From the Cover: Inhaled oxytocin increases positive social behaviors in newborn macaques

Early caregiver–infant interactions are critical for infants’ socioemotional and cognitive development. Several hormones and neuromodulators, including oxytocin, affect these interactions. Exogenous oxytocin promotes social behaviors in several species, including human and nonhuman primates. Although exogenous oxytocin increases social function in adults—including expression recognition and affiliation—it is unknown whether oxytocin can increase social interactions in infants. We hypothesized that nebulized oxytocin would increase affiliative social behaviors and such effects would be modulated by infants’ social skills, measured earlier in development. We also hypothesized that oxytocin’s effects on social behaviors may be due to its anxiolytic effects. We tested these hypotheses in a blind study by nebulizing 7- to 14-d-old macaques (n = 28) with oxytocin or saline. Following oxytocin administration, infants’ facial gesturing at a human caregiver increased, and infants’ salivary oxytocin was positively correlated with the time spent in close proximity to a caregiver. Infants’ imitative skill (measured earlier in development: 1–7 d of age) predicted oxytocin-associated increases in affiliative behaviors—lip smacking, visual attention to a caregiver, and time in close proximity to a caregiver—suggesting that infants with higher propensities for positive social interactions are more sensitive to exogenous oxytocin. Oxytocin also decreased salivary cortisol, but not stress-related behaviors (e.g., scratching), suggesting the possibility of some anxiolytic effects. To our knowledge, this study provides the first evidence that oxytocin increases positive social behaviors in newborns. This information is of critical importance for potential interventions aimed at ameliorating inadequate social behaviors in infants with higher likelihood of developing neurodevelopmental disorder.Oxytocin is a neuropeptide that has wide-ranging effects on social behaviors and social perception, including increased emotion recognition and prosocial behavior (1, 2). Animal studies present convergent evidence of oxytocin’s positive effects on social behavior (2–6), including humans (1, 7). In recent years, an increasing literature on human and nonhuman primates suggests an association between oxytocin levels—either endogenous or exogenously administered—and prosocial behaviors (8–10). In both humans and macaques, exogenous oxytocin appears to enhance social attention, prosocial behaviors, sensitivity to gaze, and sensitivity to facial expressions (for reviews, see refs. 1 and 2).Oxytocin, therefore, may be a tool for promoting social behaviors, especially in clinical populations in which social faculties are compromised (8, 11–13). In the last few years, in fact, oxytocin has been tested in autistic individuals, and it appears to increase social attention and improve emotion recognition (e.g., refs. 14–19; although see ref. 20; for a recent review, see ref. 21). Given the importance of early assessments in the diagnosis of autism (22), studies clarifying the role of oxytocin in early development are critically important. For example, human infants actively participate in face-to-face caregiver–infant interactions; failure to engage with caregivers in this way can disrupt the development of healthy emotion regulation and socioemotional skills (23–25). In both caregivers and neonates, complex cortical and limbic brain networks are prepared to sustain such exchanges (26–28), and several hormones and neuromodulators regulate the affective components of face-to-face caregiver–infant interactions (29–32). However, to our knowledge, studies investigating the role of infants’ oxytocin levels in these early intersubjective exchanges have not been carried out. Only one study to date measured endogenous oxytocin levels in newborns and reported that higher levels of oxytocin in newborns’ cerebrospinal fluid (CSF) were associated with higher levels of social engagement, including actively seeking parental social interaction for soothing and a greater interest in social interaction (33). No studies to date, however, have administered oxytocin to infants to determine its effects on social behavior, despite the fact that a more thorough understanding of oxytocin and its behavioral consequences may provide a potential tool for interventions aimed at promoting social affiliation in individuals with social impairments (11–15, 34). The necessity to fill this gap motivated the present study.Our first goal was to determine whether oxytocin influences newborn macaques’ behaviors during an interaction with a human caregiver. We predicted that oxytocin, compared with saline, would increase positive social behavior, including facial gestures [i.e., lip smacking (LPS) and tongue protrusion (TP)], visual attention to a human caregiver, and time spent in close proximity to a human caregiver (35). As adults, macaques display positive behavioral changes in response to exogenous oxytocin (2–6), as in humans (1).

Table 1.

Ethogram for 12 behaviors scored during imitation recognition

Music training alters the course of adolescent auditory development

Fundamental changes in brain structure and function during adolescence are well-characterized, but the extent to which experience modulates adolescent neurodevelopment is not. Musical experience provides an ideal case for examining this question because the influence of music training begun early in life is well-known. We investigated the effects of in-school music training, previously shown to enhance auditory skills, versus another in-school training program that did not focus on development of auditory skills (active control). We tested adolescents on neural responses to sound and language skills before they entered high school (pretraining) and again 3 y later. Here, we show that in-school music training begun in high school prolongs the stability of subcortical sound processing and accelerates maturation of cortical auditory responses. Although phonological processing improved in both the music training and active control groups, the enhancement was greater in adolescents who underwent music training. Thus, music training initiated as late as adolescence can enhance neural processing of sound and confer benefits for language skills. These results establish the potential for experience-driven brain plasticity during adolescence and demonstrate that in-school programs can engender these changes.By age six, the brain has reached 90% of its adult size (1). However, the years between childhood and young adulthood are marked by a host of subtler neural developments. Myelination and synaptic pruning (2–5) lead to a decrease in gray matter and an increase in white matter (6–13). Resting-state oscillations decline (14–16), and passive evoked responses to sound change in complex ways. Cortically, the P1, which is a positive deflection at around 50 ms generated within lateral Heschl’s gyrus (17), declines whereas the N1, a negative deflection at around 100 ms generated within primary and secondary auditory cortices (18–20), increases (21–23). Subcortically, the trial-by-trial consistency of the response declines (24, 25). An open question is how experience interacts with this developmental plasticity during adolescence. Is the transition from the plasticity of childhood to the stability of adulthood malleable by experience? And if so, what types of enrichment have the greatest impact on the development of the neural mechanisms contributing to auditory and language skills?Music training is an enrichment program commonly available to high school students, and its neural and behavioral consequences are well-understood (for a review, see ref. 26). Studies comparing nonmusicians with musicians who began training early in life have revealed a “signature” set of enhancements associated with musical experience (27, 28). Relative to nonmusician peers, musicians tend to show enhanced speech-in-noise perception (29–34), verbal memory (30–33, 35–38), phonological skills (39–45), and reading (46–50), although not without exception (51, 52). Music training has also been linked to enhancements in the encoding of sound throughout the auditory system. For example, musicians show an enhanced N1 (53–56). These enhancements extend to the subcortical auditory system, with musicians showing responses to sound that are faster (55, 57–61), are degraded less by background noise (32, 61), represent speech formant structure more robustly (32, 62–64). differentiate speech sounds to a greater extent (65–67), track stimulus pitch more accurately (68, 69), and are more consistent across trials (59, 70). In adolescence, music training leads to faster responses to speech in noise (71), but the extent to which adolescent music training can confer other aspects of the musician signature remains unknown.Motivated by a conceptual framework in which auditory enrichment interacts with the auditory processes that remain under development during adolescence, we undertook a school-based longitudinal study of adolescent auditory enrichment. We focused on objective biological measures of sound processing that (i) have shown developmental plasticity during adolescence in the absence of intervention and (ii) contribute to the “neural signature” of musicianship: the consistency of the subcortical response to speech and the magnitude of the cortical onset response to speech. Subcortical response consistency peaks in childhood, waning into young adulthood (24), coinciding with a period when learning a second language becomes more difficult than earlier in life (72). Response consistency tracks with language skills (73) and is enhanced in musicians (59, 70). Accordingly, we predicted that music training in adolescence prolongs this period of heightened auditory stability. Moreover, given that the cortical N1 onset response emerges during adolescence while the P1 response declines (17, 18, 21–23), and that N1 is enhanced in younger and older musicians (53–56), we predicted that music training during adolescence would accelerate the development of the cortical onset response.To test these hypotheses, we followed two groups of high school students longitudinally, testing them just before they entered high school (mean age 14.7) and again 4 y later during their last year of school. One group (n = 19) engaged in music training in which they performed music from written notation in a group setting whereas the active control group (n = 21) engaged in Junior Reserve Officers Training Corps (JROTC) training. Both types of training required investment of time and effort and emphasized the development of self-discipline, dedication, and determination; however, only the music training targeted auditory function. Both activities were part of the high school curriculum, which was otherwise identical for both groups. We also tested students’ language skills (phonological memory, phonological awareness, and rapid naming ability) to determine whether in-school music engendered benefits for literacy skills, a prediction consistent with cross-sectional studies (39–45). The two groups were matched demographically and on all outcome measures at the start of the study (see

INAUGURAL ARTICLE by a Recently Elected Academy Member:Positively selected FimH residues enhance virulence during urinary tract infection by altering FimH conformation

