Late Pleistocene age and archaeological context for the hominin calvaria from GvJm-22 (Lukenya Hill,Kenya)

Kenya National Museums Lukenya Hill Hominid 1 (KNM-LH 1) is a Homo sapiens partial calvaria from site GvJm-22 at Lukenya Hill, Kenya, associated with Later Stone Age (LSA) archaeological deposits. KNM-LH 1 is securely dated to the Late Pleistocene, and samples a time and region important for understanding the origins of modern human diversity. A revised chronology based on 26 accelerator mass spectrometry radiocarbon dates on ostrich eggshells indicates an age range of 23,576–22,887 y B.P. for KNM-LH 1, confirming prior attribution to the Last Glacial Maximum. Additional dates extend the maximum age for archaeological deposits at GvJm-22 to >46,000 y B.P. (>46 kya). These dates are consistent with new analyses identifying both Middle Stone Age and LSA lithic technologies at the site, making GvJm-22 a rare eastern African record of major human behavioral shifts during the Late Pleistocene. Comparative morphometric analyses of the KNM-LH 1 cranium document the temporal and spatial complexity of early modern human morphological variability. Features of cranial shape distinguish KNM-LH 1 and other Middle and Late Pleistocene African fossils from crania of recent Africans and samples from Holocene LSA and European Upper Paleolithic sites.For Late Pleistocene African populations of modern humans, the constellation of behavioral changes encapsulated in the transition from the Middle Stone Age (MSA) to the Later Stone Age (LSA) ∼70–20 kya represents a series of some of the most pronounced changes in the archaeological record before the adoption of domesticated animals and plants and the use of ceramics for cooking and storage. It is among LSA sites that the strongest parallels with ethnographic and historic foragers are observed. Typical archaeological signatures include lithic technologies focused on the production of microliths (small flakes, blades, and bladelets with one edge blunted or “backed”) from bipolar, single-, and opposed-platform cores; an increased use of ground-stone tools; and implements made of wood and bone. These new technologies occur with the appearance of material correlates of social identity and networks of long-distance exchange, including ostrich eggshell (OES) beads, ochre, and nonlocal stone raw material, as well as increased dietary breadth, all consistent with larger, more dense, or more interconnected populations (1–9).This same interval of ∼70–20 kya witnessed a number of human dispersals across Africa, with eastern Africa host to one or more candidate populations for the expansion of Homo sapiens out of Africa (10–15). However, the eastern African hominin fossil record for this interval is extremely sparse and poorly dated, and it consists largely of isolated teeth or highly fragmentary crania and postcrania (16–18). Here, we reassess the age and context of the Kenya National Museums Lukenya Hill Hominid 1 (KNM-LH 1) partial calvaria from site GvJm-22 at Lukenya Hill, Kenya, the only eastern African fossil hominin from a Last Glacial Maximum [LGM; 19–26.4 kya (19)] LSA archaeological context. We construct a revised accelerator mass spectrometry (AMS) radiocarbon chronology built on 26 new dates on OES fragments. The revised chronology confirms the LGM age of KNM-LH 1 and expands the maximum age of the site to beyond the limits of the radiocarbon method. Increased radiometric age is consistent with new technological analyses that demonstrate previously unrecognized MSA elements that indicate assemblages spanning the MSA/LSA transition from deposits underlying KNM-LH 1. Morphometric analyses using a robust comparative dataset demonstrate the variability among African Late Pleistocene hominins, including candidate populations for out-of-Africa dispersals. They indicate that KNM-LH 1 is distinct from (i) modern Africans, (ii) H. sapiens from Holocene LSA sites, and (iii) European Upper Paleolithic modern humans, suggesting that it may sample a now extinct lineage.     

Early,intensive marine resource exploitation by Middle Stone Age humans at Ysterfontein 1 rockshelter,South Africa

Modern human behavioral innovations from the Middle Stone Age (MSA) include the earliest indicators of full coastal adaptation evidenced by shell middens, yet many MSA middens remain poorly dated. We apply ²³⁰Th/U burial dating to ostrich eggshells (OES) from Ysterfontein 1 (YFT1, Western Cape, South Africa), a stratified MSA shell midden. ²³⁰Th/U burial ages of YFT1 OES are relatively precise (median ± 2.7%), consistent with other age constraints, and preserve stratigraphic principles. Bayesian age–depth modeling indicates YFT1 was deposited between 119.9 to 113.1 thousand years ago (ka) (95% CI of model ages), and the entire 3.8 m thick midden may have accumulated within ∼2,300 y. Stable carbon, nitrogen, and oxygen isotopes of OES indicate that during occupation the local environment was dominated by C₃ vegetation and was initially significantly wetter than at present but became drier and cooler with time. Integrating archaeological evidence with OES ²³⁰Th/U ages and stable isotopes shows the following: 1) YFT1 is the oldest shell midden known, providing minimum constraints on full coastal adaptation by ∼120 ka; 2) despite rapid sea-level drop and other climatic changes during occupation, relative shellfish proportions and sizes remain similar, suggesting adaptive foraging along a changing coastline; 3) the YFT1 lithic technocomplex is similar to other west coast assemblages but distinct from potentially synchronous industries along the southern African coast, suggesting human populations were fragmented between seasonal rainfall zones; and 4) accumulation rates (up to 1.8 m/ka) are much higher than previously observed for dated, stratified MSA middens, implying more intense site occupation akin to Later Stone Age middens.

The Middle Stone Age (MSA) defines an interval in which many “modern” human behaviors emerged, including the first indications of artistic and symbolic expression, the use of personal adornments, advancements in tool making, effective hunting of large mammal game, and the exploitation of harsher environments (e.g., refs. 1–3). The earliest records of humans intensely and systematically exploiting marine resources also derive from MSA sites (4), and archaeological sequences rich in marine shellfish remains provide insight into several key aspects of human behavioral and cultural evolution during the MSA. For example, marine foods such as shellfish and marine mammals (scavenged or hunted) have been proposed as uniquely rich sources of key nutrients promoting brain development and perhaps leading to enhanced cognitive development and higher reproductive fitness (5–8). Some workers have suggested that adaptation to systematic coastal foraging with its rich, geographically stable and predictable resources may have promoted behaviors considered to be unique and potent adaptations of Homo sapiens such as territoriality, intergroup competition, and high levels of nonkin cooperation (9). Differences in marine shell sizes and accumulation rates between MSA and Later Stone Age (LSA) coastal sequences have been interpreted to provide insight into, respectively, relative population size and intensity of site occupation through time (10, 11). Compared to LSA people representing comparably larger populations, smaller populations of MSA people are thought to have exploited coastal resources less intensively, more selectively, or both (4). However, without robust chronological frameworks at MSA sites, lithic technologies and evidence of behavioral innovations cannot be confidently compared between sites. We show that precise geochronology and refined paleoenvironmental information can shed light on human behavioral development, adaptations, and population sizes in the MSA.MSA shell middens occur on the South African coastline (Fig. 1) (10, 12–21), and their ages are constrained largely by diverse techniques with precision typically of the order ∼10% or more at 2σ relative uncertainty (22). Here, we apply a recently developed approach to uranium-series (hereafter U-series, or ²³⁰Th/U) dating of ostrich eggshells (OES) at Ysterfontein 1 (YFT1), a well-stratified, carefully excavated MSA shell midden on the west coast of the Western Cape Province, South Africa, hosting a record of early shellfishing (10, 12, 13).Open in a separate windowFig. 1.YFT1 site location, locations of other sites with marine shell-rich layers, and YFT1 site stratigraphy. (A) Distribution of shell middens along the coasts of South Africa, with modern winter seasonal rainfall isopleths; (B, Inset) detailed map (A, pink outline) of shell middens on the western coast in the winter rainfall zone. Maps in A and B are modified from ref. 83. (C) YFT1 composite stratigraphy, numbered LG, and placement of OES dated by ²³⁰Th/U burial dating and/or with stable isotope data, after ref. 12; Inset are the OES sampled from LG12 for stable isotopes and ²³⁰Th/U burial dating. Site acronyms for maps A and B are as follows: BBC, Blombos Cave; BNK, Byneskranskop Cave; DK1, Die Kelders 1; DKS, Diepkloof Shelter; EBC, Elands Bay Cave; HDP, Hoedjiespunt; KRM, Klasies River Mouth; NBC, Nelson Bay Cave; PP, Pinnacle Point; SH, Sea Harvest; and YFT1, Ysterfontein 1.Unlike other U-series dating of OES, “²³⁰Th/U burial dating” accounts for the secondary uptake of U in OES from soil pore water upon burial and uses geochemical criteria inherent to the ²³⁰Th/U data to help identify reliable ages (23–27). The method was first tested against radiocarbon (¹⁴C) dating in LSA contexts (23), and this paper describes results from an extension of the method into archaeological contexts beyond the range of ¹⁴C dating (∼50 thousand years ago; hereafter, ka), which has not been previously attempted. Light stable isotope–paleoenvironmental proxies from OES also provide paleoenvironmental context directly related to the archaeological site (28–31), which we apply to OES recovered from YFT1. OES fragments at YFT1 are interspersed throughout the 3.8 m stratified sequence (SI Appendix), providing abundant sample material for dating. The chronological framework and paleoenvironmental context for YFT1 presented here provides key implications for the spatiotemporal coherence of South African lithic technologies and early evidence of intensive shellfishing, a coastal adaptation thus far considered unique to modern humans (9).     

