The N-terminal globular domain of Eph receptors is sufficient for ligand binding and receptor signaling.

The Eph family of receptor protein-tyrosine kinases (RTKs) have recently been implicated in patterning and wiring events in the developing nervous system. Eph receptors are unique among other RTKs in that they fall into two large subclasses that show distinct ligand specificities and for the fact that they themselves might function as ''ligands'', thereby activating bidirectional signaling. To gain insight into the mechanisms of ligand-receptor interaction, we have mapped the ligand binding domain in Eph receptors. By using a series of deletion and domain substitution mutants, we now report that an N-terminal globular domain of the Nuk/Cek5 receptor is the ligand binding domain of the transmembrane ligand Lerk2. Using focus formation assays, we show that the Cek5 globular domain is sufficient to confer Lerk2-dependent transforming activity on the Cek9 orphan receptor. Extending our binding studies to other members of both subclasses of receptors, it became apparent that the same domain is used for binding of both transmembrane and glycosylphosphatidyl-anchored ligands. Our studies have determined the first structural elements involved in ligand-receptor interaction and will allow more fine-tuned genetic experiments to elucidate the mechanism of action of these important guidance molecules.     

Genetic and physical characterization of lysogeny by bacteriophage MX8 in Myxococcus xanthus

Myxophage MX8 can initiate a lysogenic cycle in Myxococcus xanthus. The lysogenic phage was gentically stable in vegetative cells and persisted in the latent state through many cell generations in the absence of extracellular phage reinfection. The latent state also was stable during the host developmental cycle, since myxospores transmitted latent MX8 genetic information to future progeny cells. DNA hybridization experiments to probe the structure of the lysogenic phage provided physical evidence that MX8 formed a prophage. During lysogenization, MX8 DNA was cut at a specific site (attP) on phage DNA, and we have concluded that genetic recombination between attP and a bacterial DNA site (attB) leads to integration of MX8 DNA and formation of stable MX8 prophage. The genetic and physical properties of MX8 that we describe should make MX8 useful in the analysis of development of M. xanthus by genetic methods.     

THE EFFECTS OF HOST-PLANT GENOTYPE,HYBRIDIZATION, AND ENVIRONMENT ON GALL-APHID ATTACK AND SURVIVAL IN COTTONWOOD: THE IMPORTANCE OF GENETIC STUDIES AND THE UTILITY OF RFLPS

Using restriction fragment length polymorphisms (RFLPs) we show how host-plant genotype and hybridization in cotton wood, Populus sp., affects the attack and survival of the gall-forming aphid, Pemphigus betae. Fremont cottonwoods, hybrid ???F1's and backcross ???1's were found to be highly resistant, while backcross 2's, 3's, 4's and pure narrowleafs ranged from highly resistant to highly susceptible with only a few trees being highly resistant. Results from our genetic analysis also suggest that resistance is likely polygenic, not the result of single gene resistance. In addition, genetic analysis showed that studies based upon leaf morphology alone give an inaccurate assessment of host-plant genotype ???and the extent of hybridization, altering previous views of the relationship between plant hybridization and pest attack. Previous studies assumed that narrowleafs were more resistant than backcross genotypes based upon comparisons of overall levels of resistance between the hybrid zone and the “pure” narrowleaf zone. Results from RFLP analyses, however, show that there are no significant differences in the levels of resistance between backcross genotypes (BC2's-4's) and pure narrowleafs. Furthermore, results show that the “pure” narrowleaf zone is in fact a mixture of pure and backcross genotypes, extending the zone of introgression previously reported. Experiments in combination with RFLP analyses suggest that resistance traits are differentially expressed along an environmental gradient partially explaining the previously reported differences in resistance between these two regions. In light of our results it is clear that genetic studies will be necessary to discern the true relationship between hybridization and pest resistance. Until such studies are widely conducted generalizations regarding the effects of hybridization on the structure and dynamics of pest populations will be premature at best.     