Chaperone–usher pathway pili are a widespread family of extracellular, Gram-negative bacterial fibers with important roles in bacterial pathogenesis. Type 1 pili are important virulence factors in uropathogenic Escherichia coli (UPEC), which cause the majority of urinary tract infections (UTI). FimH, the type 1 adhesin, binds mannosylated glycoproteins on the surface of human and murine bladder cells, facilitating bacterial colonization, invasion, and formation of biofilm-like intracellular bacterial communities. The mannose-binding pocket of FimH is invariant among UPEC. We discovered that pathoadaptive alleles of FimH with variant residues outside the binding pocket affect FimH-mediated acute and chronic pathogenesis of two commonly studied UPEC strains, UTI89 and CFT073. In vitro binding studies revealed that, whereas all pathoadaptive variants tested displayed the same high affinity for mannose when bound by the chaperone FimC, affinities varied when FimH was incorporated into pilus tip-like, FimCGH complexes. Structural studies have shown that FimH adopts an elongated conformation when complexed with FimC, but, when incorporated into the pilus tip, FimH can adopt a compact conformation. We hypothesize that the propensity of FimH to adopt the elongated conformation in the tip corresponds to its mannose binding affinity. Interestingly, FimH variants, which maintain a high-affinity conformation in the FimCGH tip-like structure, were attenuated during chronic bladder infection, implying that FimH’s ability to switch between conformations is important in pathogenesis. Our studies argue that positively selected residues modulate fitness during UTI by affecting FimH conformation and function, providing an example of evolutionary tuning of structural dynamics impacting in vivo survival.Urinary tract infections (UTIs) are common infections causing serious morbidity and significant expenditures in healthcare dollars and lost wages. Women are disproportionately affected, with over half of women experiencing at least one UTI during their lifetime (1). In the absence of treatment, 50–80% of women will resolve a UTI within 2 mo, but up to 60% of women may remain bacteriuric with or without symptoms for at least 5–7 wk after the initial infection (2). Furthermore, even when effective therapy is given and bacteriuria and symptoms of the acute UTI resolve, 25–40% of women experience a recurrent UTI (rUTI) (2, 3). rUTI can occur by recolonization of the urinary tract from the gastrointestinal (GI) tract or from another environmental source by the same or different strain or may be due to reactivation of the original UTI strain from a bladder reservoir (4–6). Uropathogenic Escherichia coli (UPEC) cause 80–90% of community-acquired UTI and 50% of nosocomial UTI (7). The increasing prevalence of multidrug-resistant organisms can prolong the infection (8). Thus, chronic and recurrent UTI represents a major health concern worldwide, necessitating molecular understanding of disease pathogenesis and investigations into novel diagnostics and therapies.UTI is a highly complex disease involving colonization of multiple niches, each of which presents a unique set of evolutionary pressures shaping host–microbe and microbe–microbe interactions involving a multitude of virulence factors that determine disease onset, progression, and outcome. Adhesive pili assembled by the chaperone–usher pathway (CUP), such as type 1 pili, are well-characterized UPEC UTI virulence determinants. Type 1 pili, like other CUP pili, contain an adhesin (FimH) at their tip that plays an important role in host–pathogen interactions and biofilm formation. Type 1 pili are nearly ubiquitous among clinical UPEC isolates (9, 10) as well as commensal E. coli and other Enterobacteriaceae. Expression of type 1 pili is essential for colonization of the murine urinary tract (11); however, expressing type 1 pili is not sufficient for long-term colonization, as commensal E. coli are rapidly cleared (12).Upon UPEC entrance into the bladder, FimH binds mannosylated glycoproteins, including uroplakins expressed throughout human and murine bladders (13). Subsequent to attachment, UPEC invade superficial facet cells in a FimH-dependent manner (12, 14) and replicate in the cytoplasm, forming large biofilm-like structures called intracellular bacterial communities (IBCs) (15). The formation of IBCs has been observed for numerous clinical UPEC isolates in multiple mouse models and in exfoliated uroepithelial cells in urines of patients with acute UTI, but not from healthy controls (16, 17). The process of invasion and IBC formation provides UPEC an ability to survive stringent bottlenecks during pathogenesis in the urinary tract (18, 19). Outcomes of infection range from resolution with or without accompanying quiescent intracellular reservoirs (QIRs) in the bladder tissue (4) to persistent bacteriuria and chronic cystitis (20). In C3H/HeN mice, the formation of a high number of IBCs at 6 h postinfection (hpi) and an exuberant systemic innate immune response at 24 hpi, measurable in both urine and serum, correlate with the development of chronic cystitis marked by persistent urine and bladder titers >10⁴ cfu/mL and severe bladder immunopathology (18, 20). In addition to colonizing the bladder, UPEC can ascend the ureters and infect the kidneys, leading to pyelonephritis. The connection between acute and chronic UTI is just now beginning to be characterized (21–23).Type 1 pili and the tip adhesin, FimH, are encoded by the fim operon (24, 25). Mature FimH is a 279-aa two-domain protein containing a mannose-binding lectin domain (residues 1–150) and a pilin domain (residues 159–279) with an 8-aa linker connecting the domains (Fig. 1) (26–28). The mannose-binding pocket of FimH is invariant among sequenced UPEC (26, 29); however, several residues outside the mannose-binding pocket (positions 27, 62, 66, and 163) were found to be evolving under positive selection in clinical UPEC isolates compared with fecal strains (Fig. 1) (29, 30, 31). Among four fully sequenced UPEC isolates (UTI89, CFT073, 536, and J96), differences exist in positively selected residues 27, 62, and 163 (16) and the development of chronic cystitis (20). We found that UTI89, a cystitis isolate, formed more IBCs and had higher bladder titers at 6 hpi than CFT073, a pyelonephritis isolate, in single infections and coinfections. Because of the demonstrated importance of type 1 pilus function in pathogenesis, we conducted fimH allele swap experiments to determine whether the differences in fimH between UPEC strains were responsible for the phenotypic differences. We generated CFT073 and UTI89 strains containing different fimH alleles inserted into the normal chromosomal position. We found that presence of a fimH sequence encoding FimH from UTI89 (denoted FimH::A62/V163) resulted in significant increases in IBC development and the propensity to cause chronic cystitis compared with expression of CFT073 FimH (FimH::S62/A163). In coinfections, strains expressing FimH::A62/V163 significantly outcompeted otherwise isogenic strains harboring FimH::S62/A163 in both CFT073 and UTI89. FimH complexed with its chaperone FimC adopts an elongated conformation (Fig. 1A), which binds mannose with high affinity (26, 37). When complexed with the FimG adaptor, FimH can adopt a compact conformation that binds mannose with low affinity (Fig. 1B) (37). The identity of residues at positively selected positions outside the binding pocket dramatically impacts the mannose binding affinity of FimH when in the FimCGH tip-like complex but not in the FimCH complex. Thus, we argue that the combination of residues at positively selected positions affects the propensity of FimH to adopt an elongated conformation in the tip and thus its relative mannose binding affinity. FimH alleles that retained the high-affinity binding conformation in the tip were significantly attenuated in a mouse model of UTI, suggesting that equilibrium between FimH conformations, which is modulated by positively selected residues, is critical in pathogenesis.Open in a separate windowFig. 1.FimH positively selected residues. FimH is a two-domain adhesin comprised of a lectin domain of residues 1–150 (green), a pilin domain with residues 159–279 (blue), and a linker loop (yellow) connecting them. Positively selected residues are mapped onto the structures of FimH as red spheres. (A) In the elongated FimH (V27/S62/V163) structure, mannose is observed at the distal binding pocket in white sticks (J96 FimH; PDB ID code: 1KLF; FimC removed for clarity). (B) In the compressed FimH (A27/S62/A163) structure in the absence of mannose, position 133 of the binding pocket is colored white (F18 FimH; PDB ID code: 3JWN). Note the distance of these positively selected residues from the mannose binding pocket.

Table 1.

Prevalence of positively selected FimH residues

Kinetic response of a photoperturbed allosteric protein

By covalently linking an azobenzene photoswitch across the binding groove of a PDZ domain, a conformational transition, similar to the one occurring upon ligand binding to the unmodified domain, can be initiated on a picosecond timescale by a laser pulse. The protein structures have been characterized in the two photoswitch states through NMR spectroscopy and the transition between them through ultrafast IR spectroscopy and molecular dynamics simulations. The binding groove opens on a 100-ns timescale in a highly nonexponential manner, and the molecular dynamics simulations suggest that the process is governed by the rearrangement of the water network on the protein surface. We propose this rearrangement of the water network to be another possible mechanism of allostery.Subtle conformational transitions within the folded state of highly structured proteins are often an integral aspect in their functional mechanism. These conformational transitions can occur as a result of different events, such as ligand binding, covalent modification (e.g., phosphorylation), or proteolytic cleavage. When an event or perturbation at one site in a protein changes the enzymatic activity or the binding affinity to a ligand at another distant site, this process can be described as allostery. Hemoglobin has long served as the prototypical example to study this effect, where the binding of an oxygen in one subunit modifies the affinity of binding oxygen in another subunit (1, 2). The traditional models of allostery developed by Monod et al. (3) and Koshland et al. (4) attribute allosteric effects to conformational changes by which the allosteric binding site communicates with the distant active site. There is, however, increasing evidence that allostery can be mediated also without any conformational change, relying purely on changes in internal protein dynamics (5).PDZ domains are an important class of protein interaction modules that have been studied extensively in the context of allostery. They are found in a large variety of proteins and generally bind the C termini of their targets (6–11). As scaffolding domains, they are molecular switches that play a central role in signal transduction. For several PDZ domain proteins, allosteric interactions are an important regulatory mechanism (10, 12–15).NMR spectroscopy has been particularly useful to elucidate the equilibrium dynamics of proteins on various timescales. The notion of allostery mediated through a change in dynamic properties was corroborated by a study of the third PDZ domain from the PSD-95/SAP90 protein (16). This protein contains an additional C-terminal α-helix , which shows no direct interaction with the peptide ligand. Removal of has a negligible effect on the structure of the PDZ core domain; however, it does lead to a large decrease in ligand binding affinity, which was shown to be entirely entropic in nature. Other studies have identified changes in (conformational) entropy of both backbone (17) and side-chain (18) dynamics in other systems to give rise to allosteric effects.Here, we focus on the second PDZ (PDZ2) domain from human tyrosine-phosphatase 1E (hPTP1E), which has been demonstrated to possess allosteric properties (19). The PDZ domain is a small 96-residue protein with a binding groove between the -helix and the -strand (Fig. 1B). As mentioned previously, side-chain dynamics in contiguous sectors spanning the whole protein have been the proposed allosteric mechanism (19–21), but in this case, ligand binding also results in a small but significant structural change, albeit being quite small (22–24) (Fig. 1B and 25–34).Open in a separate windowFig. 1.(A) Averaged NMR structures of the photoswitchable PDZ2 domain with the photoswitch (yellow) in the cis (Left, PDB ID 2M0Z) and trans (Right, PDB ID 2M10) conformations. (B) Overlays of the apo (blue) and holo (red) X-ray structures [3LNX and 3LNY (24)] together with the ligand (the Ras guanine nucleotide exchange factor 2 C-terminal peptide, in yellow) in the latter case. (C) The NMR structures with the photoswitch in cis (blue) and in trans (red) and (D) the averaged MD structures with the photoswitch in cis (blue) and 100 ns after switching into trans (red). For clarity, the photoswitch is not shown in C and D. The dotted lines in D indicate the C_α-C_α distances shown in Fig. 4B.

Table 1.

Structural comparison

From the Cover: Gradual decline in mobility with the adoption of food production in Europe

Increased sedentism during the Holocene has been proposed as a major cause of decreased skeletal robusticity (bone strength relative to body size) in modern humans. When and why declining mobility occurred has profound implications for reconstructing past population history and health, but it has proven difficult to characterize archaeologically. In this study we evaluate temporal trends in relative strength of the upper and lower limb bones in a sample of 1,842 individuals from across Europe extending from the Upper Paleolithic [11,000–33,000 calibrated years (Cal y) B.P.] through the 20th century. A large decline in anteroposterior bending strength of the femur and tibia occurs beginning in the Neolithic (∼4,000–7,000 Cal y B.P.) and continues through the Iron/Roman period (∼2,000 Cal y B.P.), with no subsequent directional change. Declines in mediolateral bending strength of the lower limb bones and strength of the humerus are much smaller and less consistent. Together these results strongly implicate declining mobility as the specific behavioral factor underlying these changes. Mobility levels first declined at the onset of food production, but the transition to a more sedentary lifestyle was gradual, extending through later agricultural intensification. This finding only partially supports models that tie increased sedentism to a relatively abrupt Neolithic Demographic Transition in Europe. The lack of subsequent change in relative bone strength indicates that increasing mechanization and urbanization had only relatively small effects on skeletal robusticity, suggesting that moderate changes in activity level are not sufficient stimuli for bone deposition or resorption.Declining mobility levels since the Terminal Pleistocene contributed to fundamental changes in demography, health and disease, and social organization among many human populations (1–6). The Neolithic Demographic Transition, characterized by increased fertility, population size, and density, may be partially attributable to decreased energy expenditure associated with greater sedentism (2, 7). Paradoxically, in many cases increased sedentism also may have led to declines in overall health and increased morbidity within populations by facilitating transmission of infectious diseases and through other negative consequences of more dense settlements (3, 4, 6). Reductions in mechanical loading of the skeleton associated with a more sedentary lifestyle may have contributed to the etiology of modern skeletal disorders such as osteoporosis (8–10). Declines in mobility also had significant effects on sociopolitical organization, including sexual division of labor, social hierarchy, and territoriality (1, 11, 12). However, despite its broad evolutionary significance, the timing and patterning of declining mobility during the Holocene and its relationship to changing subsistence economies has proven difficult to characterize from material archaeological remains (1, 5, 7, 13, 14), leaving many unanswered questions. For example, were declines in mobility relatively abrupt at the onset of food production in the Early Neolithic, as suggested by some demographic studies (15), or did they begin earlier, during the Mesolithic (5, 16)? Did mobility continue to decrease after the Neolithic in response to intensification of agriculture and other factors? Is there evidence for continuing declines in mobility to the present time, with recent industrialization and mechanization?An alternative approach to addressing such issues is to assess directly the evidence preserved in human skeletal remains (17). Although skeletal morphology is determined by a complex interplay between various genetic and environmental factors (18, 19), there is abundant evidence that mechanical loading during life has a strong influence on skeletal structure (20–23). Earlier studies identified declines in skeletal robusticity (strength relative to body size) in Homo throughout the Pleistocene (24, 25), but recent analyses suggest that the major decrease in robusticity occurred later, at the end of the Pleistocene, between early anatomically modern H. sapiens and Holocene populations (26, 27). This suggestion in turn strongly implicates increased sedentism as a major driver in producing the more gracile modern human skeleton (10, 25, 26) and focuses attention on the Holocene as the critical period during which this transformation took place. However, the timing and pace of this change relative to major subsistence and lifestyle transitions cannot be determined from these studies, given their sparse sampling of terminal Pleistocene and Holocene populations.Here we use a sample of 1,842 individuals distributed across Europe to investigate changes in skeletal robusticity and mobility from the Upper Paleolithic [11,000–33,000 calibrated years (Cal y) B.P.] through the 20th century (Fig. 1, Dataset S1). Europe is an appropriate region to carry out such an analysis because of the abundance of well-provenienced skeletal remains and rich archaeological context (28) over the time range of interest. The structural characteristics evaluated here are anteroposterior (A–P) and mediolateral (M–L) section moduli of the midshaft regions of long bones, which are measures of A–P and M–L bending strength (29). It is known that long bone diaphyseal cortices react to imposed mechanical loadings throughout life, changing their cross-sectional geometry to adapt to altered strain magnitudes and distributions, which in turn are dependent on behavioral use of the limbs (20–22, 30, 31). For example, vigorous exercise in humans greatly increases bending strains in the tibia (32, 33) and is associated with preferential strengthening in the direction of movement, i.e., running and jumping lead to increases in A–P/M–L strength (30, 34, 35). Increased A–P/M–L bending strength of the lower limb bones also characterizes more terrestrially mobile populations or subpopulations (36–38). Following this rationale, several previous studies have examined temporal trends in lower limb bone cross-sectional shape, as a proxy for mobility, within Late Pleistocene or Holocene archaeological samples, generally demonstrating declines in A–P/M–L (or maximum/minimum) rigidity or strength (39–42). However, all these studies were limited to particular regions (42), temporal ranges [e.g., Pleistocene through early Holocene (39) or post-Pleistocene (42)], and/or very limited population sampling within the Holocene (40, 41). In addition, none directly compared temporal changes in upper and lower limb bone strength. In this study we examine temporal changes in relative strength of the femur, tibia, and right humerus. Inclusion of the upper limb provides an important control over possible general systemic (e.g., dietary, general activity level) effects on skeletal structure.Open in a separate windowFig. 1.Location of study sites, by temporal period (see Dataset S1.

