The genomics of ecological flexibility,large brains,and long lives in capuchin monkeys revealed with fecalFACS

Ecological flexibility, extended lifespans, and large brains have long intrigued evolutionary biologists, and comparative genomics offers an efficient and effective tool for generating new insights into the evolution of such traits. Studies of capuchin monkeys are particularly well situated to shed light on the selective pressures and genetic underpinnings of local adaptation to diverse habitats, longevity, and brain development. Distributed widely across Central and South America, they are inventive and extractive foragers, known for their sensorimotor intelligence. Capuchins have among the largest relative brain size of any monkey and a lifespan that exceeds 50 y, despite their small (3 to 5 kg) body size. We assemble and annotate a de novo reference genome for Cebus imitator. Through high-depth sequencing of DNA derived from blood, various tissues, and feces via fluorescence-activated cell sorting (fecalFACS) to isolate monkey epithelial cells, we compared genomes of capuchin populations from tropical dry forests and lowland rainforests and identified population divergence in genes involved in water balance, kidney function, and metabolism. Through a comparative genomics approach spanning a wide diversity of mammals, we identified genes under positive selection associated with longevity and brain development. Additionally, we provide a technological advancement in the use of noninvasive genomics for studies of free-ranging mammals. Our intra- and interspecific comparative study of capuchin genomics provides insights into processes underlying local adaptation to diverse and physiologically challenging environments, as well as the molecular basis of brain evolution and longevity.

Large brains, long lifespans, extended juvenescence, tool use, and problem solving are hallmark characteristics of great apes, and are of enduring interest in studies of human evolution (1–4). Similar suites of traits have arisen in other lineages, including some cetaceans, corvids and, independently, in another radiation of primates, the capuchin monkeys. Like great apes, they have diverse diets, consume and seek out high-energy resources, engage in complex extractive foraging techniques (5, 6) to consume difficult-to-access invertebrates and nuts (6), and have an extended lifespan, presently recorded up to 54 y in captivity (7, 8). While they do not show evidence of some traits linked with large brain size in humans (e.g., human-like social networks and cultural and technological transmission from older to younger groupmates), their propensity for tool use and their ecological flexibility may have contributed to their convergence with the great apes (9), offering opportunities for understanding the evolution of key traits via the comparative method (10–12). Similar approaches have revealed positive selection on genes related to brain size and long lives in great apes and other mammals (13, 14), but our understanding of the genetic underpinnings of these traits remains far from complete.Capuchins also offer excellent opportunities to study local adaptation to challenging seasonal biomes. They occupy diverse habitats, including rainforests and, in the northern extent of their range, tropical dry forests. Particular challenges of the tropical dry forest are staying hydrated during the seasonally prominent droughts, high temperatures in the absence of foliage, and coping metabolically with periods of fruit dearth (Fig. 1). The long-term study of white-faced capuchins (Cebus imitator) occupying these seasonal forests has demonstrated that high infant mortality rates accompany periods of intense drought, illustrating the strength of this selective pressure (15). Furthermore, the seasonally low abundance of fruit is associated with muscular wasting and low circulating levels of urinary creatinine among these capuchins (16). Additionally, the sensory challenges of food search in dry versus humid biomes are also distinct. Odor detection and propagation is affected by temperature and humidity (17), and color vision is hypothesized to be adaptive in the search for ripe fruits and young reddish leaves against a background of thick, mature foliage (18), which is absent for long stretches in dry deciduous forests. The behavioral plasticity of capuchins is widely acknowledged as a source of their ability to adapt to these dramatically different habitats (19–21). However, physiological processes, including water balance and metabolic adaptations to low caloric intake, and sensory adaptations to food search, are also anticipated to be targets of natural selection, as seen in other mammals (22–24). Understanding population-level differences between primates inhabiting different biomes, contextualized by their demographic history, genomic diversity, and historical patterns of migration, will generate new insights.Open in a separate windowFig. 1.SSR during wet (Left) and dry (Center) seasons. (Right) Map of sampling locations in Costa Rica. The two northern sites, SSR and Cañas, have tropical dry-forest biomes, whereas the two southern sites, Quepos and Manuel Antonio, are tropical wet forests. Photos courtesy of A.D.M. Drawing of white-faced capuchin monkey by Alejandra Tejada-Martinez; map courtesy of Eric Gaba–Wikimedia Commons user: Sting.Unfortunately, high-quality biological specimens from wild capuchins are not readily available. As is the case with most of the world’s primates, many of which are rare or threatened (25), this has limited the scope of questions about their biology that can be answered. Although recent advances in noninvasive genomics have allowed for the sequencing of partial genomes by enriching the proportion of endogenous DNA in feces (26–29), it has not yet been feasible to sequence whole genomes from noninvasive samples at high coverage; this has limited the extent to which noninvasive samples can be used to generate genomic resources for nonmodel organisms, such as capuchins.Toward identifying the genetic underpinnings of local adaptation to seasonally harsh environments, large brains, and long lifespans, we assembled and annotated a reference genome of C. imitator (SI Appendix, Table S1). Additionally, we sequenced the genomes of individuals inhabiting two distinct environments in Costa Rica: Lowland evergreen rainforest (southern population) and lowland tropical dry forest (northern population). We conducted high-coverage resequencing (10× to 47×) for 10 of these individuals, and sequenced an additional 13 at low-coverage (0.1× to 4.4×). Importantly, to facilitate the population-wide analyses without the need for potentially harmful invasive sampling of wild primates, we developed a method for minimally biased, whole-genome sequencing of fecal DNA using fluorescence-activated cell sorting (fecalFACS) that we used to generate both high- and low-coverage genomes (Fig. 2). With these genomes, we assess the genetic underpinnings of capuchin-specific biology and adaptation in a comparative framework. First, we scanned the high-coverage genomes (six from the northern dry forest and four from the southern rainforest) for regions exhibiting population specific divergence to assess the extent of local adaptation to dry forest and rainforest environments. We examine how genes related to water balance, metabolism, muscular wasting, and chemosensation have diverged between populations. Second, we conduct an analysis of positive selection on the white-faced capuchin genome through codon-based models of evolution and enrichment tests focusing on genes that may underlie brain development and lifespan. Third, we identify the population structure, genomic diversity, and demographic history of the species using a mixture of traditional and noninvasive fecalFACS genomes (n = 23).Open in a separate windowFig. 2.Mapping percentages of sequencing reads from RNAlater preserved fecal DNA libraries prepared with FACS for (A) all samples (box-plot elements: center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers), and (B) individual libraries. (C) Increase in mapping rate for RNAlater preserved samples. (D) Relationship between mapped read duplication and number of cells with LOESS smoothing. The duplication rate decreases sharply once a threshold of about 1,000 cells is reached.     