Structure and polymorphism of the human gene for the interferon-induced p78 protein (MX1): evidence of association with alopecia areata in the Down syndrome region

Alopecia areata (AA) is a chronic inflammatory disease characterised by patchy hair loss with T cell infiltration of hair follicles. AA occurs in approximately 0.1% of the general population, but this is increased to 9% in Down syndrome (DS). DS is associated with an additional copy (full or partial) of chromosome 21, and the DS region may potentially include genes involved in the pathogenesis of AA. MX1 is the gene encoding the interferon-induced p78 protein (MxA). MxA protein confers resistance to influenza viruses, and we have previously shown that MxA protein is strongly expressed in lesional anagen hair bulbs from patients with AA but not in normal follicles. We therefore studied the possible involvement of MX1 in the pathogenesis of AA. To establish markers in the MX1 region which could be screened by PCR-based methods, we defined the human MX1 exon/intron organisation and screened the exons and the introns by conformation-sensitive gel electrophoresis. We found that the MX1 gene contains 17 exons extending over 33 kb. The size and sequence of the region from exon 6 to exon 16 are highly conserved between human and mouse. Screening of 4747 bp within the MX1 gene revealed four single nucleotide polymorphisms in intron 6. These polymorphisms are concentrated within 147 bp and show strong linkage disequilibrium. In a case-control association study for the MX1 (+9959) polymorphism in 165 AA patients and 510 controls we found a significant association of this marker with AA (odds ratio 1.79, 95% CI 1.21-2.66, chi2 = 8.464, P = 0.0036). The risk of disease was greater for patchy AA (mild disease) and with early age at onset (odds ratio 2.34, 95% CI 1.24-4.43, P = 0.0072), providing new evidence of genetic heterogeneity in AA. Our demonstration of genetic association between the MX1 gene and disease supports the hypothesis that this is a new candidate gene in AA.     

Herbicide response polymorphisms in wild emmer wheat: ecological and isozyme correlations

Summary We demonstrate that the scores and frequencies of chlortoluron (CT) and metoxuron (MX) resistance and susceptible phenotypes of wild emmer wheat, Triticum dicoccoides, are correlated with ecological factors and allozyme markers. Some isozyme markers located on chromosome 6B (e.g. Adh,Est-4 and Got), which also harbours the CT and MX resistance gene, provide good genetic markers for herbicide resistance breeding. Significant correlations between herbicide and photosynthetic characters suggest that the evolution of herbicide resistance polymorphisms may be related to the process of photosynthesis in nature and predated domestication of cultivated wheat.     

Myths, models and mitigation of resistance to pesticides

Resistance to pesticides in arthropod pests is a significant economic, ecological and public health problem. Although extensive research has been conducted on diverse aspects of pesticide resistance and we have learned a great deal during the past 50 years, to some degree the discussion about ''resistance management'' has been based on ''myths''. One myth involves the belief that we can manage resistance. I will maintain that we can only attempt to mitigate resistance because resistance is a natural evolutionary response to environmental stresses. As such, resistance will remain an ongoing dilemma in pest management and we can only delay the onset of resistance to pesticides. ''Resistance management'' models and tactics have been much discussed but have been tested and deployed in practical pest management programmes with only limited success. Yet the myth persists that better models will provide a ''solution'' to the problem. The reality is that success in using mitigation models is limited because these models are applied to inappropriate situations in which the critical genetic, ecological, biological or logistic assumptions cannot be met. It is difficult to predict in advance which model is appropriate to a particular situation; if the model assumptions cannot be met, applying the model sometimes can increase the rate of resistance development rather than slow it down. Are there any solutions? I believe we already have one. Unfortunately, it is not a simple or easy one to deploy. It involves employing effective agronomic practices to develop and maintain a healthy crop, monitoring pest densities, evaluating economic injury levels so that pesticides are applied only when necessary, deploying and conserving biological control agents, using host-plant resistance, cultural controls of the pest, biorational pest controls, and genetic control methods. As a part of a truly multi-tactic strategy, it is crucial to evaluate the effect of pesticides on natural enemies in order to preserve them in the cropping system. Sometimes, pesticide-resistant natural enemies are effective components of this resistance mitigation programme. Another name for this resistance mitigation model is integrated pest management (IPM). This complex model was outlined in some detail nearly 40 years ago by V. M. Stern and colleagues. To deploy the IPM resistance mitigation model, we must admit that pest management and resistance mitigation programmes are not sustainable if based on a single-tactic strategy. Delaying resistance, whether to traditional pesticides or to transgenic plants containing toxin genes from Bacillus thuringiensis, will require that we develop multi-tactic pest management programmes that incorporate all appropriate pest management approaches. Because pesticides are limited resources, and their loss can result in significant social and economic costs, they should be reserved for situations where they are truly needed--as tools to subdue an unexpected pest population outbreak. Effective multi-tactic IPM programmes delay resistance (= mitigation) because the number and rates of pesticide applications will be reduced.     

