Role of {\it ERCC1\/} in DNA repair and genetic recombination

The ERCC1 (Excision Repair Cross-Complementing-1) gene is the presumptive mammalian homolog of the Saccharomyces cerevisiae RAD10 gene. In mammalian NER, the Ercc1/XpF complex functions as an endonuclease that specifically recognizes 5$\sp\prime$ double-strand-3$\sp\prime$ single-strand structures. In yeast, the analogous function is performed by the Rad1/Rad10 complex. These observations and the conservation of amino acid homology between the Rad1 and XpF and the Rad10 and Ercc1 proteins has led to a general assumption of functional homology between these genes.^ In addition to NER, the Rad1/Rad10 endonuclease complex is also required in certain specialized mitotic recombination pathways in yeast. However, a similiar requirement for the endonuclease function of the Ercc1/XpF complex during genetic recombination in mammalian cells has not been directly demonstrated. The experiments performed in these studies were designed to determine if ERCC1 deficiency would produce recombination-deficient phenotypes in CHO cells similar to those observed in RAD10 deletion mutants, including: (1) decreased single-reciprocal exchange recombination, and (2) inability to process 5$\sp\prime$ sequence heterology in recombination intermediates.^ Specifically, these studies describe: (1) The isolation and characterization of the ERCC1 locus of Chinese hamster ovary cells; (2) The production of an ERCC1 null mutant cell line by targeted knock-out of the endogenous ERCC1 gene in a Chinese hamster ovary cell line, CHO-ATS49tg, which contains an endogenous locus, APRT, suitable as a chromosomal target for homologous recombination; (3) The characterization of mutant ERCC1 alleles from a panel of Chinese hamster ovary cell ERCC1 mutants derived by conventional mutagenesis; (4) An investigation of the effects of ERCC1 mutation on mitotic recombination through targeting of the APRT locus in an ERCC1 null background.^ The results of these studies strongly suggest that the role of ERCC1 in homologous recombination in mammalian cells is analogous to that of the yeast RAD10 gene. ^

Cloning and genetic characterization of Skb1: A novel regulator of Shk1 and mitosis in Schizosaccharomyces pombe

A partial skb1 gene was originally isolated in a yeast two-hybrid screen for Shk1-interacting polypeptides. Shk1 is one of two Schizosaccharomyces pombe p21Cdc42/Rac-activated kinases (PAKs) and is an essential component of the Ras1-dependent signal transduction pathways regulating cell morphology and mating responses in fission yeast. After cloning the skb1 gene we found the Skb1 gene product to be a novel, nonessential protein lacking homology to previously characterized proteins. However the identification of Skb1 homologs in C. elegans, S. cerevisiae, and H. sapiens reveals evolution has conserved the skb1 gene. Fission yeast cells carrying a deletion of skb1 exhibit a defect in cell size but not mating abilities. This defect is suppressed by high copy shk1. Fission yeast overexpressing skb1 were found to undergo cell division at a length 1.5X greater than normal. In the two-hybrid system, Skb1 interacts with a subdomain of the Shk1 regulatory region distinct from that with which Cdc42 interacts, and forms a ternary complex with Shk1 and Cdc42. By use of yeast genetics, we have established a role for Skb1 as a positive regulator of Shk1. Co-overexpression of shk1 with skb1 was found to suppress the morphology defect, but not the sterility, of ras1Δ fission yeast. Thus, the function of Skb1 is restricted to a morphology control pathway. We determined that Skb1 functions as a negative regulator of mitosis and does this through a Shk1-dependent mechanism. The mitotic regulatory function of Skb1 and Shk1 was also partially dependent upon Wee1, a direct negative regulator of the cyclin-dependent kinase Cdc2. The role for Skb1 and Shk1 as mitotic regulators is the first connection from a PAK protein to control of the cell cycle. Furthermore, Skb1 is the first non-Cdc42/Rac PAK modulator to be identified. ^

Plant-plant interactions, environmental gradients and plant diversity: A global synthesis of community-level studies

