The EGF Repeat-Specific O-GlcNAc-Transferase Eogt Interacts with Notch Signaling and Pyrimidine Metabolism Pathways in Drosophila

PubMed Central

Müller, Reto; Jenny, Andreas; Stanley, Pamela

2013-01-01

The O-GlcNAc transferase Eogt modifies EGF repeats in proteins that transit the secretory pathway, including Dumpy and Notch. In this paper, we show that the Notch ligands Delta and Serrate are also substrates of Eogt, that mutation of a putative UDP-GlcNAc binding DXD motif greatly reduces enzyme activity, and that Eogt and the cytoplasmic O-GlcNAc transferase Ogt have distinct substrates in Drosophila larvae. Loss of Eogt is larval lethal and disrupts Dumpy functions, but does not obviously perturb Notch signaling. To identify novel genetic interactions with eogt, we investigated dominant modification of wing blister formation caused by knock-down of eogt. Unexpectedly, heterozygosity for several members of the canonical Notch signaling pathway suppressed wing blister formation. And importantly, extensive genetic interactions with mutants in pyrimidine metabolism were identified. Removal of pyrimidine synthesis alleles suppressed wing blister formation, while removal of uracil catabolism alleles was synthetic lethal with eogt knock-down. Therefore, Eogt may regulate protein functions by O-GlcNAc modification of their EGF repeats, and cellular metabolism by affecting pyrimidine synthesis and catabolism. We propose that eogt knock-down in the wing leads to metabolic and signaling perturbations that increase cytosolic uracil levels, thereby causing wing blister formation. PMID:23671640

The putative RNA helicase Dbp6p functionally interacts with Rpl3p, Nop8p and the novel trans-acting Factor Rsa3p during biogenesis of 60S ribosomal subunits in Saccharomyces cerevisiae.

PubMed Central

de la Cruz, Jesús; Lacombe, Thierry; Deloche, Olivier; Linder, Patrick; Kressler, Dieter

2004-01-01

Ribosome biogenesis requires at least 18 putative ATP-dependent RNA helicases in Saccharomyces cerevisiae. To explore the functional environment of one of these putative RNA helicases, Dbp6p, we have performed a synthetic lethal screen with dbp6 alleles. We have previously characterized the nonessential Rsa1p, whose null allele is synthetically lethal with dbp6 alleles. Here, we report on the characterization of the four remaining synthetic lethal mutants, which reveals that Dbp6p also functionally interacts with Rpl3p, Nop8p, and the so-far-uncharacterized Rsa3p (ribosome assembly 3). The nonessential Rsa3p is a predominantly nucleolar protein required for optimal biogenesis of 60S ribosomal subunits. Both Dbp6p and Rsa3p are associated with complexes that most likely correspond to early pre-60S ribosomal particles. Moreover, Rsa3p is co-immunoprecipitated with protA-tagged Dbp6p under low salt conditions. In addition, we have established a synthetic interaction network among factors involved in different aspects of 60S-ribosomal-subunit biogenesis. This extensive genetic analysis reveals that the rsa3 null mutant displays some specificity by being synthetically lethal with dbp6 alleles and by showing some synthetic enhancement with the nop8-101 and the rsa1 null allele. PMID:15126390

Synthetic Lethal Networks for Precision Oncology: Promises and Pitfalls.

PubMed

Shen, John Paul; Ideker, Trey

2018-06-19

Synthetic lethal interactions, in which the simultaneous loss-of-function of two genes produces a lethal phenotype, are being explored as a means to therapeutically exploit cancer-specific vulnerabilities and expand the scope of precision oncology. Currently, three FDA approved drugs work by targeting the synthetic lethal interaction between BRCA1/2 and PARP. This review examines additional efforts to discover networks of synthetic lethal interactions and discusses both challenges and opportunities regarding the translation of new synthetic lethal interactions into the clinic. Copyright © 2018. Published by Elsevier Ltd.

Liver transplantation for lethal genetic syndromes: a novel model of personalized genomic medicine.

PubMed

Petrowsky, Henrik; Brunicardi, F Charles; Leow, Voon Meng; Venick, Robert S; Agopian, Vatche; Kaldas, Fady M; Zarrinpar, Ali; Markovic, Daniela; McDiarmid, Sue V; Hong, Johnny C; Farmer, Douglas G; Hiatt, Jonathan R; Busuttil, Ronald W

2013-04-01

Our aim was to analyze our single-center experience with orthotopic liver transplantation for metabolic lethal genetic syndromes in children and adults. From 1984 to 2012, all pediatric (younger than 18 years) and adult (18 years and older) patients who underwent orthotopic liver transplantation for lethal genetic disorders were identified. Data on diagnostic pathways and specific outcomes were analyzed for both groups. Outcomes measures included recurrence rate as well as graft and patient survival. Metabolic lethal genetic syndrome was the primary indication for orthotopic liver transplantation in 152 of 4,564 patients (3.3%) at University of California, Los Angeles during the study period (74 pediatric patients and 78 adults). Genetic testing was performed in only 12% of the 152 patients and in 39% of patients after 2006. Two patients (1.3%) experienced a recurrence of the genetic disease. Overall 5- and 20-year survival rates were 89% and 77% for children and 73% and 50% for adults. Survival of pediatric patients was superior to adults (log-rank p < 0.009). Multivariate analysis identified age (hazard ratio = 2.18), preoperative life support (hazard ratio = 2.68), and earlier transplantation (hazard ratio = 3.41) as independent predictors of reduced survival. Orthotopic liver transplantation achieved excellent long-term survival in pediatric and adult patients with lethal genetic syndromes and represents a model of personalized genomic medicine by providing gene therapy through solid organ transplantation. Copyright © 2013 American College of Surgeons. Published by Elsevier Inc. All rights reserved.

Genetic loci with parent-of-origin effects cause hybrid seed lethality in crosses between Mimulus species.

PubMed

Garner, Austin G; Kenney, Amanda M; Fishman, Lila; Sweigart, Andrea L

2016-07-01

In flowering plants, F1 hybrid seed lethality is a common outcome of crosses between closely related diploid species, but the genetic basis of this early-acting and potentially widespread form of postzygotic reproductive isolation is largely unknown. We intercrossed two closely related species of monkeyflower, Mimulus guttatus and Mimulus tilingii, to characterize the mechanisms and strength of postzygotic reproductive isolation. Then, using a reciprocal backcross design, we performed high-resolution genetic mapping to determine the genetic architecture of hybrid seed lethality and directly test for loci with parent-of-origin effects. We found that F1 hybrid seed lethality is an exceptionally strong isolating barrier between Mimulus species, with reciprocal crosses producing < 1% viable seeds. This form of postzygotic reproductive isolation appears to be highly polygenic, indicating that multiple incompatibility loci have accumulated rapidly between these closely related Mimulus species. It is also primarily caused by genetic loci with parent-of-origin effects, suggesting a possible role for imprinted genes in the evolution of Mimulus hybrid seed lethality. Our findings suggest that divergence in loci with parent-of-origin effects, which is probably driven by genomic coevolution within lineages, might be an important source of hybrid incompatibilities between flowering plant species. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Microbial Herd Protection Mediated by Antagonistic Interaction in Polymicrobial Communities

PubMed Central

Wong, Megan J. Q.; Liang, Xiaoye; Smart, Matt; Tang, Le; Moore, Richard; Ingalls, Brian

2016-01-01

ABSTRACT In host and natural environments, microbes often exist in complex multispecies communities. The molecular mechanisms through which such communities develop and persist, despite significant antagonistic interactions between species, are not well understood. The type VI secretion system (T6SS) is a lethal weapon commonly employed by Gram-negative bacteria to inhibit neighboring species through the delivery of toxic effectors. It is well established that intraspecies protection is conferred by immunity proteins that neutralize effector toxicities. In contrast, the mechanisms for interspecies protection are not clear. Here we use two T6SS-active antagonistic bacterial species, Aeromonas hydrophila and Vibrio cholerae, to demonstrate that interspecies protection is dependent on effectors. A. hydrophila and V. cholerae do not share conserved immunity genes but could coexist equally in a mixture. However, mutants lacking the T6SS or effectors were effectively eliminated by the competing wild-type strain. Time-lapse microscopic analyses showed that mutually lethal interactions drive the segregation of mixed species into distinct single-species clusters by eliminating interspersed single cells. Cluster formation provides herd protection by abolishing lethal interactions inside each cluster and restricting the interactions to the boundary. Using an agent-based modeling approach, we simulated the antagonistic interactions of two hypothetical species. The resulting simulations recapitulated our experimental observations. These results provide mechanistic insights regarding the general role of microbial weapons in determining the structures of complex multispecies communities. IMPORTANCE Investigating the warfare of microbes allows us to better understand the ecological relationships in complex microbial communities such as the human microbiota. Here we use the T6SS, a deadly bacterial weapon, as a model to demonstrate the importance of lethal interactions in determining community structures and the exchange of genetic materials. This simplified model elucidates a mechanism of microbial herd protection by which competing antagonistic species can coexist in the same niche, despite their diverse mutually destructive activities. Our results also suggest that antagonistic interactions impose strong selection that could promote multicellular organism-like social behaviors and contribute to the transition to multicellularity during evolution. PMID:27637882

Genetics Home Reference: Amish lethal microcephaly

MedlinePlus

... occurs in approximately 1 in 500 newborns in the Old Order Amish population of Pennsylvania. It has not been found outside this population. Related Information What information about a genetic condition can statistics provide? Why ... in the SLC25A19 gene cause Amish lethal microcephaly . The SLC25A19 ...

Microbial herd protection mediated by antagonistic interaction in polymicrobial communities.

PubMed

Wong, Megan; Liang, Xiaoye; Smart, Matt; Tang, Le; Moore, Richard; Ingalls, Brian; Dong, Tao G

2016-09-16

In the host and natural environments, microbes often exist in complex multispecies communities. The molecular mechanisms through which such communities develop and persist - despite significant antagonistic interactions between species - are not well understood. The type VI secretion system (T6SS) is a lethal weapon commonly employed by Gram-negative bacteria to inhibit neighboring species through delivery of toxic effectors. It is well established that intra-species protection is conferred by immunity proteins that neutralize effector toxicities. By contrast, the mechanisms for interspecies protection are not clear. Here we use two T6SS active antagonistic bacteria, Aeromonas hydrophila (AH) and Vibrio cholerae (VC), to demonstrate that interspecies protection is dependent on effectors. AH and VC do not share conserved immunity genes but could equally co-exist in a mixture. However, mutants lacking the T6SS or effectors were effectively eliminated by the other competing wild type. Time-lapse microscopy analyses show that mutually lethal interactions drive the segregation of mixed species into distinct single-species clusters by eliminating interspersed single cells. Cluster formation provides herd protection by abolishing lethal interaction inside each cluster and restricting it to the boundary. Using an agent-based modeling approach, we simulated the antagonistic interactions of two hypothetical species. The resulting simulations recapitulate our experimental observation. These results provide mechanistic insights for the general role of microbial weapons in determining the structures of complex multispecies communities. Investigating the warfare of microbes allows us to better understand the ecological relationships in complex microbial communities such as the human microbiota. Here we use the T6SS, a deadly bacterial weapon, as a model to demonstrate the importance of lethal interactions in determining community structures and exchange of genetic materials. This simplified model elucidates a mechanism of microbial herd protection by which competing antagonistic species coexist in the same niche despite their diverse mutually destructive activities. Our results also suggest that antagonistic interaction imposes a strong selection that could promote multicellular like social behaviors and contribute to the transition to multicellularity during evolution. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Evidence for dynamically organized modularity in the yeast protein-protein interaction network

NASA Astrophysics Data System (ADS)

Han, Jing-Dong J.; Bertin, Nicolas; Hao, Tong; Goldberg, Debra S.; Berriz, Gabriel F.; Zhang, Lan V.; Dupuy, Denis; Walhout, Albertha J. M.; Cusick, Michael E.; Roth, Frederick P.; Vidal, Marc

2004-07-01

In apparently scale-free protein-protein interaction networks, or `interactome' networks, most proteins interact with few partners, whereas a small but significant proportion of proteins, the `hubs', interact with many partners. Both biological and non-biological scale-free networks are particularly resistant to random node removal but are extremely sensitive to the targeted removal of hubs. A link between the potential scale-free topology of interactome networks and genetic robustness seems to exist, because knockouts of yeast genes encoding hubs are approximately threefold more likely to confer lethality than those of non-hubs. Here we investigate how hubs might contribute to robustness and other cellular properties for protein-protein interactions dynamically regulated both in time and in space. We uncovered two types of hub: `party' hubs, which interact with most of their partners simultaneously, and `date' hubs, which bind their different partners at different times or locations. Both in silico studies of network connectivity and genetic interactions described in vivo support a model of organized modularity in which date hubs organize the proteome, connecting biological processes-or modules -to each other, whereas party hubs function inside modules.

Conditions for success of engineered underdominance gene drive systems.

PubMed

Edgington, Matthew P; Alphey, Luke S

2017-10-07

Engineered underdominance is one of a number of different gene drive strategies that have been proposed for the genetic control of insect vectors of disease. Here we model a two-locus engineered underdominance based gene drive system that is based on the concept of mutually suppressing lethals. In such a system two genetic constructs are introduced, each possessing a lethal element and a suppressor of the lethal at the other locus. Specifically, we formulate and analyse a population genetics model of this system to assess when different combinations of release strategies (i.e. single or multiple releases of both sexes or males only) and genetic systems (i.e. bisex lethal or female-specific lethal elements and different strengths of suppressors) will give population replacement or fail to do so. We anticipate that results presented here will inform the future design of engineered underdominance gene drive systems as well as providing a point of reference regarding release strategies for those looking to test such a system. Our discussion is framed in the context of genetic control of insect vectors of disease. One of several serious threats in this context are Aedes aegypti mosquitoes as they are the primary vectors of dengue viruses. However, results are also applicable to Ae. aegypti as vectors of Zika, yellow fever and chikungunya viruses and also to the control of a number of other insect species and thereby of insect-vectored pathogens. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

The Genome-Wide Interaction Network of Nutrient Stress Genes in Escherichia coli.

PubMed

Côté, Jean-Philippe; French, Shawn; Gehrke, Sebastian S; MacNair, Craig R; Mangat, Chand S; Bharat, Amrita; Brown, Eric D

2016-11-22

Conventional efforts to describe essential genes in bacteria have typically emphasized nutrient-rich growth conditions. Of note, however, are the set of genes that become essential when bacteria are grown under nutrient stress. For example, more than 100 genes become indispensable when the model bacterium Escherichia coli is grown on nutrient-limited media, and many of these nutrient stress genes have also been shown to be important for the growth of various bacterial pathogens in vivo To better understand the genetic network that underpins nutrient stress in E. coli, we performed a genome-scale cross of strains harboring deletions in some 82 nutrient stress genes with the entire E. coli gene deletion collection (Keio) to create 315,400 double deletion mutants. An analysis of the growth of the resulting strains on rich microbiological media revealed an average of 23 synthetic sick or lethal genetic interactions for each nutrient stress gene, suggesting that the network defining nutrient stress is surprisingly complex. A vast majority of these interactions involved genes of unknown function or genes of unrelated pathways. The most profound synthetic lethal interactions were between nutrient acquisition and biosynthesis. Further, the interaction map reveals remarkable metabolic robustness in E. coli through pathway redundancies. In all, the genetic interaction network provides a powerful tool to mine and identify missing links in nutrient synthesis and to further characterize genes of unknown function in E. coli Moreover, understanding of bacterial growth under nutrient stress could aid in the development of novel antibiotic discovery platforms. With the rise of antibiotic drug resistance, there is an urgent need for new antibacterial drugs. Here, we studied a group of genes that are essential for the growth of Escherichia coli under nutrient limitation, culture conditions that arguably better represent nutrient availability during an infection than rich microbiological media. Indeed, many such nutrient stress genes are essential for infection in a variety of pathogens. Thus, the respective proteins represent a pool of potential new targets for antibacterial drugs that have been largely unexplored. We have created all possible double deletion mutants through a genetic cross of nutrient stress genes and the E. coli deletion collection. An analysis of the growth of the resulting clones on rich media revealed a robust, dense, and complex network for nutrient acquisition and biosynthesis. Importantly, our data reveal new genetic connections to guide innovative approaches for the development of new antibacterial compounds targeting bacteria under nutrient stress. Copyright © 2016 Côté et al.

Synthetic Genetic Arrays: Automation of Yeast Genetics.

PubMed

Kuzmin, Elena; Costanzo, Michael; Andrews, Brenda; Boone, Charles

2016-04-01

Genome-sequencing efforts have led to great strides in the annotation of protein-coding genes and other genomic elements. The current challenge is to understand the functional role of each gene and how genes work together to modulate cellular processes. Genetic interactions define phenotypic relationships between genes and reveal the functional organization of a cell. Synthetic genetic array (SGA) methodology automates yeast genetics and enables large-scale and systematic mapping of genetic interaction networks in the budding yeast,Saccharomyces cerevisiae SGA facilitates construction of an output array of double mutants from an input array of single mutants through a series of replica pinning steps. Subsequent analysis of genetic interactions from SGA-derived mutants relies on accurate quantification of colony size, which serves as a proxy for fitness. Since its development, SGA has given rise to a variety of other experimental approaches for functional profiling of the yeast genome and has been applied in a multitude of other contexts, such as genome-wide screens for synthetic dosage lethality and integration with high-content screening for systematic assessment of morphology defects. SGA-like strategies can also be implemented similarly in a number of other cell types and organisms, includingSchizosaccharomyces pombe,Escherichia coli, Caenorhabditis elegans, and human cancer cell lines. The genetic networks emerging from these studies not only generate functional wiring diagrams but may also play a key role in our understanding of the complex relationship between genotype and phenotype. © 2016 Cold Spring Harbor Laboratory Press.

[The genetic control of mouse coat color and its applications in genetics teaching].

PubMed

Xing, Wanjin; Morigen, Morigen

2014-10-01

Mice are the most commonly used mammalian model. The coat colors of mice are typical Mendelian traits, which have various colors such as white, black, yellow and agouti. The inheritance of mouse coat color is usually stated as an example only in teaching the knowledge of recessive lethal alleles. After searched the related literatures and summarized the molecular mechanisms of mouse coat color inheritance, we further expanded the application of this example into the introduction of the basic concepts of alleles and Mendelian laws, demonstration of the gene structure and function, regulation of gene expression, gene interaction, epigenetic modification, quantitative genetics, as well as evolutionary genetics. By running this example through the whole genetics-teaching lectures, we help the student to form a systemic and developmental view of genetic analysis. At the same time, this teaching approach not only highlights the advancement and integrity of genetics, but also results in a good teaching effect on inspiring the students' interest and attracting students' attention.

Characterization of conditionally expressed mutants affecting age-specific survival in inbred lines of Drosophila melanogaster: lethal conditions and temperature-sensitive periods.

PubMed Central

Vermeulen, C J; Bijlsma, R

2004-01-01

The specific genetic basis of inbreeding depression is poorly understood. To address this question, two conditionally expressed lethal effects that were found to cause line-specific life span reductions in two separate inbred lines of Drosophila melanogaster were characterized phenotypically and genetically in terms of whether the accelerated mortality effects are dominant or recessive. The mortality effect in one line (I4) is potentially a temperature-sensitive semilethal that expresses in adult males only and is partially dominant. The other line (I10) responds as one would expect for a recessive lethal. It requires a cold shock for expression and is cold sensitive. Flies exhibiting this lethal condition responded as pupae and freshly eclosed imagoes. The effect is recessive in both males and females. The expression of the lethal effects in both lines is highly dependent upon environmental conditions. These results will serve as a basis for more detailed and mechanistic genetic research on inbreeding depression and are relevant to sex- and environment-specific effects on life span observed in quantitative trait loci studies using inbred lines. PMID:15280238

Heterochromatin position effects on circularized sex chromosomes cause filicidal embryonic lethality in Drosophila melanogaster.

PubMed

Ferree, Patrick M; Gomez, Karina; Rominger, Peter; Howard, Dagnie; Kornfeld, Hannah; Barbash, Daniel A

2014-04-01

Some circularized X-Y chromosomes in Drosophila melanogaster are mitotically unstable and induce early embryonic lethality, but the genetic basis is unknown. Our experiments suggest that a large region of X-linked satellite DNA causes anaphase bridges and lethality when placed into a new heterochromatic environment within certain circularized X-Y chromosomes. These results reveal that repetitive sequences can be incompatible with one another in cis. The lethal phenotype also bears a remarkable resemblance to a case of interspecific hybrid lethality.

Seed-effect modeling improves the consistency of genome-wide loss-of-function screens and identifies synthetic lethal vulnerabilities in cancer cells.

PubMed

Jaiswal, Alok; Peddinti, Gopal; Akimov, Yevhen; Wennerberg, Krister; Kuznetsov, Sergey; Tang, Jing; Aittokallio, Tero

2017-06-01

Genome-wide loss-of-function profiling is widely used for systematic identification of genetic dependencies in cancer cells; however, the poor reproducibility of RNA interference (RNAi) screens has been a major concern due to frequent off-target effects. Currently, a detailed understanding of the key factors contributing to the sub-optimal consistency is still a lacking, especially on how to improve the reliability of future RNAi screens by controlling for factors that determine their off-target propensity. We performed a systematic, quantitative analysis of the consistency between two genome-wide shRNA screens conducted on a compendium of cancer cell lines, and also compared several gene summarization methods for inferring gene essentiality from shRNA level data. We then devised novel concepts of seed essentiality and shRNA family, based on seed region sequences of shRNAs, to study in-depth the contribution of seed-mediated off-target effects to the consistency of the two screens. We further investigated two seed-sequence properties, seed pairing stability, and target abundance in terms of their capability to minimize the off-target effects in post-screening data analysis. Finally, we applied this novel methodology to identify genetic interactions and synthetic lethal partners of cancer drivers, and confirmed differential essentiality phenotypes by detailed CRISPR/Cas9 experiments. Using the novel concepts of seed essentiality and shRNA family, we demonstrate how genome-wide loss-of-function profiling of a common set of cancer cell lines can be actually made fairly reproducible when considering seed-mediated off-target effects. Importantly, by excluding shRNAs having higher propensity for off-target effects, based on their seed-sequence properties, one can remove noise from the genome-wide shRNA datasets. As a translational application case, we demonstrate enhanced reproducibility of genetic interaction partners of common cancer drivers, as well as identify novel synthetic lethal partners of a major oncogenic driver, PIK3CA, supported by a complementary CRISPR/Cas9 experiment. We provide practical guidelines for improved design and analysis of genome-wide loss-of-function profiling and demonstrate how this novel strategy can be applied towards improved mapping of genetic dependencies of cancer cells to aid development of targeted anticancer treatments.

Frizzled 2 and frizzled 7 function redundantly in convergent extension and closure of the ventricular septum and palate: evidence for a network of interacting genes

PubMed Central

Yu, Huimin; Ye, Xin; Guo, Nini; Nathans, Jeremy

2012-01-01

Frizzled (Fz) 2 and Fz7, together with Fz1, form a distinct subfamily within the Frizzled family of Wnt receptors. Using targeted gene deletion, we show that: Fz7−/− mice exhibit tail truncation and kinking with 100% penetrance and ventricular septal defects (VSDs) with ~15% penetrance; Fz2+/−;Fz7−/− mice exhibit VSDs with ~50% penetrance and cleft palate with less than 10% penetrance; and Fz2−/−;Fz7−/− mice exhibit convergent extension defects and mid-gestational lethality with 100% penetrance. When Fz2 and/or Fz7 mutations are combined with mutations in Vangl2, Dvl3, Wnt3a, Wnt5a or Wnt11, an increased frequency of VSDs is observed with Dvl3, Wnt3a and Wnt11; an increased frequency of palate closure defects is observed with Vangl2; and early lethality and enhanced tail shortening are observed with Wnt5a. To assess the signaling pathways that underlie these and other Frizzled-mediated genetic interactions, we used transfected mammalian cells to analyze (1) canonical Wnt signaling induced by all pairwise combinations of the ten mouse Frizzleds and the 19 mouse Wnts and (2) localization of each Frizzled at cell-cell junctional complexes formed by mouse Celsr1, a likely indicator of competence for planar cell polarity signaling. These in vitro experiments indicate that Fz2 and Fz7 are competent to signal via the canonical pathway. Taken together, the data suggest that genetic interactions between Fz2, Fz7 and Vangl2, Dvl3 and Wnt genes reflect interactions among different signaling pathways in developmental processes that are highly sensitive to perturbations in Frizzled signaling. PMID:23095888

Structure–function analysis and genetic interactions of the SmG, SmE, and SmF subunits of the yeast Sm protein ring

PubMed Central

Schwer, Beate; Kruchten, Joshua; Shuman, Stewart

2016-01-01

A seven-subunit Sm protein ring forms a core scaffold of the U1, U2, U4, and U5 snRNPs that direct pre-mRNA splicing. Using human snRNP structures to guide mutagenesis in Saccharomyces cerevisiae, we gained new insights into structure–function relationships of the SmG, SmE, and SmF subunits. An alanine scan of 19 conserved amino acids of these three proteins, comprising the Sm RNA binding sites or inter-subunit interfaces, revealed that, with the exception of Arg74 in SmF, none are essential for yeast growth. Yet, for SmG, SmE, and SmF, as for many components of the yeast spliceosome, the effects of perturbing protein–RNA and protein–protein interactions are masked by built-in functional redundancies of the splicing machine. For example, tests for genetic interactions with non-Sm splicing factors showed that many benign mutations of SmG, SmE, and SmF (and of SmB and SmD3) were synthetically lethal with null alleles of U2 snRNP subunits Lea1 and Msl1. Tests of pairwise combinations of SmG, SmE, SmF, SmB, and SmD3 alleles highlighted the inherent redundancies within the Sm ring, whereby simultaneous mutations of the RNA binding sites of any two of the Sm subunits are lethal. Our results suggest that six intact RNA binding sites in the Sm ring suffice for function but five sites may not. PMID:27417296

INCURVATA2 Encodes the Catalytic Subunit of DNA Polymerase α and Interacts with Genes Involved in Chromatin-Mediated Cellular Memory in Arabidopsis thaliana

PubMed Central

Barrero, José María; González-Bayón, Rebeca; del Pozo, Juan Carlos; Ponce, María Rosa; Micol, José Luis

2007-01-01

Cell type–specific gene expression patterns are maintained by the stable inheritance of transcriptional states through mitosis, requiring the action of multiprotein complexes that remodel chromatin structure. Genetic and molecular interactions between chromatin remodeling factors and components of the DNA replication machinery have been identified in Schizosaccharomyces pombe, indicating that some epigenetic marks are replicated simultaneously to DNA with the participation of the DNA replication complexes. This model of epigenetic inheritance might be extended to the plant kingdom, as we report here with the positional cloning and characterization of INCURVATA2 (ICU2), which encodes the putative catalytic subunit of the DNA polymerase α of Arabidopsis thaliana. The strong icu2-2 and icu2-3 insertional alleles caused fully penetrant zygotic lethality when homozygous and incompletely penetrant gametophytic lethality, probably because of loss of DNA polymerase activity. The weak icu2-1 allele carried a point mutation and caused early flowering, leaf incurvature, and homeotic transformations of sepals into carpels and of petals into stamens. Further genetic analyses indicated that ICU2 interacts with TERMINAL FLOWER2, the ortholog of HETEROCHROMATIN PROTEIN1 of animals and yeasts, and with the Polycomb group (PcG) gene CURLY LEAF. Another PcG gene, EMBRYONIC FLOWER2, was found to be epistatic to ICU2. Quantitative RT-PCR analyses indicated that a number of regulatory genes were derepressed in the icu2-1 mutant, including genes associated with flowering time, floral meristem, and floral organ identity. PMID:17873092

A complete toolset for the study of Ustilago bromivora and Brachypodium sp. as a fungal-temperate grass pathosystem

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rabe, Franziska; Bosch, Jason; Stirnberg, Alexandra

Due to their economic relevance, the study of plant pathogen interactions is of importance. However, elucidating these interactions and their underlying molecular mechanisms remains challenging since both host and pathogen need to be fully genetically accessible organisms. Here we present milestones in the establishment of a new biotrophic model pathosystem: Ustilago bromivora and Brachypodium sp. We provide a complete toolset, including an annotated fungal genome and methods for genetic manipulation of the fungus and its host plant. This toolset will enable researchers to easily study biotrophic interactions at the molecular level on both the pathogen and the host side. Moreover,more » our research on the fungal life cycle revealed a mating type bias phenomenon. U. bromivora harbors a haplo-lethal allele that is linked to one mating type region. As a result, the identified mating type bias strongly promotes inbreeding, which we consider to be a potential speciation driver.« less

A complete toolset for the study of Ustilago bromivora and Brachypodium sp. as a fungal-temperate grass pathosystem

DOE PAGES

Rabe, Franziska; Bosch, Jason; Stirnberg, Alexandra; ...

2016-11-11

Due to their economic relevance, the study of plant pathogen interactions is of importance. However, elucidating these interactions and their underlying molecular mechanisms remains challenging since both host and pathogen need to be fully genetically accessible organisms. Here we present milestones in the establishment of a new biotrophic model pathosystem: Ustilago bromivora and Brachypodium sp. We provide a complete toolset, including an annotated fungal genome and methods for genetic manipulation of the fungus and its host plant. This toolset will enable researchers to easily study biotrophic interactions at the molecular level on both the pathogen and the host side. Moreover,more » our research on the fungal life cycle revealed a mating type bias phenomenon. U. bromivora harbors a haplo-lethal allele that is linked to one mating type region. As a result, the identified mating type bias strongly promotes inbreeding, which we consider to be a potential speciation driver.« less

A complete toolset for the study of Ustilago bromivora and Brachypodium sp. as a fungal-temperate grass pathosystem

PubMed Central

Rabe, Franziska; Bosch, Jason; Stirnberg, Alexandra; Guse, Tilo; Bauer, Lisa; Seitner, Denise; Rabanal, Fernando A; Czedik-Eysenberg, Angelika; Uhse, Simon; Bindics, Janos; Genenncher, Bianca; Navarrete, Fernando; Kellner, Ronny; Ekker, Heinz; Kumlehn, Jochen; Vogel, John P; Gordon, Sean P; Marcel, Thierry C; Münsterkötter, Martin; Walter, Mathias C; Sieber, Christian MK; Mannhaupt, Gertrud; Güldener, Ulrich; Kahmann, Regine; Djamei, Armin

2016-01-01

Due to their economic relevance, the study of plant pathogen interactions is of importance. However, elucidating these interactions and their underlying molecular mechanisms remains challenging since both host and pathogen need to be fully genetically accessible organisms. Here we present milestones in the establishment of a new biotrophic model pathosystem: Ustilago bromivora and Brachypodium sp. We provide a complete toolset, including an annotated fungal genome and methods for genetic manipulation of the fungus and its host plant. This toolset will enable researchers to easily study biotrophic interactions at the molecular level on both the pathogen and the host side. Moreover, our research on the fungal life cycle revealed a mating type bias phenomenon. U. bromivora harbors a haplo-lethal allele that is linked to one mating type region. As a result, the identified mating type bias strongly promotes inbreeding, which we consider to be a potential speciation driver. DOI: http://dx.doi.org/10.7554/eLife.20522.001 PMID:27835569

INTERACTION OF X AND ULTRAVIOLET RADIATION IN PRODUCTION OF RECESSIVE LETHALS IN DROSOPHILA MELANOGASTER (in Italian)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nicoletti, B.; Olivieri, G.

1962-01-01

The possibility that uv rays given to different biological systems before or after x rays could modify genetic or cytological effects is reviewed and discussed. Kaufmann and Hollaender's conclusions about the recovering effect of uv rays on chromosomal damage induced in Drosophila sperms by a pre-treatment of x rays are discussed and analyzed taking into accourt some general considerations. Preliminary results of similar experiments on the frequency of sex-linked recessive lethals induced after single and combined x + uv treatments in Drosophila sperms are reported. All our experiments indicate no effect of the uv treatment (at the given wave lengthsmore » and doses) in lowering the frequency of the x-ray-induced recessive lethals. On the contrary, there are some indications for a synergistic action between the two radiations. These results not in agreement with the generally accepted theory that uv rays do recover X-ray- induced chromosomal damages, could be expiained With the well established correlation between chromosomal rejoined breaks and genic mutations. (auth)« less

The Genetics of a Small Autosomal Region of DROSOPHILA MELANOGASTER Containing the Structural Gene for Alcohol Dehydrogenase. III. Hypomorphic and Hypermorphic Mutations Affecting the Expression of Hairless

PubMed Central

Ashburner, Michael

1982-01-01

A lethal locus (l(2)br7;35B6-10), near Adh on chromosome arm 2L of D. melanogaster, is identified with Plunkett's dominant suppressor of Hairless (H). Of eight new alleles, seven act as dominant suppressors of H, the eighth is a dominant enhancer of H. One of the suppressor alleles is both a leaky lethal and a weak suppressor of H. Confirming Nash (1970), deletions of l(2)br7 are dominant suppressors, and duplications are dominant enhancers of H. A simple model is proposed to account for the interaction of l(2)br7 and H, assuming that amorphic (or hypomorphic) alleles of l(2)br7 suppress H and that hypermorphic alleles enhance H. PMID:6816670

Rin4 Causes Hybrid Necrosis and Race-Specific Resistance in an Interspecific Lettuce Hybrid[W

PubMed Central

Jeuken, Marieke J.W.; Zhang, Ningwen W.; McHale, Leah K.; Pelgrom, Koen; den Boer, Erik; Lindhout, Pim; Michelmore, Richard W.; Visser, Richard G.F.; Niks, Rients E.

2009-01-01

Some inter- and intraspecific crosses may result in reduced viability or sterility in the offspring, often due to genetic incompatibilities resulting from interactions between two or more loci. Hybrid necrosis is a postzygotic genetic incompatibility that is phenotypically manifested as necrotic lesions on the plant. We observed hybrid necrosis in interspecific lettuce (Lactuca sativa and Lactuca saligna) hybrids that correlated with resistance to downy mildew. Segregation analysis revealed a specific allelic combination at two interacting loci to be responsible. The allelic interaction had two consequences: (1) a quantitative temperature-dependent autoimmunity reaction leading to necrotic lesions, lethality, and quantitative resistance to an otherwise virulent race of Bremia lactucae; and (2) a qualitative temperature-independent race-specific resistance to an avirulent race of B. lactucae. We demonstrated by transient expression and silencing experiments that one of the two interacting genes was Rin4. In Arabidopsis thaliana, RIN4 is known to interact with multiple R gene products, and their interactions result in hypersensitive resistance to Pseudomonas syringae. Site-directed mutation studies on the necrosis-eliciting allele of Rin4 in lettuce showed that three residues were critical for hybrid necrosis. PMID:19855048

Rin4 causes hybrid necrosis and race-specific resistance in an interspecific lettuce hybrid.

PubMed

Jeuken, Marieke J W; Zhang, Ningwen W; McHale, Leah K; Pelgrom, Koen; den Boer, Erik; Lindhout, Pim; Michelmore, Richard W; Visser, Richard G F; Niks, Rients E

2009-10-01

Some inter- and intraspecific crosses may result in reduced viability or sterility in the offspring, often due to genetic incompatibilities resulting from interactions between two or more loci. Hybrid necrosis is a postzygotic genetic incompatibility that is phenotypically manifested as necrotic lesions on the plant. We observed hybrid necrosis in interspecific lettuce (Lactuca sativa and Lactuca saligna) hybrids that correlated with resistance to downy mildew. Segregation analysis revealed a specific allelic combination at two interacting loci to be responsible. The allelic interaction had two consequences: (1) a quantitative temperature-dependent autoimmunity reaction leading to necrotic lesions, lethality, and quantitative resistance to an otherwise virulent race of Bremia lactucae; and (2) a qualitative temperature-independent race-specific resistance to an avirulent race of B. lactucae. We demonstrated by transient expression and silencing experiments that one of the two interacting genes was Rin4. In Arabidopsis thaliana, RIN4 is known to interact with multiple R gene products, and their interactions result in hypersensitive resistance to Pseudomonas syringae. Site-directed mutation studies on the necrosis-eliciting allele of Rin4 in lettuce showed that three residues were critical for hybrid necrosis.

Empirical complexities in the genetic foundations of lethal mutagenesis.

PubMed

Bull, James J; Joyce, Paul; Gladstone, Eric; Molineux, Ian J

2013-10-01

From population genetics theory, elevating the mutation rate of a large population should progressively reduce average fitness. If the fitness decline is large enough, the population will go extinct in a process known as lethal mutagenesis. Lethal mutagenesis has been endorsed in the virology literature as a promising approach to viral treatment, and several in vitro studies have forced viral extinction with high doses of mutagenic drugs. Yet only one empirical study has tested the genetic models underlying lethal mutagenesis, and the theory failed on even a qualitative level. Here we provide a new level of analysis of lethal mutagenesis by developing and evaluating models specifically tailored to empirical systems that may be used to test the theory. We first quantify a bias in the estimation of a critical parameter and consider whether that bias underlies the previously observed lack of concordance between theory and experiment. We then consider a seemingly ideal protocol that avoids this bias-mutagenesis of virions-but find that it is hampered by other problems. Finally, results that reveal difficulties in the mere interpretation of mutations assayed from double-strand genomes are derived. Our analyses expose unanticipated complexities in testing the theory. Nevertheless, the previous failure of the theory to predict experimental outcomes appears to reside in evolutionary mechanisms neglected by the theory (e.g., beneficial mutations) rather than from a mismatch between the empirical setup and model assumptions. This interpretation raises the specter that naive attempts at lethal mutagenesis may augment adaptation rather than retard it.

Theories of Lethal Mutagenesis: From Error Catastrophe to Lethal Defection.

PubMed

Tejero, Héctor; Montero, Francisco; Nuño, Juan Carlos

2016-01-01

RNA viruses get extinct in a process called lethal mutagenesis when subjected to an increase in their mutation rate, for instance, by the action of mutagenic drugs. Several approaches have been proposed to understand this phenomenon. The extinction of RNA viruses by increased mutational pressure was inspired by the concept of the error threshold. The now classic quasispecies model predicts the existence of a limit to the mutation rate beyond which the genetic information of the wild type could not be efficiently transmitted to the next generation. This limit was called the error threshold, and for mutation rates larger than this threshold, the quasispecies was said to enter into error catastrophe. This transition has been assumed to foster the extinction of the whole population. Alternative explanations of lethal mutagenesis have been proposed recently. In the first place, a distinction is made between the error threshold and the extinction threshold, the mutation rate beyond which a population gets extinct. Extinction is explained from the effect the mutation rate has, throughout the mutational load, on the reproductive ability of the whole population. Secondly, lethal defection takes also into account the effect of interactions within mutant spectra, which have been shown to be determinant for the understanding the extinction of RNA virus due to an augmented mutational pressure. Nonetheless, some relevant issues concerning lethal mutagenesis are not completely understood yet, as so survival of the flattest, i.e. the development of resistance to lethal mutagenesis by evolving towards mutationally more robust regions of sequence space, or sublethal mutagenesis, i.e., the increase of the mutation rate below the extinction threshold which may boost the adaptability of RNA virus, increasing their ability to develop resistance to drugs (including mutagens). A better design of antiviral therapies will still require an improvement of our knowledge about lethal mutagenesis.

Interactions between the bud emergence proteins Bem1p and Bem2p and Rho-type GTPases in yeast.

PubMed

Peterson, J; Zheng, Y; Bender, L; Myers, A; Cerione, R; Bender, A

1994-12-01

The SH3 domain-containing protein Bem1p is needed for normal bud emergence and mating projection formation, two processes that require asymmetric reorganizations of the cortical cytoskeleton in Saccharomyces cerevisiae. To identify proteins that functionally and/or physically interact with Bem1p, we screened for mutations that display synthetic lethality with a mutant allele of the BEM1 gene and for genes whose products display two-hybrid interactions with the Bem1 protein. CDC24, which is required for bud emergence and encodes a GEF (guanine-nucleotide exchange factor) for the essential Rho-type GTPase Cdc42p, was identified during both screens. The COOH-terminal 75 amino acids of Cdc24p, outside of the GEF domain, can interact with a portion of Bem1p that lacks both SH3 domains. Bacterially expressed Cdc24p and Bem1p bind to each other in vitro, indicating that no other yeast proteins are required for this interaction. The most frequently identified gene that arose from the bem1 synthetic-lethal screen was the bud-emergence gene BEM2 (Bender and Pringle. 1991. Mol. Cell Biol. 11:1295-1395), which is allelic with IPL2 (increase in ploidy; Chan and Botstein, 1993. Genetics. 135:677-691). Here we show that Bem2p contains a GAP (GTPase-activating protein) domain for Rho-type GTPases, and that this portion of Bem2p can stimulate in vitro the GTPase activity of Rho1p, a second essential yeast Rho-type GTPase. Cells deleted for BEM2 become large and multinucleate. These and other genetic, two-hybrid, biochemical, and phenotypic data suggest that multiple Rho-type GTPases control the reorganization of the cortical cytoskeleton in yeast and that the functions of these GTPases are tightly coupled. Also, these findings raise the possibility that Bem1p may regulate or be a target of action of one or more of these GTPases.

Do Gametes Woo? Evidence for Their Nonrandom Union at Fertilization.

PubMed

Nadeau, Joseph H

2017-10-01

A fundamental tenet of inheritance in sexually reproducing organisms such as humans and laboratory mice is that gametes combine randomly at fertilization, thereby ensuring a balanced and statistically predictable representation of inherited variants in each generation. This principle is encapsulated in Mendel's First Law. But exceptions are known. With transmission ratio distortion, particular alleles are preferentially transmitted to offspring. Preferential transmission usually occurs in one sex but not both, and is not known to require interactions between gametes at fertilization. A reanalysis of our published work in mice and of data in other published reports revealed instances where any of 12 mutant genes biases fertilization, with either too many or too few heterozygotes and homozygotes, depending on the mutant gene and on dietary conditions. Although such deviations are usually attributed to embryonic lethality of the underrepresented genotypes, the evidence is more consistent with genetically-determined preferences for specific combinations of egg and sperm at fertilization that result in genotype bias without embryo loss. This unexpected discovery of genetically-biased fertilization could yield insights about the molecular and cellular interactions between sperm and egg at fertilization, with implications for our understanding of inheritance, reproduction, population genetics, and medical genetics. Copyright © 2017 by the Genetics Society of America.

Structure-function analysis and genetic interactions of the SmG, SmE, and SmF subunits of the yeast Sm protein ring.

PubMed

Schwer, Beate; Kruchten, Joshua; Shuman, Stewart

2016-09-01

A seven-subunit Sm protein ring forms a core scaffold of the U1, U2, U4, and U5 snRNPs that direct pre-mRNA splicing. Using human snRNP structures to guide mutagenesis in Saccharomyces cerevisiae, we gained new insights into structure-function relationships of the SmG, SmE, and SmF subunits. An alanine scan of 19 conserved amino acids of these three proteins, comprising the Sm RNA binding sites or inter-subunit interfaces, revealed that, with the exception of Arg74 in SmF, none are essential for yeast growth. Yet, for SmG, SmE, and SmF, as for many components of the yeast spliceosome, the effects of perturbing protein-RNA and protein-protein interactions are masked by built-in functional redundancies of the splicing machine. For example, tests for genetic interactions with non-Sm splicing factors showed that many benign mutations of SmG, SmE, and SmF (and of SmB and SmD3) were synthetically lethal with null alleles of U2 snRNP subunits Lea1 and Msl1. Tests of pairwise combinations of SmG, SmE, SmF, SmB, and SmD3 alleles highlighted the inherent redundancies within the Sm ring, whereby simultaneous mutations of the RNA binding sites of any two of the Sm subunits are lethal. Our results suggest that six intact RNA binding sites in the Sm ring suffice for function but five sites may not. © 2016 Schwer et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Suppression of the lethal effect of acidic-phospholipid deficiency by defective formation of the major outer membrane lipoprotein in Escherichia coli.

PubMed Central

Asai, Y; Katayose, Y; Hikita, C; Ohta, A; Shibuya, I

1989-01-01

The Escherichia coli pgsA3 allele encoding a defective phosphatidylglycerophosphate synthase is lethal for all but certain strains. Genetic analysis of such strains has revealed that the lethal effect is fully suppressed by the lack of the major outer membrane lipoprotein that consumes phosphatidylglycerol for its maturation. Images PMID:2556377

RADIATION INDUCED VIABILITY MUTATIONS IN THE HONEY BEE

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, W.R.

The frequency of recessive detrimental mutations expressed in the haploid drone honey bee was investigated and compared with recessive and dominant lethal mutations detected in the haploid drone and diploid worker. A single queen was inseminated by a drone homozygous for three genetic markers. Viability of progeny was determined, and hybrid daughters bearing the genetic markers were stored in colonies. The spermatheca of the queen was then irradiated with 2600 r kvp x rays. Morphological defects and viability were studied in progeny and grand-progeny. A total of 92 pairs was tested during one season. Results showed that 60.8% of themore » sperm cells receiving radiation contained at least one or more dominant lethals. Correcting for the saturation effect on the assumption of independence of each dominant lethal, an average proportion of 0.94 dominant lethals were found per cell. The average reduction in embryonic viability was 28%. Forty per cent of the queens tested contained one or more recessive lethals. Corrections in procedure and plans for future work, as well as work in progress, are described. (H.M.G.)« less

The maternally expressed WRKY transcription factor TTG2 controls lethality in interploidy crosses of Arabidopsis.

PubMed

Dilkes, Brian P; Spielman, Melissa; Weizbauer, Renate; Watson, Brian; Burkart-Waco, Diana; Scott, Rod J; Comai, Luca

2008-12-09

The molecular mechanisms underlying lethality of F1 hybrids between diverged parents are one target of speciation research. Crosses between diploid and tetraploid individuals of the same genotype can result in F1 lethality, and this dosage-sensitive incompatibility plays a role in polyploid speciation. We have identified variation in F1 lethality in interploidy crosses of Arabidopsis thaliana and determined the genetic architecture of the maternally expressed variation via QTL mapping. A single large-effect QTL, DR. STRANGELOVE 1 (DSL1), was identified as well as two QTL with epistatic relationships to DSL1. DSL1 affects the rate of postzygotic lethality via expression in the maternal sporophyte. Fine mapping placed DSL1 in an interval encoding the maternal effect transcription factor TTG2. Maternal parents carrying loss-of-function mutations in TTG2 suppressed the F1 lethality caused by paternal excess interploidy crosses. The frequency of cellularization in the endosperm was similarly affected by both natural variation and ttg2 loss-of-function mutants. The simple genetic basis of the natural variation and effects of single-gene mutations suggests that F1 lethality in polyploids could evolve rapidly. Furthermore, the role of the sporophytically active TTG2 gene in interploidy crosses indicates that the developmental programming of the mother regulates the viability of interploidy hybrid offspring.