Polymorphisms in MX2 Gene Are Related with SCS in Chinese Dairy Cows

Viral infections can play direct or indirect roles in the etiology of the bovine mastitis. Mx dynamin-like GTPase 2 (MX2) gene is a main effector of the antiviral innate immune defense mediated by type I interferon (IFN I), which was demonstrated to confer positive antiviral responses to many viruses. Given the importance of the MX2 in modulating the host immune response, MX2 gene may be a suitable candidate gene for studying disease resistance in dairy cattle. Here, we scanned the sequence variation of the MX2 gene in Chinese indigenous cattle breeds. Twenty-three previously reported SNPs were identified. To further analyze the effects of SNPs detected on mastitis disease, analysis of two SNPs (g.787527 C?>?T and g.787610 T?>?C) from 297 Chinese Holstein cows revealed a significant association with somatic cell score (SCS). Although functional studies are necessary to ascertain whether these two SNPs are causal polymorphisms or merely in linkage with the true causal SNPs, implementation of these two SNPs as genetic markers in the dairy industry may be beneficial in selecting individuals with lower SCS.     

Releasing the spindle assembly checkpoint without tension

Proteins of the Bcl-2 family are critical regulators of apoptosis, but how its BH3-only members activate the essential effectors Bax and Bak remains controversial. The indirect activation model suggests that they simply must neutralize all of the prosurvival Bcl-2 family members, whereas the direct activation model proposes that Bim and Bid must activate Bax and Bak directly. As numerous in vitro studies have not resolved this issue, we have investigated Bim''s activity in vivo by a genetic approach. Because the BH3 domain determines binding specificity for Bcl-2 relatives, we generated mice having the Bim BH3 domain replaced by that of Bad, Noxa, or Puma. The mutants bound the expected subsets of prosurvival relatives but lost interaction with Bax. Analysis of the mice showed that Bim''s proapoptotic activity is not solely caused by its ability to engage its prosurvival relatives or solely to its binding to Bax. Thus, initiation of apoptosis in vivo appears to require features of both models.     

The crystal structure of S. cerevisiae Sad1, a catalytically inactive deubiquitinase that is broadly required for pre-mRNA splicing

Sad1 is an essential splicing factor initially identified in a genetic screen in Saccharomyces cerevisiae for snRNP assembly defects. Based on sequence homology, Sad1, or USP39 in humans, is predicted to comprise two domains: a zinc finger ubiquitin binding domain (ZnF-UBP) and an inactive ubiquitin-specific protease (iUSP) domain, both of which are well conserved. The role of these domains in splicing and their interaction with ubiquitin are unknown. We first used splicing microarrays to analyze Sad1 function in vivo and found that Sad1 is critical for the splicing of nearly all yeast intron-containing genes. By using in vitro assays, we then showed that it is required for the assembly of the active spliceosome. To gain structural insights into Sad1 function, we determined the crystal structure of the full-length protein at 1.8 Å resolution. In the structure, the iUSP domain forms the characteristic ubiquitin binding pocket, though with an amino acid substitution in the active site that results in complete inactivation of the enzymatic activity of the domain. The ZnF-UBP domain of Sad1 shares high structural similarly to other ZnF-UBPs; however, Sad1''s ZnF-UBP does not possess the canonical ubiquitin binding motif. Given the precedents for ZnF-UBP domains to function as activators for their neighboring USP domains, we propose that Sad1''s ZnF-UBP acts in a ubiquitin-independent capacity to recruit and/or activate Sad1''s iUSP domain to interact with the spliceosome.     

