼2,500 km) from Chaco. We know from historic accounts that such long-distance travel by Pueblo people was common, with Pueblo people exchanging turquoise and hides for macaws or their brilliantly colored feathers (13, 14). We also should note, however, the possibility that scarlet macaws were procured from more proximate, earlier Mesoamerican settlements, similar to the large 13th- to 15th-century prehispanic center of Paquimé, where scarlet macaws were bred (15).The chronology of Pueblo–Mesoamerican relations has been based exclusively on the presence of items such as macaw remains in SW archaeological contexts dated by dendrochronology or temporal changes in ceramic styles. Such indirect dating has led most scholars to conclude that these long-distance acquisition networks developed in the northern SW after A.D. 1030–1040 and were largely a result of the evolution of social complexity and hierarchy, rather than a key causal factor in the emergence of such complexity.A recent study (16), however, has demonstrated not only that such indirect dating may be inaccurate, particularly at settlements occupied for centuries, but also that an incipient form of social complexity and hierarchy developed in Chaco Canyon as early as the ninth century. As Chaco is one of the locations where scarlet macaws are concentrated, we directly accelerator mass spectrometer (AMS) ¹⁴C dated the available skeletal remains of 14 birds to reevaluate the chronology of Pueblo–Mesoamerican acquisition networks and the process by which social complexity emerged in Chaco.     

Atoms of recognition in human and computer vision

Discovering the visual features and representations used by the brain to recognize objects is a central problem in the study of vision. Recently, neural network models of visual object recognition, including biological and deep network models, have shown remarkable progress and have begun to rival human performance in some challenging tasks. These models are trained on image examples and learn to extract features and representations and to use them for categorization. It remains unclear, however, whether the representations and learning processes discovered by current models are similar to those used by the human visual system. Here we show, by introducing and using minimal recognizable images, that the human visual system uses features and processes that are not used by current models and that are critical for recognition. We found by psychophysical studies that at the level of minimal recognizable images a minute change in the image can have a drastic effect on recognition, thus identifying features that are critical for the task. Simulations then showed that current models cannot explain this sensitivity to precise feature configurations and, more generally, do not learn to recognize minimal images at a human level. The role of the features shown here is revealed uniquely at the minimal level, where the contribution of each feature is essential. A full understanding of the learning and use of such features will extend our understanding of visual recognition and its cortical mechanisms and will enhance the capacity of computational models to learn from visual experience and to deal with recognition and detailed image interpretation.The human visual system makes highly effective use of limited information (1, 2). As shown below (Fig. 1 and Figs. S1 and ​andS2),S2), it can recognize consistently subconfigurations that are severely reduced in size or resolution. Effective recognition of reduced configurations is desirable for dealing with image variability: Images of a given category are highly variable, making recognition difficult, but this variability is reduced at the level of recognizable but minimal subconfigurations (Fig. 1B). Minimal recognizable configurations (MIRCs) are useful for effective recognition, but, as shown below, they also are computationally challenging because each MIRC is nonredundant and therefore requires the effective use of all available information. We use them here as sensitive tools to identify fundamental limitations of existing models of visual recognition and directions for essential extensions.Open in a separate windowFig. 1.Reduced configurations. (A) Configurations that are reduced in size (Left) or resolution (Right) can often be recognized on their own. (B) The full images (Upper Row) are highly variable. Recognition of the common action can be obtained from local configurations (Lower Row), in which variability is reduced.Open in a separate windowFig. S1.MIRCs. Discovered MIRCs for each of the 10 original images (10 object classes) are ordered from large to small image coverage within each class. Below each MIRC are the recognition rate (Left) and size in image samples (Right).Open in a separate windowFig. S2.MIRCs coverage. Each colored frame outlines a MIRC (which may be at a reduced resolution). Together, they provide a redundant representation because recognition can be obtained from a single MIRC. Warmer colors of the MIRC frame outline areas of larger coverage.A MIRC is defined as an image patch that can be reliably recognized by human observers and which is minimal in that further reduction in either size or resolution makes the patch unrecognizable (below criterion) (Methods). To discover MIRCs, we conducted a large-scale psychophysical experiment for classification. We started from 10 greyscale images, each showing an object from a different class (Fig. S3), and tested a large hierarchy of patches at different positions and decreasing size and resolution. Each patch in this hierarchy has five descendants, obtained by either cropping the image or reducing its resolution (Fig. 2). If an image patch was recognizable, we continued to test the recognition of its descendants by additional observers. A recognizable patch in this hierarchy is identified as a MIRC if none of its five descendants reaches a recognition criterion (50% recognition; results are insensitive to criterion) (Methods and Fig. S4). Each human subject viewed a single patch from each image with unlimited viewing time and was not tested again. Testing was conducted online using the Amazon Mechanical Turk (MTurk) (3, 4) with about 14,000 subjects viewing 3,553 different patches combined with controls for consistency and presentation size (Methods). The size of the patches was measured in image samples, i.e., the number of samples required to represent the image without redundancy [twice the image frequency cutoff (5)]. For presentation to subjects, all patches were scaled to 100 × 100 pixels by standard interpolation; this scaling increases the size of the presented image smoothly without adding or losing information.Open in a separate windowFig. 2.MIRCs discovery. If an image patch was recognized by human subjects, five descendants were presented to additional observers: Four were obtained by cropping 20% of the image (Bottom Row) and one by 20% reduced resolution (Middle Row, Right). The process was repeated on all descendants until none of the descendants reached recognition criterion (50%). Detailed examples are shown in Fig. S4. The numbers next to each image indicate the fraction of subjects that correctly recognized the image.Open in a separate windowFig. S3.Original images used in the human study. The image stimuli in the human study were extracted from these 10 original images (10 object categories). In the experiment, the size of each original image was 50 × 50 image samples, or a cutoff spatial frequency of 25 cycles per image.Open in a separate windowFig. S4.MIRC hierarchical trees. Examples of MIRCs (in red boxes) and their hierarchical trees, including subimage descendants (sub-MIRCs) and superimage ancestors (super-MIRCs). At the top of each tree is a depiction of the MIRC’s position in the original image marked in a red-bordered box. The human recognition rate is shown below the image patches.     

Continental crust beneath southeast Iceland

The magmatic activity (0–16 Ma) in Iceland is linked to a deep mantle plume that has been active for the past 62 My. Icelandic and northeast Atlantic basalts contain variable proportions of two enriched components, interpreted as recycled oceanic crust supplied by the plume, and subcontinental lithospheric mantle derived from the nearby continental margins. A restricted area in southeast Iceland—and especially the Öræfajökull volcano—is characterized by a unique enriched-mantle component (EM2-like) with elevated ⁸⁷Sr/⁸⁶Sr and ²⁰⁷Pb/²⁰⁴Pb. Here, we demonstrate through modeling of Sr–Nd–Pb abundances and isotope ratios that the primitive Öræfajökull melts could have assimilated 2–6% of underlying continental crust before differentiating to more evolved melts. From inversion of gravity anomaly data (crustal thickness), analysis of regional magnetic data, and plate reconstructions, we propose that continental crust beneath southeast Iceland is part of ∼350-km-long and 70-km-wide extension of the Jan Mayen Microcontinent (JMM). The extended JMM was marginal to East Greenland but detached in the Early Eocene (between 52 and 47 Mya); by the Oligocene (27 Mya), all parts of the JMM permanently became part of the Eurasian plate following a westward ridge jump in the direction of the Iceland plume.The North Atlantic Igneous Province covers vast areas in Baffin Island, Greenland, United Kingdom, Ireland, the Faroe Islands, and offshore regions (Fig. 1). Volcanic activity commenced ∼62 Mya (1) and is attributed to the impingement of a mantle plume head on the lithosphere (2). Enriched and depleted geochemical signatures in the Paleogene to Recent basalts from Iceland reflect a combination of plume-derived and shallow asthenospheric material, representing a classic case of plume–ridge interaction. The volcanic products of the Iceland plume have signatures of recycled oceanic crust and primordial-like material with high ³He/⁴He ratios (up to 50 R_A, where R_A is the ³He/⁴He ratio of air), spanning a range of refractory to fertile compositions (3, 4). Additional shallow-level contributions include the depleted mid-ocean ridge basalt–type asthenosphere, variably mixed with subcontinental lithospheric mantle material (5–8). The proximity of Iceland to the Jan Mayen Microcontinent (JMM) (Fig. 1) raises the question of whether Iceland includes underlying fragments of continental crust (9, 10). There are unconfirmed reports of both Precambrian and Mesozoic zircons in young basalts from Iceland, but only in abstract form (9, 11). The recovery of a 1.8-Ga-old grain from the most primitive Öræfajökull basalts (9) in southeast Iceland (Figs. 1 and ​and2A)2A) is potentially very interesting, although two Jurassic zircon grains (160 Ma) uncovered in the same separation are now suspected to be due to laboratory contamination.Open in a separate windowFig. 1.Crustal thickness map based on gravity inversion and revised location of the Iceland plume (white star symbols in 10-My intervals) relative to Greenland back to 60 Ma (see Fig. 6A for details). Transition between continental and oceanic crust (COB, black lines), plate boundaries (blue lines), site locations for dated North Atlantic Igneous Province magmatism (yellow circles with black fill), and the estimated size of the “classic” Jan Mayen Microcontinent (JMM) and the extended JMM (JMM-E) are also shown. The classic JMM is ∼500 km long (200 km at its widest and shown with four continental basement ridges), and crustal thicknesses are about 18–20 km (scale at Upper Left), which suggests considerable continental stretching before drifting off Greenland. We extend JMM 350 km southwestward (∼70 km wide) beneath southeast Iceland where we calculate maximum crustal thicknesses of ∼32 km (Fig. 2B). The size of JMM-E is a conservative estimate and could be as large as the white-stippled area. Öræfajökull location is shown as a yellow star.Open in a separate windowFig. 2.(A) Simplified geological map of Iceland outlining the major rift zones (RZ), rift zone central volcanoes (black outline), flank zone central volcanoes (colored outlines), the location of the Öræfajökull central volcano, and the position of the current plume axis of Shorttle et al. (25). Yellow areas are fissure swarms. WRZ, ERZ, NRZ, and MIB: Western, Eastern, and Northern Rift Zone and Mid-Iceland Belt, respectively. The stippled west-northwest–trending line through the plume axis depicts the position of the cross sections in Fig. 9. (B) Enlarged crustal thickness map (Fig. 1) with superimposed earthquake locations (earthquake.usgs.gov/earthquakes) and contour intervals (in meters) showing that the anomalously thick crust under southeast Iceland extends offshore to the northeast suggesting that it is a southerly fragment of the Jan Mayen Microcontinent (JMM) rather than an extension of the southeast-northwest–orientated Iceland-Faroes Ridge.     