The art and science of low-energy applications in medicine: pathology perspectives

NASA Astrophysics Data System (ADS)

Thomsen, Sharon L.

2011-03-01

Applications of low energy non-ionizing irradiation result in non-lethal and lethal effects in cells, tissues and intact individuals. The effects of these applications depend on the physical parameters of the applied energies, the mechanisms of interaction of these energies on the target and the biologic status of the target. Recently, cell death has been found not to be a random accident of situation or age but a range of complicated physiological responses to various extrinsic and intrinsic events some of which are genetically programmed and/ or physiologically regulated. Therefore, cell death has been classified into three general groups: 1) Programmed cell death including apoptosis and necroptosis, cornefication and autophagy; 2) Accidental (traumatic) cell death due to the direct, immediate effects of the lethal event and 3) Necrotic cell death which is, by default, all cell death not associated with programmed or accidental cell death. Lethal low energy non-ionizing application biologic effects involve mechanisms of all three groups as compared to high energy applications that predominantly involve the mechanisms of accidental cell death. Currently, the mechanisms of all these modes of cell death are being vigorously investigated. As research and development of new low energy applications continues, the need to understand the mechanisms of cell death that they produce will be critical to the rational creation of safe, yet effective instruments.

Resistance to genetic insect control: Modelling the effects of space.

PubMed

Watkinson-Powell, Benjamin; Alphey, Nina

2017-01-21

Genetic insect control, such as self-limiting RIDL 2 (Release of Insects Carrying a Dominant Lethal) technology, is a development of the sterile insect technique which is proposed to suppress wild populations of a number of major agricultural and public health insect pests. This is achieved by mass rearing and releasing male insects that are homozygous for a repressible dominant lethal genetic construct, which causes death in progeny when inherited. The released genetically engineered ('GE') insects compete for mates with wild individuals, resulting in population suppression. A previous study modelled the evolution of a hypothetical resistance to the lethal construct using a frequency-dependent population genetic and population dynamic approach. This found that proliferation of resistance is possible but can be diluted by the introgression of susceptible alleles from the released homozygous-susceptible GE males. We develop this approach within a spatial context by modelling the spread of a lethal construct and resistance trait, and the effect on population control, in a two deme metapopulation, with GE release in one deme. Results show that spatial effects can drive an increased or decreased evolution of resistance in both the target and non-target demes, depending on the effectiveness and associated costs of the resistant trait, and on the rate of dispersal. A recurrent theme is the potential for the non-target deme to act as a source of resistant or susceptible alleles for the target deme through dispersal. This can in turn have a major impact on the effectiveness of insect population control. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

A Genetic and Molecular Analysis of the 46c Chromosomal Region Surrounding the Fmrfamide Neuropeptide Gene in Drosophila Melanogaster

PubMed Central

O'Brien, M. A.; Roberts, M. S.; Taghert, P. H.

1994-01-01

We have analyzed the FMRFamide neuropeptide gene region of Drosophila melanogaster. This gene maps to the 46C region of chromosome 2R; this interval previously was not well characterized. For this genetic and molecular analysis, we have used X-ray mutagenesis, EMS mutagenesis, and the recently reported local P element transposition method. We identified four overlapping deletions, two of which have proximal breakpoints that define a 50-60-kb region surrounding the FMRFamide gene in 46C. To this small region, we mapped three lethal complementation groups; 10 additional lethal complementation groups were mapped to more distal regions of 46CD. One of these groups corresponds to even-skipped, the other 12 are previously unidentified. Using various lines of evidence we excluded the possibility that FMRFamide corresponds to any of the three lethal complementation groups mapping to its immediate 50-60-kb vicinity. The positions of two of the three lethal complementation groups were identified with P elements using a local transposition scheme. The third lethal complementation group was excluded as being FMRFamide mutants by sequence analysis and by immunocytochemistry with proFMRFamide precursor-specific antibodies. This analysis has (1) provided a genetic map of the 46CD chromosomal region and a detailed molecular map of a portion of the 46C region and (2) provided additional evidence of the utility of local transposition for targeting nearby genes. PMID:8056304

An analysis of lethal and sublethal interactions among type I and type II pyrethroid pesticide mixtures using standard Hyalella azteca water column toxicity tests.

PubMed

Hoffmann, Krista Callinan; Deanovic, Linda; Werner, Inge; Stillway, Marie; Fong, Stephanie; Teh, Swee

2016-10-01

A novel 2-tiered analytical approach was used to characterize and quantify interactions between type I and type II pyrethroids in Hyalella azteca using standardized water column toxicity tests. Bifenthrin, permethrin, cyfluthrin, and lambda-cyhalothrin were tested in all possible binary combinations across 6 experiments. All mixtures were analyzed for 4-d lethality, and 2 of the 6 mixtures (permethrin-bifenthrin and permethrin-cyfluthrin) were tested for subchronic 10-d lethality and sublethal effects on swimming motility and growth. Mixtures were initially analyzed for interactions using regression analyses, and subsequently compared with the additive models of concentration addition and independent action to further characterize mixture responses. Negative interactions (antagonistic) were significant in 2 of the 6 mixtures tested, including cyfluthrin-bifenthrin and cyfluthrin-permethrin, but only on the acute 4-d lethality endpoint. In both cases mixture responses fell between the additive models of concentration addition and independent action. All other mixtures were additive across 4-d lethality, and bifenthrin-permethrin and cyfluthrin-permethrin were also additive in terms of subchronic 10-d lethality and sublethal responses. Environ Toxicol Chem 2016;35:2542-2549. © 2016 SETAC. © 2016 SETAC.

Embryonic lethality is not sufficient to explain hourglass-like conservation of vertebrate embryos.

PubMed

Uchida, Yui; Uesaka, Masahiro; Yamamoto, Takayoshi; Takeda, Hiroyuki; Irie, Naoki

2018-01-01

Understanding the general trends in developmental changes during animal evolution, which are often associated with morphological diversification, has long been a central issue in evolutionary developmental biology. Recent comparative transcriptomic studies revealed that gene expression profiles of mid-embryonic period tend to be more evolutionarily conserved than those in earlier or later periods. While the hourglass-like divergence of developmental processes has been demonstrated in a variety of animal groups such as vertebrates, arthropods, and nematodes, the exact mechanism leading to this mid-embryonic conservation remains to be clarified. One possibility is that the mid-embryonic period (pharyngula period in vertebrates) is highly prone to embryonic lethality, and the resulting negative selections lead to evolutionary conservation of this phase. Here, we tested this "mid-embryonic lethality hypothesis" by measuring the rate of lethal phenotypes of three different species of vertebrate embryos subjected to two kinds of perturbations: transient perturbations and genetic mutations. By subjecting zebrafish ( Danio rerio ), African clawed frog ( Xenopus laevis ), and chicken ( Gallus gallus ) embryos to transient perturbations, namely heat shock and inhibitor treatments during three developmental periods [early (represented by blastula and gastrula), pharyngula, and late], we found that the early stages showed the highest rate of lethal phenotypes in all three species. This result was corroborated by perturbation with genetic mutations. By tracking the survival rate of wild-type embryos and embryos with genetic mutations induced by UV irradiation in zebrafish and African clawed frogs, we found that the highest decrease in survival rate was at the early stages particularly around gastrulation in both these species. In opposition to the "mid-embryonic lethality hypothesis," our results consistently showed that the stage with the highest lethality was not around the conserved pharyngula period, but rather around the early period in all the vertebrate species tested. These results suggest that negative selection by embryonic lethality could not explain hourglass-like conservation of animal embryos. This highlights the potential contribution of alternative mechanisms such as the diversifying effect of positive selections against earlier and later stages, and developmental constraints which lead to conservation of mid-embryonic stages.

The Novel Application of Non-Lethal Citizen Science Tissue Sampling in Recreational Fisheries.

PubMed

Williams, Samuel M; Holmes, Bonnie J; Pepperell, Julian G

2015-01-01

Increasing fishing pressure and uncertainty surrounding recreational fishing catch and effort data promoted the development of alternative methods for conducting fisheries research. A pilot investigation was undertaken to engage the Australian game fishing community and promote the non-lethal collection of tissue samples from the black marlin Istiompax indica, a valuable recreational-only species in Australian waters, for the purpose of future genetic research. Recruitment of recreational anglers was achieved by publicizing the project in magazines, local newspapers, social media, blogs, websites and direct communication workshops at game fishing tournaments. The Game Fishing Association of Australia and the Queensland Game Fishing Association were also engaged to advertise the project and recruit participants with a focus on those anglers already involved in the tag-and-release of marlin. Participants of the program took small tissue samples using non-lethal methods which were stored for future genetic analysis. The program resulted in 165 samples from 49 participants across the known distribution of I. indica within Australian waters which was a sufficient number to facilitate a downstream population genetic analysis. The project demonstrated the potential for the development of citizen science sampling programs to collect tissue samples using non-lethal methods in order to achieve targeted research objects in recreationally caught species.

Host genetic diversity enables Ebola hemorrhagic fever pathogenesis and resistance.

PubMed

Rasmussen, Angela L; Okumura, Atsushi; Ferris, Martin T; Green, Richard; Feldmann, Friederike; Kelly, Sara M; Scott, Dana P; Safronetz, David; Haddock, Elaine; LaCasse, Rachel; Thomas, Matthew J; Sova, Pavel; Carter, Victoria S; Weiss, Jeffrey M; Miller, Darla R; Shaw, Ginger D; Korth, Marcus J; Heise, Mark T; Baric, Ralph S; de Villena, Fernando Pardo-Manuel; Feldmann, Heinz; Katze, Michael G

2014-11-21

Existing mouse models of lethal Ebola virus infection do not reproduce hallmark symptoms of Ebola hemorrhagic fever, neither delayed blood coagulation and disseminated intravascular coagulation nor death from shock, thus restricting pathogenesis studies to nonhuman primates. Here we show that mice from the Collaborative Cross panel of recombinant inbred mice exhibit distinct disease phenotypes after mouse-adapted Ebola virus infection. Phenotypes range from complete resistance to lethal disease to severe hemorrhagic fever characterized by prolonged coagulation times and 100% mortality. Inflammatory signaling was associated with vascular permeability and endothelial activation, and resistance to lethal infection arose by induction of lymphocyte differentiation and cellular adhesion, probably mediated by the susceptibility allele Tek. These data indicate that genetic background determines susceptibility to Ebola hemorrhagic fever. Copyright © 2014, American Association for the Advancement of Science.

Adapted Lethality: What We Can Learn from Guinea Pig-Adapted Ebola Virus Infection Model.

PubMed

Cheresiz, S V; Semenova, E A; Chepurnov, A A

2016-01-01

Establishment of small animal models of Ebola virus (EBOV) infection is important both for the study of genetic determinants involved in the complex pathology of EBOV disease and for the preliminary screening of antivirals, production of therapeutic heterologic immunoglobulins, and experimental vaccine development. Since the wild-type EBOV is avirulent in rodents, the adaptation series of passages in these animals are required for the virulence/lethality to emerge in these models. Here, we provide an overview of our several adaptation series in guinea pigs, which resulted in the establishment of guinea pig-adapted EBOV (GPA-EBOV) variants different in their characteristics, while uniformly lethal for the infected animals, and compare the virologic, genetic, pathomorphologic, and immunologic findings with those obtained in the adaptation experiments of the other research groups.

Drosophila nemo is an essential gene involved in the regulation of programmed cell death.

PubMed

Mirkovic, Ivana; Charish, Kristi; Gorski, Sharon M; McKnight, Kristen; Verheyen, Esther M

2002-11-01

Nemo-like kinases define a novel family of serine/threonine kinases that are involved in integrating multiple signaling pathways. They are conserved regulators of Wnt/Wingless pathways, which may coordinate Wnt with TGFbeta-mediated signaling. Drosophila nemo was identified through its involvement in epithelial planar polarity, a process regulated by a non-canonical Wnt pathway. We have previously found that ectopic expression of Nemo using the Gal4-UAS system resulted in embryonic lethality associated with defects in patterning and head development. In this study we present our analyses of the phenotypes of germline clone-derived embryos. We observe lethality associated with head defects and reduction of programmed cell death and conclude that nmo is an essential gene. We also present data showing that nmo is involved in regulating apoptosis during eye development, based on both loss of function phenotypes and on genetic interactions with the pro-apoptotic gene reaper. Finally, we present genetic data from the adult wing that suggest the activity of ectopically expressed Nemo can be modulated by Jun N-terminal kinase (JNK) signaling. Such an observation supports the model that there is cross-talk between Wnt, TGFbeta and JNK signaling at multiple stages of development. Copyright 2002 Elsevier Science Ireland Ltd.

Substance P is a determinant of lethality in diet-induced hemorrhagic pancreatitis in mice.

PubMed

Maa, J; Grady, E F; Yoshimi, S K; Drasin, T E; Kim, E H; Hutter, M M; Bunnett, N W; Kirkwood, K S

2000-08-01

The neuropeptide substance P (SP) induces plasma extravasation and neutrophil infiltration by activating the neurokinin 1-receptor (NK1-R). SP-induced neurogenic inflammation is terminated by the cell surface enzyme neutral endopeptidase (NEP), which degrades SP. We determined whether genetic deletion of the NK1-R reduces mortality and, conversely, whether genetic deletion of NEP increases mortality in a lethal model of hemorrhagic pancreatitis. Necrotizing pancreatitis was induced by feeding mice a diet deficient in choline and supplemented with ethionine. We determined the length of survival, the severity of pancreatitis (by measuring the neutrophil enzyme myeloperoxidase [MPO] and by histologic evaluation), and the severity of pancreatitis-associated lung injury (lung MPO and histology) in NK1-R (+/+)/(-/-) and NEP (+/+)/(-/-) mice. Genetic deletion of the NK1-R significantly improved survival (100% vs 8% at 120 hours, P <.001) and reduced pancreatic MPO and acinar cell necrosis. Conversely, genetic deletion of NEP significantly worsened survival (0% vs 90% at 120 hours, P <.001) and exacerbated pancreatic MPO and pancreatitis-associated lung injury. Substance P is an important determinant of lethality in this model of necrotizing pancreatitis. Defects in NEP expression could lead to uncontrolled inflammation.

The origin of Behçet's disease geoepidemiology: possible role of a dual microbial-driven genetic selection.

PubMed

Piga, Matteo; Mathieu, Alessandro

2014-01-01

It is recognised that the genetic profiles that give rise to chronic inflammatory diseases, under the influence of environmental agents, might have been implicated in the host defence mechanism against lethal infections in the past. Behçet's disease (BD) is an immune-mediated inflammatory disease, expressed as vasculitis, triggered by environmental factors in genetically susceptible individuals. We carried out a review of published data to draw up an evolutionary adaptation model, as Author's perspective, for genetic susceptibility factors and inflammatory immune response involved in BD pathogenesis. Two lethal infectious agents, Plasmodium Falciparum and Yersinia Pestis, are proposed as the putative driving forces that favoured the fixing of the major genetic susceptibility factors to BD, thus determining its geoepidemiology. Further studies are needed to confirm the validity of this evolutionary model which includes and integrates the key insights of previous hypotheses.

A Synthetic Lethal Screen Identifies a Role for Lin-44/Wnt in C. elegans Embryogenesis.

PubMed

Hartin, Samantha N; Hudson, Martin L; Yingling, Curtis; Ackley, Brian D

2015-01-01

The C. elegans proteins PTP-3/LAR-RPTP and SDN-1/Syndecan are conserved cell adhesion molecules. Loss-of-function (LOF) mutations in either ptp-3 or sdn-1 result in low penetrance embryonic developmental defects. Work from other systems has shown that syndecans can function as ligands for LAR receptors in vivo. We used double mutant analysis to test whether ptp-3 and sdn-1 function in a linear genetic pathway during C. elegans embryogenesis. We found animals with LOF in both sdn-1 and ptp-3 exhibited a highly penetrant synthetic lethality (SynLet), with only a small percentage of animals surviving to adulthood. Analysis of the survivors demonstrated that these animals had a synergistic increase in the penetrance of embryonic developmental defects. Together, these data strongly suggested PTP-3 and SDN-1 function in parallel during embryogenesis. We subsequently used RNAi to knockdown ~3,600 genes predicted to encode secreted and/or transmembrane molecules to identify genes that interacted with ptp-3 or sdn-1. We found that the Wnt ligand, lin-44, was SynLet with sdn-1, but not ptp-3. We used 4-dimensional time-lapse analysis to characterize the interaction between lin-44 and sdn-1. We found evidence that loss of lin-44 caused defects in the polarization and migration of endodermal precursors during gastrulation, a previously undescribed role for lin-44 that is strongly enhanced by the loss of sdn-1. PTP-3 and SDN-1 function in compensatory pathways during C. elegans embryonic and larval development, as simultaneous loss of both genes has dire consequences for organismal survival. The Wnt ligand lin-44 contributes to the early stages of gastrulation in parallel to sdn-1, but in a genetic pathway with ptp-3. Overall, the SynLet phenotype provides a robust platform to identify ptp-3 and sdn-1 interacting genes, as well as other genes that function in development, yet might be missed in traditional forward genetic screens.

A Synthetic Lethal Screen Identifies a Role for Lin-44/Wnt in C. elegans Embryogenesis

PubMed Central

Hartin, Samantha N.; Hudson, Martin L.; Yingling, Curtis; Ackley, Brian D.

2015-01-01

Background The C. elegans proteins PTP-3/LAR-RPTP and SDN-1/Syndecan are conserved cell adhesion molecules. Loss-of-function (LOF) mutations in either ptp-3 or sdn-1 result in low penetrance embryonic developmental defects. Work from other systems has shown that syndecans can function as ligands for LAR receptors in vivo. We used double mutant analysis to test whether ptp-3 and sdn-1 function in a linear genetic pathway during C. elegans embryogenesis. Results We found animals with LOF in both sdn-1 and ptp-3 exhibited a highly penetrant synthetic lethality (SynLet), with only a small percentage of animals surviving to adulthood. Analysis of the survivors demonstrated that these animals had a synergistic increase in the penetrance of embryonic developmental defects. Together, these data strongly suggested PTP-3 and SDN-1 function in parallel during embryogenesis. We subsequently used RNAi to knockdown ~3,600 genes predicted to encode secreted and/or transmembrane molecules to identify genes that interacted with ptp-3 or sdn-1. We found that the Wnt ligand, lin-44, was SynLet with sdn-1, but not ptp-3. We used 4-dimensional time-lapse analysis to characterize the interaction between lin-44 and sdn-1. We found evidence that loss of lin-44 caused defects in the polarization and migration of endodermal precursors during gastrulation, a previously undescribed role for lin-44 that is strongly enhanced by the loss of sdn-1. Conclusions PTP-3 and SDN-1 function in compensatory pathways during C. elegans embryonic and larval development, as simultaneous loss of both genes has dire consequences for organismal survival. The Wnt ligand lin-44 contributes to the early stages of gastrulation in parallel to sdn-1, but in a genetic pathway with ptp-3. Overall, the SynLet phenotype provides a robust platform to identify ptp-3 and sdn-1 interacting genes, as well as other genes that function in development, yet might be missed in traditional forward genetic screens. PMID:25938228

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hedrick, P.W.

A number of studies indicates that there is a high sharing of HLA antigens in couples having recurrent spontaneous abortions. The genetic hypothesis to explain this phenomenon suggests that this fetal loss results from homozygosity of recessive lethal or deleterius alleles in gametic disequilibrium with HLA antigens. Theory predicting the lethality rate is derived when antigens are shared at one, two or three loci, given the disequilibrium is absolute. In addition, the effects of partial disequilibrium, inbreeding, and segregation distortion on the lethal proportion are examined.

Non-lethal genotyping of Tribolium castaneum adults using genomic DNA extracted from wing tissue.

PubMed

Strobl, Frederic; Ross, J Alexander; Stelzer, Ernst H K

2017-01-01

The red flour beetle Tribolium castaneum has become the second most important insect model organism and is frequently used in developmental biology, genetics and pest-associated research. Consequently, the methodological arsenal increases continuously, but many routinely applied techniques for Drosophila melanogaster and other insect species are still unavailable. For example, a protocol for non-lethal genotyping has not yet been adapted but is particularly useful when individuals with known genotypes are required for downstream experiments. In this study, we present a workflow for non-lethal genotyping of T. castaneum adults based on extracting genomic DNA from wing tissue. In detail, we describe a convenient procedure for wing dissection and a custom method for wing digestion that allows PCR-based genotyping of up to fifty adults in less than an afternoon with a success rate of about 86%. The amount of template is sufficient for up to ten reactions while viability and fertility of the beetles are preserved. We prove the applicability of our protocol by genotyping the white / scarlet gene pair alleles from the black-eyed San Bernadino wild-type and white-eyed Pearl recessive mutant strains spanning four generations. Non-lethal genotyping has the potential to improve and accelerate many workflows: Firstly, during the establishment process of homozygous cultures or during stock keeping of cultures that carry recessively lethal alleles, laborious test crossing is replaced by non-lethal genotyping. Secondly, in genome engineering assays, non-lethal genotyping allows the identification of appropriate founders before they are crossed against wild-types, narrowing the efforts down to only the relevant individuals. Thirdly, non-lethal genotyping simplifies experimental strategies, in which genotype and behavior should be correlated, since the genetic configuration of potential individuals can be determined before the actual behavior assays is performed.

The changes in the reproductive barrier between hexaploid wheat (Triticum aestivum L.) and rye (Secale cereale L.): different states lead to different fates.

PubMed

Tikhenko, Natalia; Rutten, Twan; Senula, Angelika; Rubtsova, Myroslava; Keller, E R Joachim; Börner, Andreas

2017-09-01

The changes in the reproductive barrier between hexaploid wheat ( Triticum aestivum L.) and rye ( Secale cereale L.) can be induced using in situ embryo rescue of abnormal embryos, yielding stable fertile amphidiploid plants. In intergeneric crosses between hexaploid wheat (Triticum aestivum L.) and rye (Secale cereale L.), postzygotic barriers may occur at different stages of hybrid development. One such mechanism is embryo lethality, which is genetically determined by the interaction and expression of two incompatible genes in wheat (Eml-A1) and rye (Eml-R1). Using in vitro culture methods as stressors, we overcame this hybrid lethality. Normal and abnormal embryos were observed to build embryogenic calli and produce regenerated plantlets in a similar manner. The high regenerative capacity of the abnormal embryos led us to conclude that the reproductive barrier in these intergeneric hybrids may have an epigenetic origin that can be easily overcome by culturing immature embryos via callus induction. After colchicine treatment during callus culture, amphidiploid plants were obtained. However, most of these plants did not produce seeds, due mainly to sterility of the pollen but also of the embryo sacs. These findings demonstrate that hybrid sterility affects both male and female gametophytes in plants obtained from abnormal embryos. The key roles of double fertilization and stress factors in the implementation of the apical meristem formation program in embryos from incompatible intergeneric crosses between hexaploid wheat and rye during in vitro culture are discussed. We also propose a hypothetical model for a wheat-rye lethality system involving differential expression of incompatible wheat Eml-A1 and rye Eml-R1b alleles in an identical genetic background.

The light gene of Drosophila melanogaster encodes a homologue of VPS41, a yeast gene involved in cellular-protein trafficking.

PubMed

Warner, T S; Sinclair, D A; Fitzpatrick, K A; Singh, M; Devlin, R H; Honda, B M

1998-04-01

Mutations in a number of genes affect eye colour in Drosophila melanogaster; some of these "eye-colour" genes have been shown to be involved in various aspects of cellular transport processes. In addition, combinations of viable mutant alleles of some of these genes, such as carnation (car) combined with either light (lt) or deep-orange (dor) mutants, show lethal interactions. Recently, dor was shown to be homologous to the yeast gene PEP3 (VPS18), which is known to be involved in intracellular trafficking. We have undertaken to extend our earlier work on the lt gene, in order to examine in more detail its expression pattern and to characterize its gene product via sequencing of a cloned cDNA. The gene appears to be expressed at relatively high levels in all stages and tissues examined, and shows strong homology to VPS41, a gene involved in cellular-protein trafficking in yeast and higher eukaryotes. Further genetic experiments also point to a role for lt in transport processes: we describe lethal interactions between viable alleles of lt and dor, as well as phenotypic interactions (reductions in eye pigment) between allels of lt and another eye-colour gene, garnet (g), whose gene product has close homology to a subunit of the human adaptor complex, AP-3.

Dengue virus specific IgY provides protection following lethal dengue virus challenge and is neutralizing in the absence of inducing antibody dependent enhancement

PubMed Central

Williams, Katherine L.; Harris, Eva; Alvine, Travis D.; Henderson, Thomas; Schiltz, James; Nilles, Matthew L.; Bradley, David S.

2017-01-01

Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are severe disease manifestations that can occur following sequential infection with different dengue virus serotypes (DENV1-4). At present, there are no licensed therapies to treat DENV-induced disease. DHF and DSS are thought to be mediated by serotype cross-reactive antibodies that facilitate antibody-dependent enhancement (ADE) by binding to viral antigens and then Fcγ receptors (FcγR) on target myeloid cells. Using genetically engineered DENV-specific antibodies, it has been shown that the interaction between the Fc portion of serotype cross-reactive antibodies and FcγR is required to induce ADE. Additionally, it was demonstrated that these antibodies were as neutralizing as their non-modified variants, were incapable of inducing ADE, and were therapeutic following a lethal, antibody-enhanced infection. Therefore, we hypothesized that avian IgY, which do not interact with mammalian FcγR, would provide a novel therapy for DENV-induced disease. We demonstrate here that goose-derived anti-DENV2 IgY neutralized DENV2 and did not induce ADE in vitro. Anti-DENV2 IgY was also protective in vivo when administered 24 hours following a lethal DENV2 infection. We were also able to demonstrate via epitope mapping that both full-length and alternatively spliced anti-DENV2 IgY recognized different epitopes, including epitopes that have not been previously identified. These observations provide evidence for the potential therapeutic applications of goose-derived anti-DENV2 IgY. PMID:28686617

Dengue virus specific IgY provides protection following lethal dengue virus challenge and is neutralizing in the absence of inducing antibody dependent enhancement.

PubMed

Fink, Ashley L; Williams, Katherine L; Harris, Eva; Alvine, Travis D; Henderson, Thomas; Schiltz, James; Nilles, Matthew L; Bradley, David S

2017-07-01

Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are severe disease manifestations that can occur following sequential infection with different dengue virus serotypes (DENV1-4). At present, there are no licensed therapies to treat DENV-induced disease. DHF and DSS are thought to be mediated by serotype cross-reactive antibodies that facilitate antibody-dependent enhancement (ADE) by binding to viral antigens and then Fcγ receptors (FcγR) on target myeloid cells. Using genetically engineered DENV-specific antibodies, it has been shown that the interaction between the Fc portion of serotype cross-reactive antibodies and FcγR is required to induce ADE. Additionally, it was demonstrated that these antibodies were as neutralizing as their non-modified variants, were incapable of inducing ADE, and were therapeutic following a lethal, antibody-enhanced infection. Therefore, we hypothesized that avian IgY, which do not interact with mammalian FcγR, would provide a novel therapy for DENV-induced disease. We demonstrate here that goose-derived anti-DENV2 IgY neutralized DENV2 and did not induce ADE in vitro. Anti-DENV2 IgY was also protective in vivo when administered 24 hours following a lethal DENV2 infection. We were also able to demonstrate via epitope mapping that both full-length and alternatively spliced anti-DENV2 IgY recognized different epitopes, including epitopes that have not been previously identified. These observations provide evidence for the potential therapeutic applications of goose-derived anti-DENV2 IgY.

Adapted Lethality: What We Can Learn from Guinea Pig-Adapted Ebola Virus Infection Model

PubMed Central

Cheresiz, S. V.; Semenova, E. A.; Chepurnov, A. A.

2016-01-01

Establishment of small animal models of Ebola virus (EBOV) infection is important both for the study of genetic determinants involved in the complex pathology of EBOV disease and for the preliminary screening of antivirals, production of therapeutic heterologic immunoglobulins, and experimental vaccine development. Since the wild-type EBOV is avirulent in rodents, the adaptation series of passages in these animals are required for the virulence/lethality to emerge in these models. Here, we provide an overview of our several adaptation series in guinea pigs, which resulted in the establishment of guinea pig-adapted EBOV (GPA-EBOV) variants different in their characteristics, while uniformly lethal for the infected animals, and compare the virologic, genetic, pathomorphologic, and immunologic findings with those obtained in the adaptation experiments of the other research groups. PMID:26989413

Engineering species-like barriers to sexual reproduction.

PubMed

Maselko, Maciej; Heinsch, Stephen C; Chacón, Jeremy M; Harcombe, William R; Smanski, Michael J

2017-10-12

Controlling the exchange of genetic information between sexually reproducing populations has applications in agriculture, eradication of disease vectors, control of invasive species, and the safe study of emerging biotechnology applications. Here we introduce an approach to engineer a genetic barrier to sexual reproduction between otherwise compatible populations. Programmable transcription factors drive lethal gene expression in hybrid offspring following undesired mating events. As a proof of concept, we target the ACT1 promoter of the model organism Saccharomyces cerevisiae using a dCas9-based transcriptional activator. Lethal overexpression of actin results from mating this engineered strain with a strain containing the wild-type ACT1 promoter.Genetic isolation of a genetically modified organism represents a useful strategy for biocontainment. Here the authors use dCas9-VP64-driven gene expression to construct a 'species-like' barrier to reproduction between two otherwise compatible populations.

Interactions between the bud emergence proteins Bem1p and Bem2p and Rho- type GTPases in yeast

PubMed Central

1994-01-01

The SH3 domain-containing protein Bem1p is needed for normal bud emergence and mating projection formation, two processes that require asymmetric reorganizations of the cortical cytoskeleton in Saccharomyces cerevisiae. To identify proteins that functionally and/or physically interact with Bem1p, we screened for mutations that display synthetic lethality with a mutant allele of the BEM1 gene and for genes whose products display two-hybrid interactions with the Bem1 protein. CDC24, which is required for bud emergence and encodes a GEF (guanine- nucleotide exchange factor) for the essential Rho-type GTPase Cdc42p, was identified during both screens. The COOH-terminal 75 amino acids of Cdc24p, outside of the GEF domain, can interact with a portion of Bem1p that lacks both SH3 domains. Bacterially expressed Cdc24p and Bem1p bind to each other in vitro, indicating that no other yeast proteins are required for this interaction. The most frequently identified gene that arose from the bem1 synthetic-lethal screen was the bud-emergence gene BEM2 (Bender and Pringle. 1991. Mol. Cell Biol. 11:1295-1395), which is allelic with IPL2 (increase in ploidy; Chan and Botstein, 1993. Genetics. 135:677-691). Here we show that Bem2p contains a GAP (GTPase-activating protein) domain for Rho-type GTPases, and that this portion of Bem2p can stimulate in vitro the GTPase activity of Rho1p, a second essential yeast Rho-type GTPase. Cells deleted for BEM2 become large and multinucleate. These and other genetic, two-hybrid, biochemical, and phenotypic data suggest that multiple Rho-type GTPases control the reorganization of the cortical cytoskeleton in yeast and that the functions of these GTPases are tightly coupled. Also, these findings raise the possibility that Bem1p may regulate or be a target of action of one or more of these GTPases. PMID:7962098

A survey of disease connections for CD4+ T cell master genes and their directly linked genes.

PubMed

Li, Wentian; Espinal-Enríquez, Jesús; Simpfendorfer, Kim R; Hernández-Lemus, Enrique

2015-12-01

Genome-wide association studies and other genetic analyses have identified a large number of genes and variants implicating a variety of disease etiological mechanisms. It is imperative for the study of human diseases to put these genetic findings into a coherent functional context. Here we use system biology tools to examine disease connections of five master genes for CD4+ T cell subtypes (TBX21, GATA3, RORC, BCL6, and FOXP3). We compiled a list of genes functionally interacting (protein-protein interaction, or by acting in the same pathway) with the master genes, then we surveyed the disease connections, either by experimental evidence or by genetic association. Embryonic lethal genes (also known as essential genes) are over-represented in master genes and their interacting genes (55% versus 40% in other genes). Transcription factors are significantly enriched among genes interacting with the master genes (63% versus 10% in other genes). Predicted haploinsufficiency is a feature of most these genes. Disease-connected genes are enriched in this list of genes: 42% of these genes have a disease connection according to Online Mendelian Inheritance in Man (OMIM) (versus 23% in other genes), and 74% are associated with some diseases or phenotype in a Genome Wide Association Study (GWAS) (versus 43% in other genes). Seemingly, not all of the diseases connected to genes surveyed were immune related, which may indicate pleiotropic functions of the master regulator genes and associated genes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Nonpermissiveness for mouse embryonic stem (ES) cell derivation circumvented by a single backcross to 129/Sv strain: establishment of ES cell lines bearing the Omd conditional lethal mutation.

PubMed

Kress, C; Vandormael-Pournin, S; Baldacci, P; Cohen-Tannoudji, M; Babinet, C

1998-12-01

The inbred mouse strain DDK carries a conditional early embryonic lethal mutation that is manifested when DDK females are crossed to males of other inbred strains but not in the corresponding reciprocal crosses. It has been shown that embryonic lethality could be assigned to a single genetic locus called Ovum mutant (Om), on Chromosome (Chr) 11 near Syca 1. In the course of our study of the molecular mechanisms underlying the embryonic lethality, we were interested in deriving an embryonic stem cell bearing the Om mutation in the homozygous state (Omd/Omd). However, it turned out that DDK is nonpermissive for ES cell establishment, with a standard protocol. Here we show that permissiveness could be obtained using Omd/Omd blastocysts with a 75% 129/Sv and 25% DDK genetic background. Several germline-competent Omd/Omd ES cell lines have been derived from blastocysts of this genotype. Such a scenario could be extended to the generation of ES cell lines bearing any mutation present in an otherwise nonpermissive mouse strain.

Lack of genetic interaction between Tbx20 and Tbx3 in early mouse heart development.

PubMed

Gavrilov, Svetlana; Harvey, Richard P; Papaioannou, Virginia E

2013-01-01

Members of the T-box family of transcription factors are important regulators orchestrating the complex regionalization of the developing mammalian heart. Individual mutations in Tbx20 and Tbx3 cause distinct congenital heart abnormalities in the mouse: Tbx20 mutations result in failure of heart looping, developmental arrest and lack of chamber differentiation, while hearts of Tbx3 mutants progress further, loop normally but show atrioventricular convergence and outflow tract defects. The two genes have overlapping areas of expression in the atrioventricular canal and outflow tract of the heart but their potential genetic interaction has not been previously investigated. In this study we produced compound mutants to investigate potential genetic interactions at the earliest stages of heart development. We find that Tbx20; Tbx3 double heterozygous mice are viable and fertile with no apparent abnormalities, while double homozygous mutants are embryonic lethal by midgestation. Double homozygous mutant embryos display abnormal cardiac morphogenesis, lack of heart looping, expression patterns of cardiac genes and time of death that are indistinguishable from Tbx20 homozygous mutants. Prior to death, the double homozygotes show an overall developmental delay similar to Tbx3 homozygous mutants. Thus the effects of Tbx20 are epistatic to Tbx3 in the heart but Tbx3 is epistatic to Tbx20 with respect to developmental delay.

Estimation of genetic parameters and genotype-by-environment interactions related to acute ammonia stress in Pacific white shrimp (Litopenaeus vannamei) juveniles at two different salinity levels

PubMed Central

Lu, Xia; Luan, Sheng; Cao, Baoxiang; Meng, Xianhong; Sui, Juan; Dai, Ping; Luo, Kun; Shi, Xiaoli; Hao, Dengchun; Han, Guomin; Kong, Jie

2017-01-01

Regarding the practical farming of Litopenaeus vannamei, the deterioration of water quality from intensive culture systems and environmental pollution is a common but troublesome problem in the cultivation of this species. The toxicities that result from deteriorating water quality, such as that from ammonia stress, have lethal effects on juvenile shrimp and can increase their susceptibility to pathogens. The toxicity of ammonia plays an important role in the frequently high mortality during the early stage on shrimp farms. However, little information is available regarding the genetic parameters of the ammonia tolerance of juveniles in the early stage, but this information is necessary to understand the potential for the genetic improvement of this trait. Considering the euryhalinity of L. vannamei and the fact that low salinity can increase the toxicity of ammonia stress, we estimated the heritability of ammonia tolerance in juveniles in 30‰ (normal) and 5‰ (low) salinity in this study using the survival time (ST) at individual level and the survival status at the half-lethal time (SS50) at the family level. In the normal and low salinity conditions and for the merged data, the heritability estimates of the ST (0.784±0.070, 0.575±0.068, and 0.517±0.058, respectively) and SS50 (0.402±0.061, 0.216±0.050, and 0.264±0.050, respectively) were all significantly greater than zero, which indicates that the ammonia-tolerance of shrimp can be greatly improved. So it might provide an alternative method to reduce mortality, help to enhance resistance to pathogens and reduce the occurrence of infectious diseases. The significant positive genetic correlation between ST and body length suggested that ammonia is more toxic to shrimp in the early stage. The medium-strength genetic correlations of the ST and SS50 between the two environments (0.394±0.097 and 0.377±0.098, respectively) indicate a strong genotype-by-environment (G×E) interaction for ammonia tolerance between the different salinity levels. Therefore, salinity-specific breeding programs for ammonia tolerance in shrimp should be purposefully implemented. PMID:28328986

Estimation of genetic parameters and genotype-by-environment interactions related to acute ammonia stress in Pacific white shrimp (Litopenaeus vannamei) juveniles at two different salinity levels.

PubMed

Lu, Xia; Luan, Sheng; Cao, Baoxiang; Meng, Xianhong; Sui, Juan; Dai, Ping; Luo, Kun; Shi, Xiaoli; Hao, Dengchun; Han, Guomin; Kong, Jie

2017-01-01

Regarding the practical farming of Litopenaeus vannamei, the deterioration of water quality from intensive culture systems and environmental pollution is a common but troublesome problem in the cultivation of this species. The toxicities that result from deteriorating water quality, such as that from ammonia stress, have lethal effects on juvenile shrimp and can increase their susceptibility to pathogens. The toxicity of ammonia plays an important role in the frequently high mortality during the early stage on shrimp farms. However, little information is available regarding the genetic parameters of the ammonia tolerance of juveniles in the early stage, but this information is necessary to understand the potential for the genetic improvement of this trait. Considering the euryhalinity of L. vannamei and the fact that low salinity can increase the toxicity of ammonia stress, we estimated the heritability of ammonia tolerance in juveniles in 30‰ (normal) and 5‰ (low) salinity in this study using the survival time (ST) at individual level and the survival status at the half-lethal time (SS50) at the family level. In the normal and low salinity conditions and for the merged data, the heritability estimates of the ST (0.784±0.070, 0.575±0.068, and 0.517±0.058, respectively) and SS50 (0.402±0.061, 0.216±0.050, and 0.264±0.050, respectively) were all significantly greater than zero, which indicates that the ammonia-tolerance of shrimp can be greatly improved. So it might provide an alternative method to reduce mortality, help to enhance resistance to pathogens and reduce the occurrence of infectious diseases. The significant positive genetic correlation between ST and body length suggested that ammonia is more toxic to shrimp in the early stage. The medium-strength genetic correlations of the ST and SS50 between the two environments (0.394±0.097 and 0.377±0.098, respectively) indicate a strong genotype-by-environment (G×E) interaction for ammonia tolerance between the different salinity levels. Therefore, salinity-specific breeding programs for ammonia tolerance in shrimp should be purposefully implemented.

Expression and Genetic Variation in Neuroendocrine Signaling Pathways in Lethal and Nonlethal Prostate Cancer among Men Diagnosed with Localized Disease.

PubMed

Lu, Donghao; Carlsson, Jessica; Penney, Kathryn L; Davidsson, Sabina; Andersson, Swen-Olof; Mucci, Lorelei A; Valdimarsdóttir, Unnur; Andrén, Ove; Fang, Fang; Fall, Katja

2017-12-01

Background: Recent data suggest that neuroendocrine signaling pathways may play a role in the progression of prostate cancer, particularly for early-stage disease. We aimed to explore whether expression of selected genes in the adrenergic, serotoninergic, glucocorticoid, and dopaminergic pathways differs in prostate tumor tissue from men with lethal disease compared with men with nonlethal disease. Methods: On the basis of the Swedish Watchful Waiting Cohort, we included 511 men diagnosed with incidental prostate cancer through transurethral resection of the prostate during 1977-1998 with follow-up up to 30 years. For those with tumor tissue ( N = 262), we measured mRNA expression of 223 selected genes included in neuroendocrine pathways. Using DNA from normal prostate tissue ( N = 396), we genotyped 36 SNPs from 14 receptor genes. Lethal prostate cancer was the primary outcome in analyses with pathway gene expression and genetic variants. Results: Differential expression of genes in the serotoninergic pathway was associated with risk of lethal prostate cancer ( P = 0.007); similar but weaker associations were noted for the adrenergic ( P = 0.014) and glucocorticoid ( P = 0.020) pathways. Variants of the HTR2A (rs2296972; P = 0.002) and NR3CI (rs33388; P = 0.035) genes (within the serotoninergic and glucocorticoid pathways) were associated with lethal cancer in overdominant models. These genetic variants were correlated with expression of several genes in corresponding pathways ( P < 0.05). Conclusions: Our findings lend support to hypothesis that the neuroendocrine pathways, particularly serotoninergic pathway, are associated with lethal outcome in the natural course of localized prostate cancer. Impact: This study provides evidence of the role of neuroendocrine pathways in prostate cancer progression that may have clinical utility. Cancer Epidemiol Biomarkers Prev; 26(12); 1781-7. ©2017 AACR . ©2017 American Association for Cancer Research.

Virulence of Melissococcus plutonius and secondary invaders associated with European foulbrood disease of the honey bee.

PubMed

Lewkowski, Oleg; Erler, Silvio

2018-05-24

European foulbrood is a globally distributed brood disease affecting honey bees. It may lead to lethal infections of larvae and, in severe cases, even to colony collapse. Lately, a profound genetic and phenotypic diversity was documented for the causative agent Melissococcus plutonius. However, experimental work on the impact of diverse M. plutonius strains on hosts with different genetic background is completely lacking and the role of secondary invaders is poorly understood. Here, we address these issues and elucidate the impact and interaction of both host and pathogen on one another. Moreover, we try to unravel the role of secondary bacterial invasions in foulbrood-diseased larvae. We employed in vitro infections with honey bee larvae from queens with different genetic background and three different M. plutonius strains. Larvae infection experiments showed host-dependent survival dynamics although M. plutonius strain 49.3 consistently had the highest virulence. This pattern was also reflected in significantly reduced weights of 49.3 strain-infected larvae compared to the other treatments. No difference was found in groups additionally inoculated with a secondary invader (Enterococcus faecalis or Paenibacillus alvei) neither in terms of larval survival nor weight. These results suggest that host background contributes markedly to the course of the disease but virulence is mainly dependent on pathogen genotype. Secondary invaders following a M. plutonius infection do not increase disease lethality and therefore may just be a colonization of weakened and immunodeficient, or dead larvae. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Interactive lethal and mutagenic effects of ultraviolet light and bleomycin in yeast: synergism or antagonism?

PubMed

Lillo, O L; Severgnini, A A; Nunes, E M

1997-11-01

The mutagenic interactions of ultraviolet light and bleomycin in haploid populations of Saccharomyces cerevisiae were analyzed. Survival and mutation frequency as a function of different bleomycin concentrations after one conditioning dose of UV radiation were determined. Furthermore, corresponding interaction functions and sensitization factors were calculated. A synergistic interaction between UV light and bleomycin was shown for both lethal and mutagenic events when the cells were in nutrient broth during the treatments. Conversely, the interaction between UV light and bleomycin was antagonistic when the cells were in deionized water during the treatment. The magnitude of lethal and mutagenic interactions depends on dose, and thus presumably on the number of lesions. The observed interactions between UV light and bleomycin suggest that the mechanism that is most likely involved is the induction of repair systems with different error probabilities during the delay of cell division.

De Novo Emergence of Genetically Resistant Mutants of Mycobacterium tuberculosis from the Persistence Phase Cells Formed against Antituberculosis Drugs In Vitro

PubMed Central

Sebastian, Jees; Swaminath, Sharmada; Nair, Rashmi Ravindran; Jakkala, Kishor; Pradhan, Atul

2016-01-01

ABSTRACT Bacterial persisters are a subpopulation of cells that can tolerate lethal concentrations of antibiotics. However, the possibility of the emergence of genetically resistant mutants from antibiotic persister cell populations, upon continued exposure to lethal concentrations of antibiotics, remained unexplored. In the present study, we found that Mycobacterium tuberculosis cells exposed continuously to lethal concentrations of rifampin (RIF) or moxifloxacin (MXF) for prolonged durations showed killing, RIF/MXF persistence, and regrowth phases. RIF-resistant or MXF-resistant mutants carrying clinically relevant mutations in the rpoB or gyrA gene, respectively, were found to emerge at high frequency from the RIF persistence phase population. A Luria-Delbruck fluctuation experiment using RIF-exposed M. tuberculosis cells showed that the rpoB mutants were not preexistent in the population but were formed de novo from the RIF persistence phase population. The RIF persistence phase M. tuberculosis cells carried elevated levels of hydroxyl radical that inflicted extensive genome-wide mutations, generating RIF-resistant mutants. Consistent with the elevated levels of hydroxyl radical-mediated genome-wide random mutagenesis, MXF-resistant M. tuberculosis gyrA de novo mutants could be selected from the RIF persistence phase cells. Thus, unlike previous studies, which showed emergence of genetically resistant mutants upon exposure of bacteria for short durations to sublethal concentrations of antibiotics, our study demonstrates that continuous prolonged exposure of M. tuberculosis cells to lethal concentrations of an antibiotic generates antibiotic persistence phase cells that form a reservoir for the generation of genetically resistant mutants to the same antibiotic or another antibiotic. These findings may have clinical significance in the emergence of drug-resistant tubercle bacilli. PMID:27895008

FIRST RESULTS ON X-RAY-INDUCED GENETIC DAMAGE IN ARTEMIA SALINA LEACH

DOE Office of Scientific and Technical Information (OSTI.GOV)

Metalli, P.; Ballardin, E.