上皮细胞黏附分子增强细胞间紧密连接促进乳腺癌细胞耐药

乳腺癌是致死率很高的恶性肿瘤,由ABCG2 (ATP-binding cassette G2)介导的多药耐药(multidrug resistance,MDR)是导致其化疗失败的重要原因,探讨ABCG2介导的耐药机制并探寻其关键分子是当前亟待解决的难题。上皮细胞黏附分子(epithelial cell adhesion molecule,EpCAM)参与多种肿瘤耐药,且与乳腺癌MDR密切相关,但它在ABCG2介导的乳腺癌耐药中的作用尚未阐明。本研究目的在于探究EpCAM对于ABCG2介导的乳腺癌细胞的多药耐药的调节作用及其机制。CCK8细胞毒性结果证实,相对于人乳腺癌药物敏感株MCF-7,耐药株MCF-7/MX对米托蒽醌(mitoxantrone,MX)的耐药性显著增强;Western 印迹结果显示,与MCF-7相比,MCF-7/MX细胞中ABCG2高表达,EpCAM表达上调。siRNA法敲低MCF-7/MX细胞中EpCAM可下调其ABCG2表达,并恢复对MX的敏感性。倒置显微镜观察细胞形态,发现敲低EpCAM可减少MCF-7/MX细胞间连接。免疫荧光双染法观察到EpCAM与密封蛋白1(claudin 1)在MCF-7/MX细胞共定位;进一步Western 印迹结果表明,敲低EpCAM减少MCF-7/MX细胞中密封蛋白1表达。综上所述,EpCAM可能通过与密封蛋白1相互作用,增强细胞间紧密连接,促进ABCG2介导的乳腺癌多药耐药。     

Conserving threatened riparian ecosystems in the American West: Precipitation gradients and river networks drive genetic connectivity and diversity in a foundation riparian tree (Populus angustifolia)

Gene flow is an evolutionary process that supports genetic connectivity and contributes to the capacity of species to adapt to environmental change. Yet, for most species, little is known about the specific environmental factors that influence genetic connectivity, or their effects on genetic diversity and differentiation. We used a landscape genetic approach to understand how geography and climate influence genetic connectivity in a foundation riparian tree (Populus angustifolia), and their relationships with specieswide patterns of genetic diversity and differentiation. Using multivariate restricted optimization in a reciprocal causal modelling framework, we quantified the relative contributions of riparian network connectivity, terrestrial upland resistance and climate gradients on genetic connectivity. We found that (i) all riparian corridors, regardless of river order, equally facilitated connectivity, while terrestrial uplands provided 2.5× more resistance to gene flow than riparian corridors. (ii) Cumulative differences in precipitation seasonality and precipitation of the warmest quarter were the primary climatic factors driving genetic differentiation; furthermore, maximum climate resistance was 45× greater than riparian resistance. (iii) Genetic diversity was positively correlated with connectivity (R² = 0.3744, p = .0019), illustrating the utility of resistance models for identifying landscape conditions that can support a species' ability to adapt to environmental change. From these results, we present a map highlighting key genetic connectivity corridors across P. angustifolia's range that if disrupted could have long‐term ecological and evolutionary consequences. Our findings provide recommendations for conservation and restoration management of threatened riparian ecosystems throughout the western USA and the high biodiversity they support.     

Genetic Architecture of Intrinsic Antibiotic Susceptibility

Background

Antibiotic exposure rapidly selects for more resistant bacterial strains, and both a drug''s chemical structure and a bacterium''s cellular network affect the types of mutations acquired.

Methodology/Principal Findings

To better characterize the genetic determinants of antibiotic susceptibility, we exposed a transposon-mutagenized library of Escherichia coli to each of 17 antibiotics that encompass a wide range of drug classes and mechanisms of action. Propagating the library for multiple generations with drug concentrations that moderately inhibited the growth of the isogenic parental strain caused the abundance of strains with even minor fitness advantages or disadvantages to change measurably and reproducibly. Using a microarray-based genetic footprinting strategy, we then determined the quantitative contribution of each gene to E. coli''s intrinsic antibiotic susceptibility. We found both loci whose removal increased general antibiotic tolerance as well as pathways whose down-regulation increased tolerance to specific drugs and drug classes. The beneficial mutations identified span multiple pathways, and we identified pairs of mutations that individually provide only minor decreases in antibiotic susceptibility but that combine to provide higher tolerance.