Table 1.

Study sample sizes by temporal period

Rationale and mechanism for the low photoinactivation rate of bacteria in plasma

The rate of bacterial photoinactivation in plasma by methylene blue (MB), especially for Gram-negative bacteria, has been reported to be lower, by about an order of magnitude, than the rate of inactivation in PBS and water solutions. This low inactivation rate we attribute to the bleaching of the 660-nm absorption band of MB in plasma that results in low yields of MB triplet states and consequently low singlet oxygen generation. We have recorded the change of the MB 660-nm-band optical density in plasma, albumin, and cysteine solutions, as a function of time, after 661-nm excitation. The transient triplet spectra were recorded and the singlet oxygen generated in these solutions was determined by the rate of decrease in the intensity of the 399-nm absorption band of 9, 10-anthracene dipropionic acid. We attribute the bleaching of MB, low singlet oxygen yield, and consequently the low inactivation rate of bacteria in plasma to the attachment of a hydrogen atom, from the S–H group of cysteine, to the central nitrogen atom of MB and formation of cysteine dimer.Even though infections from bacterially contaminated plasma are thought to be very few (1) and the frequency of patient sepsis caused by transfusion of blood contaminated by bacteria is as low or less than the transfusion-associated hepatitis C virus infection (2), the possibility of such infection cannot be entirely discounted in view of the fact that new or even more resistant strains of known bacteria are being encountered. To a large extent, pooled human plasma is sterilized by the solvent–detergent method (3–5), whereas single-donor human plasma is often decontaminated by methylene blue (MB) photoexcitation, which generates reactive oxygen species that safely inactivate many viruses (6, 7). However, several Gram-positive and especially Gram-negative bacteria are found to be very resistant to photoinactivating agents such as porphyrins and thiazine dyes, possibly because the outer walls of Gram-negative bacteria, such as Serratia marcescens (SM), are composed of negatively charged lipopolysaccharides, which are resistant to photoinactivation by these dyes (8).Gram-positive bacteria and several Gram-negative bacteria are relatively easily inactivated, especially in high-pH MB–PBS solutions, after irradiation with 661-nm light for a rather short period, i.e., 10 min (9). Gram-negative bacteria usually require longer exposure times for inactivation. 10). This inactivation deficiency, observed in plasma, cannot be attributed to the decrease in light intensity due to absorption or scattering, because the fresh human plasma used is clear and neither absorbs in the 661-nm excitation wavelength region nor scatters the LED light. Singlet oxygen is the dominant active species responsible for the photoinactivation of bacteria (11–21), with MB being the primary photosensitizing agent used for the generation of singlet oxygen and the sterilization of plasma (22–27). To that effect we investigated possible reactions between plasma and MB that may be responsible for decrease in singlet oxygen generation that would explain the low bacteria inactivation rate in plasma.

Table 1.

Inactivation of Gram-positive and Gram-negative bacteria in MB–PBS and MB–plasma solutions as a function of irradiation time with 6.8-mW, 661-nm LED light

Mobile phone data highlights the role of mass gatherings in the spreading of cholera outbreaks

The spatiotemporal evolution of human mobility and the related fluctuations of population density are known to be key drivers of the dynamics of infectious disease outbreaks. These factors are particularly relevant in the case of mass gatherings, which may act as hotspots of disease transmission and spread. Understanding these dynamics, however, is usually limited by the lack of accurate data, especially in developing countries. Mobile phone call data provide a new, first-order source of information that allows the tracking of the evolution of mobility fluxes with high resolution in space and time. Here, we analyze a dataset of mobile phone records of ∼150,000 users in Senegal to extract human mobility fluxes and directly incorporate them into a spatially explicit, dynamic epidemiological framework. Our model, which also takes into account other drivers of disease transmission such as rainfall, is applied to the 2005 cholera outbreak in Senegal, which totaled more than 30,000 reported cases. Our findings highlight the major influence that a mass gathering, which took place during the initial phase of the outbreak, had on the course of the epidemic. Such an effect could not be explained by classic, static approaches describing human mobility. Model results also show how concentrated efforts toward disease control in a transmission hotspot could have an important effect on the large-scale progression of an outbreak.Human mobility is undisputedly one of the main spreading mechanisms of infectious diseases. Understanding the propagation of an epidemic in a population at any spatial scale of analysis inevitably calls for the understanding of the underlying mobility patterns (1–6). Researchers have commonly focused on infectious diseases transmitted through direct contact between persons (e.g., refs. 1–4). The key role of human mobility has only recently been acknowledged also for water-related diseases (where transmission is mediated by water, which influences the habitat’s suitability for the pathogen and/or its possible intermediate hosts), as highlighted by the development and widespread application of spatially explicit epidemiological models (7–10). Such models translate our comprehension of the mechanisms driving disease transmission [such as rainfall (10)] and spread [such as hydrologic transport of pathogens (8, 11) besides human mobility] into a simplified mathematical form. They may be used not only to predict the spatiotemporal pattern of the spread of a disease (12–14) but also to test alternative model implementations (15), or to evaluate the effects of various interventions on disease dynamics (16–18).To include population movement in epidemiological models, researchers often rely on approaches such as gravity (e.g., ref. 19) or radiation (20) models, where the fluxes between any two sites are expressed as a function of their relative distance and the embedded population distribution. Such models have primarily been developed and tested for countries in the western world, where transportation networks are dense and efficient, supraregional travel is cheap, and regular commuting patterns are predominant. Lack of data has so far frustrated a thorough validation of such models in the developing world, where mobility drivers and patterns may be different than those of western countries. In some applications, the absence of information about mobility fluxes has been circumvented by inferring the parameters of the mobility model directly from epidemiological data (9, 10, 17). This, however, contributes to increasing uncertainty in model identification because many different factors concur in the spreading of an epidemic. Another important shortcoming of current mobility models is their inability to adapt to seasonal and subseasonal changes in mobility patterns.With the increasing diffusion of mobile phones, which have become very widely used even in developing countries (21, 22), a new source of information about human mobility has emerged. Each time a phone emits or receives a call or text message, the antenna that the cell phone is logged into is registered by the service provider, along with the time of the event (23). It is thus possible to track the movement of cell phone users as they advance from antenna to antenna. Suitably aggregated and properly anonymized to prevent privacy issues (24), a sample of this data can be used to estimate fluxes of people between areas in a region by assigning a set of antennas to each geographical area in the study domain (e.g., based on administrative boundaries). The resolution in time can be as high as the typical frequency of calls allows, whereas the spatial resolution is limited only by the typical distance between two antennas (23). Using mobile phone records of a sufficiently large number of users, one can thus estimate human mobility fluxes with high accuracy, including spatiotemporal variability across a variety of scales (24), and without resorting to any particular model.A number of recent studies focus on the use of mobile phone data to extract human mobility patterns in developing countries at different scales in space and time (25–27). Others compare the movement patterns extracted from mobile records to traditional data sources such as censuses (28) and surveys (29). Several studies deal with the comparison with human mobility models (21, 30). In the context of infectious disease spread in developing countries, this new source of information enables previously unseen kinds of analyses. Examples are the derivation of magnitude and destination of population fluxes following a sudden outbreak (25, 31), and the quantification of the importance of human mobility and its seasonal variations on the spread of disease in terms of increased outbreak risk in and infectious pressure on connected areas (5, 30, 32–34).Mass gatherings, such as pilgrimages, sport events, or music festivals, can be critical in the spread of infectious diseases via various transmission routes (35, 36). When it comes to orofecally transmitted diseases, such as shigellosis (37) or cholera (38, 39), insufficient safe drinking water supply and sanitary infrastructure related to overcrowding are often the main causes of local disease outbreaks and subsequent spread by homecoming infected attendees. To model the effect of mass gatherings, one needs to account for the spatiotemporal dynamics of human mobility and the associated short-term fluctuations of population distribution. Mobility models and static data sources, such as censuses or surveys, are therefore unsuitable. Conversely, mobile phone records contain all required information at the desired timescales and thus represent an excellent new data source for epidemiological models.Here, we study the cholera epidemic that spread throughout Senegal in 2005. A distinctive feature of this outbreak was its sudden flare. It started from the order of magnitude of hundreds of cases per week during the first 3 mo of the year, localized in the region of Diourbel and surroundings, and abruptly jumped to thousands of cases at the end of March, rapidly spreading to 10 out of 11 regions of the country, with over 27,000 reported cases (38, 40, 41) suggests that this first peak was related to a religious pilgrimage, the Grand Magal de Touba (GMdT), that took place in late March when an estimated 3 million pilgrims traveled to Touba in the region of Diourbel. During later stages, the outbreak evolved, showing distinct dynamics in different regions of the country, rainfall and the associated floods being important drivers, especially in the capital city of Dakar (39).

Table 1.