1962-01-01

Prophase oocytes of diploid and tetraploid pantenogenetic Antemia salina were x irradiated with 1000 r and damage was scored as oocyte or embryo lethal mutations at the first (X/sub 1/) and at the second (X/sub 2/) generations after irradiation. Dominant lethality shown at X/sub 1/ was much greater for the diploid strain than for the tetraploid; lethality observed at X/sub 2/ was increased with respect to X/sub 1/ in the diploid strain, while in the tetraploid it remained unmodified. (auth)

'Sex and crime' in evolution - why sexuality was so successful.

PubMed

Schubert, Ingo

2011-01-01

Components of sexuality have fore-runners already in prokaryotes, for instance conjugation, recombination- repair and the molecular constituents needed for nuclear division. For eukaryotes, the basic and predominant mode of propagation is via sexuality, although it is highly complex and costly. Many interactions between individual cells are detrimental for one partner and might be considered as a 'criminal' act performed by the active partner. For instance, the irreversible and non-reciprocal processes of phagocytosis or endocellular parasitism but also the irreversible, asymmetric and sustainable endosymbiosis with a benefit bias in favour of the active partner represent such events. Contrary to this, sexuality in general represents an indirectly reversible, reciprocal, sustainable (by reiteration) and mutually beneficial interaction between equal-ranking cells. After fertilization, a doubled set of genetic information protects against loss of essential genes, while the haplophase allows ridding lethal mutations. Resorting of parental chromosome sets, recombination between homologous chromosomes during meiosis, and new combination of alleles during fertilization, mediate a high genetic variability at a minimum risk of deleterious variants, thus promoting evolutionary adaptability.

Computers in Biological Education: Simulation Approaches. Genetics and Evolution. CAL Research Group Technical Report No. 13.

ERIC Educational Resources Information Center

Murphy, P. J.

Three examples of genetics and evolution simulation concerning Mendelian inheritance, genetic mapping, and natural selection are used to illustrate the use of simulations in modeling scientific/natural processes. First described is the HERED series, which illustrates such phenomena as incomplete dominance, multiple alleles, lethal alleles,…

Suitability of non-lethal marker and marker-free systems for development of transgenic crop plants: present status and future prospects.

PubMed

Manimaran, P; Ramkumar, G; Sakthivel, K; Sundaram, R M; Madhav, M S; Balachandran, S M

2011-01-01

Genetically modified crops are one of the prudent options for enhancing the production and productivity of crop plants by safeguarding from the losses due to biotic and abiotic stresses. Agrobacterium-mediated and biolistic transformation methods are used to develop transgenic crop plants in which selectable marker genes (SMG) are generally deployed to identify 'true' transformants. The commonly used SMG obtained from prokaryotic sources when employed in transgenic plants pose risks due to their lethal nature during selection process. In the recent past, some non-lethal SMGs have been identified and used for selection of transformants with increased precision and high selection efficiency. Considering the concerns related to bio-safety of the environment, it is desirable to remove the SMG in order to maximize the commercial success through wide adoption and public acceptance of genetically modified (GM) food crops. In this review, we examine the availability, and the suitability of wide range of non-lethal selection markers and elimination of SMG methods to develop marker-free transgenics for achieving global food security. As the strategies for marker-free plants are still in proof-of-concept stage, adaptation of new genomics tools for identification of novel non-lethal marker systems and its application for developing marker-free transgenics would further strengthen the crop improvement program. Copyright © 2011 Elsevier Inc. All rights reserved.

The pathogenesis of Ebola hemorrhagic fever.

PubMed

Takada, A; Kawaoka, Y

2001-10-01

Ebola virus causes lethal hemorrhagic disease in humans, yet there are still no satisfactory biological explanations to account for its extreme virulence. This review focuses on recent findings relevant to understanding the pathogenesis of Ebola virus infection and developing vaccines and effective therapy. The available data suggest that the envelope glycoprotein and the interaction of some viral proteins with the immune system are likely to play important roles in the extraordinary pathogenicity of this virus. There are also indications that genetically engineered vaccines, including plasmid DNA and viral vectors expressing Ebola virus proteins, and passive transfer of neutralizing antibodies could be feasible options for the control of Ebola virus-associated disease.

Use of the Polymerase Chain Reaction and Complementary DNA Probes in the Detection of Duchenne Muscular Dystrophy Carriers

DTIC Science & Technology

1990-01-01

dominant or X-linked mutations, for example DMD and lethal osteogenesis imperfecta (1, 97). This phenomenon is the result of a dual population of...of the mutations. Am J Hum Genet 1988; 43: 620-29. 97. Cohn DH, Starman B, Blumberg B, Byers PH. Recurrence of lethal osteogenesis imperfecta due to

Epistatic Interactions within the Influenza A Virus Polymerase Complex Mediate Mutagen Resistance and Replication Fidelity

PubMed Central

Pauly, Matthew D.; Lyons, Daniel M.; Fitzsimmons, William J.

2017-01-01

ABSTRACT Lethal mutagenesis is a broad-spectrum antiviral strategy that employs mutagenic nucleoside analogs to exploit the high mutation rate and low mutational tolerance of many RNA viruses. Studies of mutagen-resistant viruses have identified determinants of replicative fidelity and the importance of mutation rate to viral population dynamics. We have previously demonstrated the effective lethal mutagenesis of influenza A virus using three nucleoside analogs as well as the virus’s high genetic barrier to mutagen resistance. Here, we investigate the mutagen-resistant phenotypes of mutations that were enriched in drug-treated populations. We find that PB1 T123A has higher replicative fitness than the wild type, PR8, and maintains its level of genome production during 5-fluorouracil (2,4-dihydroxy-5-fluoropyrimidine) treatment. Surprisingly, this mutagen-resistant variant also has an increased baseline rate of C-to-U and G-to-A mutations. A second drug-selected mutation, PA T97I, interacts epistatically with PB1 T123A to mediate high-level mutagen resistance, predominantly by limiting the inhibitory effect of nucleosides on polymerase activity. Consistent with the importance of epistatic interactions in the influenza virus polymerase, our data suggest that nucleoside analog resistance and replication fidelity are strain dependent. Two previously identified ribavirin {1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1H-1,2,4-triazole-3-carboxamide} resistance mutations, PB1 V43I and PB1 D27N, do not confer drug resistance in the PR8 background, and the PR8-PB1 V43I polymerase exhibits a normal baseline mutation rate. Our results highlight the genetic complexity of the influenza A virus polymerase and demonstrate that increased replicative capacity is a mechanism by which an RNA virus can counter the negative effects of elevated mutation rates. IMPORTANCE RNA viruses exist as genetically diverse populations. This standing genetic diversity gives them the potential to adapt rapidly, evolve resistance to antiviral therapeutics, and evade immune responses. Viral mutants with altered mutation rates or mutational tolerance have provided insights into how genetic diversity arises and how it affects the behavior of RNA viruses. To this end, we identified variants within the polymerase complex of influenza virus that are able to tolerate drug-mediated increases in viral mutation rates. We find that drug resistance is highly dependent on interactions among mutations in the polymerase complex. In contrast to other viruses, influenza virus counters the effect of higher mutation rates primarily by maintaining high levels of genome replication. These findings suggest the importance of maintaining large population sizes for viruses with high mutation rates and show that multiple proteins can affect both mutation rate and genome synthesis. PMID:28815216

Linear-quadratic dose kinetics or dose-dependent repair/misrepair

DOE Office of Scientific and Technical Information (OSTI.GOV)

Braby, L.A.; Nelson, J.M.

1991-09-01

Models for the response of cells exposed to low LET radiation can be grouped into three general types on the basis of assumptions about the nature of the interaction which results in the shoulder of the survival curve. The three forms of interaction are (1) sublethal damage becoming lethal, (2) potentially lethal damage becoming irreparable, and (3) potentially lethal damage saturating'' a repair system. The effects that these three forms of interaction would have on the results of specific types of experiments are investigated. Comparisons with experimental results indicate that only the second type is significant in determining the responsemore » of typical cultured mammalian cells. 5 refs., 2 figs.« less

Genetics Home Reference: bladder cancer

MedlinePlus

... events in bladder tumors. Researchers believe that several genes that control cell growth and division are probably located on chromosome 9 . ... Kwast TH, Zwarthoff EC, Radvanyi F. Novel fibroblast growth factor receptor ... identified in non-lethal skeletal disorders. Eur J Hum Genet. 2002 Dec;10( ...

Synergistic Lethality of Mifepristone and LY294002 in Ovarian Cancer Cells

PubMed Central

Wempe, Stacy L.; Gamarra-Luques, Carlos D.; Telleria, Carlos M.

2013-01-01

We have previously shown that the antiprogestin and antiglucocorticoid mifepristone inhibits the growth of ovarian cancer cells. In this work, we hypothesized that cellular stress caused by mifepristone is limited to cytostasis and that cell killing is avoided as a consequence of the persistent activity of the PI3K/Akt survival pathway. To investigate the role of this pathway in mifepristone-induced growth inhibition, human ovarian cancer cells of various histological subtypes and genetic backgrounds were exposed to cytostatic doses of mifepristone in the presence or absence of the PI3K inhibitor, LY294002. The activation of Akt in ovarian cancer cells, as marked by its phosphorylation on Ser473, was not modified by cytostatic concentrations of mifepristone, but it was blocked upon treatment with LY294002. The combination mifepristone/LY294002, but not the individual drugs, killed ovarian cancer cells via apoptosis, as attested by genomic DNA fragmentation and cleavage of caspase-3, and the concomitant downregulation of antiapoptotic proteins Bcl-2 and XIAP. From a pharmacological standpoint, when assessing cell growth inhibition using a median-dose analysis algorithm, the interaction between mifepristone and LY294002 was synergistic. The lethality caused by the combination mifepristone/LY294004 in 2-dimensional cell cultures was recapitulated in organized, 3-dimensional spheroids. This study demonstrates that mifepristone and LY294002 when used individually cause cell growth arrest; yet, when combined, they cause lethality. PMID:23420486

A synthetic lethal screen identifies ATR-inhibition as a novel therapeutic approach for POLD1-deficient cancers

PubMed Central

Hocke, Sandra; Guo, Yang; Job, Albert; Orth, Michael; Ziesch, Andreas; Lauber, Kirsten; De Toni, Enrico N; Gress, Thomas M.; Herbst, Andreas; Göke, Burkhard; Gallmeier, Eike

2016-01-01

The phosphoinositide 3-kinase-related kinase ATR represents a central checkpoint regulator and mediator of DNA-repair. Its inhibition selectively eliminates certain subsets of cancer cells in various tumor types, but the underlying genetic determinants remain enigmatic. Here, we applied a synthetic lethal screen directed against 288 DNA-repair genes using the well-defined ATR knock-in model of DLD1 colorectal cancer cells to identify potential DNA-repair defects mediating these effects. We identified a set of DNA-repair proteins, whose knockdown selectively killed ATR-deficient cancer cells. From this set, we further investigated the profound synthetic lethal interaction between ATR and POLD1. ATR-dependent POLD1 knockdown-induced cell killing was reproducible pharmacologically in POLD1-depleted DLD1 cells and a panel of other colorectal cancer cell lines by using chemical inhibitors of ATR or its major effector kinase CHK1. Mechanistically, POLD1 depletion in ATR-deficient cells caused caspase-dependent apoptosis without preceding cell cycle arrest and increased DNA-damage along with impaired DNA-repair. Our data could have clinical implications regarding tumor genotype-based cancer therapy, as inactivating POLD1 mutations have recently been identified in small subsets of colorectal and endometrial cancers. POLD1 deficiency might thus represent a predictive marker for treatment response towards ATR- or CHK1-inhibitors that are currently tested in clinical trials. PMID:26755646

Expression of Hygromycin Phosphotransferase Alters Virulence of Histoplasma capsulatum▿

PubMed Central

Smulian, A. George; Gibbons, Reta S.; Demland, Jeffery A.; Spaulding, Deborah T.; Deepe, George S.

2007-01-01

The Escherichia coli hygromycin phosphotransferase (hph) gene, which confers hygromycin resistance, is commonly used as a dominant selectable marker in genetically modified bacteria, fungi, plants, insects, and mammalian cells. Expression of the hph gene has rarely been reported to induce effects other than those expected. Hygromycin B is the most common dominant selectable marker used in the molecular manipulation of Histoplasma capsulatum in the generation of knockout strains of H. capsulatum or as a marker in mutant strains. hph-expressing organisms appear to have no defect in long-term in vitro growth and survival and have been successfully used to exploit host-parasite interaction in short-term cell culture systems and animal experiments. We introduced the hph gene as a selectable marker together with the gene encoding green fluorescent protein into wild-type strains of H. capsulatum. Infection of mice with hph-expressing H. capsulatum yeast cells at sublethal doses resulted in lethality. The lethality was not attributable to the site of integration of the hph construct into the genomes or to the method of integration and was not H. capsulatum strain related. Death of mice was not caused by altered cytokine profiles or an overwhelming fungal burden. The lethality was dependent on the kinase activity of hygromycin phosphotransferase. These results should raise awareness of the potential detrimental effects of the hph gene. PMID:17873086

Expression of hygromycin phosphotransferase alters virulence of Histoplasma capsulatum.

PubMed

Smulian, A George; Gibbons, Reta S; Demland, Jeffery A; Spaulding, Deborah T; Deepe, George S

2007-11-01

The Escherichia coli hygromycin phosphotransferase (hph) gene, which confers hygromycin resistance, is commonly used as a dominant selectable marker in genetically modified bacteria, fungi, plants, insects, and mammalian cells. Expression of the hph gene has rarely been reported to induce effects other than those expected. Hygromycin B is the most common dominant selectable marker used in the molecular manipulation of Histoplasma capsulatum in the generation of knockout strains of H. capsulatum or as a marker in mutant strains. hph-expressing organisms appear to have no defect in long-term in vitro growth and survival and have been successfully used to exploit host-parasite interaction in short-term cell culture systems and animal experiments. We introduced the hph gene as a selectable marker together with the gene encoding green fluorescent protein into wild-type strains of H. capsulatum. Infection of mice with hph-expressing H. capsulatum yeast cells at sublethal doses resulted in lethality. The lethality was not attributable to the site of integration of the hph construct into the genomes or to the method of integration and was not H. capsulatum strain related. Death of mice was not caused by altered cytokine profiles or an overwhelming fungal burden. The lethality was dependent on the kinase activity of hygromycin phosphotransferase. These results should raise awareness of the potential detrimental effects of the hph gene.

Biological responses of Habrobracon to spaceflight.

PubMed

von Borstel, R C; Smith, R H; Whiting, A R; Grosch, D S

1970-01-01

Since the interaction of the parasitic wasp Habrobracon with the space environment could not be prejudged, we decided to test approximately 30 different parameters of a genetic, mutational, biochemical, behavioral, and physiological character in the one spaceflight we had at our disposal. These parameters were examined at six different exposures of gamma-radiation (including 0 dose) in flight, resulting in about 180 different endpoints in all. The most profound effects of spaceflight in conjunction with radiation were decreased hatchability and enhanced fecundity of eggs exposed to spaceflight at different stages of oogenesis. The interpretation we favor is that these two endpoints are reflections of chromosomal non-disjunction in the former case and inhibition of cell division in the latter. Our most comprehensive study of mutagenesis was on sperm, where dominant lethality, recessive lethality, translocations, and visible mutations were assayed; the only effect found was a threefold enhancement of the recessive lethal mutation frequency in the non-irradiated sperm in the orbited Habrobracon males. Behavioral and biochemical differences were found. Mating activity of orbited males was severely disrupted and xanthine dehydrogenase activity was sharply decreased in the irradiated flight animals, an unexpected observation. Postflight experiments were like the ground-based control experiments in all aspects but one. Under conditions of vibration similar to those encountered during the launch and re-entry, the mutation frequency in the sperm increased by a factor of three over that of the non-vibrated control.

O-chromosome lethal frequencies in Serbian and Montenegrin Drosophila subobscura populations.

PubMed

Zivanovic, G; Arenas, C; Mestres, F

2011-10-01

Lethal chromosomal frequencies were obtained from three Drosophila subobscura samples from the Mt. Avala (Serbia) population in September 2003 (0.218), June 2004 (0.204) and September 2004 (0.250). These values and those from other Balkan populations studied previously (Petnica, Kamariste, Zanjic and Djerdap) were used to analyze the possible effect of population, year, month and altitude above sea level on lethal chromosomal frequencies. According to ANOVAS no effect were observed. Furthermore, the lethal frequencies of the Balkan populations did not vary according to latitude. This is probably due to the relative proximity and high gene flow between these populations. From a joint study of all the Palearctic D. subobscura populations so far analyzed, it can be deduced that the Balkan populations are located in the central area of the species distribution. Finally, it seems that lethal chromosomal frequencies are a consequence of the genetic structure of the populations.

Murine Toxicity of Cochliobolus carbonum1

PubMed Central

Hamilton, Pat B.; Nelson, R. R.; Harris, B. S. H.

1968-01-01

Seventeen wild-type strains of the phytopathogenic fungus Cochliobolus carbonum, tested by intraperitoneal injection into mice, were lethal within 48 hr. The lethal effect appeared to be a toxic rather than an infectious process, because death occurred within 3 hr after injection of two of the isolates and heat-killed cultures were lethal. Assays of ascospore progeny from two crosses involving three isolates indicated that the toxic metabolites were under genetic control and quantitative regulation. Studies of the toxicological, cultural, and chemical characteristics of these three strains indicated that more than one murine toxin was present. PMID:16349821

RAP-1 and the RAL-1/exocyst pathway coordinate hypodermal cell organization in Caenorhabditis elegans

PubMed Central

Frische, Ester W; Pellis-van Berkel, Wendy; van Haaften, Gijs; Cuppen, Edwin; Plasterk, Ronald H A; Tijsterman, Marcel; Bos, Johannes L; Zwartkruis, Fried J T

2007-01-01

The small Ras-like GTPase Rap1 has been identified as a regulator of integrin activation and cadherin-mediated cell–cell contacts. Surprisingly, null mutants of RAP-1 in Caenorhabditis elegans are viable and fertile. In a synthetic lethal RNAi screen with C. elegans rap-1 mutants, the Ras-like GTPase ral-1 emerged as one of seven genes specifically required for viability. Depletion of exoc-8 and sec-5, encoding two putative RAL-1 effectors and members of the exocyst complex, also caused lethality of rap-1 mutants, but did not affect wild-type worms. The RAP-1 and the RAL-1/exocyst pathway appear to coordinate hypodermal cell movement and elongation during embryonic development. They mediate their effect in part through targeting the α-catenin homologue HMP-1 to the lateral membrane. Genetic interactions show that the RAP-1 and RAL-1/exocyst pathway also act in parallel during larval stages. Together these data provide in vivo evidence for the exocyst complex as a downstream RAL-1 effector in cell migration. PMID:17989692

RAP-1 and the RAL-1/exocyst pathway coordinate hypodermal cell organization in Caenorhabditis elegans.

PubMed

Frische, Ester W; Pellis-van Berkel, Wendy; van Haaften, Gijs; Cuppen, Edwin; Plasterk, Ronald H A; Tijsterman, Marcel; Bos, Johannes L; Zwartkruis, Fried J T

2007-12-12

The small Ras-like GTPase Rap1 has been identified as a regulator of integrin activation and cadherin-mediated cell-cell contacts. Surprisingly, null mutants of RAP-1 in Caenorhabditis elegans are viable and fertile. In a synthetic lethal RNAi screen with C. elegans rap-1 mutants, the Ras-like GTPase ral-1 emerged as one of seven genes specifically required for viability. Depletion of exoc-8 and sec-5, encoding two putative RAL-1 effectors and members of the exocyst complex, also caused lethality of rap-1 mutants, but did not affect wild-type worms. The RAP-1 and the RAL-1/exocyst pathway appear to coordinate hypodermal cell movement and elongation during embryonic development. They mediate their effect in part through targeting the alpha-catenin homologue HMP-1 to the lateral membrane. Genetic interactions show that the RAP-1 and RAL-1/exocyst pathway also act in parallel during larval stages. Together these data provide in vivo evidence for the exocyst complex as a downstream RAL-1 effector in cell migration.

Comparative studies on the lethal, mutagenic, and recombinogenic effects of ultraviolet -A, -B, -C, and visible light with and without 8-methoxypsoralen in Saccharomyces cerevisiae.

PubMed

Mondon, P; Shahin, M M

1992-05-01

Genetic effects of UV-A, UV-B, UV-C, and the combination of 8-methoxypsoralen (8-MOP) with UV-A or visible light were studied in the haploid strain XV185-14C and diploid strain D5 of Saccharomyces cerevisiae. The induction of his+, lys+, and hom+ reverse mutations was measured in strain XV185-14C. In strain D5 we measured the induction of genetically altered colonies, particularly twin spot colonies arising from a mitotic crossing-over. UV-C and UV-B induced point mutations at the three loci in the haploid strain and mitotic crossing-over and other genetic alterations in the diploid strain. UV-C was more mutagenic and recombinogenic than UV-B. UV-A or visible light alone did not induce genotoxic effects at the doses tested. However, UV-A plus 8-MOP produced lethal and mutagenic effects in the haploid strain XV185-14C, although mutagenic activity was less than that of UV-B. Visible light plus 8-MOP also induced genotoxic effects in strain XV185-14C. In the diploid strain D5, UV-A plus 8-MOP induced a higher frequency of genetic alterations than UV-B at comparative doses. Visible light plus 8-MOP was also genetically active in strain D5. The haploid strain was more sensitive to the lethal effects of UV-C, UV-B, UV-A, and impure visible light plus 8-MOP than the diploid strain.

Mutants Resistant to anti-Microtubule Herbicides Map to a Locus on the Uni Linkage Group in Chlamydomonas Reinhardtii

PubMed Central

James, S. W.; Ranum, LPW.; Silflow, C. D.; Lefebvre, P. A.

1988-01-01

We have used genetic analysis to study the mode of action of two anti-microtubule herbicides, amiprophos-methyl (APM) and oryzalin (ORY). Over 200 resistant mutants were selected by growth on APM- or ORY-containing plates. The 21 independently isolated mutants examined in this study are 3- to 8-fold resistant to APM and are strongly cross-resistant to ORY and butamiphos, a close analog of APM. Two Mendelian genes, apm1 and apm2, are defined by linkage and complementation analysis. There are 20 alleles of apm1 and one temperature-sensitive lethal (33°) allele of apm2. Mapping by two-factor crosses places apm1 6.5 cM centromere proximal to uni1 and within 4 cM of pf7 on the uni linkage group, a genetically circular linkage group comprising genes which affect flagellar assembly or function; apm2 maps near the centromere of linkage group VIII. Allele-specific synthetic lethality is observed in crosses between apm2 and alleles of apm1. Also, self crosses of apm2 are zygotic lethal, whereas crosses of nine apm1 alleles inter se result in normal germination and tetrad viability. The mutants are recessive to their wild-type alleles but doubly heterozygous diploids (apm1 +/+ apm2) made with apm2 and any of 15 apm1 alleles display partial intergenic noncomplementation, expressed as intermediate resistance. Diploids homozygous for mutant alleles of apm1 are 4-6-fold resistant to APM and ORY; diploids homozygous for apm2 are ts(-) and 2-fold resistant to the herbicides. Doubly heterozygous diploids complement the ts(-) phenotype of apm2, but they are typically 1.5-2-fold resistant to APM and ORY. From the results described we suggest that the gene products of apm1 and apm2 may interact directly or function in the same structure or process. PMID:8608924

CARFILZOMIB INTERACTS SYNERGISTICALY WITH HISTONE DEACETYLASE INHIBITORS IN MANTLE CELL LYMPHOMA CELLS IN VITRO AND IN VIVO

PubMed Central

Dasmahapatra, Girija; Lembersky, Dmitry; Son, Minkyeong P.; Attkisson, Elisa; Dent, Paul; Fisher, Richard. I.; Friedberg, Jonathan W.; Grant, Steven

2011-01-01

Interactions between the proteasome inhibitor carfilzomib and the HDAC inhibitors vorinostat and SNDX-275 were examined in mantle cell lymphoma (MCL) cells in vitro and in vivo. Co-administration of very low, marginally toxic carfilzomib concentrations (e.g., 3–4 nM) with minimally lethal vorinostat or SNDX-275 concentrations induced sharp increases in mitochondrial injury and apoptosis in multiple MCL cell lines and primary MCL cells. Enhanced lethalitly was associated with JNK1/2 activation, increased DNA damage (induction of λH2A.X), and ERK1/2 and AKT1/2 inactivation. Co-administration of carfilzomib and HDACIs induced a marked increase in ROS generation, and G2M arrest. Significantly, the free radical scavenger TBAP blocked carfilzomib/HDACI-mediated ROS generation, λH2A.X formation, JNK1/2 activation, and lethality. Genetic (shRNA) knock down of JNK1/2 significantly attenuated carfilzomib/HDACI-induced apoptosis, but did not prevent ROS generation or DNA damage. Carfilzomib/HDACI regimens were also active against bortezomib-resistant MCL cells. Finally, carfilzomib/vorinostat co-administrationo resulted in a pronounced reduction in tumor growth compared to single agent treatment in a MCL xenograft model associated with enhanced apoptosis, λH2A.X formation, and JNK activation. Collectively, these findings suggest that carfilzomib/HDACI regimens warrants attention in MCL. PMID:21750224

Social Network Analysis of Crowds

DTIC Science & Technology

2009-08-06

crowd responses to non-lethal weapons d tan sys ems – Prior, existing social relationships – Real time social interactions – Formal/informal...Crowd Behavior Testbed Layout Video Cameras on Trusses Importance of Social Factors • Response to non-lethal weapons fire depends on social ... relationships among crowd members – Pre-existing Personal Relationships – Ongoing Real Time Social Interactions – Formal/Informal Hierarchies • Therefore

[Hematological and immunological parameters during Ebola virus passages in guinea-pigs].

PubMed

Dadaeva, A A; Sizikova, L P; Subbotina, E L; Chepurnov, A A

2006-01-01

The trend in hematological and immunological parameters during Ebola virus passages in guinea-pigs indicated that pathophysiological changes occurred just during the second passage and further became stronger. The increase of some parameters and their correlation with the occurrence of fatal outcomes allowed the authors to reveal the most significant changes as increased juvenile platelets, whole blood virus appearance, higher echinocytes, a rise in the pro mil of blast cells and megakaryocytes in the bone marrow, and decreased neutrophilic phagocytic activity. Viral acquisition of the properties of lethality to guinea-pigs depends on the fine mechanisms responsible for viral interaction with host cells, which may lead to viral genetic changes during passages.

Pervasive and largely lineage-specific adaptive protein evolution in the dosage compensation complex of Drosophila melanogaster.

PubMed

Levine, Mia T; Holloway, Alisha K; Arshad, Umbreen; Begun, David J

2007-11-01

Dosage compensation refers to the equalization of X-linked gene transcription among heterogametic and homogametic sexes. In Drosophila, the dosage compensation complex (DCC) mediates the twofold hypertranscription of the single male X chromosome. Loss-of-function mutations at any DCC protein-coding gene are male lethal. Here we report a population genetic analysis suggesting that four of the five core DCC proteins--MSL1, MSL2, MSL3, and MOF--are evolving under positive selection in D. melanogaster. Within these four proteins, several domains that range in function from X chromosome localization to protein-protein interactions have elevated, D. melanogaster-specific, amino acid divergence.

[Mutagenic and antimutagenic properties of bemitil].

PubMed

Seredenin, S B; Bobkov, Iu G; Durnev, A D; Dubovskaia, O Iu

1986-07-01

Complex research of the genetic activity of a new 2-mercaptobenzimidazole derivative bemythyl has shown that the drug failed to induce recessive, age-related lethal mutations in drosophila, dominant lethal mutations in germ mammalian cells and chromosomal damage in murine bone marrow cells and human peripheral blood cell cultures. The experiments on mice have demonstrated that therapeutic bemythyl doses caused a two-fold decrease in the level of aberrant cells induced by alkylating agents--fotrin and fopurin.

Enhancing the stability and ecological safety of mass-reared transgenic strains for field release by redundant conditional lethality systems

USDA-ARS?s Scientific Manuscript database

Advances in the genetic manipulation of agriculturally important insects now allows the development of genetic sexing and male sterility systems for more highly efficient biologically-based population control programs, most notably SIT, in fruit pests throughout the world. Potentially, these condit...

RNA Virus Reverse Genetics and Vaccine Design

PubMed Central

Stobart, Christopher C.; Moore, Martin L.

2014-01-01

RNA viruses are capable of rapid spread and severe or potentially lethal disease in both animals and humans. The development of reverse genetics systems for manipulation and study of RNA virus genomes has provided platforms for designing and optimizing viral mutants for vaccine development. Here, we review the impact of RNA virus reverse genetics systems on past and current efforts to design effective and safe viral therapeutics and vaccines. PMID:24967693

Novel synthetic lethality screening method identifies TIP60-dependent radiation sensitivity in the absence of BAF180.

PubMed

Hopkins, Suzanna R; McGregor, Grant A; Murray, Johanne M; Downs, Jessica A; Savic, Velibor

2016-10-01

In recent years, research into synthetic lethality and how it can be exploited in cancer treatments has emerged as major focus in cancer research. However, the lack of a simple to use, sensitive and standardised assay to test for synthetic interactions has been slowing the efforts. Here we present a novel approach to synthetic lethality screening based on co-culturing two syngeneic cell lines containing individual fluorescent tags. By associating shRNAs for a target gene or control to individual fluorescence labels, we can easily follow individual cell fates upon siRNA treatment and high content imaging. We have demonstrated that the system can recapitulate the functional defects of the target gene depletion and is capable of discovering novel synthetic interactors and phenotypes. In a trial screen, we show that TIP60 exhibits synthetic lethality interaction with BAF180, and that in the absence of TIP60, there is an increase micronuclei dependent on the level of BAF180 loss, significantly above levels seen with BAF180 present. Moreover, the severity of the interactions correlates with proxy measurements of BAF180 knockdown efficacy, which may expand its usefulness to addressing synthetic interactions through titratable hypomorphic gene expression. Copyright © 2016. Published by Elsevier B.V.

Lethality of Sortase Depletion in Actinomyces oris Caused by Excessive Membrane Accumulation of a Surface Glycoprotein

PubMed Central

Wu, Chenggang; Huang, I-Hsiu; Chang, Chungyu; Reardon-Robinson, Melissa Elizabeth; Das, Asis; Ton-That, Hung

2014-01-01

Sortase, a cysteine-transpeptidase conserved in Gram-positive bacteria, anchors on the cell wall many surface proteins that facilitate bacterial pathogenesis and fitness. Genetic disruption of the housekeeping sortase in several Gram-positive pathogens reported thus far attenuates virulence, but not bacterial growth. Paradoxically, we discovered that depletion of the housekeeping sortase SrtA was lethal for Actinomyces oris; yet, all of its predicted cell wall-anchored protein substrates (AcaA-N) were individually dispensable for cell viability. Using Tn5-transposon mutagenesis to identify factors that upend lethality of srtA deletion, we uncovered a set of genetic suppressors harboring transposon insertions within genes of a locus encoding AcaC and a LytR-CpsA-Psr (LCP)-like protein. AcaC was shown to be highly glycosylated and dependent on LCP for its glycosylation. Upon SrtA depletion, the glycosylated form of AcaC, hereby renamed GspA, was accumulated in the membrane. Overexpression of GspA in a mutant lacking gspA and srtA was lethal; conversely, cells overexpressing a GspA mutant missing a membrane-localization domain were viable. The results reveal a unique glycosylation pathway in A. oris that is coupled to cell wall anchoring catalyzed by sortase SrtA. Significantly, this novel phenomenon of glyco-stress provides convenient cell-based assays for developing a new class of inhibitors against Gram-positive pathogens. PMID:25230351

Calpain chronicle--an enzyme family under multidisciplinary characterization.

PubMed

Sorimachi, Hiroyuki; Hata, Shoji; Ono, Yasuko

2011-01-01

Calpain is an intracellular Ca2+-dependent cysteine protease (EC 3.4.22.17; Clan CA, family C02) discovered in 1964. It was also called CANP (Ca2+-activated neutral protease) as well as CASF, CDP, KAF, etc. until 1990. Calpains are found in almost all eukaryotes and a few bacteria, but not in archaebacteria. Calpains have a limited proteolytic activity, and function to transform or modulate their substrates' structures and activities; they are therefore called, "modulator proteases." In the human genome, 15 genes--CAPN1, CAPN2, etc.--encode a calpain-like protease domain. Their products are calpain homologs with divergent structures and various combinations of functional domains, including Ca2+-binding and microtubule-interaction domains. Genetic studies have linked calpain deficiencies to a variety of defects in many different organisms, including lethality, muscular dystrophies, gastropathy, and diabetes. This review of the study of calpains focuses especially on recent findings about their structure-function relationships. These discoveries have been greatly aided by the development of 3D structural studies and genetic models.

Attitudes Towards Prenatal Genetic Counseling, Prenatal Genetic Testing, and Termination of Pregnancy among Southeast and East Asian Women in the United States.

PubMed

Tsai, Ginger J; Cameron, Carrie A; Czerwinski, Jennifer L; Mendez-Figueroa, Hector; Peterson, Susan K; Noblin, Sarah Jane

2017-10-01

Recognizing the heterogeneity of the Asian population with regards to acculturation, education, health awareness, and cultural values is vital for tailoring culturally sensitive and appropriate care. Prior studies show that cultural values influence perceptions of genetics within Asian populations. The reputation of the family unit factors into decisions such as pregnancy termination and disclosure of family medical history, and the nondirective model of American genetic counseling may conflict with the historical Asian model of paternalistic health care. Previous studies also provide conflicting evidence regarding correlations between education, acculturation, age, and awareness and perceptions of genetic testing. The aims of this study were to describe attitudes towards prenatal genetics among Southeast and East Asian women living in the United States for varying amounts of time and to explore sociocultural factors influencing those attitudes. Twenty-three Asian women who were members of Asian cultural organizations in the United States were interviewed via telephone about their attitudes towards prenatal genetic counseling, prenatal genetic testing, and termination of pregnancy. Responses were transcribed and coded for common themes using a thematic analysis approach. Four major themes emerged. In general, participants: (1) had diverse expectations for genetic counselors; (2) tended to weigh risks and benefits with regards to genetic testing decisions; (3) had mixed views on termination for lethal and non-lethal genetic conditions; and (4) identified cultural factors which influenced testing and termination such as lack of available resources, societal shame and stigma, and family pressure. These findings may allow prenatal genetic counselors to gain a richer, more nuanced understanding of their Asian patients and to offer culturally tailored prenatal genetic counseling.

Unifying measures of gene function and evolution.

PubMed

Wolf, Yuri I; Carmel, Liran; Koonin, Eugene V

2006-06-22

Recent genome analyses revealed intriguing correlations between variables characterizing the functioning of a gene, such as expression level (EL), connectivity of genetic and protein-protein interaction networks, and knockout effect, and variables describing gene evolution, such as sequence evolution rate (ER) and propensity for gene loss. Typically, variables within each of these classes are positively correlated, e.g. products of highly expressed genes also have a propensity to be involved in many protein-protein interactions, whereas variables between classes are negatively correlated, e.g. highly expressed genes, on average, evolve slower than weakly expressed genes. Here, we describe principal component (PC) analysis of seven genome-related variables and propose biological interpretations for the first three PCs. The first PC reflects a gene's 'importance', or the 'status' of a gene in the genomic community, with positive contributions from knockout lethality, EL, number of protein-protein interaction partners and the number of paralogues, and negative contributions from sequence ER and gene loss propensity. The next two PCs define a plane that seems to reflect the functional and evolutionary plasticity of a gene. Specifically, PC2 can be interpreted as a gene's 'adaptability' whereby genes with high adaptability readily duplicate, have many genetic interaction partners and tend to be non-essential. PC3 also might reflect the role of a gene in organismal adaptation albeit with a negative rather than a positive contribution of genetic interactions; we provisionally designate this PC 'reactivity'. The interpretation of PC2 and PC3 as measures of a gene's plasticity is compatible with the observation that genes with high values of these PCs tend to be expressed in a condition- or tissue-specific manner. Functional classes of genes substantially vary in status, adaptability and reactivity, with the highest status characteristic of the translation system and cytoskeletal proteins, highest adaptability seen in cellular processes and signalling genes, and top reactivity characteristic of metabolic enzymes.

Genetics of pancreatic cancer and implications for therapy.

PubMed

Bhosale, Priya; Cox, Veronica; Faria, Silvana; Javadi, Sanaz; Viswanathan, Chitra; Koay, Eugene; Tamm, Eric

2018-02-01

Pancreatic cancer is a highly lethal disease with a dismal 5-year prognosis. Knowledge of its genetics may help in identifying new methods for patient screening, and cancer treatment. In this review, we will describe the most common mutations responsible for the genesis of pancreatic cancer and their impact on screening, patterns of disease progression, and therapy.

Altering host resistance to infections through microbial transplantation.

PubMed

Willing, Benjamin P; Vacharaksa, Anjalee; Croxen, Matthew; Thanachayanont, Teerawat; Finlay, B Brett

2011-01-01

Host resistance to bacterial infections is thought to be dictated by host genetic factors. Infections by the natural murine enteric pathogen Citrobacter rodentium (used as a model of human enteropathogenic and enterohaemorrhagic E. coli infections) vary between mice strains, from mild self-resolving colonization in NIH Swiss mice to lethality in C3H/HeJ mice. However, no clear genetic component had been shown to be responsible for the differences observed with C. rodentium infections. Because the intestinal microbiota is important in regulating resistance to infection, and microbial composition is dependent on host genotype, it was tested whether variations in microbial composition between mouse strains contributed to differences in "host" susceptibility by transferring the microbiota of resistant mice to lethally susceptible mice prior to infection. Successful transfer of the microbiota from resistant to susceptible mice resulted in delayed pathogen colonization and mortality. Delayed mortality was associated with increased IL-22 mediated innate defense including antimicrobial peptides Reg3γ and Reg3β, and immunono-neutralization of IL-22 abrogated the beneficial effect of microbiota transfer. Conversely, depletion of the native microbiota in resistant mice by antibiotics and transfer of the susceptible mouse microbiota resulted in reduced innate defenses and greater pathology upon infection. This work demonstrates the importance of the microbiota and how it regulates mucosal immunity, providing an important factor in susceptibility to enteric infection. Transfer of resistance through microbial transplantation (bacteriotherapy) provides additional mechanisms to alter "host" resistance, and a novel means to alter enteric infection and to study host-pathogen interactions.

Molecular and Genetic Characterization of the Drosophila Melanogaster 87e Actin Gene Region

PubMed Central

Manseau, L. J.; Ganetzky, B.; Craig, E. A.

1988-01-01

A combined molecular and genetic analysis of the 87E actin gene (Act87E) in Drosophila melanogaster was undertaken. A clone of Act87E was isolated and characterized. The Act87E transcription unit is 1.57 kb and includes a 556-base intervening sequence in the 5' leader of the gene. The protein-coding region is contiguous and encodes a protein that is >93% identical to the other Drosophila actins. By in situ hybridization with a series of deficiencies that break in 87E, Act87E was localized to a region encompassing one to three faint, polytene chromosome bands. The region between the deficiency endpoints that flank the actin gene was isolated and measures approximately 24-30 kb. The closest proximal deficiency endpoint lies 8-10 kb 5' to the actin gene; the closest distal deficiency endpoint lies 16-20 kb 3' to the actin gene. A single, recessive lethal complementation group lies between the deficiency endpoints that flank the actin gene. An EMS mutagenesis screen produced four additional members of this recessive lethal complementation group. Molecular analysis of the members of this complementation group indicated that two of the newly induced mutations have deletions of approximately 1 kb in a transcribed region 4-5 kb 3' (distal) to the actin gene. This result suggests that the recessive lethal complementation group represents a gene separate from and distal to the actin gene. The mutagenesis screen failed to identify additional recessive lethal complementation groups in the actin gene-containing region. The implications of the failure to identify recessive lethal mutations in the actin gene are discussed in reference to studies of other conserved multigene families and other muscle protein mutations. PMID:2840338

Synthetic lethality in DNA repair network: A novel avenue in targeted cancer therapy and combination therapeutics.

PubMed

Bhattacharjee, Sonali; Nandi, Saikat

2017-12-01

Synthetic lethality refers to a lethal phenotype that results from the simultaneous disruptions of two genes, while the disruption of either gene alone is viable. Many DNA double strand break repair (DSBR) genes have synthetic lethal relationships with oncogenes and tumor suppressor genes, which can be exploited for targeted cancer therapy, an approach referred to as combination therapy. DNA double-strand breaks (DSBs) are one of the most toxic lesions to a cell and can be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). HR and NHEJ genes are particularly attractive targets for cancer therapy because these genes have altered expression patterns in cancer cells when compared with normal cells and these genetic abnormalities can be targeted for selectively killing cancer cells. Here, we review recent advances in the development of small molecule inhibitors against HR and NHEJ genes to induce synthetic lethality and address the future directions and clinical relevance of this approach. © 2017 IUBMB Life, 69(12):929-937, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

A Common Suite of Coagulation Proteins Function in Drosophila Muscle Attachment.

PubMed

Green, Nicole; Odell, Nadia; Zych, Molly; Clark, Cheryl; Wang, Zong-Heng; Biersmith, Bridget; Bajzek, Clara; Cook, Kevin R; Dushay, Mitchell S; Geisbrecht, Erika R

2016-11-01

The organization and stability of higher order structures that form in the extracellular matrix (ECM) to mediate the attachment of muscles are poorly understood. We have made the surprising discovery that a subset of clotting factor proteins are also essential for muscle attachment in the model organism Drosophila melanogaster One such coagulation protein, Fondue (Fon), was identified as a novel muscle mutant in a pupal lethal genetic screen. Fon accumulates at muscle attachment sites and removal of this protein results in decreased locomotor behavior and detached larval muscles. A sensitized genetic background assay reveals that fon functions with the known muscle attachment genes Thrombospondin (Tsp) and Tiggrin (Tig). Interestingly, Tig is also a component of the hemolymph clot. We further demonstrate that an additional clotting protein, Larval serum protein 1γ (Lsp1γ), is also required for muscle attachment stability and accumulates where muscles attach to tendons. While the local biomechanical and organizational properties of the ECM vary greatly depending on the tissue microenvironment, we propose that shared extracellular protein-protein interactions influence the strength and elasticity of ECM proteins in both coagulation and muscle attachment. Copyright © 2016 by the Genetics Society of America.

A geometric model of mortality and crop protection for insects feeding on discrete toxicant deposits.

PubMed

Ebert, Timothy; Derksen, Richard

2004-04-01

Current theory governing the biological effectiveness of toxicants stresses the dose-response relationship and focuses on uniform toxicant distributions in the insect's environment. However, toxicants are seldom uniformly dispersed under field conditions. Toxicant distribution affects bioavailability, but the mechanics of such interactions is not well documented. We present a geometric model of the interactions between insects and heterogeneously distributed toxicants. From the model, we conclude the following: 1) There is an optimal droplet size, and droplets both smaller and larger than this optimum will decrease efficacy. 2) There is an ideal droplet distribution. Droplets should be spaced based on two criteria: calculate the allowable damage, double this quantity, and one lethal deposit should be placed in this area; and define the quantity of leaf the larva could eat before the toxicant decays below the lethal level and place one lethal deposit within this area. 3) Distributions of toxicant where deposits are sublethal will often be ineffective, but the application is wasteful if deposits contain more than a lethal dose. 4) Insect behavior both as individuals and collectively influences the level of crop production provided by an application. This conclusion has implications for both crop protection and natural plant-insect interactions. The effective utilization of new more environmentally sensitive toxicants may depend on how well we understand how heterogeneous toxicant distributions interact with insect behavior to determine the biological outcome.

Genetics Home Reference: oculofaciocardiodental syndrome

MedlinePlus

... the signs and symptoms of OFCD syndrome. In males (who have only one X chromosome ), mutations result ... be lethal very early in development, so no males are born with OFCD syndrome. Related Information What ...

Transcript map of the Ovum mutant (Om) locus: isolation by exon trapping of new candidate genes for the DDK syndrome.

PubMed

Le Bras, Stéphanie; Cohen-Tannoudji, Michel; Guyot, Valérie; Vandormael-Pournin, Sandrine; Coumailleau, Franck; Babinet, Charles; Baldacci, Patricia

2002-08-21

The DDK syndrome is defined as the embryonic lethality of F1 mouse embryos from crosses between DDK females and males from other strains (named hereafter as non-DDK strains). Genetically controlled by the Ovum mutant (Om) locus, it is due to a deleterious interaction between a maternal factor present in DDK oocytes and the non-DDK paternal pronucleus. Therefore, the DDK syndrome constitutes a unique genetic tool to study the crucial interactions that take place between the parental genomes and the egg cytoplasm during mammalian development. In this paper, we present an extensive analysis performed by exon trapping on the Om region. Twenty-seven trapped sequences were from genes in the databases: beta-adaptin, CCT zeta2, DNA LigaseIII, Notchless, Rad51l3 and Scya1. Twenty-eight other sequences presented similarities with expressed sequence tags and genomic sequences whereas 57 did not. The pattern of expression of 37 of these markers was established. Importantly, five of them are expressed in DDK oocytes and are candidate genes for the maternal factor, and 20 are candidate genes for the paternal factor since they are expressed in testis. This data is an important step towards identifying the genes responsible for the DDK syndrome.

Synthetic dosage lethality in the human metabolic network is highly predictive of tumor growth and cancer patient survival.

PubMed

Megchelenbrink, Wout; Katzir, Rotem; Lu, Xiaowen; Ruppin, Eytan; Notebaart, Richard A

2015-09-29

Synthetic dosage lethality (SDL) denotes a genetic interaction between two genes whereby the underexpression of gene A combined with the overexpression of gene B is lethal. SDLs offer a promising way to kill cancer cells by inhibiting the activity of SDL partners of activated oncogenes in tumors, which are often difficult to target directly. As experimental genome-wide SDL screens are still scarce, here we introduce a network-level computational modeling framework that quantitatively predicts human SDLs in metabolism. For each enzyme pair (A, B) we systematically knock out the flux through A combined with a stepwise flux increase through B and search for pairs that reduce cellular growth more than when either enzyme is perturbed individually. The predictive signal of the emerging network of 12,000 SDLs is demonstrated in five different ways. (i) It can be successfully used to predict gene essentiality in shRNA cancer cell line screens. Moving to clinical tumors, we show that (ii) SDLs are significantly underrepresented in tumors. Furthermore, breast cancer tumors with SDLs active (iii) have smaller sizes and (iv) result in increased patient survival, indicating that activation of SDLs increases cancer vulnerability. Finally, (v) patient survival improves when multiple SDLs are present, pointing to a cumulative effect. This study lays the basis for quantitative identification of cancer SDLs in a model-based mechanistic manner. The approach presented can be used to identify SDLs in species and cell types in which "omics" data necessary for data-driven identification are missing.