Conclusions/Significance

Our results illustrate that a wide-range of mutations can modulate the activity of many cellular resistance processes and demonstrate that E. coli has a large mutational target size for increasing antibiotic tolerance. Furthermore, the work suggests that clinical levels of antibiotic resistance might develop through the sequential accumulation of chromosomal mutations of small individual effect.     

Homodimerization of the Wnt Receptor DERAILED Recruits the Src Family Kinase SRC64B

Ryk pseudokinase receptors act as important transducers of Wnt signals, particularly in the nervous system. Little is known, however, of their interactions at the cell surface. Here, we show that a Drosophila Ryk family member, DERAILED (DRL), forms cell surface homodimers and can also heterodimerize with the two other fly Ryks, DERAILED-2 and DOUGHNUT ON 2. DERAILED homodimerization levels increase significantly in the presence of its ligand, WNT5. In addition, DERAILED displays ligand-independent dimerization mediated by a motif in its transmembrane domain. Increased dimerization of DRL upon WNT5 binding or upon the replacement of DERAILED''s extracellular domain with the immunoglobulin Fc domain results in an increased recruitment of the Src family kinase SRC64B, a previously identified downstream pathway effector. Formation of the SRC64B/DERAILED complex requires SRC64B''s SH2 domain and DERAILED''s PDZ-binding motif. Mutations in DERAILED''s inactive tyrosine kinase-homologous domain also disrupt the formation of DERAILED/SRC64B complexes, indicating that its conformation is likely important in facilitating its interaction with SRC64B. Finally, we show that DERAILED''s function during embryonic axon guidance requires its Wnt-binding domain, a putative juxtamembrane extracellular tetrabasic cleavage site, and the PDZ-binding domain, indicating that DERAILED''s activation involves a complex set of events including both dimerization and proteolytic processing.     

The use of molecular xenomonitoring for surveillance of mosquito-borne diseases

The scientific community recognizes that molecular xenomonitoring (MX) can allow infected mosquitoes to serve as a proxy for human infection in vector-borne disease surveillance, but developing reliable MX systems for programmatic use has been challenging. The primary aim of this article is to examine the available evidence to recommend how MX can best be used for various purposes. Although much of the literature published within the last 20 years focuses on using MX for lymphatic filariasis elimination, a growing body of evidence supports its use in early warning systems for emerging infectious diseases (EIDs). An MX system design must consider the goal and target (e.g. diseases targeted for elimination versus EIDs), mosquito and pathogen characteristics, and context (e.g. setting and health system). MX is currently used as a ‘supplement’ to human surveillance and will not be considered as a ‘replacement’ until the correlation between pathogen-infection rates in human and mosquito populations is better understood. Establishing such relationships may not be feasible in elimination scenarios, due to increasingly dwindling human infection prevalence after successful control, but may still be possible for EIDs and in integrated disease surveillance systems.This article is part of the theme issue ‘Novel control strategies for mosquito-borne diseases''.     

Meloxicam Taste-Masked Oral Disintegrating Tablet with Dissolution Enhanced by Ion Exchange Resins and Cyclodextrin

The purpose of this study was to develop taste-masked oral disintegrating tablets (ODTs) using the combination of ion exchange resin and cyclodextrin, to mask the bitter taste and enhance drug dissolution. Meloxicam (MX) was selected as a model drug with poor water solubility and a bitter taste. Formulations containing various forms of MX (free drug, MX-loaded resin or resinate, complexes of MX and 2-hydroxypropyl-β-cyclodextrin (HPβCD) or MX/HPβCD complexes, and a mixture of resinate and MX/HPβCD complexes) were made and tablets were prepared by direct compression. The ODTs were evaluated for weight variation, thickness, diameter, hardness, friability, disintegration time, wetting time, MX content, MX release, degree of bitter taste, and stability. The results showed that thickness, diameter, weight, and friability did not differ significantly for all of these formulations. The tablet hardness was approximately 3 kg/in.², and the friability was less than 1%. Tablets formulated with resinate and the mixture of resinate and MX/HPβCD complexes disintegrated rapidly within 60 s, which is the acceptable limit for ODTs. These results corresponded to the in vivo disintegration and wetting times. However, only tablets containing the mixture of resinate and MX/HPβCD complexes provided complete MX dissolution and successfully masked the bitter taste of MX. In addition, this tablet was stable at least 6 months. The results from this study suggest that the appropriate combination of ion exchange resin and cyclodextrin could be used in ODTs to mask the bitter taste of drug and enhance the dissolution of drugs that are weakly soluble in water.     

