The developing bird pelvis passes through ancestral dinosaurian conditions.

Living birds (Aves) have bodies substantially modified from the ancestral reptilian condition. The avian pelvis in particular experienced major changes during the transition from early archosaurs to living birds1,2. This stepwise transformation is well documented by an excellent fossil record2-4; however, the ontogenetic alterations that underly it are less well understood. We used embryological imaging techniques to examine the morphogenesis of avian pelvic tissues in three dimensions, allowing direct comparison with the fossil record. Many ancestral dinosaurian features2 (for example, a forward-facing pubis, short ilium and pubic 'boot') are transiently present in the early morphogenesis of birds and arrive at their typical 'avian' form after transitioning through a prenatal developmental sequence that mirrors the phylogenetic sequence of character acquisition. We demonstrate quantitatively that avian pelvic ontogeny parallels the non-avian dinosaur-to-bird transition and provide evidence for phenotypic covariance within the pelvis that is conserved across Archosauria. The presence of ancestral states in avian embryos may stem from this conserved covariant relationship. In sum, our data provide evidence that the avian pelvis, whose early development has been little studied5-7, evolved through terminal addition-a mechanism8-10 whereby new apomorphic states are added to the end of a developmental sequence, resulting in expression8,11 of ancestral character states earlier in that sequence. The phenotypic integration we detected suggests a previously unrecognized mechanism for terminal addition and hints that retention of ancestral states in development is common during evolutionary transitions.

Increasing crop field size does not consistently exacerbate insect pest problems.

Increasing diversity on farms can enhance many key ecosystem services to and from agriculture, and natural control of arthropod pests is often presumed to be among them. The expectation that increasing the size of monocultural crop plantings exacerbates the impact of pests is common throughout the agroecological literature. However, the theoretical basis for this expectation is uncertain; mechanistic mathematical models suggest instead that increasing field size can have positive, negative, neutral, or even nonlinear effects on arthropod pest densities. Here, we report a broad survey of crop field-size effects: across 14 pest species, 5 crops, and 20,000 field years of observations, we quantify the impact of field size on pest densities, pesticide applications, and crop yield. We find no evidence that larger fields cause consistently worse pest impacts. The most common outcome (9 of 14 species) was for pest severity to be independent of field size; larger fields resulted in less severe pest problems for four species, and only one species exhibited the expected trend of larger fields worsening pest severity. Importantly, pest responses to field size strongly correlated with their responses to the fraction of the surrounding landscape planted to the focal crop, suggesting that shared ecological processes produce parallel responses to crop simplification across spatial scales. We conclude that the idea that larger field sizes consistently disrupt natural pest control services is without foundation in either the theoretical or empirical record.

The dinosaurian femoral head experienced a morphogenetic shift from torsion to growth along the avian stem.

Significant evolutionary shifts in locomotor behaviour often involve comparatively subtle anatomical transitions. For dinosaurian and avian evolution, medial overhang of the proximal femur has been central to discussions. However, there is an apparent conflict with regard to the evolutionary origin of the dinosaurian femoral head, with neontological and palaeontological data suggesting seemingly incongruent hypotheses. To reconcile this, we reconstructed the evolutionary history of morphogenesis of the proximal end of the femur from early archosaurs to crown birds. Embryological comparison of living archosaurs (crocodylians and birds) suggests the acquisition of the greater overhang of the femoral head in dinosaurs results from additional growth of the proximal end in the medial-ward direction. On the other hand, the fossil record suggests that this overhang was acquired by torsion of the proximal end, which projected in a more rostral direction ancestrally. We reconcile this apparent conflict by inferring that the medial overhang of the dinosaur femoral head was initially acquired by torsion, which was then superseded by mediad growth. Details of anatomical shifts in fossil forms support this hypothesis, and their biomechanical implications are congruent with the general consensus regarding broader morpho-functional evolution on the avian stem.

The causes and consequences of pest population variability in agricultural landscapes.