A 2,300-year-old architectural and astronomical complex in the Chincha Valley,Peru

Recent archaeological research on the south coast of Peru discovered a Late Paracas (ca. 400–100 BCE) mound and geoglyph complex in the middle Chincha Valley. This complex consists of linear geoglyphs, circular rock features, ceremonial mounds, and settlements spread over a 40-km² area. A striking feature of this culturally modified landscape is that the geoglyph lines converge on mounds and habitation sites to form discrete clusters. Likewise, these clusters contain a number of paired line segments and at least two U-shaped structures that marked the setting sun of the June solstice in antiquity. Excavations in three mounds confirm that they were built in Late Paracas times. The Chincha complex therefore predates the better-known Nasca lines to the south by several centuries and provides insight into the development and use of geoglyphs and platform mounds in Paracas society. The data presented here indicate that Paracas peoples engineered a carefully structured, ritualized landscape to demarcate areas and times for key ritual and social activities.The Chincha Valley, located 200 km south of Lima, was one of the largest and most productive regions of southern coastal Peru (Fig. 1). Previous research identified a rich prehispanic history in the valley, beginning at least in the early first millennium BCE and continuing through the Inca period in the 16th century CE (1–3). The earliest settled villages were part of the Paracas culture, a widespread political and social entity that began around 800 BCE and continued up to around 100 BCE. Previous field surveys identified at least 30 major Paracas period sites in the valley (1, 3, 4), making Chincha one of the main centers of development for this early Andean civilization (5). As such, it is an ideal area to test models of social evolution in general and to define the strategies that early peoples used to construct complex social organizations within the opportunities and constraints provided by their environments.Open in a separate windowFig. 1.Map showing location of the Chincha Valley, southern coastal Peru.Previous research demonstrated a dense Paracas settlement in the lower valley that focused on large platform mound complexes (Fig. 2) (4, 6). Three seasons of systematic, intensive survey and excavations by our team confirm the existence of a rich and complex Paracas occupation in the midvalley area as well, including both mound clusters and associated geoglyph features. In short, our data indicate that (i): the Chincha geoglyphs predate the better-known Nasca drainage ones by at least three centuries; (ii) Paracas period peoples created a complex landscape by constructing linear geoglyphs that converge on key settlements; and (iii) solstice marking was one component underlying the logic of geoglyph and platform mound construction and use in the Chincha Valley during the Paracas period.Open in a separate windowFig. 2.Distribution of archaeological sites linked to Paracas period settlement in Chincha, Peru. Redrawn from Canziani (4).     

Metagenomic natural product discovery in lichen provides evidence for a family of biosynthetic pathways in diverse symbioses

Bacteria are a major source of natural products that provide rich opportunities for both chemical and biological investigation. Although the vast majority of known bacterial metabolites derive from free-living organisms, increasing evidence supports the widespread existence of chemically prolific bacteria living in symbioses. A strategy based on bioinformatic prediction, symbiont cultivation, isotopic enrichment, and advanced analytics was used to characterize a unique polyketide, nosperin, from a lichen-associated Nostoc sp. cyanobacterium. The biosynthetic gene cluster and the structure of nosperin, determined from 30 μg of compound, are related to those of the pederin group previously known only from nonphotosynthetic bacteria associated with beetles and marine sponges. The presence of this natural product family in such highly dissimilar associations suggests that some bacterial metabolites may be specific to symbioses with eukaryotes and encourages exploration of other symbioses for drug discovery and better understanding of ecological interactions mediated by complex bacterial metabolites.Symbiosis, defined by de Bary (1) as the “living together of two organisms,” includes a broad range of partnerships, from loose associations to obligate interdependencies and host–parasite interactions. Many involve microbes, with perhaps the most successful—between bacteria and early nucleated cells in the Precambrian—leading to mitochondria and chloroplasts in modern eukaryotes (2). Symbiotic interactions are being examined with increasing molecular detail, focusing not only on attributes that may be beneficial for each organism individually but also on what might be important for the association. It is increasingly being recognized that biosynthetic pathways leading to synthesis of specialized metabolites may play key roles in the biology of symbiosis (3).Lichens are ancient and physiologically highly integrated symbioses between heterotrophic filamentous fungi (mycobionts) and cyanobacteria or coccoidal green algae (photobionts) that may date as far back as 600 Mya (4). The morphology of the characteristic and stable macroscopic body of a lichen, the thallus, typically bears little resemblance to the individual organisms that form it and, in many cases, can be highly organized: fungal cells on the periphery for physical support and protection and photobiont cells inside, providing photosynthate or fixed nitrogen or both (5) (Fig. 1 A–C). Although the photobionts can often be isolated in pure culture (Fig. 1D), most mycobionts (almost exclusively from the Ascomycota) are refractory to propagation in vitro by standard methods, and intact lichens cannot be maintained artificially for long. Nevertheless, such limitations are gradually being overcome using advanced analytical platforms, e.g., metagenomics in the characterization of mycobiont lectin genes (6), and PCR-based phylogenetics in investigation of intrathalline bacterial diversity (7).Open in a separate windowFig. 1.The foliose lichen Peltigera membranacea and Nostoc symbiont. (A) Lichen in situ. (Scale bar, 5 cm.) (B) Rhizines (Rhi) on lower surface and apothecia (Apo) protruding from thallus edge. (C) Thallus cross section illustrating stratified internal structure including photosynthetic cyanobiont layer (shown with arrows) between cortical and medullary mycobiont layers (above and below, respectively). (Scale bar, 100 μm.) (D) Nostoc sp. N6 in culture. (Scale bar, 100 μm.) (photograph for Fig. 1C, courtesy of Martin Grube).In a number of bacterial–eukaryote symbioses, bacterial partners have been implicated in the production of complex molecules derived from polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) pathways (3, 8, 9). Examples include pederin, made by bacteria that live in rove beetles of the genus Paederus, and structurally related metabolites, the onnamides and psymberin, produced by bacteria that live in marine sponges (Fig. 2). In general, metabolites known or suspected to be of symbiont origin show remarkably low structural overlap with natural products discovered in screening programs from free-living bacteria (10). This phenomenon raises the intriguing question of whether symbiont chemistry might encompass structural scaffolds covering distinctive regions of chemical space.Open in a separate windowFig. 2.Pederin family compounds and symbioses. (Upper Left) Image of Paederus fuscipes courtesy of Christoph Benisch (www.kerbtier.de). (Upper Right) Image of Theonella swinhoei courtesy of Yoichi Nakao. (Lower Right) Image of Psammocinia aff. bulbosa adapted with permission from ref. 15. Copyright 2007 American Chemical Society. (Lower Left) Image of Mycale hentscheli courtesy of Mike Page.In this study, we applied a combination of metagenomic and natural product discovery methods to identify nosperin, the first member of the pederin family from a lichenized cyanobacterium and a further example toward the emerging concept of symbiosis-associated natural product pathways (10).     

4D multiple-cathode ultrafast electron microscopy

Four-dimensional multiple-cathode ultrafast electron microscopy is developed to enable the capture of multiple images at ultrashort time intervals for a single microscopic dynamic process. The dynamic process is initiated in the specimen by one femtosecond light pulse and probed by multiple packets of electrons generated by one UV laser pulse impinging on multiple, spatially distinct, cathode surfaces. Each packet is distinctly recorded, with timing and detector location controlled by the cathode configuration. In the first demonstration, two packets of electrons on each image frame (of the CCD) probe different times, separated by 19 picoseconds, in the evolution of the diffraction of a gold film following femtosecond heating. Future elaborations of this concept to extend its capabilities and expand the range of applications of 4D ultrafast electron microscopy are discussed. The proof-of-principle demonstration reported here provides a path toward the imaging of irreversible ultrafast phenomena of materials, and opens the door to studies involving the single-frame capture of ultrafast dynamics using single-pump/multiple-probe, embedded stroboscopic imaging.In 4D ultrafast electron microscopy (UEM), ultrafast light pulses generate electron packets by photoemission at the cathode of an electron microscope, and these are used to probe a dynamic process initiated by heating or exciting the microscopic specimen with a second, synchronized ultrafast light pulse (1, 2). In conventional implementations, each pump pulse on the specimen is accompanied by one suitably delayed laser pulse on the cathode to generate one packet of electrons probing a single time point in the evolution of the specimen. A record of the full course of temporal evolution of the specimen is then constructed by repeating the experiment multiple times with variation of the delay time between the two light pulses, reading out a separate CCD image for each delay time. Thus, information about different time points in the dynamic response of the specimen is obtained from different excitation events. This implementation is ideally suited for a specimen that undergoes irreversible but sufficiently well-defined dynamics to allow a new specimen area to be used for each time point (Fig. 1A), or for a specimen that recovers fully to allow repeated identical excitations of the same area (Fig. 1B); see also Methodology. The applications of these two approaches are numerous, as highlighted in a recent review account of the work (3).Open in a separate windowFig. 1.Variant implementations of UEM. (A) Single-pulse UEM, which enables single-shot imaging of homogeneous specimens. (B) Stroboscopic UEM. (D) Multiple-cathode UEM. For comparison, we include, in C, the single-cathode, deflection method. See text for details.For the study of completely nonrepetitive dynamics, for example, a stochastic process in a heterogeneous sample that does not return to its initial configuration, a series of snapshots following a single excitation event can provide the only direct and detailed view of the evolution. Observing the effects of a single excitation pulse with video-mode imaging can currently reach millisecond-scale time resolution, far short of the time scale for many phenomena of interest in nanoscale materials science, chemistry, and physics. Nanosecond resolution has been reached (4) by combining one excitation pulse with a train of light pulses on a single cathode, with deflection of the imaging electrons after passing the specimen plane to direct each successive pulse to a new region of the detector (Fig. 1C). This nanosecond method has been successfully used with a high-speed electrostatic deflector array to obtain time sequences of irreversible and stochastic processes (5, 6).Here we demonstrate a technique that removes any limit on time resolution imposed by image deflection and in a single frame enables the capture of ultrafast phenomena. With this approach, it is possible to probe and distinctly record multiple time points in a dynamic process following a single initiation pulse. The probing electron packets are all generated by a single light pulse that impinges on multiple, spatially distinct, cathode surfaces (Fig. 1D). Time separations between packets in the electron-pulse train are adjusted by the cathode spatial and electrostatic configuration. In the present application, two electron packets, generated from two source locations at the same potential and separated in time by 19 picoseconds (ps), are recorded on each CCD frame after undergoing diffraction in a gold film following femtosecond heating. The packets originate from different cathode locations, pass through the same area of the specimen, and are recorded at distinct locations on the detector, thereby encoding two different time points in the evolution of the specimen. The results obtained provide the basis for exploration of expanded application of the multiple-cathode concept.     