Previous syntheses on the effects of environmental conditions on the outcome of plant-plant interactions summarize results from pairwise studies. However, the upscaling to the community-level of such studies is problematic because of the existence of multiple species assemblages and species-specific responses to both the environmental conditions and the presence of neighbors. We conducted the first global synthesis of community-level studies from harsh environments, which included data from 71 alpine and 137 dryland communities to: (i) test how important are facilitative interactions as a driver of community structure, (ii) evaluate whether we can predict the frequency of positive plant-plant interactions across differing environmental conditions and habitats, and (iii) assess whether thresholds in the response of plant-plant interactions to environmental gradients exists between ``moderate'' and ``extreme'' environments. We also used those community-level studies performed across gradients of at least three points to evaluate how the average environmental conditions, the length of the gradient studied, and the number of points sampled across such gradient affect the form and strength of the facilitation-environmental conditions relationship. Over 25% of the species present were more spatially associated to nurse plants than expected by chance in both alpine and chyland areas, illustrating the high importance of positive plant-plant interactions for the maintenance of plant diversity in these environments. Facilitative interactions were more frequent, and more related to environmental conditions, in alpine than in dryland areas, perhaps because drylands are generally characterized by a larger variety of environmental stress factors and plant functional traits. The frequency of facilitative interactions in alpine communities peaked at 1000 mm of annual rainfall, and globally decreased with elevation. The frequency of positive interactions in dtyland communities decreased globally with water scarcity or temperature annual range. Positive facilitation-drought stress relationships are more likely in shorter regional gradients, but these relationships are obscured in regions with a greater species turnover or with complex environmental gradients. By showing the different climatic drivers and behaviors of plant-plant interactions in dryland and alpine areas, our results will improve predictions regarding the effect of facilitation on the assembly of plant communities and their response to changes in environmental conditions.

Disentangling the role of phenotypic plasticity and genetic divergence in contemporary ecotype formation during a biological invasion

The occurrence of contemporary ecotype formation through adaptive divergence of populations within the range of an invasive species typically requires standing genetic variation but can be facilitated by phenotypic plasticity. The relative contributions of both of these to adaptive trait differentiation have rarely been simultaneously quantified in recently diverging vertebrate populations. Here we study a case of intraspecific divergence into distinct lake and stream ecotypes of threespine stickleback that evolved in the past 140 years within the invasive range in Switzerland. Using a controlled laboratory experiment with full-sib crosses and treatments mimicking a key feature of ecotypic niche divergence, we test if the phenotypic divergence that we observe in the wild results from phenotypic plasticity or divergent genetic predisposition. Our experimental groups show qualitatively similar phenotypic divergence as those observed among wild adults. The relative contribution of plasticity and divergent genetic predisposition differs among the traits studied, with traits related to the biomechanics of feeding showing a stronger genetic predisposition, whereas traits related to locomotion are mainly plastic. These results implicate that phenotypic plasticity and standing genetic variation interacted during contemporary ecotype formation in this case.

Metabolomics in plant-herbivore interactions: challenges and applications

Metabolomics as the study of the entire set of metabolites of a given organism is an important frontier in life sciences. As a tool that captures the ‘front end’ of cellular machineries, metabolomics is particularly suited to investigate biotic interactions, including for instance the interplay between plants and insects. In this review, we discuss the opportunities and challenges of metabolomics to study plant–herbivore interactions. We first present a brief overview of the typical analytical workflows used in metabolomics and their associated issues, in particular those related to metabolome coverage and compound identification. Second, recent advances in the field of plant–herbivore relationships that are promoted by non-targeted approaches are reviewed, with examples ranging from classical herbivore resistance patterns to plant-mediated interactions across different spatial scales and volatile-mediated tritrophic interactions. Through general considerations and the discussion of a few selected case studies, our review highlights the potential and challenges of metabolomics as a research approach to understand biological interfaces.

Linking altered central pain processing and genetic polymorphism to drug efficacy in chronic low back pain.