Variability in population densities is key to the ecology of natural systems but also has great implications for agriculture. Farmers' decisions are heavily influenced by their risk aversion to pest outbreaks that result in major yield losses. However, the need for long-term pest population data across many farms has prevented researchers from exploring the drivers and implications of pest population variability (PV). Here, we demonstrate the critical importance of PV for sustainable farming by analyzing 13 years of pest densities across >1300 Spanish olive groves and vineyards. Variable populations were more likely to cause major yield losses, but also occasionally created temporal windows when densities fell below insecticide spray thresholds. Importantly, environmental factors regulating pest variability were very distinct from factors regulating mean density, suggesting variability needs to be uniquely managed. Finally, we found diversifying landscapes may be a win-win situation for conservation and farmers, as diversified landscapes promote less abundant and less variable pest populations. Therefore, we encourage agricultural stakeholders to increase the complexity of the landscapes surrounding their farms through conserving/restoring natural habitat and/or diversifying crops.

Parasitoids of Lobesia botrana (Lepidoptera: Tortricidae) in the Douro Demarcated Region vineyards and the prospects for enhancing conservation biological control.

The more restrictive regulations of pesticides in Europe have led to an increase in conservation biological control (CBC) research. However, little attention has been paid to the main determinants of Lobesia botrana parasitism. The Douro Demarcated Region landscape offers scope for the use of CBC. The study was conducted between 2002 and 2015 aiming at: (i) identifying parasitoids associated with L. botrana and evaluating their impact as biological control agents in each generation of the pest, and (ii) evaluating the effect of both the proportion of ecological infrastructures (EI) near the vineyards, and the impact of management practices (chemical treatments and ground cover) on the parasitism of L. botrana. A total of 3226 larvae/pupae of L. botrana were collected (15% were parasitized and 485 parasitoids emerged). A complex of 16 taxa of parasitoids was identified, the majority belonging to Hymenoptera. The most abundant were Elachertus sp. (Eulophidae), Campoplex capitator Aubert (Ichneumonidae), and Brachymeria tibialis (Walker) (Chalcididae), which represented 62.5, 12.6, and 12.0% of the total assemblage of parasitoids which emerged, respectively. The percentage of parasitism ranged from 0.0 to 61.5% (first generation), from 0.0 to 36.8% (second generation), and from 0.0 to 12.1% (third generation). Importantly, it was found that the parasitism rate was higher in vineyards with ground cover. In addition, EI in the area surrounding the vineyards produced a significant increase in parasitism. These results suggest potential for CBC of L. botrana if EI around vineyards, and ground cover with native perennial plants within vineyards, are encouraged.

A developmental staging system and musculoskeletal development sequence of the common musk turtle (Sternotherus odoratus).

BACKGROUND: The extremely derived body plan of turtles has sparked a great interest in studying their developmental biology. Here, we describe the embryonic development of the Stinkpot, or common musk turtle (Sternotherus odoratus), a small aquatic turtle from the family Kinosternidae. RESULTS: We identify 20 distinct developmental stages, some comparable to stages described by previous studies on other turtles and some in between these, improving the resolution of the generalities of turtle development. We provide a detailed account of both the external morphology and skeletal development, as well as a general look at the early stages of muscular development until the attainment of the adult muscular anatomical pattern. CONCLUSIONS: Several potential skeletal and muscular apomorphies of turtles are identified or elaborated. The musk turtle, with its small size and hard-shelled egg, could become an important species for the study of turtle evolution and development, suitable for in ovo experimentation and late stage imaging of well-advanced anatomical features.

Efficacy of cap-assisted forward-viewing endoscopy as a method for the evaluation of the ampulla of Vater.

INTRODUCTION: endoscopic evaluation of the ampulla of Vater (AV), although routinely recommended, is not always possible due to its anatomic configuration, which can hide it from the visual field of the forward-viewing endoscope. Cap-assisted forward-viewing endoscopy has been proposed as a useful alternative to facilitate the examination of this structure. OBJECTIVES: to assess the efficacy of cap-assisted forward-viewing endoscopy for the complete evaluation of the AV. Secondary outcomes were to assess AV morphology, search and total procedure times and technique safety. METHODS: a prospective, single-arm study. Patients who were selected for elective upper endoscopy were included. Patients with advanced neoplasia, modified anatomy, upper gastrointestinal stenosis or obstructions were excluded. OUTCOMES: ninety patients were included, 36 males (40 %) and 54 females (60 %). Fifteen percent had a history of hereditary colon cancer syndrome. Technical success of cap-assisted, forward-viewing endoscopy was 98.8 %. AV was classified as type 1 (classic) in 49.4 %, type 2 (small) in 16.8 %, type 3 (protruding) in 11.2 % and type 4 (ridged) in 22.4 %. The mean search time was 37.7 seconds (s) (SD ± 31.6) and the total procedure time was 487.4 s (SD ± 206.2). No adverse events were reported. CONCLUSIONS: cap-assisted forward-viewing endoscopy is an effective and safe technique for the complete visualization and morphologic characterization of the ampulla of Vater.