High thermoelectric performance by resonant dopant indium in nanostructured SnTe

From an environmental perspective, lead-free SnTe would be preferable for solid-state waste heat recovery if its thermoelectric figure-of-merit could be brought close to that of the lead-containing chalcogenides. In this work, we studied the thermoelectric properties of nanostructured SnTe with different dopants, and found indium-doped SnTe showed extraordinarily large Seebeck coefficients that cannot be explained properly by the conventional two-valence band model. We attributed this enhancement of Seebeck coefficients to resonant levels created by the indium impurities inside the valence band, supported by the first-principles simulations. This, together with the lower thermal conductivity resulting from the decreased grain size by ball milling and hot pressing, improved both the peak and average nondimensional figure-of-merit (ZT) significantly. A peak ZT of ∼1.1 was obtained in 0.25 atom % In-doped SnTe at about 873 K.Good thermoelectric (TE) materials should not only have high figure-of-merit (Z), but also be environmentally friendly and cost-effective (1–5). The nondimensional figure-of-merit (ZT) is defined as ZT = [S²σ/(κ_L+κ_e)]T, where S is the Seebeck coefficient, σ the electrical conductivity, κ_L the lattice thermal conductivity, κ_e the electronic thermal conductivity, and T the absolute temperature. Lead chalcogenides and their alloys can be engineered to exhibit high ZTs; however, environmental concern regarding Pb prevents their deployment in large-scale applications (6–10). Tin telluride (SnTe), a lead-free IV–VI narrow band-gap semiconductor has not been considered favorably as a good thermoelectric material because of its low ZT due to the relatively low Seebeck coefficient and high electronic thermal conductivity caused by intrinsic Sn vacancies (11–13), although SnTe has been used to alloy with other tellurides for better TE properties (14–26). Even though there has been no real success in achieving good TE properties of lead-free SnTe, the similarity between the electronic band structure of SnTe and that of PbTe and PbSe (27–31) suggests it has the potential to be a good TE material, especially given the two valence bands (light-hole and heavy-hole bands) that contribute to the hole density of states. The main difficulty here, however, is the fact that the separation between the light-hole and heavy-hole band edges in SnTe is estimated to be in the range of ∼0.3 to ∼0.4 eV (27, 29), larger than those of PbTe or PbSe (9), rendering the benefit of the heavier mass for the Seebeck coefficient less significant.In this paper, we prepared In-doped SnTe by high-energy ball milling and hot pressing and measured the samples up to 873 K without experiencing any mechanical strength issues. We show, based on both experiments and first-principles simulation, that a small amount of In-doping helps create resonant states around the Fermi level inside the valence band, which increases the Seebeck coefficient, especially at room temperature, leading to improvements in both average ZT and peak ZT, combined with the decreased lattice thermal conductivity due to the increased density of grain boundaries (32–34). Peak ZT value reaches ∼1.1 at about 873 K for SnTe doped with 0.25 atom % In.Single-phased In-doped SnTe was obtained by ball milling and hot pressing. Fig. 1 presents the X-ray diffraction (XRD) patterns of In_xSn_1-xTe (x = 0, 0.0025, 0.005, and 0.01). All the peaks can be indexed to the face-centered structure (space group Fmm). No impurity phase was found, despite the increasing content of In. First-principles calculations (Table S1) indicated it is energetically favorable for In to substitute for Sn, which is consistent with the case in In-doped PbTe and PbSe. In previous work, we found In substitutes for Pb in PbTe and PbSe, which is the same with In-doped SnTe, but it is n-type doping in In_xPb_1-xTe and In_xPb_1-xSe, which is different from p-type doping by In in SnTe, as we are reporting in this work (35, 36).Open in a separate windowFig. 1.XRD patterns for In_xSn_1-xTe (x = 0, 0.0025, 0.005, and 0.01) prepared by ball milling and hot pressing.The electrical conductivities decrease with increasing temperature, as shown in Fig. 2A, showing the typical behavior of degenerate semiconductors. With increasing content of In, the electrical conductivity decreases, especially at room temperature, from ∼7 × 10⁵ S⋅m⁻¹ to ∼2 ×10⁵ S⋅m⁻¹. The hole concentration indicated by the Hall measurement, however, changes in an interesting way with increasing In content: it drops below the intrinsic value at the beginning and starts to rise after x ≥ 0.0025 (as shown in Fig. 3A). Based on this observation, we conclude In atoms should be p-type dopants and explain the change of the carrier concentration as follows. The intrinsic SnTe is p-type because of the Sn vacancies (19). Those vacancies create empty electronic states and behave like p-type dopants. If we dope SnTe with In, In atoms first fill the Sn vacancies. Despite being p-type dopants, they are not as “strong” as the vacancies, in the sense that they induce fewer holes (examined by the simulation shown in Table S1); thus, at low doping levels, the p-type charge concentration decreases. However, as the doping level is increased, at some point all the Sn vacancies are filled with In, and beyond that point, excessive In atoms substitute for Sn, and the p-type charge concentration increases again (Fig. 3A). However, when In is more than the solubility limit in SnTe, the extra In atoms act as donors, which decreases the hole carrier concentration (x = 0.01) (37). The fact that the electrical conductivity decreases all the way indicates that the In dopants affected the hole mobility significantly (shown in Fig. 3B), as the result of both increased effective mass and impurity scattering. The Seebeck coefficients increase with temperature in the whole temperature range and also increase with In content, as shown in Fig. 2B. No bipolar effect is evident, even up to 873 K, in all the compositions despite the small band gap ∼0.18 eV for SnTe (29, 31). All the measured Seebeck coefficients are positive, consistent with the density of states (DOS) calculation presented in Fig. 4 and different from In-doped PbTe and PbSe, in which In turned out to be an n-type dopant (36, 38). Fig. 2C shows the power factors for undoped and In-doped SnTe. The highest power factor reaches ∼2.0 × 10⁻³ W⋅m⁻¹⋅K⁻² at about 873 K, higher than all the reported power factors of doped PbTe and PbSe at this temperature (9, 39–41). Most importantly, the average power factor is increased a great deal by In doping. Compared with the undoped SnTe prepared by melting and hand milling (M+HM) (broken line), the electrical properties of the ball-milled samples are not different.Open in a separate windowFig. 2.Temperature dependence of (A) electrical conductivity, (B) the Seebeck coefficient, and (C) the power factor for In_xSn_1-xTe (x = 0, 0.0025, 0.005, and 0.01). The undoped SnTe prepared by melting, hand milling, and hot pressing (M+HM) is shown for comparison (broken line).Open in a separate windowFig. 3.Hall carrier concentration (A) and Hall mobility (B) at room temperature with respect to the doping content x. ○, undoped SnTe; ●, In-doped SnTe.Open in a separate windowFig. 4.Comparison of DOS for undoped SnTe (broken line), Bi-doped SnTe (solid line), and In-doped SnTe (bold solid line). Sharp features are observed in the DOS of In-doped SnTe near the band edge, to which the abnormal Seebeck coefficient might be attributed. The simulated supercell configuration corresponds to 3 atom % In concentration, which is higher than that achieved in the experiment. The Fermi level in the simulation resides at 6.207 eV, slightly below the DOS hump. With the experimental In concentration, the Fermi level is expected to sit around the DOS peak.Fig. 5 shows variation of the Seebeck coefficient vs. carrier concentration for both pure SnTe and In-doped SnTe. The Seebeck coefficients of undoped SnTe with different hole concentrations (2 × 10²⁰ to 1.8 × 10²¹ cm⁻³) were obtained previously by annealing under different conditions (open circles) (27). The carrier concentration obtained in this work is ∼2.35 × 10²⁰ cm⁻³ (filled circle). Unlike PbTe and PbSe (7, 9, 36, 39, 40), the Seebeck coefficient of SnTe shows abnormal variation with increasing carrier concentration, which was qualitatively explained previously by two parabolic band models (27) and density functional theory (DFT) calculations (31). The valence band model (VBM), which takes into account the nonparabolicity of the light-hole band (solid line), provides a quantitative fit to all the Seebeck coefficient data, except for those of In-doped samples, and thus is expected to best depict the contribution from the intrinsic band structure of SnTe (29). The model details for TE transport of p-type SnTe may be found in SI Text. Compared with the same model we used for PbTe and PbSe (9, 36), two major differences should be stated. The L point energy gap, Eg, is smaller for SnTe, making the nonparabolicity larger. This makes the Seebeck coefficient drop faster with increasing concentration, as seen in Fig. S1. The light-hole–heavy-hole band edge energy difference is 0.12 eV for PbTe, 0.26 eV for PbSe, and 0.35 eV for SnTe (9, 29, 36); thus, the heavy-hole contribution is relatively weaker for SnTe. This may be seen from the fact that there is not much difference between the predictions of VBM and those of the two-band Kane model (which ignores the heavy-hole band contribution) at room temperature for SnTe, until 10 × 10¹⁹ cm⁻³. However, the contribution from the heavy-hole band gradually increases at higher temperatures (Fig. S2) as for PbSe (9, 36), helping improve the Seebeck coefficient at high temperature and suppress the bipolar effect. Although the Seebeck coefficients of bismuth- (Bi-) and Cu-doped samples agree well with the VBM model, as shown in Fig. 5, indicating pure doping effects, the deviation of the In-doped samples from the VBM model implies that there must be mechanisms through which In dopants significantly alter the band structure of pure SnTe near the band edge. One of the possible mechanisms is the introduction of resonant levels (6, 42–44) into the valence band. Fig. 4 shows the DOS of pure SnTe, Bi-doped SnTe, and In-doped SnTe near the top of the valence band. A well-defined peak is observed in the DOS of In-doped SnTe that may contribute to the large deviation of the Seebeck coefficient from the VBM model. One may question whether the observed features are a result of the limited size of the supercell and thus the artificial interactions between In atoms. Similar features, however, are not observed in Bi-doped SnTe with the same supercell size. Therefore, we believe the added feature originates from the interactions of the In atoms with the host atoms. Because of the limitation of computing resources, a sufficiently dense k-mesh for calculating transport properties for the supercells is not possible at this stage; also, the simulated supercells are too small to represent a realistic doping concentration. [The simulated supercell corresponds to 3% In concentration, with a Fermi level located slightly below the DOS “hump.” With the doping concentration achieved in the experiments, the Fermi level is expected to reside close to the DOS peak. An alternative simulation method, such as a Korringa–Kohn–Rostoker coherent-potential-approximation (KKR-CPA) calculation (44), is required in cases of more dilute doping concentrations.] Thus, a direct evaluation of the effect of the features in DOS on the Seebeck coefficient is not available for now. However, the rich features introduced by In atoms are speculated to play an important role in the enhanced TE properties.Open in a separate windowFig. 5.Room temperature Pisarenko plot for ball-milled and hot-pressed In_xSn_1-xTe (x = 0, shown by ●; x = 0.001, 0.0015, 0.0025, 0.005, 0.0075, and 0.01, shown by ▲) in comparison with reported data on undoped SnTe (○), Bi-doped SnTe (□), and Cu-doped SnTe (♢) by Brebrick and Strauss (27). The solid curve is based on the VBM (light nonparabolic band and heavy parabolic band) with the heavy-hole effective mass of SnTe m*/m_e = 1.92.The other problem we should resolve is the high thermal conductivity induced by intrinsic Sn vacancies, causing very high electrical conductivity. By In doping, the decreased electrical conductivity results in a reduced electronic part of the thermal conductivity determined by the Wiedemann–Franz law (κ_e = LσT), where L is the Lorenz number. The Lorenz number is calculated using the VBM in a way similar to that of the Seebeck coefficient, including contributions from both nonparabolic light-hole and parabolic heavy-hole bands. The detailed expressions used are included in SI Text. Fig. 6 A–C gives the temperature dependences of the thermal diffusivity, specific heat, total thermal conductivity, and lattice thermal conductivity (obtained by subtracting the electronic contribution from the total thermal conductivity) of the undoped and In-doped SnTe, respectively. With increasing temperature, the total thermal conductivity decreases rapidly without showing any bipolar effect, consistent with the behavior of the Seebeck coefficient in Fig. 2B. The total thermal conductivities of all In-doped SnTe are lower than the undoped sample. Compared with the undoped SnTe prepared by melting and hot pressing (dotted line), the samples prepared by ball milling and hot pressing exhibit lower lattice thermal conductivity, which may be attributed to the increased density of grain boundaries by ball milling. In Fig. 7, the representative microstructure of ball-milled and hot-pressed In-doped SnTe is presented. Scanning electron microscopic (SEM) images shown in Fig. 7A indicate that the In_0.0025Sn_0.9975Te samples consist of both big grains with diameters of several tens of microns and small grains. The observed small cavities may contribute to the lower lattice thermal conductivity. The densities of all the samples are listed in Table S2. The size of the small grains is about 1 μm, as shown in Fig. 7B, less than one tenth that of the big grains. Nanograins in the samples also are observed via transmission electron microscopy (TEM). Fig. 7C shows a typical bright-field TEM image of the nanograins, with sizes around 100 nm. As a result, the lattice thermal conductivity of the samples is greatly reduced by significantly enhanced boundary scatterings of the phonons, as shown in Fig. 6C. Selected area electron diffraction and high-resolution TEM (HRTEM) images show that all the grains, whether in microns or nanometers, are single crystals with clean boundaries and good crystallinity, as shown in Fig. 7D. The crystalline grains and boundaries would benefit the transport of charge carriers, as observed in nanograined Bi_xSb_2-xTe₃ bulks (45), without degrading the electronic properties (Fig. 2).Open in a separate windowFig. 6.Temperature dependence of (A) thermal diffusivity (the undoped SnTe prepared by melting and hot pressing is shown by the broken line), (B) specific heat (the specific heat of sample x = 0 is used for the undoped SnTe prepared by melting and hot pressing), and (C) total thermal conductivity and lattice thermal conductivity for In_xSn_1-xTe (x = 0, 0.0025, 0.005, and 0.01) (the undoped SnTe prepared by melting and hot pressing is shown by the broken line).Open in a separate windowFig. 7.Representative SEM (A and B), TEM (C), and HRTEM (D) images for as-prepared In_0.0025Sn_0.9975Te samples by ball milling and hot pressing.Fig. 8 summarizes the ZT values of different samples. The two intrinsic valence bands contribute to the peak ZT value ∼0.7 at about 873 K for the undoped SnTe. The decreased lattice thermal conductivity by ball milling further boosts the peak ZT value to ∼0.8. However, the ZT values in both cases are quite low, below 600 K, resulting in low average ZTs. The enhanced Seebeck coefficient by resonant states increased both the peak and average ZTs in the In-doped nanostructured SnTe. A peak ZT ∼1.1 is obtained at about 873 K in In_0.0025Sn_0.9975Te.Open in a separate windowFig. 8.Temperature dependence of ZT for In_xSn_1-xTe (x = 0, 0.0025, 0.005, and 0.01) compared with the reported data on undoped SnTe (0.5–2.0 atom % Te excess) (■) by Vedeneev et al. (25) and codoped SnTe (0.5–2.0 atom % Te excess) with 1 atom % In and 1 atom % Ag (●) by Vedeneev et al. (25). The undoped SnTe prepared by melting and hot pressing is included for comparison (broken line).In summary, nanostructured In-doped SnTe with a ZT >1 has been prepared by ball milling and hot pressing. The improved ZT (peaked around 1.1 at about 873 K in 0.25 atom % In-doped SnTe) incorporates both the high Seebeck coefficient resulting from the two valence bands and the local resonant states around Fermi level created by In-doping and the lowered lattice thermal conductivity owing to the increased phonon interface scattering. This lead-free TE material is a potential candidate to replace lead chalcogenides used at medium to high temperatures for waste heat recovery applications. Further improvement is expected by adding suitable nanoinclusions or alloying with SnSe and SnS to decrease the thermal conductivity and increase the Seebeck coefficient.     