BACKGROUND Inability to predict the therapeutic effect of a drug in individual pain patients prolongs the process of drug and dose finding until satisfactory pharmacotherapy can be achieved. Many chronic pain conditions are associated with hypersensitivity of the nervous system or impaired endogenous pain modulation. Pharmacotherapy often aims at influencing these disturbed nociceptive processes. Its effect might therefore depend on the extent to which they are altered. Quantitative sensory testing (QST) can evaluate various aspects of pain processing and might therefore be able to predict the analgesic efficacy of a given drug. In the present study three drugs commonly used in the pharmacological management of chronic low back pain are investigated. The primary objective is to examine the ability of QST to predict pain reduction. As a secondary objective, the analgesic effects of these drugs and their effect on QST are evaluated. METHODS/DESIGN In this randomized, double blinded, placebo controlled cross-over study, patients with chronic low back pain are randomly assigned to imipramine, oxycodone or clobazam versus active placebo. QST is assessed at baseline, 1 and 2 h after drug administration. Pain intensity, side effects and patients' global impression of change are assessed in intervals of 30 min up to two hours after drug intake. Baseline QST is used as explanatory variable to predict drug effect. The change in QST over time is analyzed to describe the pharmacodynamic effects of each drug on experimental pain modalities. Genetic polymorphisms are analyzed as co-variables. DISCUSSION Pharmacotherapy is a mainstay in chronic pain treatment. Antidepressants, anticonvulsants and opioids are frequently prescribed in a "trial and error" fashion, without knowledge however, which drug suits best which patient. The present study addresses the important need to translate recent advances in pain research to clinical practice. Assessing the predictive value of central hypersensitivity and endogenous pain modulation could allow for the implementation of a mechanism-based treatment strategy in individual patients. TRIAL REGISTRATION Clinicaltrials.gov, NCT01179828.

Roots under attack: contrasting plant responses to below- and aboveground insect herbivory

The distinctive ecology of root herbivores, the complexity and diversity of root–microbe interactions, and the physical nature of the soil matrix mean that plant responses to root herbivory extrapolate poorly from our understanding of responses to aboveground herbivores. For example, root attack induces different changes in phytohormones to those in damaged leaves, including a lower but more potent burst of jasmonates in several plant species. Root secondary metabolite responses also differ markedly, although patterns between roots and shoots are harder to discern. Root defences must therefore be investigated in their own ecophysiological and evolutionary context, specifically one which incorporates root microbial symbionts and antagonists, if we are to better understand the battle between plants and their hidden herbivores.

Sociodemographic, behavioral and genetic determinants of allostatic load in a Swiss population-based study.

Allostatic load (AL) is a marker of physiological dysregulation which reflects exposure to chronic stress. High AL has been related to poorer health outcomes including mortality. We examine here the association of socioeconomic and lifestyle factors with AL. Additionally, we investigate the extent to which AL is genetically determined. We included 803 participants (52% women, mean age 48±16years) from a population and family-based Swiss study. We computed an AL index aggregating 14 markers from cardiovascular, metabolic, lipidic, oxidative, hypothalamus-pituitary-adrenal and inflammatory homeostatic axes. Education and occupational position were used as indicators of socioeconomic status. Marital status, stress, alcohol intake, smoking, dietary patterns and physical activity were considered as lifestyle factors. Heritability of AL was estimated by maximum likelihood. Women with a low occupational position had higher AL (low vs. high OR=3.99, 95%CI [1.22;13.05]), while the opposite was observed for men (middle vs. high OR=0.48, 95%CI [0.23;0.99]). Education tended to be inversely associated with AL in both sexes(low vs. high OR=3.54, 95%CI [1.69;7.4]/OR=1.59, 95%CI [0.88;2.90] in women/men). Heavy drinking men as well as women abstaining from alcohol had higher AL than moderate drinkers. Physical activity was protective against AL while high salt intake was related to increased AL risk. The heritability of AL was estimated to be 29.5% ±7.9%. Our results suggest that generalized physiological dysregulation, as measured by AL, is determined by both environmental and genetic factors. The genetic contribution to AL remains modest when compared to the environmental component, which explains approximately 70% of the phenotypic variance.