Antibiotics induce redox-related physiological alterations as part of their lethality

PubMed Central

Dwyer, Daniel J.; Belenky, Peter A.; Yang, Jason H.; MacDonald, I. Cody; Martell, Jeffrey D.; Takahashi, Noriko; Chan, Clement T. Y.; Lobritz, Michael A.; Braff, Dana; Schwarz, Eric G.; Ye, Jonathan D.; Pati, Mekhala; Vercruysse, Maarten; Ralifo, Paul S.; Allison, Kyle R.; Khalil, Ahmad S.; Ting, Alice Y.; Walker, Graham C.; Collins, James J.

2014-01-01

Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part by inducing production of damaging reactive species. This notion has been supported by many groups but has been challenged recently. Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular H2O2 sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species to demonstrate that antibiotics broadly induce redox stress. Subsequent gene-expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supraphysiological levels of H2O2. We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions but could be enhanced by exposure to molecular oxygen or by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality. PMID:24803433

Gene Mutations and Genomic Rearrangements in the Mouse as a Result of Transposon Mobilization from Chromosomal Concatemers

PubMed Central

Geurts, Aron M; Collier, Lara S; Geurts, Jennifer L; Oseth, Leann L; Bell, Matthew L; Mu, David; Lucito, Robert; Godbout, Susan A; Green, Laura E; Lowe, Scott W; Hirsch, Betsy A; Leinwand, Leslie A; Largaespada, David A

2006-01-01

Previous studies of the Sleeping Beauty (SB) transposon system, as an insertional mutagen in the germline of mice, have used reverse genetic approaches. These studies have led to its proposed use for regional saturation mutagenesis by taking a forward-genetic approach. Thus, we used the SB system to mutate a region of mouse Chromosome 11 in a forward-genetic screen for recessive lethal and viable phenotypes. This work represents the first reported use of an insertional mutagen in a phenotype-driven approach. The phenotype-driven approach was successful in both recovering visible and behavioral mutants, including dominant limb and recessive behavioral phenotypes, and allowing for the rapid identification of candidate gene disruptions. In addition, a high frequency of recessive lethal mutations arose as a result of genomic rearrangements near the site of transposition, resulting from transposon mobilization. The results suggest that the SB system could be used in a forward-genetic approach to recover interesting phenotypes, but that local chromosomal rearrangements should be anticipated in conjunction with single-copy, local transposon insertions in chromosomes. Additionally, these mice may serve as a model for chromosome rearrangements caused by transposable elements during the evolution of vertebrate genomes. PMID:17009875

A genetic screen for zygotic embryonic lethal mutations affecting cuticular morphology in the wasp Nasonia vitripennis.

PubMed Central

Pultz, M A; Zimmerman, K K; Alto, N M; Kaeberlein, M; Lange, S K; Pitt, J N; Reeves, N L; Zehrung, D L

2000-01-01

We have screened for zygotic embryonic lethal mutations affecting cuticular morphology in Nasonia vitripennis (Hymenoptera; Chalcidoidea). Our broad goal was to investigate the use of Nasonia for genetically surveying conservation and change in regulatory gene systems, as a means to understand the diversity of developmental strategies that have arisen during the course of evolution. Specifically, we aim to compare anteroposterior patterning gene functions in two long germ band insects, Nasonia and Drosophila. In Nasonia, unfertilized eggs develop as haploid males while fertilized eggs develop as diploid females, so the entire genome can be screened for recessive zygotic mutations by examining the progeny of F1 females. We describe 74 of >100 lines with embryonic cuticular mutant phenotypes, including representatives of coordinate, gap, pair-rule, segment polarity, homeotic, and Polycomb group functions, as well as mutants with novel phenotypes not directly comparable to those of known Drosophila genes. We conclude that Nasonia is a tractable experimental organism for comparative developmental genetic study. The mutants isolated here have begun to outline the extent of conservation and change in the genetic programs controlling embryonic patterning in Nasonia and Drosophila. PMID:10866651

Leapfrogging: primordial germ cell transplantation permits recovery of CRISPR/Cas9-induced mutations in essential genes

PubMed Central

Fish, Margaret B.; Cho, Ken W. Y.

2016-01-01

CRISPR/Cas9 genome editing is revolutionizing genetic loss-of-function analysis but technical limitations remain that slow progress when creating mutant lines. First, in conventional genetic breeding schemes, mosaic founder animals carrying mutant alleles are outcrossed to produce F1 heterozygotes. Phenotypic analysis occurs in the F2 generation following F1 intercrosses. Thus, mutant analyses will require multi-generational studies. Second, when targeting essential genes, efficient mutagenesis of founders is often lethal, preventing the acquisition of mature animals. Reducing mutagenesis levels may improve founder survival, but results in lower, more variable rates of germline transmission. Therefore, an efficient approach to study lethal mutations would be useful. To overcome these shortfalls, we introduce ‘leapfrogging’, a method combining efficient CRISPR mutagenesis with transplantation of mutated primordial germ cells into a wild-type host. Tested using Xenopus tropicalis, we show that founders containing transplants transmit mutant alleles with high efficiency. F1 offspring from intercrosses between F0 animals that carry embryonic lethal alleles recapitulate loss-of-function phenotypes, circumventing an entire generation of breeding. We anticipate that leapfrogging will be transferable to other species. PMID:27385011

Lethality of sortase depletion in Actinomyces oris caused by excessive membrane accumulation of a surface glycoprotein.

PubMed

Wu, Chenggang; Huang, I-Hsiu; Chang, Chungyu; Reardon-Robinson, Melissa Elizabeth; Das, Asis; Ton-That, Hung

2014-12-01

Sortase, a cysteine-transpeptidase conserved in Gram-positive bacteria, anchors on the cell wall many surface proteins that facilitate bacterial pathogenesis and fitness. Genetic disruption of the housekeeping sortase in several Gram-positive pathogens reported thus far attenuates virulence, but not bacterial growth. Paradoxically, we discovered that depletion of the housekeeping sortase SrtA was lethal for Actinomyces oris; yet, all of its predicted cell wall-anchored protein substrates (AcaA-N) were individually dispensable for cell viability. Using Tn5-transposon mutagenesis to identify factors that upend lethality of srtA deletion, we uncovered a set of genetic suppressors harbouring transposon insertions within genes of a locus encoding AcaC and a LytR-CpsA-Psr (LCP)-like protein. AcaC was shown to be highly glycosylated and dependent on LCP for its glycosylation. Upon SrtA depletion, the glycosylated form of AcaC, hereby renamed GspA, was accumulated in the membrane. Overexpression of GspA in a mutant lacking gspA and srtA was lethal; conversely, cells overexpressing a GspA mutant missing a membrane-localization domain were viable. The results reveal a unique glycosylation pathway in A. oris that is coupled to cell wall anchoring catalysed by sortase SrtA. Significantly, this novel phenomenon of glyco-stress provides convenient cell-based assays for developing a new class of inhibitors against Gram-positive pathogens. © 2014 John Wiley & Sons Ltd.

Germline mutations in RYR1 are associated with foetal akinesia deformation sequence/lethal multiple pterygium syndrome.

PubMed

McKie, Arthur B; Alsaedi, Atif; Vogt, Julie; Stuurman, Kyra E; Weiss, Marjan M; Shakeel, Hassan; Tee, Louise; Morgan, Neil V; Nikkels, Peter G J; van Haaften, Gijs; Park, Soo-Mi; van der Smagt, Jasper J; Bugiani, Marianna; Maher, Eamonn R

2014-12-05

Foetal akinesia deformation sequence syndrome (FADS) is a genetically heterogeneous disorder characterised by the combination of foetal akinesia and developmental defects which may include pterygia (joint webbing). Traditionally multiple pterygium syndrome (MPS) has been divided into two forms: prenatally lethal (LMPS) and non-lethal Escobar type (EVMPS) types. Interestingly, FADS, LMPS and EVMPS may be allelic e.g. each of these phenotypes may result from mutations in the foetal acetylcholine receptor gamma subunit gene (CHRNG). Many cases of FADS and MPS do not have a mutation in a known FADS/MPS gene and we undertook molecular genetic studies to identify novel causes of these phenotypes. After mapping a novel locus for FADS/LMPS to chromosome 19, we identified a homozygous null mutation in the RYR1 gene in a consanguineous kindred with recurrent LMPS pregnancies. Resequencing of RYR1 in a cohort of 66 unrelated probands with FADS/LMPS/EVMPS (36 with FADS/LMPS and 30 with EVMPS) revealed two additional homozygous mutations (in frame deletions). The overall frequency of RYR1 mutations in probands with FADS/LMPS was 8.3%. Our findings report, for the first time, a homozygous RYR1 null mutation and expand the range of RYR1-related phenotypes to include early lethal FADS/LMPS. We suggest that RYR1 mutation analysis should be performed in cases of severe FADS/LMPS even in the absence of specific histopathological indicators of RYR1-related disease.

Genetics Home Reference: multiple pterygium syndrome

MedlinePlus

... Lethal multiple pterygium syndrome Sources for This Page Cox PM, Brueton LA, Bjelogrlic P, Pomroy P, Sewry ... article on PubMed Central Morgan NV, Brueton LA, Cox P, Greally MT, Tolmie J, Pasha S, Aligianis IA, ...

Transcriptomic study on persistence and survival of Listeria monocytogenes following lethal treatment with Nisin.

PubMed

Wu, Shuyan; Yu, Pak-Lam; Wheeler, Dave; Flint, Steve

2018-06-19

The aim of this study was to determine the gene expression associated with the persistence of a Listeria monocytogenes stationary phase population when facing lethal nisin treatment METHODS: RNA Seq analysis was used for gene expression profiling of the persister cells in rich medium (persister TN) compared with untreated cells (non-persister).The results were confirmed using RT PCR. Functional genes associated with the persister populations were identified in multiple systems, such as heat shock related stress response, cell wall synthesis, ATP-binding cassette (ABC) transport system, phosphotransferase system (PTS system), and SOS/DNA repair. This study pointed to genetic regulation of persister cells exposed to lethal nisin and provides some insight into possible mechanisms of impeding bacterial persistence. Copyright © 2018. Published by Elsevier Ltd.

Bacterial Signaling Nucleotides Inhibit Yeast Cell Growth by Impacting Mitochondrial and Other Specifically Eukaryotic Functions

PubMed Central

Vergnano, Marta; Wan, Chris

2017-01-01

ABSTRACT We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP), cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA) and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR) inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection. PMID:28743817

Mutations in GLDN, Encoding Gliomedin, a Critical Component of the Nodes of Ranvier, Are Responsible for Lethal Arthrogryposis.

PubMed

Maluenda, Jérôme; Manso, Constance; Quevarec, Loic; Vivanti, Alexandre; Marguet, Florent; Gonzales, Marie; Guimiot, Fabien; Petit, Florence; Toutain, Annick; Whalen, Sandra; Grigorescu, Romulus; Coeslier, Anne Dieux; Gut, Marta; Gut, Ivo; Laquerrière, Annie; Devaux, Jérôme; Melki, Judith

2016-10-06

Arthrogryposis multiplex congenita (AMC) is a developmental condition characterized by multiple joint contractures resulting from reduced or absent fetal movements. Through linkage analysis, homozygosity mapping, and exome sequencing in four unrelated families affected by lethal AMC, we identified biallelic mutations in GLDN in the affected individuals. GLDN encodes gliomedin, a secreted cell adhesion molecule involved in the formation of the nodes of Ranvier. Transmission electron microscopy of the sciatic nerve from one of the affected individuals showed a marked lengthening defect of the nodes. The GLDN mutations found in the affected individuals abolish the cell surface localization of gliomedin and its interaction with its axonal partner, neurofascin-186 (NF186), in a cell-based assay. The axoglial contact between gliomedin and NF186 is essential for the initial clustering of Na + channels at developing nodes. These results indicate a major role of gliomedin in node formation and the development of the peripheral nervous system in humans. These data indicate that mutations of GLDN or CNTNAP1 (MIM: 616286), encoding essential components of the nodes of Ranvier and paranodes, respectively, lead to inherited nodopathies, a distinct disease entity among peripheral neuropathies. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Pharmacological targeting of MYC-regulated IRE1/XBP1 pathway suppresses MYC-driven breast cancer.

PubMed

Zhao, Na; Cao, Jin; Xu, Longyong; Tang, Qianzi; Dobrolecki, Lacey E; Lv, Xiangdong; Talukdar, Manisha; Lu, Yang; Wang, Xiaoran; Hu, Dorothy Z; Shi, Qing; Xiang, Yu; Wang, Yunfei; Liu, Xia; Bu, Wen; Jiang, Yi; Li, Mingzhou; Gong, Yingyun; Sun, Zheng; Ying, Haoqiang; Yuan, Bo; Lin, Xia; Feng, Xin-Hua; Hartig, Sean M; Li, Feng; Shen, Haifa; Chen, Yiwen; Han, Leng; Zeng, Qingping; Patterson, John B; Kaipparettu, Benny Abraham; Putluri, Nagireddy; Sicheri, Frank; Rosen, Jeffrey M; Lewis, Michael T; Chen, Xi

2018-04-02

The unfolded protein response (UPR) is a cellular homeostatic mechanism that is activated in many human cancers and plays pivotal roles in tumor progression and therapy resistance. However, the molecular mechanisms for UPR activation and regulation in cancer cells remain elusive. Here, we show that oncogenic MYC regulates the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) branch of the UPR in breast cancer via multiple mechanisms. We found that MYC directly controls IRE1 transcription by binding to its promoter and enhancer. Furthermore, MYC forms a transcriptional complex with XBP1, a target of IRE1, and enhances its transcriptional activity. Importantly, we demonstrate that XBP1 is a synthetic lethal partner of MYC. Silencing of XBP1 selectively blocked the growth of MYC-hyperactivated cells. Pharmacological inhibition of IRE1 RNase activity with small molecule inhibitor 8866 selectively restrained the MYC-overexpressing tumor growth in vivo in a cohort of preclinical patient-derived xenograft models and genetically engineered mouse models. Strikingly, 8866 substantially enhanced the efficacy of docetaxel chemotherapy, resulting in rapid regression of MYC-overexpressing tumors. Collectively, these data establish the synthetic lethal interaction of the IRE1/XBP1 pathway with MYC hyperactivation and provide a potential therapy for MYC-driven human breast cancers.

ATM loss leads to synthetic lethality in BRCA1 BRCT mutant mice associated with exacerbated defects in homology-directed repair.

PubMed

Chen, Chun-Chin; Kass, Elizabeth M; Yen, Wei-Feng; Ludwig, Thomas; Moynahan, Mary Ellen; Chaudhuri, Jayanta; Jasin, Maria

2017-07-18

BRCA1 is essential for homology-directed repair (HDR) of DNA double-strand breaks in part through antagonism of the nonhomologous end-joining factor 53BP1. The ATM kinase is involved in various aspects of DNA damage signaling and repair, but how ATM participates in HDR and genetically interacts with BRCA1 in this process is unclear. To investigate this question, we used the Brca1 S1598F mouse model carrying a mutation in the BRCA1 C-terminal domain of BRCA1. Whereas ATM loss leads to a mild HDR defect in adult somatic cells, we find that ATM inhibition leads to severely reduced HDR in Brca1 S1598F cells. Consistent with a critical role for ATM in HDR in this background, loss of ATM leads to synthetic lethality of Brca1 S1598F mice. Whereas both ATM and BRCA1 promote end resection, which can be regulated by 53BP1, 53bp1 deletion does not rescue the HDR defects of Atm mutant cells, in contrast to Brca1 mutant cells. These results demonstrate that ATM has a role in HDR independent of the BRCA1-53BP1 antagonism and that its HDR function can become critical in certain contexts.

Idiosyncratic Mòjiāng virus attachment glycoprotein directs a host-cell entry pathway distinct from genetically related henipaviruses.

PubMed

Rissanen, Ilona; Ahmed, Asim A; Azarm, Kristopher; Beaty, Shannon; Hong, Patrick; Nambulli, Sham; Duprex, W Paul; Lee, Benhur; Bowden, Thomas A

2017-07-12

In 2012, cases of lethal pneumonia among Chinese miners prompted the isolation of a rat-borne henipavirus (HNV), Mòjiāng virus (MojV). Although MojV is genetically related to highly pathogenic bat-borne henipaviruses, the absence of a conserved ephrin receptor-binding motif in the MojV attachment glycoprotein (MojV-G) indicates a differing host-cell recognition mechanism. Here we find that MojV-G displays a six-bladed β-propeller fold bearing limited similarity to known paramyxoviral attachment glycoproteins, in particular at host receptor-binding surfaces. We confirm the inability of MojV-G to interact with known paramyxoviral receptors in vitro, indicating an independence from well-characterized ephrinB2/B3, sialic acid and CD150-mediated entry pathways. Furthermore, we find that MojV-G is antigenically distinct, indicating that MojV would less likely be detected in existing large-scale serological screening studies focused on well-established HNVs. Altogether, these data indicate a unique host-cell entry pathway for this emerging and potentially pathogenic HNV.

The Yeast Environmental Stress Response Regulates Mutagenesis Induced by Proteotoxic Stress

PubMed Central

Shor, Erika; Fox, Catherine A.; Broach, James R.

2013-01-01

Conditions of chronic stress are associated with genetic instability in many organisms, but the roles of stress responses in mutagenesis have so far been elucidated only in bacteria. Here, we present data demonstrating that the environmental stress response (ESR) in yeast functions in mutagenesis induced by proteotoxic stress. We show that the drug canavanine causes proteotoxic stress, activates the ESR, and induces mutagenesis at several loci in an ESR-dependent manner. Canavanine-induced mutagenesis also involves translesion DNA polymerases Rev1 and Polζ and non-homologous end joining factor Ku. Furthermore, under conditions of chronic sub-lethal canavanine stress, deletions of Rev1, Polζ, and Ku-encoding genes exhibit genetic interactions with ESR mutants indicative of ESR regulating these mutagenic DNA repair processes. Analyses of mutagenesis induced by several different stresses showed that the ESR specifically modulates mutagenesis induced by proteotoxic stress. Together, these results document the first known example of an involvement of a eukaryotic stress response pathway in mutagenesis and have important implications for mechanisms of evolution, carcinogenesis, and emergence of drug-resistant pathogens and chemotherapy-resistant tumors. PMID:23935537

PARP inhibition as a prototype for synthetic lethal screens.

PubMed

Liu, Xuesong

2013-01-01

Although DNA damaging chemotherapy and radiation therapy remain the main stay of current treatments for cancer patient, these therapies usually have toxic side effect and narrow therapeutic window. One of the challenges in cancer drug discovery is how to identify drugs that selectively kill cancer cells while leaving the normal cell intact. Recently, synthetic lethality has been applied to cancer drug discovery in various settings, and has become a promising approach for identifying novel agents for the treatment of cancer. A prototypical example is the synthetic lethal interaction between PARP inhibition and BRCA deficiency. PARP inhibitors represent the most advanced clinical agents targeting specifically DNA repair mechanisms in cancer therapy. In this chapter, I will review the molecular mechanism for this synthetic lethality and the clinical applications for PARP inhibitors. I will also discuss the formats of synthetic lethal screens, current progress on the utilization of these screens, and some of the advantages and challenges of synthetic lethal screens in cancer drug discovery.

Reanalysis of parabiosis of obesity mutants in the age of leptin.

PubMed

Zeng, Wenwen; Lu, Yi-Hsueh; Lee, Jonah; Friedman, Jeffrey M

2015-07-21

In this study we set out to explain the differing effects of parabiosis with genetically diabetic (db) mice versus administration of recombinant leptin. Parabiosis of db mutant, which overexpress leptin, to wildtype (WT) or genetically obese (ob) mice has been reported to cause death by starvation, whereas leptin infusions do not produce lethality at any dose or mode of delivery tested. Leptin is not posttranslationally modified other than a single disulphide bond, raising the possibility that it might require additional factor(s) to exert the maximal appetite-suppressing effect. We reconfirmed the lethal effect of parabiosis of db mutant on WT mice and further showed that this lethality could not be rescued by administration of ghrelin or growth hormone. We then initiated a biochemical fractionation of a high-molecular-weight leptin complex from human plasma and identified clusterin as a major component of this leptin-containing complex. However, in contrast to previous reports, we failed to observe a leptin-potentiating effect of either exogenous or endogenous clusterin, and parabiosis of db clusterin(-/-) double-mutant to WT mice still caused lethality. Intriguingly, in parabiotic pairs of two WT mice, leptin infusion into one of the mice led to an enhanced starvation response during calorie restriction as evidenced by increased plasma ghrelin and growth-hormone levels. Moreover, leptin treatment resulted in death of the parabiotic pairs. These data suggest that the appetite suppression in WT mice after parabiosis to db mutants is the result of induced hyperleptinemia combined with the stress or other aspect(s) of the parabiosis procedure.

Reanalysis of parabiosis of obesity mutants in the age of leptin

PubMed Central

Zeng, Wenwen; Lu, Yi-Hsueh; Lee, Jonah; Friedman, Jeffrey M.

2015-01-01

In this study we set out to explain the differing effects of parabiosis with genetically diabetic (db) mice versus administration of recombinant leptin. Parabiosis of db mutant, which overexpress leptin, to wildtype (WT) or genetically obese (ob) mice has been reported to cause death by starvation, whereas leptin infusions do not produce lethality at any dose or mode of delivery tested. Leptin is not posttranslationally modified other than a single disulphide bond, raising the possibility that it might require additional factor(s) to exert the maximal appetite-suppressing effect. We reconfirmed the lethal effect of parabiosis of db mutant on WT mice and further showed that this lethality could not be rescued by administration of ghrelin or growth hormone. We then initiated a biochemical fractionation of a high-molecular-weight leptin complex from human plasma and identified clusterin as a major component of this leptin-containing complex. However, in contrast to previous reports, we failed to observe a leptin-potentiating effect of either exogenous or endogenous clusterin, and parabiosis of db clusterin−/− double-mutant to WT mice still caused lethality. Intriguingly, in parabiotic pairs of two WT mice, leptin infusion into one of the mice led to an enhanced starvation response during calorie restriction as evidenced by increased plasma ghrelin and growth-hormone levels. Moreover, leptin treatment resulted in death of the parabiotic pairs. These data suggest that the appetite suppression in WT mice after parabiosis to db mutants is the result of induced hyperleptinemia combined with the stress or other aspect(s) of the parabiosis procedure. PMID:26150485

Topology of evolving, mutagenized viral populations: quasispecies expansion, compression, and operation of negative selection.

PubMed

Ojosnegros, Samuel; Agudo, Rubén; Sierra, Macarena; Briones, Carlos; Sierra, Saleta; González-López, Claudia; Domingo, Esteban; Cristina, Juan

2008-07-17

The molecular events and evolutionary forces underlying lethal mutagenesis of virus (or virus extinction through an excess of mutations) are not well understood. Here we apply for the first time phylogenetic methods and Partition Analysis of Quasispecies (PAQ) to monitor genetic distances and intra-population structures of mutant spectra of foot-and-mouth disease virus (FMDV) quasispecies subjected to mutagenesis by base and nucleoside analogues. Phylogenetic and PAQ analyses have revealed a highly dynamic variation of intrapopulation diversity of FMDV quasispecies. The population diversity first suffers striking expansions in the presence of mutagens and then compressions either when the presence of the mutagenic analogue was discontinued or when a mutation that decreased sensitivity to a mutagen was selected. The pattern of mutations found in the populations was in agreement with the behavior of the corresponding nucleotide analogues with FMDV in vitro. Mutations accumulated at preferred genomic sites, and dn/ds ratios indicate the operation of negative (or purifying) selection in populations subjected to mutagenesis. No evidence of unusually elevated genetic distances has been obtained for FMDV populations approaching extinction. Phylogenetic and PAQ analysis provide adequate procedures to describe the evolution of viral sequences subjected to lethal mutagenesis. These methods define the changes of intra-population structure more precisely than mutation frequencies and Shannon entropies. PAQ is very sensitive to variations of intrapopulation genetic distances. Strong negative (or purifying) selection operates in FMDV populations subjected to enhanced mutagenesis. The quantifications provide evidence that extinction does not imply unusual increases of intrapopulation complexity, in support of the lethal defection model of virus extinction.

Incontinentia pigmenti

MedlinePlus

IP is caused by an X-linked dominant genetic defect that occurs on a gene known as IKBKG. Because the gene defect occurs on the X chromosome, the condition is most often seen in females. When it occurs in males, it is usually lethal.

A Plasmodium yoelii HECT-like E3 ubiquitin ligase regulates parasite growth and virulence.

PubMed

Nair, Sethu C; Xu, Ruixue; Pattaradilokrat, Sittiporn; Wu, Jian; Qi, Yanwei; Zilversmit, Martine; Ganesan, Sundar; Nagarajan, Vijayaraj; Eastman, Richard T; Orandle, Marlene S; Tan, John C; Myers, Timothy G; Liu, Shengfa; Long, Carole A; Li, Jian; Su, Xin-Zhuan

2017-08-09

Infection of mice with strains of Plasmodium yoelii parasites can result in different pathology, but molecular mechanisms to explain this variation are unclear. Here we show that a P. yoelii gene encoding a HECT-like E3 ubiquitin ligase (Pyheul) influences parasitemia and host mortality. We genetically cross two lethal parasites with distinct disease phenotypes, and identify 43 genetically diverse progeny by typing with microsatellites and 9230 single-nucleotide polymorphisms. A genome-wide quantitative trait loci scan links parasite growth and host mortality to two major loci on chromosomes 1 and 7 with LOD (logarithm of the odds) scores = 6.1 and 8.1, respectively. Allelic exchange of partial sequences of Pyheul in the chromosome 7 locus and modification of the gene expression alter parasite growth and host mortality. This study identifies a gene that may have a function in parasite growth, virulence, and host-parasite interaction, and therefore could be a target for drug or vaccine development.Many strains of Plasmodium differ in virulence, but factors that control these distinctions are not known. Here the authors comparatively map virulence loci using the offspring from a P. yoelii YM and N67 genetic cross, and identify a putative HECT E3 ubiquitin ligase that may explain the variance.

Estimating the Economic Value of Lethal Versus Nonlethal Deer Control in Suburban Communities

Treesearch

J. Michael Bowker; David H. Newman; Robert J. Warren; David W. Henderson

2003-01-01

Negative people/wildlife interaction has raised public interest in wildlife population control. We present a contingent valuation study of alternative deer control measures considered for Hilton Head Island, SC. Lethal control usig sharpshooters and nonlethal immuno-contraception techniques are evaluated. A mail-back survey was used to collect resident willingness-to-...

Cystic fibrosis genetics: from molecular understanding to clinical application.

PubMed

Cutting, Garry R

2015-01-01

The availability of the human genome sequence and tools for interrogating individual genomes provide an unprecedented opportunity to apply genetics to medicine. Mendelian conditions, which are caused by dysfunction of a single gene, offer powerful examples that illustrate how genetics can provide insights into disease. Cystic fibrosis, one of the more common lethal autosomal recessive Mendelian disorders, is presented here as an example. Recent progress in elucidating disease mechanism and causes of phenotypic variation, as well as in the development of treatments, demonstrates that genetics continues to play an important part in cystic fibrosis research 25 years after the discovery of the disease-causing gene.

Phage Lambda P Protein: Trans-Activation, Inhibition Phenotypes and their Suppression

PubMed Central

Hayes, Sidney; Erker, Craig; Horbay, Monique A.; Marciniuk, Kristen; Wang, Wen; Hayes, Connie

2013-01-01

The initiation of bacteriophage λ replication depends upon interactions between the oriλ DNA site, phage proteins O and P, and E. coli host replication proteins. P exhibits a high affinity for DnaB, the major replicative helicase for unwinding double stranded DNA. The concept of P-lethality relates to the hypothesis that P can sequester DnaB and in turn prevent cellular replication initiation from oriC. Alternatively, it was suggested that P-lethality does not involve an interaction between P and DnaB, but is targeted to DnaA. P-lethality is assessed by examining host cells for transformation by ColE1-type plasmids that can express P, and the absence of transformants is attributed to a lethal effect of P expression. The plasmid we employed enabled conditional expression of P, where under permissive conditions, cells were efficiently transformed. We observed that ColE1 replication and plasmid establishment upon transformation is extremely sensitive to P, and distinguish this effect from P-lethality directed to cells. We show that alleles of dnaB protect the variant cells from P expression. P-dependent cellular filamentation arose in ΔrecA or lexA[Ind-] cells, defective for SOS induction. Replication propagation and restart could represent additional targets for P interference of E. coli replication, beyond the oriC-dependent initiation step. PMID:23389467

Interaction of metronidazole with resistant and susceptible Bacteroides fragilis.

PubMed Central

McLafferty, M A; Koch, R L; Goldman, P

1982-01-01

The kinetics of the lethal action of metronidazole and the formation of acetamide have been studied in a strain of Bacteroides fragilis which is relatively resistant to metronidazole. As with a susceptible strain of B. fragilis, the data are consistent with a model in which a labile intermediate in metronidazole metabolism interacts either with water to form acetamide or with a bacterium to cause its death. Although the relatively resistant strain grows more slowly than the susceptible one and is killed less rapidly by metronidazole, the resistant strain displays the same relationship between the lethal action of metronidazole and metronidazole metabolism to acetamide. The relatively resistant strain, like the susceptible one, has an enhanced lethal response to metronidazole in the presence of a strain of Escherichia coli. The results suggest that the proposed labile reactive intermediate of metronidazole forms more slowly in the resistant strains. PMID:7081970

An orally available, small-molecule polymerase inhibitor shows efficacy against a lethal morbillivirus infection in a large animal model.

PubMed

Krumm, Stefanie A; Yan, Dan; Hovingh, Elise S; Evers, Taylor J; Enkirch, Theresa; Reddy, G Prabhakar; Sun, Aiming; Saindane, Manohar T; Arrendale, Richard F; Painter, George; Liotta, Dennis C; Natchus, Michael G; von Messling, Veronika; Plemper, Richard K

2014-04-16

Measles virus is a highly infectious morbillivirus responsible for major morbidity and mortality in unvaccinated humans. The related, zoonotic canine distemper virus (CDV) induces morbillivirus disease in ferrets with 100% lethality. We report an orally available, shelf-stable pan-morbillivirus inhibitor that targets the viral RNA polymerase. Prophylactic oral treatment of ferrets infected intranasally with a lethal CDV dose reduced viremia and prolonged survival. Ferrets infected with the same dose of virus that received post-infection treatment at the onset of viremia showed low-grade viral loads, remained asymptomatic, and recovered from infection, whereas control animals succumbed to the disease. Animals that recovered also mounted a robust immune response and were protected against rechallenge with a lethal CDV dose. Drug-resistant viral recombinants were generated and found to be attenuated and transmission-impaired compared to the genetic parent virus. These findings may pioneer a path toward an effective morbillivirus therapy that could aid measles eradication by synergizing with vaccination to close gaps in herd immunity due to vaccine refusal.

Atypical myxomatosis--virus isolation, experimental infection of rabbits and restriction endonuclease analysis of the isolate.

PubMed

Psikal, I; Smíd, B; Rodák, L; Valícek, L; Bendová, J

2003-08-01

Atypical form of myxomatosis, which caused non-lethal and clinically mild disease in domestic rabbits 1 month after immunization with a commercially available vaccine MXT, is described. The isolated myxoma virus designated as Litovel 2 (Li-2) did not induce systemic disease following subcutaneous and intradermal applications in susceptible experimental rabbits but led to the immune response demonstrated by ELISA. No severe disease was induced in those Li-2 inoculated rabbits by challenge with the virulent strains Lausanne (Lu) or Sanar (SA), while the control animals showed nodular form of myxomatosis with lethal course of the illness. Restriction fragment length polymorphism (RFLP) of genomic DNA with KpnI and BamHI endonucleases was used for genetic characterization of the Li-2 isolate, the vaccine strain MXT and both virulent strains Lu and SA, respectively. In general, RFLP analysis has shown to be informative for inferring genetic relatedness between myxoma viruses. Based on restriction endonuclease DNA fragment size distribution, it was evident that the pathogenic strain SA is genetically related to the reference strain Lu and the isolate Li-2 is more related, but not identical, to the vaccination strain MXT.

Origins based clinical and molecular complexities of epithelial ovarian cancer.

PubMed

Muinao, Thingreila; Pal, Mintu; Boruah, Hari Prasanna Deka

2018-06-08

Ovarian cancer is the most lethal of all common gynaecological malignancies in women worldwide. Ovarian cancer comprises of >15 distinct tumor types and subtypes characterized by histopathological features, environmental and genetic risk factors, precursor lesions and molecular events during oncogenesis. Recent studies on gene signatures profiling of different subtypes of ovarian cancer have revealed significant genetic heterogeneity between and within each ovarian cancer histological subtype. Thus, an immense interest have shown towards a more personalized medicine for understanding the clinical and molecular complexities of four major types of epithelial ovarian cancer (serous, endometrioid, clear cell, and mucinous). As such, further in depth studies are needed for identification of molecular signalling network complexities associated with effective prognostication and targeted therapies to prevent or treat metastasis. Therefore, understanding the metastatic potential of primary ovarian cancer and therapeutic interventions against lethal ovarian cancer for the development of personalized therapies is very much indispensable. Consequently, in this review we have updated the key dysregulated genes of four major subtypes of epithelial carcinomas. We have also highlighted the recent advances and current challenges in unravelling the complexities of the origin of tumor as well as genetic heterogeneity of ovarian cancer. Copyright © 2017. Published by Elsevier B.V.

Genetics Home Reference: platyspondylic lethal skeletal dysplasia, Torrance type

MedlinePlus

... type of collagen in the body. Instead of forming collagen molecules, the abnormal COL2A1 protein builds up ... Y, Nagai T, Yamaguchi T, Kosaki R, Ohashi H, Makita Y, Ikegawa S. Identification of COL2A1 mutations in ...

Imaginal Disc Abnormalities in Lethal Mutants of Drosophila

PubMed Central

Shearn, Allen; Rice, Thomas; Garen, Alan; Gehring, Walter

1971-01-01

Late lethal mutants of Drosophila melanogaster, dying after the larval stage of development, were isolated. The homozygous mutant larvae were examined for abnormal imaginal disc morphology, and the discs were injected into normal larval hosts to test their capacities to differentiate into adult structures. In about half of the mutants analyzed, disc abnormalities were found. Included among the abnormalities were missing discs, small discs incapable of differentiating, morphologically normal discs with limited capacities for differentiation, and discs with homeotic transformations. In some mutants all discs were affected, and in others only certain discs. The most extreme abnormal phenotype is a class of “discless” mutants. The viability of these mutant larvae indicates that the discs are essential only for the development of an adult and not of a larva. The late lethals are therefore a major source of mutants for studying the genetic control of disc formation. Images PMID:5002822

Synthetic Lethal Therapy for KRAS Mutant Non-small-cell Lung Carcinoma with Nanoparticle-mediated CDK4 siRNA Delivery

PubMed Central

Mao, Cheng-Qiong; Xiong, Meng-Hua; Liu, Yang; Shen, Song; Du, Xiao-Jiao; Yang, Xian-Zhu; Dou, Shuang; Zhang, Pei-Zhuo; Wang, Jun

2014-01-01

The KRAS mutation is present in ~20% of lung cancers and has not yet been effectively targeted for therapy. This mutation is associated with a poor prognosis in non-small-cell lung carcinomas (NSCLCs) and confers resistance to standard anticancer treatment drugs, including epidermal growth factor receptor tyrosine kinase inhibitors. In this study, we exploited a new therapeutic strategy based on the synthetic lethal interaction between cyclin-dependent kinase 4 (CDK4) downregulation and the KRAS mutation to deliver micellar nanoparticles (MNPs) containing small interfering RNA targeting CDK4 (MNPsiCDK4) for treatment in NSCLCs harboring the oncogenic KRAS mutation. Following MNPsiCDK4 administration, CDK4 expression was decreased, accompanied by inhibited cell proliferation, specifically in KRAS mutant NSCLCs. However, this intervention was harmless to normal KRAS wild-type cells, confirming the proposed mechanism of synthetic lethality. Moreover, systemic delivery of MNPsiCDK4 significantly inhibited tumor growth in an A549 NSCLC xenograft murine model, with depressed expression of CDK4 and mutational KRAS status, suggesting the therapeutic promise of MNPsiCDK4 delivery in KRAS mutant NSCLCs via a synthetic lethal interaction between KRAS and CDK4. PMID:24496383

Sequence editing by Apolipoprotein B RNA-editing catalytic component-B and epidemiological surveillance of transmitted HIV-1 drug resistance

PubMed Central

Gifford, Robert J.; Rhee, Soo-Yon; Eriksson, Nicolas; Liu, Tommy F.; Kiuchi, Mark; Das, Amar K.; Shafer, Robert W.

2008-01-01

Design Promiscuous guanine (G) to adenine (A) substitutions catalysed by apolipoprotein B RNA-editing catalytic component (APOBEC) enzymes are observed in a proportion of HIV-1 sequences in vivo and can introduce artifacts into some genetic analyses. The potential impact of undetected lethal editing on genotypic estimation of transmitted drug resistance was assessed. Methods Classifiers of lethal, APOBEC-mediated editing were developed by analysis of lentiviral pol gene sequence variation and evaluated using control sets of HIV-1 sequences. The potential impact of sequence editing on genotypic estimation of drug resistance was assessed in sets of sequences obtained from 77 studies of 25 or more therapy-naive individuals, using mixture modelling approaches to determine the maximum likelihood classification of sequences as lethally edited as opposed to viable. Results Analysis of 6437 protease and reverse transcriptase sequences from therapy-naive individuals using a novel classifier of lethal, APOBEC3G-mediated sequence editing, the polypeptide-like 3G (APOBEC3G)-mediated defectives (A3GD) index’, detected lethal editing in association with spurious ‘transmitted drug resistance’ in nearly 3% of proviral sequences obtained from whole blood and 0.2% of samples obtained from plasma. Conclusion Screening for lethally edited sequences in datasets containing a proportion of proviral DNA, such as those likely to be obtained for epidemiological surveillance of transmitted drug resistance in the developing world, can eliminate rare but potentially significant errors in genotypic estimation of transmitted drug resistance. PMID:18356601

Topology of evolving, mutagenized viral populations: quasispecies expansion, compression, and operation of negative selection

PubMed Central

2008-01-01

Background The molecular events and evolutionary forces underlying lethal mutagenesis of virus (or virus extinction through an excess of mutations) are not well understood. Here we apply for the first time phylogenetic methods and Partition Analysis of Quasispecies (PAQ) to monitor genetic distances and intra-population structures of mutant spectra of foot-and-mouth disease virus (FMDV) quasispecies subjected to mutagenesis by base and nucleoside analogues. Results Phylogenetic and PAQ analyses have revealed a highly dynamic variation of intrapopulation diversity of FMDV quasispecies. The population diversity first suffers striking expansions in the presence of mutagens and then compressions either when the presence of the mutagenic analogue was discontinued or when a mutation that decreased sensitivity to a mutagen was selected. The pattern of mutations found in the populations was in agreement with the behavior of the corresponding nucleotide analogues with FMDV in vitro. Mutations accumulated at preferred genomic sites, and dn/ds ratios indicate the operation of negative (or purifying) selection in populations subjected to mutagenesis. No evidence of unusually elevated genetic distances has been obtained for FMDV populations approaching extinction. Conclusion Phylogenetic and PAQ analysis provide adequate procedures to describe the evolution of viral sequences subjected to lethal mutagenesis. These methods define the changes of intra-population structure more precisely than mutation frequencies and Shannon entropies. PAQ is very sensitive to variations of intrapopulation genetic distances. Strong negative (or purifying) selection operates in FMDV populations subjected to enhanced mutagenesis. The quantifications provide evidence that extinction does not imply unusual increases of intrapopulation complexity, in support of the lethal defection model of virus extinction. PMID:18637173

Advances in Skeletal Dysplasia Genetics

PubMed Central

Geister, Krista A.; Camper, Sally A.

2017-01-01

Skeletal dysplasias result from disruptions in normal skeletal growth and development and are a major contributor to severe short stature. They occur in approximately 1/5,000 births, and some are lethal. Since the most recent publication of the Nosology and Classification of Genetic Skeletal Disorders, genetic causes of 56 skeletal disorders have been uncovered. This remarkable rate of discovery is largely due to the expanded use of high-throughput genomic technologies. In this review, we discuss these recent discoveries and our understanding of the molecular mechanisms behind these skeletal dysplasia phenotypes. We also cover potential therapies, unusual genetic mechanisms, and novel skeletal syndromes both with and without known genetic causes. The acceleration of skeletal dysplasia genetics is truly spectacular, and these advances hold great promise for diagnostics, risk prediction, and therapeutic design. PMID:25939055

Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis.

PubMed Central

Schnorr, J D; Holdcraft, R; Chevalier, B; Berg, C A

2001-01-01

Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype. PMID:11606538

Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis.

PubMed

Schnorr, J D; Holdcraft, R; Chevalier, B; Berg, C A

2001-10-01

Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype.

RADIATION-INDUCED GENETIC DAMAGE IN THE MEXICAN TOAD (BUFO VALLICEPS)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blair, W.F.

1960-10-01

Lines of Mexican toads (Bufo valliceps) bearing x-ray induced genetic damage were established by mating normal females with males that had received gonadal x-ray doses ranging from 300 to 3000 r. Survival in the first generation was inversely proportional to dose,-as was expected. Toads of the 300-r and l000- r lines were inbred, and toads of these lines and of the 700-r line were outcrossed to normal ones. Two crosses were made between toads of the 500-r and 1000-r lines. Developmental abnormalities of various kinds appeared at life history stages rangthg from early embryonic development to post-metamorphic life in bothmore » inbred and outcross generations. These included abnormal gastrulation and neurulation, larval and post-metamorphic edema, abnormally positioned or missing limbs, optical deficiencies, prognathous jaw due to excessive elongation of the lower jaw, and melanin deficiency. The prognathous jaw, in its extreme expression, would probably be lethal in natural populations because of difficulty of feeding. The melanin deficiency, in its extreme expression, is lethal as metamorphosis fails to occur, and in lesser expression, it appears to be lethal or detrimental. The various abnormalities do not appear to be inherited in any simple way, but instead they vary in expression both within and between generations, possibly in relation to genotype and environment. (auth)« less

Nuclear pore proteins are involved in the biogenesis of functional tRNA.

PubMed

Simos, G; Tekotte, H; Grosjean, H; Segref, A; Sharma, K; Tollervey, D; Hurt, E C

1996-05-01

Los1p and Pus1p, which are involved in tRNA biogenesis, were found in a genetic screen for components interacting with the nuclear pore protein Nsp1p. LOS1, PUS1 and NSP1 interact functionally, since the combination of mutations in the three genes causes synthetic lethality. Pus1p is an intranuclear protein which exhibits a nucleotide-specific and intron-dependent tRNA pseudouridine synthase activity. Los1p was shown previously to be required for efficient pre-tRNA splicing; we report here that Los1p localizes to the nuclear pores and is linked functionally to several components of the tRNA biogenesis machinery including Pus1p and Tfc4p. When the formation of functional tRNA was analyzed by an in vivo assay, the los1(-) pus1(-) double mutant, as well as several thermosensitive nucleoporin mutants including nsp1, nup116, nup133 and nup85, exhibited loss of suppressor tRNA activity even at permissive temperatures. These data suggest that nuclear pore proteins are required for the biogenesis of functional tRNA.

Functional analyses of interacting factors involved in both pre-mRNA splicing and cell cycle progression in Saccharomyces cerevisiae.

PubMed Central

Russell, C S; Ben-Yehuda, S; Dix, I; Kupiec, M; Beggs, J D

2000-01-01

Through a genetic screen to search for factors that interact with Prp17/Cdc40p, a protein involved in both cell cycle progression and pre-mRNA splicing, we identify three novel factors, which we call Syf1p, Syf2p, and Syf3 (SYnthetic lethal with cdc Forty). Here we present evidence that all three proteins are spliceosome associated, that they associate weakly or transiently with U6 and U5 snRNAs, and that Syf1p and Syf3p (also known as Clf1p) are required for pre-mRNA splicing. In addition we show that depletion of Syf1p or Syf3p results in cell cycle arrest at the G2/M transition. Thus, like Prp17/Cdc40p, Syf1p and Syf3p are involved in two distinct cellular processes. We discuss the likelihood that Syf1p, Syf2p, and Syf3p are components of a protein complex that assembles into spliceosomes and also regulates cell cycle progression. PMID:11105756

Functional analyses of interacting factors involved in both pre-mRNA splicing and cell cycle progression in Saccharomyces cerevisiae.

PubMed

Russell, C S; Ben-Yehuda, S; Dix, I; Kupiec, M; Beggs, J D

2000-11-01

Through a genetic screen to search for factors that interact with Prp17/Cdc40p, a protein involved in both cell cycle progression and pre-mRNA splicing, we identify three novel factors, which we call Syf1p, Syf2p, and Syf3 (SYnthetic lethal with cdc Forty). Here we present evidence that all three proteins are spliceosome associated, that they associate weakly or transiently with U6 and U5 snRNAs, and that Syf1p and Syf3p (also known as Clf1p) are required for pre-mRNA splicing. In addition we show that depletion of Syf1p or Syf3p results in cell cycle arrest at the G2/M transition. Thus, like Prp17/Cdc40p, Syf1p and Syf3p are involved in two distinct cellular processes. We discuss the likelihood that Syf1p, Syf2p, and Syf3p are components of a protein complex that assembles into spliceosomes and also regulates cell cycle progression.

Transcriptomic analysis of inbreeding depression in cold-sensitive Drosophila melanogaster shows upregulation of the immune response.