Direct coupling of haptic signals between hands

Although motor actions can profoundly affect the perceptual interpretation of sensory inputs, it is not known whether the combination of sensory and movement signals occurs only for sensory surfaces undergoing movement or whether it is a more general phenomenon. In the haptic modality, the independent movement of multiple sensory surfaces poses a challenge to the nervous system when combining the tactile and kinesthetic signals into a coherent percept. When exploring a stationary object, the tactile and kinesthetic signals come from the same hand. Here we probe the internal structure of haptic combination by directing the two signal streams to separate hands: one hand moves but receives no tactile stimulation, while the other hand feels the consequences of the first hand’s movement but remains still. We find that both discrete and continuous tactile and kinesthetic signals are combined as if they came from the same hand. This combination proceeds by direct coupling or transfer of the kinesthetic signal from the moving to the feeling hand, rather than assuming the displacement of a mediating object. The combination of signals is due to perception rather than inference, because a small temporal offset between the signals significantly degrades performance. These results suggest that the brain simplifies the complex coordinate transformation task of remapping sensory inputs to take into account the movements of multiple body parts in haptic perception, and they show that the effects of action are not limited to moving sensors.Motor and kinesthetic signals arising from the movement of the eyes in the head, and translation of the eyes and ears in space due to head and body movements, have been shown to play an important role in visual (1–3) and auditory (4–6) perception. However, because of the small number of sensory surfaces in these modalities, and the rigid constraints on their movement, the number of kinesthetic degrees of freedom is limited. In active touch or the haptic modality (7–13), the large number of sensory surfaces, and the nearly unlimited ways these surfaces can move, lead to the question of how movement can be represented and associated with the cutaneous or tactile signals. To study how tactile and kinesthetic cues are combined to haptically perceive object shape and size, we created a novel haptic stimulus in which these cues were completely dissociated. This stimulus consisted of simulated triangles felt through a narrow slit, as in anorthoscopic perception in vision (14–16) or haptic perception (17), and as illustrated in Fig. 1.Open in a separate windowFig. 1.Tactile stimuli and movement conditions. (A) An example of the tactile stimulus, here an expanding bar. The stimulus is shown as a series of “snapshots,” with the time running to the Right—the actual stimulus was continuous. The red rectangle represents the vibrating pins. (B) SAME condition: the same hand moves and experiences the tactile consequences of the movement. The hand moves the tactile display (green rectangle) mounted on the slider. In this example, a forward movement (away from the participant, upward in the figure) together with an expanding bar simulates a backward-pointing triangle (shown in red), felt through a slit. (C) DIFF condition: one hand moves, while the other hand, immobile, receives the tactile stimulus. The tactile signal (expanding bar) and kinesthetic signal (forward movement) are the same as in the previous example. Will the observer perceive simply an expanding bar or triangle in space? If a triangle is perceived, in which direction will it point? (D) IMMOB condition: the tactile signal is presented alone, with no movement. The expanding or contracting bar’s width as a function of time is a replay of a previous trial. (E) Correct spatial orientation of the triangle in the SAME condition, as a function of the movement direction (forward or backward) and the tactile stimulus (expansion or contraction). This truth table is an exclusive-or function, which has null correlations with its two input signals. Using either of the two signals alone will result in chance performance.The tactile signal consisted of a line that expanded or contracted on the index finger, delivered using a tactile display composed of pins that could vibrate independently, as shown in Fig. 1A. In one condition (SAME), the display was mounted on a slider that experimental participants slid along a track perpendicular to the tactile expansion or contraction, as shown in Fig. 1B. The slider’s position was used to update the tactile display to simulate stationary triangles of various lengths, oriented toward or away from the participant, felt through a virtual slit that moved with the finger. Would participants perceive an extended triangular shape, rather than the proximal stimulus consisting of expansion or contraction, and if so, which orientation would they perceive? For example, a backward-pointing triangle could result from a tactile expansion coupled with a forward movement (as shown in Fig. 1B, where forward motions and orientations—away from the participant—are shown as upward), or a contraction coupled with a backward movement—and vice versa for a forward-pointing triangle (Fig. 1E). Thus, the directions of the tactile stimulus and of the finger movement are insufficient by themselves to yield veridical perception of triangle orientation, and must be combined in an exclusive-or function (Fig. 1E) to yield veridical perception of object orientation. There are no purely tactile shape cues: the triangle’s edges are not slanted in our tactile stimulus (in contrast to the edges of an actual triangle behind a slit). Therefore, from the functional point of view, the only way to obtain triangle orientation is for the nervous system to make use of the exclusive-or rule on some level. Below we will show that this rule is applied perceptually rather than through cognitive inference.Crucially, we separated tactile and kinesthetic stimuli in the different (DIFF) condition. Participants moved the slider with one hand, and received the tactile stimulation through the stationary fingertip of the other hand (Fig. 1C). The position of the moving was measured and used to update the tactile display as in the SAME condition. Finally, in the immobile (IMMOB) condition, neither hand moved, and the durations of the tactile expansions and contractions were the same as in the other conditions (Fig. 1D). Thus, in both the DIFF and IMMOB conditions, we simulated a moving triangle felt through a stationary slit. In all conditions, participants reported both the perceived orientation and size of the triangle using a visual probe at the end of each trial. Participants closed their eyes during the movement and tactile stimulation, and therefore could not see the display during the crucial part of each trial. In the movement conditions (SAME and DIFF) the direction and speed of finger movement varied from trial to trial. Each of the three conditions was performed in separate blocks for different hands, with the blocks in random order.     

