Massively Parallel Sequencing Reveals the Complex Structure of an Irradiated Human Chromosome on a Mouse Background in the Tc1 Model of Down Syndrome

Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and presents a complex phenotype that arises from abnormal dosage of genes on this chromosome. However, the individual dosage-sensitive genes underlying each phenotype remain largely unknown. To help dissect genotype – phenotype correlations in this complex syndrome, the first fully transchromosomic mouse model, the Tc1 mouse, which carries a copy of human chromosome 21 was produced in 2005. The Tc1 strain is trisomic for the majority of genes that cause phenotypes associated with DS, and this freely available mouse strain has become used widely to study DS, the effects of gene dosage abnormalities, and the effect on the basic biology of cells when a mouse carries a freely segregating human chromosome. Tc1 mice were created by a process that included irradiation microcell-mediated chromosome transfer of Hsa21 into recipient mouse embryonic stem cells. Here, the combination of next generation sequencing, array-CGH and fluorescence in situ hybridization technologies has enabled us to identify unsuspected rearrangements of Hsa21 in this mouse model; revealing one deletion, six duplications and more than 25 de novo structural rearrangements. Our study is not only essential for informing functional studies of the Tc1 mouse but also (1) presents for the first time a detailed sequence analysis of the effects of gamma radiation on an entire human chromosome, which gives some mechanistic insight into the effects of radiation damage on DNA, and (2) overcomes specific technical difficulties of assaying a human chromosome on a mouse background where highly conserved sequences may confound the analysis. Sequence data generated in this study is deposited in the ENA database, Study Accession number: ERP000439.     

Neutering Dogs: Effects on Joint Disorders and Cancers in Golden Retrievers

In contrast to European countries, the overwhelming majority of dogs in the U.S. are neutered (including spaying), usually done before one year of age. Given the importance of gonadal hormones in growth and development, this cultural contrast invites an analysis of the multiple organ systems that may be adversely affected by neutering. Using a single breed-specific dataset, the objective was to examine the variables of gender and age at the time of neutering versus leaving dogs gonadally intact, on all diseases occurring with sufficient frequency for statistical analyses. Given its popularity and vulnerability to various cancers and joint disorders, the Golden Retriever was chosen for this study. Veterinary hospital records of 759 client-owned, intact and neutered female and male dogs, 1–8 years old, were examined for diagnoses of hip dysplasia (HD), cranial cruciate ligament tear (CCL), lymphosarcoma (LSA), hemangiosarcoma (HSA), and mast cell tumor (MCT). Patients were classified as intact, or neutered early (<12 mo) or late (≥12 mo). Statistical analyses involved survival analyses and incidence rate comparisons. Outcomes at the 5 percent level of significance are reported. Of early-neutered males, 10 percent were diagnosed with HD, double the occurrence in intact males. There were no cases of CCL diagnosed in intact males or females, but in early-neutered males and females the occurrences were 5 percent and 8 percent, respectively. Almost 10 percent of early-neutered males were diagnosed with LSA, 3 times more than intact males. The percentage of HSA cases in late-neutered females (about 8 percent) was 4 times more than intact and early-neutered females. There were no cases of MCT in intact females, but the occurrence was nearly 6 percent in late-neutered females. The results have health implications for Golden Retriever companion and service dogs, and for oncologists using dogs as models of cancers that occur in humans.     

Inhibition of Dopamine Transporter Activity by G Protein βγ Subunits

Uptake through the Dopamine Transporter (DAT) is the primary mechanism of terminating dopamine signaling within the brain, thus playing an essential role in neuronal homeostasis. Deregulation of DAT function has been linked to several neurological and psychiatric disorders including ADHD, schizophrenia, Parkinson’s disease, and drug addiction. Over the last 15 years, several studies have revealed a plethora of mechanisms influencing the activity and cellular distribution of DAT; suggesting that fine-tuning of dopamine homeostasis occurs via an elaborate interplay of multiple pathways. Here, we show for the first time that the βγ subunits of G proteins regulate DAT activity. In heterologous cells and brain tissue, a physical association between Gβγ subunits and DAT was demonstrated by co-immunoprecipitation. Furthermore, in vitro pull-down assays using purified proteins established that this association occurs via a direct interaction between the intracellular carboxy-terminus of DAT and Gβγ. Functional assays performed in the presence of the non-hydrolyzable GTP analog GTP-γ-S, Gβγ subunit overexpression, or the Gβγ activator mSIRK all resulted in rapid inhibition of DAT activity in heterologous systems. Gβγ activation by mSIRK also inhibited dopamine uptake in brain synaptosomes and dopamine clearance from mouse striatum as measured by high-speed chronoamperometry in vivo. Gβγ subunits are intracellular signaling molecules that regulate a multitude of physiological processes through interactions with enzymes and ion channels. Our findings add neurotransmitter transporters to the growing list of molecules regulated by G-proteins and suggest a novel role for Gβγ signaling in the control of dopamine homeostasis.     