PubMed

Vermeulen, C J; Sørensen, P; Kirilova Gagalova, K; Loeschcke, V

2013-09-01

In sexually reproducing species, increased homozygosity often causes a decline in fitness, called inbreeding depression. Recently, researchers started describing the functional genomic changes that occur during inbreeding, both in benign conditions and under environmental stress. To further this aim, we have performed a genome-wide gene expression study of inbreeding depression, manifesting as cold sensitivity and conditional lethality. Our focus was to describe general patterns of gene expression during inbreeding depression and to identify specific processes affected in our line. There was a clear difference in gene expression between the stressful restrictive environment and the benign permissive environment in both the affected inbred line and the inbred control line. We noted a strong inbreeding-by-environment interaction, whereby virtually all transcriptional differences between lines were found in the restrictive environment. Functional annotation showed enrichment of transcripts coding for serine proteases and their inhibitors (serpins and BPTI/Kunitz family), which indicates activation of the innate immune response. These genes have previously been shown to respond transcriptionally to cold stress, suggesting the conditional lethal effect is associated with an exaggerated cold stress response. The set of differentially expressed genes significantly overlapped with those found in three other studies of inbreeding depression, demonstrating that it is possible to detect a common signature across different genetic backgrounds. © 2013 The Authors. Journal of Evolutionary Biology © 2013 European Society For Evolutionary Biology.

ATM loss leads to synthetic lethality in BRCA1 BRCT mutant mice associated with exacerbated defects in homology-directed repair

PubMed Central

Chen, Chun-Chin; Kass, Elizabeth M.; Yen, Wei-Feng; Ludwig, Thomas; Moynahan, Mary Ellen; Chaudhuri, Jayanta; Jasin, Maria

2017-01-01

BRCA1 is essential for homology-directed repair (HDR) of DNA double-strand breaks in part through antagonism of the nonhomologous end-joining factor 53BP1. The ATM kinase is involved in various aspects of DNA damage signaling and repair, but how ATM participates in HDR and genetically interacts with BRCA1 in this process is unclear. To investigate this question, we used the Brca1S1598F mouse model carrying a mutation in the BRCA1 C-terminal domain of BRCA1. Whereas ATM loss leads to a mild HDR defect in adult somatic cells, we find that ATM inhibition leads to severely reduced HDR in Brca1S1598F cells. Consistent with a critical role for ATM in HDR in this background, loss of ATM leads to synthetic lethality of Brca1S1598F mice. Whereas both ATM and BRCA1 promote end resection, which can be regulated by 53BP1, 53bp1 deletion does not rescue the HDR defects of Atm mutant cells, in contrast to Brca1 mutant cells. These results demonstrate that ATM has a role in HDR independent of the BRCA1–53BP1 antagonism and that its HDR function can become critical in certain contexts. PMID:28659469

Sex-Specific Sub-Lethal Effects and Immune Response in Ceratitis capitata Wied. (Diptera: Tephritidae) Challenged with Spinosad.

PubMed

Mura, Maria Elena; Ruiu, Luca

2018-06-21

The main objective of this study was to investigate the effects of the insecticidal compound spinosad on the survival, reproduction, and immune functions of the Mediterranean fruit fly. The lethal and sub-lethal effects were determined on Ceratitis capitata Wied. (Diptera: Tephritidae) challenged with different concentrations of spinosad. A median lethal concentration of 0.28 ppm was observed on flies feeding for 5 days on a treated diet. A significant and concentration-dependent decrease in fecundity, egg hatch rate, and lifespan was also detected in treated compared with control flies. Gene expression analyses conducted on treated insects by RT-qPCR revealed an immunomodulatory action of sub-lethal concentrations of spinosad. Target transcripts included several genes involved in medfly immunity and male or female reproductive functions. While a significant upregulation was detected in treated males a short time after spinosad ingestion, most target genes were downregulated in treated females. Our study confirmed the high toxicity of spinosad to C. capitata , highlighting an indirect effect on insect lifespan and reproductive performance at sub-lethal doses. In addition to defining the acute and sub-lethal toxicity of spinosad against the fly, this study provides new insights on the interaction of this compound with insect physiology.

Sec34p, a Protein Required for Vesicle Tethering to the Yeast Golgi Apparatus, Is in a Complex with Sec35p

PubMed Central

VanRheenen, Susan M.; Cao, Xiaochun; Sapperstein, Stephanie K.; Chiang, Elbert C.; Lupashin, Vladimir V.; Barlowe, Charles; Waters, M. Gerard

1999-01-01

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393–406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)–associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an ∼750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well. PMID:10562277

Synthetic Lethality Reveals Mechanisms of Mycobacterium tuberculosis Resistance to β-Lactams

PubMed Central

Lun, Shichun; Miranda, David; Kubler, Andre; Guo, Haidan; Maiga, Mariama C.; Winglee, Kathryn; Pelly, Shaaretha

2014-01-01

ABSTRACT Most β-lactam antibiotics are ineffective against Mycobacterium tuberculosis due to the microbe’s innate resistance. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has prompted interest to repurpose this class of drugs. To identify the genetic determinants of innate β-lactam resistance, we carried out a synthetic lethality screen on a transposon mutant library for susceptibility to imipenem, a carbapenem β-lactam antibiotic. Mutations in 74 unique genes demonstrated synthetic lethality. The majority of mutations were in genes associated with cell wall biosynthesis. A second quantitative real-time PCR (qPCR)-based synthetic lethality screen of randomly selected mutants confirmed the role of cell wall biosynthesis in β-lactam resistance. The global transcriptional response of the bacterium to β-lactams was investigated, and changes in levels of expression of cell wall biosynthetic genes were identified. Finally, we validated these screens in vivo using the MT1616 transposon mutant, which lacks a functional acyl-transferase gene. Mice infected with the mutant responded to β-lactam treatment with a 100-fold decrease in bacillary lung burden over 4 weeks, while the numbers of organisms in the lungs of mice infected with wild-type bacilli proliferated. These findings reveal a road map of genes required for β-lactam resistance and validate synthetic lethality screening as a promising tool for repurposing existing classes of licensed, safe, well-characterized antimicrobials against tuberculosis. PMID:25227469

Genetic control of cuticle formation during embryonic development of Drosophila melanogaster.

PubMed Central

Ostrowski, Stephen; Dierick, Herman A; Bejsovec, Amy

2002-01-01

The embryonic cuticle of Drosophila melanogaster is deposited by the epidermal epithelium during stage 16 of development. This tough, waterproof layer is essential for maintaining the structural integrity of the larval body. We have characterized mutations in a set of genes required for proper deposition and/or morphogenesis of the cuticle. Zygotic disruption of any one of these genes results in embryonic lethality. Mutant embryos are hyperactive within the eggshell, resulting in a high proportion reversed within the eggshell (the "retroactive" phenotype), and all show poor cuticle integrity when embryos are mechanically devitellinized. This last property results in embryonic cuticle preparations that appear grossly inflated compared to wild-type cuticles (the "blimp" phenotype). We find that one of these genes, krotzkopf verkehrt (kkv), encodes the Drosophila chitin synthase enzyme and that a closely linked gene, knickkopf (knk), encodes a novel protein that shows genetic interaction with the Drosophila E-cadherin, shotgun. We also demonstrate that two other known mutants, grainy head (grh) and retroactive (rtv), show the blimp phenotype when devitellinized, and we describe a new mutation, called zeppelin (zep), that shows the blimp phenotype but does not produce defects in the head cuticle as the other mutations do. PMID:12019232

A Common Suite of Coagulation Proteins Function in Drosophila Muscle Attachment

PubMed Central

Green, Nicole; Odell, Nadia; Zych, Molly; Clark, Cheryl; Wang, Zong-Heng; Biersmith, Bridget; Bajzek, Clara; Cook, Kevin R.; Dushay, Mitchell S.; Geisbrecht, Erika R.

2016-01-01

The organization and stability of higher order structures that form in the extracellular matrix (ECM) to mediate the attachment of muscles are poorly understood. We have made the surprising discovery that a subset of clotting factor proteins are also essential for muscle attachment in the model organism Drosophila melanogaster. One such coagulation protein, Fondue (Fon), was identified as a novel muscle mutant in a pupal lethal genetic screen. Fon accumulates at muscle attachment sites and removal of this protein results in decreased locomotor behavior and detached larval muscles. A sensitized genetic background assay reveals that fon functions with the known muscle attachment genes Thrombospondin (Tsp) and Tiggrin (Tig). Interestingly, Tig is also a component of the hemolymph clot. We further demonstrate that an additional clotting protein, Larval serum protein 1γ (Lsp1γ), is also required for muscle attachment stability and accumulates where muscles attach to tendons. While the local biomechanical and organizational properties of the ECM vary greatly depending on the tissue microenvironment, we propose that shared extracellular protein–protein interactions influence the strength and elasticity of ECM proteins in both coagulation and muscle attachment. PMID:27585844

Identification of Novel, Inherited Genetic Markers for Aggressive PCa in European and African Americans Using Whole Genome Sequencing

DTIC Science & Technology

2013-09-01

DATES COVERED (From - To) 22 August 2012 – 21 August 2013 4. TITLE AND SUBTITLE Identification of Novel, Inherited Genetic Markers for Aggressive... Inherited markers of aggressive PCa could be used for screening and diagnosis of aggressive PCa at an early stage while reducing over-diagnosis and...treatment for others. The overall hypothesis is that inherited sequence variants in the genome are associated with a lethal (aggressive) form of PCa but not

Bacterial Signaling Nucleotides Inhibit Yeast Cell Growth by Impacting Mitochondrial and Other Specifically Eukaryotic Functions.

PubMed

Hesketh, Andy; Vergnano, Marta; Wan, Chris; Oliver, Stephen G

2017-07-25

We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP), cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA) and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR) inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection. IMPORTANCE During infections, pathogenic bacteria can release nucleotides into the cells of their eukaryotic hosts. These nucleotides are recognized as signals that contribute to the initiation of defensive immune responses that help the infected cells recover. Despite the importance of this process, the broader impact of bacterial nucleotides on the functioning of eukaryotic cells remains poorly defined. To address this, we genetically modified cells of the eukaryote Saccharomyces cerevisiae (baker's yeast) to produce three of these molecules (cdiAMP, cdiGMP, and ppGpp) and used the engineered strains as model systems to characterize the effects of the molecules on the cells. In addition to demonstrating that the nucleotides are each capable of adversely affecting yeast cell function and growth, we also identified the cellular functions important for mitigating the damage caused, suggesting possible modes of action. This study expands our understanding of the molecular interactions that can take place between bacterial and eukaryotic cells. Copyright © 2017 Hesketh et al.

Prediction of protein-peptide interactions: application of the XPairIt API to anthrax lethal factor and substrates

NASA Astrophysics Data System (ADS)

Hurley, Margaret M.; Sellers, Michael S.

2013-05-01

As software and methodology develop, key aspects of molecular interactions such as detailed energetics and flexibility are continuously better represented in docking simulations. In the latest iteration of the XPairIt API and Docking Protocol, we perform a blind dock of a peptide into the cleavage site of the Anthrax lethal factor (LF) metalloprotein. Molecular structures are prepared from RCSB:1JKY and we demonstrate a reasonably accurate docked peptide through analysis of protein motion and, using NCI Plot, visualize and characterize the forces leading to binding. We compare our docked structure to the 1JKY crystal structure and the more recent 1PWV structure, and discuss both captured and overlooked interactions. Our results offer a more detailed look at secondary contact and show that both van der Waals and electrostatic interactions from peptide residues further from the enzyme's catalytic site are significant.

Genetic doping and health damages.

PubMed

Fallahi, Aa; Ravasi, Aa; Farhud, Dd

2011-01-01

Use of genetic doping or gene transfer technology will be the newest and the lethal method of doping in future and have some unpleasant consequences for sports, athletes, and outcomes of competitions. The World Anti-Doping Agency (WADA) defines genetic doping as "the non-therapeutic use of genes, genetic elements, and/or cells that have the capacity to enhance athletic performance ". The purpose of this review is to consider genetic doping, health damages and risks of new genes if delivered in athletes. This review, which is carried out by reviewing relevant publications, is primarily based on the journals available in GOOGLE, ELSEVIER, PUBMED in fields of genetic technology, and health using a combination of keywords (e.g., genetic doping, genes, exercise, performance, athletes) until July 2010. There are several genes related to sport performance and if they are used, they will have health risks and sever damages such as cancer, autoimmunization, and heart attack.

Genetic Doping and Health Damages

PubMed Central

Fallahi, AA; Ravasi, AA; Farhud, DD

2011-01-01

Background: Use of genetic doping or gene transfer technology will be the newest and the lethal method of doping in future and have some unpleasant consequences for sports, athletes, and outcomes of competitions. The World Anti-Doping Agency (WADA) defines genetic doping as “the non-therapeutic use of genes, genetic elements, and/or cells that have the capacity to enhance athletic performance ”. The purpose of this review is to consider genetic doping, health damages and risks of new genes if delivered in athletes. Methods: This review, which is carried out by reviewing relevant publications, is primarily based on the journals available in GOOGLE, ELSEVIER, PUBMED in fields of genetic technology, and health using a combination of keywords (e.g., genetic doping, genes, exercise, performance, athletes) until July 2010. Conclusion: There are several genes related to sport performance and if they are used, they will have health risks and sever damages such as cancer, autoimmunization, and heart attack. PMID:23113049

Improved mutagen-testing systems in mice: Progress report, June 1, 1988--May 31, 1989

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roderick, T.H.

1989-01-01

In December we injected 20 mice with 250 mg/kg of ENU. Half of the mice were males of C57BL/6J and half were males of strains AEJ/GnRk. The strains were chosen to include a standard widely used strain (C57BL/6J) but also to include a strain (AEJ/GnRk) that has an unusually large litter size (mean = 10) to promote better survival of embryos with induced recessives. Sentinal females are in with the treated males to determine return to fertility of the injected males. All males will be mated with the new lethal test system. Lethal No. 1 has now been clearly determinedmore » to be just distal to the distal breakpoint of inversion In(1)1Rk. All other lethals, including the new lethal No. 8 are crossed with our specially constructed stocks for linkage analysis. Also, the lethals are being crossed with known recessive markers along the inverted segment to determine possible allelism. All lethals have been intercrossed to determine whether they complement each other. Wild type animals should not be recovered if the lethals are overlapping. In a new study we are examining the wild type animals from each of these crosses carefully for general phenotypic conformation, eye disorders, feet, body weight and gait. Although these lethals may be complementary, it is possible that some interactive effects could be produced in an animal doubly heterozygous for different deletions.« less

Mechanism of Ovarian Epithelial Tumor Predispostion in Individuals Carrying Germline BRCA1 Mutations

DTIC Science & Technology

2005-01-01

corresponds to the luteal phase. Vaginal smears obtained at the diestrus phase show primarily inflammatory cells. Immature (green) epithelial cells start...Koller, B.H. (1996). BRCA1 deficiency results in early embryonic lethality characterized by neuroepithelial abnormalities. Nat. Genet. 12, 191-194. 22

Unlocking the Bottleneck in Forward Genetics Using Whole-Genome Sequencing and Identity by Descent to Isolate Causative Mutations

PubMed Central

Siggs, Owen M.; Miosge, Lisa A.; Roots, Carla M.; Enders, Anselm; Bertram, Edward M.; Crockford, Tanya L.; Whittle, Belinda; Potter, Paul K.; Simon, Michelle M.; Mallon, Ann-Marie; Brown, Steve D. M.; Beutler, Bruce; Goodnow, Christopher C.; Lunter, Gerton; Cornall, Richard J.

2013-01-01

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis. PMID:23382690

Genetically obese (ob/ob) mice are resistant to the lethal effects of thioacetamide hepatotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Won, Young-Suk; Song, Ji-Won; Lim, Jong-Hwan

Obesity increases the risk of chronic liver diseases, including viral hepatitis, alcohol-induced liver disease, and non-alcoholic steatohepatitis. In this study, we investigated the effects of obesity in acute hepatic failure using a murine model of thioacetamide (TA)-induced liver injury. Genetically obese ob/ob mice, together with non-obese ob/+ littermates, were subjected to a single intraperitoneal injection of TA, and examined for signs of hepatic injury. ob/ob mice showed a significantly higher survival rate, lower levels of serum alanine aminotransferase and aspartate aminotransferase, and less hepatic necrosis and apoptosis, compared with ob/+ mice. In addition, ob/ob mice exhibited significantly lower levels ofmore » malondialdehyde and significantly higher levels of glutathione and antioxidant enzyme activities compared with their ob/+ counterparts. Bioactivation analyses revealed reduced plasma clearance of TA and covalent binding of [{sup 14}C]TA to liver macromolecules in ob/ob mice. Together, these data demonstrate that genetically obese mice are resistant to TA-induced acute liver injury through diminished bioactivation of TA and antioxidant effects. - Highlights: • ob/ob mice are resistant to lethal doses of thioacetamide, compared to ob/+ mice. • ob/ob mice show reduced oxidative stress and enhanced antioxidant enzyme activity. • ob/ob mice exhibit diminished bioactivation of thioacetamide.« less

Sudden cardiac death: the pro-arrhythmic interaction of an acute loading with an underlying substrate.

PubMed

Sutherland, George R

2017-10-21

Sudden cardiac death (SCD) is a complex phenomenon, occurring either in apparently normal individuals or in those where there is a recognized underlying cardiac abnormality. In both groups, the lethal arrhythmia has frequently been related to the physiologic trigger of either exercise or stress. Prior research into SCD has focused mainly on a combination of identifying either vulnerable myocardial substrates; pharmacological approaches to altering electrical activation/repolarisation in substrates; or the suppression of induced lethal arrhythmias with implantable defibrillators. However, it has been suggested that in a significant number of cases, the interaction of a transient induced trigger with a pre-existing electrical or mechanical substrate is the basis for the induction of the sustained lethal arrhythmia. In this manuscript we will discuss the precise mechanisms whereby one of such potential physiologic trigger: an acute change in systolic blood pressure, can induce a sequence of alterations in global and local cardiac mechanics which in turn result in regional left ventricular post-systolic deformation which, mediated (through stretch-induced changes in local mechano-electrical coupling) provokes local electrical after-depolarisations which can spill over into complex runs of premature ventricular beats. These local acute pressure/stretch induced runs of ventricular ectopy originate in either basal or apical normal myocardium and, in combination with a co-existing distal pro-arrhymic substrate, can interact to induce a lethal arrhythmia. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For Permissions, please email: journals.permissions@oup.com.

Genetic architecture of a feeding adaptation: garter snake (Thamnophis) resistance to tetrodotoxin bearing prey.

PubMed

Feldman, Chris R; Brodie, Edmund D; Brodie, Edmund D; Pfrender, Michael E

2010-11-07

Detailing the genetic basis of adaptive variation in natural populations is a first step towards understanding the process of adaptive evolution, yet few ecologically relevant traits have been characterized at the genetic level in wild populations. Traits that mediate coevolutionary interactions between species are ideal for studying adaptation because of the intensity of selection and the well-characterized ecological context. We have previously described the ecological context, evolutionary history and partial genetic basis of tetrodotoxin (TTX) resistance in garter snakes (Thamnophis). Derived mutations in a voltage-gated sodium channel gene (Na(v)1.4) in three garter snake species are associated with resistance to TTX, the lethal neurotoxin found in their newt prey (Taricha). Here we evaluate the contribution of Na(v)1.4 alleles to TTX resistance in two of those species from central coastal California. We measured the phenotypes (TTX resistance) and genotypes (Na(v)1.4 and microsatellites) in a local sample of Thamnophis atratus and Thamnophis sirtalis. Allelic variation in Na(v)1.4 explains 23 per cent of the variation in TTX resistance in T. atratus while variation in a haphazard sample of the genome (neutral microsatellite markers) shows no association with the phenotype. Similarly, allelic variation in Na(v)1.4 correlates almost perfectly with TTX resistance in T. sirtalis, but neutral variation does not. These strong correlations suggest that Na(v)1.4 is a major effect locus. The simple genetic architecture of TTX resistance in garter snakes may significantly impact the dynamics of phenotypic coevolution. Fixation of a few alleles of major effect in some garter snake populations may have led to the evolution of extreme phenotypes and an 'escape' from the arms race with newts.

Soluble factor(s) from bone marrow cells can rescue lethally irradiated mice by protecting endogenous hematopoietic stem cells.

PubMed

Zhao, Yi; Zhan, Yuxia; Burke, Kathleen A; Anderson, W French

2005-04-01

Ionizing radiation-induced myeloablation can be rescued via bone marrow transplantation (BMT) or administration of cytokines if given within 2 hours after radiation exposure. There is no evidence for the existence of soluble factors that can rescue an animal after a lethal dose of radiation when administered several hours postradiation. We established a system that could test the possibility for the existence of soluble factors that could be used more than 2 hours postirradiation to rescue animals. Animals with an implanted TheraCyte immunoisolation device (TID) received lethal-dose radiation and then normal bone marrow Lin- cells were loaded into the device (thereby preventing direct interaction between donor and recipient cells). Animal survival was evaluated and stem cell activity was tested with secondary bone marrow transplantation and flow cytometry analysis. Donor cell gene expression of five antiapoptotic cytokines was examined. Bone marrow Lin- cells rescued lethally irradiated animals via soluble factor(s). Bone marrow cells from the rescued animals can rescue and repopulate secondary lethally irradiated animals. Within the first 6 hours post-lethal-dose radiation, there is no significant change of gene expression of the known radioprotective factors TPO, SCF, IL-3, Flt-3 ligand, and SDF-1. Hematopoietic stem cells can be protected in lethally irradiated animals by soluble factors produced by bone marrow Lin- cells.

A Model Framework to Estimate Impact and Cost of Genetics-Based Sterile Insect Methods for Dengue Vector Control

PubMed Central

Alphey, Nina; Alphey, Luke; Bonsall, Michael B.

2011-01-01

Vector-borne diseases impose enormous health and economic burdens and additional methods to control vector populations are clearly needed. The Sterile Insect Technique (SIT) has been successful against agricultural pests, but is not in large-scale use for suppressing or eliminating mosquito populations. Genetic RIDL technology (Release of Insects carrying a Dominant Lethal) is a proposed modification that involves releasing insects that are homozygous for a repressible dominant lethal genetic construct rather than being sterilized by irradiation, and could potentially overcome some technical difficulties with the conventional SIT technology. Using the arboviral disease dengue as an example, we combine vector population dynamics and epidemiological models to explore the effect of a program of RIDL releases on disease transmission. We use these to derive a preliminary estimate of the potential cost-effectiveness of vector control by applying estimates of the costs of SIT. We predict that this genetic control strategy could eliminate dengue rapidly from a human community, and at lower expense (approximately US$ 2∼30 per case averted) than the direct and indirect costs of disease (mean US$ 86–190 per case of dengue). The theoretical framework has wider potential use; by appropriately adapting or replacing each component of the framework (entomological, epidemiological, vector control bio-economics and health economics), it could be applied to other vector-borne diseases or vector control strategies and extended to include other health interventions. PMID:21998654

CYP2C9*3 polymorphism presenting as lethal subdural hematoma with low-dose warfarin

PubMed Central

Karnik, Niteen D.; Sridharan, Kannan; Tiwari, D.; Gupta, V.

2014-01-01

Warfarin is the most common and cheap oral anticoagulant currently used in clinical practice. A high inter-individual variation is seen in the response to warfarin. Recently, pharmacogenetics has gained importance in managing patients on warfarin, both in predicting the optimum required dose as well as in decreasing the risk of bleeding. This case report is a description of a 49-year-old patient who had a lethal subdural hematoma with low-dose warfarin. He was subsequently found to have CYP2C9 gene polymorphism (*1/*3). This case report stresses the importance of pre-prescription assessment of genetic analysis for those initiated on warfarin. PMID:25298588

RIPK3 Mediates Necroptosis during Embryonic Development and Postnatal Inflammation in Fadd-Deficient Mice.

PubMed

Zhao, Qun; Yu, XianJun; Zhang, HaiWei; Liu, YongBo; Zhang, XiXi; Wu, XiaoXia; Xie, Qun; Li, Ming; Ying, Hao; Zhang, Haibing

2017-04-25

RIPK3 mediates cell death and regulates inflammatory responses. Although genetic studies have suggested that RIPK3-MLKL-mediated necroptosis leads to embryonic lethality in Fadd or Caspase-8-deficient mice, the exact mechanisms are not fully understood. Here, we generated Ripk3 mutant mice by altering the RIPK3 kinase domain (Ripk3 Δ/Δ mice), thus abolishing its kinase activity. Ripk3 Δ/Δ cells were resistant to necroptosis stimulation in vitro, and Ripk3 Δ/Δ mice were protected from necroptotic diseases. Although the Ripk3 Δ/Δ mutation rescued embryonic lethality in Fadd -/- embryos, Fadd -/- Ripk3 Δ/Δ mice died within 1 day after birth due to massive inflammation. These results indicate that Ripk3 ablation rescues embryonic lethality in Fadd-deficient mice by suppressing two RIPK3-mediating processes: necroptosis during embryogenesis and inflammation during postnatal development in Fadd -/- mice. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Altered trophoblast proliferation is insufficient to account for placental dysfunction in Egfr null embryos

PubMed Central

Dackor, J.; Strunk, K. E.; Wehmeyer, M. M.; Threadgill, D. W.

2007-01-01

Homozygosity for the Egfrtm1Mag null allele in mice leads to genetic background dependent placental abnormalities and embryonic lethality. Molecular mechanisms or genetic modifiers that differentiate strains with surviving versus non-surviving Egfr nullizygous embryos have yet to be identified. Egfr transcripts in wildtype placenta was quantified by ribonuclease protection assay (RPA) and the lowest level of Egfr mRNA expression was found to coincide with Egfrtm1Mag homozygous lethality. Immunohistochemical analysis of ERBB family receptors, ERBB2, ERBB3, and ERBB4, showed similar expression between Egfr wildtype and null placentas indicating that Egfr null trophoblast do not up-regulate these receptors to compensate for EGFR deficiency. Significantly fewer numbers of bromodeoxyuridine (BrdU) positive trophoblast were observed in Egfr nullizygous placentas and Cdc25a and Myc, genes associated with proliferation, were significantly down-regulated in null placentas. However, strains with both mild and severe placental phenotypes exhibit reduced proliferation suggesting that this defect alone does not account for strain-specific embryonic lethality. Consistent with this hypothesis, intercrosses generating mice null for cell cycle checkpoint genes (Trp53, Rb1, Cdkn1a, Cdkn1b or Cdkn2c) in combination with Egfr deficiency did not increase survival of Egfr nullizygous embryos. Since complete development of the spongiotrophoblast compartment is not required for survival of Egfr nullizygous embryos, reduction of this layer that is commonly observed in Egfr nullizygous placentas likely accounts for the decrease in proliferation. PMID:17822758

Chitin synthase III: synthetic lethal mutants and "stress related" chitin synthesis that bypasses the CSD3/CHS6 localization pathway.

PubMed

Osmond, B C; Specht, C A; Robbins, P W

1999-09-28

We screened Saccharomyces strains for mutants that are synthetically lethal with deletion of the major chitin synthase gene CHS3. In addition to finding, not surprisingly, that mutations in major cell wall-related genes such as FKS1 (glucan synthase) and mutations in any of the Golgi glycosylation complex genes (MNN9 family) are lethal in combination with chs3Delta, we found that a mutation in Srv2p, a bifunctional regulatory gene, is notably lethal in the chs3 deletion. In extending studies of fks1-chitin synthase 3 interactions, we made the surprising discovery that deletion of CSD3/CHS6, a gene normally required for Chs3p delivery and activity in vivo, was not lethal with fks1 and, in fact, that lack of Csd3p/Chs6p did not decrease the high level of stress-related chitin made in the fks1 mutant. This finding suggests that "stress response" chitin synthesis proceeds through an alternate Chs3p targeting pathway.

Chitin synthase III: Synthetic lethal mutants and “stress related” chitin synthesis that bypasses the CSD3/CHS6 localization pathway

PubMed Central

Osmond, Barbara C.; Specht, Charles A.; Robbins, Phillips W.

1999-01-01

We screened Saccharomyces strains for mutants that are synthetically lethal with deletion of the major chitin synthase gene CHS3. In addition to finding, not surprisingly, that mutations in major cell wall-related genes such as FKS1 (glucan synthase) and mutations in any of the Golgi glycosylation complex genes (MNN9 family) are lethal in combination with chs3Δ, we found that a mutation in Srv2p, a bifunctional regulatory gene, is notably lethal in the chs3 deletion. In extending studies of fks1-chitin synthase 3 interactions, we made the surprising discovery that deletion of CSD3/CHS6, a gene normally required for Chs3p delivery and activity in vivo, was not lethal with fks1 and, in fact, that lack of Csd3p/Chs6p did not decrease the high level of stress-related chitin made in the fks1 mutant. This finding suggests that “stress response” chitin synthesis proceeds through an alternate Chs3p targeting pathway. PMID:10500155

The lethal interaction of x ray and penicillin induced lesions following x-irradiation of Escherichia coli B/r in the presence of hypoxic cell sensitizers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gillies, N.E.; Obioha, F.I.

When Escherichia coli B/r were x-irradiated under anoxia in the presence of different electron-affinic sensitizers and then incubated in broth containing penicillin (at a concentration that did not kill unirradiated cells) additional killing of the bacteria occurred provided the sensitizers were of relatively high lipophilicity. The overall effect was to increase the efficiency of these sensitizers. It is concluded that sensitizer-dependent latent radiation lesions(s) are produced in membrane components of the cell envelope that interact with damage caused by penicillin in the peptidoglycan layer and this causes the additional lethality.

Saturation Mutagenesis of 5S rRNA in Saccharomyces cerevisiae

PubMed Central

Smith, Maria W.; Meskauskas, Arturas; Wang, Pinger; Sergiev, Petr V.; Dinman, Jonathan D.

2001-01-01

rRNAs are the central players in the reactions catalyzed by ribosomes, and the individual rRNAs are actively involved in different ribosome functions. Our previous demonstration that yeast 5S rRNA mutants (called mof9) can impact translational reading frame maintenance showed an unexpected function for this ubiquitous biomolecule. At the time, however, the highly repetitive nature of the genes encoding rRNAs precluded more detailed genetic and molecular analyses. A new genetic system allows all 5S rRNAs in the cell to be transcribed from a small, easily manipulated plasmid. The system is also amenable for the study of the other rRNAs, and provides an ideal genetic platform for detailed structural and functional studies. Saturation mutagenesis reveals regions of 5S rRNA that are required for cell viability, translational accuracy, and virus propagation. Unexpectedly, very few lethal alleles were identified, demonstrating the resilience of this molecule. Superimposition of genetic phenotypes on a physical map of 5S rRNA reveals the existence of phenotypic clusters of mutants, suggesting that specific regions of 5S rRNA are important for specific functions. Mapping these mutants onto the Haloarcula marismortui large subunit reveals that these clusters occur at important points of physical interaction between 5S rRNA and the different functional centers of the ribosome. Our analyses lead us to propose that one of the major functions of 5S rRNA may be to enhance translational fidelity by acting as a physical transducer of information between all of the different functional centers of the ribosome. PMID:11713264

Genetic structure of Cantharellus formosus populations in a second-growth temperate rain forest of the Pacific Northwest

USGS Publications Warehouse

Redman, Regina S.; Ranson, Judith; Rodriguez, Rusty J.

2006-01-01

Cantharellus formosus growing on the Olympic Peninsula of the Pacific Northwest was sampled from September – November 1995 for genetic analysis. A total of ninety-six basidiomes from five clusters separated from one another by 3 - 25 meters were genetically characterized by PCR analysis of 13 arbitrary loci and rDNA sequences. The number of basidiomes in each cluster varied from 15 to 25 and genetic analysis delineated 15 genets among the clusters. Analysis of variance utilizing thirteen apPCR generated genetic molecular markers and PCR amplification of the ribosomal ITS regions indicated that 81.41% of the genetic variation occurred between clusters and 18.59% within clusters. Proximity of the basidiomes within a cluster was not an indicator of genotypic similarity. The molecular profiles of each cluster were distinct and defined as unique populations containing 2 - 6 genets. The monitoring and analysis of this species through non-lethal sampling and future applications is discussed.

Drugging the Cancers Addicted to DNA Repair.

PubMed

Nickoloff, Jac A; Jones, Dennie; Lee, Suk-Hee; Williamson, Elizabeth A; Hromas, Robert

2017-11-01

Defects in DNA repair can result in oncogenic genomic instability. Cancers occurring from DNA repair defects were once thought to be limited to rare inherited mutations (such as BRCA1 or 2). It now appears that a clinically significant fraction of cancers have acquired DNA repair defects. DNA repair pathways operate in related networks, and cancers arising from loss of one DNA repair component typically become addicted to other repair pathways to survive and proliferate. Drug inhibition of the rescue repair pathway prevents the repair-deficient cancer cell from replicating, causing apoptosis (termed synthetic lethality). However, the selective pressure of inhibiting the rescue repair pathway can generate further mutations that confer resistance to the synthetic lethal drugs. Many such drugs currently in clinical use inhibit PARP1, a repair component to which cancers arising from inherited BRCA1 or 2 mutations become addicted. It is now clear that drugs inducing synthetic lethality may also be therapeutic in cancers with acquired DNA repair defects, which would markedly broaden their applicability beyond treatment of cancers with inherited DNA repair defects. Here we review how each DNA repair pathway can be attacked therapeutically and evaluate DNA repair components as potential drug targets to induce synthetic lethality. Clinical use of drugs targeting DNA repair will markedly increase when functional and genetic loss of repair components are consistently identified. In addition, future therapies will exploit artificial synthetic lethality, where complementary DNA repair pathways are targeted simultaneously in cancers without DNA repair defects. © The Author 2017. Published by Oxford University Press.

A non-lethal means to identify spermiating D&B strain of blue catfish, Ictalurus furcatus

USDA-ARS?s Scientific Manuscript database

US farm-raised catfish production was 334 million pounds in 2013, the first annual increase documented in a decade. This positive development is due to the increased adoption of channel x blue hybrid catfish in the industry. Research and development efforts to improve the genetics of channel catfi...

Deletion of the thymidine kinase gene induces complete attenuation of the Georgia isolate of African swine fever virus

USDA-ARS?s Scientific Manuscript database

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs. There are no vaccines to control Africa swine fever (ASF). Experimental vaccines have been developed using genetically modified live attenuated ASFVs obtained by specifically de...

Lethal effects of Clostridium perfringens epsilon toxin are potentiated by alpha and perfringolysin-O toxins in a mouse model.

PubMed

Fernandez-Miyakawa, Mariano E; Jost, B Helen; Billington, Stephen J; Uzal, Francisco A

2008-03-18

Epsilon toxin (ETX) is the most important virulence factor of Clostridium perfringens type D. Two other important toxins, alpha toxin (CPA) and perfringolysin-O (PFO), are encoded and potentially produced by most C. perfringens type D isolates. The biological effects of these toxins are dissimilar although they are all lethal. Since the possible interaction of these toxins during infection is unknown, the effects of CPA and PFO on the lethal activity of ETX were studied in a mouse model. Mice were injected intravenously or intragastrically with CPA or PFO with or without ETX. Sublethal doses of CPA or PFO did not affect the lethality of ETX when either was injected together with the latter intravenously. However, sublethal or lethal doses of CPA or PFO resulted in reduction of the survival time of mice injected simultaneously with ETX when compared with the intravenous effect of ETX injected alone. When PFO was inoculated intragastrically with ETX, a reduction of the survival time was observed. CPA did not alter the survival time when inoculated intragastrically with ETX. The results of the present study suggest that both CPA and PFO have the potential to enhance the ETX lethal effects during enterotoxemia in natural hosts such as sheep and goats.

MLKL and FADD Are Critical for Suppressing Progressive Lymphoproliferative Disease and Activating the NLRP3 Inflammasome.

PubMed

Zhang, Xixi; Fan, Cunxian; Zhang, Haiwei; Zhao, Qun; Liu, Yongbo; Xu, Chengxian; Xie, Qun; Wu, Xiaoxia; Yu, Xianjun; Zhang, Jianke; Zhang, Haibing

2016-09-20

MLKL, a key component downstream of RIPK3, is suggested to be a terminal executor of necroptosis. Genetic studies have revealed that Ripk3 ablation rescues embryonic lethality in Fadd- or Caspase-8-deficient mice. Given that RIPK3 has also been implicated in non-necroptotic pathways including apoptosis and inflammatory signaling, it remains unclear whether the lethality in Fadd(-/-) mice is indeed caused by necropotosis. Here, we show that genetic deletion of Mlkl rescues the developmental defect in Fadd-deficient mice and that Fadd(-/-)Mlkl(-/-) mice are viable and fertile. Mlkl(-/-)Fadd(-/-) mice display significantly accelerated lymphoproliferative disease characterized by lymphadenopathy and splenomegaly when compared to Ripk3(-/-)Fadd(-/-) mice. Mlkl(-/-)Fadd(-/-) bone-marrow-derived macrophages and dendritic cells have impaired NLRP3 inflammasome activation associated with defects in ASC speck formation and NF-κB-dependent NLRP3 transcription. Our findings reveal that MLKL and FADD play critical roles in preventing lymphoproliferative disease and activating the NLRP3 inflammasome. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Field and laboratory studies reveal interacting effects of stream oxygenation and warming on aquatic ectotherms.

PubMed

Verberk, Wilco C E P; Durance, Isabelle; Vaughan, Ian P; Ormerod, Steve J

2016-05-01

Aquatic ecological responses to climatic warming are complicated by interactions between thermal effects and other environmental stressors such as organic pollution and hypoxia. Laboratory experiments have demonstrated how oxygen limitation can set heat tolerance for some aquatic ectotherms, but only at unrealistic lethal temperatures and without field data to assess whether oxygen shortages might also underlie sublethal warming effects. Here, we test whether oxygen availability affects both lethal and nonlethal impacts of warming on two widespread Eurasian mayflies, Ephemera danica, Müller 1764 and Serratella ignita (Poda 1761). Mayfly nymphs are often a dominant component of the invertebrate assemblage in streams, and play a vital role in aquatic and riparian food webs. In the laboratory, lethal impacts of warming were assessed under three oxygen conditions. In the field, effects of oxygen availability on nonlethal impacts of warming were assessed from mayfly occurrence in 42 293 UK stream samples where water temperature and biochemical oxygen demand were measured. Oxygen limitation affected both lethal and sublethal impacts of warming in each species. Hypoxia lowered lethal limits by 5.5 °C (±2.13) and 8.2 °C (±0.62) for E. danica and S. ignita respectively. Field data confirmed the importance of oxygen limitation in warmer waters; poor oxygenation drastically reduced site occupancy, and reductions were especially pronounced under warm water conditions. Consequently, poor oxygenation lowered optimal stream temperatures for both species. The broad concordance shown here between laboratory results and extensive field data suggests that oxygen limitation not only impairs survival at thermal extremes but also restricts species abundance in the field at temperatures well below upper lethal limits. Stream oxygenation could thus control the vulnerability of aquatic ectotherms to global warming. Improving water oxygenation and reducing pollution can provide key facets of climate change adaptation for running waters. © 2016 The Authors. Global Change Biology Published by John Wiley & Sons Ltd.

P-Element Insertion Alleles of Essential Genes on the Third Chromosome of Drosophila Melanogaster: Correlation of Physical and Cytogenetic Maps in Chromosomal Region 86e-87f

PubMed Central

Deak, P.; Omar, M. M.; Saunders, RDC.; Pal, M.; Komonyi, O.; Szidonya, J.; Maroy, P.; Zhang, Y.; Ashburner, M.; Benos, P.; Savakis, C.; Siden-Kiamos, I.; Louis, C.; Bolshakov, V. N.; Kafatos, F. C.; Madueno, E.; Modolell, J.; Glover, D. M.

1997-01-01

We have established a collection of 2460 lethal or semi-lethal mutant lines using a procedure thought to insert single P elements into vital genes on the third chromosome of Drosophila melanogaster. More than 1200 randomly selected lines were examined by in situ hybridization and 90% found to contain single insertions at sites that mark 89% of all lettered subdivisions of the Bridges' map. A set of chromosomal deficiencies that collectively uncover ~25% of the euchromatin of chromosome 3 reveal lethal mutations in 468 lines corresponding to 145 complementation groups. We undertook a detailed analysis of the cytogenetic interval 86E-87F and identified 87 P-element-induced mutations falling into 38 complementation groups, 16 of which correspond to previously known genes. Twenty-one of these 38 complementation groups have at least one allele that has a P-element insertion at a position consistent with the cytogenetics of the locus. We have rescued P elements and flanking chromosomal sequences from the 86E-87F region in 35 lines with either lethal or genetically silent P insertions, and used these as probes to identify cosmids and P1 clones from the Drosophila genome projects. This has tied together the physical and genetic maps and has linked 44 previously identified cosmid contigs into seven ``supercontigs'' that span the interval. STS data for sequences flanking one side of the P-element insertions in 49 lines has identified insertions in the αγ element at 87C, two known transposable elements, and the open reading frames of seven putative single copy genes. These correspond to five known genes in this interval, and two genes identified by the homology of their predicted products to known proteins from other organisms. PMID:9409831

Lethality of mice bearing a knockout of the Ngly1-gene is partially rescued by the additional deletion of the Engase gene

PubMed Central

Fujihira, Haruhiko; Masahara-Negishi, Yuki; Tamura, Masaru; Huang, Chengcheng; Harada, Yoichiro; Wakana, Shigeharu; Takakura, Daisuke; Kawasaki, Nana; Taniguchi, Naoyuki; Kondoh, Gen; Yamashita, Tadashi; Funakoshi, Yoko; Suzuki, Tadashi

2017-01-01

The cytoplasmic peptide:N-glycanase (Ngly1 in mammals) is a de-N-glycosylating enzyme that is highly conserved among eukaryotes. It was recently reported that subjects harboring mutations in the NGLY1 gene exhibited severe systemic symptoms (NGLY1-deficiency). While the enzyme obviously has a critical role in mammals, its precise function remains unclear. In this study, we analyzed Ngly1-deficient mice and found that they are embryonic lethal in C57BL/6 background. Surprisingly, the additional deletion of the gene encoding endo-β-N-acetylglucosaminidase (Engase), which is another de-N-glycosylating enzyme but leaves a single GlcNAc at glycosylated Asn residues, resulted in the partial rescue of the lethality of the Ngly1-deficient mice. Additionally, we also found that a change in the genetic background of C57BL/6 mice, produced by crossing the mice with an outbred mouse strain (ICR) could partially rescue the embryonic lethality of Ngly1-deficient mice. Viable Ngly1-deficient mice in a C57BL/6 and ICR mixed background, however, showed a very severe phenotype reminiscent of the symptoms of NGLY1-deficiency subjects. Again, many of those defects were strongly suppressed by the additional deletion of Engase in the C57BL/6 and ICR mixed background. The defects observed in Ngly1/Engase-deficient mice (C57BL/6 background) and Ngly1-deficient mice (C57BL/6 and ICR mixed background) closely resembled some of the symptoms of patients with an NGLY1-deficiency. These observations strongly suggest that the Ngly1- or Ngly1/Engase-deficient mice could serve as a valuable animal model for studies related to the pathogenesis of the NGLY1-deficiency, and that cytoplasmic ENGase represents one of the potential therapeutic targets for this genetic disorder. PMID:28426790

Comparison of cellular lethality in DNA repair-proficient or -deficient cell lines resulting from exposure to 70 MeV/n protons or 290 MeV/n carbon ions.

PubMed

Genet, Stefan C; Maeda, Junko; Fujisawa, Hiroshi; Yurkon, Charles R; Fujii, Yoshihiro; Romero, Ashley M; Genik, Paula C; Fujimori, Akira; Kitamura, Hisashi; Kato, Takamitsu A

2012-11-01

Charged particle therapy utilizing protons or carbon ions has been rapidly intensifying over recent years. The present study was designed to jointly investigate these two charged particle treatment modalities with respect to modeled anatomical depth-dependent dose and linear energy transfer (LET) deliveries to cells with either normal or compromised DNA repair phenotypes. We compared cellular lethality in response to dose, LET and Bragg peak location for accelerated protons and carbon ions at 70 and 290 MeV/n, respectively. A novel experimental live cell irradiation OptiCell™ in vitro culture system using three different Chinese hamster ovary (CHO) cells as a mammalian model was conducted. A wild-type DNA repair-competent CHO cell line (CHO 10B2) was compared to two other CHO cell lines (51D1 and xrs5), each genetically deficient with respect to one of the two major DNA repair pathways (homologous recombination and non-homologous end joining pathways, respectively) following genotoxic insults. We found that wild-type and homologous recombination-deficient (Rad51D) cellular lethality was dependent on both the dose and LET of the carbon ions, whereas it was only dependent on dose for protons. The non-homologous end joining deficient cell line (Ku80 mutant) showed nearly identical dose-response profiles for both carbon ions and protons. Our results show that the increasingly used modality of carbon ions as charged particle therapy is advantageous to protons in a radiotherapeutic context, primarily for tumor cells proficient in non-homologous end joining DNA repair where cellular lethality is dependent not only on the dose as in the case of more common photon therapeutic modalities, but more importantly on the carbon ion LETs. Genetic characterization of patient tumors would be key to individualize and optimize the selection of radiation modality, clinical outcome and treatment cost.

Utilization of a quantitative mammalian cell mutation system, CHO/HGPRT, in experimental mutagenesis and genetic toxicology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsie, A. W.; Couch, D. B.; O'Neill, J. P.

1977-01-01

Development of the CHO/HGPRT system is described and a host-mediated CHO/HGPRT assay is discussed. The following topics are discussed: evidence for the genetic origin of mutation induction in the CHO/HGPRT system; dose-response relationship for EMS-mediated mutation induction and cell lethality; apparent dosimetry of EMS-induced mutagenesis; structure-activity relationship of alkylating agents and ICR compounds; mutagenicity and cytotoxicity of congeners of two classes of nitrosi compounds; and preliminary validation of the CHO/HGPRT assay in predicting chemical carcinogenicity. (HLW)

A Type I Signal Peptidase Is Required for Pilus Assembly in the Gram-Positive, Biofilm-Forming Bacterium Actinomyces oris

PubMed Central

Siegel, Sara D.