Giant and switchable surface activity of liquid metal via surface oxidation

We present a method to control the interfacial tension of a liquid alloy of gallium via electrochemical deposition (or removal) of the oxide layer on its surface. In sharp contrast with conventional surfactants, this method provides unprecedented lowering of surface tension (∼500 mJ/m² to near zero) using very low voltage, and the change is completely reversible. This dramatic change in the interfacial tension enables a variety of electrohydrodynamic phenomena. The ability to manipulate the interfacial properties of the metal promises rich opportunities in shape-reconfigurable metallic components in electronic, electromagnetic, and microfluidic devices without the use of toxic mercury. This work suggests that the wetting properties of surface oxides—which are ubiquitous on most metals and semiconductors—are intrinsic “surfactants.” The inherent asymmetric nature of the surface coupled with the ability to actively manipulate its energetics is expected to have important applications in electrohydrodynamics, composites, and melt processing of oxide-forming materials.The ability to control interfacial energy is an effective approach for manipulating fluids at submillimeter length scales due to the dominance of these forces at these small length scales and can be accomplished using a wide variety of methods including temperature (1, 2), light (3), surface chemistry (4–6), or electrostatics (7). These techniques are effective for many organic and aqueous solutions, but they have limited utility for manipulating high interfacial tension liquids, such as liquid metals. Liquid metals offer new opportunities for soft, stretchable, and shape-reconfigurable electronic and electromagnetic components (8–12). Although it is possible to mechanically manipulate these fluids at submillimeter length scales (13), electrical methods (14, 15) are preferable due to the ease of miniaturization, control, and integration. Existing electrohydrodynamic techniques can modestly tune the interfacial tension of metals but either limit the shape of liquid metals to plugs (e.g., continuous electrowetting) (16) or necessitate excessive potentials to achieve actuation on a limited scale (e.g., electrowetting) (17). Here, we demonstrate that the surface oxide on a liquid metal can be formed or removed in situ using low voltages (<1 V) and behaves like a surfactant that can significantly lower its interfacial tension from ∼500 mJ/m² to near zero. In contrast, conventional molecular surfactants effect only modest changes in interfacial tension (changes of ∼20–50 mJ/m²) and are difficult to remove rapidly on demand (18). Our approach relies on the electrical control of surface oxidation, which is simple, requires minimal energy, and provides rapid and reversible control of interfacial tension over an enormous range, independent of the properties of the substrate upon which it rests. Furthermore, this method avoids the use of toxic mercury and the ensuing modulation of surface tension is compatible with microfluidics.Fig. 1A contains a series of images that illustrate how liquid metal alloys of gallium [here, eutectic gallium indium (EGaIn), 75 wt % Ga, 25 wt % In (19)] spread dramatically in electrolyte in response to modest voltages (Movies S1 and S2). Fig. 1 reports the potentials relative to a saturated Ag–AgCl reference electrode in which the open-circuit potential is approximately −1.5 V. Spreading occurs in a variety of electrolytes, over a wide range of pH (over all pH attempted from 0 to 14), with and without dissolved oxygen, over a range of electrolyte concentrations, and on a wide variety of substrates including glass, Teflon, polystyrene, and tungsten. The spreading observed here has been reported previously in the literature, but was attributed to electrocapillarity (20). We show here that the mechanism goes beyond electrocapillarity and the electrochemical formation of the surface oxide plays an essential role.Open in a separate windowFig. 1.Spreading enabled by a surface oxide. (A) Oxidative spreading of a bead of liquid metal in 1 M NaOH solution. (Left) A needle serves as a top electrical contact to the droplet. (Right) A wire serves as a bottom electrical contact to the droplet (see Fig. S2 for details). (A, i) The drop assumes a spherical shape initially due to its large surface tension; (A, ii) upon application of an oxidative potential, the metal assumes a new equilibrium shape; (A, iii) above a critical potential, the metal flattens and spreads without bound and ultimately forms fingering patterns that further increase its surface area and destabilize the metal. (B–D) The unshaded region denotes oxidative potentials. (B) The areal footprint of a drop of EGaIn as a function of time and potential identifies the critical potential above which spreading occurs without bound (solid circles) and below which the droplet adopts equilibrium shapes (hashed symbols). (C) An electrocapillary curve of EGaIn measured by sessile drop profile in 1 M NaOH. (D) A cyclic voltammogram of EGaIn. (E) The measured capacitance and calculated capacitive energy of EGaIn from impedance spectroscopy.Fig. 1B plots the areal footprint of a spreading EGaIn drop as a function of time and potential (for example, see Fig. S1). The shape of the metal represents a balance between the interfacial tension and gravity. Lower interfacial tensions therefore correspond to an increased areal footprint. Above a critical potential (∼−0.6 V relative to a saturated Ag–AgCl reference electrode), the metal spreads initially as a disk that eventually breaks into a fingering morphology (Fig. 1 A, iii) that continues to increase in area with respect to time until it becomes unstable and separates from the electrode entirely. Below this critical potential (i.e., at potentials that are less oxidative), the drop adopts equilibrium shapes, as indicated by the plateaus in the normalized area in Fig. 1B.The change in surface tension of a liquid drop with respect to potential—that is, electrocapillarity—has been used previously to alter the shape of liquid metals (21), albeit less dramatically than that seen in Fig. 1A. Electrocapillarity lowers the interfacial tension of the metal γ from its maximum value γ₀ due to capacitive effects arising from the potential E as shown in Eq. 1 (22),γ = γ₀ ? ½C(E?E_PZC)², [1]where C is the capacitance per unit area and E_PZC is the potential of zero charge (PZC). The PZC is the potential at which the excess surface charge is zero and the surface tension is at its maximum value. One implication of Eq. 1 is that any change in potential from the E_PZC, whether positive or negative, will result in a symmetric decrease in the surface tension. In contrast, the spreading in Fig. 1A only occurs at oxidative potentials. Normally, undesirable electrochemical reactions, such as electrolysis, limit electrocapillarity to a small range of potentials (E) and thus a modest range of interfacial tension, whereas here the electrochemical reactions further lower the interfacial tension.Fig. 1C plots the interfacial tension of the metal measured by analyzing the shape of multiple sessile droplets (viewed from the side) versus potential. Between −2 and −1.4 V, in the absence of the oxide or electrochemical reactions, a droplet of EGaIn behaves in a manner that is consistent with classic electrocapillarity. Fitting this portion of the curve yields a maximum surface tension of 509 mJ/m² and an E_PZC of −0.91 V, consistent with previous electrocapillary measurements (23). At potentials more oxidative than −1.4 V, the oxide layer begins to form on the surface (24), as confirmed by the cyclic voltammogram in Fig. 1D. This oxidation causes a sudden and substantial drop in the surface tension, despite the decrease of measured capacitance and calculated capacitive energy over this potential range, as shown in Fig. 1E.The remarkable implication of the data summarized in Fig. 1 is that the oxide layer acts like a surfactant on the surface of the metal. In the absence of the oxide, the interfacial tension is large, as expected for a metal in contact with electrolyte. When the oxide forms, it replaces this high-energy interface with two new interfaces: metal–metal oxide and metal oxide–electrolyte. Most oxides, including gallium oxide, form hydroxyl groups on their exterior surface, rendering them hydrophilic (confirmed by a near-zero contact angle of water on the oxide surface). The interior surface of gallium oxide, however, consists of Ga atoms (25). The absence of any notable change in contact angle between the droplet and substrate during spreading and the independence of spreading relative to substrate composition further suggests spreading is due primarily to liquid metal–oxide–electrolyte interactions.Oxygen adsorption is known to lower the interfacial tension of molten metals as a function of the partial pressure of oxygen (26). At partial pressures in which the oxide forms fully, the interfacial tension of the metal becomes challenging to measure because it is encased in an oxide with mechanical properties that prevent the liquid from adopting an equilibrium shape. Alloys of gallium are no exception; the oxide can stabilize the metal in nonequilibrium shapes (27) despite being quite thin [∼1 nm in dry air (25, 28, 29)]. To achieve spreading, an electrochemical driving force is necessary. Although it is conceivable that spreading could be due to mechanical stresses arising from oxide growth, the ability of the drop to return to equilibrium shapes after physical perturbation at subcritical potentials (Movie S3), the lack of hysteresis in the “electrocapillary” curve, and the retardation of spreading by the formation of thicker oxides in pH neutral electrolytes (Movie S4), suggest otherwise. In NaOH(aq.), dissolution of the oxide (30) competes with the electrochemical formation of the oxide (31), which likely allows the metal to behave as a fluid with minimal mechanical hindrance.The change in surface tension in response to potential is completely reversible, with limited hysteretic effects. Fig. 2 demonstrates the ability to reversibly and rapidly switch the interfacial tension over a large range by only varying the potential modestly in steps of 0.25 or 0.5 V. In each case, the drop returns to the expected interfacial tension in <1 s, despite the kinetics of oxide growth and dissolution.Open in a separate windowFig. 2.Interfacial tension of the metal may be tuned dramatically, rapidly, and reversibly in response to small changes in potential, as shown using multistep chronoamperometry of a sessile EGaIn drop in 1 M NaOH. Increasing the electrochemical potential results in a corresponding decrease of the interfacial tension, consistent with the values in Fig. 1C.The interfacial tension undergoes a large step change when the oxide forms at −1.4 V, but continues to decrease with increased potential according to Fig. 1C. The dynamic interfacial behavior in this regime is difficult to analyze, but the decrease in the measured capacitance values from −1.4 to −0.7 V suggests that (i) electrocapillary behavior cannot solely explain the decrease in interfacial tension with respect to potential and (ii) the effectiveness of the oxide for separating the electrolyte from the metal likely improves with respect to potential.At potentials above the critical voltage (−0.6 V), two self-consistent observations suggest behavior resembling that of very low surface tension: (i) the area increases without bound until it becomes unstable, and (ii) the interfacial tension projects toward zero at this critical voltage (compare Fig. 1C). It is difficult to measure the interfacial tension beyond −0.6 V because the metal spreads without ever reaching an equilibrium shape and sessile drop analysis fails. To approximate the interfacial tension at potentials greater than −0.6 V, we use a scaling analysis based on the capillary length because the shape of the metal represents a balance between gravity and interfacial tension. Comparing the critical dimension (height) during spreading to the critical dimension at voltages of known tension provides an estimate, as described in SI Materials and Methods. First, comparing the capillary length of the oxide-coated metal when it is nearly spherical (−1.3 V) to when it spreads critically (−0.6 V) suggests the interfacial tension is ∼2 mN/m at −0.6 V. Likewise, comparing the capillary length of the oxide-coated metal at the last measurable point in Fig. 1C (i.e., −0.7 V) to the critical voltage (−0.6 V) also suggests the interfacial tension is ∼2 mN/m at −0.6 V. Although these two values are above zero, we refer to them as “near zero” because (i) the values are only estimates and are remarkably low considering the enormous tension of the bare metal, (ii) the metal never reaches an equilibrium shape at this potential, and (iii) the oxide may provide some minor mechanical impediment to spreading, in which case the liquid tension would be even lower.The spreading of EGaIn at oxidative potentials can be exploited to manipulate the shape of the metal in both closed and open systems. For example, oxidative potentials can induce the metal to flow uphill into capillaries containing electrolyte, as shown in Fig. 3A (Movie S5). Removal of the oxide chemically or electrochemically reverses the direction of flow, making it possible to impart reversible flow. Likewise, the position of a counterelectrode in electrolyte directs the movement of the metal in an open channel, as shown in Fig. 3B (Movie S6), because the spreading metal moves preferentially toward the counterelectrode. Oxidative potentials can also cause the metal to form metastable fibers as it extrudes from a syringe, as shown in Fig. 3D (Movie S7). In the absence of applied potential, metal pumped out of the end of a capillary forms beads that fall periodically due to the force of gravity, as shown in Fig. 3C.Open in a separate windowFig. 3.(A) Inducing liquid metal into an upward-tilted capillary channel (∼0.9 mm i.d.) by application of a voltage in the presence of 1 M NaOH. (B) Controlling the shape and direction of a metal drop into an open T-shaped Plexiglas channel submerged in 1 M NaOH solution using only voltage. Switching the position of the counterelectrode at different points (B, ii-iv), guides the direction of the metal droplet. (C) Side view of a small droplet of EGaIn pumped out of a 0.5-mm-i.d. polymer tube at 20-mL/h in 1 M NaOH. The metal forms droplets in the absence of potential. (D) Formation of an oxide-coated liquid metal fiber coming out of the tube at 5 V.It is also possible to remove the oxide skin using modest reductive potentials (e.g., −1 V applied) to return the metal to a state of large interfacial tension, induce capillary behavior, change the contact angle, and alter the rheological properties on demand; we call this behavior “recapillarity” (reductive capillarity). Before reduction, the oxide layer stabilizes the metal droplet in a nonequilibrium shape in pH-neutral electrolytes and gives it non-Newtonian rheological properties (32). In the absence of the stabilizing skin, the metal becomes Newtonian, decreases its footprint, and increases its contact angle (Fig. 4A). The experiments reported in Fig. 4 began by placing a droplet of the metal on a dry glass substrate before submerging the substrate in pH-neutral electrolyte. The presence of the oxide pins the drop to the dry substrate and allows the drop to be physically manipulated to a low initial contact angle before applying potential. The ability to mechanically manipulate the contact angle is indicative of the hysteretic nature of the wetting of the oxide-coated metal on dry surfaces (33). Removing the oxide allows the metal to dewet the substrate and return to an equilibrium contact angle. Recapillarity can turn this dewetting process on or off by electrochemically removing the oxide in pH-neutral solutions. Dewetting can occur asymmetrically by removing oxide preferentially from one side of the drop. Fig. 4B shows sequential images of a drop of metal in which the contact angle changes asymmetrically from 31° to 86° to 124° incrementally by inducing recapillarity in discrete steps on one side of the drop (Movie S8). The contact angle on the other side of the drop increases due to the combination of conservation of mass in the drop and the ability of the underlying oxide to pin the drop to the substrate.Open in a separate windowFig. 4.Recapillarity can manipulate the shape and local contact angle of droplets placed on dry substrates. (A) A droplet of liquid metal with a surface oxide (Left) supported by a glass slide. Electrochemical reduction of the oxide causes the metal to bead up (Right). Removal of the electrode before imaging creates the lobe on the top of the drop. (B) Three sequential side views (B, i–iii) of an EGaIn drop that asymmetrically dewets a glass slide in deionized water upon application of reductive potentials relative to a counterelectrode placed on the right side of the drop. Recapillarity causes the metal to move away from the anode, which is outside the frame of the image. The cathode gently touches the top of the drop to minimize perturbation of the shape of the metal.In the absence of potential, the oxide mechanically stabilizes the metal in microchannels (Fig. 5B). The adhesion of the oxide to the walls of dry channels makes it difficult to remove the metal by pressure differentials without leaving residue (34). In contrast, recapillarity induces liquid metal to withdraw controllably, smoothly, and rapidly (up to 20 cm/s) from microchannels via capillary action without leaving metal and oxide residue (Fig. 5 and Movie S9). The use of a pH-neutral electrolyte (NaF) ensures that segments of the metal that are not in the electrical path remain stable because of the presence of oxide skin (Fig. 5C).Open in a separate windowFig. 5.Recapillarity-induced withdrawal of liquid metal from microchannels. (A) The electrochemical reduction of gallium oxide in pH-neutral electrolyte induces retreat of the metal from the channel by applying a reductive potential. (B) Top-down photograph of a T-shaped microfluidic channel (1 mm wide, 100 µm tall, 65 mm long horizontally). The surface oxide stabilizes the liquid metal in the channel. (C) A reductive bias (−1 V) applied to the metal relative to a drop of electrolyte (0.01 M NaF) at the anode removes the oxide and the liquid metal retreats toward the cathode. (D) The metal continues to withdraw until the applied voltage ceases. Metal that is not in the electrical pathway remains stable.This paper demonstrates that surface oxides behave as excellent surfactants for metals and may be removed or deposited to rapidly and reversibly tune the interfacial tension of a low-toxicity liquid metal from ∼500 mJ/m² to near zero using modest voltages. This capability enables previously unidentified types of electrohydrodynamic phenomena to manipulate the shape of metal, which is attractive for a wide range of applications including microelectromechanical systems (MEMS) switches and conductive microcomponents (35), microactuators and pumps (36), adaptive electronic skins (37), tunable antennas and apertures (38, 39), fluidic optical components and displays (40, 41), field-programmable circuits (42), and metamaterials with reversible cloaking. The necessity of electrolyte and the reliance on electrochemical reactions may present some practical limitations for long-term switchability, although batteries operate with similar restrictions. The spreading behavior induced by a surface oxide provides the most significant evidence to date that buried oxide interfaces—which exist ubiquitously on most metals and semiconductors, yet are difficult to probe—lower interfacial energy of the underlying substrate significantly. The ability to rapidly remove and deposit oxides on a wide range of materials may enable new methods to control interfacial phenomena on both liquids and solids.     