Regions of Senegal (as of 2005) with their population (2005 estimates), total number of reported cases during the epidemic, cumulative incidence, and mobile phone sample size (relative to 2013 population)

Electrocorticography reveals the temporal dynamics of posterior parietal cortical activity during recognition memory decisions

Theories of the neurobiology of episodic memory predominantly focus on the contributions of medial temporal lobe structures, based on extensive lesion, electrophysiological, and imaging evidence. Against this backdrop, functional neuroimaging data have unexpectedly implicated left posterior parietal cortex (PPC) in episodic retrieval, revealing distinct activation patterns in PPC subregions as humans make memory-related decisions. To date, theorizing about the functional contributions of PPC has been hampered by the absence of information about the temporal dynamics of PPC activity as retrieval unfolds. Here, we leveraged electrocorticography to examine the temporal profile of high gamma power (HGP) in dorsal PPC subregions as participants made old/new recognition memory decisions. A double dissociation in memory-related HGP was observed, with activity in left intraparietal sulcus (IPS) and left superior parietal lobule (SPL) differing in time and sign for recognized old items (Hits) and correctly rejected novel items (CRs). Specifically, HGP in left IPS increased for Hits 300–700 ms poststimulus onset, and decayed to baseline ∼200 ms preresponse. By contrast, HGP in left SPL increased for CRs early after stimulus onset (200−300 ms) and late in the memory decision (from 700 ms to response). These memory-related effects were unique to left PPC, as they were not observed in right PPC. Finally, memory-related HGP in left IPS and SPL was sufficiently reliable to enable brain-based decoding of the participant’s memory state at the single-trial level, using multivariate pattern classification. Collectively, these data provide insights into left PPC temporal dynamics as humans make recognition memory decisions.The ability to remember past events—episodic memory—is known to critically depend on medial temporal lobe (MTL) structures (1, 2) and their interaction with prefrontal cortex (3). Neuroimaging studies of humans making memory-based decisions, although advancing understanding of MTL and prefrontal mnemonic function (4–8), consistently and unexpectedly demonstrate that activity in left lateral posterior parietal cortex (PPC) also varies with episodic memory outcomes (9–14). In particular, functional MRI (fMRI) data reveal dissociable effects of memory on activity in left intraparietal sulcus (IPS), superior parietal lobule (SPL), and angular gyrus (AG), wherein activity tracks perceived memory strength, retrieval decision uncertainty, and episodic recollection, respectively (9–14). For example, during recognition memory decisions, activity in left lateral IPS is greater during higher confidence hits and monotonically decreases across lower confidence hits to lower confidence correct rejections (CRs) to higher confidence CRs (15–17). By contrast, activity in left SPL is greater during lower confidence recognition decisions (for both hits and correct rejections) relative to higher confidence decisions (11, 16, 17).Studies of patients with PPC lesions demonstrate a complex pattern of effects on memory, with performance spared on some measures of episodic memory, but impaired as measured by retrieval confidence, memory for source details, and cued recall (18–23). For example, a recent study of two patients with bilateral IPS lesions revealed unimpaired recognition memory accuracy, but a reduction in high confidence hits and false alarms relative to matched controls (22). Importantly, this decline in high confidence recognition decisions was only observed for items perceived as old, and not for items perceived as novel. As with fMRI measures of left lateral IPS activity, these lesion data suggest that the role of IPS in memory varies as a function of whether the test probe is perceived as old or new.The role of lateral PPC in episodic retrieval is posited to relate to broader (i.e., nonmnemonic) functions. Extant evidence indicates that dorsal PPC is involved in other cognitive domains, such as perceptual decision making (24–28). For example, human fMRI studies of two-choice perceptual decisions have demonstrated that IPS activity tracks the strength of perceptual evidence (24, 25), independent of response modality (25). A mechanistic interpretation of such activity is that IPS neurons act as evidence accumulators, with a distinct population of IPS neurons accumulating evidence toward each of the two decision bounds. When the evidence reaches one of the bounds, a perceptual decision it thought to be reached. Such results motivated the hypothesis that left IPS might serve as a mnemonic accumulator during old/new recognition decisions (9, 16).By contrast, SPL is a core component of the dorsal frontoparietal network that implements top-down visual attention (29, 30). Parallel lines of work in the memory and perception literatures demonstrate that left SPL activity is greater during uncertain memory decisions (17) and uncertain perceptual decisions (31). Such findings suggest that decision uncertainty, be it in the mnemonic or perceptual realm, results in increased engagement of top-down visual attention to driving inputs (17).Although the preceding suggests a possible functional differentiation between IPS and SPL during decision making, an alternative account posits that IPS and SPL activity during retrieval reflects a common attention function (11). In this view, dorsal PPC retrieval activity is seen as a reflection of top-down attention, acting to maintain retrieval goals and to monitor memory signals that presumably emerge from MTL computations. One challenge for this perspective is the distinct patterns of fMRI blood oxygen level-dependent (BOLD) signal in left IPS and SPL during episodic retrieval (16, 17, 32).Theorizing about the distinct memory-related response profiles in dorsal PPC subregions observed with fMRI—that is, in IPS and SPL—has been hampered by the absence of precise information about the temporal dynamics of lateral parietal activity. Although scalp-recorded event-related potentials (ERPs) demonstrate a differential positivity over left parietal cortex as a function of recognition memory (hits > CRs, ∼400–800 ms postretrieval cue onset) (33), the anatomical source(s) of this parietal “old/new” effect and its relation to the multiple memory-related PPC effects observed with fMRI remain unknown. A recent effort to use simultaneous EEG−fMRI to advance understanding of the temporal profile of parietal activity during retrieval did not reveal a link between ERP and fMRI BOLD effects in any lateral parietal region (34). Importantly, the hypothesis that IPS and SPL subserve distinct computations predicts that these subregions will display different temporal response patterns during memory-based decisions; these patterns promise to directly inform hypotheses about their functional roles. More broadly, understanding the temporal profile of responses in these subregions of PPC during decision making may inform theories of dorsal PPC function across cognitive domains, including perceptual decision making and attention.A correlate of fMRI BOLD activity is provided by the high-frequency range of local field potentials (LFPs) (35–38), which reflects local population activity (39–41), providing a means to specify and dissociate the temporal dynamics of memory effects in specific PPC regions. Accordingly, we used electrocorticography (ECoG) to directly record cortical LFPs from human left IPS and SPL while participants made recognition memory decisions. During the encoding phase, participants viewed a series of individually presented nouns, making abstract/concrete judgments on each. Subsequently, participants made old/new recognition judgments about test items, half studied and half novel (Fig. S1; see Materials and Methods). LFPs were recorded in eight participants (Fig. S2 and Open in a separate windowFig. S1.Experiment design. Encoding phase: 20 nouns were serially presented at the center of the screen, and the participant had to make an abstract/concrete decision on each word. Retrieval phase: 40 nouns (20 from the previous list/ 20 new) were presented serially at the center of the screen. Participants were asked to indicate whether each word was old or new.Open in a separate windowFig. S2.Electrode coverage on each participant. Two renderings per participant, one in native space (left) and the second projected into the Montreal Neurological Institute (MNI) standard template space (right). Color-coded are the anatomical labels based on the native space rendering and postoperative CT scans.

Table S1.

Participant characteristics and clinical data

NO binding kinetics in myoglobin investigated by picosecond Fe K-edge absorption spectroscopy

Diatomic ligands in hemoproteins and the way they bind to the active center are central to the protein’s function. Using picosecond Fe K-edge X-ray absorption spectroscopy, we probe the NO-heme recombination kinetics with direct sensitivity to the Fe-NO binding after 532-nm photoexcitation of nitrosylmyoglobin (MbNO) in physiological solutions. The transients at 70 and 300 ps are identical, but they deviate from the difference between the static spectra of deoxymyoglobin and MbNO, showing the formation of an intermediate species. We propose the latter to be a six-coordinated domed species that is populated on a timescale of ∼200 ps by recombination with NO ligands. This work shows the feasibility of ultrafast pump–probe X-ray spectroscopic studies of proteins in physiological media, delivering insight into the electronic and geometric structure of the active center.Diatomic molecules, such as CO, NO, and O₂, are the receptors that bind to and activate heme proteins. Among them, NO has been highlighted as a key biological messenger (1) and its level controls various physiological responses, such as NO synthases, message transduction (soluble guanylyl cyclases) (2, 3), NO transport and oxidation [hemoglobin, myoglobin (Mb), and nitrophorin] (4–6), and regulation of the NO/O₂ balance (neuroglobin) (7, 8). In all of these cases, the heme group that binds the NO ligand is chemically identical, and therefore, variations in the reactivity and function are thought to be closely related to the spin, electronic configuration, and geometric structure on binding (9) and/or suggestive of different steric and electronic interactions of the bound NO with neighboring protein residues (10). Consequently, there is great interest in understanding the nature of NO binding to heme proteins and its biochemical role.The binding kinetics of NO in Mb have been studied by a variety of time-resolved spectroscopic techniques. Ligand dissociation from the heme iron was triggered by excitation into either the Soret or the Q bands, whereas the ensuing dynamics were probed using transient absorption (TA) in the UV visible (UV-Vis) (11–20), the near IR (21–23), and the mid-IR (10, 23, 24) or by resonance Raman spectroscopy (20). For UV-Vis and near-IR TA spectroscopy, the signals are dominated by the π-orbitals of the porphyrin, whereas mid-IR TA of the NO stretch mode is sensitive to the orientation of the NO dipole. Resonance Raman spectroscopy maps several vibrational modes of the porphyrin, but most studies have focused on the important Fe-N stretch vibration (at 220 cm⁻¹) with the proximal histidine (20, 25–27), which is sensitive to the position of the iron atom out of the heme plane and to the strain that the protein exerts on the heme through movements of the helices (28, 29).All of the TA studies report multiexponential recombination kinetics with time constants spanning from subpicoseconds to several hundreds of picoseconds or even longer. 10, 12, 13, 16–18, 20, 21, 24). Based on temperature-dependent UV-Vis TA studies, Champion and coworkers (17) argued that the transition state associated with the fast recombination kinetics (8–15 ps) has no barrier and is caused by rebinding of NO from the center of the distal pocket very close to the iron. A barrierless recombination is thought to occur, because the unpaired NO electron forms a transition state with the antibonding d_z² orbitals without structural distortions of the protein matrix (17). The slow component in their work (170–200 ps) was assigned to recombination of NO from the more distant Xe4 pocket (30). The rebinding occurs on a similar timescale as the structural fluctuations of the protein architecture (13, 23, 28, 29), and therefore, relaxation of the active site after dissociation gives rise to a small time-dependent barrier (∼3 kJ mol⁻¹) (17). From their mid-IR TA studies, Lim and coworkers (23, 24) similarly concluded that the slow recombination component (133 ps in their case) is caused by the protein environment surrounding the distal side of the heme, and conformational relaxation of the protein after photolysis may raise the barrier to NO rebinding. In both cases (long and short components), it was hypothesized that NO binds to an out-of-plane iron (domed porphyrin), in agreement with theoretical calculations (31–33). Finally, τ₄ in ProbeExcitation wavelength (nm)τ₁ (ps)τ₂ (ps)τ₃ (ps)τ₄ (ns)SourceUV-Vis TA^*57427.6 (52%)279.3 (48%)12UV-Vis TA^*Times change with mutation3–518.9 (41%)126.4 (49%)∞ (10%)11UV-Vis TA^†5709.1 (40%)200 (50%)∞ (10%)14UV-Vis TA^*5642–413 (40%)148 (50%)∞ (10%)Resonance Raman^†560–57030 ± 1020Near-IR TA^‡560–5702–427.5 ± 5 (42%)293 ± 30 (33%)∞ (25%)21Visible TA^‡5642–412 ± 3 (40%)205 ± 30 (37%)∞ (23%)21Mid-IR TA—1 and 4 (35%)42 (29%)238 (36%)10Mid-IR TA5805.3 (54%)133 (46%)24UV-Vis TA^§4001.8 (42%)13.8 (24%)200 (34%)∞ (<5%)16UV-Vis TA^§5801.1 (50%)8 (30%)170 (20%)16UV-Vis TA40013.8 (41%)200 (59%)17UV-Vis TA5808 (60%)170 (40%)17X-ray absorption532192 ± 44 (75%)>1.3 (25%)This workOpen in a separate window^*Single-wavelength detection at 480 nm (Soret band).^†Single-wavelength detection at 435 nm (Soret band).^‡Single-wavelength detection at 615 nm (Q bands).^§White light continuum detection.More recent studies by Negrerie and coworkers (20, 21) combined UV-Vis, near-IR (in the region of band III) TA, and resonance Raman measurements. The resonance Raman mainly probed the Fe-N_histidine mode that is sensitive to the heme iron out-of-plane position, just like the near-IR band III (27). Although the UV-Vis TA results in the Soret and Q-band regions agree with the results in refs. 16 and 18, the resonance Raman results and the TA studies of band III reveal an additional ∼30-ps component [also reported in one of the mid-IR studies (10) and one UV-Vis TA study (12)]. Negrerie and coworkers (21) also concluded that GR leads to the formation of a transient six-coordinated domed heme, with a rise time of ∼10 ps and a decay of 30 ± 10 ps, corresponding to the return to the planar form. Thus, the transition from domed to planar is not a prompt one [contrary to the reverse process (34)], and Negrerie and coworkers (21) attributed its timescale to the constraints that the protein exerts on the porphyrin. Indeed, using picosecond UV resonance Raman studies of MbCO, Mizutani and coworkers (29) found that, on photodissociation of CO, the signal of the Tryptophan situated on the A helix showed a prompt decrease followed by a recovery in ∼50 ps that was attributed to changes in the protein tertiary structure that exerts strain on the heme–protein link. Negrerie and coworkers (20) studied other heme proteins and found that the timescale of the primary domed to planar heme transition was ∼15 ps for hemoglobin, ∼7 ps for dehaloperoxidase, and ∼6 ps for Cytochrome c (Cytc), and they attributed these timescales to the constraints exerted by the protein structure on the heme cofactor (20). Negrerie and coworkers (20) noted, however, that dehaloperoxidase and Cytc have similar time constants, despite their different structure and heme linking, revealing that several factors, not only the protein strain, can influence the heme response kinetics. Similar time constants were reported using IR TA by Lim and coworkers (22) for Cytc and a model heme, microperoxidase-8, but they attributed the faster ligand rebinding in Cytc compared with Mb to the fact that the former does not have a primary docking site-like structure that slows down NO rebinding.On the theory side, Franzen (31) calculated potential curves along the Fe-NO distance for the different spin states of a compound consisting of an imidazole (Im) ligand bound to iron-porphine (FeP) trans to the diatomic NO ligand (Im-FeP-NO): the electronic ground state (S = 1/2) and the excited quartet (S = 3/2) and sextet (S = 5/2) states. For the planar geometry, Franzen (31) found, as expected for the ground state, that the doublet is the most stable configuration. The quartet is somewhat less binding, whereas the sextet state is even less binding with an Fe-NO equilibrium distance above 2 Å. Franzen (31) attributed the ∼10-ps component to the S = 5/2→S = 3/2 relaxation and the >100-ps timescale to a sequential relaxation S = 5/2→S = 3/2→S = 1/2 process. More refined calculations by Strickland and Harvey (32) found that the quartet state is the most likely state to be populated on recombination. Contrary to the works in refs. 16, 17, 23, and 24, these calculations exclusively invoke intramolecular electronic relaxation, neglect the role of the environment, and do not include the strain on the porphyrin.Because the measured time constants somewhat vary with the observable (35–37). However, solid samples are far from the physiological conditions under which proteins operate, and it is desirable to investigate the ligand dynamics of heme proteins in physiological solutions. In addition, the latter can be flowed continuously to ensure the renewal of the sample and to decrease the X-ray dose on it. Adopting such an approach, X-ray scattering studies of MbCO in solution with a 100-ps resolution were recently reported (38–40), but the spatial resolution is such that tertiary and global structural changes can be probed but not atomic-scale changes. In addition, this approach does not deliver information about the electronic structure of the active site, which plays a central role in the biochemistry and reactivity of heme proteins (41, 42). The valence 3d electrons of the iron atom are significantly delocalized over the porphyrin ligand π*-orbitals, and the ability of the heme to redistribute charge and spin density plays an important role in the formation and stabilization of a variety of intermediates important for biological function (42–44). Time-resolved X-ray absorption spectroscopy (XAS) (45) offers the advantage of interrogating the electronic and geometric structure of the biochemically active center of the system with elemental selectivity (i.e., the Fe atom). It was used to investigate the recombination of CO to Mb after its photodissociation from MbCO (46–48). In this case, ligand recombination occurs on times up to milliseconds (49), and the transient XAS was recorded using alternating intervals of data acquisition for the laser-excited sample at a 100-ps time delay and the unexcited sample, contrary to the pulse-to-pulse data acquisition where the unexcited and excited XASs are recorded sequentially (50). The resulting transient reflects the formation of the deoxy-Mb species (46), indicating the absence of GR of CO at this time delay. Very recently, Levantino et al. (51) reported a femtosecond XAS study of MbCO at the Fe K edge using the Stanford Free Electron Laser LCLS. Levantino et al. (51) presented kinetic traces exhibiting two time components (∼70 and ∼400 fs), which they tentatively attributed to a structural rearrangement induced by photolysis involving essentially only the heme chromophore and a residual Fe motion out of the heme plane that is coupled to the displacement of Mb F helix, respectively. However, without transient spectra, these conclusions are highly speculative.The high-repetition rate scheme for picosecond XAS studies (46) was originally developed to investigate photoinduced processes in highly dilute media (52), such as proteins in physiological solutions, which have concentrations (1–4 mM) that are one to two orders of magnitude lower than those of the metal complexes that we investigated (50). Here, we show for the first time, to our knowledge, its implementation to address the nature of the recombination of NO to the porphyrin Fe atom with 70-ps resolution. Nitrosyl-Mb (MbNO) is excited at 532 nm into the Q bands, and the system is probed at the Fe K edge near 7.12 keV. We carried out two series of experiments under somewhat different conditions, which are both described in SI Experimental Details, Figs. S1–S3, and 16 and 17, forming the domed hexacoordinated species, which relaxes to the planar configuration in ∼30 ps (20, 21). The experimental setups and sample preparation are described in SI Experimental Details. We also performed simulations of the transient X-ray absorption near-edge structure (XANES) spectra using multiple scattering theory (MST) (details in ref. 42 and SI Simulations of Transient XANES Spectra).Open in a separate windowFig. S1.Comparison of the time-resolved XAS signal at 70-ps time of the different series of measurements: series I (red) and series II (black). The static difference spectrum is in blue. Norm. Trans. Abs., normalized transient absorption.Open in a separate windowFig. S3.ei Sets of data points making up a full transient spectrum: e1 (Green), e2 (red), e3 (blue), and e4 (yellow). Note that not all datasets have been measured a similar number of times (details in SeriesSample type (window thickness, optical path; μm)Laser focus (μm²)Laser fluence (mJ/cm²)X-ray focus (μm²)Incident X-ray photons per pulseDetected X-ray (photons per point)Sample stabilityIFlat diamond flow cell (50, 250)85 × 656540 × 402 × 10⁴5 × 10⁶12 hII^*Cylindrical quartz capillary (10–20, 300)70 × 702430 × 302 × 10⁴5 × 10⁶24 h^†Open in a separate window^*Similar values for 300 ps.^†The sample was rereduced every 4 h, and each time, the UV-Vis spectrum immediately showed characteristic Soret and Q bands of MbNO. After 24 h, a new sample was used.