Landscape simplification increases vineyard pest outbreaks and insecticide use.

Diversifying agricultural landscapes may mitigate biodiversity declines and improve pest management. Yet landscapes are rarely managed to suppress pests, in part because researchers seldom measure key variables related to pest outbreaks and insecticides that drive management decisions. We used a 13-year government database to analyse landscape effects on European grapevine moth (Lobesia botrana) outbreaks and insecticides across c. 400 Spanish vineyards. At harvest, we found pest outbreaks increased four-fold in simplified, vineyard-dominated landscapes compared to complex landscapes in which vineyards are surrounded by semi-natural habitats. Similarly, insecticide applications doubled in vineyard-dominated landscapes but declined in vineyards surrounded by shrubland. Importantly, pest population stochasticity would have masked these large effects if numbers of study sites and years were reduced to typical levels in landscape pest-control studies. Our results suggest increasing landscape complexity may mitigate pest populations and insecticide applications. Habitat conservation represents an economically and environmentally sound approach for achieving sustainable grape production.

Hidden limbs in the "limbless skink" Brachymeles lukbani: Developmental observations.

Reduced limbs and limblessness have evolved independently in many lizard clades. Scincidae exhibit a wide range of limb-reduced morphologies, but only some species have been used to study the embryology of limb reduction (e.g., digit reduction in Chalcides and limb reduction in Scelotes). The genus Brachymeles, a Southeast Asian clade of skinks, includes species with a range of limb morphologies, from pentadactyl to functionally and structurally limbless species. Adults of the small, snake-like species Brachymeles lukbani show no sign of external limbs in the adult except for small depressions where they might be expected to occur. Here, we show that embryos of B. lukbani in early stages of development, on the other hand, show a truncated but well-developed limb with a stylopod and a zeugopod, but no signs of an autopod. As development proceeds, the limb's small size persists even while the embryo elongates. These observations are made based on external morphology. We used florescent whole-mount immunofluorescence to visualize the morphology of skeletal elements and muscles within the embryonic limb of B. lukabni. Early stages have a humerus and separated ulna and radius cartilages; associated with these structures are dorsal and ventral muscle masses as those found in the embryos of other limbed species. While the limb remains small, the pectoral girdle grows in proportion to the rest of the body, with well-developed skeletal elements and their associated muscles. In later stages of development, we find the small limb is still present under the skin, but there are few indications of its presence, save for the morphology of the scale covering it. By use of CT scanning, we find that the adult morphology consists of a well-developed pectoral girdle, small humerus, extremely reduced ulna and radius, and well-developed limb musculature connected to the pectoral girdle. These muscles form in association with a developing limb during embryonic stages, a hint that "limbless" lizards that possess these muscles may have or have had at least transient developing limbs, as we find in B. lukbani. Overall, this newly observed pattern of ontogenetic reduction leads to an externally limbless adult in which a limb rudiment is hidden and covered under the trunk skin, a situation called cryptomelia. The results of this work add to our growing understanding of clade-specific patterns of limb reduction and the convergent evolution of limbless phenotypes through different developmental processes.

KCNH2-3.1 mediates aberrant complement activation and impaired hippocampal-medial prefrontal circuitry associated with working memory deficits.