BIOCHEMICAL CHARACTERIZATION AND GENETIC MAPPING OF WILD TYPE AND MUTANT GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE ALLELES IN CHINESE HAMSTER OVARY CELLS

A UV-induced mutation of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPD) was characterized in the CHO clone A24. The asymmetric 4-banded zymogram and an in vitro GAPD activity equal to that of wild type cells were not consistent with models of a mutant heterozygote producing equal amounts of wild type and either catalytically active or inactive mutant subunits that interacted randomly. Cumulative evidence indicated that the site of the mutation was the GAPD structural locus expressed in CHO wild type cells, and that the mutant allele coded for a subunit that differed from the wild type subunit in stability and kinetics. The evidence included the appearance of a fifth band, the putative mutant homotetramer, after addition of the substrate glyceraldehyde-3-phosphate (GAP) to the gel matrix; dilution experiments indicating stability differences between the subunits; experiments with subsaturating levels of GAP indicating differences in affinity for the substrate; GAPD zymograms of A24 x mouse hybrids that were consistent with the presence of two distinct A24 subunits; independent segregation of A24 wild type and mutant electrophoretic bands from the hybrids, which was inconsistent with models of mutation of a locus involved in posttranslational modification; the mapping of both wild type and mutant forms of GAPD to chromosome 8; and the failure to detect any evidence of posttranslational modification (of other A24 isozymes, or through mixing of homogenates of A24 and mouse).^ The extent of skewing of the zymogram toward the wild type band, and the unreduced in vitro activity were inconsistent with models based solely on differences in activity of the two subunits. Comparison of wild type homotetramer bands in wild type cells and A24 suggested the latter had a preponderance of wild type subunits over mutant subunits, and had more GAPD tetramers than did CHO controls.^ Two CHO linkages, GAPD-triose phosphate isomerase, and acid phosphatase 2-adenosine deaminase were reported provisionally, and several others were confirmed. ^

Biochemical and genetic analysis of two protein kinases JNKK2 and MEKK3

Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryotic signaling modules consisting of a MAPK, a MAPKK and a MAP3K. MAPK cascades are involved in many cellular responses including proliferation, differentiation, apoptosis, stress and immune responses. ^ The first part of this thesis describes the cloning and biochemical analysis of JNKK2, a member of MAPKK gene family. Our results demonstrate that JNKK2 is a specific JNK activator and activates the JNK-dependent signal transduction pathway in vivo by inducing c-Jun and ATF2-mediated gene expression. We also found that JNKK2 is specifically activated by a MAP3K MEKK2 through formation of MEKK2-JNKK2-JNK1 triple complex module. JNKK2 is likely to mediate specific upstream signals to activate JNK cascade. ^ The second part of this thesis describes biochemical and gene disruption analysis of MEKK3, a member of MAP3K gene family. We showed that overexpression of MEKK3 strongly activates both JNK and p38 MAPKs but only weakly activates ERK. MEKK−/− embryos die at about embryonic day (E) 11. MEKK3−/− embryos displayed defects in blood vessel development in the yolk sacs, and in the myocardium and endocardium development at E9.5. The angiogenesis in the head, intersomitic region and placenta was also abnormal. These results demonstrate that MEKK3, a member of MAP3K MEKK/STE11 subgene family, is essential for early embryonic cardiovascular development. Furthermore, it was found that disruption of MEKK3 did not alter the expression of vascular endothelial growth factor-1 (VEGF-1), angiopoietin-1, -2 and their respective receptors Flt-1, Flk-1, Tie-1, Tie-2. Finally, MEKK3 was shown to activate myocyte-specific enhancer factor 2C (MEF2C), a crucial transcription factor for early embryonic cardiovascular development through the p38 MAPK cascade, suggesting that MEF2C is one of the key targets of the MEEKK3 signaling pathway during early embryonic cardiovascular development. ^

Are functional and genetic components of platelets linked to coronary artery disease: A case-control study