From the Cover: Radiometric 81Kr dating identifies 120,000-year-old ice at Taylor Glacier,Antarctica

We present successful ⁸¹Kr-Kr radiometric dating of ancient polar ice. Krypton was extracted from the air bubbles in four ∼350-kg polar ice samples from Taylor Glacier in the McMurdo Dry Valleys, Antarctica, and dated using Atom Trap Trace Analysis (ATTA). The ⁸¹Kr radiometric ages agree with independent age estimates obtained from stratigraphic dating techniques with a mean absolute age offset of 6 ± 2.5 ka. Our experimental methods and sampling strategy are validated by (i) ⁸⁵Kr and ³⁹Ar analyses that show the samples to be free of modern air contamination and (ii) air content measurements that show the ice did not experience gas loss. We estimate the error in the ⁸¹Kr ages due to past geomagnetic variability to be below 3 ka. We show that ice from the previous interglacial period (Marine Isotope Stage 5e, 130–115 ka before present) can be found in abundance near the surface of Taylor Glacier. Our study paves the way for reliable radiometric dating of ancient ice in blue ice areas and margin sites where large samples are available, greatly enhancing their scientific value as archives of old ice and meteorites. At present, ATTA ⁸¹Kr analysis requires a 40–80-kg ice sample; as sample requirements continue to decrease, ⁸¹Kr dating of ice cores is a future possibility.Ice cores from the Greenland and Antarctic ice sheets provide highly resolved, well-dated climate records of past polar temperatures, atmospheric composition, and aerosol loading up to 800 ka before present (1–3). In addition to deep ice cores, old ice can also be found at ice margin sites and blue ice areas (BIAs) where it is exposed due to local ice dynamics and ablation (4–6). Antarctic BIAs have attracted much attention for their high concentration of meteorites, which accumulate at the surface over time (7). More recently, BIAs have also been used for paleoclimate studies, as large quantities of old ice are available at the surface where it can be sampled with relative ease (8, 9). Because the ice stratigraphy is exposed laterally along the BIA surface, such ice records are often referred to as horizontal ice cores.Determining the age of the ablating ice is the main difficulty in using BIAs for climate reconstructions (4). The most reliable method is stratigraphic matching, where dust, atmospheric composition, or water-stable isotopes of the horizontal core are compared with well-dated, regular ice core records to construct a chronology (10, 11). This technique, however, requires extensive sampling along the ice surface and relatively undisturbed stratigraphy, cannot be used past 800 ka B.P. (the current limit of the ice core record), and can be ambiguous during some time intervals. Several radiometric methods have been applied to ice dating, all of which have distinct limitations. Radiocarbon dating of trapped CO₂ suffers from in situ cosmogenic ¹⁴C production in the ice (12). Other methods rely on the incidental inclusion of datable material, such as sufficiently thick Tephra layers (13) or meteorites (7). The terrestrial age of meteorites is not likely to be representative of the surrounding ice, however, because they accumulate near the BIA surface as the ice ablates. A promising new technique uses the accumulated recoil ²³⁴U in the ice matrix from ²³⁸U decay in dust grains as an age marker (14); currently, the method still has a fairly large age uncertainty (16–300 ka). Flow modeling can provide useful constraints on the age of BIAs (15–18), but large errors are introduced by the limited availability of field data and necessary assumptions about past flow and ice thickness. Finally, the continued degassing of ⁴⁰Ar from the solid Earth allows dating of the ancient air trapped in the ice by evaluating the δ⁴⁰Ar/³⁸Ar and δ⁴⁰Ar/³⁶Ar stable isotope ratios (19), but the current uncertainty in this dating method is also large (180 ka or 11% relative age, whichever is greater).There is significant scientific interest in obtaining glacial ice dating beyond 800 ka, as such an archive would extend the ice core record further back in time, providing valuable constraints on the evolution of past climate, atmospheric composition, and the Antarctic ice sheet (20). Of particular interest is the middle Pleistocene transition (1200–800 ka B.P.) and the role of CO₂ forcing therein (21, 22). Such old ice can potentially be found in Antarctic BIAs such as the Allan Hills site (23), providing a strong impetus to developing reliable (absolute) dating tools for glacial ice.Krypton is a noble gas present in the atmosphere at a mixing ratio of around 1 ppm (24) and has two long-lived radioisotopes of interest to earth sciences (25, 26): ⁸¹Kr (t_1/2 = 2.29 × 10⁵ y) and ⁸⁵Kr (t_1/2 = 10.76 y). Kr-85 is produced in nuclear fission and released into the atmosphere primarily by nuclear fuel reprocessing plants. Kr-81 is naturally produced in the upper atmosphere by cosmic ray interactions with the stable isotopes of Kr, primarily through spallation and thermal neutron capture (27). The long half-life of ⁸¹Kr allows for radiometric dating in the 50–1,500-ka age range (28), well past the reach of radiocarbon dating. Kr-81–Kr dating has already been used to determine the residence time of groundwater in old aquifers (29–31) and for several reasons has great potential for applications in dating polar ice as well. First, krypton is not chemically reactive. Second, due to its long residence time, ⁸¹Kr is well-mixed in the atmosphere. Third, the method does not rely on sporadically occurring tephra, meteorite, or organic inclusions in the ice but is widely applicable, as all glacial ice contains trapped air. Fourth, it does not require a continuous or undisturbed ice stratigraphy. Finally, in contrast to ¹⁴C, ⁸¹Kr does not suffer from in situ cosmogenic production in the ice (12). What has precluded its use in ice core science to date is the large sample requirement owing to the small ⁸¹Kr abundance (⁸¹Kr/Kr = 5 × 10⁻¹³). Recent technological advances in Atom Trace Trap Analysis [ATTA (32–34)] have reduced sample requirements to 40–80 kg of ice, which can realistically be obtained from BIAs and ice margins.An earlier attempt at ⁸¹Kr dating of ice gave inconclusive results owing to ∼50% gas loss and substantial but poorly quantified (≥20%) contamination with modern air (35). The single sample in that study was extracted by chainsaw from the surface of the Allan Hills BIA site in Antarctica, and the sample quality was compromised by “extensive fracturing of the highly strained ice” (ref. 35, p 1825). In addition, a reliable method for ⁸¹Kr/Kr analysis was lacking at the time. Here we describe the successful ⁸¹Kr radiometric dating of polar ice using air extracted from four ice samples from the Taylor Glacier blue ice area in Antarctica. The ice at this site is heavily fractured to a depth of ∼3 m with sporadic cracks extending to 5 m, and consequently, we sampled ice in the 5–15-m depth range. Using ⁸⁵Kr we demonstrate that our samples are uncontaminated by modern air. We independently date our samples using stratigraphic matching techniques and show an excellent agreement with the ⁸¹Kr radiometric ages. Our study shows that ice from the previous interglacial period [Marine Isotope Stage (MIS) 5e, 130–115 ka B.P.] can be found in abundance near the surface of the Taylor Glacier BIA.     