Table S4.

Statistics of the two series of measurements

Multitarget,quantitative nanoplasmonic electrical field-enhanced resonating device (NE2RD) for diagnostics

Recent advances in biosensing technologies present great potential for medical diagnostics, thus improving clinical decisions. However, creating a label-free general sensing platform capable of detecting multiple biotargets in various clinical specimens over a wide dynamic range, without lengthy sample-processing steps, remains a considerable challenge. In practice, these barriers prevent broad applications in clinics and at patients’ homes. Here, we demonstrate the nanoplasmonic electrical field-enhanced resonating device (NE²RD), which addresses all these impediments on a single platform. The NE²RD employs an immunodetection assay to capture biotargets, and precisely measures spectral color changes by their wavelength and extinction intensity shifts in nanoparticles without prior sample labeling or preprocessing. We present through multiple examples, a label-free, quantitative, portable, multitarget platform by rapidly detecting various protein biomarkers, drugs, protein allergens, bacteria, eukaryotic cells, and distinct viruses. The linear dynamic range of NE²RD is five orders of magnitude broader than ELISA, with a sensitivity down to 400 fg/mL This range and sensitivity are achieved by self-assembling gold nanoparticles to generate hot spots on a 3D-oriented substrate for ultrasensitive measurements. We demonstrate that this precise platform handles multiple clinical samples such as whole blood, serum, and saliva without sample preprocessing under diverse conditions of temperature, pH, and ionic strength. The NE²RD’s broad dynamic range, detection limit, and portability integrated with a disposable fluidic chip have broad applications, potentially enabling the transition toward precision medicine at the point-of-care or primary care settings and at patients’ homes.Biosensing platforms have enabled various applications in different fields of clinical medicine such as biomarker/drug discovery and initiation and monitoring of therapy (1–3). However, material cost, accessibility, ease of operation, lack of portability, and complexity in readout remain major challenges for developing robust diagnostic assays (SI Appendix, Table S1). Recent advances in nanotechnology and biosensing have created new avenues to address these issues (4–9). Technically, they have provided integration of high-throughput sampling with readout systems for quantitative detection of disease-specific biotargets. Therefore, they have demonstrated great potential to revolutionize medical diagnostics. However, from a clinical and technological perspective, existing platforms still face several challenges. First, lengthy assay time hinders physicians from making early clinical decisions. Second, examining clinical samples with diverse pH range, ionic content, and ionic strength requires extensive sample preprocessing steps to avoid signal fluctuations and inaccuracies, and this requirement increases the complexity of current systems. Third, temperature is an extrinsic factor that needs to be tightly controlled for reliable measurements, thus resulting in additional cost for developing diagnostic technologies. Fourth, a wide dynamic linear range is needed to distinguish concentrations of biotargets reliably. Fifth, sensitivity is hindered by the complex composition of biological specimens, thus requiring labeling (10, 11), which further increases assay cost significantly. Sixth, since a panel of parameters is monitored for complex diseases, detection of multiple biotargets is critically needed. Seventh, portability is another important parameter to deploy a biosensing platform at point-of-care (POC) settings. These practical barriers seriously reduce the broad clinical applicability of current platforms for diagnostic implementations.The development of various sensitive detection modalities has addressed some of these requirements. The mass-sensitive piezoelectric and microcantilever-based systems that use surface oscillations or changes in surface stress for molecular interactions and mass input (12, 13) have simplified sample-preparation steps. Likewise, electrical detection systems such as electrochemical sensors and interdigitated electrodes sense changes in molecular charges (14–17) and can provide affordable, simple, and high-throughput measurements. Further, plasmonic-based platforms that use an electrical field around metal surfaces/nanoparticles coupled with light (5, 18–22) have minimized readout complexity and demonstrated quantitative and sensitive measurements (23–26). In particular, surface plasmons on 3D-oriented surfaces generate highly sensitive spots that provide a sevenfold stronger electrical field than 2D-oriented plasmonic sensors (27–29). However, these advances have only reduced the effort required for sample preparation and the readout complexity, and some critical challenges remain unaddressed as summarized in ParametersMagneto-nanosensor chip (51)SMC (Singulex) (52)Simoa (Quanterix) (52)Bio-barcode assay (53)ChIP-NMR biosensor (54)Advanced SPR biosensor (55)Plasmonic ELISA (56)Plasmonic gold chip (36)NE²RDTargetLactoferrin, survivin, CEA, VEGF, EpCAM, G-CSF, TNF-α, and eotaxincTnI, cytokines, amyloid-β, IL-22PSA, TNF-α, TauPSAS. aureus, mouse macrophages, breast cancer cells, and multiple protein biomarkersE. coli O157:H7PSA and HIV-1 capsid antigen p24Insulin Ab, GAD65 Ab, and IA2 AbIFN-γ, carbamazepine, casein, E. coli, human lung adenocarcinoma epithelial cells, HBV, DENV-1, DENV-2, TVV-1, KSHV and HIV-1Assay time>2 h 20 minFour 96-well plates per day (for single assay, not multiplex)Five 96-well plates per day (for single assay, not multiplex)>3 h 30 min30 min for incubation and 10 min for analysis30 min>5 h 30 min<2 h1 h for incubation and 10 min for analysisMultiple biotarget detectionOnly protein biomarkersNot available10-plexNot availableBacteria, mammalian cells, and protein biomarkersNot availableOnly two protein biomarkersOnly autoimmune antibodiesProtein biomarkers, protein allergens, drugs, bacteria, eukaryotic cells, and virusesReadoutMagnetic field-based detectionFluorescent-based system (digital and analog at high concentrations)Fluorescent-based system (digital and analog at high concentrations)Scanometric detection for DNANMR signalLong-range surface plasmons enhanced by magnetic nanoparticlesOptical-based detectionNIR-FEPlasmonic signal on a 3D-oriented substrateLimit of detection1 pg/mL or 5 fM (with mono labeling)fg–pg/mLfg–pg/mL330 fg/mL5 pg/mL (∼1 ng in 5 µL)50 cfu/mL1 ag/mL0.1 kU/mL400 fg/mL and ∼100 copies/mL10 fg/mL or 50 aM (with dual labeling)Linear dynamic rangeSix orders of magnitudeFour orders of magnitudeFour orders of magnitudeTwo to three orders of magnitudeFour orders of magnitudeThree to four orders of magnitudeThree to four orders of magnitudeFour to five orders of magnitudeEight orders of magnitudeSample typePBS, serum, urine, and salivaPlasma, serum, CSF, cell lysate, urine, human brain tissue homogenateSerumPBS and serumPBS and serumPBSPBSWhole serum or bloodPBS, whole saliva, serum, and whole bloodOpen in a separate windowCEA, carcinoembryonic antigen; CFS, cerebrospinal fluid; EpCAM, epithelial cell adhesion molecule; GAD65, glutamic acid decarboxylase-65; G-CSF, granulocyte-colony stimulating factor; IA2, islet antigen-2; NIR-FE, near-infrared fluorescence-enhanced spectroscopy; PSA, prostate-specific antigen; TNF-α, tumor necrosis factor-alpha; VEGF, vascular endothelial growth factor.Here, we report the development of an innovative device, the nanoplasmonic electrical field-enhanced resonating device (NE²RD), which addresses all these challenges on a single platform. A comparison of the NE²RD capability with that of existing technologies is presented in SI Appendix, Tables S1 and S2. The NE²RD employs a label-free optical readout that precisely measures collective oscillations of nanoparticles leading to an unprecedented platform sensitivity that is enabled by hot-spot regions on the surface, where the internanoparticle distance is reduced by self-assembly (Fig. 1). This strategy uniquely enables highly sensitive detection to monitor binding and capture events of biotargets. Thus, each well or chip surface of the current prototypes individually measures a change in spectral color that is translated into a quantitative biotarget concentration in a multitarget manner. Further, the NE²RD can be implemented into a disposable fluidic chip format, and the measurements are collected using a customized portable measurement tool. The NE²RD uses only fingerprick volumes of clinical samples without the need for sample preprocessing steps, thus providing a rapid assay and potentially enabling direct translation to the clinic.Open in a separate windowFig. 1.Representation of the NE²RD platform. (A) Gold nanoparticles are immobilized on polystyrene surfaces using poly-l-lysine for fabrication of the NE²RD. (B) In surface chemistry steps, several activators and antibody anchors are used to immobilize antibodies onto the surface. (C) Biological samples such as whole blood are applied to the surfaces without sample preprocessing. (D) Unbound cells and cellular components are washed out with physiological buffer. (E) Multiple biotargets of interest are captured specifically, depending on the antibodies immobilized on the surface, and spectral measurements are performed to monitor capture events, which change the spectral color of the surfaces in terms of both wavelength and extinction intensity parameters. Yusuf Yesil drew all schematics in Fig. 1.     