Acquired homozygosity of the rearranged bcr allele during the acute leukemic phase of a patient with Ph-negative chronic myeloid leukemia

A 45-year-old male patient with Ph-negative chronic myeloid leukemia (CML) had rearranged bcr-3' and bcr-5' gene regions in Southern blot studies when leukemia was diagnosed. During development of terminal blast crisis, successive blood samples showed a progressive decrease in the amount of germline bcr DNA and its complete loss by full blast crisis. There were also increased amounts of rearranged bcr DNA consistent with acquired homozygosity. A similar result was obtained with an IgV lambda probe and indicated homozygosity of a significant part of chromosome 22. The bcr-abl gene complex behaves as a somatic dominant in CML, and we suggest that its acquired homozygosity is a mechanism of bcr-abl amplification similar to duplication of the Ph chromosome commonly found in the blast crisis of CML.     

Hepatitis A virus and non-A, non-B virus superinfections in HBsAg chronic carriers

Clinical course, morphologic changes, immunohistochemical localization of HBV-associated markers (HBsAg, HBcAg), and serum HBV-DNA production are described in 2 chronic HBsAg carriers superinfected with HAV and hepatitis non-A, non-B virus. Our data suggest that the superinfections do not cause more severe disease and do not influence the clinical course of the HBsAg chronic carriers. Our observations indicate that a careful diagnosis of hepatocytolysis is necessary in HBV chronic infection, in order to discriminate causes that are able to induce severe damage in underlying disease.     

Autosomal recessive hypercholesterolemia in a Sicilian kindred harboring the 432insA mutation of the ARH gene

We describe a Sicilian family presenting a recessive form of hypercholesterolemia harboring a mutation of the autosomal recessive hypercholesterolemia (ARH) gene. In two of the three sibs, a 26-year-old male and a 22-year-old female, a severe hypercholesterolemia was diagnosed with very high levels of plasma cholesterol (15.9 and 12.2 mmol/l, respectively); tendon xanthomatas and xanthelasms were present and in the male proband was documented a diffuse coronary atherosclerotic disease with a rapid and fatal progression. Both the parents had normal or slightly increased levels of plasma cholesterol. All causes of secondary hypercholesterolemia were ruled out as well as an involvement of the LDL receptor or apoB genes. Beta-Sitosterol plasma levels were in the normal range. Cultured fibroblasts from skin biopsy from parents and the two probands displayed a normal ability to bind and degrade 125I-LDL. Direct sequencing of ARH gene demonstrated the presence of a 432insA mutation in homozygosis in the two probands; parents were heterozygotes for the same mutation. This mutation is the first report of a mutation of the ARH gene responsible for recessive forms of hypercholesterolemia in Sicily.     

Evolution of social organization: a reappraisal for primates by using phylogenetic methods.