A rock engraving made by Neanderthals in Gibraltar

The production of purposely made painted or engraved designs on cave walls—a means of recording and transmitting symbolic codes in a durable manner—is recognized as a major cognitive step in human evolution. Considered exclusive to modern humans, this behavior has been used to argue in favor of significant cognitive differences between our direct ancestors and contemporary archaic hominins, including the Neanderthals. Here we present the first known example of an abstract pattern engraved by Neanderthals, from Gorham’s Cave in Gibraltar. It consists of a deeply impressed cross-hatching carved into the bedrock of the cave that has remained covered by an undisturbed archaeological level containing Mousterian artifacts made by Neanderthals and is older than 39 cal kyr BP. Geochemical analysis of the epigenetic coating over the engravings and experimental replication show that the engraving was made before accumulation of the archaeological layers, and that most of the lines composing the design were made by repeatedly and carefully passing a pointed lithic tool into the grooves, excluding the possibility of an unintentional or utilitarian origin (e.g., food or fur processing). This discovery demonstrates the capacity of the Neanderthals for abstract thought and expression through the use of geometric forms.Considerable debate surrounds the Neanderthals'' cognitive abilities (1–7), and the view that the Neanderthals did not have the same cognitive capacities as modern humans persists in the literature (8) despite evidence to the contrary (9–15). One of the arguments against Neanderthals’ modern cognition is their apparent inability to generate cave art (16–19). The earliest evidence of rock art is typically associated with the arrival of modern humans (MH) in Western Europe ∼40 kyr (20, 21). The dating of calcitic layers covering painted dots at El Castillo Cave, Spain has pushed back this starting point beyond 41 kyr, opening the possibility of a Neanderthal authorship (22). Possible hypotheses include (i) the earliest rock art was produced by MH before their arrival in Europe but remains unidentified; (ii) rock art was created by Neanderthals or other archaic hominins and predated the arrival of MH; (iii) MH developed rock art on arrival in Europe; and (iv) rock art was developed in Europe after the arrival of MH.The lack of associated archaeological remains precludes assigning the El Castillo paintings to a specific population. Other factors contributing to the difficulty in testing the foregoing hypotheses include persistent uncertainties in the chronology of archaeological sites at the so called Middle-to-Upper Paleolithic transition in Europe (23–25) and in the taxonomic affiliation of their inhabitants during this period (26–28).Recent excavations at Gorham’s Cave led to the discovery in an area at the back of the cavity, below basal archaeological level IV, of an abstract pattern engraved into the bedrock. Level IV is an archaeological horizon containing exclusively Mousterian artifacts (29–31) deposited between 38.5 and 30.5 cal kyr BP (29, 32) (SI Appendix, Table S1). In this paper, we describe this engraving, provide additional contextual data demonstrating its attribution to Mousterian Neanderthals, reconstruct how it was created, and discuss implications of our findings for Neanderthal culture and cognition.     

From the Cover: Canine sexual dimorphism in Ardipithecus ramidus was nearly human-like

Body and canine size dimorphism in fossils inform sociobehavioral hypotheses on human evolution and have been of interest since Darwin’s famous reflections on the subject. Here, we assemble a large dataset of fossil canines of the human clade, including all available Ardipithecus ramidus fossils recovered from the Middle Awash and Gona research areas in Ethiopia, and systematically examine canine dimorphism through evolutionary time. In particular, we apply a Bayesian probabilistic method that reduces bias when estimating weak and moderate levels of dimorphism. Our results show that Ar. ramidus canine dimorphism was significantly weaker than in the bonobo, the least dimorphic and behaviorally least aggressive among extant great apes. Average male-to-female size ratios of the canine in Ar. ramidus are estimated as 1.06 and 1.13 in the upper and lower canines, respectively, within modern human population ranges of variation. The slightly greater magnitude of canine size dimorphism in the lower than in the upper canines of Ar. ramidus appears to be shared with early Australopithecus, suggesting that male canine reduction was initially more advanced in the behaviorally important upper canine. The available fossil evidence suggests a drastic size reduction of the male canine prior to Ar. ramidus and the earliest known members of the human clade, with little change in canine dimorphism levels thereafter. This evolutionary pattern indicates a profound behavioral shift associated with comparatively weak levels of male aggression early in human evolution, a pattern that was subsequently shared by Australopithecus and Homo.

A small canine tooth with little sexual dimorphism is a well-known hallmark of the human condition. The small and relatively nonprojecting deciduous canine of the first known fossil of Australopithecus, the Taung child skull, was a key feature used by Raymond Dart for his inference that the fossil represented an early stage of human evolution (1). However, recovery of additional Australopithecus fossils led to the canine of Australopithecus africanus to be characterized as large (compared to that of humans or “robust australopithecines”) and its morphology primitive, based on a projecting main cusp and crown structures lacking or hardly expressed in Homo (2). Later, the perception of a large and primitive canine was enhanced by the discovery and recognition of Australopithecus afarensis and Australopithecus anamensis (3–8), the latter species extending back in time to 4.2 million years ago (Ma). Although assessments of canine size variation and sexual dimorphism in Au. afarensis were hampered by limited sample sizes (9, 10), some suggested that the species had a more dimorphic canine than do humans, equivalent in degree to the bonobo (11) or to chimpanzees and orangutans (12). Initially, Au. anamensis was suggested to express greater canine dimorphism than did Au. afarensis (13, 14). However, based on a somewhat larger sample size, this is now considered to be the case with the tooth root but not necessarily its crown (15–17).Throughout the 1990s and 2000s, a pre-Australopithecus record of fossils spanning >6.0 to 4.4 Ma revealed that the canines of these earlier forms did not necessarily exceed those of Au. afarensis or Au. anamensis in general size (18–28). However, all these taxa apparently possessed canine crowns on average about 30% larger than in modern humans, which makes moderately high levels of sexual dimorphism potentially possible. Canine sexual dimorphism, combined with features such as body size dimorphism, inform sociobehavioral and ecological adaptations of past and present primates, and therefore have been of considerable interest since Darwin’s 1871 considerations (29–57). In particular, the relationship of canine size dimorphism (and/or male and female relative canine sizes) with reproductive strategies and aggression/competition levels in primate species have been a continued focus of interest (14, 33, 35–45, 49–56). Conspecific-directed agonistic behavior in primates related to mate and/or resource competition can be particularly intense among males both within and between groups (14, 44, 57). It is widely recognized that a large canine functions as a weapon in intra- and intergroup incidences of occasional lethal aggression (45, 58–61), and a large, tall canine has been shown or inferred to significantly enhance male fitness (50, 56). Hence, canine size and dimorphism levels in fossil species provide otherwise unavailable insights into their adaptive strategies.Here, we apply a recently developed method of estimating sexual size dimorphism from fossil assemblages of unknown sex compositions, the posterior density peak (pdPeak) method (62), and reexamine canine sexual dimorphism in Ardipithecus ramidus at ∼4.5 Ma. We include newly available fossils recovered from the Middle Awash and Gona paleoanthropological research areas in the Afar Rift, Ethiopia (26, 63, 64) in order to obtain the most reliable dimorphism estimates currently possible. We apply the same method to Australopithecus, Homo, and selected fossil apes, and evaluate canine sexual dimorphism through evolutionary time.We operationally define canine sexual dimorphism as the ratio between male and female means of basal canine crown diameters (the m/f ratio). Because the canines of Ar. ramidus, Au. anamensis, and extant and fossil apes are variably asymmetric in crown shape, we examine the maximum basal dimension of the crown. This can be either the mesiodistal crown diameter or a maximum diameter taken from the distolingual to mesiobuccal crown base (7, 27, 65). In the chronologically later Au. afarensis and all other species of Australopithecus sensu lato and Homo, we examine the more widely available conventional metric of buccolingual breadth, which corresponds to or approximates the maximum basal crown diameter. In anthropoid primates, canine height is more informative than basal canine diameter as a functional indicator of aggression and/or related display (14, 41–44). We therefore also examine available unworn and minimally worn fossil canines with reliable crown heights.     