Variation in the schedules of somite and neural development in frogs

The timing of notochord, somite, and neural development was analyzed in the embryos of six different frog species, which have been divided into two groups, according to their developmental speed. Rapid developing species investigated were Xenopus laevis (Pipidae), Engystomops coloradorum, and Engystomops randi (Leiuperidae). The slow developers were Epipedobates machalilla and Epipedobates tricolor (Dendrobatidae) and Gastrotheca riobambae (Hemiphractidae). Blastopore closure, notochord formation, somite development, neural tube closure, and the formation of cranial neural crest cell-streams were detected by light and scanning electron microscopy and by immuno-histochemical detection of somite and neural crest marker proteins. The data were analyzed using event pairing to determine common developmental aspects and their relationship to life-history traits. In embryos of rapidly developing frogs, elongation of the notochord occurred earlier relative to the time point of blastopore closure in comparison with slowly developing species. The development of cranial neural crest cell-streams relative to somite formation is accelerated in rapidly developing frogs, and it is delayed in slowly developing frogs. The timing of neural tube closure seemed to be temporally uncoupled with somite formation. We propose that these changes are achieved through differential timing of developmental modules that begin with the elongation of the notochord during gastrulation in the rapidly developing species. The differences might be related to the necessity of developing a free-living tadpole quickly in rapid developers.The transformation of the spherical egg into the elongated body of the larva and adult is a major event in amphibian development. In Xenopus laevis, body elongation begins in the mid-gastrula with the formation and elongation of the notochord (1). The process of body elongation in X. laevis is guided by convergent extension (2). Convergent extension is the process by which the presumptive notochord and neural plate lengthen and narrow owing to the morphogenetic movements of mediolateral cell intercalation (2), and it involves the genetic control of planar cell polarity pathway genes in mesoderm and neural tissues (1, 3–7). Planar cell polarity signaling mediated by dishevelled and strabismus (also called lpp1, ltap, stb1, stbm, and vangl2) is required for elongation of the notochord and neural plate and for the initiation of neural tube closure (3, 6, 8, 9). However, different components of the Wnt signaling network are involved in X. laevis convergent extension of the mesoderm and in the closure of the neural tube (3). Besides X. laevis, the molecular control of convergent extension has been investigated in the zebrafish (9). In addition, the onset of convergent extension, detected by the expression of the proteins brachyury and lhx1 (previously known as Lim1) in the notochord, is known for several species of anurans (10–12). We investigated the variation in the number of somites and in the advancement of neural development in frogs that differ in their reproductive strategies, developmental rate, and in the timing of notochord elongation (13). In contrast, the poison-arrow frogs Epipedobates machalilla and Epipedobates tricolor deposit fewer eggs in terrestrial nests (14, 15). In the case of the marsupial frog Gastrotheca riobambae, the developing embryos are transported in a pouch of integument by the mother (14, 15). The analyzed frogs belong to four different families and have notable differences in egg size (13, 15, 16). In the rapidly developing embryos of E. coloradorum and E. randi, elongation of the notochord was detected at the mid-gastrula stage, as in the embryos of X. laevis (12, 15, 17). In contrast, in the slowly developing embryos of E. machalilla, E. tricolor, and G. riobambae, elongation of the notochord and consequently of the body begins only after the process of gastrulation is completed and the blastopore is closed (15, 17, 18) (8, 10). In particular, convergent extension is a module that can be superimposed or separated from other gastrulation morphogenetic movements in the embryogenesis of different frogs (14, 15, 18). Elongation of the notochord occurred earlier relative to the time point of blastopore closure in rapidly developing embryos, in contrast with slowly developing species. Moreover, the development of cranial neural crest cell-streams is accelerated relative to somite formation in rapidly developing frogs, and it is delayed in slowly developing frogs. Acceleration of body elongation and of cranial neural crest cell-stream development occurs in embryos of frog species that require the rapid development of free-living tadpoles.

Table 1.

Developmental characteristics of the frogs

High risk of near-crash driving events following night-shift work

Night-shift workers are at high risk of drowsiness-related motor vehicle crashes as a result of circadian disruption and sleep restriction. However, the impact of actual night-shift work on measures of drowsiness and driving performance while operating a real motor vehicle remains unknown. Sixteen night-shift workers completed two 2-h daytime driving sessions on a closed driving track at the Liberty Mutual Research Institute for Safety: (i) a postsleep baseline driving session after an average of 7.6 ± 2.4 h sleep the previous night with no night-shift work, and (ii) a postnight-shift driving session following night-shift work. Physiological measures of drowsiness were collected, including infrared reflectance oculography, electroencephalography, and electrooculography. Driving performance measures included lane excursions, near-crash events, and drives terminated because of failure to maintain control of the vehicle. Eleven near-crashes occurred in 6 of 16 postnight-shift drives (37.5%), and 7 of 16 postnight-shift drives (43.8%) were terminated early for safety reasons, compared with zero near-crashes or early drive terminations during 16 postsleep drives (Fishers exact: P = 0.0088 and P = 0.0034, respectively). Participants had a significantly higher rate of lane excursions, average Johns Drowsiness Scale, blink duration, and number of slow eye movements during postnight-shift drives compared with postsleep drives (3.09/min vs. 1.49/min; 1.71 vs. 0.97; 125 ms vs. 100 ms; 35.8 vs. 19.1; respectively, P < 0.05 for all). Night-shift work increases driver drowsiness, degrading driving performance and increasing the risk of near-crash drive events. With more than 9.5 million Americans working overnight or rotating shifts and one-third of United States commutes exceeding 30 min, these results have implications for traffic and occupational safety.Between 2009 and 2013 in the United States, a drowsy driver was involved in an estimated 21% of fatal crashes and 13% of crashes causing severe injury, consistent with earlier estimates that drowsy driving causes 20% of serious motor vehicle crash injuries resulting in hospitalization or death, and is associated with a four- to sixfold increase in crash/near-crash risk (1–4). These data indicate that drowsiness is likely one of the most prevalent causes of preventable road crashes worldwide (5). Most drivers admit to driving while drowsy, with 28% reporting falling asleep while driving within the past year (6). The nation’s 9.5 million shift workers, comprising 15% of the workforce (7), are at particular risk of drowsy driving (8). Night-shift work increases the risk for drowsy driving crashes, especially on the morning commute home from overnight work (9–11), when elevated homeostatic sleep pressure interacts with the peak of circadian sleep propensity to create a critical zone of performance vulnerability (10–12).Despite the high prevalence of drowsy driving and sleep-related crashes, little is known about the characteristics of driver impairment preceding or during a drowsiness-related critical driving event. Increased weaving in the lane and episodes of lane crossing occur during prolonged nocturnal driving, similar to behaviors observed in drivers with elevated blood alcohol concentrations (13). Small steering corrections are reduced in drowsy drivers, who make infrequent large steering corrections (14). Physiological signs of drowsiness identified during night-time driving include brief sleep episodes and partial eyelid closure with slow eye movements (15). However, few studies have related these physiological changes to behavioral signs of impaired driving. After a night shift, increased out-of-lane events, subjective sleepiness, and blink duration are evident during simulated driving (16, 17).Drowsiness and crash risk also increase with drive duration (13, 18–23). During simulated driving, sleep-deprived drivers show increased subjective sleepiness, reaction time, and improper lane crossings with increasing drive duration (18). Corrective steering wheel movements (22) and variation in car speed and lateral position (23) deteriorate over time, with severe driving impairment during nighttime highway driving after 2 h (13). Although few studies have evaluated the impact of drive duration on physiological indices of drowsiness, ocular measures of drowsiness appear to increase during the latter part of the commute home after the night shift (21). As such, the effects of circadian disruption and sleep restriction exhibited in night-shift workers are likely to be exacerbated as the homeward commute becomes prolonged.The goals of this study were to test the hypotheses that night-shift work results in increased objective and subjective drowsiness and impaired driving performance, and that drive duration increases the risk of these events. To test these hypotheses, actual night-shift workers (VariableMean (SD)RangeAge (y)48.7 (14.8)19–65Driving experience (y)27.4 (16.5)3–50Weight (lbs)184.3 (32.0)151–245Height (inches)62.9 (17.2)61–76Body mass index29.1 (6.0)19.2–40.7ChronotypeMorning type (n = 2)Intermediate type (n = 11)Evening type (n = 2)Risk of sleep apnea5 of 16Open in a separate window     

Self-assembly of amphiphilic Janus dendrimers into uniform onion-like dendrimersomes with predictable size and number of bilayers