Tracking Resilience to Infections by Mapping Disease Space

Infected hosts differ in their responses to pathogens; some hosts are resilient and recover their original health, whereas others follow a divergent path and die. To quantitate these differences, we propose mapping the routes infected individuals take through “disease space.” We find that when plotting physiological parameters against each other, many pairs have hysteretic relationships that identify the current location of the host and predict the future route of the infection. These maps can readily be constructed from experimental longitudinal data, and we provide two methods to generate the maps from the cross-sectional data that is commonly gathered in field trials. We hypothesize that resilient hosts tend to take small loops through disease space, whereas nonresilient individuals take large loops. We support this hypothesis with experimental data in mice infected with Plasmodium chabaudi, finding that dying mice trace a large arc in red blood cells (RBCs) by reticulocyte space as compared to surviving mice. We find that human malaria patients who are heterozygous for sickle cell hemoglobin occupy a small area of RBCs by reticulocyte space, suggesting this approach can be used to distinguish resilience in human populations. This technique should be broadly useful in describing the in-host dynamics of infections in both model hosts and patients at both population and individual levels.     

Hawksbill turtle terra incognita: conservation genetics of?eastern Pacific rookeries

Prior to 2008 and the discovery of several important hawksbill turtle (Eretmochelys imbricata) nesting colonies in the EP (Eastern Pacific), the species was considered virtually absent from the region. Research since that time has yielded new insights into EP hawksbills, salient among them being the use of mangrove estuaries for nesting. These recent revelations have raised interest in the genetic characterization of hawksbills in the EP, studies of which have remained lacking to date. Between 2008 and 2014, we collected tissue samples from 269 nesting hawksbills at nine rookeries across the EP and used mitochondrial DNA sequences (766 bp) to generate the first genetic characterization of rookeries in the region. Our results inform genetic diversity, population differentiation, and phylogeography of the species. Hawksbills in the EP demonstrate low genetic diversity: We identified a total of only seven haplotypes across the region, including five new and two previously identified nesting haplotypes (pooled frequencies of 58.4% and 41.6%, respectively), the former only evident in Central American rookeries. Despite low genetic diversity, we found strong stock structure between the four principal rookeries, suggesting the existence of multiple populations and warranting their recognition as distinct management units. Furthermore, haplotypes EiIP106 and EiIP108 are unique to hawksbills that nest in mangrove estuaries, a behavior found only in hawksbills along Pacific Central America. The detected genetic differentiation supports the existence of a novel mangrove estuary “reproductive ecotype” that may warrant additional conservation attention. From a phylogeographic perspective, our research indicates hawksbills colonized the EP via the Indo‐Pacific, and do not represent relict populations isolated from the Atlantic by the rising of the Panama Isthmus. Low overall genetic diversity in the EP is likely the combined result of few rookeries, extremely small reproductive populations and evolutionarily recent colonization events. Additional research with larger sample sizes and variable markers will help further genetic understanding of hawksbill turtles in the EP.     

High-Density Lipoprotein-Associated miR-223 Is Altered after Diet-Induced Weight Loss in Overweight and Obese Males

Background and Aims

microRNAs (miRNAs) are small, endogenous non-coding RNAs that regulate metabolic processes, including obesity. The levels of circulating miRNAs are affected by metabolic changes in obesity, as well as in diet-induced weight loss. Circulating miRNAs are transported by high-density lipoproteins (HDL) but the regulation of HDL-associated miRNAs after diet-induced weight loss has not been studied. We aim to determine if HDL-associated miR-16, miR-17, miR-126, miR-222 and miR-223 levels are altered by diet-induced weight loss in overweight and obese males.

Methods

HDL were isolated from 47 subjects following 12 weeks weight loss comparing a high protein diet (HP, 30% of energy) with a normal protein diet (NP, 20% of energy). HDL-associated miRNAs (miR-16, miR-17, miR-126, miR-222 and miR-223) at baseline and after 12 weeks of weight loss were quantified by TaqMan miRNA assays. HDL particle sizes were determined by non-denaturing polyacrylamide gradient gel electrophoresis. Serum concentrations of human HDL constituents were measured immunoturbidometrically or enzymatically.

Results

miR-16, miR-17, miR-126, miR-222 and miR-223 were present on HDL from overweight and obese subjects at baseline and after 12 weeks of the HP and NP weight loss diets. The HP diet induced a significant decrease in HDL-associated miR-223 levels (p = 0.015), which positively correlated with changes in body weight (r = 0.488, p = 0.032). Changes in miR-223 levels were not associated to changes in HDL composition or size.

Conclusion

HDL-associated miR-223 levels are significantly decreased after HP diet-induced weight loss in overweight and obese males. This is the first study reporting changes in HDL-associated miRNA levels with diet-induced weight loss.