Adaptive divergence despite strong genetic drift: genomic analysis of the evolutionary mechanisms causing genetic differentiation in the island fox (Urocyon littoralis)

The evolutionary mechanisms generating the tremendous biodiversity of islands have long fascinated evolutionary biologists. Genetic drift and divergent selection are predicted to be strong on islands and both could drive population divergence and speciation. Alternatively, strong genetic drift may preclude adaptation. We conducted a genomic analysis to test the roles of genetic drift and divergent selection in causing genetic differentiation among populations of the island fox (Urocyon littoralis). This species consists of six subspecies, each of which occupies a different California Channel Island. Analysis of 5293 SNP loci generated using Restriction‐site Associated DNA (RAD) sequencing found support for genetic drift as the dominant evolutionary mechanism driving population divergence among island fox populations. In particular, populations had exceptionally low genetic variation, small N_e (range = 2.1–89.7; median = 19.4), and significant genetic signatures of bottlenecks. Moreover, islands with the lowest genetic variation (and, by inference, the strongest historical genetic drift) were most genetically differentiated from mainland grey foxes, and vice versa, indicating genetic drift drives genome‐wide divergence. Nonetheless, outlier tests identified 3.6–6.6% of loci as high F_ST outliers, suggesting that despite strong genetic drift, divergent selection contributes to population divergence. Patterns of similarity among populations based on high F_ST outliers mirrored patterns based on morphology, providing additional evidence that outliers reflect adaptive divergence. Extremely low genetic variation and small N_e in some island fox populations, particularly on San Nicolas Island, suggest that they may be vulnerable to fixation of deleterious alleles, decreased fitness and reduced adaptive potential.     

Quantification of Cellular NEMO Content and Its Impact on NF-κB Activation by Genotoxic Stress

NF-κB essential modulator, NEMO, plays a key role in canonical NF-κB signaling induced by a variety of stimuli, including cytokines and genotoxic agents. To dissect the different biochemical and functional roles of NEMO in NF-κB signaling, various mutant forms of NEMO have been previously analyzed. However, transient or stable overexpression of wild-type NEMO can significantly inhibit NF-κB activation, thereby confounding the analysis of NEMO mutant phenotypes. What levels of NEMO overexpression lead to such an artifact and what levels are tolerated with no significant impact on NEMO function in NF-κB activation are currently unknown. Here we purified full-length recombinant human NEMO protein and used it as a standard to quantify the average number of NEMO molecules per cell in a 1.3E2 NEMO-deficient murine pre-B cell clone stably reconstituted with full-length human NEMO (C5). We determined that the C5 cell clone has an average of 4 x 10⁵ molecules of NEMO per cell. Stable reconstitution of 1.3E2 cells with different numbers of NEMO molecules per cell has demonstrated that a 10-fold range of NEMO expression (0.6–6x10⁵ molecules per cell) yields statistically equivalent NF-κB activation in response to the DNA damaging agent etoposide. Using the C5 cell line, we also quantified the number of NEMO molecules per cell in several commonly employed human cell lines. These results establish baseline numbers of endogenous NEMO per cell and highlight surprisingly normal functionality of NEMO in the DNA damage pathway over a wide range of expression levels that can provide a guideline for future NEMO reconstitution studies.     

Elevated Intracranial Pressure and Cerebral Edema following Permanent MCA Occlusion in an Ovine Model

Introduction

Malignant middle cerebral artery (MCA) stroke has a disproportionately high mortality due to the rapid development of refractory space-occupying cerebral edema. Animal models are essential in developing successful anti-edema therapies; however to date poor clinical translation has been associated with the predominately used rodent models. As such, large animal gyrencephalic models of stroke are urgently needed. The aim of the study was to characterize the intracranial pressure (ICP) response to MCA occlusion in our recently developed ovine stroke model.

Materials and Methods

30 adult female Merino sheep (n = 8–12/gp) were randomized to sham surgery, temporary or permanent proximal MCA occlusion. ICP and brain tissue oxygen were monitored for 24 hours under general anesthesia. MRI, infarct volume with triphenyltetrazolium chloride (TTC) staining and histology were performed.