Coronary artery disease (CAD) is a multifactorial disease process involving behavioral, inflammatory, clinical, thrombotic, and genetic components. Previous epidemiologic studies focused on identifying behavioral and demographic risk factors of CAD, but none focused on platelets. Current platelet literature lacks the known effects of platelet function and platelet receptor polymorphisms on CAD. This case-control analysis addressed these issues by analyzing data collected for a previous study. Cases were individuals who had undergone CABG and thus had been diagnosed with CAD, while the controls were volunteers presumed to be CAD free. The platelet function variables analyzed included fibrinogen Von Willebrand Factor activity (VWF), shear-induced platelet aggregation (SIPA), sCD40L, and mean platelet volume; and the platelet polymorphisms studied included PIA, α2 807, Ko, Kozak, and VNTR. Univariate analysis found fibrinogen, VWF, SIPA, and PIA to be independent risk factors of CAD. Logistic regression was used to build a predictive model for CAD using the platelet function and platelet polymorphism data adjusted for age, sex, race, and current smoking status. A model containing only platelet polymorphisms and their respective receptor densities, found polymorphisms within GPIbα to be associated with CAD, yielding an 86% (95% C.I. 0.97–3.55) increased risk with the presence of at least 1 polymorphism in Ko, Kozak, or VNTR. Another model included both platelet function and platelet polymorphism data. Fibrinogen, the receptor density of GPIbα, and the polymorphism in GPIa-IIa (α2 807) were all associated with CAD with odds ratios of 1.10, 1.04, and 2.30 for fibrinogen (10mg/dl increase), GPIbα receptors (1 MFI increase), and GPIa-IIa, respectively. In addition, risk estimates and 99% confidence intervals adjusted for race were calculated to determine if the presence of a platelet receptor polymorphism was associated with CAD. The results were as follows: PIA (1.64, 0.74–3.65); α2 807 (1.35, 0.77–2.37); Ko (1.71, 0.70–4.16); Kozak (1.17, 0.54–2.52); and VNTR (1.24, 0.52–2.91). Although not statistically significant, all platelet polymorphisms were associated with an increased risk for CAD. These exploratory findings indicate that platelets do appear to have a role in atherosclerosis and that anti-platelet drugs targeting GPI-IIa and GPIbα may be better treatment candidates for individuals with CAD. ^

Molecular and genetic analyses of Fgf signaling during cardiac outflow tract development

Epithelial-mesenchymal tissue interactions regulate the development of derivatives of the caudal pharyngeal arches (PAs) to govern the ultimate morphogenesis of the aortic arch and outflow tract (OFT) of the heart. Disruption of these signaling pathways is thought to contribute to the pathology of a significant proportion of congenital cardiovascular defects in humans. In this study, I tested whether Fibroblast Growth Factor 15 (Fgf15), a secreted signaling molecule expressed within the PAs, is an extracellular mediator of tissue interactions during PA and OFT development. Analyses of Fgf15−/− mouse embryonic hearts revealed abnormalities primarily localized to the OFT, correlating with aberrant cardiac neural crest cell behavior. The T-box-containing transcription factor Tbx1 has been implicated in the cardiovascular defects associated with the human 22q11 Deletion Syndromes, and regulates the expression of other Fgf family members within the mouse PAs. However, expression and genetic interaction studies incorporating mice deficient for Tbx1, its upstream regulator, Sonic Hedgehog (Shh), or its putative downstream effector, Fgf8, indicated that Fgf15 functions during OFT development in a manner independent of these factors. Rather, analyses of compound mutant mice indicated that Fgf15 and Fgf9, an additional Fgf family member expressed within the PAs, genetically interact, providing insight into the factors acting in conjunction with Fgf15 during OFT development. Finally, in an effort to further characterize this Fgf15-mediated developmental pathway, promoter deletion analyses were employed to isolate a 415bp sequence 7.1Kb 5′ to the Fgf15 transcription start site both necessary and sufficient to drive reporter gene expression within the epithelium of the PAs. Sequence comparisons among multiple mammalian species facilitated the identification of evolutionarily conserved potential trans-acting factor binding sites within this fragment. Subsequent studies will investigate the molecular pathway(s) through which Fgf15 functions via identification of factors that bind to this element to govern Fgf15 gene expression. Furthermore, targeted deletion of this element will establish the developmental requirement for pharyngeal epithelium-derived Fgf15 signaling function. Taken as a whole, these data demonstrate that Fgf15 is a component of a novel, Tbx1-independent molecular pathway, functioning within the PAs in a manner cooperative with Fgf9, required for proper development of the cardiac OFT. ^