2016-01-01

ABSTRACT The Gram-positive bacterium Actinomyces oris, a key colonizer in the development of oral biofilms, contains 18 LPXTG motif-containing proteins, including fimbrillins that constitute two fimbrial types critical for adherence, biofilm formation, and polymicrobial interactions. Export of these protein precursors, which harbor a signal peptide, is thought to be mediated by the Sec machine and require cleavage of the signal peptide by type I signal peptidases (SPases). Like many Gram-positive bacteria, A. oris expresses two SPases, named LepB1 and LepB2. The latter has been linked to suppression of lethal “glyco-stress,” caused by membrane accumulation of the LPXTG motif-containing glycoprotein GspA when the housekeeping sortase srtA is genetically disrupted. Consistent with this finding, we show here that a mutant lacking lepB2 and srtA was unable to produce high levels of glycosylated GspA and hence was viable. However, deletion of neither lepB1 nor lepB2 abrogated the signal peptide cleavage and glycosylation of GspA, indicating redundancy of SPases for GspA. In contrast, the lepB2 deletion mutant failed to assemble the wild-type levels of type 1 and 2 fimbriae, which are built by the shaft fimbrillins FimP and FimA, respectively; this phenotype was attributed to aberrant cleavage of the fimbrillin signal peptides. Furthermore, the lepB2 mutants, including the catalytically inactive S101A and K169A variants, exhibited significant defects in polymicrobial interactions and biofilm formation. Conversely, lepB1 was dispensable for the aforementioned processes. These results support the idea that LepB2 is specifically utilized for processing of fimbrial proteins, thus providing an experimental model with which to study the basis of type I SPase specificity. IMPORTANCE Sec-mediated translocation of bacterial protein precursors across the cytoplasmic membrane involves cleavage of their signal peptide by a signal peptidase (SPase). Like many Gram-positive bacteria, A. oris expresses two SPases, LepB1 and LepB2. The latter is a genetic suppressor of lethal “glyco-stress” caused by membrane accumulation of glycosylated GspA when the housekeeping sortase srtA is genetically disrupted. We show here that LepB1 and LepB2 are capable of processing GspA, whereas only LepB2 is required for cleavage of fimbrial signal peptides. This is the first example of a type I SPase dedicated to LPXTG motif-containing fimbrial proteins. Thus, A. oris provides an experimental model with which to investigate the specificity mechanism of type I SPases. PMID:27215787

A Type I Signal Peptidase Is Required for Pilus Assembly in the Gram-Positive, Biofilm-Forming Bacterium Actinomyces oris.

PubMed

Siegel, Sara D; Wu, Chenggang; Ton-That, Hung

2016-08-01

The Gram-positive bacterium Actinomyces oris, a key colonizer in the development of oral biofilms, contains 18 LPXTG motif-containing proteins, including fimbrillins that constitute two fimbrial types critical for adherence, biofilm formation, and polymicrobial interactions. Export of these protein precursors, which harbor a signal peptide, is thought to be mediated by the Sec machine and require cleavage of the signal peptide by type I signal peptidases (SPases). Like many Gram-positive bacteria, A. oris expresses two SPases, named LepB1 and LepB2. The latter has been linked to suppression of lethal "glyco-stress," caused by membrane accumulation of the LPXTG motif-containing glycoprotein GspA when the housekeeping sortase srtA is genetically disrupted. Consistent with this finding, we show here that a mutant lacking lepB2 and srtA was unable to produce high levels of glycosylated GspA and hence was viable. However, deletion of neither lepB1 nor lepB2 abrogated the signal peptide cleavage and glycosylation of GspA, indicating redundancy of SPases for GspA. In contrast, the lepB2 deletion mutant failed to assemble the wild-type levels of type 1 and 2 fimbriae, which are built by the shaft fimbrillins FimP and FimA, respectively; this phenotype was attributed to aberrant cleavage of the fimbrillin signal peptides. Furthermore, the lepB2 mutants, including the catalytically inactive S101A and K169A variants, exhibited significant defects in polymicrobial interactions and biofilm formation. Conversely, lepB1 was dispensable for the aforementioned processes. These results support the idea that LepB2 is specifically utilized for processing of fimbrial proteins, thus providing an experimental model with which to study the basis of type I SPase specificity. Sec-mediated translocation of bacterial protein precursors across the cytoplasmic membrane involves cleavage of their signal peptide by a signal peptidase (SPase). Like many Gram-positive bacteria, A. oris expresses two SPases, LepB1 and LepB2. The latter is a genetic suppressor of lethal "glyco-stress" caused by membrane accumulation of glycosylated GspA when the housekeeping sortase srtA is genetically disrupted. We show here that LepB1 and LepB2 are capable of processing GspA, whereas only LepB2 is required for cleavage of fimbrial signal peptides. This is the first example of a type I SPase dedicated to LPXTG motif-containing fimbrial proteins. Thus, A. oris provides an experimental model with which to investigate the specificity mechanism of type I SPases. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Dominant Drop mutants are gain-of-function alleles of the muscle segment homeobox gene (msh) whose overexpression leads to the arrest of eye development.

PubMed

Mozer, B A

2001-05-15

Dominant Drop (Dr) mutations are nearly eyeless and have additional recessive phenotypes including lethality and patterning defects in eye and sensory bristles due to cis-regulatory lesions in the cell cycle regulator string (stg). Genetic analysis demonstrates that the dominant small eye phenotype is the result of separate gain-of-function mutations in the closely linked muscle segment homeobox (msh) gene, encoding a homeodomain transcription factor required for patterning of muscle and nervous system. Reversion of the Dr(Mio) allele was coincident with the generation of lethal loss-of-function mutations in msh in cis, suggesting that the dominant eye phenotype is the result of ectopic expression. Molecular genetic analysis revealed that two dominant Dr alleles contain lesions upstream of the msh transcription start site. In the Dr(Mio) mutant, a 3S18 retrotransposon insertion is the target of second-site mutations (P-element insertions or deletions) which suppress the dominant eye phenotype following reversion. The pattern of 3S18 expression and the absence of msh in eye imaginal discs suggest that transcriptional activation of the msh promoter accounts for ectopic expression. Dr dominant mutations arrest eye development by blocking the progression of the morphogenetic furrow leading to photoreceptor cell loss via apoptosis. Gal4-mediated ubiquitous expression of msh in third-instar larvae was sufficient to arrest the morphogenetic furrow in the eye imaginal disc and resulted in lethality prior to eclosion. Dominant mutations in the human msx2 gene, one of the vertebrate homologs of msh, are associated with craniosynostosis, a disease affecting cranial development. The Dr mutations are the first example of gain-of-function mutations in the msh/msx gene family identified in a genetically tractible model organism and may serve as a useful tool to identify additional genes that regulate this class of homeodomain proteins. Copyright 2001 Academic Press.

Opposing roles of p38 and JNK in a Drosophila model of TDP-43 proteinopathy reveal oxidative stress and innate immunity as pathogenic components of neurodegeneration.

PubMed

Zhan, Lihong; Xie, Qijing; Tibbetts, Randal S

2015-02-01

Pathological aggregation and mutation of the 43-kDa TAR DNA-binding protein (TDP-43) are strongly implicated in the pathogenesis amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 neurotoxicity has been extensively modeled in mice, zebrafish, Caenorhabditis elegans and Drosophila, where selective expression of TDP-43 in motoneurons led to paralysis and premature lethality. Through a genetic screen aimed to identify genetic modifiers of TDP-43, we found that the Drosophila dual leucine kinase Wallenda (Wnd) and its downstream kinases JNK and p38 influenced TDP-43 neurotoxicity. Reducing Wnd gene dosage or overexpressing its antagonist highwire partially rescued TDP-43-associated premature lethality. Downstream of Wnd, the JNK and p38 kinases played opposing roles in TDP-43-associated neurodegeneration. LOF alleles of the p38b gene as well as p38 inhibitors diminished TDP-43-associated premature lethality, whereas p38b GOF caused phenotypic worsening. In stark contrast, disruptive alleles of Basket (Bsk), the Drosophila homologue of JNK, exacerbated longevity shortening, whereas overexpression of Bsk extended lifespan. Among possible mechanisms, we found motoneuron-directed expression of TDP-43 elicited oxidative stress and innate immune gene activation that were exacerbated by p38 GOF and Bsk LOF, respectively. A key pathologic role for innate immunity in TDP-43-associated neurodegeneration was further supported by the finding that genetic suppression of the Toll/Dif and Imd/Relish inflammatory pathways dramatically extended lifespan of TDP-43 transgenic flies. We propose that oxidative stress and neuroinflammation are intrinsic components of TDP-43-associated neurodegeneration and that the balance between cytoprotective JNK and cytotoxic p38 signaling dictates phenotypic outcome to TDP-43 expression in Drosophila. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Cationic lipid-assisted polymeric nanoparticle mediated GATA2 siRNA delivery for synthetic lethal therapy of KRAS mutant non-small-cell lung carcinoma.

PubMed

Shen, Song; Mao, Chong-Qiong; Yang, Xian-Zhu; Du, Xiao-Jiao; Liu, Yang; Zhu, Yan-Hua; Wang, Jun

2014-08-04

Synthetic lethal interaction provides a conceptual framework for the development of wiser cancer therapeutics. In this study, we exploited a therapeutic strategy based on the interaction between GATA binding protein 2 (GATA2) downregulation and the KRAS mutation status by delivering small interfering RNA targeting GATA2 (siGATA2) with cationic lipid-assisted polymeric nanoparticles for treatment of non-small-cell lung carcinoma (NSCLC) harboring oncogenic KRAS mutations. Nanoparticles carrying siGATA2 (NPsiGATA2) were effectively taken up by NSCLC cells and resulted in targeted gene suppression. NPsiGATA2 selectively inhibited cell proliferation and induced cell apoptosis in KRAS mutant NSCLC cells. However, this intervention was harmless to normal KRAS wild-type NSCLC cells and HL7702 hepatocytes, confirming the advantage of synthetic lethality-based therapy. Moreover, systemic delivery of NPsiGATA2 significantly inhibited tumor growth in the KRAS mutant A549 NSCLC xenograft murine model, suggesting the therapeutic promise of NPsiGATA2 delivery in KRAS mutant NSCLC therapy.

A combined parasitological molecular approach for noninvasive characterization of parasitic nematode communities in wild hosts.

PubMed

Budischak, Sarah A; Hoberg, Eric P; Abrams, Art; Jolles, Anna E; Ezenwa, Vanessa O

2015-09-01

Most hosts are concurrently or sequentially infected with multiple parasites; thus, fully understanding interactions between individual parasite species and their hosts depends on accurate characterization of the parasite community. For parasitic nematodes, noninvasive methods for obtaining quantitative, species-specific infection data in wildlife are often unreliable. Consequently, characterization of gastrointestinal nematode communities of wild hosts has largely relied on lethal sampling to isolate and enumerate adult worms directly from the tissues of dead hosts. The necessity of lethal sampling severely restricts the host species that can be studied, the adequacy of sample sizes to assess diversity, the geographic scope of collections and the research questions that can be addressed. Focusing on gastrointestinal nematodes of wild African buffalo, we evaluated whether accurate characterization of nematode communities could be made using a noninvasive technique that combined conventional parasitological approaches with molecular barcoding. To establish the reliability of this new method, we compared estimates of gastrointestinal nematode abundance, prevalence, richness and community composition derived from lethal sampling with estimates derived from our noninvasive approach. Our noninvasive technique accurately estimated total and species-specific worm abundances, as well as worm prevalence and community composition when compared to the lethal sampling method. Importantly, the rate of parasite species discovery was similar for both methods, and only a modest number of barcoded larvae (n = 10) were needed to capture key aspects of parasite community composition. Overall, this new noninvasive strategy offers numerous advantages over lethal sampling methods for studying nematode-host interactions in wildlife and can readily be applied to a range of study systems. © 2015 John Wiley & Sons Ltd.

Nuclear pore proteins are involved in the biogenesis of functional tRNA.

PubMed Central

Simos, G; Tekotte, H; Grosjean, H; Segref, A; Sharma, K; Tollervey, D; Hurt, E C

1996-01-01

Los1p and Pus1p, which are involved in tRNA biogenesis, were found in a genetic screen for components interacting with the nuclear pore protein Nsp1p. LOS1, PUS1 and NSP1 interact functionally, since the combination of mutations in the three genes causes synthetic lethality. Pus1p is an intranuclear protein which exhibits a nucleotide-specific and intron-dependent tRNA pseudouridine synthase activity. Los1p was shown previously to be required for efficient pre-tRNA splicing; we report here that Los1p localizes to the nuclear pores and is linked functionally to several components of the tRNA biogenesis machinery including Pus1p and Tfc4p. When the formation of functional tRNA was analyzed by an in vivo assay, the los1(-) pus1(-) double mutant, as well as several thermosensitive nucleoporin mutants including nsp1, nup116, nup133 and nup85, exhibited loss of suppressor tRNA activity even at permissive temperatures. These data suggest that nuclear pore proteins are required for the biogenesis of functional tRNA. Images PMID:8641292

K-RasV14I recapitulates Noonan syndrome in mice

PubMed Central

Hernández-Porras, Isabel; Fabbiano, Salvatore; Schuhmacher, Alberto J.; Aicher, Alexandra; Cañamero, Marta; Cámara, Juan Antonio; Cussó, Lorena; Desco, Manuel; Heeschen, Christopher; Mulero, Francisca; Bustelo, Xosé R.; Guerra, Carmen; Barbacid, Mariano

2014-01-01

Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. NS also is associated with a risk for developing myeloproliferative disorders (MPD), including juvenile myelomonocytic leukemia (JMML). Mutations responsible for NS occur in at least 11 different loci including KRAS. Here we describe a mouse model for NS induced by K-RasV14I, a recurrent KRAS mutation in NS patients. K-RasV14I–mutant mice displayed multiple NS-associated developmental defects such as growth delay, craniofacial dysmorphia, cardiac defects, and hematologic abnormalities including a severe form of MPD that resembles human JMML. Homozygous animals had perinatal lethality whose penetrance varied with genetic background. Exposure of pregnant mothers to a MEK inhibitor rescued perinatal lethality and prevented craniofacial dysmorphia and cardiac defects. However, Mek inhibition was not sufficient to correct these defects when mice were treated after weaning. Interestingly, Mek inhibition did not correct the neoplastic MPD characteristic of these mutant mice, regardless of the timing at which the mice were treated, thus suggesting that MPD is driven by additional signaling pathways. These genetically engineered K-RasV14I–mutant mice offer an experimental tool for studying the molecular mechanisms underlying the clinical manifestations of NS. Perhaps more importantly, they should be useful as a preclinical model to test new therapies aimed at preventing or ameliorating those deficits associated with this syndrome. PMID:25359213

Severe inbreeding depression and no evidence of purging in an extremely inbred wild species--the Chatham Island black robin.

PubMed

Kennedy, Euan S; Grueber, Catherine E; Duncan, Richard P; Jamieson, Ian G

2014-04-01

Although evidence of inbreeding depression in wild populations is well established, the impact of genetic purging in the wild remains controversial. The contrasting effects of inbreeding depression, fixation of deleterious alleles by genetic drift, and the purging of deleterious alleles via natural selection mean that predicting fitness outcomes in populations subjected to prolonged bottlenecks is not straightforward. We report results from a long-term pedigree study of arguably the world's most inbred wild species of bird: the Chatham Island black robin Petroica traversi, in which conditions were ideal for purging to occur. Contrary to expectations, black robins showed a strong, negative relationship between inbreeding and juvenile survival, yielding lethal equivalents (2B) of 6.85. We also determined that the negative relationship between inbreeding and survival did not appear to be mediated by levels of ancestral inbreeding and may be attributed in part to unpurged lethal recessives. Although the black robin demographic history provided ideal conditions for genetic purging, our results show no clear evidence of purging in the major life-history trait of juvenile survival. Our results also show no evidence of fixation of deleterious alleles in juvenile survival, but do confirm that continued high levels of contemporary inbreeding in a historically inbred population could lead to additional severe inbreeding depression. © 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.

Genetic background has a major effect on the penetrance and severity of craniofacial defects in mice heterozygous for the gene encoding the nucleolar protein Treacle.

PubMed

Dixon, Jill; Dixon, Michael James

2004-04-01

Treacher Collins syndrome (TCS) is a craniofacial disorder that results from mutations in TCOF1, which encodes the nucleolar protein Treacle. The severity of the clinical features exhibits wide variation and includes hypoplasia of the mandible and maxilla, abnormalities of the external ears and middle ear ossicles, and cleft palate. To determine the in vivo function of Treacle, we previously generated Tcof1 heterozygous mice on a mixed C57BL/6 and 129 background. These mice exhibited a lethal phenotype, which included abnormal development of the maxilla, absence of the eyes and nasal passages, and neural tube defects. Here, we show that placing the mutation onto different genetic backgrounds has a major effect on the penetrance and severity of the craniofacial and other defects. The offspring exhibit markedly variable strain-dependent phenotypes that range from extremely severe and lethal in a mixed CBA/Ca and 129 background, to apparently normal and viable in a mixed BALB/c and 129 background. In the former case, in addition to a profoundly severe craniofacial phenotype, CBA-derived heterozygous mice also exhibited delayed ossification of the long bones, rib fusions, and digit anomalies. The results of our studies indicate that factors in the different genetic backgrounds contribute extensively to the Tcof1 phenotype. Copyright 2004 Wiley-Liss, Inc.

Duchenne Muscular Dystrophy: a Survey of Perspectives on Carrier Testing and Communication Within the Family.

PubMed

Hayes, Brenna; Hassed, Susan; Chaloner, Jae Lindsay; Aston, Christopher E; Guy, Carrie

2016-06-01

Carrier testing is widely available for multiple genetic conditions, and several professional organizations have created practice guidelines regarding appropriate clinical application and the testing of minors. Previous research has focused on carrier screening, predictive testing, and testing for X-linked conditions. However, family perspectives on carrier testing for X-linked lethal diseases have yet to be described. In this study, we explored communication within the family about carrier testing and the perspectives of mothers of sons with an X-linked lethal disease, Duchenne muscular dystrophy (DMD). Twenty-five mothers of sons with DMD participated in an anonymous online survey. Survey questions included multiple choice, Likert scale, and open ended, short answer questions. Analysis of the multiple choice and Likert scale questions revealed that most mothers preferred a gradual style of communication with their daughters regarding risk status. In addition, most participants reported having consulted with a genetic counselor and found it helpful. Comparisons between groups, analyzed using Fisher's exact tests, found no differences in preferred style due to mother's carrier status or having a daughter. Thematic analysis was conducted on responses to open ended questions. Themes identified included the impact of family implications, age and maturity, and a desire for autonomy regarding the decision to discuss and undergo carrier testing with at-risk daughters, particularly timing of these discussions. Implications for genetic counseling practice are discussed.

Genomewide Screen for Synthetic Lethal Interactions with Mutant KRAS in Lung Cancer

DTIC Science & Technology

2017-11-01

proposed work uses the same concept but with a novel approach. 15. SUBJECT TERMS CRISPR /Cas9, synthetic lethality, KRAS 16. SECURITY CLASSIFICATION OF: 17...essential in cells bearing an activated RAS. We used CRISPR /Cas9-based screening approach for this purpose. Body The overall objective was to identify...of the inducible hCas9 into the AAVS1 site through CRISPR /Cas9-mediated knockin approach. Shown in the top is gRNA sequence for AAVS1. Shown in the

Isolation of sochi virus from a fatal case of hantavirus disease with fulminant clinical course.

PubMed

Dzagurova, Tamara K; Witkowski, Peter T; Tkachenko, Evgeniy A; Klempa, Boris; Morozov, Vyacheslav G; Auste, Brita; Zavora, Dmitriy L; Iunicheva, Iulia V; Mutnih, Elena S; Kruger, Detlev H

2012-01-01

Sochi virus, a novel genetic variant of Dobrava-Belgrade virus, was isolated in cell culture from a fulminant lethal case of hantavirus disease presenting with shock and combined kidney and lung failure. Sochi virus is transmitted to humans from host reservoir Apodemus ponticus and must be considered a life-threatening emerging agent.

In-situ genetic conservation of white ash (Fraxinus americana) at the Allegheny national forest

Treesearch

Charles E. Flower; Elijah Aubihl; Jeremie Fant; Stephen Forry; Andrea Hille; Kathleen S. Knight; William K. Oldland; Alejandro A. Royo; Richard M. Turcotte

2017-01-01

The emerald ash borer (EAB, Agrilus planipennis) is a non-native forest pest that has been sweeping across North America causing widespread mortality of trees in the genus Fraxinus, which includes the economically valuable white ash (F. americana). The rapid spread and lethality of EAB, paired with low levels of natural...

Diverse chromosomal locations of quantitative trait loci for tolerance to maize chlorotic mottle in five maize populations

USDA-ARS?s Scientific Manuscript database

The recent rapid emergence of maize lethal necrosis (MLN), caused by coinfection of maize with maize chlorotic mottle virus (MCMV) and a second virus usually from the family Potyviridae, is causing extensive losses for farmers in East Africa, Southeast Asia and South America. Although the genetic ba...

White pine blister rust resistance in limber pine: Evidence for a major gene

Treesearch

A. W. Schoettle; R. A. Sniezko; A. Kegley; K. S. Burns

2014-01-01

Limber pine (Pinus flexilis) is being threatened by the lethal disease white pine blister rust caused by the non-native pathogen Cronartium ribicola. The types and frequencies of genetic resistance to the rust will likely determine the potential success of restoration or proactive measures. These first extensive inoculation trials using individual tree seed collections...

Pharmacokinetic-Pharmacodynamic Assessment of Faropenem in a Lethal Murine Bacillus anthracis Inhalation Postexposure Prophylaxis Model

DTIC Science & Technology

2010-05-01

Inhalation Postexposure Prophylaxis Model Stanley C. Gill,1* Christopher M. Rubino,2 Jennifer Bassett,3 Lynda Miller,3 Paul G. Ambrose,2 Sujata M. Bhavnani,2...tandem repeat analysis reveals genetic relationships within Bacillus anthracis. J. Bacteriol. 182:2928–2936. 14. Mohammed, M. J., C. K. Marston , T

A Synthetic Lethal Screen Identifies DNA Repair Pathways that Sensitize Cancer Cells to Combined ATR Inhibition and Cisplatin Treatments

PubMed Central

Mohni, Kareem N.; Thompson, Petria S.; Luzwick, Jessica W.; Glick, Gloria G.; Pendleton, Christopher S.; Lehmann, Brian D.; Pietenpol, Jennifer A.; Cortez, David

2015-01-01

The DNA damage response kinase ATR may be a useful cancer therapeutic target. ATR inhibition synergizes with loss of ERCC1, ATM, XRCC1 and DNA damaging chemotherapy agents. Clinical trials have begun using ATR inhibitors in combination with cisplatin. Here we report the first synthetic lethality screen with a combination treatment of an ATR inhibitor (ATRi) and cisplatin. Combination treatment with ATRi/cisplatin is synthetically lethal with loss of the TLS polymerase ζ and 53BP1. Other DNA repair pathways including homologous recombination and mismatch repair do not exhibit synthetic lethal interactions with ATRi/cisplatin, even though loss of some of these repair pathways sensitizes cells to cisplatin as a single-agent. We also report that ATRi strongly synergizes with PARP inhibition, even in homologous recombination-proficient backgrounds. Lastly, ATR inhibitors were able to resensitize cisplatin-resistant cell lines to cisplatin. These data provide a comprehensive analysis of DNA repair pathways that exhibit synthetic lethality with ATR inhibitors when combined with cisplatin chemotherapy, and will help guide patient selection strategies as ATR inhibitors progress into the cancer clinic. PMID:25965342

No evidence for female discrimination against male house mice carrying a selfish genetic element

PubMed Central

Lindholm, Anna K.

2016-01-01

Meiotic drivers distort transmission to the next generation in their favor, with detrimental effects on the fitness of their homologues and the rest of the genome. Male carriers of meiotic drivers commonly inflict costs on their mates through genetic incompatibility, reduced fecundity, or biased brood sex ratios. Given these costs, evidence for female discrimination against male carriers is surprisingly rare. One of few examples is the t haplotype in house mice, a meiotic driver that shows strong transmission distortion in males and is typically homozygote lethal. As a consequence, mating between 2 t heterozygous (+/t) mice leads to high embryo mortality. Previous experiments showing that +/t females avoid this incompatibility cost by preferring +/+ versus +/t males have inferred preference based on olfactory cues or brief social interactions. Evidence from mating contexts in laboratory settings and semi-natural populations has been inconclusive. Here, we investigated female choice from a large number of no-choice mating trials. We found no evidence for discrimination against +/t males based on mating, remating, and copulatory behavior. Further, we found no evidence for avoidance of incompatibility through selective interactions between gametes. The likelihood of mating showed significant effects of female weight and genotype, suggesting that our test paradigm enabled females to exhibit mate choice. We discuss the strengths and limitations of our approach. By explicitly considering selection at both the individual and gene level, we argue why precopulatory female discrimination by +/t females may be less evolutionarily stable than discrimination by all females based on postcopulatory mechanisms. PMID:29491955

No evidence for female discrimination against male house mice carrying a selfish genetic element.

PubMed

Sutter, Andreas; Lindholm, Anna K

2016-12-01

Meiotic drivers distort transmission to the next generation in their favor, with detrimental effects on the fitness of their homologues and the rest of the genome. Male carriers of meiotic drivers commonly inflict costs on their mates through genetic incompatibility, reduced fecundity, or biased brood sex ratios. Given these costs, evidence for female discrimination against male carriers is surprisingly rare. One of few examples is the t haplotype in house mice, a meiotic driver that shows strong transmission distortion in males and is typically homozygote lethal. As a consequence, mating between 2 t heterozygous (+/ t ) mice leads to high embryo mortality. Previous experiments showing that +/ t females avoid this incompatibility cost by preferring +/+ versus +/ t males have inferred preference based on olfactory cues or brief social interactions. Evidence from mating contexts in laboratory settings and semi-natural populations has been inconclusive. Here, we investigated female choice from a large number of no-choice mating trials. We found no evidence for discrimination against +/ t males based on mating, remating, and copulatory behavior. Further, we found no evidence for avoidance of incompatibility through selective interactions between gametes. The likelihood of mating showed significant effects of female weight and genotype, suggesting that our test paradigm enabled females to exhibit mate choice. We discuss the strengths and limitations of our approach. By explicitly considering selection at both the individual and gene level, we argue why precopulatory female discrimination by +/ t females may be less evolutionarily stable than discrimination by all females based on postcopulatory mechanisms.

Field Measurements Indicate Unexpected, Serious Underestimation of Mussel Heart Rates and Thermal Tolerance by Laboratory Studies

PubMed Central

Tagliarolo, Morgana; McQuaid, Christopher D.

2016-01-01

Attempts to predict the response of species to long-term environmental change are generally based on extrapolations from laboratory experiments that inevitably simplify the complex interacting effects that occur in the field. We recorded heart rates of two genetic lineages of the brown mussel Perna perna over a full tidal cycle in-situ at two different sites in order to evaluate the cardiac responses of the two genetic lineages present on the South African coast to temperature and the immersion/emersion cycle. “Robomussel” temperature loggers were used to monitor thermal conditions at the two sites over one year. Comparison with live animals showed that robomussels provided a good estimate of mussel body temperatures. A significant difference in estimated body temperatures was observed between the sites and the results showed that, under natural conditions, temperatures regularly approach or exceed the thermal limits of P. perna identified in the laboratory. The two P. perna lineages showed similar tidal and diel patterns of heart rate, with higher cardiac activity during daytime immersion and minimal values during daytime emersion. Comparison of the heart rates measured in the field with data previously measured in the laboratory indicates that laboratory results seriously underestimate heart rate activity, by as much as 75%, especially during immersion. Unexpectedly, field estimates of body temperatures indicated an ability to tolerate temperatures considered lethal on the basis of laboratory measurements. This suggests that the interaction of abiotic conditions in the field does not necessarily raise vulnerability to high temperatures. PMID:26840775

Freezing: an underutilized food safety technology?

PubMed

Archer, Douglas L

2004-01-15

Freezing is an ancient technology for preserving foods. Freezing halts the activities of spoilage microorganisms in and on foods and can preserve some microorganisms for long periods of time. Frozen foods have an excellent overall safety record. The few outbreaks of food-borne illness associated with frozen foods indicate that some, but not all human pathogens are killed by commercial freezing processes. Freezing kills microorganisms by physical and chemical effects and possibly through induced genetic changes. Research is needed to better understand the physical and chemical interactions of various food matrices with the microbial cell during freezing and holding at frozen temperatures. The literature suggests that many pathogenic microorganisms may be sublethally injured by freezing, so research should be done to determine how to prevent injured cells from resuscitating and becoming infectious. Studies on the genetics of microbial stress suggest that the induction of resistance to specific stresses may be counteracted by, for example, simple chemicals. Research is needed to better understand how resistance to the lethal effect of freezing is induced in human pathogens and means by which it can be counteracted in specific foods. Through research, it seems possible that freezing may in the future be used to reliably reduce populations of food-borne pathogens as well as to preserve foods.

DNAJB1–PRKACA fusion kinase interacts with β-catenin and the liver regenerative response to drive fibrolamellar hepatocellular carcinoma

PubMed Central

Kastenhuber, Edward R.; Lalazar, Gadi; Houlihan, Shauna L.; Tschaharganeh, Darjus F.; Baslan, Timour; Chen, Chi-Chao; Requena, David; Tian, Sha; Bosbach, Benedikt; Wilkinson, John E.; Simon, Sanford M.; Lowe, Scott W.

2017-01-01

A segmental deletion resulting in DNAJB1–PRKACA gene fusion is now recognized as the signature genetic event of fibrolamellar hepatocellular carcinoma (FL-HCC), a rare but lethal liver cancer that primarily affects adolescents and young adults. Here we implement CRISPR-Cas9 genome editing and transposon-mediated somatic gene transfer to demonstrate that expression of either the endogenous fusion protein or a chimeric cDNA leads to the formation of indolent liver tumors in mice that closely resemble human FL-HCC. Notably, overexpression of the wild-type PRKACA was unable to fully recapitulate the oncogenic activity of DNAJB1–PRKACA, implying that FL-HCC does not simply result from enhanced PRKACA expression. Tumorigenesis was significantly enhanced by genetic activation of β-catenin, an observation supported by evidence of recurrent Wnt pathway mutations in human FL-HCC, as well as treatment with the hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine, which causes tissue injury, inflammation, and fibrosis. Our study validates the DNAJB1–PRKACA fusion kinase as an oncogenic driver and candidate drug target for FL-HCC, and establishes a practical model for preclinical studies to identify strategies to treat this disease. PMID:29162699

A single genetic locus in the phytopathogen Pantoea stewartii enables gut colonization and pathogenicity in an insect host.

PubMed

Stavrinides, John; No, Alexander; Ochman, Howard

2010-01-01

Aphids are typically exposed to a variety of epiphytic and phytopathogenic bacteria, many of which have entomopathogenic potential. Here we describe the interaction between Pantoea stewartii ssp. stewartii DC283 (DC283), an enteric phytopathogen and causal agent of Stewart's wilt, and the pea aphid, Acyrthosiphon pisum. When ingested by aphids, DC283 establishes and aggregates in the crop and gut, preventing honeydew flow and excretion, resulting in aphid death in 72 h. A mutagenesis screen identified a single locus, termed ucp1 (youcannot pass), whose disruption abolishes aphid pathogenicity. Moreover, the expression of ucp1 in Escherichia coli is sufficient to mediate the hindgut aggregation phenotype by this normally avirulent species. Ucp1 is related to six other proteins in the DC283 genome, each having a common N-terminal region and a divergent C-terminus, but only ucp1 has a role in pathogenicity. Based on predicted motifs and secondary structure, Ucp1 is a membrane-bound protein that functions in bacterial adhesion and promotes the formation of aggregates that are lethal to the insect host. These results illustrate that the enteric plant pathogenic bacteria have the capacity to exploit alternative non-plant hosts, and retain genetic determinants for colonizing the gut.

Alternatives to animal testing: research, trends, validation, regulatory acceptance.

PubMed

Huggins, Jane

2003-01-01

Current trends and issues in the development of alternatives to the use of animals in biomedical experimentation are discussed in this position paper. Eight topics are considered and include refinement of acute toxicity assays; eye corrosion/irritation alternatives; skin corrosion/irritation alternatives; contact sensitization alternatives; developmental/reproductive testing alternatives; genetic engineering (transgenic) assays; toxicogenomics; and validation of alternative methods. The discussion of refinement of acute toxicity assays is focused primarily on developments with regard to reduction of the number of animals used in the LD(50) assay. However, the substitution of humane endpoints such as clinical signs of toxicity for lethality in these assays is also evaluated. Alternative assays for eye corrosion/irritation as well as those for skin corrosion/irritation are described with particular attention paid to the outcomes, both successful and unsuccessful, of several validation efforts. Alternative assays for contact sensitization and developmental/reproductive toxicity are presented as examples of methods designed for the examination of interactions between toxins and somewhat more complex physiological systems. Moreover, genetic engineering and toxicogenomics are discussed with an eye toward the future of biological experimentation in general. The implications of gene manipulation for research animals, specifically, are also examined. Finally, validation methods are investigated as to their effectiveness, or lack thereof, and suggestions for their standardization and improvement, as well as implementation are reviewed.

Minimal Contribution of APOBEC3-Induced G-to-A Hypermutation to HIV-1 Recombination and Genetic Variation

PubMed Central

Nikolaitchik, Olga A.; Burdick, Ryan C.; Gorelick, Robert J.; Keele, Brandon F.; Hu, Wei-Shau; Pathak, Vinay K.

2016-01-01

Although the predominant effect of host restriction APOBEC3 proteins on HIV-1 infection is to block viral replication, they might inadvertently increase retroviral genetic variation by inducing G-to-A hypermutation. Numerous studies have disagreed on the contribution of hypermutation to viral genetic diversity and evolution. Confounding factors contributing to the debate include the extent of lethal (stop codon) and sublethal hypermutation induced by different APOBEC3 proteins, the inability to distinguish between G-to-A mutations induced by APOBEC3 proteins and error-prone viral replication, the potential impact of hypermutation on the frequency of retroviral recombination, and the extent to which viral recombination occurs in vivo, which can reassort mutations in hypermutated genomes. Here, we determined the effects of hypermutation on the HIV-1 recombination rate and its contribution to genetic variation through recombination to generate progeny genomes containing portions of hypermutated genomes without lethal mutations. We found that hypermutation did not significantly affect the rate of recombination, and recombination between hypermutated and wild-type genomes only increased the viral mutation rate by 3.9 × 10−5 mutations/bp/replication cycle in heterozygous virions, which is similar to the HIV-1 mutation rate. Since copackaging of hypermutated and wild-type genomes occurs very rarely in vivo, recombination between hypermutated and wild-type genomes does not significantly contribute to the genetic variation of replicating HIV-1. We also analyzed previously reported hypermutated sequences from infected patients and determined that the frequency of sublethal mutagenesis for A3G and A3F is negligible (4 × 10−21 and1 × 10−11, respectively) and its contribution to viral mutations is far below mutations generated during error-prone reverse transcription. Taken together, we conclude that the contribution of APOBEC3-induced hypermutation to HIV-1 genetic variation is substantially lower than that from mutations during error-prone replication. PMID:27186986

Minimal Contribution of APOBEC3-Induced G-to-A Hypermutation to HIV-1 Recombination and Genetic Variation.

PubMed

Delviks-Frankenberry, Krista A; Nikolaitchik, Olga A; Burdick, Ryan C; Gorelick, Robert J; Keele, Brandon F; Hu, Wei-Shau; Pathak, Vinay K

2016-05-01

Although the predominant effect of host restriction APOBEC3 proteins on HIV-1 infection is to block viral replication, they might inadvertently increase retroviral genetic variation by inducing G-to-A hypermutation. Numerous studies have disagreed on the contribution of hypermutation to viral genetic diversity and evolution. Confounding factors contributing to the debate include the extent of lethal (stop codon) and sublethal hypermutation induced by different APOBEC3 proteins, the inability to distinguish between G-to-A mutations induced by APOBEC3 proteins and error-prone viral replication, the potential impact of hypermutation on the frequency of retroviral recombination, and the extent to which viral recombination occurs in vivo, which can reassort mutations in hypermutated genomes. Here, we determined the effects of hypermutation on the HIV-1 recombination rate and its contribution to genetic variation through recombination to generate progeny genomes containing portions of hypermutated genomes without lethal mutations. We found that hypermutation did not significantly affect the rate of recombination, and recombination between hypermutated and wild-type genomes only increased the viral mutation rate by 3.9 × 10-5 mutations/bp/replication cycle in heterozygous virions, which is similar to the HIV-1 mutation rate. Since copackaging of hypermutated and wild-type genomes occurs very rarely in vivo, recombination between hypermutated and wild-type genomes does not significantly contribute to the genetic variation of replicating HIV-1. We also analyzed previously reported hypermutated sequences from infected patients and determined that the frequency of sublethal mutagenesis for A3G and A3F is negligible (4 × 10-21 and1 × 10-11, respectively) and its contribution to viral mutations is far below mutations generated during error-prone reverse transcription. Taken together, we conclude that the contribution of APOBEC3-induced hypermutation to HIV-1 genetic variation is substantially lower than that from mutations during error-prone replication.

Influence of Protein Abundance on High-Throughput Protein-Protein Interaction Detection

DTIC Science & Technology

2009-06-05

the interaction data sets we determined, via comparisons with strict randomized simulations , the propensity for essential proteins to selectively...and analysis of high- quality PPI data sets. Materials and Methods We analyzed protein interaction networks for yeast and E. coli determined from Y2H...we reinvestigated the centrality-lethality rule, which implies that proteins having more interactions are more likely to be essential. From analysis

Lethal coalitionary aggression and long-term alliance formation among Yanomamö men.

PubMed

Macfarlan, Shane J; Walker, Robert S; Flinn, Mark V; Chagnon, Napoleon A

2014-11-25

Some cross-cultural evidence suggests lethal coalitionary aggression in humans is the product of residence and descent rules that promote fraternal interest groups, i.e., power groups of coresident males bonded by kinship. As such, human lethal coalitions are hypothesized to be homologous to chimpanzee (Pan troglodytes) border patrols. However, humans demonstrate a unique metagroup social structure in which strategic alliances allow individuals to form coalitions transcending local community boundaries. We test predictions derived from the fraternal interest group and strategic alliance models using lethal coalition data from a lowland South American population, the Yanomamö. Yanomamö men who kill an enemy acquire a special status, termed unokai. We examine the social characteristics of co-unokais or men who jointly kill others. Analyses indicate co-unokais generally are (i) from the same population but from different villages and patrilines, (ii) close age mates, and (iii) maternal half-first cousins. Furthermore, the incident rate for co-unokai killings increases if men are similar in age, from the same population, and from different natal communities. Co-unokais who have killed more times in the past and who are more genetically related to each other have a higher probability of coresidence in adulthood. Last, a relationship exists between lethal coalition formation and marriage exchange. In this population, internal warfare unites multiple communities, and co-unokais strategically form new residential groups and marriage alliances. These results support the strategic alliance model of coalitionary aggression, demonstrate the complexities of human alliance formation, and illuminate key differences in social structure distinguishing humans from other primates.

Lethal coalitionary aggression and long-term alliance formation among Yanomamö men

PubMed Central

Macfarlan, Shane J.; Walker, Robert S.; Flinn, Mark V.; Chagnon, Napoleon A.

2014-01-01

Some cross-cultural evidence suggests lethal coalitionary aggression in humans is the product of residence and descent rules that promote fraternal interest groups, i.e., power groups of coresident males bonded by kinship. As such, human lethal coalitions are hypothesized to be homologous to chimpanzee (Pan troglodytes) border patrols. However, humans demonstrate a unique metagroup social structure in which strategic alliances allow individuals to form coalitions transcending local community boundaries. We test predictions derived from the fraternal interest group and strategic alliance models using lethal coalition data from a lowland South American population, the Yanomamö. Yanomamö men who kill an enemy acquire a special status, termed unokai. We examine the social characteristics of co-unokais or men who jointly kill others. Analyses indicate co-unokais generally are (i) from the same population but from different villages and patrilines, (ii) close age mates, and (iii) maternal half-first cousins. Furthermore, the incident rate for co-unokai killings increases if men are similar in age, from the same population, and from different natal communities. Co-unokais who have killed more times in the past and who are more genetically related to each other have a higher probability of coresidence in adulthood. Last, a relationship exists between lethal coalition formation and marriage exchange. In this population, internal warfare unites multiple communities, and co-unokais strategically form new residential groups and marriage alliances. These results support the strategic alliance model of coalitionary aggression, demonstrate the complexities of human alliance formation, and illuminate key differences in social structure distinguishing humans from other primates. PMID:25349394

Increased H+ efflux is sufficient to induce dysplasia and necessary for viability with oncogene expression

PubMed Central

Grillo-Hill, Bree K; Choi, Changhoon; Jimenez-Vidal, Maite; Barber, Diane L

2015-01-01

Intracellular pH (pHi) dynamics is increasingly recognized as an important regulator of a range of normal and pathological cell behaviors. Notably, increased pHi is now acknowledged as a conserved characteristic of cancers and in cell models is confirmed to increase proliferation and migration as well as limit apoptosis. However, the significance of increased pHi for cancer in vivo remains unresolved. Using Drosophila melanogaster, we show that increased pHi is sufficient to induce dysplasia in the absence of other transforming cues and potentiates growth and invasion with oncogenic Ras. Using a genetically encoded biosensor we also confirm increased pHi in situ. Moreover, in Drosophila models and clonal human mammary cells we show that limiting H+ efflux with oncogenic Raf or Ras induces acidosis and synthetic lethality. Further, we show lethality in invasive primary tumor cell lines with inhibiting H+ efflux. Synthetic lethality with reduced H+ efflux and activated oncogene expression could be exploited therapeutically to restrain cancer progression while limiting off-target effects. DOI: http://dx.doi.org/10.7554/eLife.03270.001 PMID:25793441

Mutations in KIAA0586 Cause Lethal Ciliopathies Ranging from a Hydrolethalus Phenotype to Short-Rib Polydactyly Syndrome.

PubMed

Alby, Caroline; Piquand, Kevin; Huber, Céline; Megarbané, André; Ichkou, Amale; Legendre, Marine; Pelluard, Fanny; Encha-Ravazi, Ferechté; Abi-Tayeh, Georges; Bessières, Bettina; El Chehadeh-Djebbar, Salima; Laurent, Nicole; Faivre, Laurence; Sztriha, László; Zombor, Melinda; Szabó, Hajnalka; Failler, Marion; Garfa-Traore, Meriem; Bole, Christine; Nitschké, Patrick; Nizon, Mathilde; Elkhartoufi, Nadia; Clerget-Darpoux, Françoise; Munnich, Arnold; Lyonnet, Stanislas; Vekemans, Michel; Saunier, Sophie; Cormier-Daire, Valérie; Attié-Bitach, Tania; Thomas, Sophie

2015-08-06

KIAA0586, the human ortholog of chicken TALPID3, is a centrosomal protein that is essential for primary ciliogenesis. Its disruption in animal models causes defects attributed to abnormal hedgehog signaling; these defects include polydactyly and abnormal dorsoventral patterning of the neural tube. Here, we report homozygous mutations of KIAA0586 in four families affected by lethal ciliopathies ranging from a hydrolethalus phenotype to short-rib polydactyly. We show defective ciliogenesis, as well as abnormal response to SHH-signaling activation in cells derived from affected individuals, consistent with a role of KIAA0586 in primary cilia biogenesis. Whereas centriolar maturation seemed unaffected in mutant cells, we observed an abnormal extended pattern of CEP290, a centriolar satellite protein previously associated with ciliopathies. Our data show the crucial role of KIAA0586 in human primary ciliogenesis and subsequent abnormal hedgehog signaling through abnormal GLI3 processing. Our results thus establish that KIAA0586 mutations cause lethal ciliopathies. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Host-Selected Amino Acid Changes at the Sialic Acid Binding Pocket of the Parvovirus Capsid Modulate Cell Binding Affinity and Determine Virulence

PubMed Central

López-Bueno, Alberto; Rubio, Mari-Paz; Bryant, Nathan; McKenna, Robert; Agbandje-McKenna, Mavis; Almendral, José M.

2006-01-01

The role of receptor recognition in the emergence of virulent viruses was investigated in the infection of severe combined immunodeficient (SCID) mice by the apathogenic prototype strain of the parvovirus minute virus of mice (MVMp). Genetic analysis of isolated MVMp viral clones (n = 48) emerging in mice, including lethal variants, showed only one of three single changes (V325M, I362S, or K368R) in the common sequence of the two capsid proteins. As was found for the parental isolates, the constructed recombinant viruses harboring the I362S or the K368R single substitutions in the capsid sequence, or mutations at both sites, showed a large-plaque phenotype and lower avidity than the wild type for cells in the cytotoxic interaction with two permissive fibroblast cell lines in vitro and caused a lethal disease in SCID mice when inoculated by the natural oronasal route. Significantly, the productive adsorption of MVMp variants carrying any of the three mutations selected through parallel evolution in mice showed higher sensitivity to the treatment of cells by neuraminidase than that of the wild type, indicating a lower affinity of the viral particle for the sialic acid component of the receptor. Consistent with this, the X-ray crystal structure of the MVMp capsids soaked with sialic acid (N-acetyl neuraminic acid) showed the sugar allocated in the depression at the twofold axis of symmetry (termed the dimple), immediately adjacent to residues I362 and K368, which are located on the wall of the dimple, and approximately 22 Å away from V325 in a threefold-related monomer. This is the first reported crystal structure identifying an infectious receptor attachment site on a parvovirus capsid. We conclude that the affinity of the interactions of sialic-acid-containing receptors with residues at or surrounding the dimple can evolutionarily regulate parvovirus pathogenicity and adaptation to new hosts. PMID:16415031

Public acceptance of management methods under different human-wildlife conflict scenarios.

PubMed

Liordos, Vasilios; Kontsiotis, Vasileios J; Georgari, Marina; Baltzi, Kerasia; Baltzi, Ioanna

2017-02-01

Wildlife management seeks to minimise public controversy for successful application of wildlife control methods. Human dimensions research in wildlife seeks a better understanding of public preferences for effective human-wildlife conflict resolution. In face to face interviews, 630 adults in Greece were asked to rate on a 5-point Likert-like scale their acceptance of 3 management methods, i.e., do nothing, non-lethal control, and lethal control, in the context of 5 human-wildlife conflict scenarios: 1) corvids damage crops; 2) starlings damage crops; 3) starlings foul urban structures; 4) coypus damage crops; and 5) coypus transfer disease. Univariate GLMs determined occupation, hunting membership and their interaction as the stronger predictors of public acceptance, generating 4 stakeholder groups: the general public, farmers, hunters, and farmers-hunters. Differences in acceptance and consensus among stakeholder groups were assessed using the Potential for Conflict Index 2 (PCI 2 ). All 4 stakeholder groups agreed that doing nothing was unacceptable and non-lethal control acceptable in all 5 scenarios, with generally high consensus within and between groups. The lethal control method was more controversial and became increasingly more acceptable as the severity of scenarios was increased and between non-native and native species. Lethal control was unacceptable for the general public in all scenarios. Farmers accepted lethal methods in the corvids and starlings scenarios, were neutral in the coypus damage crops scenario, whereas they accepted lethal control when coypus transfer disease. Hunters' opinion was neutral in the corvids, starlings and coypus damage crops and starlings foul urban structures scenarios, but they accepted lethal methods in the coypus transfer disease scenario. Farmers-hunters considered lethal control acceptable in all 5 scenarios. Implications from this study could be used for designing a socio-ecological approach which incorporates wildlife management with public interests. The studied species have a wide distribution, therefore present findings might also prove useful elsewhere. Copyright © 2016 Elsevier B.V. All rights reserved.