Results

No increase in ICP, radiological evidence of ischemia within the MCA territory but without space-occupying edema, and TTC infarct volumes of 7.9+/-5.1% were seen with temporary MCAO. Permanent MCAO resulted in significantly elevated ICP, accompanied by 30% mortality, radiological evidence of space-occupying cerebral edema and TTC infarct volumes of 27.4+/-6.4%.

Conclusions

Permanent proximal MCAO in the sheep results in space-occupying cerebral edema, raised ICP and mortality similar to human malignant MCA stroke. This animal model may prove useful for pre-clinical testing of anti-edema therapies that have shown promise in rodent studies.     

Polymorphism of 9p21.3 Locus Is Associated with 5-Year Survival in High-Risk Patients with Myocardial Infarction

Objective

The rs10757278, rs1333049 and rs4977574 are single nucleotide polymorphisms (SNPs) of chromosome 9p21 locus associated with a prevalence of acute coronary syndromes (ACS). Reports concerning their association with long-term outcome after an ACS are equivocal. The aim of our study was to investigate the association of the 9p21.3 locus with 5-year overall mortality in patients with ST-elevation myocardial infarction (STEMI).

Materials and methods

We performed a retrospective analysis of data collected prospectively in 2 independent registries of consecutive patients with STEMI (derivation and validation group). Genotyping was performed with the TaqMan method. The analyzed end-point was total mortality.

Results

The derivation group comprised 589 patients: 25.3% female (n = 149), mean age 62.4±12.0 years, total 5-year mortality 16.6% (n = 98). When all the study group was analyzed, no significant differences in mortality were found between the genotypes. However, in high-risk patients (GRACE risk score ≥155 points, n = 238), homozygotes associated with higher risk for ACS had significantly better 5-year survival compared to other genotypes. The hazard ratio associated with the high-risk genotype (a homozygote of high risk for ACS or a heterozygote) was: HR = 2.2 (1.15–4.2) for the rs10757278 polymorphism, HR = 2.7 (95% CI 1.3–5.4) for the rs4977574 one and HR = 2.3 (1.2–4.5) for the rs1333049 one (Cox proportional hazards model). Survival analysis in the validation group (n = 365) showed a clear trend towards better prognosis in GG homozygotes of the rs10757278 SNP, which confirms our initial results (p = 0.09, log-rank test).

Conclusions

The 9p21.3 locus is associated with 5-year mortality in high-risk patients with STEMI. The genotypes associated with higher risk for ACS show a protective effect in terms of further survival (instead of a deteriorating prognosis, as reported previously). This finding, due to the very high size of the effect, could potentially be applied to clinical practice, if appropriate methods are elaborated.     

Local selection underlies the geographic distribution of sex-ratio drive in Drosophila neotestacea

"Selfish" genetic elements promote their own transmission to the next generation, often at a cost to the host individual. A sex-ratio (SR) driving X chromosome prevents the maturation of Y-bearing sperm, and as a result is transmitted to 100% of the offspring, all of which are female. Because the spread of a SR chromosome can result in a female-biased population sex ratio, the ecological and evolutionary consequences of harboring this selfish element can be severe. In this study, we show that the prevalence of SR drive in Drosophila neotestacea varies between 0% and 30% among populations, and is common in the south whereas rare in the north. The prevalence of SR is not associated with the presence of suppressors of drive, geographic distance, or genetic distance based on autosomal microsatellite loci. Instead, our results indicate that ecological selection on SR drive varies among populations, as the prevalence of SR is highly correlated with climatic factors, with the severity of winter the best determinant of SR frequency. Thus, ecological and demographic factors may have significant consequences for the short and long term evolutionary dynamics of selfish elements and the manner with which they coevolve with the rest of the genome.     