Propagation of prions causing synucleinopathies in cultured cells

Increasingly, evidence argues that many neurodegenerative diseases, including progressive supranuclear palsy (PSP), are caused by prions, which are alternatively folded proteins undergoing self-propagation. In earlier studies, PSP prions were detected by infecting human embryonic kidney (HEK) cells expressing a tau fragment [TauRD(LM)] fused to yellow fluorescent protein (YFP). Here, we report on an improved bioassay using selective precipitation of tau prions from human PSP brain homogenates before infection of the HEK cells. Tau prions were measured by counting the number of cells with TauRD(LM)–YFP aggregates using confocal fluorescence microscopy. In parallel studies, we fused α-synuclein to YFP to bioassay α-synuclein prions in the brains of patients who died of multiple system atrophy (MSA). Previously, MSA prion detection required ∼120 d for transmission into transgenic mice, whereas our cultured cell assay needed only 4 d. Variation in MSA prion levels in four different brain regions from three patients provided evidence for three different MSA prion strains. Attempts to demonstrate α-synuclein prions in brain homogenates from Parkinson’s disease patients were unsuccessful, identifying an important biological difference between the two synucleinopathies. Partial purification of tau and α-synuclein prions facilitated measuring the levels of these protein pathogens in human brains. Our studies should facilitate investigations of the pathogenesis of both tau and α-synuclein prion disorders as well as help decipher the basic biology of those prions that attack the CNS.James Parkinson first described a progressive deterioration of the nervous system in 1817 and called it “shaking palsy” (1). Almost one century later, Friederich Heinrich Lewy described the neuropathological hallmark now known as Lewy bodies (LBs) (2). Progress toward discerning the etiology of Parkinson’s disease (PD) was achieved 85 years later when the first of several studies identified mutations in or multiplications of the gene encoding α-synuclein, SNCA, in inherited cases of PD (3–5). These studies were corroborated by immunostaining for α-synuclein in brain sections from PD patients (6) and subsequently from dementia with Lewy bodies (DLB) cases (7, 8), which found that LBs are surrounded by a halo of α-synuclein polymers.Along with point mutations in SNCA (3), and duplication and triplication of the gene (4, 5) as causes of inherited PD, meta-analysis of genome-wide association studies (9) have identified common variations in SNCA as a risk factor for sporadic PD cases. Combined, these data strongly support an etiological role for α-synuclein in the pathogenesis of both the inherited and sporadic forms of PD.In 1998, brain sections from cases classified as multiple system atrophy (MSA) were analyzed for α-synuclein. Although no LBs were found, abundant immunostaining in the cytoplasm of glial cells was identified (8, 10, 11). A decade earlier, these large immunopositive deposits of α-synuclein were called glial cytoplasmic inclusions (GCIs) based on silver staining (12); they are primarily found in oligodendrocytes but have been occasionally observed in astrocytes and neurons. Limited ultrastructural studies performed on GCIs suggest that they are collections of poorly organized bundles of α-synuclein fibrils (8).In addition to the accumulation of α-synuclein into LBs in PD and GCIs in MSA, depigmentation of the substantia nigra pars compacta is a hallmark of both PD and the majority of MSA cases (13). This loss of dopaminergic neurons results in diminished input to the basal ganglia that is reflected in the motor deficits exhibited by patients. In the 1990s, fetal tissue transplants into the substantia nigra of PD patients were performed in an attempt to counteract the effects of dopamine loss. Strikingly, upon autopsy of patients that survived at least 10 years posttransplant, LBs were found in the grafted fetal tissue. Because these grafts were no more than 16 years old, the findings argued for host-to-graft transmission of LBs (14, 15). The results of these transplant studies offered evidence supporting the hypothesis that PD is a prion disease, characterized by a misfolded protein that self-propagates and gives rise to progressive neurodegeneration (16, 17). Additional support for this hypothesis came from studies on the spread of α-synuclein deposits from the substantia nigra to other regions of the CNS in PD patients (18).Even more convincing support for α-synuclein prions came from animal studies demonstrating the transmissibility of an experimental synucleinopathy. The first report used transgenic (Tg) mice expressing human α-synuclein containing the A53T mutation found in familial PD; the mice were designated TgM83 (19). Homozygous mice (TgM83^+/+) were found to develop spontaneous motor deficits along with increased amounts of insoluble phosphorylated α-synuclein throughout the brain between 8–16 months of age. Ten years later, Mougenot et al. (20) intracerebrally inoculated brain homogenates from sick TgM83^+/+ mice into ∼2-months-old TgM83^+/+ mice and found a substantial reduction in the survival time with incubation periods of ∼130 days. Similar observations were reported from two other groups using either homozygous TgM83^+/+ (21) or hemizygous TgM83^+/− (22) mice.Although our initial attempts to transmit PD to TgM83^+/− mice failed (23), the transmission of MSA to the same mouse line was the first demonstration of α-synuclein prions in human brain (22). The TgM83^+/− mice, which differ from their homozygous counterparts by not developing spontaneous disease, exhibited progressive CNS dysfunction ∼120 days following intrathalamic inoculation of brain homogenates from two MSA patients. Inoculation of brain fractions enriched for LBs from PD patients into wild-type (WT) mice and macaque monkeys induced aberrant α-synuclein deposits, but neither species developed neurological disease (24). In a similar approach, inoculation of WT mice with the insoluble protein fraction isolated from DLB patients also induced phosphorylated α-synuclein pathology after 15 months, but it failed to induce neurological disease characteristic of DLB (25).Because α-synuclein prions from MSA patients were transmissible to TgM83^+/− mice, we asked whether a more rapid cell-based bioassay could be developed to characterize the MSA prions. With the cell bioassay for progressive supranuclear palsy (PSP) in mind (26, 27), we began by constructing WT and mutant α-synuclein cDNAs fused to yellow fluorescent protein (YFP) (28–30) and expressed these in human embryonic kidney (HEK) cells. By testing the cells with full-length recombinant mutant human α-syn140*A53T fibrils, we induced aggregate formation in HEK cells expressing WT and mutant human SNCA transgenes. To expand these findings beyond synthetic prions and to examine natural prions, we report here that phosphotungstic acid (PTA) (31) can be used to selectively precipitate α-synuclein from MSA patients. Screening PTA-precipitated brain homogenate with our cellular bioassay, we detected MSA prions in all six of the cases examined. By measuring the distribution of prions in the substantia nigra, basal ganglia, cerebellum, and temporal gyrus, we found evidence to suggest that at least three different strains of α-synuclein prions may give rise to MSA. We also found that after enrichment by PTA precipitation, ∼6 million α-synuclein molecules comprised an infectious unit of MSA prions in cell culture. Importantly, we transmitted neurodegenerative disease to TgM83^+/− mice using PTA-precipitated brain homogenate from an MSA patient, confirming that the aggregate isolation methods used successfully purify prions from patient samples.     

Age constraints on the dispersal of dinosaurs in the Late Triassic from magnetochronology of the Los Colorados Formation (Argentina)

A measured magnetozone sequence defined by 24 sampling sites with normal polarity and 28 sites with reverse polarity characteristic magnetizations was established for the heretofore poorly age-constrained Los Colorados Formation and its dinosaur-bearing vertebrate fauna in the Ischigualasto–Villa Union continental rift basin of Argentina. The polarity pattern in this ∼600-m-thick red-bed section can be correlated to Chrons E7r to E15n of the Newark astrochronological polarity time scale. This represents a time interval from 227 to 213 Ma, indicating that the Los Colorados Formation is predominantly Norian in age, ending more than 11 My before the onset of the Jurassic. The magnetochronology confirms that the underlying Ischigualasto Formation and its vertebrate assemblages including some of the earliest known dinosaurs are of Carnian age. The oldest dated occurrences of vertebrate assemblages with dinosaurs in North America (Chinle Formation) are younger (Norian), and thus the rise of dinosaurs was diachronous across the Americas. Paleogeography of the Ischigualasto and Los Colorados Formations indicates prolonged residence in the austral temperate humid belt where a provincial vertebrate fauna with early dinosaurs may have incubated. Faunal dispersal across the Pangean supercontinent in the development of more cosmopolitan vertebrate assemblages later in the Norian may have been in response to reduced contrasts between climate zones and lowered barriers resulting from decreasing atmospheric pCO₂ levels.The leading candidate for the oldest known occurrence of dinosaurs is the tetrapod assemblage of the Ischigualasto Formation of Argentina (1–4) where a preferred recalculated ⁴⁰Ar/³⁹Ar date of 231.4 ± 0.3 Ma (1σ analytical uncertainty as reported) (5) on the Herr Toba tuff from near the base of the formation points to a Carnian age for the dinosaur-bearing fauna. However, two recent studies of high-precision U-Pb zircon dates from the Chinle Formation in the American Southwest, practically the only other Late Triassic strata with radioisotopic age constraints on vertebrate assemblages, arrive at very different interpretations of the timing of the dispersal of dinosaurs depending on the accepted degree of total uncertainty for the Ischigualasto ⁴⁰Ar/³⁹Ar data.The study by Irmis et al. (6) advocated a diachronous rise of dinosaurs, starting in the Ischigualasto Formation of Argentina in the Southern Hemisphere and appearing only later in the Chinle Formation. They pointed to a new U-Pb zircon date of 211.9 ± 0.7 Ma (2σ uncertainty as conventionally reported for U-Pb dates) for a dinosaur-bearing vertebrate assemblage in Hayden Quarry at Ghost Ranch, New Mexico, that was considerably younger than the nominal dates from the Ischigualasto Formation. The Placerias Quarry in northeastern Arizona apparently contains even older dinosaurs from the Chinle Formation (7), and although it was not dated directly, Irmis et al. (6) suggested that a new U-Pb zircon date of 218.1 ± 0.7 Ma from presumably age-correlative strata in New Mexico would make even the Placerias assemblage much younger than the Ischigualastian fauna if the Herr Toba date is taken at face value.In contrast, Ramezani et al. (8) suggested that the rise of dinosaurs may have occurred at about the same time across the Americas. Their new U-Pb zircon dates for seven tuffaceous horizons in the Chinle Formation at Petrified Forest National Park, Arizona, indicated that the entire succession spans from ∼225.0 to 207.8 Ma (or younger), with the Adamanian–Revueltian faunal transition (9) between 219 and 213 Ma. More pertinently, their assessment of the full dating error envelope for the Ischigualasto Formation, including ⁴⁰Ar/³⁹Ar data in a thesis (10), suggested that an age of ∼218 Ma (or younger) cannot be excluded for its contact with the overlying Los Colorados Formation. Such a younger age would allow a closer temporal correspondence between the geographically separated assemblages, signifying that the Adamanian was effectively the age equivalent of the Ischigualastian (11) and thus that there was virtually parallel development of early dinosaurs across the Americas. Olsen et al. (12) also expressed doubts about the reliability of the dating of the Ischigualastian vertebrate assemblages that would necessarily make them of Carnian age.There are only two dated levels to formally constrain the numerical age of the Late Triassic epoch: a 230.1 ± 0.06 Ma U-Pb zircon date on a volcanic ash in late Carnian marine strata from southern Italy (13) and an age of 201.3 ± 0.18 Ma calculated for the Triassic–Jurassic boundary from U-Pb zircon dates on volcanic ashes bracketing the boundary in ammonite-bearing sediments from Peru (14). In the current absence of other reliable radioisotopic age controls on fossiliferous marine strata that are the basis for a global chronostratigraphy, correlations to the Newark astrochronological polarity time scale [APTS (15)] have provided important constraints on ages for standard subdivisions of the Late Triassic (16–18), which have largely been adopted in recently published geologic time scales (19, 20). High-precision U-Pb geochronology on earliest Jurassic volcanics of the Central Atlantic Magmatic Province (CAMP) interbedded with sediments in the upper part of the Newark continental rift sequence strongly affirms the astrochronological methodology (21).Over the entire ∼35-My-long Late Triassic epoch, there are only four land vertebrate biozones recognized in North America and just two in South America (11). The low temporal resolution combined with endemism of faunas for the Late Triassic make it difficult to disentangle temporal and spatial components governing the distribution of dispersed vertebrate assemblages and for progress requires age control aside from biostratigraphy. In this regard, dating of the Los Colorados Formation would help determine if the underlying Ischigualasto Formation extends into the Norian or is confined to the Carnian and if the temporal range of the dinosaur-bearing Coloradian fauna actually extends to the end of the Triassic as sometimes supposed (e.g., 11). The apparent absence of volcanic ash layers suitable for radioisotopic dating in the Los Colorados Formation motivated this magnetostratigraphic study of the unit and enabled us to address these objectives.     