Increased expression of the 3.1 isoform of the KCNH2 potassium channel has been associated with cognitive dysfunction and with schizophrenia, yet little is known about the underlying pathophysiological mechanisms. Here, by using in vivo wireless local field potential recordings during working memory processing, in vitro brain slice whole-cell patching recordings and in vivo stereotaxic hippocampal injection of AAV-encoded expression, we identified specific and delayed disruption of hippocampal-mPFC synaptic transmission and functional connectivity associated with reductions of SERPING1, CFH, and CD74 in the KCNH2-3.1 overexpression transgenic mice. The differentially expressed genes in mice are enriched in neurons and microglia, and reduced expression of these genes dysregulates the complement cascade, which has been previously linked to synaptic plasticity. We find that knockdown of these genes in primary neuronal-microglial cocultures from KCNH2-3.1 mice impairs synapse formation, and replenishing reduced CFH gene expression rescues KCNH2-3.1-induced impaired synaptogenesis. Translating to humans, we find analogous dysfunctional interactions between hippocampus and prefrontal cortex in coupling of the fMRI blood oxygen level-dependent (BOLD) signal during working memory in healthy subjects carrying alleles associated with increased KCNH2-3.1 expression in brain. Our data uncover a previously unrecognized role of the truncated KCNH2-3.1 potassium channel in mediating complement activation, which may explain its association with altered hippocampal-prefrontal connectivity and synaptic function. These results provide a potential molecular link between increased KCNH2-3.1 expression, synapse alterations, and hippocampal-prefrontal circuit abnormalities implicated in schizophrenia.

Echinacea Angustifolia DC Extract Induces Apoptosis and Cell Cycle Arrest and Synergizes with Paclitaxel in the MDA-MB-231 and MCF-7 Human Breast Cancer Cell Lines.

BACKGROUND: Echinacea spp. displays different biological activities, such as antiviral, immunomodulatory, and anticancer activities. Currently, high sales of hydroalcoholic extracts of Echinacea have been reported; hence, the importance of studies on Echinacea. AIM: To establish the effects of Echinacea angustifolia DC extract obtained with ethyl acetate (Ea-AcOEt) in breast cancer cell lines. METHODS: Cytotoxicity, cell cycle arrest, and cell death were evaluated. Besides, the safety of the extract, as well as its effect in combination with paclitaxel were investigated. RESULTS: The echinacoside and caffeic acid content in the Ea-AcOEt extract were quantified by HPLC, and its antioxidant activity was assessed. The Ea-AcOEt extract showed cytotoxic activity on breast cancer MDA-MB-231 cells (IC50 28.18 ± 1.14 µg/ml) and MCF-7 cells (19.97 ± 2.31 µg/ml). No effect was observed in normal breast MCF-10 cells. The Ea-AcOEt extract induced cell cycle arrest in the G1 phase and caspase-mediated apoptosis. No genotoxicity was found in vitro or in vivo, and the extract showed no signs of toxicity or death at 2,000 mg/kg in rodents. In vitro, the combination of Ea-AcOEt extract and paclitaxel showed a synergistic effect on both cancer cell lines. CONCLUSION: The Ea-AcOEt extract is a potential candidate for breast cancer treatment.

Integrated Metabolomics and Proteomics Analyses in the Local Milieu of Islet Allografts in Rejection versus Tolerance.

An understanding of the immune mechanisms that lead to rejection versus tolerance of allogeneic pancreatic islet grafts is of paramount importance, as it facilitates the development of innovative methods to improve the transplant outcome. Here, we used our established intraocular islet transplant model to gain novel insight into changes in the local metabolome and proteome within the islet allograft's immediate microenvironment in association with immune-mediated rejection or tolerance. We performed integrated metabolomics and proteomics analyses in aqueous humor samples representative of the graft's microenvironment under each transplant outcome. The results showed that several free amino acids, small primary amines, and soluble proteins related to the Warburg effect were upregulated or downregulated in association with either outcome. In general, the observed shifts in the local metabolite and protein profiles in association with rejection were consistent with established pro-inflammatory metabolic pathways and those observed in association with tolerance were immune regulatory. Taken together, the current findings further support the potential of metabolic reprogramming of immune cells towards immune regulation through targeted pharmacological and dietary interventions against specific metabolic pathways that promote the Warburg effect to prevent the rejection of transplanted islets and promote their immune tolerance.

Inhibitory designer receptors aggravate memory loss in a mouse model of down syndrome.