A constitutional isomeric library synthesized by a modular approach has been used to discover six amphiphilic Janus dendrimer primary structures, which self-assemble into uniform onion-like vesicles with predictable dimensions and number of internal bilayers. These vesicles, denoted onion-like dendrimersomes, are assembled by simple injection of a solution of Janus dendrimer in a water-miscible solvent into water or buffer. These dendrimersomes provide mimics of double-bilayer and multibilayer biological membranes with dimensions and number of bilayers predicted by the Janus compound concentration in water. The simple injection method of preparation is accessible without any special equipment, generating uniform vesicles, and thus provides a promising tool for fundamental studies as well as technological applications in nanomedicine and other fields.Most living organisms contain single-bilayer membranes composed of lipids, glycolipids, cholesterol, transmembrane proteins, and glycoproteins (1). Gram-negative bacteria (2, 3) and the cell nucleus (4), however, exhibit a strikingly special envelope that consists of a concentric double-bilayer membrane. More complex membranes are also encountered in cells and their various organelles, such as multivesicular structures of eukaryotic cells (5) and endosomes (6), and multibilayer structures of endoplasmic reticulum (7, 8), myelin (9, 10), and multilamellar bodies (11, 12). This diversity of biological membranes inspired corresponding biological mimics. Liposomes (Fig. 1) self-assembled from phospholipids are the first mimics of single-bilayer biological membranes (13–16), but they are polydisperse, unstable, and permeable (14). Stealth liposomes coassembled from phospholipids, cholesterol, and phospholipids conjugated with poly(ethylene glycol) exhibit improved stability, permeability, and mechanical properties (17–20). Polymersomes (21–24) assembled from amphiphilic block copolymers exhibit better mechanical properties and permeability, but are not always biocompatible and are polydisperse. Dendrimersomes (25–28) self-assembled from amphiphilic Janus dendrimers and minidendrimers (26–28) have also been elaborated to mimic single-bilayer biological membranes. Amphiphilic Janus dendrimers take advantage of multivalency both in their hydrophobic and hydrophilic parts (23, 29–32). Dendrimersomes are assembled by simple injection (33) of a solution of an amphiphilic Janus dendrimer (26) in a water-soluble solvent into water or buffer and produce uniform (34), impermeable, and stable vesicles with excellent mechanical properties. In addition, their size and properties can be predicted by their primary structure (27). Amphiphilic Janus glycodendrimers self-assemble into glycodendrimersomes that mimic the glycan ligands of biological membranes (35). They have been demonstrated to be bioactive toward biomedically relevant bacterial, plant, and human lectins, and could have numerous applications in nanomedicine (20).Open in a separate windowFig. 1.Strategies for the preparation of single-bilayer vesicles and multibilayer onion-like vesicles.More complex and functional cell mimics such as multivesicular vesicles (36, 37) and multibilayer onion-like vesicles (38–40) have also been discovered. Multivesicular vesicles compartmentalize a larger vesicle (37) whereas multibilayer onion-like vesicles consist of concentric alternating bilayers (40). Currently multibilayer vesicles are obtained by very complex and time-consuming methods that do not control their size (39) and size distribution (40) in a precise way. Here we report the discovery of “single–single” (28) amphiphilic Janus dendrimer primary structures that self-assemble into uniform multibilayer onion-like dendrimersomes (Fig. 1) with predictable size and number of bilayers by simple injection of their solution into water or buffer.     

Global biogeography of human infectious diseases

The distributions of most infectious agents causing disease in humans are poorly resolved or unknown. However, poorly known and unknown agents contribute to the global burden of disease and will underlie many future disease risks. Existing patterns of infectious disease co-occurrence could thus play a critical role in resolving or anticipating current and future disease threats. We analyzed the global occurrence patterns of 187 human infectious diseases across 225 countries and seven epidemiological classes (human-specific, zoonotic, vector-borne, non–vector-borne, bacterial, viral, and parasitic) to show that human infectious diseases exhibit distinct spatial grouping patterns at a global scale. We demonstrate, using outbreaks of Ebola virus as a test case, that this spatial structuring provides an untapped source of prior information that could be used to tighten the focus of a range of health-related research and management activities at early stages or in data-poor settings, including disease surveillance, outbreak responses, or optimizing pathogen discovery. In examining the correlates of these spatial patterns, among a range of geographic, epidemiological, environmental, and social factors, mammalian biodiversity was the strongest predictor of infectious disease co-occurrence overall and for six of the seven disease classes examined, giving rise to a striking congruence between global pathogeographic and “Wallacean” zoogeographic patterns. This clear biogeographic signal suggests that infectious disease assemblages remain fundamentally constrained in their distributions by ecological barriers to dispersal or establishment, despite the homogenizing forces of globalization. Pathogeography thus provides an overarching context in which other factors promoting infectious disease emergence and spread are set.The distributions of the vast majority of infectious agents causing disease in humans have not been resolved (1). However, unknown and poorly known agents make a significant contribution to the global burden of disease (e.g., ref. 2) and are likely to underlie many future disease impacts (e.g., ref. 3). In the absence of disease- or pathogen-specific data, existing patterns of infectious disease occurrence may provide the first insights into the spatial distributions of many disease risks. These insights could be leveraged to research, survey, define, or manage burgeoning or poorly understood infectious disease risks more efficiently (4–8).Previous studies have found some striking patterns in the geographic distributions of human infectious diseases, including richness gradients (e.g., more diseases in the tropics relative to higher latitudes, more diseases on larger islands relative to smaller ones) (9–11), nestedness patterns (e.g., disease assemblages at higher latitudes are subsets of larger assemblages toward the tropics) (9), and varying geographic range sizes according to latitude and for varying types of diseases (12–14). Despite these patterns, the biogeography of human infectious diseases remains poorly explored in comparison to other biological taxa (15–18) and biogeographic insights into human infectious diseases have been little explored for public or global health applications.Biogeographic patterns have routinely underpinned efforts to discover, monitor, and manage global biodiversity for almost 150 y (19–21). Better understanding the broad biogeographical patterns of human infectious diseases, why diseases occur in some places but not others, or how the presence of a disease in one place might relate to the likelihood of its presence in another thus have considerable potential for decomposing, monitoring, and managing the risks currently faced by the global health community (4). Potential applications range from focusing outbreak investigations, pathogen discovery strategies, risk assessments, and disease surveillance to disease management and mitigation (5, 7, 8).Here, we make use of the most comprehensive infectious disease occurrence database currently available at a global scale (22, 23) to analyze the occurrence patterns of 187 human infectious diseases in 225 geopolitical regions (hereafter countries) and for seven nonexclusive disease classes with varying epidemiological features: human-specific, zoonotic, vector-borne, non–vector-borne, bacterial, viral, and parasitic diseases (16, 24) to represent the change in infectious disease assemblages among countries spatially at a global level. From this metric, we derive a region-specific beta diversity measure, which we term the co-zone layer, for its ability to illustrate the connectivity in existing disease occurrence patterns among regions. We then test whether these patterns could be leveraged for baseline infectious disease risk assessments in the absence of disease-specific data, using outbreaks of Ebola virus as a topical model system for which data are available for validation (25). Finally, we explore a range of environmental (climate, latitude, land area, and mammalian biodiversity) and social (human flight traffic, health expenditure, population size, and biases in observation effort) factors for their abilities to explain these biogeographic patterns after controlling for spatial dependence (26), providing insights into the potential drivers of shared disease risks.

Table 1.

Composition of infectious disease data analyzed in this study

Lipid domains control myelin basic protein adsorption and membrane interactions between model myelin lipid bilayers

The surface forces apparatus and atomic force microscope were used to study the effects of lipid composition and concentrations of myelin basic protein (MBP) on the structure of model lipid bilayers, as well as the interaction forces and adhesion between them. The lipid bilayers had a lipid composition characteristic of the cytoplasmic leaflets of myelin from “normal” (healthy) and “disease-like” [experimental allergic encephalomyelitis (EAE)] animals. They showed significant differences in the adsorption mechanism of MBP. MBP adsorbs on normal bilayers to form a compact film (3–4 nm) with strong intermembrane adhesion (∼0.36 mJ/m²), in contrast to its formation of thicker (7–8 nm) swelled films with weaker intermembrane adhesion (∼0.13 mJ/m²) on EAE bilayers. MBP preferentially adsorbs to liquid-disordered submicron domains within the lipid membranes, attributed to hydrophobic attractions. These results show a direct connection between the lipid composition of membranes and membrane–protein adsorption mechanisms that affects intermembrane spacing and adhesion and has direct implications for demyelinating diseases.Myelin is an asymmetric multilamellar membrane wrapped around the axons of the central nervous system (CNS) and consists of alternating extracellular and cytoplasmic leaflets (1–3). The bilayer-associated proteins, mainly myelin basic protein (MBP) and proteolipid protein, play an essential role in stabilizing and maintaining the myelin structure. The bilayers are in close contact (∼3 nm separation between lipid headgroup–water interfaces), providing a low dielectric constant through the compact bilayers, which is essential for efficient and fast saltatory propagation of nerve impulses. Any structural changes of the myelin sheath in the CNS, including lesion formation, loss of adhesion, swelling of the water gaps, vacuolization, vesiculation, and complete delamination (demyelination) of the myelin sheath (4–6), are signatures of several inflammatory neurological disorders. These types of disorders are characterized by a broad spectrum of neurological symptoms, such as physical and cognitive disabilities, with multiple sclerosis (MS) being one of the most common demyelinating diseases (2).The primary cause of structural changes in the myelin is still under debate; however, morphological changes of the myelin structure due to diseases such as MS are well known. A well-studied and accepted animal model for MS is the experimental allergic encephalomyelitis (EAE) of the marmoset (2, 6). Using this model, recent studies conducted with the surface forces apparatus (SFA) have shown that a loss of adhesion force (7) and structural changes of model membranes with lipid composition characteristic of myelin accompanied compositional alterations of the lipid species (8), as well as an electric charge imbalance between lipid molecules and MBP (9). These alterations also change the lateral distribution and stability of phase-separated lipid domains (or rafts) within model myelin membranes (8, 10).MBP is one of the most abundant proteins in the CNS and is an intrinsically unstructured (disordered) protein (11). MBP acts as an intermembrane adhesion protein between the cytoplasmic leaflets of the myelin sheath. The predominant size and charge isoform of MBP in healthy and mature myelin has a molecular weight of 18.5 kDa and a net positive charge of 19 (12, 13). Several studies conducted with model and extracted myelin bilayers (9, 14–19) showed that because of its high content of positively charged residues, MBP binds to the negatively charged lipids of the cytoplasmic leaflets of the bilayer via electrostatic interaction in addition to hydrophobic interactions. However, studies have shown that the MBP charge component composition, as well as the balance between charged lipids and MBP charge components, changes in EAE (as well as in MS) tissues (20, 21). Other studies (9, 15) demonstrated the importance of the hydrophobic interaction between MBP and lipid membranes by showing that MBP specifically binds to lipid domain boundaries (defects), altering the lateral organization of the model myelin bilayers. A recent study also showed that MBP’s association with the cytoplasmic leaflet of the myelin membrane induces a phase transition into a cohesive mesh-like protein network (22, 23).One open question regarding the stability of myelin membranes is how the concentration of MBP affects the interaction forces between myelin bilayers. We reconstructed and used two types of supported model monolayers with a lipid composition characteristic of “normal” or “healthy” and “disease-like” EAE myelin deposited on a dipalmitoylphosphatidylethanolamine (DPPE) monolayer to examine the effect of lipid composition, domains, and fluidity on the interaction forces, film viscosity, and MBP adsorption mechanism between these model myelin bilayers. The lipid composition used is based on data from Inouye and Kirschner (1, 24) and Ohler et al. (6) (25), and the interaction forces were measured using surface forces apparatus (SFA) model 2000 (26), with the capacity of measuring interaction forces and separation distances between macroscopic surfaces with a resolution of 10 nN and 0.1 nm, respectively. The MBP adsorption mechanism onto the bilayers also was examined using an atomic force microscope (AFM). This study aims to establish the relationship between the structure of the model lipid myelin bilayers and protein adsorption and also to quantify the effect of MBP concentration on the intermembrane interaction forces.

Table 1.