Genetic data reveal wintering ground affiliation of humpback whales from the Mexican Central Pacific

Two groups of humpback whales inhabit the waters off the Pacific coast of Mexico the coastal wintering aggregation in the north (MX), and the southern Mexico/Central America wintering aggregation (S-MX/CEA) in the south. However, along the coast of the Mexican Central Pacific (MCP), the population affiliation of humpback whales is uncertain. Some studies have concluded that the MCP whales are part of S-MX/CEA, while others have suggested that the MCP may represent an overlap zone between the two wintering aggregations. In this study, data from 354 biopsy samples were collected over a 12-year period, to provide insight from genetic information into the affiliation of MCP whales to and the boundaries between the wintering aggregations. Using mitochondrial control region sequences, we found that the majority (73%) of MCP whales are part of MX, but that the boundary between the two wintering aggregations may shift latitudinally depending on environmental conditions. The high haplotypic (h ± SD = 0.859 ± 0.0138) and nucleotide diversity (π ± SD = 0.0145 ± 0.0075) of the MCP whales are also consistent with our sample, including animals from both wintering aggregations. More research is needed to better describe the ranges of the MX and S-MX/CEA wintering aggregations to ensure their successful conservation and management.     

Unravelling the Relationship between Animal Growth and Immune Response during Micro-Parasitic Infections

Background

Both host genetic potentials for growth and disease resistance, as well as nutrition are known to affect responses of individuals challenged with micro-parasites, but their interactive effects are difficult to predict from experimental studies alone.

Methodology/Principal Findings

Here, a mathematical model is proposed to explore the hypothesis that a host''s response to pathogen challenge largely depends on the interaction between a host''s genetic capacities for growth or disease resistance and the nutritional environment. As might be expected, the model predicts that if nutritional availability is high, hosts with higher growth capacities will also grow faster under micro-parasitic challenge, and more resistant animals will exhibit a more effective immune response. Growth capacity has little effect on immune response and resistance capacity has little effect on achieved growth. However, the influence of host genetics on phenotypic performance changes drastically if nutrient availability is scarce. In this case achieved growth and immune response depend simultaneously on both capacities for growth and disease resistance. A higher growth capacity (achieved e.g. through genetic selection) would be detrimental for the animal''s ability to cope with pathogens and greater resistance may reduce growth in the short-term.

Significance

Our model can thus explain contradicting outcomes of genetic selection observed in experimental studies and provides the necessary biological background for understanding the influence of selection and/or changes in the nutritional environment on phenotypic growth and immune response.     

MX 2 is a novel regulator of cell cycle in melanoma cells

MX2 protein is a dynamin‐like GTPase2 that has recently been identified as an interferon‐induced restriction factor of HIV‐1 and other primate lentiviruses. A single nucleotide polymorphism (SNP), rs45430, in an intron of the MX2 gene, was previously reported as a novel melanoma susceptibility locus in genome‐wide association studies. Functionally, however, it is still unclear whether and how MX2 contributes to melanoma susceptibility and tumorigenesis. Here, we show that MX2 is differentially expressed in melanoma tumors and cell lines, with most metastatic cell lines showing lower MX2 expression than primary melanoma cell lines and melanocytes. Furthermore, high expression of MX2 RNA in primary melanoma tumors is associated with better patient survival. Overexpression of MX2 reduces in vivo proliferation partially through inhibition of AKT activation, suggesting that it can act as a tumor suppressor in melanoma. However, we have also identified a subset of melanoma cell lines with high endogenous MX2 expression where downregulation of MX2 leads to reduced proliferation. In these cells, MX2 downregulation interfered with DNA replication and cell cycle processes. Collectively, our data for the first time show that MX2 is functionally involved in the regulation of melanoma proliferation but that its function is context‐dependent.