The pontine nucleus locus coeruleus (LC) is the primary source of noradrenergic (NE) projections to the brain and is important for working memory, attention, and cognitive flexibility. Individuals with Down syndrome (DS) develop Alzheimer's disease (AD) with high penetrance and often exhibit working memory deficits coupled with degeneration of LC-NE neurons early in the progression of AD pathology. Designer receptors exclusively activated by designer drugs (DREADDs) are chemogenetic tools that allow targeted manipulation of discrete neuronal populations in the brain without the confounds of off-target effects. We utilized male Ts65Dn mice (a mouse model for DS), and male normosomic (NS) controls to examine the effects of inhibitory DREADDs delivered via an AAV vector under translational control of the synthetic PRSx8, dopamine ß hydroxylase (DßH) promoter. This chemogenetic tool allowed LC inhibition upon administration of the inert DREADD ligand, clozapine-N-oxide (CNO). DREADD-mediated LC inhibition impaired performance in a novel object recognition task and reversal learning in a spatial task. DREADD-mediated LC inhibition gave rise to an elevation of α-adrenoreceptors both in NS and in Ts65Dn mice. Further, microglial markers showed that the inhibitory DREADD stimulation led to increased microglial activation in the hippocampus in Ts65Dn but not in NS mice. These findings strongly suggest that LC signaling is important for intact memory and learning in Ts65Dn mice and disruption of these neurons leads to increased inflammation and dysregulation of adrenergic receptors.

Greater Growth of Proximal Metatarsals in Bird Embryos and the Evolution of Hallux Position in the Grasping Foot.

In early theropod dinosaurs-the ancestors of birds-the hallux (digit 1) had an elevated position within the foot and had lost the proximal portion of its metatarsal. It no longer articulated with the ankle, but was attached at about mid-length of metatarsal 2 (mt2). In adult birds, the hallux is articulated closer to the distal end of mt2 at ground level with the other digits. However, on chick embryonic day 7, its position is as in early theropods at half-length of mt2. The adult distal location is acquired during embryonic days 8-10. To assess how the adult phenotype is acquired, we produced fate maps of the metatarsals of day 6 chicken embryos injecting the lipophilic tracer DiI. The fates of these marks indicate a larger expansion of the metatarsals at their proximal end, which creates the illusory effect that d1 moves distally. This larger proximal expansion occurs concomitantly with growth and early differentiation of cartilage. Histological analysis of metatarsals shows that the domains of flattened and prehypertrophic chondrocytes are larger toward the proximal end. The results suggest that the distal position of the hallux in the avian foot evolved as a consequence of an embryological period of expansion of the metatarsus toward the proximal end. It also brings attention to the developmental mechanisms leading to differential growth between epiphyses and their evolutionary consequences.

Ecological regime shift drives declining growth rates of sea turtles throughout the West Atlantic.

Somatic growth is an integrated, individual-based response to environmental conditions, especially in ectotherms. Growth dynamics of large, mobile animals are particularly useful as bio-indicators of environmental change at regional scales. We assembled growth rate data from throughout the West Atlantic for green turtles, Chelonia mydas, which are long-lived, highly migratory, primarily herbivorous mega-consumers that may migrate over hundreds to thousands of kilometers. Our dataset, the largest ever compiled for sea turtles, has 9690 growth increments from 30 sites from Bermuda to Uruguay from 1973 to 2015. Using generalized additive mixed models, we evaluated covariates that could affect growth rates; body size, diet, and year have significant effects on growth. Growth increases in early years until 1999, then declines by 26% to 2015. The temporal (year) effect is of particular interest because two carnivorous species of sea turtles-hawksbills, Eretmochelys imbricata, and loggerheads, Caretta caretta-exhibited similar significant declines in growth rates starting in 1997 in the West Atlantic, based on previous studies. These synchronous declines in productivity among three sea turtle species across a trophic spectrum provide strong evidence that an ecological regime shift (ERS) in the Atlantic is driving growth dynamics. The ERS resulted from a synergy of the 1997/1998 El Niño Southern Oscillation (ENSO)-the strongest on record-combined with an unprecedented warming rate over the last two to three decades. Further support is provided by the strong correlations between annualized mean growth rates of green turtles and both sea surface temperatures (SST) in the West Atlantic for years of declining growth rates (r = -.94) and the Multivariate ENSO Index (MEI) for all years (r = .74). Granger-causality analysis also supports the latter finding. We discuss multiple stressors that could reinforce and prolong the effect of the ERS. This study demonstrates the importance of region-wide collaborations.

New developmental evidence clarifies the evolution of wrist bones in the dinosaur-bird transition.

