Exome sequencing of hepatocellular carcinoma in lemurs identifies potential cancer drivers: A pilot study.

Background and objectives: Hepatocellular carcinoma occurs frequently in prosimians, but the cause of these liver cancers in this group is unknown. Characterizing the genetic changes associated with hepatocellular carcinoma in prosimians may point to possible causes, treatments and methods of prevention, aiding conservation efforts that are particularly crucial to the survival of endangered lemurs. Although genomic studies of cancer in non-human primates have been hampered by a lack of tools, recent studies have demonstrated the efficacy of using human exome capture reagents across primates. Methodology: In this proof-of-principle study, we applied human exome capture reagents to tumor-normal pairs from five lemurs with hepatocellular carcinoma to characterize the mutational landscape of this disease in lemurs. Results: Several genes implicated in human hepatocellular carcinoma, including ARID1A, TP53 and CTNNB1, were mutated in multiple lemurs, and analysis of cancer driver genes mutated in these samples identified enrichment of genes involved with TP53 degradation and regulation. In addition to these similarities with human hepatocellular carcinoma, we also noted unique features, including six genes that contain mutations in all five lemurs. Interestingly, these genes are infrequently mutated in human hepatocellular carcinoma, suggesting potential differences in the etiology and/or progression of this cancer in lemurs and humans. Conclusions and implications: Collectively, this pilot study suggests that human exome capture reagents are a promising tool for genomic studies of cancer in lemurs and other non-human primates. Lay Summary: Hepatocellular carcinoma occurs frequently in prosimians, but the cause of these liver cancers is unknown. In this proof-of-principle study, we applied human DNA sequencing tools to tumor-normal pairs from five lemurs with hepatocellular carcinoma and compared the lemur mutation profiles to those of human hepatocellular carcinomas.

On the modulation and maintenance of hibernation in captive dwarf lemurs.

In nature, photoperiod signals environmental seasonality and is a strong selective "zeitgeber" that synchronizes biological rhythms. For animals facing seasonal environmental challenges and energetic bottlenecks, daily torpor and hibernation are two metabolic strategies that can save energy. In the wild, the dwarf lemurs of Madagascar are obligate hibernators, hibernating between 3 and 7 months a year. In captivity, however, dwarf lemurs generally express torpor for periods far shorter than the hibernation season in Madagascar. We investigated whether fat-tailed dwarf lemurs (Cheirogaleus medius) housed at the Duke Lemur Center (DLC) could hibernate, by subjecting 8 individuals to husbandry conditions more in accord with those in Madagascar, including alternating photoperiods, low ambient temperatures, and food restriction. All dwarf lemurs displayed daily and multiday torpor bouts, including bouts lasting ~ 11 days. Ambient temperature was the greatest predictor of torpor bout duration, and food ingestion and night length also played a role. Unlike their wild counterparts, who rarely leave their hibernacula and do not feed during hibernation, DLC dwarf lemurs sporadically moved and ate. While demonstrating that captive dwarf lemurs are physiologically capable of hibernation, we argue that facilitating their hibernation serves both husbandry and research goals: first, it enables lemurs to express the biphasic phenotypes (fattening and fat depletion) that are characteristic of their wild conspecifics; second, by "renaturalizing" dwarf lemurs in captivity, they will emerge a better model for understanding both metabolic extremes in primates generally and metabolic disorders in humans specifically.

Applications of 3D printing in small animal magnetic resonance imaging.

Three-dimensional (3D) printing has significantly impacted the quality, efficiency, and reproducibility of preclinical magnetic resonance imaging. It has vastly expanded the ability to produce MR-compatible parts that readily permit customization of animal handling, achieve consistent positioning of anatomy and RF coils promptly, and accelerate throughput. It permits the rapid and cost-effective creation of parts customized to a specific imaging study, animal species, animal weight, or even one unique animal, not routinely used in preclinical research. We illustrate the power of this technology by describing five preclinical studies and specific solutions enabled by different 3D printing processes and materials. We describe fixtures, assemblies, and devices that were created to ensure the safety of anesthetized lemurs during an MR examination of their brain or to facilitate localized, contrast-enhanced measurements of white blood cell concentration in a mouse model of pancreatitis. We illustrate expansive use of 3D printing to build a customized birdcage coil and components of a ventilator to enable imaging of pulmonary gas exchange in rats using hyperpolarized Xe 129 . Finally, we present applications of 3D printing to create high-quality, dual RF coils to accelerate brain connectivity mapping in mouse brain specimens and to increase the throughput of brain tumor examinations in a mouse model of pituitary adenoma.