Dislocation is a developmental mechanism in Dictyostelium and vertebrates

The excitable cells of Dictyostelium discoideum show traveling waves of signaling and generate a variety of complex wave forms during their morphogenesis. Important among these wave forms is the 3D spiral or scroll wave, which has been proposed previously to have a twisted variant: the “turbine wave.” Herein we argue that a D. discoideum scroll or concentric wave territory containing prespore and prestalk cell types can undergo “dislocation”: a wave field that initially controls aggregation of a whole developing population of Dictyostelium cells splits into two. This process leads to discontinuity between two connected domains of wave propagation and to specific phenomena, including high-frequency concentric pacemaker activity by the slime mold’s scroll-wave tip. The resulting morphogenetic events reveal a unique mechanism in morphogenesis.The organism Dictyostelium discoideum has attracted eminent mathematicians, theoretical physicists, and computer scientists to biology. Pioneering observations by John Bonner, Theo Konijn, and their colleagues (1, 2), and by Brian Shaffer (3), respectively, in the 1960s and 1970s demonstrated that chemotaxis to cAMP and cAMP signal relay, respectively, are the key cell properties that mediate aggregation in this organism. These early insights have only recently fully borne fruit. The complex relayed waves of cAMP signaling and chemotaxis generated by these two simple properties mean that a mass of D. discoideum cells behaves as an isotropic excitable medium (4) and generates a range of complex and beautiful behaviors and structures: notably, structures based on scroll waves. It was thought initially that these waves mediated only the first (aggregation) stage of D. discoideum development, but it became evident that they organize morphogenesis of all multicellular stages (5–8). The waveforms are mathematically predictable and the structures they organize presumably are as well (Fig. 1). The prevalent waveform is the Archimedean spiral, a form that can arise when wavefronts are broken; its 3D manifestation is the scroll wave (6, 8) (Fig. 1 A–C). Investigation of these waves reveals how two simple cellular properties can generate very complex morphogenesis. This article explores unique inferences about an aspect of this process, reasoning that late D. discoideum aggregates manifest dislocation: The spontaneous functional separation of a wave field with smoothly varying properties into two or more connected domains. These may or may not be domains that support the propagation of distinct waveforms. A single homogeneous territory breaks up spontaneously into two or more, a topological bifurcation. The domains remain connected by function and sometimes by wave propagation. We can expect different behaviors and different morphogenetic consequences in the different domains. This article shows below that this is a developmental mechanism in D. discoideum. The concern herein is the evolution of an original D. discoideum scroll or concentric wave domain to connected scroll prestalk and concentric prespore subdomains, but dislocation may be relevant for other cases. It is interesting that exactly the same concept proposed herein from experimental observations in D. discoideum was deduced previously from theoretical considerations, indicating that a wave field containing a frequency gradient can disintegrate, generating subdomains entrained to pacemakers with different frequencies (P 59 in ref. 9). This type of process is therefore likely to have general importance for morphogenesis. The article also identifies an example of dislocation in vertebrate somitogenesis, below.Open in a separate windowFig. 1.Waveforms in D. discoideum and in Beloussov–Zhabotinsky reagent, a chemical excitable medium, to illustrate the waveforms made in simple excitable media. (A) Concentric ring waves in Zhabotinsky reagent. The waves spread outward from a periodic point source pacemaker. New waves are initiated at regular intervals, leading to a target pattern of regularly spaced waves. (B) Zhabotinsky spirals. When a target ring is broken by shaking the medium, the wave ends propagate around to the back of their own refractory zone. They continue to curl around as the wave propagates out, generating a self-sustaining involute spiral that runs at the minimum period. (C) Spiral and concentric waves in an early D. discoideum aggregation field. The spirals can be high frequency, leading to closely packed waves. The concentrics are lower frequency. (Magnification: A and B, 1–10×; C, 10×.) (D) Spirals become scroll waves in three dimensions. Some of these scroll waves are bent and can also have their ends joined in torus rings. (E) A torus ring scroll wave in a late D. discoideum aggregate. The aggregating cells move from the inside outward over the top of the ring shaped aggregate. (F) Proposed scroll waves in slugs. (Lower) A D. mucoroides slug is proposed to contain a twisted scroll wave. (Upper) A D. discideum slug is known to contain a scroll wave in its tip and plane waves in its back part.     