Biochemical and genetic characterization of the Saccharomyces cerevisiae Hsp110 molecular chaperone Sse1

The Ssel/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a carboxyl-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an amino-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Δ phenotypes. Surprisingly, all mutants predicted to abolish ATP hydrolysis complemented the temperature sensitivity of sse1Δ, whereas mutations in predicted ATP binding residues were non-functional. Remarkably, the two domains of Ssel when expressed in trans functionally complement the sse1Δ growth phenotype and interact by coimmunoprecipitation analysis, indicative of a novel type of interdomain communication. ^ Relatively little is known regarding the interactions and cellular functions of Ssel. Through co-immunoprecipitation analysis, we found that Ssel forms heterodimeric complexes with the abundant cytosolic Hsp70s Ssa and Ssb in vivo. Furthermore, these complexes can be efficiently reconstituted in vitro using purified proteins. The ATPase domains of Ssel and the Hsp70s were found to be critical for interaction as inactivating point mutations severely reduced interaction efficiency. Ssel stimulated Ssal ATPase activity synergistically with the co-chaperone Ydj1 via a novel nucleotide exchange activity. Furthermore, FES1, another Ssa nucleotide exchange factor, can functionally substitute for SSE1/2 when overexpressed, suggesting that Hsp70 nucleotide exchange is the fundamental role of the Sse proteins in yeast, and by extension, the Hsp110 homologs in mammals. ^ Cells lacking SSE1 were found to accumulate prepro-α-factor, but not the cotranslationally imported protein Kar2, similar to mutants in the Ssa chaperones. This indicates that the interaction between Ssel and Ssa is functionally significant in vivo. In addition, sse10 cells are compromised for cell wall strength, likely a result of decreased Hsp90 chaperone activity with the cell integrity MAP kinase SIC. Taken together, this work established that the Hsp110 family must be considered an essential component of Hsp70 chaperone biology in the eukaryotic cell.^

Immune and genetic risk factors in glioma

Despite extensive research, the etiology of adult glioma remains largely unknown. We sought to further explore the role of immune and genetic factors in glioma etiology using data from the Harris County Brain Tumor Study and the first U.S. genome-wide association study of glioma. First, using a case-control study design, we examined the association between adult glioma risk and surrogates of the timing and frequency of common early childhood infections, birth order and sibship size, respectively. We found that each one-unit increase in birth order was associated with a 12% decreased risk of glioma development in adulthood (OR=0.88, 95% CI=0.81-0.96); however, sibship size was not associated with adult glioma risk (OR=0.96, 95% CI=0.91-1.02). Second, we used a multi-strategic approach to explore the relationships between glioma risk, history of asthma/allergies, and 23 functional SNPs in 11 inflammation genes. We found three inflammation gene SNPs to be significantly associated with glioma risk (COX2/PTGS2 rs20417 [OR=1.41]; IL10 rs1800896 [OR=1.57]; and IL13 rs20541 [OR=0.39]). Joint effects analysis of the risk-conferring alleles of these three SNPs revealed a trend of increasing risk with increasing number of adverse alleles among those without asthma/allergies (p<0.0001). Finally, we conducted a case-only study to explore pairwise SNP-SNP interactions in immune-related pathways among a population of 1304 non-Hispanic white glioma cases. After correction for multiple comparisons, we found 279 significant SNP-SNP interactions among polymorphisms of immune-related genes, many of which have not been previously examined. Our results, cumulatively, suggest an important role for immune and genetic factors in glioma etiology and provide several new hypotheses for future studies.^