Atmospheric Ar and Ne returned from mantle depths to the Earth’s surface by forearc recycling

In subduction zones, sediments, hydrothermally altered lithosphere, fluids, and atmospheric gases are transported into the mantle, where ultrahigh-pressure (UHP) metamorphism takes place. However, the extent to which atmospheric noble gases are trapped in minerals crystallized during UHP metamorphism is unknown. We measured Ar and Ne trapped in phengite and omphacite from the youngest known UHP terrane on Earth to determine the composition of Ar and Ne returned from mantle depths to the surface by forearc recycling. An ⁴⁰Ar/³⁹Ar age [7.93 ± 0.10 My (1σ)] for phengite is interpreted as the timing of crystallization at mantle depths and indicates that ⁴⁰Ar/³⁹Ar phengite ages reliably record the timing of UHP metamorphism. Both phengite and omphacite yielded atmospheric ³⁸Ar/³⁶Ar and ²⁰Ne/²²Ne. Our study provides the first documentation, to our knowledge, of entrapment of atmospheric Ar and Ne in phengite and omphacite. Results indicate that a subduction barrier for atmospheric-derived noble gases does not exist at mantle depths associated with UHP metamorphism. We show that the crystallization age together with the isotopic composition of nonradiogenic noble gases trapped in minerals formed during subsolidus crystallization at mantle depths can be used to unambiguously assess forearc recycling of atmospheric noble gases. The flux of atmospheric noble gas entering the deep Earth through subduction and returning to the surface cannot be fully realized until the abundances of atmospheric noble gases trapped in exhumed UHP rocks are known.It has long been known that water and CO₂ can be transported into the deep Earth by subduction of sediments and hydrothermally altered oceanic crust (1–3). Water is carried from the surface into the upper mantle by hydrous minerals in the uppermost 10–12 km subducting lithosphere to depths of at least 400 km (4). However, to what extent are atmospheric noble gases transported into the deep Earth and returned to the surface in the forearcs of subduction zones? The isotopic compositions of Ar and Ne can be used as tracers of atmospheric recycling in subduction zones (5), because they are chemically inert at conditions relevant to processes on Earth (6). Serpentinite subduction has been proposed as a viable mechanism for high abundances of noble gas to be transported into the mantle (7), and hydration of oceanic lithosphere has been proposed as a mechanism to release argon into the atmosphere (8). To determine the flux of atmospheric noble gases recycled into the mantle in subduction zones requires knowing when, at what depth, and how atmospheric Ar and Ne are trapped in minerals crystallized at mantle depths and subsequently, exhumed to the surface. This flux calculation requires interpreting noble gas concentrations in minerals with respect to their pressure–temperature–time–deformation (P-T-t-D) histories.Studies that have addressed recycling of atmospheric noble gases in the Earth’s mantle have largely focused on volcanic rocks in arcs, backarcs, ocean island basalts, and midocean ridge basalts (MORBs) (5). However, atmospheric-derived noble gases have been shown to contaminate mantle noble gas signatures in volcanic rocks, irrespective of eruption setting (underwater, under ice, or in atmosphere) (9). Indeed, ³⁸Ar/³⁶Ar values for mantle-derived volcanic rocks generally have been found to be indistinguishable from atmospheric ratios (10, 11). The possibility of atmospheric contamination during eruption or interaction with meteoric fluids and seawater exists for any mineral/rock formed, or altered, at or near the Earth’s surface.Noble gas studies of peridotites (12) and serpentinized peridotites (13) have also argued for recycling of atmospheric noble gases into Earth’s mantle; however, linking the conditions of noble gas entrapment to specific parts of P-T-t-D histories in these lithologies is challenging. Peridotites are readily altered on the seafloor at temperatures <500 °C, where olivine and orthopyroxene react to form serpentine minerals. Serpentinite forms in many tectonic settings (14) and is stable over a wide range of pressure–temperature conditions (2). High-pressure serpentinites have been used to investigate noble gas and halogen compositions interpreted to have been trapped during subseafloor alteration and subsequent dehydration metamorphism (7). Noble gas and halogen studies of the Higashi-akaishi peridotite body of the Sanbagawa metamorphic terrane argue for the subduction and survival of marine pore fluid to depths of at least 100 km (15). The prograde pressure–temperature–deformation path of the peridotite is not well-constrained, and it is unclear whether early deformation is related to subduction (16). Therefore, several interpretations are possible to explain when and how entrapment of noble gases occurred (e.g., during prograde metamorphism, peak metamorphism, exhumation, or obduction).This study investigates noble gases, specifically Ar and Ne, trapped in minerals that formed during ultrahigh-pressure (UHP) metamorphism. Exhumed UHP rocks have been shown to preserve a record of the geochemical and fluid transport of material from mantle depths to the Earth’s surface (17–19). The forearc subduction channel provides a pathway for “input” lithologies (continental and oceanic crust and sediments) to be recycled, because they are metamorphosed at mantle depths where diagnostic assemblages (e.g., coesite and diamond) can crystallize and trap noble gases during UHP metamorphism. When UHP rocks are subsequently returned (exhumed) to the surface, their mineral assemblages provide clues to the changing conditions along their P-T-t-D paths. Thus, noble gases trapped in material entering (in crust and sediments) and exiting (in high-pressure and UHP metamorphic rocks) forearcs can provide insight into the recycling of atmospheric noble gases within the subduction channel. Previous studies that have investigated noble gases in exhumed high-pressure and UHP rocks focused on (i) ⁴⁰Ar/³⁹Ar age determination on irradiated K-bearing minerals (20), (ii) He and Ne isotopic studies aimed at documenting noble gas compositions trapped during diamond formation (21), and (iii) noble gas and halogen studies of serpentinite and peridotite (7, 12, 15). Here, we investigate whether atmospheric Ar and Ne are trapped in minerals during crystallization at mantle depths during subduction zone metamorphism. We present Ar and Ne isotopic data for omphacite and phengite from Late Miocene coesite eclogite that document both the ⁴⁰Ar/³⁹Ar age of phengite crystallization and the isotopic composition of Ar and Ne trapped in these minerals during UHP metamorphism.     