Lipid compositions used for the reconstituted cytoplasmic myelin lipid monolayers

Intrasperm vertical symbiont transmission

Symbiotic bacteria are commonly associated with cells and tissues of diverse animals and other organisms, which affect hosts’ biology in a variety of ways. Most of these symbionts are present in the cytoplasm of host cells and maternally transmitted through host generations. The paucity of paternal symbiont transmission is likely relevant to the extremely streamlined sperm structure: the head consisting of condensed nucleus and the tail made of microtubule bundles, without the symbiont-harboring cytoplasm that is discarded in the process of spermatogenesis. Here, we report a previously unknown mechanism of paternal symbiont transmission via an intrasperm passage. In the leafhopper Nephotettix cincticeps, a facultative Rickettsia symbiont was found not only in the cytoplasm but also in the nucleus of host cells. In male insects, strikingly, most sperm heads contained multiple intranuclear Rickettsia cells. The Rickettsia infection scarcely affected the host fitness including normal sperm functioning. Mating experiments revealed both maternal and paternal transmission of the Rickettsia symbiont through host generations. When cultured with mosquito and silkworm cell lines, the Rickettsia symbiont was preferentially localized within the insect cell nuclei, indicating that the Rickettsia symbiont itself must have a mechanism for targeting nucleus. The mechanisms underlying the sperm head infection without disturbing sperm functioning are, although currently unknown, of both basic and applied interest.Endocellular bacterial symbionts are commonly found in diverse eukaryotes including animals, plants, fungi, and protists (1–7). In the majority of these cases, the symbionts are located in the cytoplasm of the host cells. Whereas the cytoplasmic symbionts are simply passed to daughter cells through host cell division in unicellular protists (6, 7), sex-related asymmetry in vertical symbiont transmission is generally observed in multicellular metazoans with sexual reproduction. Namely, the symbionts are transmitted vertically to the next host generation via infection to eggs in the maternal body, but not via infection to sperms (8, 9). Exceptional reports of paternal symbiont transmission are venereal transmission cases of several symbiotic bacteria (10, 11) and biparental transmission cases of some symbiotic viruses (12). Oocytes accumulate a large quantity of cytoplasm that provide a room for symbiont infection, whereas sperms discard their cytoplasm (together with inhabiting symbiotic bacteria) during spermatogenesis and transform into a streamlined shape with the small head consisting of condensed nucleus and the slender tail made of microtubule bundles for motility (13, 14). Therefore, if such symbiotic bacterial cells can be transmitted via sperm, a possible target may be the sperm head nucleus. Intranuclear bacterial symbionts, such as Holospora and Caedibacter, have been relatively well-documented from unicellular ciliates (6, 15), but reported only rarely from multicellular metazoans (16, 17). In insects and other arthropods, intracellular Rickettsia and Orientia pathogens/symbionts are sometimes observed to localize not only to the cytoplasm, but also to the nucleus of the host cells (18–21). In bathymodiolin mussels inhabiting hydrothermal vents and cold seeps, intranuclear bacterial parasites “Candidatus Endonucleobacter bathymodioli” have been described (16). Thus far, no case of paternal symbiont transmission via intrasperm passage has been reported. Considering the extremely streamlined sperm structure, bacterial infection to the sperm head nucleus is expected to impair genetic material and normal functioning of the sperm and, thus, intrasperm vertical symbiont transmission may seem unlikely to occur. In this study, however, we demonstrate that such a case exists in an insect.The green rice leafhopper Nephotettix cincticeps (Uhler) (Hemiptera: Cicadellidae) (Fig. 1A), known as a notorious pest of rice in East Asia, is associated with two bacteriome-associated obligate symbionts, Sulcia and Nasuia, and a facultative symbiont of the genus Rickettsia (22). The Rickettsia symbiont of N. cincticeps represents a basal lineage of the genus Rickettsia (Fig. 1B) and exhibits high infection frequencies among N. cincticeps strains established from natural populations in Japan (23, 24). Here we report that, in N. cincticeps, the Rickettsia symbiont efficiently targets and infects the host’s cell nuclei including sperm head nuclei, and vertically transmitted to the next host generation not only maternally via ovarial passage but also paternally via intrasperm passage with high fidelity.Open in a separate windowFig. 1.The green rice leafhopper N. cincticeps and its Rickettsia symbiont. (A) An adult male of N. cincticeps. (B) Phylogenetic placement of the Rickettsia symbiont of N. cincticeps on the basis of 16S rRNA gene sequence. A Bayesian phylogeny inferred from 1,296 aligned nucleotide sites is shown. Posterior probabilities for the Bayesian phylogeny and bootstrap probabilities for the maximum likelihood phylogeny at 50% or higher are shown at the nodes, whereas asterisks indicate support values lower than 50%. Sequence accession numbers are shown in brackets. Major Rickettsia groups (40) are indicated on the right side.

Table 1.

Rickettsia infection frequencies in laboratory strains of N. cincticeps derived from different natural populations in Japan

Engineering super mycovirus donor strains of chestnut blight fungus by systematic disruption of multilocus vic genes

Transmission of mycoviruses that attenuate virulence (hypovirulence) of pathogenic fungi is restricted by allorecognition systems operating in their fungal hosts. We report the use of systematic molecular gene disruption and classical genetics for engineering fungal hosts with superior virus transmission capabilities. Four of five diallelic virus-restricting allorecognition [vegetative incompatibility (vic)] loci were disrupted in the chestnut blight fungus Cryphonectria parasitica using an adapted Cre-loxP recombination system that allowed excision and recycling of selectable marker genes (SMGs). SMG-free, quadruple vic mutant strains representing both allelic backgrounds of the remaining vic locus were then produced through mating. In combination, these super donor strains were able to transmit hypoviruses to strains that were heteroallelic at one or all of the virus-restricting vic loci. These results demonstrate the feasibility of modulating allorecognition to engineer pathogenic fungi for more efficient transmission of virulence-attenuating mycoviruses and enhanced biological control potential.Mycovirus infections have been reported to reduce virulence (hypovirulence) for a wide range of plant pathogenic fungi, providing potential for biological disease control (1–6). For hypovirulence to be effective, the virulence-attenuating viruses must be efficiently transmitted from infected hypovirulent strains to uninfected virulent strains (5, 7). Mycoviruses generally have evolved exclusive intracellular lifestyles (8). With very few exceptions (9), mycovirus infections cannot be initiated by exposure of uninfected hyphae to cell extracts or secretions from infected fungal isolates. Transmission is limited to intracellular mechanisms, vertical transmission through asexual spores, and horizontal transmission through anastomosis (fusion of hyphae).Horizontal mycovirus transmission to uninfected isolates of the same fungal species is complicated by nonself allorecognition genetic systems, termed “heterokaryon” or “vegetative incompatibility” (vic), which operate widely in filamentous fungi (10). Interactions between genetically distinct individuals of the same species result in an incompatible reaction that triggers localized programmed cell death (PCD), forming a line of demarcation, or barrage, along the zone of contact (10–12) and restricting cytoplasmic transmission of viruses and other cytoplasmic elements (1, 13–15).A negative correlation between vic diversity and virus transmission has been reported for several fungal hosts (1, 16), but has most extensively been demonstrated for the chestnut blight fungus Cryphonectria parasitica infected with virulence-attenuating hypoviruses (7, 17–20). Genetic analyses have defined six diallelic vic genetic loci for C. parasitica (21). These loci and associated genes were recently identified at the molecular level through a comparative genomics approach (22, 23) (22, 23) provided formal confirmation that five of the six loci contribute to restriction of virus transmission; an allelic difference at vic4 was shown previously not to restrict virus transmission (24). Moreover, all but one of these mutations resulted in no observable phenotypic change other than increased virus transmission or loss of the incompatibility reaction (22, 23). We now report the use of sequential vic gene disruption (23, 25) and mating approaches to engineer C. parasitica super hypovirus donor strains. The results demonstrate the feasibility of global modulation of fungal host allorecognition systems to remove restrictions to mycovirus transmission.

Table 1.

Summary of C. parasitica vic genetic loci and associated genes

Chronological evidence fails to support claim of an isochronous widespread layer of cosmic impact indicators dated to 12,800 years ago

According to the Younger Dryas Impact Hypothesis (YDIH), ∼12,800 calendar years before present, North America experienced an extraterrestrial impact that triggered the Younger Dryas and devastated human populations and biotic communities on this continent and elsewhere. This supposed event is reportedly marked by multiple impact indicators, but critics have challenged this evidence, and considerable controversy now surrounds the YDIH. Proponents of the YDIH state that a key test of the hypothesis is whether those indicators are isochronous and securely dated to the Younger Dryas onset. They are not. We have examined the age basis of the supposed Younger Dryas boundary layer at the 29 sites and regions in North and South America, Europe, and the Middle East in which proponents report its occurrence. Several of the sites lack any age control, others have radiometric ages that are chronologically irrelevant, nearly a dozen have ages inferred by statistically and chronologically flawed age–depth interpolations, and in several the ages directly on the supposed impact layer are older or younger than ∼12,800 calendar years ago. Only 3 of the 29 sites fall within the temporal window of the YD onset as defined by YDIH proponents. The YDIH fails the critical chronological test of an isochronous event at the YD onset, which, coupled with the many published concerns about the extraterrestrial origin of the purported impact markers, renders the YDIH unsupported. There is no reason or compelling evidence to accept the claim that a cosmic impact occurred ∼12,800 y ago and caused the Younger Dryas.The Younger Dryas Impact Hypothesis (YDIH) proposes that at 12,800 ± 150 (or 12,900 ± 100) calendar years before present (cal B.P.), North America experienced an extraterrestrial event variously described as an impact (or impacts), airburst (or airbursts), or some combination thereof (1, 2). [In earlier publications, 12,900 ± 100 calendar years before present was identified as the age of the YDB, based on the IntCal04 radiocarbon calibration curve. YDIH proponents subsequently changed the YDB date after introduction of IntCal09. Such a change is fully appropriate, given the refinements in the calibration (IntCal09 has now been superseded by IntCal13, but we do not use the latter to insure analytical comparability [Methods]). In discussions of specific sites we use the same calibration curve as the original work. Where relevant, radiocarbon ages are presented both as ¹⁴C years B.P. and in calibrated years (cal B.P.).] This event is claimed to have been so significant that it abruptly triggered the global onset of the Younger Dryas (YD), a millennium-long cooling episode that interrupted the warming that had been taking place as the Pleistocene came to an end and in North America ostensibly ignited continent-wide wildfires, caused the extinction of several dozen genera of Pleistocene mammals, and led to the termination of the Clovis culture (ref. 2, p. 16021), the earliest archaeologically well-documented group occupying the continent. First introduced in a popular book in 2006 (1) and soon thereafter to the scientific community (2), YDIH proponents have offered geological and geochemical evidence of an extraterrestrial impact from localities in and outside of North America (3–11). Independent investigators have provided support as well (12, 13).According to YDIH proponents, that cosmic event is marked in terrestrial deposits by magnetic grains with iridium, magnetic microspherules, charcoal, soot, carbon spherules, glass-like carbon containing nanodiamonds, and fullerenes with extraterrestrial helium (cf. refs. 14–15). High concentrations of these indicators are said to be found in “a thin, sedimentary layer (usually <5 cm)” that they label the Younger Dryas boundary (YDB) layer (ref. 2, p. 16,017). The YDB is reportedly capped at some sites by a carbon-rich layer likened to the black mat previously identified by Haynes (16). The YDB is said to represent the extraterrestrial impact, and the black mat is said to represent subsequent impact-related processes “such as climate change and biomass burning” (ref. 2, p. 16,019). YDIH proponents claim that the supposed YDB layer is securely dated to a 300-y span centered on 12,800 cal B.P. (or a 200-y span centered on 12,900 cal B.P. depending on the calibration used).From the outset the YDIH has been highly controversial. The reproducibility, reliability, and validity of the impact indicators have been challenged, not least because many of these may be terrestrial in origin (possibly volcanic, organic, or detrital) and have been found in deposits younger and older than the YD (14, 15, 17–27). Others have questioned the physics of the supposed impact and whether it could or did have consequences for Late Pleistocene environments, animals, and people (26, 28–32).One key test of the YDIH, however, has been largely lacking: whether the supposed YDB layer securely dates to the Younger Dryas onset (27, 33). Knowing its precise age and demonstrating that it is isochronous across sites that reportedly extend from North America to South America and Europe (34, p. 2531). We provide just such an analysis here.

Table 1.

Sites reportedly dated to the onset of the Younger Dryas and yielding impact markers