Hibernation in a primate: does sleep occur?

During hibernation, critical physiological processes are downregulated and thermogenically induced arousals are presumably needed periodically to fulfil those physiological demands. Among the processes incompatible with a hypome tabolic state is sleep. However, one hibernating primate, the dwarf lemur Cheirogaleus medius, experiences rapid eye movement (REM)-like states during hibernation, whenever passively reaching temperatures above 30°C, as occurs when it hibernates in poorly insulated tree hollows under tropical conditions. Here, we report electroencephalographic (EEG) recordings, temperature data and metabolic rates from two related species (C. crossleyi and C. sibreei), inhabiting high-altitude rainforests and hibernating underground, conditions that mirror, to some extent, those experienced by temperate hibernators. We compared the physiology of hibernation and spontaneous arousals in these animals to C. medius, as well as the much more distantly related non-primate hibernators, such as Arctic, golden-mantled and European ground squirrels. We observed a number of commonalities with non-primate temperate hibernators including: (i) monotonous ultra-low voltage EEG during torpor bouts in these relatively cold-weather hibernators, (ii) the absence of sleep during torpor bouts, (iii) the occurrence of spontaneous arousals out of torpor, during which sleep regularly occurred, (iv) relatively high early EEG non-REM during the arousal, and (v) a gradual transition to the torpid EEG state from non-REM sleep. Unlike C. medius, our study species did not display sleep-like states during torpor bouts, but instead exclusively exhibited them during arousals. During these short euthermic periods, non-REM as well as REM sleep-like stages were observed. Differences observed between these two species and their close relative, C. medius, for which data have been published, presumably reflect differences in hibernaculum temperature.

Environmental and physiological factors contributing to outbreaks of cryptosporidium in Coquerel's sifaka (Propithecus coquereli) at the Duke Lemur Center: 1999-2007.

This population-based, retrospective study examined the susceptibility of a prosimian primate, Coquerel's sifaka (Propithecus coquereli), to Cryptosporidium spp. over a 9-yr period from 1999 to 2007 at the Duke Lemur Center (DLC) located in Durham, North Carolina. The investigation examined potential epidemiologic risk factors that could be correlated to infectious outbreaks at the center, such as prevalence, signalment (species, age, and sex), seasonality of occurrence, recurrence rate, family lineage, parturition, clinical signs, and concurrent diseases or health conditions. Findings included Propithecus spp. being the only lemur species at the DLC showing clinical signs of infection, with age being an important factor in susceptibility, and showing a strong correlation between temperature and seasonality with shedding of Cryptosporidium oocysts. These findings present new information regarding cryptosporidiosis in captive prosimians.

Survey of wildlife rehabilitators regarding rabies vector species.

OBJECTIVE: To evaluate the risks associated with wildlife rehabilitation and the reemergence of wildlife rabies in North Carolina through assessment of the status of knowledge and attitudes of licensed in-state wildlife rehabilitators about rabies and rabies vector species (RVS). DESIGN: Questionnaire survey. SAMPLE POPULATION: 672 North Carolina licensed wildlife rehabilitators registered in 1999. PROCEDURE: Wildlife rehabilitators were contacted by mail to determine their status of knowledge and attitudes regarding rabies and RVS. The questionnaire was designed to determine rehabilitators' recent experiences with RVS, attitudes toward regulations, and knowledge of rabies virus transmission. Results were analyzed by use of the chi2 test. RESULTS: Questionnaire responses were provided by 210 of the 672 (31.3%) wildlife rehabilitators. Among rehabilitators, there were some inconsistencies in their knowledge base regarding rabies (eg, 25% reported that they did not know at what age animals were capable of transmitting rabies virus). Most respondents were amenable to all proposed licensing prerequisites for handling RVS (ie, record keeping, additional training, and veterinarian support). Respondents reported > 580 calls annually about rehabilitating RVS, and 80% believed at least some of their peers were rehabilitating RVS illegally. CONCLUSIONS AND CLINICAL RELEVANCE: With the establishment of rabies as a disease that is endemic among wildlife species in North Carolina, educational efforts directed at wildlife rehabilitators (a subpopulation of residents potentially at high risk of rabies virus infection) would have direct and indirect public health benefits; similar efforts may be useful to public health communities elsewhere in the United States.