Consortium for Osteogenesis Imperfecta Mutations in the Helical Domain of Type I Collagen: Regions Rich in Lethal Mutations Align With Collagen Binding Sites for Integrins and Proteoglycans

PubMed Central

Marini, Joan C.; Forlino, Antonella; Cabral, Wayne A.; Barnes, Aileen M.; San Antonio, James D.; Milgrom, Sarah; Hyland, James C.; Körkkö, Jarmo; Prockop, Darwin J.; De Paepe, Anne; Coucke, Paul; Symoens, Sofie; Glorieux, Francis H.; Roughley, Peter J.; Lund, Alan M.; Kuurila-Svahn, Kaija; Hartikka, Heini; Cohn, Daniel H.; Krakow, Deborah; Mottes, Monica; Schwarze, Ulrike; Chen, Diana; Yang, Kathleen; Kuslich, Christine; Troendle, James; Dalgleish, Raymond; Byers, Peter H.

2014-01-01

Osteogenesis imperfecta (OI) is a generalized disorder of connective tissue characterized by fragile bones and easy susceptibility to fracture. Most cases of OI are caused by mutations in type I collagen. We have identified and assembled structural mutations in type I collagen genes (COL1A1 and COL1A2, encoding the proα1(I) and proα2(I) chains, respectively) that result in OI. Quantitative defects causing type I OI were not included. Of these 832 independent mutations, 682 result in substitution for glycine residues in the triple helical domain of the encoded protein and 150 alter splice sites. Distinct genotype–phenotype relationships emerge for each chain. One-third of the mutations that result in glycine substitutions in α1(I) are lethal, especially when the substituting residues are charged or have a branched side chain. Substitutions in the first 200 residues are nonlethal and have variable outcome thereafter, unrelated to folding or helix stability domains. Two exclusively lethal regions (helix positions 691–823 and 910–964) align with major ligand binding regions (MLBRs), suggesting crucial interactions of collagen monomers or fibrils with integrins, matrix metalloproteinases (MMPs), fibronectin, and cartilage oligomeric matrix protein (COMP). Mutations in COL1A2 are predominantly nonlethal (80%). Lethal substitutions are located in eight regularly spaced clusters along the chain, supporting a regional model. The lethal regions align with proteoglycan binding sites along the fibril, suggesting a role in fibril–matrix interactions. Recurrences at the same site in α2(I) are generally concordant for outcome, unlike α1(I). Splice site mutations comprise 20% of helical mutations identified in OI patients, and may lead to exon skipping, intron inclusion, or the activation of cryptic splice sites. Splice site mutations in COL1A1 are rarely lethal; they often lead to frameshifts and the mild type I phenotype. In α2(I), lethal exon skipping events are located in the carboxyl half of the chain. Our data on genotype–phenotype relationships indicate that the two collagen chains play very different roles in matrix integrity and that phenotype depends on intracellular and extracellular events. PMID:17078022

Genetic testing in heritable cardiac arrhythmia syndromes: differentiating pathogenic mutations from background genetic noise.

PubMed

Giudicessi, John R; Ackerman, Michael J

2013-01-01

In this review, we summarize the basic principles governing rare variant interpretation in the heritable cardiac arrhythmia syndromes, focusing on recent advances that have led to disease-specific approaches to the interpretation of positive genetic testing results. Elucidation of the genetic substrates underlying heritable cardiac arrhythmia syndromes has unearthed new arrhythmogenic mechanisms and given rise to a number of clinically meaningful genotype-phenotype correlations. As such, genetic testing for these disorders now carries important diagnostic, prognostic, and therapeutic implications. Recent large-scale systematic studies designed to explore the background genetic 'noise' rate associated with these genetic tests have provided important insights and enhanced how positive genetic testing results are interpreted for these potentially lethal, yet highly treatable, cardiovascular disorders. Clinically available genetic tests for heritable cardiac arrhythmia syndromes allow the identification of potentially at-risk family members and contribute to the risk-stratification and selection of therapeutic interventions in affected individuals. The systematic evaluation of the 'signal-to-noise' ratio associated with these genetic tests has proven critical and essential to assessing the probability that a given variant represents a rare pathogenic mutation or an equally rare, yet innocuous, genetic bystander.

Evaluation of the Insecticidal Efficacy of Wild Type and Recombinant Baculoviruses.

PubMed

Popham, Holly J R; Ellersieck, Mark R; Li, Huarong; Bonning, Bryony C

2016-01-01

A considerable amount of work has been undertaken to genetically enhance the efficacy of baculovirus insecticides. Following construction of a genetically altered baculovirus, laboratory bioassays are used to quantify various parameters of insecticidal activity such as the median lethal concentration (or dose) required to kill 50 % of infected larvae (LC50 or LD50), median survival of larvae infected (ST50), and feeding damage incurred by infected larvae. In this chapter, protocols are described for a variety of bioassays and the corresponding data analyses for assessment of the insecticidal activity of baculovirus insecticides.

Pancreatic cancer stromal biology and therapy

PubMed Central

Xie, Dacheng; Xie, Keping

2015-01-01

Pancreatic cancer is one of the most lethal malignancies. Significant progresses have been made in understanding of pancreatic cancer pathogenesis, including appreciation of precursor lesions or premalignant pancreatic intraepithelial neoplasia (PanINs), description of sequential transformation from normal pancreatic tissue to invasive pancreatic cancer and identification of major genetic and epigenetic events and the biological impact of those events on malignant behavior. However, the currently used therapeutic strategies targeting tumor epithelial cells, which are potent in cell culture and animal models, have not been successful in the clinic. Presumably, therapeutic resistance of pancreatic cancer is at least in part due to its drastic desmoplasis, which is a defining hallmark for and circumstantially contributes to pancreatic cancer development and progression. Improved understanding of the dynamic interaction between cancer cells and the stroma is important to better understanding pancreatic cancer biology and to designing effective intervention strategies. This review focuses on the origination, evolution and disruption of stromal molecular and cellular components in pancreatic cancer, and their biological effects on pancreatic cancer pathogenesis. PMID:26114155

Mutant KRAS as a critical determinant of the therapeutic response of colorectal cancer

PubMed Central

Knickelbein, Kyle; Zhang, Lin

2014-01-01

Mutations in the KRAS oncogene represent one of the most prevalent genetic alterations in colorectal cancer (CRC), the third leading cause of cancer-related death in the US. In addition to their well-characterized function in driving tumor progression, KRAS mutations have been recognized as a critical determinant of the therapeutic response of CRC. Recent studies demonstrate that KRAS-mutant tumors are intrinsically insensitive to clinically-used epidermal growth factor receptor (EGFR) targeting antibodies, including cetuximab and panitumumab. Acquired resistance to the anti-EGFR therapy was found to be associated with enrichment of KRAS-mutant tumor cells. However, the underlying molecular mechanism of mutant-KRAS-mediated therapeutic resistance has remained unclear. Despite intensive efforts, directly targeting mutant KRAS has been largely unsuccessful. This review summarizes the recent advances in understanding the biological function of KRAS mutations in determining the therapeutic response of CRC, highlighting several recently developed agents and strategies for targeting mutant KRAS, such as synthetic lethal interactions. PMID:25815366

The embryonic mouse hindbrain as a qualitative and quantitative model for studying the molecular and cellular mechanisms of angiogenesis.

PubMed

Fantin, Alessandro; Vieira, Joaquim M; Plein, Alice; Maden, Charlotte H; Ruhrberg, Christiana

2013-02-01

The mouse embryo hindbrain is a robust and adaptable model for studying sprouting angiogenesis. It permits the spatiotemporal analysis of organ vascularization in normal mice and in mouse strains with genetic mutations that result in late embryonic or perinatal lethality. Unlike postnatal models such as retinal angiogenesis or Matrigel implants, there is no requirement for the breeding of conditional knockout mice. The unique architecture of the hindbrain vasculature allows whole-mount immunolabeling of blood vessels and high-resolution imaging, as well as easy quantification of angiogenic sprouting, network density and vessel caliber. The hindbrain model also permits the visualization of ligand binding to blood vessels in situ and the analysis of blood vessel growth within a natural multicellular microenvironment in which endothelial cells (ECs) interact with non-ECs to refine the 3D organ architecture. The entire procedure, from embryo isolation to imaging and through to results analysis, takes approximately 4 d.

Genetic Studies of the Prp17 Gene of Saccharomyces Cerevisiae: A Domain Essential for Function Maps to a Nonconserved Region of the Protein

PubMed Central

Seshadri, V.; Vaidya, V. C.; Vijayraghavan, U.

1996-01-01

The PRP17 gene product is required for the second step of pre-mRNA splicing reactions. The C-terminal half of this protein bears four repeat units with homology to the β transducin repeat. Missense mutations in three temperature-sensitive prp17 mutants map to a region in the N-terminal half of the protein. We have generated, in vitro, 11 missense alleles at the β transducin repeat units and find that only one affects function in vivo. A phenotypically silent missense allele at the fourth repeat unit enhances the slow-growing phenotype conferred by an allele at the third repeat, suggesting an interaction between these domains. Although many missense mutations in highly conserved amino acids lack phenotypic effects, deletion analysis suggests an essential role for these units. Only mutations in the N-terminal nonconserved domain of PRP17 are synthetically lethal in combination with mutations in PRP16 and PRP18, two other gene products required for the second splicing reaction. A mutually allele-specific interaction between prp17 and snr7, with mutations in U5 snRNA, was observed. We therefore suggest that the functional region of Prp17p that interacts with Prp18p, Prp16p, and U5 snRNA is in the N terminal region of the protein. PMID:8722761

Accumulation of peptidyl tRNA is lethal to Escherichia coli.

PubMed Central

Menninger, J R

1979-01-01

A mutant strain of Escherichia coli with temperature-sensitive peptidyl-tRNA hydrolase grows at 30 degrees C but, when shifted to 40 degrees C, dies at rates affected by physiological, pharmacological, and genetical perturbations. The rate of killing correlates with the relative accumulation of peptidyl-tRNA, suggesting that it is responsible for the death of the cells. PMID:368041

Designing Trojan Horses | Center for Cancer Research

Cancer.gov

Waging battle against cancer cells without inflicting damage on normal tissue has long been a goal for cancer treatment. A new type of drug called immunotoxins may help make this goal a reality. Much like the Greeks used a wooden horse to get soldiers inside the gates of Troy, immunotoxins use clever genetic engineering to get a lethal toxin inside cancer cells. Each

To Know or Not to Know? Integrating Ethical Aspects of Genomic Healthcare in the Education of Health Professionals

ERIC Educational Resources Information Center

Eriksen, Kathrine Krageskov

2015-01-01

Novel possibilities for employing genetic testing as part of the diagnostic process for a wide variety of diseases and conditions are emerging almost every day. This development brings prospects of more efficient treatment and prevention of serious and often lethal conditions. However, it also raises ethical questions concerning the issue of…

Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens into the cytosol to induce T cell immunity

NASA Astrophysics Data System (ADS)

Lu, Yichen; Friedman, Rachel; Kushner, Nicholas; Doling, Amy; Thomas, Lawrence; Touzjian, Neal; Starnbach, Michael; Lieberman, Judy

2000-07-01

Bacillus anthrax lethal toxin can be engineered to deliver foreign proteins to the cytosol for antigen presentation to CD8 T cells. Vaccination with modified toxins carrying 8-9 amino acid peptide epitopes induces protective immunity in mice. To evaluate whether large protein antigens can be used with this system, recombinant constructs encoding several HIV antigens up to 500 amino acids were produced. These candidate HIV vaccines are safe in animals and induce CD8 T cells in mice. Constructs encoding gag p24 and nef stimulate gag-specific CD4 proliferation and a secondary cytotoxic T lymphocyte response in HIV-infected donor peripheral blood mononuclear cells in vitro. These results lay the foundation for future clinical vaccine studies.

Establishment of conditional vectors for hairpin siRNA knockdowns

PubMed Central

Matsukura, Shiro; Jones, Peter A.; Takai, Daiya

2003-01-01

Small interference RNA (siRNA) is an emerging methodology in reverse genetics. Here we report the development of a new tetracycline-inducible vector-based siRNA system, which uses a tetracycline-responsive derivative of the U6 promoter and the tetracycline repressor for conditional in vivo transcription of short hairpin RNA. This method prevents potential lethality immediately after transfection of a vector when the targeted gene is indispensable, or the phenotype of the knockdown is lethal or results in a growth abnormality. We show that the controlled knockdown of DNA methyltransferase 1 (DNMT1) in human cancer resulted in growth arrest. Removal of the inducer, doxycycline, from treated cells led to re-expression of the targeted gene. Thus the method allows for a highly controlled approach to gene knockdown. PMID:12888529

Neonatal mucosal immunization with a non-living, non-genetically modified Lactococcus lactis vaccine carrier induces systemic and local Th1-type immunity and protects against lethal bacterial infection

PubMed Central

Ramirez, Karina; Ditamo, Yanina; Rodriguez, Liliana; Picking, Wendy L.; van Roosmalen, Maarten L.; Leenhouts, Kees; Pasetti, Marcela F.

2010-01-01

Safe and effective immunization of newborns and infants can significantly reduce childhood mortality, yet conventional vaccines have been largely unsuccessful in stimulating the neonatal immune system. We explored the capacity of a novel mucosal antigen delivery system consisting of non-living, non-genetically modified Lactococcus lactis particles, designated Gram-positive Enhancer Matrix (GEM), to induce immune responses in the neonatal setting. Yersinia pestis LcrV, used as model protective antigen, was displayed on the GEM particles. Newborn mice immunized intranasally with GEM-LcrV developed LcrV-specific antibodies, Th1-type cell-mediated immunity, and were protected against lethal Y. pestis (plague) infection. The GEM particles activated and enhanced the maturation of neonatal dendritic cells both in vivo and in vitro. These dendritic cells showed increased capacities for secretion of pro-inflammatory and Th1-cell polarizing cytokines, antigen presentation and stimulation of CD4+ and CD8+ T cells. These data show that mucosal immunization with L. lactis GEM particles carrying vaccine antigens represents a promising approach to prevent infectious diseases early in life. PMID:19924118

A genetic screen for temperature-sensitive cell-division mutants of Caenorhabditis elegans.

PubMed Central

O'Connell, K F; Leys, C M; White, J G

1998-01-01

A novel screen to isolate conditional cell-division mutants in Caenorhabditis elegans has been developed. The screen is based on the phenotypes associated with existing cell-division mutations: some disrupt postembryonic divisions and affect formation of the gonad and ventral nerve cord-resulting in sterile, uncoordinated animals-while others affect embryonic divisions and result in lethality. We obtained 19 conditional mutants that displayed these phenotypes when shifted to the restrictive temperature at the appropriate developmental stage. Eighteen of these mutations have been mapped; 17 proved to be single alleles of newly identified genes, while 1 proved to be an allele of a previously identified gene. Genetic tests on the embryonic lethal phenotypes indicated that for 13 genes, embryogenesis required maternal expression, while for 6, zygotic expression could suffice. In all cases, maternal expression of wild-type activity was found to be largely sufficient for embryogenesis. Cytological analysis revealed that 10 mutants possessed embryonic cell-division defects, including failure to properly segregate DNA, failure to assemble a mitotic spindle, late cytokinesis defects, prolonged cell cycles, and improperly oriented mitotic spindles. We conclude that this approach can be used to identify mutations that affect various aspects of the cell-division cycle. PMID:9649522

The locus Om, responsible for the DDK syndrome, maps close to Sigje on mouse chromosome 11.

PubMed

Baldacci, P A; Richoux, V; Renard, J P; Guénet, J L; Babinet, C

1992-01-01

The DDK inbred strain of mouse has a striking particularity: when DDK females are crossed to males of other strains they exhibit a reduced fertility, whereas the reciprocal crosses (non-DDK females x DDK males) are fertile (Wakasugi et al. 1967; Wakasugi 1973). The low fertility results from an early embryonic lethality, the F1 embryos dying near the late morula-early blastocyst stage. Genetic analyses (Wakasugi 1974) and nuclear and cytoplasmic transfers (Renard and Babinet 1986; Babinet et al. 1990; Mann 1986), have shown that the failure of the embroys to develop is due to an incompatibility between a DDK maternally encoded cytoplasmic product and the non-DDK paternal genome. In order to elucidate the genetic determinism of this embryonic lethality, we have analyzed the fertility of male progeny from a backcross BALB/c females x (BALB/c x DDK)F1 males and that of males from a set of recombinant inbred (RI) strains, established from DDK and BALB/c progenitors, when mated with DDK females. Our results indicate that a single locus, Om, is responsible for the DDK syndrome and is located on Chromosome (Chr) 11, very close to the Sigje locus.

Scalability of Robotic Controllers: An Evaluation of Controller Options-Experiment II

DTIC Science & Technology

2011-09-01

for the Soldier, to ensure mission success while maximizing the survivability and lethality through the synergistic interaction of equipment...based touch interface for gloved finger interactions . This interface had to have larger-than-normal touch-screen buttons for commanding the robot...C.; Hill, S.; Pillalamarri, K. Extreme Scalability: Designing Interfaces and Algorithms for Soldier-Robotic Swarm Interaction , Year 2; ARL- TR

Arginine-glycine-aspartic acid motif is critical for human parechovirus 1 entry.

PubMed

Boonyakiat, Y; Hughes, P J; Ghazi, F; Stanway, G

2001-10-01

The human parechovirus 1 RGD motif in VP1 was studied by mutagenesis. An RGD-to-RGE change gave only revertant viruses with a restored RGD, while deletion of GD was lethal and nonrevertable. Mutations at the +1 and +2 positions had some effect on growth properties and a +1 M-to-P change was lethal. These studies indicate that the RGD motif plays a critical role in infectivity, presumably by interacting with integrins, and that downstream amino acids can have an influence on function.

Effects of BCG infection on the susceptibility of mouse macrophages to endotoxin.

PubMed Central

Peavy, D L; Baughn, R E; Musher, D M; Musher, D M

1979-01-01

Mice infected intravenously with Mycobacterium bovis (BCG) are 100 to 1,000 times more sensitive to the lethal effects of bacterial lipopolysaccharides (LPS). Since BCG infection results in macrophage activation and LPS may cause pathophysiological effects through interaction with this cell type, it was of interest to determine whether macrophages from BCG-infected animals were more susceptible to the toxic effects of LPS in vitro. When LPS-susceptible, C57BL/6 mice were infected with BCG, a significant reduction in the 50% lethal dose of LPS was first observed after 7 days and persisted for several weeks. Macrophages from these animals had greatly increased susceptibility to LPS in vitro, which correlated with the development of acquired cellular resistance as determined by their ability to inhibit the growth of Listeria monocytogenes. In contrast, BCG infection of C3H/HeJ mice, a strain resistant to LPS, did not alter the 50% lethal dose of LPS for these animals or increase the sensitivity of their peritoneal macrophages to LPS in vitro. These results indicate that susceptibility of BCG-infected mice to the lethal effects of LPS parallels the susceptibility of their macrophages in vitro; release of vasoactive substances from LPS-susceptible activated macrophages in vivo may be, in part, responsible for lethality. PMID:378847

The Yeast Gene, MDM20, Is Necessary for Mitochondrial Inheritance and Organization of the Actin Cytoskeleton

PubMed Central

Hermann, Greg J.; King, Edward J.; Shaw, Janet M.

1997-01-01

In Saccharomyces cerevisiae, the growing bud inherits a portion of the mitochondrial network from the mother cell soon after it emerges. Although this polarized transport of mitochondria is thought to require functions of the cytoskeleton, there are conflicting reports concerning the nature of the cytoskeletal element involved. Here we report the isolation of a yeast mutant, mdm20, in which both mitochondrial inheritance and actin cables (bundles of actin filaments) are disrupted. The MDM20 gene encodes a 93-kD polypeptide with no homology to other characterized proteins. Extra copies of TPM1, a gene encoding the actin filament–binding protein tropomyosin, suppress mitochondrial inheritance defects and partially restore actin cables in mdm20Δ cells. Synthetic lethality is also observed between mdm20 and tpm1 mutant strains. Overexpression of a second yeast tropomyosin, Tpm2p, rescues mutant phenotypes in the mdm20 strain to a lesser extent. Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process. MDM20 and TPM1 also exhibit the same pattern of genetic interactions; mutations in MDM20 are synthetically lethal with mutations in BEM2 and MYO2 but not SAC6. Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents. Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton. PMID:9105043

Exploiting the synthetic lethality between terminal respiratory oxidases to kill Mycobacterium tuberculosis and clear host infection

PubMed Central

Kalia, Nitin P.; Hasenoehrl, Erik J.; Ab Rahman, Nurlilah B.; Koh, Vanessa H.; Ang, Michelle L. T.; Sajorda, Dannah R.; Hards, Kiel; Grüber, Gerhard; Alonso, Sylvie; Cook, Gregory M.; Berney, Michael; Pethe, Kevin

2017-01-01

The recent discovery of small molecules targeting the cytochrome bc1:aa3 in Mycobacterium tuberculosis triggered interest in the terminal respiratory oxidases for antituberculosis drug development. The mycobacterial cytochrome bc1:aa3 consists of a menaquinone:cytochrome c reductase (bc1) and a cytochrome aa3-type oxidase. The clinical-stage drug candidate Q203 interferes with the function of the subunit b of the menaquinone:cytochrome c reductase. Despite the affinity of Q203 for the bc1:aa3 complex, the drug is only bacteriostatic and does not kill drug-tolerant persisters. This raises the possibility that the alternate terminal bd-type oxidase (cytochrome bd oxidase) is capable of maintaining a membrane potential and menaquinol oxidation in the presence of Q203. Here, we show that the electron flow through the cytochrome bd oxidase is sufficient to maintain respiration and ATP synthesis at a level high enough to protect M. tuberculosis from Q203-induced bacterial death. Upon genetic deletion of the cytochrome bd oxidase-encoding genes cydAB, Q203 inhibited mycobacterial respiration completely, became bactericidal, killed drug-tolerant mycobacterial persisters, and rapidly cleared M. tuberculosis infection in vivo. These results indicate a synthetic lethal interaction between the two terminal respiratory oxidases that can be exploited for anti-TB drug development. Our findings should be considered in the clinical development of drugs targeting the cytochrome bc1:aa3, as well as for the development of a drug combination targeting oxidative phosphorylation in M. tuberculosis. PMID:28652330

JIL-1 and Su(var)3-7 Interact Genetically and Counteract Each Other's Effect on Position-Effect Variegation in Drosophila

PubMed Central

Deng, Huai; Cai, Weili; Wang, Chao; Lerach, Stephanie; Delattre, Marion; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M.

2010-01-01

The essential JIL-1 histone H3S10 kinase is a key regulator of chromatin structure that functions to maintain euchromatic domains while counteracting heterochromatization and gene silencing. In the absence of the JIL-1 kinase, two of the major heterochromatin markers H3K9me2 and HP1a spread in tandem to ectopic locations on the chromosome arms. Here we address the role of the third major heterochromatin component, the zinc-finger protein Su(var)3-7. We show that the lethality but not the chromosome morphology defects associated with the null JIL-1 phenotype to a large degree can be rescued by reducing the dose of the Su(var)3-7 gene and that Su(var)3-7 and JIL-1 loss-of-function mutations have an antagonistic and counterbalancing effect on position-effect variegation (PEV). Furthermore, we show that in the absence of JIL-1 kinase activity, Su(var)3-7 gets redistributed and upregulated on the chromosome arms. Reducing the dose of the Su(var)3-7 gene dramatically decreases this redistribution; however, the spreading of H3K9me2 to the chromosome arms was unaffected, strongly indicating that ectopic Su(var)3-9 activity is not a direct cause of lethality. These observations suggest a model where Su(var)3-7 functions as an effector downstream of Su(var)3-9 and H3K9 dimethylation in heterochromatic spreading and gene silencing that is normally counteracted by JIL-1 kinase activity. PMID:20457875

Identification of a nucleotide in 5′ untranslated region contributing to virus replication and virulence of Coxsackievirus A16

PubMed Central

Li, Zhaolong; Liu, Xin; Wang, Shaohua; Li, Jingliang; Hou, Min; Liu, Guanchen; Zhang, Wenyan; Yu, Xiao-Fang

2016-01-01

Coxsackievirus A16 (CA16) and enterovirus 71 (EV71) are two main causative pathogens of hand, foot and mouth disease (HFMD). Unlike EV71, virulence determinants of CA16, particularly within 5′ untranslated region (5′UTR), have not been investigated until now. Here, a series of nucleotides present in 5′UTR of lethal but not in non-lethal CA16 strains were screened by aligning nucleotide sequences of lethal circulating Changchun CA16 and the prototype G10 as well as non-lethal SHZH05 strains. A representative infectious clone based on a lethal Changchun024 sequence and infectious mutants with various nucleotide alterations in 5′UTR were constructed and further investigated by assessing virus replication in vitro and virulence in neonatal mice. Compared to the lethal infectious clone, the M2 mutant with a change from cytosine to uracil at nucleotide 104 showed weaker virulence and lower replication capacity. The predicted secondary structure of the 5′UTR of CA16 RNA showed that M2 mutant located between the cloverleaf and stem-loop II, affected interactions between the 5′UTR and the heterogeneous nuclear ribonucleoprotein K (hnRNP K) and A1 (hnRNP A1) that are important for translational activity. Thus, our research determined a virulence-associated site in the 5′UTR of CA16, providing a crucial molecular target for antiviral drug development. PMID:26861413

Effective lethal mutagenesis of influenza virus by three nucleoside analogs.

PubMed

Pauly, Matthew D; Lauring, Adam S

2015-04-01

Lethal mutagenesis is a broad-spectrum antiviral strategy that exploits the high mutation rate and low mutational tolerance of many RNA viruses. This approach uses mutagenic drugs to increase viral mutation rates and burden viral populations with mutations that reduce the number of infectious progeny. We investigated the effectiveness of lethal mutagenesis as a strategy against influenza virus using three nucleoside analogs, ribavirin, 5-azacytidine, and 5-fluorouracil. All three drugs were active against a panel of seasonal H3N2 and laboratory-adapted H1N1 strains. We found that each drug increased the frequency of mutations in influenza virus populations and decreased the virus' specific infectivity, indicating a mutagenic mode of action. We were able to drive viral populations to extinction by passaging influenza virus in the presence of each drug, indicating that complete lethal mutagenesis of influenza virus populations can be achieved when a sufficient mutational burden is applied. Population-wide resistance to these mutagenic agents did not arise after serial passage of influenza virus populations in sublethal concentrations of drug. Sequencing of these drug-passaged viral populations revealed genome-wide accumulation of mutations at low frequency. The replicative capacity of drug-passaged populations was reduced at higher multiplicities of infection, suggesting the presence of defective interfering particles and a possible barrier to the evolution of resistance. Together, our data suggest that lethal mutagenesis may be a particularly effective therapeutic approach with a high genetic barrier to resistance for influenza virus. Influenza virus is an RNA virus that causes significant morbidity and mortality during annual epidemics. Novel therapies for RNA viruses are needed due to the ease with which these viruses evolve resistance to existing therapeutics. Lethal mutagenesis is a broad-spectrum strategy that exploits the high mutation rate and the low mutational tolerance of most RNA viruses. It is thought to possess a higher barrier to resistance than conventional antiviral strategies. We investigated the effectiveness of lethal mutagenesis against influenza virus using three different drugs. We showed that influenza virus was sensitive to lethal mutagenesis by demonstrating that all three drugs induced mutations and led to an increase in the generation of defective viral particles. We also found that it may be difficult for resistance to these drugs to arise at a population-wide level. Our data suggest that lethal mutagenesis may be an attractive anti-influenza strategy that warrants further investigation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Introgression and susceptibility to disease in a wild population of rainbow trout

USGS Publications Warehouse

Currens, K.P.; Hemmingsen, A.R.; French, R.A.; Buchanan, D.V.; Schreck, C.B.; Li, H.W.

1997-01-01

We examined susceptibility of wild rainbow trout Oncorhynchus mykiss from the Metolius River, a tributary of the Deschutes River, Oregon, to genetic introgression and ceratomyxosis as a result of stocking nonnative hatchery rainbow trout. Ceratomyxa shasta, an enzootic myxosporean parasite that can be lethal to nonnative hatchery rainbow trout, might have been limiting the interbreeding of hatchery and wild rainbow trout in the river. However, rainbow trout from the Metolius River had allozyme frequencies intermediate between those of wild and hatchery fish at LDH-B2* and sSOD-1*, two diagnostic genetic loci that allow the inland subspecies of rainbow trout to be distinguished from hatchery strains of coastal origin. They also had notable frequencies of ADA-1*85, an allele documented in hatchery rainbow trout but rarely seen in wild populations. We also found that rainbow trout in the Metolius River averaged 138.9 scales in the lateral series, intermediate between the counts for 9 coastal or nonnative hatchery populations, which always had fewer than 140 scales, and 10 inland populations, which always had more than 140 scales. Disease challenges revealed that rainbow trout from the Metolius River had much greater susceptibility to C. shasta than rainbow trout from the Deschutes River, which have genetic resistance to the lethal disease. Based on these data, we concluded that introgression with nonnative hatchery rainbow trout has reduced the abilities of wild rainbow trout in the Metolius River to survive when conditions for ceratomyxosis infection occur.

Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer

PubMed Central

Bortolussi, Giulia; Zentilin, Lorena; Baj, Gabriele; Giraudi, Pablo; Bellarosa, Cristina; Giacca, Mauro; Tiribelli, Claudio; Muro, Andrés F.

2012-01-01

Crigler-Najjar type I (CNI) syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by uridine diphosphoglucuronosyltransferase 1A1 (UGT1A1) deficiency. The disease is lethal due to bilirubin-induced neurological damage unless phototherapy is applied from birth. However, treatment becomes less effective during growth, and liver transplantation is required. To investigate the pathophysiology of the disease and therapeutic approaches in mice, we generated a mouse model by introducing a premature stop codon in the UGT1a1 gene, which results in an inactive enzyme. Homozygous mutant mice developed severe jaundice soon after birth and died within 11 d, showing significant cerebellar alterations. To rescue neonatal lethality, newborns were injected with a single dose of adeno-associated viral vector 9 (AAV9) expressing the human UGT1A1. Gene therapy treatment completely rescued all AAV-treated mutant mice, accompanied by lower plasma bilirubin levels and normal brain histology and motor coordination. Our mouse model of CNI reproduces genetic and phenotypic features of the human disease. We have shown, for the first time, the full recovery of the lethal effects of neonatal hyperbilirubinemia. We believe that, besides gene-addition-based therapies, our mice could represent a very useful model to develop and test novel technologies based on gene correction by homologous recombination.—Bortolussi, G., Zentilin, L., Baj, G., Giraudi, P., Bellarosa, C., Giacca, M., Tiribelli, C., Muro, A. F. Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer. PMID:22094718

Epidermolysis bullosa with congenital pyloric atresia: novel mutations in the beta 4 integrin gene (ITGB4) and genotype/phenotype correlations.

PubMed

Nakano, A; Pulkkinen, L; Murrell, D; Rico, J; Lucky, A W; Garzon, M; Stevens, C A; Robertson, S; Pfendner, E; Uitto, J

2001-05-01

Epidermolysis bullosa with pyloric atresia (EB-PA: OMIM 226730), also known as Carmi syndrome, is a rare autosomal recessive genodermatosis that manifests with neonatal mucocutaneous fragility associated with congenital pyloric atresia. The disease is frequently lethal within the first year, but nonlethal cases have been reported. Mutations in the genes encoding subunit polypeptides of the alpha 6 beta 4 integrin (ITGA6 and ITGB4) have been demonstrated in EB-PA patients. To extend the repertoire of mutations and to identify genotype-phenotype correlations, we examined seven new EB-PA families, four with lethal and three with nonlethal disease variants. DNA from patients was screened for mutations using heteroduplex analysis followed by nucleotide sequencing of PCR products spanning all beta 4 integrin-coding sequences. Mutation analysis disclosed 12 distinct mutations, 11 of them novel. Four mutations predicted a premature termination codon as a result of nonsense mutations or small out-of-frame insertions or deletions, whereas seven were missense mutations. This brings the total number of distinct ITGB4 mutations to 33. The mutation database indicates that premature termination codons are associated predominantly with the lethal EB-PA variants, whereas missense mutations are more prevalent in nonlethal forms. However, the consequences of the missense mutations are position dependent, and substitutions of highly conserved amino acids may have lethal consequences. In general, indirect immunofluorescence studies of affected skin revealed negative staining for beta 4 integrin in lethal cases and positive, but attenuated, staining in nonlethal cases and correlated with clinical phenotype. The data on specific mutations in EB-PA patients allows prenatal testing and preimplantation genetic diagnosis in families at risk.

Relation of the Allelic Variants of Multidrug Resistance Gene to Agranulocytosis Associated With Clozapine.

PubMed

Anıl Yağcioğlu, A Elif; Yoca, Gökhan; Ayhan, Yavuz; Karaca, R Özgür; Çevik, Lokman; Müderrisoğlu, Ahmet; Göktaş, Mustafa T; Eni, Nurhayat; Yazıcı, M Kâzım; Bozkurt, Atilla; Babaoğlu, Melih O

2016-06-01

Clozapine use is associated with leukopenia and more rarely agranulocytosis, which may be lethal. The drug and its metabolites are proposed to interact with the multidrug resistance transporter (ABCB1/MDR1) gene product, P-glycoprotein (P-gp). Among various P-glycoprotein genetic polymorphisms, nucleotide changes in exons 26 (C3435T), 21 (G2677T), and 12 (C1236T) have been implicated for changes in pharmacokinetics and pharmacodynamics of many substrate drugs. In this study, we aimed to investigate the association between these specific ABCB1 polymorphisms and clozapine-associated agranulocytosis (CAA). Ten patients with a history of CAA and 91 control patients without a history of CAA, despite 10 years of continuous clozapine use, were included. Patient recruitment and blood sample collection were conducted at the Hacettepe University Faculty of Medicine, Department of Psychiatry, in collaboration with the members of the Schizophrenia and Other Psychotic Disorders Section of the Psychiatric Association of Turkey, working in various psychiatry clinics. After DNA extraction from peripheral blood lymphocytes, genotyping was performed using polymerase chain reaction and endonuclease digestion. Patients with CAA had shorter duration of clozapine use but did not show any significant difference in other clinical, sociodemographic characteristics and in genotypic or allelic distributions of ABCB1 variants and haplotypes compared with control patients. Among the 10 patients with CAA, none carried the ABCB1 all-variant haplotype (TT-TT-TT), whereas the frequency of this haplotype was approximately 12% among the controls. Larger sample size studies and thorough genetic analyses may reveal both genetic risk and protective factors for this serious adverse event.

Imp2, the PSTPIP homolog in fission yeast, affects sensitivity to the immunosuppressant FK506 and membrane trafficking in fission yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kita, Ayako; Higa, Mari; Doi, Akira

Cytokinesis is a highly ordered process that divides one cell into two cells, which is functionally linked to the dynamic remodeling of the plasma membrane coordinately with various events such as membrane trafficking. Calcineurin is a highly conserved serine/threonine protein phosphatase, which regulates multiple biological functions, such as membrane trafficking and cytokinesis. Here, we isolated imp2-c3, a mutant allele of the imp2{sup +} gene, encoding a homolog of the mouse PSTPIP1 (proline-serine-threonine phosphatase interacting protein 1), using a genetic screen for mutations that are synthetically lethal with calcineurin deletion in fission yeast. The imp2-c3 mutants showed a defect in cytokinesis withmore » multi-septated phenotypes, which was further enhanced upon treatment with the calcineurin inhibitor FK506. Notably, electron micrographs revealed that the imp2-c3 mutant cells accumulated aberrant multi-lamella Golgi structures and putative post-Golgi secretory vesicles, and exhibited fragmented vacuoles in addition to thickened septa. Consistently, imp2-c3 mutants showed a reduced secretion of acid phosphatase and defects in vacuole fusion. The imp2-c3 mutant cells exhibited a weakened cell wall, similar to the membrane trafficking mutants identified in the same genetic screen such as ypt3-i5. These findings implicate the PSTPIP1 homolog Imp2 in Golgi/vacuole function, thereby affecting various cellular processes, including cytokinesis and cell integrity. - Highlights: • We isolated imp2-c3, in a synthetic lethal screen with calcineurin in fission yeast. • The imp2{sup +} gene encodes a component of the actin contractile ring similar to Cdc15. • The imp2-c3 mutants showed defects in cytokinesis, which were exacerbated by FK506. • The imp2-c3 mutants were defective in membrane trafficking and cell wall integrity. • Our study revealed a novel role for Imp2 in the Golgi/vacuolar membrane trafficking.« less

Discovery of an ebolavirus-like filovirus in europe.

PubMed

Negredo, Ana; Palacios, Gustavo; Vázquez-Morón, Sonia; González, Félix; Dopazo, Hernán; Molero, Francisca; Juste, Javier; Quetglas, Juan; Savji, Nazir; de la Cruz Martínez, Maria; Herrera, Jesus Enrique; Pizarro, Manuel; Hutchison, Stephen K; Echevarría, Juan E; Lipkin, W Ian; Tenorio, Antonio

2011-10-01

Filoviruses, amongst the most lethal of primate pathogens, have only been reported as natural infections in sub-Saharan Africa and the Philippines. Infections of bats with the ebolaviruses and marburgviruses do not appear to be associated with disease. Here we report identification in dead insectivorous bats of a genetically distinct filovirus, provisionally named Lloviu virus, after the site of detection, Cueva del Lloviu, in Spain.

Clonal Evaluation of Prostate Cancer by ERG/SPINK1 Status to Improve Prognosis Prediction

DTIC Science & Technology

2016-10-01

clinical oncology , cancer genetics, genomic science/bioinformatics, clinical pathology, social and behavioral sciences, and bioethics in order to...Interpret and translate sequence variants into clinical oncology setting; 5) Assess and evaluate costs associated with clinical sequencing. Role...Lethal Prostate Cancer Goal(s): Radiation Therapy Oncology Group (RTOG) 96-01 represents a phase III trial of of salvage radiation therapy (RT) alone

Discovery of an Ebolavirus-Like Filovirus in Europe

PubMed Central

Vázquez-Morón, Sonia; González, Félix; Dopazo, Hernán; Molero, Francisca; Juste, Javier; Quetglas, Juan; Savji, Nazir; de la Cruz Martínez, Maria; Herrera, Jesus Enrique; Pizarro, Manuel; Hutchison, Stephen K.; Echevarría, Juan E.; Lipkin, W. Ian; Tenorio, Antonio

2011-01-01

Filoviruses, amongst the most lethal of primate pathogens, have only been reported as natural infections in sub-Saharan Africa and the Philippines. Infections of bats with the ebolaviruses and marburgviruses do not appear to be associated with disease. Here we report identification in dead insectivorous bats of a genetically distinct filovirus, provisionally named Lloviu virus, after the site of detection, Cueva del Lloviu, in Spain. PMID:22039362

Mechanism of Innate Resistance to Viral Encephalitis.

DTIC Science & Technology

1976-12-01

contrast, local inhabitants would likely be immune to infection from previous contact. Moreover, effective vaccination procedures for most neurotropic...individuals and, 3) developing a rational procedure for vaccin - ation. Prevailing explanations for innate genetic resistance to lethal flaviviral...He mice infected the same way (Table 7). We began to examine the responses of He and RV mice to immunization with formalinized Banzi virus vaccine (BV

Invasive Threats to the American Homeland

DTIC Science & Technology

2004-04-01

The second example is the glassy-winged sharpshooter, an invasive insect that hosts the bacterium Xylella Fastidiosa . The insect was first de- tected... Xylella Fastidiosa causes Pierce’s Disease in grapes, which infects and kills the grapevine. The glassy-winged sharpshooter transmits and spreads the...netic processes.” Adversaries of the United States may modify the genetics of an invasive species to increase its competitiveness, virulence , lethality

Invasive Species - A Threat to the Homeland?

DTIC Science & Technology

2003-04-07

sharpshooter, an invasive insect that hosts the bacterium Xylella Fastidiosa . The insect was first detected in California in 1990. Although it is...uncertain how it arrived in California, it is believed to have arrived on plants imported from an infected area. The bacterium Xylella Fastidiosa causes...United States may modify the genetics of an invasive species to increase its competitiveness, virulence , lethality, or resistance to control measures

Progressive adaptation of a Georgian isolate of African swine fever virus to vero cells leads to a gradual attenuation of virulence in swine corresponding to major changes of the viral genome

USDA-ARS?s Scientific Manuscript database

African swine fever virus (ASFV) causes a contagious and often lethal disease of feral and domestic swine. Experimental vaccines derived from naturally occurring, genetically modified or cell culture-adapted ASFV have been evaluated but no commercial vaccine is available to control African Swine Fev...

Chemical, Biochemical, and Genetic Approaches to Arsenic Metabolism" -An overview of arsenic metabolism and toxicity- A series of five papers to appear together in Current Protocols in Toxicology

EPA Science Inventory

The toxic properties of arsenic (As) were recognized long before Albertus Magnus in the 13th century prepared its elemental form (Buchanan, 1962). Its use as a poison has played lethal and decisive roles in domestic and dynastic intrigues throughout history (Cullen, 2008). Inorga...

The App-Runx1 Region Is Critical for Birth Defects and Electrocardiographic Dysfunctions Observed in a Down Syndrome Mouse Model

PubMed Central

Raveau, Matthieu; Lignon, Jacques M.; Nalesso, Valérie; Duchon, Arnaud; Groner, Yoram; Sharp, Andrew J.; Dembele, Doulaye; Brault, Véronique; Hérault, Yann

2012-01-01

Down syndrome (DS) leads to complex phenotypes and is the main genetic cause of birth defects and heart diseases. The Ts65Dn DS mouse model is trisomic for the distal part of mouse chromosome 16 and displays similar features with post-natal lethality and cardiovascular defects. In order to better understand these defects, we defined electrocardiogram (ECG) with a precordial set-up, and we found conduction defects and modifications in wave shape, amplitudes, and durations in Ts65Dn mice. By using a genetic approach consisting of crossing Ts65Dn mice with Ms5Yah mice monosomic for the App-Runx1 genetic interval, we showed that the Ts65Dn viability and ECG were improved by this reduction of gene copy number. Whole-genome expression studies confirmed gene dosage effect in Ts65Dn, Ms5Yah, and Ts65Dn/Ms5Yah hearts and showed an overall perturbation of pathways connected to post-natal lethality (Coq7, Dyrk1a, F5, Gabpa, Hmgn1, Pde10a, Morc3, Slc5a3, and Vwf) and heart function (Tfb1m, Adam19, Slc8a1/Ncx1, and Rcan1). In addition cardiac connexins (Cx40, Cx43) and sodium channel sub-units (Scn5a, Scn1b, Scn10a) were found down-regulated in Ts65Dn atria with additional down-regulation of Cx40 in Ts65Dn ventricles and were likely contributing to conduction defects. All these data pinpoint new cardiac phenotypes in the Ts65Dn, mimicking aspects of human DS features and pathways altered in the mouse model. In addition they highlight the role of the App-Runx1 interval, including Sod1 and Tiam1, in the induction of post-natal lethality and of the cardiac conduction defects in Ts65Dn. These results might lead to new therapeutic strategies to improve the care of DS people. PMID:22693452

Heterozygous Ambra1 Deficiency in Mice: A Genetic Trait with Autism-Like Behavior Restricted to the Female Gender

PubMed Central

Dere, Ekrem; Dahm, Liane; Lu, Derek; Hammerschmidt, Kurt; Ju, Anes; Tantra, Martesa; Kästner, Anne; Chowdhury, Kamal; Ehrenreich, Hannelore

2014-01-01

Autism-spectrum disorders (ASD) are heterogeneous, highly heritable neurodevelopmental conditions affecting around 0.5% of the population across cultures, with a male/female ratio of approximately 4:1. Phenotypically, ASD are characterized by social interaction and communication deficits, restricted interests, repetitive behaviors, and reduced cognitive flexibility. Identified causes converge at the level of the synapse, ranging from mutation of synaptic genes to quantitative alterations in synaptic protein expression, e.g., through compromised transcriptional or translational control. We wondered whether reduced turnover and degradation of synapses, due to deregulated autophagy, would lead to similar phenotypical consequences. Ambra1, strongly expressed in cortex, hippocampus, and striatum, is a positive regulator of Beclin1, a principal player in autophagosome formation. While homozygosity of the Ambra1 null mutation causes embryonic lethality, heterozygous mice with reduced Ambra1 expression are viable, reproduce normally, and lack any immediately obvious phenotype. Surprisingly, comprehensive behavioral characterization of these mice revealed an autism-like phenotype in Ambra1+/− females only, including compromised communication and social interactions, a tendency of enhanced stereotypies/repetitive behaviors, and impaired cognitive flexibility. Reduced ultrasound communication was found in adults as well as pups, which achieved otherwise normal neurodevelopmental milestones. These features were all absent in male Ambra1+/− mice. As a first hint explaining this gender difference, we found a much stronger reduction of Ambra1 protein in the cortex of Ambra1+/− females compared to males. To conclude, Ambra1 deficiency can induce an autism-like phenotype. The restriction to the female gender of autism-generation by a defined genetic trait is unique thus far and warrants further investigation. PMID:24904333

Heterozygous ambra1 deficiency in mice: a genetic trait with autism-like behavior restricted to the female gender.

