Double-Strand Break Repair by Interchromosomal Recombination: An In Vivo Repair Mechanism Utilized by Multiple Somatic Tissues in Mammals

Homologous recombination (HR) is essential for accurate genome duplication and maintenance of genome stability. In eukaryotes, chromosomal double strand breaks (DSBs) are central to HR during specialized developmental programs of meiosis and antigen receptor gene rearrangements, and form at unusual DNA structures and stalled replication forks. DSBs also result from exposure to ionizing radiation, reactive oxygen species, some anti-cancer agents, or inhibitors of topoisomerase II. Literature predicts that repair of such breaks normally will occur by non-homologous end-joining (in G1), intrachromosomal HR (all phases), or sister chromatid HR (in S/G²). However, no in vivo model is in place to directly determine the potential for DSB repair in somatic cells of mammals to occur by HR between repeated sequences on heterologs (i.e., interchromosomal HR). To test this, we developed a mouse model with three transgenes—two nonfunctional green fluorescent protein (GFP) transgenes each containing a recognition site for the I-SceI endonuclease, and a tetracycline-inducible I-SceI endonuclease transgene. If interchromosomal HR can be utilized for DSB repair in somatic cells, then I-SceI expression and induction of DSBs within the GFP reporters may result in a functional GFP+ gene. Strikingly, GFP+ recombinant cells were observed in multiple organs with highest numbers in thymus, kidney, and lung. Additionally, bone marrow cultures demonstrated interchromosomal HR within multiple hematopoietic subpopulations including multi-lineage colony forming unit–granulocyte-erythrocyte-monocyte-megakaryocte (CFU-GEMM) colonies. This is a direct demonstration that somatic cells in vivo search genome-wide for homologous sequences suitable for DSB repair, and this type of repair can occur within early developmental populations capable of multi-lineage differentiation.     

The Association between Genetic Variations of CHI3L1, Levels of the Encoded Glycoprotein YKL-40 and the Lipid Profile in a Danish Population

Background

The inflammatory biomarker YKL-40 seems to play a role in atherosclerosis and is elevated in patients with obesity, cardiovascular disease and type 2 diabetes. Single nucleotide polymorphisms (SNPs) of the YKL-40 encoding gene, CHI3L1, are associated with inter-individual YKL-40 levels. One study has described an association between a promoter polymorphism of CHI3L1 and levels of low density lipoprotein. The objective of this study was to evaluate the influence of YKL-40 on lipid parameters by determining the association between polymorphisms of CHI3L1, serum YKL-40 and levels of the differentiated lipid profile in a Danish general population.

Methodology/Principle Findings

12 SNPs of CHI3L1 were genotyped, and serum YKL-40 and parameters of the lipid profile were measured in 2,656 Danes. Lipid profile and genotypes were available in another Danish population (n = 6,784) for replication. Cholesterol and triglyceride levels increased with increasing YKL-40 quartile (both p<0.0001), and YKL-40 correlated with triglyceride levels (β = 0.15, p<0.0001). Low density lipoprotein levels increased slightly from the 1^st to the 3^rd quartile (p = 0.006). The highest YKL-40 quartile was associated with a greater risk of hypercholesterolemia compared to the lowest YKL-40 quartile (odds ratio 1.36, p = 0.009). Minor homozygosity of rs12123883 was associated with higher triglyceride levels (p = 0.022) and a higher prevalence of low high density lipoprotein (p = 0.012), but these associations could not be confirmed in the replication population.

Conclusions/Significance

Serum YKL-40 correlates with triglyceride levels in a representative group of the general Danish population. No consistent associations between SNPs of CHI3L1 and lipid levels could be documented.     

Tracking the emergence of a new breed using 49,034 SNP in sheep

Domestic animals are unique in that they have been organised into managed populations called breeds. The strength of genetic divergence between breeds may vary dependent on the age of the breed, the scenario under which it emerged and the strength of reproductive isolation it has from other breeds. In this study, we investigated the Gulf Coast Native breed of sheep to determine if it contains lines of animals that are sufficiently divergent to be considered separate breeds. Allele sharing and principal component analysis (PCA) using nearly 50,000 SNP loci revealed a clear genetic division that corresponded with membership of either the Florida or Louisiana Native lines. Subsequent analysis aimed to determine if the strength of the divergence exceeded that found between recognised breed pairs. Genotypes from 14 breeds sampled from Europe and Asia were used to obtain estimates of pair-wise population divergence measured as F _ST. The divergence separating the Florida and Louisiana Native (F _ST = 6.2%) was approximately 50% higher than the average divergence separating breeds developed within the same region of Europe (F _ST = 4.2%). This strongly indicated that the two Gulf Coast Native lines are sufficiently different to be considered separate breeds. PCA using small SNP sets successfully distinguished between the Florida and Louisiana Native animals, suggesting that allele frequency differences have accumulated across the genome. This is consistent with a population history involving geographic separation and genetic drift. Suggestive evidence was detected for divergence at the poll locus on sheep chromosome 10; however drift at neutral markers has been the largest contributor to the genetic separation observed. These results document the emergence of populations that can be considered separate breeds, with practical consequences for bio-conservation priorities, animal registration and the establishment of separate breed societies.     

Variations of CHI3L1, Levels of the Encoded Glycoprotein YKL-40 and Prediction of Fatal and Non-fatal Ischemic Stroke

Background

Polymorphisms of CHI3L1 are associated with inter-individual YKL-40 levels and YKL-40 is associated with an increased mortality and is elevated in patients with cardiovascular disease. We investigated the association between single nucleotide polymorphisms (SNPs) of CHI3L1, serum YKL-40 levels and all-cause and cardiovascular mortality and first-time incidence of myocardial infarction, ischemic heart disease (IHD) and stroke.