Ancient DNA at the edge of the world: Continental immigration and the persistence of Neolithic male lineages in Bronze Age Orkney

Orkney was a major cultural center during the Neolithic, 3800 to 2500 BC. Farming flourished, permanent stone settlements and chambered tombs were constructed, and long-range contacts were sustained. From ∼3200 BC, the number, density, and extravagance of settlements increased, and new ceremonial monuments and ceramic styles, possibly originating in Orkney, spread across Britain and Ireland. By ∼2800 BC, this phenomenon was waning, although Neolithic traditions persisted to at least 2500 BC. Unlike elsewhere in Britain, there is little material evidence to suggest a Beaker presence, suggesting that Orkney may have developed along an insular trajectory during the second millennium BC. We tested this by comparing new genomic evidence from 22 Bronze Age and 3 Iron Age burials in northwest Orkney with Neolithic burials from across the archipelago. We identified signals of inward migration on a scale unsuspected from the archaeological record: As elsewhere in Bronze Age Britain, much of the population displayed significant genome-wide ancestry deriving ultimately from the Pontic-Caspian Steppe. However, uniquely in northern and central Europe, most of the male lineages were inherited from the local Neolithic. This suggests that some male descendants of Neolithic Orkney may have remained distinct well into the Bronze Age, although there are signs that this had dwindled by the Iron Age. Furthermore, although the majority of mitochondrial DNA lineages evidently arrived afresh with the Bronze Age, we also find evidence for continuity in the female line of descent from Mesolithic Britain into the Bronze Age and even to the present day.

Benefiting from the tail end of the Holocene climatic optimum, the British Early Neolithic spread rapidly through Britain and Ireland from the south over 300 to 400 y from ∼4050 BC (1–3). The settlers brought with them domesticated wheat, barley, sheep, and cattle, as well as knowledge of carinated bowl ceramics and causewayed enclosures (1–5), pointing to a likely source in northern France or Belgium.The Orkney Islands, lying to the north of the Scottish mainland, flourished during the Neolithic (3800 to 2500 BC), becoming a major cultural center (6–9). Underpinned by a successful farming economy and long-range contacts, the earliest permanent settlements were constructed in wood, followed by stone-built dwellings from 3300 cal. (calibrated) BC onward (9, 10). The use of stone appears to have been a conscious design choice (9, 11, 12) and has resulted in an extraordinary level of archaeological preservation.While recent genome-wide studies (13) have demonstrated the extent and tempo of continental migration into Britain during the Beaker period, after 2500 BC, there has so far been little or no recognition of the archaeological implications of this for Orkney. The paucity of Beakers and associated material culture in the archaeological record has been taken as an indication that the cultural and population shifts occurring elsewhere in Britain at this time had little direct impact in Orkney (8, 14–18) and indeed may have been locally resisted (6). As a result, Orkney has been seen to have developed along a largely insular trajectory during the second millennium BC.Significant changes in funerary practice did begin to emerge at this time, and research has concentrated on funerary remains. Barrow cemeteries, some of the largest in northern Britain, appeared in Orkney around the end of the third millennium BC. These earthen mounds contained multiple burials, added sequentially and most frequently comprising cremated remains in pits or stone-lined cists (18). Flat cist cemeteries were also in use for both inhumation and cremation burials, and often graves contained the remains of several individuals, but grave goods were infrequent.Until recently, the low visibility of settlement sites had led to the idea that this was a period of environmental and cultural recession (19). The balance has begun to be redressed through focused environmental analyses (20) and reports on settlements such as at Crossiecrown (9) and Tofts Ness (21). Opportunities to correlate settlement and funerary remains are very rare, and few sites extend across the Neolithic and Bronze Age (BA) periods, making it difficult to draw a coherent picture of change over time. In this respect, the ongoing investigations at the Links of Noltland (LoN) are providing valuable new insights.The LoN is located on Westray, the northwesterly most island of the Orkney archipelago. The exceptional conditions have preserved extensive settlement and cemetery remains dating from at least 3300 cal. BC up to about 500 BC (22–25). While no direct overlap has yet been detected between Neolithic and BA phases of settlement, there is no evidence for a major hiatus in occupation. The BA settlement, distinguished on architectural grounds and dating from ∼2500 to 1200 cal. BC, includes three separate conglomerations of domestic and ancillary buildings, which, like their Neolithic counterparts, were spread across a contemporary farmed landscape. Built from a mix of stone and earthen banks, often arranged in pairs, they were in use until at least 1200 cal. BC. A cemetery located among these settlements, used between at least 2150 BC and 850 BC, comprised >50 burials, including >100 individuals. Both cremation and inhumation were practiced, at times contemporaneously, and multiple burials within a single grave were common. Material evidence of the “Beaker complex,” seen across mainland Britain, is scant in Orkney; a few sherds from two Beaker vessels were recovered from the wider area (19), dated to ∼2265 to1975 cal. BC, but no further pottery or recognizable artifacts have been found in association with the cemetery or settlement.The study of ancient genomes has shown that across much of Europe, including mainland Britain, the arrival of Metal Age culture was accompanied by the introduction of new ancestry from the Pontic-Caspian Steppe and a predominance of Y-chromosomal haplogroup R1b-M269 (13, 26–31). We investigated genomic variation in the Orkney archipelago within the context of this framework. Genome-wide SNP (single–nucleotide polymorphism) capture and shotgun data were available from 21 Early Neolithic Orcadians (13, 32), but only one from the BA (13). To investigate BA Orkney, we generated whole-genome shotgun sequence data from 22 samples from the LoN cemetery and analyzed them alongside these published data. We also included new data from three Iron Age (IA) samples from the multiperiod ritual complex and cemetery site of Knowe of Skea (KoS), on the west coast of Westray, and 12 further prehistoric samples from Scotland and northern England.     

Redox potential of the terminal quinone electron acceptor QB in photosystem II reveals the mechanism of electron transfer regulation