From early dinosaurs with as many as nine wrist bones, modern birds evolved to develop only four ossifications. Their identity is uncertain, with different labels used in palaeontology and developmental biology. We examined embryos of several species and studied chicken embryos in detail through a new technique allowing whole-mount immunofluorescence of the embryonic cartilaginous skeleton. Beyond previous controversy, we establish that the proximal-anterior ossification develops from a composite radiale+intermedium cartilage, consistent with fusion of radiale and intermedium observed in some theropod dinosaurs. Despite previous claims that the development of the distal-anterior ossification does not support the dinosaur-bird link, we found its embryonic precursor shows two distinct regions of both collagen type II and collagen type IX expression, resembling the composite semilunate bone of bird-like dinosaurs (distal carpal 1+distal carpal 2). The distal-posterior ossification develops from a cartilage referred to as "element x," but its position corresponds to distal carpal 3. The proximal-posterior ossification is perhaps most controversial: It is labelled as the ulnare in palaeontology, but we confirm the embryonic ulnare is lost during development. Re-examination of the fossil evidence reveals the ulnare was actually absent in bird-like dinosaurs. We confirm the proximal-posterior bone is a pisiform in terms of embryonic position and its development as a sesamoid associated to a tendon. However, the pisiform is absent in bird-like dinosaurs, which are known from several articulated specimens. The combined data provide compelling evidence of a remarkable evolutionary reversal: A large, ossified pisiform re-evolved in the lineage leading to birds, after a period in which it was either absent, nonossified, or very small, consistently escaping fossil preservation. The bird wrist provides a modern example of how developmental and paleontological data illuminate each other. Based on all available data, we introduce a new nomenclature for bird wrist ossifications.

The developmental origin of zygodactyl feet and its possible loss in the evolution of Passeriformes.

The zygodactyl orientation of toes (digits II and III pointing forwards, digits I and IV pointing backwards) evolved independently in different extant bird taxa. To understand the origin of this trait in modern birds, we investigated the development of the zygodactyl foot of the budgerigar (Psittaciformes). We compared its muscular development with that of the anisodactyl quail (Galliformes) and show that while the musculus abductor digiti IV (ABDIV) becomes strongly developed at HH36 in both species, the musculus extensor brevis digiti IV (EBDIV) degenerates and almost disappears only in the budgerigar. The asymmetric action of those muscles early in the development of the budgerigar foot causes retroversion of digit IV (dIV). Paralysed budgerigar embryos do not revert dIV and are anisodactyl. Both molecular phylogenetic analysis and palaeontological information suggest that the ancestor of passerines could have been zygodactyl. We followed the development of the zebra finch (Passeriformes) foot muscles and found that in this species, both the primordia of the ABDIV and of the EBDIV fail to develop. These data suggest that loss of asymmetric forces of muscular activity exerted on dIV, caused by the absence of the ABDIV, could have resulted in secondary anisodactyly in Passeriformes.

GS 455534 selectively suppresses binge eating of palatable food and attenuates dopamine release in the accumbens of sugar-bingeing rats.

Binge eating palatable foods has been shown to have behavioral and neurochemical similarities to drug addiction. GS 455534 is a highly selective reversible aldehyde dehydrogenase 2 inhibitor that has been shown to reduce alcohol and cocaine intake in rats. Given the overlaps between binge eating and drug abuse, we examined the effects of GS 455534 on binge eating and subsequent dopamine release. Sprague-Dawley rats were maintained on a sugar (experiment 1) or fat (experiment 2) binge eating diet. After 25 days, GS 455534 was administered at 7.5 and 15 mg/kg by an intraperitoneal injection, and food intake was monitored. In experiment 3, rats with cannulae aimed at the nucleus accumbens shell were maintained on the binge sugar diet for 25 days. Microdialysis was performed, during which GS 455534 15 mg/kg was administered, and sugar was available. Dialysate samples were analyzed to determine extracellular levels of dopamine. In experiment 1, GS 455534 selectively decreased sugar intake food was made available in the Binge Sugar group but not the Ad libitum Sugar group, with no effect on chow intake. In experiment 2, GS 455534 decreased fat intake in the Binge Fat group, but not the Ad libitum Fat group, however, it also reduced chow intake. In experiment 3, GS 455534 attenuated accumbens dopamine release by almost 50% in binge eating rats compared with the vehicle injection. The findings suggest that selective reversible aldehyde dehydrogenase 2 inhibitors may have the therapeutic potential to reduce binge eating of palatable foods in clinical populations.