Human (Clovis)–gomphothere (Cuvieronius sp.) association ～13,390 calibrated yBP in Sonora,Mexico

The earliest known foragers to populate most of North America south of the glaciers [∼11,500 to ≥ ∼10,800 ¹⁴C yBP; ∼13,300 to ∼12,800 calibrated (Cal) years] made distinctive “Clovis” artifacts. They are stereotypically characterized as hunters of Pleistocene megamammals (mostly mammoth) who entered the continent via Beringia and an ice-free corridor in Canada. The origins of Clovis technology are unclear, however, with no obvious evidence of a predecessor to the north. Here we present evidence for Clovis hunting and habitation ∼11,550 yBP (∼13,390 Cal years) at “El Fin del Mundo,” an archaeological site in Sonora, northwestern Mexico. The site also includes the first evidence to our knowledge for gomphothere (Cuvieronius sp.) as Clovis prey, otherwise unknown in the North American archaeological record and terminal Pleistocene paleontological record. These data (i) broaden the age and geographic range for Clovis, establishing El Fin del Mundo as one of the oldest and southernmost in situ Clovis sites, supporting the hypothesis that Clovis had its origins well south of the gateways into the continent, and (ii) expand the make-up of the North American megafauna community just before extinction.Clovis is the oldest well-established archaeological technocomplex in North America and is documented across much of the continent. The fundamental characteristics of this earliest well-defined foraging group to occupy North America south of the glaciers remain unclear, however. The timing of their appearance, the geographical origins of their distinctive Clovis projectile points, and their subsistence base, are the subject of continued debate (1–5). Archaeological excavations at El Fin del Mundo in northwestern Mexico (Fig. 1) provide new data and insights for all of these issues.Open in a separate windowFig. 1.El Fin del Mundo with location of locality 1 (black area at east end is the excavation), nonarchaeological localities 3 and 4 (1 and 3 are in an arroyo system), and the areas of the upland Clovis camp (2, 5, 8, 9, 10, 22). Inset shows location of the site (El Fin del Mundo, FdM) in northern Mexico relative to the Clovis mammoth kills in the upper San Pedro Valley (SPV) of southern Arizona.In situ Clovis sites are known primarily from the Great Plains and southeastern Arizona (3). The latter represents the densest concentration of in situ Clovis sites, with four Clovis–mammoth sites along a 20-km reach of the upper San Pedro River and two other Clovis sites in the same area with probable mammoth associations (Fig. 1) (6). In 2007, following reports from a local rancher, we discovered Clovis artifacts and Proboscidean remains eroding from an arroyo wall at El Fin del Mundo. Excavations and surveys during 2007–2012 documented Clovis artifacts in association with the remains of two gomphotheres, a Clovis camp on the surface of the surrounding uplands, and nearby sources of raw material for manufacture of stone tools. This paper focuses on the Clovis–gomphothere bone bed, to our knowledge the first in situ Clovis finds reported south of the international border.El Fin del Mundo is located in an intermontane basin within a chain of volcanic hills in the Sonoran Desert ∼100 km northwest of Hermosillo, in the Mexican state of Sonora (Fig. 1). The site is exposed by an arroyo system along the distal edge of a large bajada composed of Pleistocene and older basin fill. The drainage is part of the Rio Bacoachi. Dissection left the local basin fill exposed in head cuts and in a series of erosional islands (Fig. 1). The bone bed, artifacts, and their containing strata are associated with only one of these islands (locality 1) (Fig. 1 and SI Appendix, Site Stratigraphy and Formation Processes and Fig. S1). Locality 1 is isolated from and stratigraphically different from all other exposures in the area. Geomorphic and stratigraphic relations, therefore, cannot be fully reconstructed across the site.     

Predicting experimentally stable allotropes: Instability of penta-graphene

In recent years, a plethora of theoretical carbon allotropes have been proposed, none of which has been experimentally isolated. We discuss here criteria that should be met for a new phase to be potentially experimentally viable. We take as examples Haeckelites, 2D networks of sp²-carbon–containing pentagons and heptagons, and “penta-graphene,” consisting of a layer of pentagons constructed from a mixture of sp²- and sp³-coordinated carbon atoms. In 2D projection appearing as the “Cairo pattern,” penta-graphene is elegant and aesthetically pleasing. However, we dispute the author’s claims of its potential stability and experimental relevance.One of the joys of carbon research is the huge flexibility of carbon bonding (1–4), resulting in many varied allotropes that have already been experimentally identified. Computational modeling opens the floor to predicting many more, and tools such as graph theory (5) and evolutionary algorithms (6) allow systematic exploration of potential bonding networks. New computationally proposed phases are typically identified as metastable via positive phonon modes, and sometimes via molecular-dynamics (MD) simulations showing lattice coherence at experimental operating temperatures. However, there are common criteria beyond these two tests that link those allotropes that have been experimentally isolated.First, they occupy deep potential wells in the surrounding energetic landscape. Additionally, the surrounding energy wells are all higher in energy, “funneling” toward the stable structural form. Finally, barriers to subsequent conversion to alternative structures are typically high. Buckminsterfullerene, I_h-C₆₀, is a good example. The disconnectivity graph for C₆₀ connecting the 1,812 isomers with pentagonal and hexagonal faces via branches whose height indicates the transformation barrier has a “willow tree pattern,” with a gentle funnel running toward the stable I_h-C₆₀ isomer (7) (Fig. 1A). The relatively high barriers are accessible during high-temperature growth and alternatively can be catalyzed via the presence of impurities or carbon interstitial atoms (8–10).Open in a separate windowFig. 1.(A) “Willow tree” pattern of different C₆₀ isomers, with the lower points of each vertical bar representing the calculated formation enthalpy relative to I_h-C₆₀, and bar heights representing the calculated barrier to transformation. This shows that I_h-C₆₀ is significantly more stable than other isomers and lies at the center of an “energetic funnel.” Adapted from ref. 7, with permission from Macmillan Publishers Ltd.: Nature, copyright 1998. (B) Calculated formation enthalpies of B₄₀, showing the D_2d cage structure is significantly more stable than alternative isomers. Reproduced from ref. 12, with permission from Macmillan Publishers: Nature Chemistry, copyright 2014.In contrast, attempts to experimentally isolate higher-order boron fullerenes have been largely unsuccessful to date. For the proposed fullerene B₈₀, this can be understood because the energy landscape was shown to feature many closely related isomers with similar (and sometimes lower) energies (11). In contrast, calculations for B₄₀, for which there are first experimental indications (12), show a single (D_2d, 1A1) cage isomer, energetically well separated from alternative isomers (Fig. 1B). This behavior is consistent with the rules discussed above.We apply here a similar analysis for experimental viability to other proposed phases, starting with “penta-graphene,” a 2D carbon allotrope proposed by Zhang et al. (4). The structure can be viewed as a series of out-of-plane distorted ethylene units connected via tetrahedral sp³-carbon linkers. The result is a corrugated layer that in projection matches the “Cairo pattern” of distorted pentagons (Fig. 2A).Open in a separate windowFig. 2.A calculated structural transformation route from (A) penta-graphene to (D) graphene; each step is exothermic. Red arrows indicate direction of motion of atoms for 90° rotation of carbon–carbon bonds. Red (blue) lines indicate C–C bonds that are broken (formed). Note that structures A–C were constrained within orthogonal unit cells; this constraint was lifted for step C to D. The final structure, graphene, is 0.761 eV per atom more stable than A. Unit cells are marked with dotted lines; calculated cell dimensions are (A) 5.095 × 5.095 Å, (B) 4.769 × 5.510 Å, (C) 4.888 × 5.318 Å, and (D) 4.883 × 6.476 Å, α = 100.88°.     

Rapid seafloor changes associated with the degradation of Arctic submarine permafrost

Repeated high-resolution bathymetric surveys of the shelf edge of the Canadian Beaufort Sea during 2- to 9-y-long survey intervals reveal rapid morphological changes. New steep-sided depressions up to 28 m in depth developed, and lateral retreat along scarp faces occurred at multiple sites. These morphological changes appeared between 120-m and 150-m water depth, near the maximum limit of the submerged glacial-age permafrost, and are attributed to permafrost thawing where ascending groundwater is concentrated along the relict permafrost boundary. The groundwater is produced by the regional thawing of the permafrost base due to the shift in the geothermal gradient as a result of the interglacial transgression of the shelf. In contrast, where groundwater discharge is reduced, sediments freeze at the ambient sea bottom temperature of ∼−1.4 °C. The consequent expansion of freezing sediment creates ice-cored topographic highs or pingos, which are particularly abundant adjacent to the discharge area.