Photosystem II (PSII) extracts electrons from water at a Mn₄CaO₅ cluster using light energy and then transfers them to two plastoquinones, the primary quinone electron acceptor Q_A and the secondary quinone electron acceptor Q_B. This forward electron transfer is an essential process in light energy conversion. Meanwhile, backward electron transfer is also significant in photoprotection of PSII proteins. Modulation of the redox potential (E_m) gap of Q_A and Q_B mainly regulates the forward and backward electron transfers in PSII. However, the full scheme of electron transfer regulation remains unresolved due to the unknown E_m value of Q_B. Here, for the first time (to our knowledge), the E_m value of Q_B reduction was measured directly using spectroelectrochemistry in combination with light-induced Fourier transform infrared difference spectroscopy. The E_m(Q_B⁻/Q_B) was determined to be approximately +90 mV and was virtually unaffected by depletion of the Mn₄CaO₅ cluster. This insensitivity of E_m(Q_B⁻/Q_B), in combination with the known large upshift of E_m(Q_A⁻/Q_A), explains the mechanism of PSII photoprotection with an impaired Mn₄CaO₅ cluster, in which a large decrease in the E_m gap between Q_A and Q_B promotes rapid charge recombination via Q_A⁻.In oxygenic photosynthesis in plants and cyanobacteria, photosystem II (PSII) has an important function in light-driven water oxidation, a process that leads to the generation of electrons and protons for CO₂ reduction and ATP synthesis, respectively (1–3). Photosynthetic water oxidation also produces molecular oxygen as a byproduct, which is the source of atmospheric oxygen and sustains virtually all life on Earth. PSII reactions are initiated by light-induced charge separation between a chlorophyll (Chl) dimer (P680) and a pheophytin (Pheo) electron acceptor, leading to the formation of a P680⁺Pheo⁻ radical pair (4, 5). An electron hole on P680⁺ is transferred to a Mn₄CaO₅ cluster, the catalytic center of water oxidation, via the redox-active tyrosine, Y_Z (D1-Tyr161). At the Mn₄CaO₅ cluster, water oxidation proceeds through a cycle of five intermediates denoted S_n states (n = 0–4) (6, 7). On the electron acceptor side, the electron is transferred from Pheo⁻ to the primary quinone electron acceptor Q_A and then to the secondary quinone electron acceptor Q_B (8, 9). Q_A and Q_B have many similarities: they consist of plastoquinone (PQ), are located symmetrically around a nonheme iron center, and interact with D2 and D1 proteins, respectively, in a similar manner (Fig. 1) (10, 11). However, they play significantly different roles in PSII (8, 9). Q_A is only singly reduced to transfer an electron to Q_B, whereas Q_B accepts one or two electrons. When Q_B is doubly reduced, the resultant Q_B²⁻ takes up two protons to form plastoquinol (PQH₂), which is then released into thylakoid membranes. Differences between Q_A and Q_B could be caused by differences in the molecular interactions of PQ with surrounding proteins in Q_A and Q_B pockets, although the detailed mechanism remains to be clarified (12, 13).Open in a separate windowFig. 1.Redox cofactors in PSII and the electron transfer pathway (blue arrows). For the PSII structure, the X-ray crystallographic structure at 1.9-Å resolution (Protein Data Bank ID code 3ARC) (9) was used. The electron acceptor side is expanded, showing the arrangements of Q_A, Q_B, and the nonheme iron with their molecular interactions. Nearby carboxylic groups are also shown.Electron transfer reactions in PSII are highly regulated by the spatial localization of redox components and their redox potentials (E_m values). Both forward and backward electron transfers are important; backward electron transfers control charge recombination in PSII, and this serves as photoprotection for PSII proteins (5, 14–17). PSII involves specific mechanisms to regulate forward and backward electron transfer reactions in response to environmental changes. For instance, in strong light, some species of cyanobacteria increase the E_m of Pheo to facilitate charge recombination. Specifically, they exchange D1 subunits originating from different psbA genes to change the hydrogen bond interactions of Pheo (16–20). On the other hand, it was found that impairment of the Mn₄CaO₅ cluster led to a significant increase in the E_m of Q_A by ∼150 mV (21–27). This potential increase was thought to inhibit forward electron transfer to Q_B to promote direct relaxation of Q_A⁻ without forming triplet-state Chl, a precursor of harmful singlet oxygen (2, 5, 14, 15, 17, 23). In addition, charge recombination of Q_A⁻ with P680⁺ prevents oxidative damage by high-potential P680⁺ (5). However, the full mechanism of photoprotection by the regulation of the quinone electron acceptor E_m values remains to be resolved, because the E_m of Q_B has not been determined conclusively, and the effect of Mn₄CaO₅ cluster inactivation on it has not been examined (5).Although the E_m of the single reduction of Q_B has been estimated to be ∼80 mV higher than that of Q_A from kinetic and thermodynamic data (28–32), so far no reports have measured the E_m of Q_B directly. In contrast, the E_m of Q_A was measured extensively using chemical or electrochemical titrations and determined to be approximately −100 mV for oxygen-evolving PSII (21–27). The main reason for this difference is due to the fact that the Q_A reaction can be monitored readily by fluorescence measurement in that an increase in fluorescence indicates Q_A⁻ formation (8, 33–35). However, the fluorescence method cannot be used easily to monitor Q_B reduction. Although UV-Vis absorption and electron spin resonance have also been used to monitor Q_A⁻ in redox titration (summarized in ref. 22), so far these methods have not been used to monitor the titration of Q_B, likely because Q_A⁻ and Q_B⁻ give similar signals (36–39). Another spectroscopic method that can be used to monitor Q_A and Q_B reactions is Fourier transform infrared (FTIR) difference spectroscopy, which detects reaction-induced changes in the molecular vibrations of a cofactor and its environment in proteins (40–45). It was previously shown that comparison of FTIR difference spectra upon Q_A⁻ and Q_B⁻ formation showed some characteristic differences in spectral features (46). In particular, bands at 1,721 and 1,745 cm⁻¹, which were assigned to ester C=O vibrations of nearby Pheo molecules affected by the reduction of Q_A and Q_B, respectively, were suggested to be good markers for discriminating between Q_A and Q_B reactions (46).In this study, we directly measured the E_m of Q_B in PSII using spectroelectrochemistry and light-induced FTIR difference spectroscopy. The effect of Mn₄CaO₅ cluster depletion on the E_m value was also examined. Spectroelectrochemistry has been used to accurately measure the E_m values of cofactors in various redox proteins (47, 48) including redox cofactors in PSII (24, 25, 49, 50). FTIR spectroelectrochemistry, which has the additional merit of being able to provide structural information, has also been used to investigate redox reactions of biomolecules and proteins (48, 50–54). This method was recently applied to the nonheme iron center of PSII to examine the effect of Mn depletion on the E_m value and obtain structural information around the nonheme iron (50). The results in our study showed that the E_m of the first reduction of Q_B [E_m(Q_B⁻/Q_B)] was much higher than previously estimated, and the E_m of the second reduction [E_m(PQH₂/Q_B⁻)] was higher than the first reduction. Furthermore, we showed that Mn depletion hardly affected the E_m values of Q_B, in contrast to the large change in the E_m of Q_A (21–27). With these results, the mechanism of photoprotection of PSII when the Mn₄CaO₅ cluster is inactivated is now clearly explained.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Synergistic regulation of nonbinary molecular switches by protonation and light

We report a molecular switching ensemble whose states may be regulated in synergistic fashion by both protonation and photoirradiation. This allows hierarchical control in both a kinetic and thermodynamic sense. These pseudorotaxane-based molecular devices exploit the so-called Texas-sized molecular box (cyclo[2]-(2,6-di(1H-imidazol-1-yl)pyridine)[2](1,4-dimethylenebenzene); 1⁴⁺, studied as its tetrakis-PF₆ ⁻ salt) as the wheel component. Anions of azobenzene-4,4′-dicarboxylic acid (2H⁺•2) or 4,4′-stilbenedicarboxylic acid (2H⁺•3) serve as the threading rod elements. The various forms of 2 and 3 (neutral, monoprotonated, and diprotonated) interact differently with 1⁴⁺, as do the photoinduced cis or trans forms of these classic photoactive guests. The net result is a multimodal molecular switch that can be regulated in synergistic fashion through protonation/deprotonation and photoirradiation. The degree of guest protonation is the dominating control factor, with light acting as a secondary regulatory stimulus. The present dual input strategy provides a complement to more traditional orthogonal stimulus-based approaches to molecular switching and allows for the creation of nonbinary stimulus-responsive functional materials.

Multifactor regulation of biomolecular machines is essential to their ability to carry out various biological functions (1 –11). Construction of artificial molecular devices with multifactor regulation features may allow us to understand and simulate biological systems more effectively (12 –31). However, creating and controlling such synthetic constructs remains challenging (16, 32 –37). Most known systems involving multifactor regulation, including most so-called molecular switches and logic devices (38 –43), have been predicated on an orthogonal strategy wherein the different control factors that determine the distribution of accessible states do not affect one another (20, 44 –56). However, in principle, a greater level of control can be achieved by using two separate regulatory inputs that operate in synergistic fashion. Ideally, this could lead to hierarchical control where different states are specifically accessed by means of appropriately selected nonorthogonal inputs. However, to our knowledge, only a limited number of reports detailing controlled hierarchical systems have appeared (57). Furthermore, the balance between specific effects (e.g., kinetics vs. thermodynamics) under conditions of stimulus regulation is still far from fully understood (54). There is thus a need for new systems that can provide further insights into the underlying design determinants. Here we report a set of pseudorotaxane molecular shuttles that act as multimodal chemical switches subject to hierarchical control.     

Coulomb interaction,phonons, and superconductivity in twisted bilayer graphene

The polarizability of twisted bilayer graphene, due to the combined effect of electron–hole pairs, plasmons, and acoustic phonons, is analyzed. The screened Coulomb interaction allows for the formation of Cooper pairs and superconductivity in a significant range of twist angles and fillings. The tendency toward superconductivity is enhanced by the coupling between longitudinal phonons and electron–hole pairs. Scattering processes involving large momentum transfers, Umklapp processes, play a crucial role in the formation of Cooper pairs. The magnitude of the superconducting gap changes among the different pockets of the Fermi surface.

Twisted bilayer graphene (TBG) shows a complex phase diagram which combines superconducting and insulating phases (1, 2) and resembles strongly correlated materials previously encountered in condensed matter physics (3–6). On the other hand, superconductivity seems more prevalent in TBG (7–11), while in other strongly correlated materials magnetic phases are dominant.The pairing interaction responsible for superconductivity in TBG has been intensively studied. Among other possible pairing mechanisms, the effect of phonons (12–19) (see also ref. 20), the proximity of the chemical potential to a van Hove singularity in the density of states (DOS) (21–25) and excitations of insulating phases (26–28) (see also refs. 29–31), and the role of electronic screening (32–35) have been considered.In the following, we analyze how the screened Coulomb interaction induces pairing in TBG. The calculation is based on the Kohn–Luttinger formalism (36) for the study of anisotropic superconductivity via repulsive interactions. The screening includes electron–hole pairs (37), plasmons (38), and phonons (note that acoustic phonons overlap with the electron–hole continuum in TBG). Our results show that the repulsive Coulomb interaction, screened by plasmons and electron–hole pairs only, leads to anisotropic superconductivity, although with critical temperatures of order T_c ∼ 10⁻³ to 10⁻² K. The inclusion of phonons in the screening function substantially enhances the critical temperature, to T_c ∼ 1 to 10 K.     