Methodology/Principal Findings

12 SNPs of CHI3L1 were genotyped and serum YKL-40 was measured in 2656 Danes representative of the general population. Median follow-up period was 15 (0–16) years. Admission data and deaths were ascertained from registers from the Danish National Board of Health. Fourth quartile YKL-40 levels were associated with an increased mortality risk of ischemic stroke (HR 2.44 (1.01–5.88), p = 0.041) and so were homozygotes of the minor allele of rs872129 (HR 9.35 (1.25–69.87, p = 0.022)). Both continuous YKL-40 levels and 4^th quartile YKL-40 values (>85 ng/ml) were associated with all-cause mortality (HRs 1.22 (95% CI, 1.10–1.35), p<0.0001, and 1.40 (1.15–1.71), p<0.0001), an increased risk of first-time stroke (HR 1.16 (1.01–1.33), p = 0.04, and 1.63 (1.23–2.16), p = 0.001) and a decreased risk of incidence of IHD (HR 0.77 (0.65–0.91), p = 0.002, and 0.61 (0.44–0.85), p = 0.003).

Conclusions/Signficance

High YKL-40 levels (>85 ng/ml) and rs872129 were associated with an increased mortality risk of ischemic stroke, but high YKL-40 levels were also inverse related with the risk of incidence of IHD. This could be a chance finding but could also elucidate that YKL-40 plays different roles in development of thromboembolisms versus the formation of local thrombosis.     

Dopamine and the Development of Executive Dysfunction in Autism Spectrum Disorders

Persons with autism regularly exhibit executive dysfunction (ED), including problems with deliberate goal-directed behavior, planning, and flexible responding in changing environments. Indeed, this array of deficits is sufficiently prominent to have prompted a theory that executive dysfunction is at the heart of these disorders. A more detailed examination of these behaviors reveals, however, that some aspects of executive function remain developmentaly appropriate. In particular, while people with autism often have difficulty with tasks requiring cognitive flexibility, their fundamental cognitive control capabilities, such as those involved in inhibiting an inappropriate but relatively automatic response, show no significant impairment on many tasks. In this article, an existing computational model of the prefrontal cortex and its role in executive control is shown to explain this dichotomous pattern of behavior by positing abnormalities in the dopamine-based modulation of frontal systems in individuals with autism. This model offers excellent qualitative and quantitative fits to performance on standard tests of cognitive control and cognitive flexibility in this clinical population. By simulating the development of the prefrontal cortex, the computational model also offers a potential explanation for an observed lack of executive dysfunction early in life.     

The effects of 1,25-dihydroxyvitamin D3 on human T lymphocyte activation and proliferation: a cell cycle analysis

Recent studies have demonstrated that 1,25-dihydroxyvitamin D3 (calcitriol), the most biologically active metabolite of vitamin D, is a potent inhibitor of both lectin- and antigen-driven human T lymphocyte proliferation. To better characterize this effect, we performed cell cycle analysis of both untreated and calcitriol-treated peripheral blood mononuclear cells after PHA stimulation. By using the metachromatic dye acridine orange and flow cytometry, we found that calcitriol blocks the transition from the early, low RNA compartment of G1 (G1A) to the late, higher RNA compartment of G1 (G1B). Consistent with this observation was the inability of exogenous IL 1 or phorbol myristic acetate to overcome calcitriol's suppression of DNA synthesis. Indomethacin slightly reversed calcitriol's inhibition of transition from early to late G1, suggesting a minor, prostaglandin-dependent component to calcitriol's antiproliferative activity. Finally, by using the monoclonal antibodies anti-Tac and OKT9, we found that calcitriol had no effect on IL 2 receptor expression, an early G1 event, but markedly inhibited transferrin receptor expression, an IL 2-dependent, late G1 event. Thus, analysis of calcitriol's effects on the expression of these T cell activation antigens provides further evidence of the cell cycle specificity of calcitriol's action in regulating human T lymphocyte proliferation.     

Transplantation survival is maintained by granzyme B+ regulatory cells and adaptive regulatory T cells

Granzyme B (GZB) has been implicated as an effector mechanism in regulatory T cells (T(reg)) suppression. In a model of T(reg)-dependent graft tolerance, it is shown that GZB- deficient mice are unable to establish long-term tolerance. Moreover, mice overexpressing the inhibitor of GZB, serine protease inhibitor 6, are also resistant to tolerization to alloantigen. Graft survival was shorter in bone marrow-mixed chimeras reconstituted with GZB-deficient T(reg) as compared with wild-type T(reg). Whereas there was no difference in graft survival in mixed chimeras reconstituted with wild-type, perforin-deficient, or Fas ligand-deficient T(reg). Finally, data also show that if alloreactive effectors cannot express FoxP3 and be induced to convert in the presence of competent T(reg), then graft tolerance is lost. Our data are the first in vivo data to implicate GZB expression by T(reg) in sustaining long-lived graft survival.     

Intrahepatic Genetic Inoculation of Hepatitis C Virus RNA Confers Cross-Protective Immunity

Naturally occurring hepatitis C virus (HCV) infection has long been thought to induce a weak immunity which is insufficient to protect an individual from subsequent infections and has cast doubt on the ability to develop effective vaccines. A series of intrahepatic genetic inoculations (IHGI) with type 1a HCV RNA were performed in a chimpanzee to determine whether a form of genetic immunization might stimulate protective immunity. We demonstrate that the chimpanzee not only developed protective immunity to the homologous type 1a RNA after rechallenge by IHGI but was also protected from chronic HCV infection after sequential rechallenge with 100 50% chimpanzee infectious doses of a heterologous type 1a (H77) and 1b (HC-J4) whole-virus inoculum. These results offer encouragement to pursue the development of HCV vaccines.