For many animal taxa, the extent to which phylogeny can account for the form of species' social systems has seldom been investigated formally. A quantitative phylogenetic analysis of social systems in the order Primates reveals that social organization may be strongly conserved in some lineages, even in the face of considerable ecological variability. This result has important implications for efforts to understand the evolution of animal societies and for attempts to reconstruct the social organization of early humans.     

Detection of human herpesvirus 8 in cervicovaginal secretions and seroprevalence in human immunodeficiency virus type 1-seropositive and -seronegative women

Epidemiologic studies suggest that human herpesvirus 8 (HHV-8) may be sexually transmitted. To study the potential for HHV-8 transmission through cervicovaginal (CV) secretions, the presence of HHV-8 DNA was investigated by nested polymerase chain reaction in the cellular fraction of CV secretions from 36 human immunodeficiency virus type 1 (HIV-1)-seropositive and 29 HIV-1-seronegative women. The same patients were tested for antibodies to two defined HHV-8 antigens (latency-associated nuclear antigen and open-reading frame 65-encoded structural protein) and for HHV-8 DNA in their peripheral blood mononuclear cells (PBMC). The findings were compared with the rate of HHV-8 detection in semen samples of 20 HIV-1-infected men. HHV-8 DNA was detected in the CV samples from only 1 HHV-8-seropositive AIDS patient, in 3 PBMC samples (1/29 HIV-1-seronegative patients, 1/3 AIDS patients with Kaposi's sarcoma, and 1/19 AIDS patients), and in 1 of 20 semen samples. HHV-8 infection was more common in HIV-1-infected than uninfected women. Thus HHV-8 DNA is only rarely detectable in CV secretions and semen of HHV-8-infected individuals.     

Palliative esophageal stent placement using endoscopic guidance without fluoroscopy

AIMS: Fluoroscopy is not available in every endoscopic unit. This situation leads to delays in treatment or to transfer of patients to other centres for stent insertion. We assessed safety and effectiveness of expandable esophageal metal stent placement under endoscopic control without fluoroscopy using a thin gastroscope. PATIENTS AND METHODS: From October 2002 to June 2004, thirty-three consecutive patients have been included for esophageal stent placement under endoscopic control alone with a nasogastroscope (5.9 mm). A proximal release covered stent (Ultraflex; Boston Scientific Microvasive) was used. Indications were malignant esophageal stricture (N = 26), malignant extrinsic compression (N = 2 ) and esophago-respiratory neoplastic fistulae (N = 5). RESULTS: Stent placement using endoscopic control alone was successful in 30/33 (90%) patients. Complications occurred in 11 patients. Early complications (<7 days) included one death from pulmonary embolism, severe retrosternal pain needing transient morphinic treatment (N = 2) and GERD despite antisecretory therapy (N = 1). Late complications included: food impaction (N = 1), tumour overgrowth-related obstruction of the stent (N = 5) and one late esophago-respiratory fistula at 4 months at the proximal end of the stent. Relief of dysphagia was obtained for all patients at 48 hours and dysphagia score decreased from 3.1 before stent to 1.2 at 1 month (P < 0.05). CONCLUSION: Expandable esophageal stents can be accurately and safely placed using endoscopy with a thin gastrosocope. This method obviates the requirement of fluoroscopic access, lacking in many centres, and avoids exposure to X-ray.     

Use of porcine factor VIII in the treatment of patients with acquired hemophilia

Data have been collected from 47 centers in Europe and North America on the treatment with porcine factor VIII concentrate of 74 acute bleeding episodes in 65 patients with acquired hemophilia. The median initial anti-human factor VIII auto-antibody inhibitor level was 38 Bethesda unit (BU)/mL (range 1.2 to 1,024) whereas that against porcine was 1 BU/mL (range 0 to 15). The mean initial dose of porcine factor VIII infused was 84 IU/kg, which increased the plasma factor VIII:C activity by 0.85 IU/mL. Therapy was continued for a mean of 8.5 days during which time the average number of infusions was 11. Objective clinical responses were rated as good or excellent in 78% of recipients. Side effects were uncommon; only one patient experienced a severe anaphylactic reaction necessitating the discontinuation of porcine FVIII therapy. After therapy, no increase in the median level of anti- human FVIII or anti-porcine antibody was noted in the group as a whole, although 13 patients showed individual increases in either anti-human or anti-porcine antibody levels or both of more than 10 BU/mL. Of the 7 patients who subsequently rebled, 5 were successfully re-treated and 2 did not respond to further porcine factor VIII treatment. Porcine factor VIII is safe and clinically effective treatment for bleeding episodes associated with acquired hemophilia and should be considered as first-line therapy for patients whose acquired anti-factor VIII:C antibody cross-reacts with porcine factor VIII:C at low levels.