The effects of on-going terrestrial permafrost degradation (1–3) have been appraised by comparison of sequential images of Arctic landscapes that show geomorphic changes attributed primarily to thermokarst activity induced by recent atmospheric warming and ongoing natural periglacial processes (4–8). While the existence of extensive relict submarine permafrost on the continental shelves in the Arctic has been known for years (9, 10), the dynamics of submarine permafrost growth and decay and consequent modifications of seafloor morphology are largely unexplored.Throughout the Pleistocene, much of the vast continental shelf areas of the Arctic Ocean experienced marine transgressions and regressions associated with ∼125-m global sea level changes (11). Extensive terrestrial permafrost formed during sea-level low stands when the mean annual air temperatures of the exposed shelves were less than −15 °C (11, 12). Exploration wells drilled on the continental shelf in the Canadian Beaufort Sea show that relict terrestrial permafrost occurs in places to depths >600 m below seafloor (mbsf) and forms a seaward-thinning wedge beneath the outer shelf (10, 13, 14) (Fig. 1 A and B). The hydrography of the Canadian Beaufort Sea slows the degradation of the relict permafrost because a cold-water layer with temperatures usually near -1.4 °C blankets the seafloor from midshelf depths down to ∼200-m water depth (mwd) (15, 16) (Fig. 1B). As the freezing point temperature of interstitial waters is also controlled by salinity and sediment grain size, partially frozen sediments occur in a zone delimited by the ∼−2 °C and 0 °C isotherms (17, 18) (Fig. 1B).Open in a separate windowFig. 1.Map and cross-section showing the relationship between shelf edge morphology and the subsurface thermal structure along the shelf edge in the Canadian Beaufort Sea. (A) Shows the location of the study area with respect to estimates of submarine permafrost density (see key) and thickness, modified after 14. Thin contours indicate permafrost thickness in meters. Thicker contour is 120-m isobath marking the shelf edge. Area of repeat mapping coverage shown in Fig. 2 is indicated with a red box. (B) Shows a schematic cross-section with contours of selected subsurface isotherms modified after 15 along line x-x’ in A. The dotted blue line illustrates a thermal minimum (T-min) running through the relict permafrost isotherm and Beaufort Sea waters (16). Green shading indicates relict permafrost. Turquoise arrows show inferred flow of water from permafrost thawing along the base of the relict permafrost to the seafloor. The brown area indicates the zone where relict Pleistocene permafrost is predicted to have thawed with consequent movement of liberated groundwater, associated latent heat transfer and thaw consolidation causing surface settlement. Dashed brown lines define the subbottom limits for methane hydrate stability zone (MHSZ) which starts at ∼240 m below the sea surface and extends into the subsurface depending on the pressure and temperature gradient. The red box indicates the area shown in more detail in C with the same color scheme. The area of denuded seafloor in C is flanked by PLFs (dark-blue fill). Red arrows indicate the direction of heat transfer along the seaward edge of relict permafrost wedge.Distinctive surface morphologies characterize terrestrial permafrost areas. Conical hills (3 to 100 m in diameter) called pingos are common in the Arctic (19, 20). Pingos are formed due to freezing of groundwater. They characteristically contain lenses of nearly pure ground ice that cause heaving of the ground surface. Positive relief features with similar dimensions, referred to as pingo-like features (PLFs), are scattered across the Canadian Beaufort shelf (21, 22). On land, permafrost thawing, where there is ground ice in excess of the sediment pore space, can induce sediment consolidation (23), and surface subsidence results in widespread thermokarst landforms. Among the more dramatic occurrences are retrogressive thaw slumps (4–8, 24). These form where ice-rich permafrost experiences surface thaw causing thaw settlement and release of liquified sediment flows. Because of the loss of volume associated with thawing of massive ground ice, thaw slumps can quickly denude permafrost landscapes.During the first systematic multibeam mapping surveys in 2010 covering part of the shelf edge and slope in the Canadian Beaufort Sea, a band of unusually rough seafloor morphology between ∼120 and ∼200 mwds (25) was discovered along a ∼95-km-long stretch of the shelf. Subsequently, three additional multibeam surveys covering small characteristic areas (Fig. 2A) were conducted to understand the processes responsible for the observed morphologies. Here, we document the unique morphologies and seafloor change in this area and explore how the seafloor features may be related to subsea permafrost degradation and formation.Open in a separate windowFig. 2.(A) Shows bathymetry of a small section of the shelf edge indicated in Fig. 1A, with a color scale going from white (128 m) to blue (200 m) and contours at 120, 140, 170, and 200 mbsf. Outlines of areas resurveyed in 2013 (blue), 2017 (turquoise), and 2019 (green) are superimposed on the 2019 and regional 2010 survey. Colored symbols indicate locations of cores with porewater data using same key as Fig. 5B. The red star indicates the location of a temperature tripod deployed in the period 2015 to 2016. The location of ROV dive tracks (blue paths) are indicated. (B) Covers the same area as A with polygons identifying sites where changes were noted between surveys as follows: 2010 to 2013 (green), 2013 to 2017 (purple), 2017 to 2019 (black), and 2010 and 2019 (red). (C) Shows the same area, colored according to the difference in bathymetry between the 2019 survey and an idealized smooth surface extending between the top of the shelf edge scarp and the layered sediments occurring between the numerous PLFs. This is used to estimate the volume of material that eroded assuming the earlier Holocene seafloor corresponded with this idealized surface. Three zones of topography are labeled. Red boxes are locations of Figs. 3 and ​and5.5. (D) Shows Chirp profiles with the position of profiles shown in C and Fig. 5A. Light-green backdrop in X-X’ indicates possible void produced by retrogressive slide retreat used to calculated volume loss. Also indicated are TL, tilted layers; P, pingo-like-feature; and DR, diffuse reflector.     

Structure of a prereaction complex between the nerve agent sarin,its biological target acetylcholinesterase,and the antidote HI-6

Organophosphorus nerve agents interfere with cholinergic signaling by covalently binding to the active site of the enzyme acetylcholinesterase (AChE). This inhibition causes an accumulation of the neurotransmitter acetylcholine, potentially leading to overstimulation of the nervous system and death. Current treatments include the use of antidotes that promote the release of functional AChE by an unknown reactivation mechanism. We have used diffusion trap cryocrystallography and density functional theory (DFT) calculations to determine and analyze prereaction conformers of the nerve agent antidote HI-6 in complex with Mus musculus AChE covalently inhibited by the nerve agent sarin. These analyses reveal previously unknown conformations of the system and suggest that the cleavage of the covalent enzyme–sarin bond is preceded by a conformational change in the sarin adduct itself. Together with data from the reactivation kinetics, this alternate conformation suggests a key interaction between Glu202 and the O-isopropyl moiety of sarin. Moreover, solvent kinetic isotope effect experiments using deuterium oxide reveal that the reactivation mechanism features an isotope-sensitive step. These findings provide insights into the reactivation mechanism and provide a starting point for the development of improved antidotes. The work also illustrates how DFT calculations can guide the interpretation, analysis, and validation of crystallographic data for challenging reactive systems with complex conformational dynamics.A protein’s structure can be described in terms of a multidimensional energy landscape where different conformational states are separated by energy barriers (1, 2). The protein moves constantly across this landscape because of thermally driven fluctuations and interactions with ligands and substrates. The motions and dynamics of proteins are typically studied using techniques, such as NMR and/or molecular dynamics simulations. However, conformational ensembles are also often encountered in X-ray crystallography, enabling structural analysis of the spatial component of dynamics (3). Although such ensembles of conformers can be critical for understanding a protein’s function, they can also generate electron density maps that are difficult to interpret because of spatial averaging. This problem has been encountered in crystallographic studies on enzyme reactivation mediated by nerve agent antidotes (i.e., nucleophilic compounds that are used to treat intoxication by chemical warfare agents, such as sarin, VX, or tabun). Nerve agents potently inhibit the essential cholinergic enzyme acetylcholinesterase (AChE; EC 3.1.1.7) by phosphonylating its catalytic serine residue (Ser203) and therefore, rendering it incapable of hydrolyzing the neurotransmitter acetylcholine (4). This inhibition causes acetylcholine to accumulate, leading to overstimulation of the nervous system and eventually, death. Treatment of nerve agent-inactivated AChE with an antidote causes the release of the functional enzyme and the formation of a phosphonyl oxime (Fig. 1A) (4, 5). Although the process has been studied for decades, detailed mechanistic information is still lacking.Open in a separate windowFig. 1.(A) The catalytic S203 (blue) of mAChE is phosphonylated by sarin. The adduct is reactive and can undergo multiple chemical reactions. (B) The nerve agent reactivator HI-6 binds close to the adduct in (C) the deep, narrow, and highly aromatic active site gorge of mAChE. (D) In previous crystal structures (PDB ID code 2WHP), the O-isopropyl of the sarin adduct sterically shields the phosphorus, and the electron density maps do not define the position or conformation of the nucleophilic oxime moiety. Oxygen, nitrogen, and phosphorus are shown in red, blue, and orange, respectively.We have previously used X-ray crystallography to study the complex between the nerve agent antidote HI-6 and Mus musculus acetylcholinesterase (mAChE) phosphonylated by the nerve agent sarin (sarin–mAChE) (Fig. 1 B–D) (6). The binary sarin–mAChE complex and the ternary complex HI-6•sarin–mAChE can undergo (multiple) chemical reactions in solution (Fig. 1A) (7, 8) as well as in the crystal phase (9, 10). The addition of HI-6 to the sarin–AChE complex leads to a reaction that cleaves the bond between the oxygen of Ser203 and the phosphorus atom of sarin (Fig. 1A) (4, 5, 11). Furthermore, the sarin adduct can undergo a dealkylation reaction, termed “aging,” that renders the enzyme resistant to reactivation by HI-6 (7). Because of the system’s reactivity, the composition of crystals obtained by trapping the productive complex of sarin–mAChE and HI-6 is highly time-dependent, and the crystals inevitably contain several conformers and chemical species. These features make the system particularly challenging to study. Analyses of the previously reported ternary complex [Protein Data Bank (PDB) ID code 2WHP] revealed that HI-6 interacted with the arenes of Tyr124 and Trp286 to form a well-defined sandwich structure and that the sarin O-isopropyl moiety adopted a “closed” conformation that sterically shields the phosphorus atom against nucleophilic attack (Fig. 1C) (6). However, the electron density maps did not define the conformation or position of the reactive oxime moiety of HI-6, presumably because of spatial averaging (Fig. 1D) (6). These findings were supported by molecular dynamics simulations of the system, which revealed significant conformational dynamics of the nucleophilic pyridinium-oxime ring of HI-6 (6). Thus, in addition to the difficulties in defining the oxime moiety, the structure did not reveal how the nucleophile approaches the phosphorus atom of the sarin adduct and could not be used to support a detailed analysis of the reactivation mechanism.Ligands that are challenging to model in the electron density map are not unique to the system described above (12). For example, a computational method for redefining noise and interpreting weak electron density features revealed a previously hidden conformation of the HIV capsid protein (13). We recently presented a study in which we integrated conventional crystallographic refinement with a quantum chemical cluster approach using implicit dispersion-corrected density functional theory (DFT) calculations to refine and analyze the structure of a complex between mAChE and a pair of enantiomeric ligands (14, 15). In conventional crystallographic refinement, the experimental data are supported by molecular mechanics force fields that ensure that the bond lengths and angles of the protein and ligands are realistic (16). However, in the enantiomeric study, the experimental data did not unambiguously define the conformations and intermolecular contacts of the studied complexes. Combining crystallographic data with DFT calculations made it possible to identify low-energy conformations that are chemically plausible and consistent with experimentally derived electron density maps. Here, we describe the use of a similar refinement strategy that combines diffusion trap cryocrystallography and DFT calculations to identify low-energy conformations of the complex between HI-6 and mAChE phosphonylated by sarin (sarin–mAChE). The structure reveals previously unknown conformations of the system and a plausible preattack coordination of the nucleophile. The mechanistic implications of the structural analysis were investigated using site-directed mutagenesis, studies on the enzyme’s reactivation kinetics, and measurements of solvent isotope effects using deuterium oxide.