Microscopic origins of the crystallographically preferred growth in evaporation-induced colloidal crystals

Unlike crystalline atomic and ionic solids, texture development due to crystallographically preferred growth in colloidal crystals is less studied. Here we investigate the underlying mechanisms of the texture evolution in an evaporation-induced colloidal assembly process through experiments, modeling, and theoretical analysis. In this widely used approach to obtain large-area colloidal crystals, the colloidal particles are driven to the meniscus via the evaporation of a solvent or matrix precursor solution where they close-pack to form a face-centered cubic colloidal assembly. Via two-dimensional large-area crystallographic mapping, we show that the initial crystal orientation is dominated by the interaction of particles with the meniscus, resulting in the expected coalignment of the close-packed direction with the local meniscus geometry. By combining with crystal structure analysis at a single-particle level, we further reveal that, at the later stage of self-assembly, however, the colloidal crystal undergoes a gradual rotation facilitated by geometrically necessary dislocations (GNDs) and achieves a large-area uniform crystallographic orientation with the close-packed direction perpendicular to the meniscus and parallel to the growth direction. Classical slip analysis, finite element-based mechanical simulation, computational colloidal assembly modeling, and continuum theory unequivocally show that these GNDs result from the tensile stress field along the meniscus direction due to the constrained shrinkage of the colloidal crystal during drying. The generation of GNDs with specific slip systems within individual grains leads to crystallographic rotation to accommodate the mechanical stress. The mechanistic understanding reported here can be utilized to control crystallographic features of colloidal assemblies, and may provide further insights into crystallographically preferred growth in synthetic, biological, and geological crystals.

As an analogy to atomic crystals, colloidal crystals are highly ordered structures formed by colloidal particles with sizes ranging from 100 nm to several micrometers (1–6). In addition to engineering applications such as photonics, sensing, and catalysis (4, 5, 7, 8), colloidal crystals have also been used as model systems to study some fundamental processes in statistical mechanics and mechanical behavior of crystalline solids (9–14). Depending on the nature of interparticle interactions, many equilibrium and nonequilibrium colloidal self-assembly processes have been explored and developed (1, 4). Among them, the evaporation-induced colloidal self-assembly presents a number of advantages, such as large-size fabrication, versatility, and cost and time efficiency (3–5, 15–18). In a typical synthesis where a substrate is immersed vertically or at an angle into a colloidal suspension, the colloidal particles are driven to the meniscus by the evaporation-induced fluid flow and subsequently self-assemble to form a colloidal crystal with the face-centered cubic (fcc) lattice structure and the close-packed {111} plane parallel to the substrate (2, 3, 19–23) (see Fig. 1A for a schematic diagram of the synthetic setup).Open in a separate windowFig. 1.Evaporation-induced coassembly of colloidal crystals. (A) Schematic diagram of the evaporation-induced colloidal coassembly process. “G”, “M”, and “N” refer to “growth,” “meniscus,” and “normal” directions, respectively. The reaction solution contains silica matrix precursor (tetraethyl orthosilicate, TEOS) in addition to colloids. (B) Schematic diagram of the crystallographic system and orientations used in this work. (C and D) Optical image (Top Left) and scanning electron micrograph (SEM) (Bottom Left) of a typical large-area colloidal crystal film before (C) and after (D) calcination. (Right) SEM images of select areas (yellow rectangles) at different magnifications. Corresponding fast-Fourier transform (see Inset in Middle in C) shows the single-crystalline nature of the assembled structure. (E) The 3D reconstruction of the colloidal crystal (left) based on FIB tomography data and (right) after particle detection. (F) Top-view SEM image of the colloidal crystal with crystallographic orientations indicated.While previous research has focused on utilizing the assembled colloidal structures for different applications (4, 5, 7, 8), considerably less effort is directed to understand the self-assembly mechanism itself in this process (17, 24). In particular, despite using the term “colloidal crystals” to highlight the microstructures’ long-range order, an analogy to atomic crystals, little is known regarding the crystallographic evolution of colloidal crystals in relation to the self-assembly process (3, 22, 25). The underlying mechanisms for the puzzling—yet commonly observed—phenomenon of the preferred growth along the close-packed <110> direction in evaporation-induced colloidal crystals are currently not understood (3, 25–29). The <110> growth direction has been observed in a number of processes with a variety of particle chemistries, evaporation rates, and matrix materials (3, 25–28, 30), hinting at a universal underlying mechanism. This behavior is particularly intriguing as the colloidal particles are expected to close-pack parallel to the meniscus, which should lead to the growth along the <112> direction and perpendicular to the <110> direction (16, 26, 31)^*.Preferred growth along specific crystallographic orientations, also known as texture development, is commonly observed in crystalline atomic solids in synthetic systems, biominerals, and geological crystals. While current knowledge recognizes mechanisms such as the oriented nucleation that defines the future crystallographic orientation of the growing crystals and competitive growth in atomic crystals (32–34), the underlying principles for texture development in colloidal crystals remain elusive. Previous hypotheses based on orientation-dependent growth speed and solvent flow resistance are inadequate to provide a universal explanation for different evaporation-induced colloidal self-assembly processes (3, 25–29). A better understanding of the crystallographically preferred growth in colloidal self-assembly processes may shed new light on the crystal growth in atomic, ionic, and molecular systems (35–37). Moreover, mechanistic understanding of the self-assembly processes will allow more precise control of the lattice types, crystallography, and defects to improve the performance and functionality of colloidal assembly structures (38–40).     

Protons are a neurotransmitter that regulates synaptic plasticity in the lateral amygdala

Stimulating presynaptic terminals can increase the proton concentration in synapses. Potential receptors for protons are acid-sensing ion channels (ASICs), Na⁺- and Ca²⁺-permeable channels that are activated by extracellular acidosis. Those observations suggest that protons might be a neurotransmitter. We found that presynaptic stimulation transiently reduced extracellular pH in the amygdala. The protons activated ASICs in lateral amygdala pyramidal neurons, generating excitatory postsynaptic currents. Moreover, both protons and ASICs were required for synaptic plasticity in lateral amygdala neurons. The results identify protons as a neurotransmitter, and they establish ASICs as the postsynaptic receptor. They also indicate that protons and ASICs are a neurotransmitter/receptor pair critical for amygdala-dependent learning and memory.Although homeostatic mechanisms generally maintain the brain’s extracellular pH within narrow limits, neural activity can induce transient and localized pH fluctuations. For example, acidification may occur when synaptic vesicles, which have a pH of ∼5.2–5.7 (1–3), release their contents into the synapse. Studies of mammalian cone photoreceptors showed that synaptic vesicle exocytosis rapidly reduced synaptic cleft pH by an estimated 0.2–0.6 units (4–6). Transient synaptic cleft acidification also occurred with GABAergic transmission (7). Some, but not all, studies also reported that high-frequency stimulation (HFS) transiently acidified hippocampal brain slices, likely as a result of the release of synaptic vesicle contents (8, 9). Neurotransmission also induces a slower, more prolonged alkalinization (10, 11). In addition to release of synaptic vesicle protons, neuronal and glial H⁺ and HCO₃⁻ transporters, channels, H⁺-ATPases, and metabolism might influence extracellular pH (10–12).ASICs are potential targets of reduced extracellular pH. ASICs are Na⁺-permeable and, to a lesser extent, Ca²⁺-permeable channels that are activated by extracellular acidosis (13–19). In the brain, ASICs consist of homotrimeric and heterotrimeric complexes of ASIC1a, ASIC2a, and ASIC2b. The ASIC1a subunit is required for acid-activation in the physiological range (>pH 5.0) (20, 21). Several observations indicate that ASIC are located postsynaptically. ASICs are located on dendritic spines. Although similar to glutamate receptors, they are also present on dendrites and cell bodies (20, 22–24). ASIC subunits interact with postsynaptic scaffolding proteins, including postsynaptic density protein 95 and protein interacting with C-kinase-1 (20, 24–29). In addition, ASICs are enriched in synaptosome-containing brain fractions (20, 24, 30).Although these observations raised the possibility that protons might be a neurotransmitter, postsynaptic ASIC currents have not been detected in cultured hippocampal neurons (31, 32), and whether localized pH transients might play a signaling role in neuronal communication remains unclear. In previous studies of hippocampal brain slices, extracellular field potential recordings suggested impaired hippocampal long-term potentiation (LTP) in ASIC1a^−/− mice (20), although another study did not detect an effect of ASIC1a (33). Another study using microisland cultures of hippocampal neurons suggested that the probability of neurotransmitter release increased in ASIC1a^−/− mice (32).Here, we tested the hypothesis that protons are a neurotransmitter and that ASICs are the receptor. Criteria to identify substances as neurotransmitters have been proposed (34). Beg and colleagues (35) used these criteria to conclude that protons are a transmitter released from Caenorhabditis elegans intestine to cause muscle contraction. Key questions about whether protons meet criteria for a neurotransmitter are: Does presynaptic stimulation increase the extracellular proton concentration? Do protons activate currents in postsynaptic cells? Can exogenously applied protons reproduce effects of endogenous protons? What is the postsynaptic proton receptor? We studied lateral amygdala brain slices because amygdala-dependent fear-related behavior depends on a pH reduction (36). In addition, ASICs are abundantly expressed there, and ASIC1a^−/− mice have impaired fear-like behavior (36–38).