RAGE signaling by alveolar macrophages influences tobacco smoke-induced inflammation

Receptors for advanced glycation end-products (RAGE) are multiligand cell surface receptors of the immunoglobin family expressed by epithelium and macrophages, and expression increases following exposure to cigarette smoke extract (CSE). The present study sought to characterize the proinflammatory contributions of RAGE expressed by alveolar macrophages (AMs) following CSE exposure. Acute exposure of mice to CSE via nasal instillation revealed diminished bronchoalveolar lavage (BAL) cellularity and fewer AMs in RAGE knockout (KO) mice compared with controls. Primary AMs were obtained from BAL, exposed to CSE in vitro, and analyzed. CSE significantly increased RAGE expression by wild-type AMs. Employing ELISAs, wild-type AMs exposed to CSE had increased levels of active Ras, a small GTPase that perpetuates proinflammatory signaling. Conversely, RAGE KO AMs had less Ras activation compared with wild-type AMs after exposure to CSE. In RAGE KO AMs, assessment of p38 MAPK and NF-κB, important intracellular signaling intermediates induced during an inflammatory response, revealed that CSE-induced inflammation may occur in part via RAGE signaling. Lastly, quantitative RT-PCR revealed that the expression of proinflammatory cytokines including TNF-α and IL-1β were detectably decreased in RAGE KO AMs exposed to CSE compared with CSE-exposed wild-type AMs. These results reveal that primary AMs orchestrate CSE-induced inflammation, at least in part, via RAGE-mediated mechanisms.     

Tumor suppressor Tsc1 is a new Hsp90 co‐chaperone that facilitates folding of kinase and non‐kinase clients

The tumor suppressors Tsc1 and Tsc2 form the tuberous sclerosis complex (TSC), a regulator of mTOR activity. Tsc1 stabilizes Tsc2; however, the precise mechanism involved remains elusive. The molecular chaperone heat‐shock protein 90 (Hsp90) is an essential component of the cellular homeostatic machinery in eukaryotes. Here, we show that Tsc1 is a new co‐chaperone for Hsp90 that inhibits its ATPase activity. The C‐terminal domain of Tsc1 (998–1,164 aa) forms a homodimer and binds to both protomers of the Hsp90 middle domain. This ensures inhibition of both subunits of the Hsp90 dimer and prevents the activating co‐chaperone Aha1 from binding the middle domain of Hsp90. Conversely, phosphorylation of Aha1‐Y223 increases its affinity for Hsp90 and displaces Tsc1, thereby providing a mechanism for equilibrium between binding of these two co‐chaperones to Hsp90. Our findings establish an active role for Tsc1 as a facilitator of Hsp90‐mediated folding of kinase and non‐kinase clients—including Tsc2—thereby preventing their ubiquitination and proteasomal degradation.     

T cell-induced secretion of MHC class II-peptide complexes on B cell exosomes

Antigen-specific interactions between B cells and T cells are essential for the generation of an efficient immune response. Since this requires peptide–MHC class II complexes (pMHC-II) on the B cell to interact with TCR on antigen-specific T cells, we have examined the mechanisms regulating the persistence, loss, and secretion of specific pMHC-II complexes on activated B cells. Using a mAb that recognizes specific pMHC-II, we found that activated B cells degrade approximately 50% of pMHC-II every day and release 12% of these pMHC-II from the cell on small membrane vesicles termed exosomes. These exosomes directly stimulate primed, but not naïve, CD4 T cells. Interestingly, engagement of antigen-loaded B cells with specific CD4 T cells stimulates exosome release in a manner that can be mimicked by pMHC-II crosslinking. Biochemical studies revealed that the pMHC-II released on exosomes was previously expressed on the plasma membrane of the B cells, suggesting that regulated exosome release from activated B cells is a mechanism to allow pMHC-II to escape intracellular degradation and decorate secondary lymphoid organs with membrane-associated pMHC-II complexes.     

Role of copper in tumour angiogenesis--clinical implications.

The formation of new blood vessels is the initial step in progressive tumour development and metastasis. The first stage in tumour angiogenesis is the activation of endothelial cells. Copper ions stimulate proliferation and migration of endothelial cells. It has been shown that serum copper concentration increases as the cancer disease progresses and correlates with tumour incidence and burden. Copper ions also activate several proangiogenic factors, e.g., vascular endothelial growth factor, basic fibroblast growth factor, tumour necrosis factor alpha and interleukin 1. This review concerns a brief introduction into the basics of tumour blood vessel development as well as the regulatory mechanisms of this process. The role of copper ions in tumour angiogenesis is discussed. The new antiangiogenic therapies based on a reduction of copper levels in tumour microenvironment are reviewed.