PubMed

Dere, Ekrem; Dahm, Liane; Lu, Derek; Hammerschmidt, Kurt; Ju, Anes; Tantra, Martesa; Kästner, Anne; Chowdhury, Kamal; Ehrenreich, Hannelore

2014-01-01

Autism-spectrum disorders (ASD) are heterogeneous, highly heritable neurodevelopmental conditions affecting around 0.5% of the population across cultures, with a male/female ratio of approximately 4:1. Phenotypically, ASD are characterized by social interaction and communication deficits, restricted interests, repetitive behaviors, and reduced cognitive flexibility. Identified causes converge at the level of the synapse, ranging from mutation of synaptic genes to quantitative alterations in synaptic protein expression, e.g., through compromised transcriptional or translational control. We wondered whether reduced turnover and degradation of synapses, due to deregulated autophagy, would lead to similar phenotypical consequences. Ambra1, strongly expressed in cortex, hippocampus, and striatum, is a positive regulator of Beclin1, a principal player in autophagosome formation. While homozygosity of the Ambra1 null mutation causes embryonic lethality, heterozygous mice with reduced Ambra1 expression are viable, reproduce normally, and lack any immediately obvious phenotype. Surprisingly, comprehensive behavioral characterization of these mice revealed an autism-like phenotype in Ambra1 (+/-) females only, including compromised communication and social interactions, a tendency of enhanced stereotypies/repetitive behaviors, and impaired cognitive flexibility. Reduced ultrasound communication was found in adults as well as pups, which achieved otherwise normal neurodevelopmental milestones. These features were all absent in male Ambra1 (+/-) mice. As a first hint explaining this gender difference, we found a much stronger reduction of Ambra1 protein in the cortex of Ambra1 (+/-) females compared to males. To conclude, Ambra1 deficiency can induce an autism-like phenotype. The restriction to the female gender of autism-generation by a defined genetic trait is unique thus far and warrants further investigation.

Context-dependent interactive effects of non-lethal predation on larvae impact adult longevity and body composition.

PubMed

Chandrasegaran, Karthikeyan; Kandregula, Samyuktha Rao; Quader, Suhel; Juliano, Steven A

2018-01-01

Predation impacts development, behavior and morphology of prey species thereby shaping their abundances, distribution and community structure. Non-lethal threat of predation, specifically, can have a strong influence on prey lifehistory characteristics. While investigations often focus on the impact of predation threat on prey in isolation, tests of its interactive effects with food availability and resource competition on prey survival and fitness can improve understanding of costs, benefits and trade-offs of anti-predator strategies. This study, involving Aedes aegypti mosquitoes as a model organism, investigates both simple and interactive effects of predation threat during the larval stage on survival, size at and time to maturity, stored teneral reserves of glycogen, protein and lipid in adults, and adult longevity. Our results show that development times of mosquito larvae were increased (by 14.84% in males and by 97.63% in females), and size of eclosing adults decreased (by 62.30% in males and by 58.33% in females) when exposed to lowered nutrition and elevated intraspecific competition, but that predation had no detectable effect on these simple traits. Teneral reserves of glycogen, protein and lipid and adult longevity were positively correlated with adult body size. Non-lethal predation threat had significant interactive effects with nutrition and larval competition on teneral reserves in males and adult longevity in males and females. The sexes responded differently to conditions encountered as larvae, with the larval environment affecting development and adult characteristics more acutely for females than for males. The outcome of this study shows how threat of predation on juveniles can have long-lasting effects on adults that are likely to impact mosquito population dynamics and that may impact disease transmission.

Chemical genomics in plant biology.

PubMed

Sadhukhan, Ayan; Sahoo, Lingaraj; Panda, Sanjib Kumar

2012-06-01

Chemical genomics is a newly emerged and rapidly progressing field in biology, where small chemical molecules bind specifically and reversibly to protein(s) to modulate their function(s), leading to the delineation and subsequent unravelling of biological processes. This approach overcomes problems like lethality and redundancy of classical genetics. Armed with the powerful techniques of combinatorial synthesis, high-throughput screening and target discovery chemical genomics expands its scope to diverse areas in biology. The well-established genetic system of Arabidopsis model allows chemical genomics to enter into the realm of plant biology exploring signaling pathways of growth regulators, endomembrane signaling cascades, plant defense mechanisms and many more events.

Transferability of MCR-1/2 Polymyxin Resistance: Complex Dissemination and Genetic Mechanism.

PubMed

Feng, Youjun

2018-03-09

Polymyxins, a group of cationic antimicrobial polypeptides, act as a last-resort defense against lethal infections by carbapenem-resistant Gram-negative pathogens. Recent emergence and fast spread of mobilized colistin resistance determinant mcr-1 argue the renewed interest of colistin in clinical therapies, threatening global public health and agriculture production. This mini-review aims to present an updated overview of mcr-1, covering its global dissemination, the diversity of its hosts/plasmid reservoirs, the complexity in the genetic environment adjacent to mcr-1, the appearance of new mcr-like genes, and the molecular mechanisms for mobilized colistin resistance determinant 1/2 (MCR-1/2).

Overview of Genetically Engineered Mouse Models of Papillary and Anaplastic Thyroid Cancers: Enabling Translational Biology for Patient Care Improvement.

PubMed

Charles, Roch-Philippe

2015-06-01

The prognosis from thyroid cancer subtypes in humans covers a spectrum from "cured at almost 90%" to "100% lethal." Invasive and poorly differentiated forms of thyroid cancer are among the most aggressive human cancers, and there are few effective therapeutic options. Genetically engineered mice, based on mutations observed in patients, can accurately recapitulate the human disease and its progression, providing invaluable tools for the preclinical evaluation of novel therapeutic approaches. This overview details models developed to date as well as their uses for identifying novel anticancer agents. Copyright © 2013 John Wiley & Sons, Inc. All rights reserved.

Therapy-Related Myeloid Neoplasms: When Genetics and Environment Collide

PubMed Central

McNerney, Megan E.; Godley, Lucy A.; Le Beau, Michelle M.

2018-01-01

Therapy-related myeloid neoplasms (t-MN) arise as a late-effect of chemotherapy and/or radiation administered for a primary condition, typically a malignant disease, solid organ transplant, or autoimmune disease. Survival is measured in months, not years, making t-MN one of the most aggressive and lethal cancers. In this review, we discuss recent developments that reframe our understanding of the genetic and environmental etiology of t-MN. Emerging data illuminate who is at highest risk for developing t-MN, why t-MN is chemoresistant, and how we may use this information to treat and ultimately prevent this dreaded disease. PMID:28835720

Exposure to Glycolytic Carbon Sources Reveals a Novel Layer of Regulation for the MalT Regulon

PubMed Central

Reimann, Sylvia A.; Wolfe, Alan J.

2011-01-01

Bacteria adapt to changing environments by means of tightly coordinated regulatory circuits. The use of synthetic lethality, a genetic phenomenon in which the combination of two nonlethal mutations causes cell death, facilitates identification and study of such circuitry. In this study, we show that the E. coli ompR malT con double mutant exhibits a synthetic lethal phenotype that is environmentally conditional. MalTcon, the constitutively active form of the maltose system regulator MalT, causes elevated expression of the outer membrane porin LamB, which leads to death in the absence of the osmoregulator OmpR. However, the presence and metabolism of glycolytic carbon sources, such as sorbitol, promotes viability and unveils a novel layer of regulation within the complex circuitry that controls maltose transport and metabolism. PMID:21912549

Exposure to Glycolytic Carbon Sources Reveals a Novel Layer of Regulation for the MalT Regulon.

PubMed

Reimann, Sylvia A; Wolfe, Alan J

2011-01-01

Bacteria adapt to changing environments by means of tightly coordinated regulatory circuits. The use of synthetic lethality, a genetic phenomenon in which the combination of two nonlethal mutations causes cell death, facilitates identification and study of such circuitry. In this study, we show that the E. coli ompR malT(con) double mutant exhibits a synthetic lethal phenotype that is environmentally conditional. MalT(con), the constitutively active form of the maltose system regulator MalT, causes elevated expression of the outer membrane porin LamB, which leads to death in the absence of the osmoregulator OmpR. However, the presence and metabolism of glycolytic carbon sources, such as sorbitol, promotes viability and unveils a novel layer of regulation within the complex circuitry that controls maltose transport and metabolism.

Inferring the distribution of mutational effects on fitness in Drosophila.

PubMed

Loewe, Laurence; Charlesworth, Brian

2006-09-22

The properties of the distribution of deleterious mutational effects on fitness (DDME) are of fundamental importance for evolutionary genetics. Since it is extremely difficult to determine the nature of this distribution, several methods using various assumptions about the DDME have been developed, for the purpose of parameter estimation. We apply a newly developed method to DNA sequence polymorphism data from two Drosophila species and compare estimates of the parameters of the distribution of the heterozygous fitness effects of amino acid mutations for several different distribution functions. The results exclude normal and gamma distributions, since these predict too few effectively lethal mutations and power-law distributions as a result of predicting too many lethals. Only the lognormal distribution appears to fit both the diversity data and the frequency of lethals. This DDME arises naturally in complex systems when independent factors contribute multiplicatively to an increase in fitness-reducing damage. Several important parameters, such as the fraction of effectively neutral non-synonymous mutations and the harmonic mean of non-neutral selection coefficients, are robust to the form of the DDME. Our results suggest that the majority of non-synonymous mutations in Drosophila are under effective purifying selection.

The Wiggle Index: An Open Source Bioassay to Assess Sub-Lethal Insecticide Response in Drosophila melanogaster

PubMed Central

Denecke, Shane; Nowell, Cameron J.; Fournier-Level, Alexandre; Perry, Trent; Batterham, Phil

2015-01-01

Toxicological assays measuring mortality are routinely used to describe insecticide response, but sub-lethal exposures to insecticides can select for resistance and yield additional biological information describing the ways in which an insecticide impacts the insect. Here we present the Wiggle Index (WI), a high-throughput method to quantify insecticide response by measuring the reduction in motility during sub-lethal exposures in larvae of the vinegar fly Drosophila melanogaster. A susceptible wild type strain was exposed to the insecticides chlorantraniliprole, imidacloprid, spinosad, and ivermectin. Each insecticide reduced larval motility, but response times and profiles differed among insecticides. Two sets of target site mutants previously identified in mortality studies on the basis of imidacloprid or spinosad resistance phenotypes were tested. In each case the resistant mutant responded significantly less than the control. The WI was also able to detect a spinosad response in the absence of the primary spinosad target site. This response was not detected in mortality assays suggesting that spinosad, like many other insecticides, may have secondary targets affecting behaviour. The ability of the WI to detect changes in insecticide metabolism was confirmed by overexpressing the imidacloprid metabolizing Cyp6g1 gene in digestive tissues or the central nervous system. The data presented here validate the WI as an inexpensive, generic, sub-lethal assay that can complement information gained from mortality assays, extending our understanding of the genetic basis of insecticide response in D. melanogaster. PMID:26684454

A Strong Loss-of-Function Mutation in RAN1 Results in Constitutive Activation of the Ethylene Response Pathway as Well as a Rosette-Lethal Phenotype

PubMed Central

Woeste, Keith E.; Kieber, Joseph J.

2000-01-01

A recessive mutation was identified that constitutively activated the ethylene response pathway in Arabidopsis and resulted in a rosette-lethal phenotype. Positional cloning of the gene corresponding to this mutation revealed that it was allelic to responsive to antagonist1 (ran1), a mutation that causes seedlings to respond in a positive manner to what is normally a competitive inhibitor of ethylene binding. In contrast to the previously identified ran1-1 and ran1-2 alleles that are morphologically indistinguishable from wild-type plants, this ran1-3 allele results in a rosette-lethal phenotype. The predicted protein encoded by the RAN1 gene is similar to the Wilson and Menkes disease proteins and yeast Ccc2 protein, which are integral membrane cation-transporting P-type ATPases involved in copper trafficking. Genetic epistasis analysis indicated that RAN1 acts upstream of mutations in the ethylene receptor gene family. However, the rosette-lethal phenotype of ran1-3 was not suppressed by ethylene-insensitive mutants, suggesting that this mutation also affects a non-ethylene-dependent pathway regulating cell expansion. The phenotype of ran1-3 mutants is similar to loss-of-function ethylene receptor mutants, suggesting that RAN1 may be required to form functional ethylene receptors. Furthermore, these results suggest that copper is required not only for ethylene binding but also for the signaling function of the ethylene receptors. PMID:10715329

A strong loss-of-function mutation in RAN1 results in constitutive activation of the ethylene response pathway as well as a rosette-lethal phenotype

NASA Technical Reports Server (NTRS)

Woeste, K. E.; Kieber, J. J.; Evans, M. L. (Principal Investigator)

2000-01-01

A recessive mutation was identified that constitutively activated the ethylene response pathway in Arabidopsis and resulted in a rosette-lethal phenotype. Positional cloning of the gene corresponding to this mutation revealed that it was allelic to responsive to antagonist1 (ran1), a mutation that causes seedlings to respond in a positive manner to what is normally a competitive inhibitor of ethylene binding. In contrast to the previously identified ran1-1 and ran1-2 alleles that are morphologically indistinguishable from wild-type plants, this ran1-3 allele results in a rosette-lethal phenotype. The predicted protein encoded by the RAN1 gene is similar to the Wilson and Menkes disease proteins and yeast Ccc2 protein, which are integral membrane cation-transporting P-type ATPases involved in copper trafficking. Genetic epistasis analysis indicated that RAN1 acts upstream of mutations in the ethylene receptor gene family. However, the rosette-lethal phenotype of ran1-3 was not suppressed by ethylene-insensitive mutants, suggesting that this mutation also affects a non-ethylene-dependent pathway regulating cell expansion. The phenotype of ran1-3 mutants is similar to loss-of-function ethylene receptor mutants, suggesting that RAN1 may be required to form functional ethylene receptors. Furthermore, these results suggest that copper is required not only for ethylene binding but also for the signaling function of the ethylene receptors.

Physiological response and microRNA expression profiles in head kidney of genetically improved farmed tilapia (GIFT, Oreochromis niloticus) exposed to acute cold stress.

PubMed

Qiang, Jun; Cui, Yan T; Tao, Fan Y; Bao, Wen J; He, Jie; Li, Xia H; Xu, Pao; Sun, Lan Y

2018-01-09

Cold stress has a serious impact on the overwintering survival and yield of genetically improved farmed tilapia (GIFT, Oreochromis niloticus). Understanding the physiological and molecular regulation mechanisms of low-temperature adaptation is necessary to help breed new tolerant strains. The semi-lethal low temperature of juvenile GIFT at 96 h was determined as 9.4 °C. We constructed and sequenced two small RNA libraries from head kidney tissues, one for the control (CO) group and one for the 9.4 °C-stressed (LTS) group, and identified 1736 and 1481 known microRNAs (miRNAs), and 164 and 152 novel miRNAs in the CO and LTS libraries, respectively. We verify the expression of nine up-regulated miRNAs and eight down-regulation miRNAs by qRT-PCR, and found their expression patterns were consistent with the sequencing results. We found that cold stress may have produced dysregulation of free radical and lipid metabolism, decreased superoxide dismutase activity, reduced respiratory burst and phagocytic activity of macrophages, increased malondialdehyde content, and adversely affected the physiological adaptation of GIFT, eventually leading to death. This study revealed interactions among miRNAs and signal regulated pathways in GIFT under cold stress that may help to understand the pathways involved in cold resistance.

DNA repair targeted therapy: the past or future of cancer treatment?

PubMed Central

Gavande, Navnath S.; VanderVere-Carozza, Pamela S.; Hinshaw, Hilary D.; Jalal, Shadia I.; Sears, Catherine R.; Pawelczak, Katherine S.; Turchi, John J.

2016-01-01

The repair of DNA damage is a complex process that relies on particular pathways to remedy specific types of damage to DNA. The range of insults to DNA includes small, modest changes in structure including mismatched bases and simple methylation events to oxidized bases, intra- and interstrand DNA crosslinks, DNA double strand breaks and protein-DNA adducts. Pathways required for the repair of these lesions include mismatch repair, base excision repair, nucleotide excision repair, and the homology directed repair/Fanconi anemia pathway. Each of these pathways contributes to genetic stability, and mutations in genes encoding proteins involved in these pathways have been demonstrated to promote genetic instability and cancer. In fact, it has been suggested all cancers display defects in DNA repair. It has also been demonstrated that the ability of cancer cells to repair therapeutically induced DNA damage impacts therapeutic efficacy. This has led to targeting DNA repair pathways and proteins to develop anti-cancer agents that will increase sensitivity to traditional chemotherapeutics. While initial studies languished and were plagued by a lack of specificity and a defined mechanism of action, more recent approaches to exploit synthetic lethal interaction and develop high affinity chemical inhibitors have proven considerably more effective. In this review we will highlight recent advances and discuss previous failures in targeting DNA repair to pave the way for future DNA repair targeted agents and their use in cancer therapy. PMID:26896565

A resource for functional profiling of noncoding RNA in the yeast Saccharomyces cerevisiae.

PubMed

Parker, Steven; Fraczek, Marcin G; Wu, Jian; Shamsah, Sara; Manousaki, Alkisti; Dungrattanalert, Kobchai; de Almeida, Rogerio Alves; Estrada-Rivadeneyra, Diego; Omara, Walid; Delneri, Daniela; O'Keefe, Raymond T

2017-08-01

Eukaryotic genomes are extensively transcribed, generating many different RNAs with no known function. We have constructed 1502 molecular barcoded ncRNA gene deletion strains encompassing 443 ncRNAs in the yeast Saccharomyces cerevisiae as tools for ncRNA functional analysis. This resource includes deletions of small nuclear RNAs (snRNAs), transfer RNAs (tRNAs), small nucleolar RNAs (snoRNAs), and other annotated ncRNAs as well as the more recently identified stable unannotated transcripts (SUTs) and cryptic unstable transcripts (CUTs) whose functions are largely unknown. Specifically, deletions have been constructed for ncRNAs found in the intergenic regions, not overlapping genes or their promoters (i.e., at least 200 bp minimum distance from the closest gene start codon). The deletion strains carry molecular barcodes designed to be complementary with the protein gene deletion collection enabling parallel analysis experiments. These strains will be useful for the numerous genomic and molecular techniques that utilize deletion strains, including genome-wide phenotypic screens under different growth conditions, pooled chemogenomic screens with drugs or chemicals, synthetic genetic array analysis to uncover novel genetic interactions, and synthetic dosage lethality screens to analyze gene dosage. Overall, we created a valuable resource for the RNA community and for future ncRNA research. © 2017 Parker et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Superinfection exclusion and the long-term survival of honey bees in Varroa-infested colonies

PubMed Central

Mordecai, Gideon J; Brettell, Laura E; Martin, Stephen J; Dixon, David; Jones, Ian M; Schroeder, Declan C

2016-01-01

Over the past 50 years, many millions of European honey bee (Apis mellifera) colonies have died as the ectoparasitic mite, Varroa destructor, has spread around the world. Subsequent studies have indicated that the mite's association with a group of RNA viral pathogens (Deformed Wing Virus, DWV) correlates with colony death. Here, we propose a phenomenon known as superinfection exclusion that provides an explanation of how certain A. mellifera populations have survived, despite Varroa infestation and high DWV loads. Next-generation sequencing has shown that a non-lethal DWV variant ‘type B' has become established in these colonies and that the lethal ‘type A' DWV variant fails to persist in the bee population. We propose that this novel stable host-pathogen relationship prevents the accumulation of lethal variants, suggesting that this interaction could be exploited for the development of an effective treatment that minimises colony losses in the future. PMID:26505829

[Detection of the lethal process in plankton noctiluca by means of a forbidden transition of ESR of Mn2+ ion].

PubMed

Kamenev, S E; Kopvillem, U Kh; Pasynkov, A S; Sharipov, R Z

1981-01-01

A forbidden ESR line of Mn2+ that is connected with the penetration of Mn into the plancton organism and binding it to a marcomolecule is selected from the experiment. A method for saturating the plancton organism with paramagnetic ions is proposed. It is shown that the constant of the axial electric field in the spin hamiltonian of Mn2+ ion described the dynamics of a selforganizing system. It is tested that the lethal process in the plancton with paramagnetic ion enrichment originated from boson avalanche. Experiments are performed with plancton noctiluca which illustrate the occurrence of avalancheline lethal process in the case of paramagnetic ion enrichment with limiting concentration. The meaning of these results for the problems of oceanology and pollution-ocean inhabitants interaction in the case of paramagnetic ions is discussed.

Daily ingestion of the probiotic Lactobacillus paracasei ST11 decreases Vaccinia virus dissemination and lethality in a mouse model.

PubMed

Dos Santos Pereira Andrade, A C; Lima, M Teixeira; Oliveira, G Pereira; Calixto, R Silva; de Sales E Souza, É Lorenna; da Glória de Souza, D; de Almeida Leite, C M; Ferreira, J M Siqueira; Kroon, E G; de Oliveira, D Bretas; Dos Santos Martins, F; Abrahão, J S

2017-02-07

Vaccinia virus (VACV) is an important pathogen. Although studies have shown relationships between probiotics and viruses, the effect of probiotics on VACV infection is unknown. Therefore, this work aims to investigate the probiotics effects on VACV infection. Mice were divided into four groups, two non-infected groups, one receiving the probiotic, the other one not receiving it, and two groups infected intranasally with VACV Western Reserve (VACV-WR) receiving or not receiving the probiotic. Viral titres in organs and cytokine production in the lungs were analysed. Lung samples were also subjected to histological analysis. The intake of probiotic results in reduction in viral spread with a significant decrease of VACV titer on lung, liver and brain of treated group. In addition,treatment with the probiotic results in attenuated mice lung inflammation showing fewer lesions on histological findings and decreased lethality in mice infected with VACV. The ingestion of Lactobacillus paracasei ST11 (LPST11) after VACV infection resulted in 2/9 animal lethality compared with 4/9 in the VACV group. This is the first study on probiotics and VACV interactions, providing not only information about this interaction, but also proposing a model for future studies involving probiotics and other poxvirus.

Lethal/sublethal responses of Daphnia magna to acute norfloxacin contamination and changes in phytoplankton-zooplankton interactions induced by this antibiotic

NASA Astrophysics Data System (ADS)

Pan, Ying; Yan, Shi-Wei; Li, Ruo-Zhu; Hu, Yi-Wen; Chang, Xue-Xiu

2017-01-01

Although the well-known antibiotic norfloxacin (NOR) is recognized as an important environmental pollutant, little is known about its impacts on ecological processes, particularly on species interactions. In this paper, we quantified Daphnia magna (Crustacea, Cladocera) responses in mortality rate at lethal NOR concentrations (0, 25, 50, 100, 200, 300 and 400 mg L-1), and in heartbeat rate, swimming behavior and feeding rate (on the green alga Chlorella pyrenoidosa) at sublethal NOR concentrations (0, 25, 50 and 100 mg L-1) to determine the effects of this antibiotic in plankton systems. In 96-h-long lethal experiment, mortality rates of D. magna increased significantly with increasing NOR concentration and exposure time. In sublethal experiments, heartbeat rate decreased, while time ratio of vertical to horizontal swimming (TVH) and the duration of quiescence increased in D. magna individuals exposed to increasing NOR concentrations after 4 and 12 h of exposure. These collectively led to decreases in both average swimming ability and feeding rate, consistent with the positive relationship between average swimming ability and feeding rate. Overall, results indicate that, by affecting zooplankton heartbeat rate and behavior, NOR decreased feeding efficiency of D. magna even at low doses, therefore, it might seriously compromise ecosystem health and function.

The Holstein Friesian Lethal Haplotype 5 (HH5) Results from a Complete Deletion of TBF1M and Cholesterol Deficiency (CDH) from an ERV-(LTR) Insertion into the Coding Region of APOB

PubMed Central

Schütz, Ekkehard; Wehrhahn, Christin; Wanjek, Marius; Bortfeld, Ralf; Wemheuer, Wilhelm E.; Beck, Julia; Brenig, Bertram

2016-01-01

Background With the availability of massive SNP data for several economically important cattle breeds, haplotype tests have been performed to identify unknown recessive disorders. A number of so-called lethal haplotypes, have been uncovered in Holstein Friesian cattle and, for at least seven of these, the causative mutations have been identified in candidate genes. However, several lethal haplotypes still remain elusive. Here we report the molecular genetic causes of lethal haplotype 5 (HH5) and cholesterol deficiency (CDH). A targeted enrichment for the known genomic regions, followed by massive parallel sequencing was used to interrogate for causative mutations in a case/control approach. Methods Targeted enrichment for the known genomic regions, followed by massive parallel sequencing was used in a case/control approach. PCRs for the causing mutations were developed and compared to routine imputing in 2,100 (HH5) and 3,100 (CDH) cattle. Results HH5 is caused by a deletion of 138kbp, spanning position 93,233kb to 93,371kb on chromosome 9 (BTA9), harboring only dimethyl-adenosine transferase 1 (TFB1M). The deletion breakpoints are flanked by bovine long interspersed nuclear elements Bov-B (upstream) and L1ME3 (downstream), suggesting a homologous recombination/deletion event. TFB1M di-methylates adenine residues in the hairpin loop at the 3’-end of mitochondrial 12S rRNA, being essential for synthesis and function of the small ribosomal subunit of mitochondria. Homozygous TFB1M-/- mice reportedly exhibit embryonal lethality with developmental defects. A 2.8% allelic frequency was determined for the German HF population. CDH results from a 1.3kbp insertion of an endogenous retrovirus (ERV2-1-LTR_BT) into exon 5 of the APOB gene at BTA11:77,959kb. The insertion is flanked by 6bp target site duplications as described for insertions mediated by retroviral integrases. A premature stop codon in the open reading frame of APOB is generated, resulting in a truncation of the protein to a length of only <140 amino acids. Such early truncations have been shown to cause an inability of chylomicron excretion from intestinal cells, resulting in malabsorption of cholesterol. The allelic frequency of this mutation in the German HF population was 6.7%, which is substantially higher than reported so far. Compared to PCR assays inferring the genetic variants directly, the routine imputing used so far showed a diagnostic sensitivity of as low as 91% (HH5) and 88% (CDH), with a high specificity for both (≥99.7%). Conclusion With the availability of direct genetic tests it will now be possible to more effectively reduce the carrier frequency and ultimately eliminate the disorders from the HF populations. Beside this, the fact that repetitive genomic elements (RE) are involved in both diseases, underline the evolutionary importance of RE, which can be detrimental as here, but also advantageous over generations. PMID:27128314

Evolution to pathogenicity of the parvovirus minute virus of mice in immunodeficient mice involves genetic heterogeneity at the capsid domain that determines tropism.

PubMed

López-Bueno, Alberto; Segovia, José C; Bueren, Juan A; O'Sullivan, M Gerard; Wang, Feng; Tattersall, Peter; Almendral, José M

2008-02-01

Very little is known about the role that evolutionary dynamics plays in diseases caused by mammalian DNA viruses. To address this issue in a natural host model, we compared the pathogenesis and genetics of the attenuated fibrotropic and the virulent lymphohematotropic strains of the parvovirus minute virus of mice (MVM), and of two invasive fibrotropic MVM (MVMp) variants carrying the I362S or K368R change in the VP2 major capsid protein, in the infection of severe combined immunodeficient (SCID) mice. By 14 to 18 weeks after oronasal inoculation, the I362S and K368R viruses caused lethal leukopenia characterized by tissue damage and inclusion bodies in hemopoietic organs, a pattern of disease found by 7 weeks postinfection with the lymphohematotropic MVM (MVMi) strain. The MVMp populations emerging in leukopenic mice showed consensus sequence changes in the MVMi genotype at residues G321E and A551V of VP2 in the I362S virus infections or A551V and V575A changes in the K368R virus infections, as well as a high level of genetic heterogeneity within a capsid domain at the twofold depression where these residues lay. Amino acids forming this capsid domain are important MVM tropism determinants, as exemplified by the switch in MVMi host range toward mouse fibroblasts conferred by coordinated changes of some of these residues and by the essential character of glutamate at residue 321 for maintaining MVMi tropism toward primary hemopoietic precursors. The few viruses within the spectrum of mutants from mice that maintained the respective parental 321G and 575V residues were infectious in a plaque assay, whereas the viruses with the main consensus sequences exhibited low levels of fitness in culture. Consistent with this finding, a recombinant MVMp virus carrying the consensus sequence mutations arising in the K368R virus background in mice failed to initiate infection in cell lines of different tissue origins, even though it caused rapid-course lethal leukopenia in SCID mice. The parental consensus genotype prevailed during leukopenia development, but plaque-forming viruses with the reversion of the 575A residue to valine emerged in affected organs. The disease caused by the DNA virus in mice, therefore, involves the generation of heterogeneous viral populations that may cooperatively interact for the hemopoietic syndrome. The evolutionary changes delineate a sector of the surface of the capsid that determines tropism and that surrounds the sialic acid receptor binding domain.

Characterization of Neurofibromas of the Skin and Spinal Roots in a Mouse Model

DTIC Science & Technology

2011-02-01

renewal program of stem/progenitor cells can cause tumorigenesis. By utilizing genetically engineered mouse models of neurofibromatosis type 1 (NF1...pathetic ganglia and adrenal medulla and died at birth (Gitler et al., 2003). To circumvent early lethality of the Nf1NC mice, we utilized a previously...Supplemental experimental procedures Tissue Processing For histological analysis, we utilized both paraffin sections and frozen sections. For both

Preclinical Mouse Models of Neurofibromatosis

DTIC Science & Technology

2005-11-01

and NF2-deficient human cells and in cells from Nf1 and Nf2 mutant mice. Genetic analysis of human and murine tumors has provided compelling...lethal myeloproliferative disorder (MPD) characterized by over-production of infiltrative myeloid cells (13). JMML has been modeled in mice by...tumor development for 18 months after exposure. Pathologic analysis was performed on 91% of the Shannon, K.M. 11 study cohort, including 95 of 104

Computational Sensing and in vitro Classification of GMOs and Biomolecular Events

DTIC Science & Technology

2008-12-01

COMPUTATIONAL SENSING AND IN VITRO CLASSIFICATION OF GMOs AND BIOMOLECULAR EVENTS Elebeoba May1∗, Miler T. Lee2†, Patricia Dolan1, Paul Crozier1...modified organisms ( GMOs ) in the pres- ence of non-lethal agents. Using an information and coding- theoretic framework we develop a de novo method for...high through- put screening, distinguishing genetically modified organisms ( GMOs ), molecular computing, differentiating biological mark- ers

Uncovering the Role of BMP Signaling in Melanocyte Development and Melanoma Tumorigenesis

DTIC Science & Technology

2015-06-01

Undergrad. student 2013 (W. New England University) Dissertation committees: Shawna Guillemette, UMass Medical School, Cancer Biology Program Tomoko...SUPPLEMENTARY NOTES 14. ABSTRACT Melanoma is the most aggressive and lethal form of skin cancer . In 2013 over 75,000 Americans were diagnosed with...skin cancer , and every year it kills nearly 10,000 Americans and roughly 60,000 people worldwide. A greater understanding of the genetic basis for

Maintaining Genome Stability: The Role of Helicases and Deaminases

DTIC Science & Technology

2006-07-01

functional for replication. Then, we screened the surviving transformants for sensitivity to hydroxyurea . Our first screen was conducted under conditions...that are lethal for checkpoint mutants such as ∆rad3. We did not observe any hydroxyurea -sensitive MCM clones. However, following the old genetics...this kinase. In particular, we observe that an mcm4ts mutant blocked in hydroxyurea , and then released to restrictive temperature, is competent to

Maintaining Genome Stability: The Role of Helicases and Deaminases

DTIC Science & Technology

2007-07-01

transformants for sensitivity to hydroxyurea . Our first screen was conducted under conditions that are lethal for checkpoint mutants such as ∆rad3...We did not observe any hydroxyurea -sensitive MCM clones. However, following the old genetics adage “absence of evidence is not evidence of... hydroxyurea , and then released to restrictive temperature, is competent to complete replication and go on to divide. In contrast, this mutant shifted

Properties and natural occurrence of maternal-effect selfish genes ('Medea' factors) in the red flour beetle, tribolium castaneum

PubMed

Beeman; Friesen

1999-05-01

Maternally acting selfish genes, termed 'Medea' factors, were found to be widespread in wild populations of Tribolium castaneum collected in Europe, North and South America, Africa and south-east Asia, but were rare or absent in populations from Australia and the Indian subcontinent. We detected at least four distinct genetic loci in at least two different linkage groups that exhibit the Medea pattern of differential mortality of genotypes within maternal families. Although each M factor tested had similar properties of maternal lethality to larvae and zygotic self-rescue, M factors representing distinct loci did not show cross-rescue. Alleles at two of these loci, M1 and M4, were by far the most prevalent, M4 being the predominant type. M2 and M3 were each found only once, in Pakistan and Japan, respectively. Although M1 could be genetically segregated from M4 and maintained as a purified stock, the M1 factor invariably co-occurred with M4 in field populations, whereas M4 usually occurred in the absence of other Medea factors. The dominant maternal lethal action of M1 could be selectively inactivated (reverted) by gene-knockout gamma irradiation with retention of zygotic rescue activity.

Inheritance patterns of morphological laterality in mouth opening of zebrafish, Danio rerio.

PubMed

Hata, Hiroki; Hori, Michio

2012-01-01

The inheritance patterns of asymmetry in mouth opening in zebrafish were investigated using crossing experiments. Zebrafish exhibit asymmetric laterality in mouth opening, with each individual having either a leftward (righty) or rightward (lefty) bias. All righty incrosses produced only righty F(1), whereas all lefty incrosses resulted in an F(1) L:R ratio of 2:1. All test crosses between lefty and righty individuals resulted in an F(1) L:R=1:1. These results were consistent with the hereditary pattern for Japanese medaka, three Tanganyikan cichlids, and a Japanese riverine goby. The pattern suggests a one-locus two-allele Mendelian model of inheritance, with the lefty allele being dominant over righty and the dominant homozygote being lethal. To determine the reason for the absence of lefty homozygotes, the survival rates of the offspring were examined according to developmental stage. Survival did not differ among combinations of parent laterality. Thus the mechanism underlying the lethality of the dominant homozygote remains unclear. This study showed that the mouth-opening laterality of zebrafish is genetically determined and that the direction follows a Mendelian inheritance pattern that is shared among cypriniform zebrafish, beloniform medaka, perciform cichlids, and a goby, suggesting a common genetic background in mouth-opening laterality among these species.

Associations between malaria and MHC genes in a migratory songbird

PubMed Central

Westerdahl, Helena; Waldenström, Jonas; Hansson, Bengt; Hasselquist, Dennis; von Schantz, Torbjörn; Bensch, Staffan

2005-01-01

Malaria parasites are a widespread and species-rich group infecting many wild populations of mammals, birds and reptiles. Studies on humans have demonstrated that genetic factors play a key role in the susceptibility and outcome of malaria infections. Until the present study, it has not been examined whether genetic variation in hosts is important for the outcome of malaria infections in natural avian populations. We investigated associations between major histocompatibility complex (MHC) genes and prevalence of three different avian malaria parasites (Haemoproteus payevskyi (GRW1), Plasmodium sp. (GRW2) and Plasmodium sp. (GRW4)) in a long-term study of great reed warblers Acrocephalus arundinaceus. We hypothesized that the MHC genes could either give full protection against a malaria infection, or confer protection against lethal malaria and direct the infection towards being milder. We found a positive association between numbers of MHC class I alleles (a measure of level of heterozygosity) and prevalence of the GRW2 parasite, suggesting the latter scenario. There was also a positive association between a specific MHC allele (B4b), previously shown to be under frequency-dependent selection in the study population, and prevalence of GRW2. These associations suggest that individuals carrying either a large number of MHC alleles or a specific MHC allele are protected against lethal malaria infections. PMID:16011927

Associations between malaria and MHC genes in a migratory songbird.

PubMed

Westerdahl, Helena; Waldenström, Jonas; Hansson, Bengt; Hasselquist, Dennis; von Schantz, Torbjörn; Bensch, Staffan

2005-07-22

Malaria parasites are a widespread and species-rich group infecting many wild populations of mammals, birds and reptiles. Studies on humans have demonstrated that genetic factors play a key role in the susceptibility and outcome of malaria infections. Until the present study, it has not been examined whether genetic variation in hosts is important for the outcome of malaria infections in natural avian populations. We investigated associations between major histocompatibility complex (MHC) genes and prevalence of three different avian malaria parasites (Haemoproteus payevskyi (GRW1), Plasmodium sp. (GRW2) and Plasmodium sp. (GRW4)) in a long-term study of great reed warblers Acrocephalus arundinaceus. We hypothesized that the MHC genes could either give full protection against a malaria infection, or confer protection against lethal malaria and direct the infection towards being milder. We found a positive association between numbers of MHC class I alleles (a measure of level of heterozygosity) and prevalence of the GRW2 parasite, suggesting the latter scenario. There was also a positive association between a specific MHC allele (B4b), previously shown to be under frequency-dependent selection in the study population, and prevalence of GRW2. These associations suggest that individuals carrying either a large number of MHC alleles or a specific MHC allele are protected against lethal malaria infections.

A data-mining approach to rank candidate protein-binding partners-The case of biogenesis of lysosome-related organelles complex-1 (BLOC-1).

PubMed

Rodriguez-Fernandez, I A; Dell'Angelica, E C

2009-04-01

The study of protein-protein interactions is a powerful approach to uncovering the molecular function of gene products associated with human disease. Protein-protein interaction data are accumulating at an unprecedented pace owing to interactomics projects, although it has been recognized that a significant fraction of these data likely represents false positives. During our studies of biogenesis of lysosome-related organelles complex-1 (BLOC-1), a protein complex involved in protein trafficking and containing the products of genes mutated in Hermansky-Pudlak syndrome, we faced the problem of having too many candidate binding partners to pursue experimentally. In this work, we have explored ways of efficiently gathering high-quality information about candidate binding partners and presenting the information in a visually friendly manner. We applied the approach to rank 70 candidate binding partners of human BLOC-1 and 102 candidates of its counterpart from Drosophila melanogaster. The top candidate for human BLOC-1 was the small GTPase encoded by the RAB11A gene, which is a paralogue of the Rab38 and Rab32 proteins in mammals and the lightoid gene product in flies. Interestingly, genetic analyses in D. melanogaster uncovered a synthetic sick/lethal interaction between Rab11 and lightoid. The data-mining approach described herein can be customized to study candidate binding partners for other proteins or possibly candidates derived from other types of 'omics' data.

Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae.

PubMed Central

Ben-Yehuda, S; Dix, I; Russell, C S; McGarvey, M; Beggs, J D; Kupiec, M

2000-01-01

The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Delta allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression. PMID:11102353

Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae.

PubMed

Ben-Yehuda, S; Dix, I; Russell, C S; McGarvey, M; Beggs, J D; Kupiec, M

2000-12-01

The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Delta allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression.

Evidence of infection by H5N2 highly pathogenic avian influenza viruses in healthy wild waterfowl

USGS Publications Warehouse

Gaidet, N.; Cattoli, G.; Hammoumi, S.; Newman, S.H.; Hagemeijer, W.; Takekawa, John Y.; Cappelle, J.; Dodman, T.; Joannis, T.; Gil, P.; Monne, I.; Fusaro, A.; Capua, I.; Manu, S.; Micheloni, P.; Ottosson, U.; Mshelbwala, J.H.; Lubroth, J.; Domenech, J.; Monicat, F.

2008-01-01

The potential existence of a wild bird reservoir for highly pathogenic avian influenza (HPAI) has been recently questioned by the spread and the persisting circulation of H5N1 HPAI viruses, responsible for concurrent outbreaks in migratory and domestic birds over Asia, Europe, and Africa. During a large-scale surveillance programme over Eastern Europe, the Middle East, and Africa, we detected avian influenza viruses of H5N2 subtype with a highly pathogenic (HP) viral genotype in healthy birds of two wild waterfowl species sampled in Nigeria. We monitored the survival and regional movements of one of the infected birds through satellite telemetry, providing a rare evidence of a non-lethal natural infection by an HP viral genotype in wild birds. Phylogenetic analysis of the H5N2 viruses revealed close genetic relationships with H5 viruses of low pathogenicity circulating in Eurasian wild and domestic ducks. In addition, genetic analysis did not reveal known gallinaceous poultry adaptive mutations, suggesting that the emergence of HP strains could have taken place in either wild or domestic ducks or in non-gallinaceous species. The presence of coexisting but genetically distinguishable avian influenza viruses with an HP viral genotype in two cohabiting species of wild waterfowl, with evidence of non-lethal infection at least in one species and without evidence of prior extensive circulation of the virus in domestic poultry, suggest that some strains with a potential high pathogenicity for poultry could be maintained in a community of wild waterfowl.

Electromagnetic pulse (EMP) coupling codes for use with the vulnerability/lethality (VIL) taxonomy. Final report, June-October 1984

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mar, M.H.

1995-07-01

Based on the vulnerability Lethality (V/L) taxonomy developed by the Ballistic Vulnerability Lethality Division (BVLD) of the Survivability Lethality Analysis Directorate (SLAD), a nuclear electromagnetic pulse (EMP) coupling V/L analysis taxonomy has been developed. A nuclear EMP threat to a military system can be divided into two levels: (1) coupling to a system level through a cable, antenna, or aperture; and (2) the component level. This report will focus on the initial condition, which includes threat definition and target description, as well as the mapping process from the initial condition to damaged components state. EMP coupling analysis at a systemmore » level is used to accomplish this. This report introduces the nature of EMP threat, interaction between the threat and target, and how the output of EMP coupling analysis at a system level becomes the input to the component level analysis. Many different tools (EMP coupling codes) will be discussed for the mapping process, which correponds to the physics of phenomenology. This EMP coupling V/L taxonomy and the models identified in this report will provide the tools necessary to conduct basic V/L analysis of EMP coupling.« less

Wild worm embryogenesis harbors ubiquitous polygenic modifier variation.

PubMed

Paaby, Annalise B; White, Amelia G; Riccardi, David D; Gunsalus, Kristin C; Piano, Fabio; Rockman, Matthew V

2015-08-22

Embryogenesis is an essential and stereotypic process that nevertheless evolves among species. Its essentiality may favor the accumulation of cryptic genetic variation (CGV) that has no effect in the wild-type but that enhances or suppresses the effects of rare disruptions to gene function. Here, we adapted a classical modifier screen to interrogate the alleles segregating in natural populations of Caenorhabditis elegans: we induced gene knockdowns and used quantitative genetic methodology to examine how segregating variants modify the penetrance of embryonic lethality. Each perturbation revealed CGV, indicating that wild-type genomes harbor myriad genetic modifiers that may have little effect individually but which in aggregate can dramatically influence penetrance. Phenotypes were mediated by many modifiers, indicating high polygenicity, but the alleles tend to act very specifically, indicating low pleiotropy. Our findings demonstrate the extent of conditional functionality in complex trait architecture.

Adaptive response in animals exposed to non-ionizing radiofrequency fields: some underlying mechanisms.

PubMed

Cao, Yi; Tong, Jian

2014-04-22

During the last few years, our research group has been investigating the phenomenon of adaptive response in animals exposed to non-ionizing radiofrequency fields. The results from several separate studies indicated a significant increase in survival, decreases in genetic damage as well as oxidative damage and, alterations in several cellular processes in mice pre-exposed to radiofrequency fields and subsequently subjected to sub-lethal or lethal doses of γ-radiation or injected with bleomycin, a radiomimetic chemical mutagen. These observations indicated the induction of adaptive response providing the animals the ability to resist subsequent damage. Similar studies conducted by independent researchers in mice and rats have supported our observation on increased survival. In this paper, we have presented a brief review of all of our own and other independent investigations on radiofrequency fields-induced adaptive response and some underlying mechanisms discussed.

Adaptive Response in Animals Exposed to Non-Ionizing Radiofrequency Fields: Some Underlying Mechanisms

PubMed Central

Cao, Yi; Tong, Jian

2014-01-01

During the last few years, our research group has been investigating the phenomenon of adaptive response in animals exposed to non-ionizing radiofrequency fields. The results from several separate studies indicated a significant increase in survival, decreases in genetic damage as well as oxidative damage and, alterations in several cellular processes in mice pre-exposed to radiofrequency fields and subsequently subjected to sub-lethal or lethal doses of γ-radiation or injected with bleomycin, a radiomimetic chemical mutagen. These observations indicated the induction of adaptive response providing the animals the ability to resist subsequent damage. Similar studies conducted by independent researchers in mice and rats have supported our observation on increased survival. In this paper, we have presented a brief review of all of our own and other independent investigations on radiofrequency fields-induced adaptive response and some underlying mechanisms discussed. PMID:24758897

Sex determination in insects: a binary decision based on alternative splicing.

PubMed

Salz, Helen K

2011-08-01

The gene regulatory networks that control sex determination vary between species. Despite these differences, comparative studies in insects have found that alternative splicing is reiteratively used in evolution to control expression of the key sex-determining genes. Sex determination is best understood in Drosophila where activation of the RNA binding protein-encoding gene Sex-lethal is the central female-determining event. Sex-lethal serves as a genetic switch because once activated it controls its own expression by a positive feedback splicing mechanism. Sex fate choice in is also maintained by self-sustaining positive feedback splicing mechanisms in other dipteran and hymenopteran insects, although different RNA binding protein-encoding genes function as the binary switch. Studies exploring the mechanisms of sex-specific splicing have revealed the extent to which sex determination is integrated with other developmental regulatory networks. Copyright © 2011 Elsevier Ltd. All rights reserved.

Production of maternal-zygotic mutant zebrafish by germ-line replacement.

PubMed

Ciruna, Brian; Weidinger, Gilbert; Knaut, Holger; Thisse, Bernard; Thisse, Christine; Raz, Erez; Schier, Alexander F

2002-11-12

We report a generally applicable strategy for transferring zygotic lethal mutations through the zebrafish germ line. By using a morpholino oligonucleotide that blocks primordial germ cell (PGC) development, we generate embryos devoid of endogenous PGCs to serve as hosts for the transplantation of germ cells derived from homozygous mutant donors. Successful transfers are identified by the localization of specifically labeled donor PGCs to the region of the developing gonad in chimeric embryos. This strategy, which results in the complete replacement of the host germ line with donor PGCs, was validated by the generation of maternal and maternal-zygotic mutants for the miles apart locus. This germ-line replacement technique provides a powerful tool for studying the maternal effects of zygotic lethal mutations. Furthermore, the ability to generate large clutches of purely mutant embryos will greatly facilitate embryological, genetic, genomic, and biochemical studies.

Production of maternal-zygotic mutant zebrafish by germ-line replacement

PubMed Central

Ciruna, Brian; Weidinger, Gilbert; Knaut, Holger; Thisse, Bernard; Thisse, Christine; Raz, Erez; Schier, Alexander F.

2002-01-01

We report a generally applicable strategy for transferring zygotic lethal mutations through the zebrafish germ line. By using a morpholino oligonucleotide that blocks primordial germ cell (PGC) development, we generate embryos devoid of endogenous PGCs to serve as hosts for the transplantation of germ cells derived from homozygous mutant donors. Successful transfers are identified by the localization of specifically labeled donor PGCs to the region of the developing gonad in chimeric embryos. This strategy, which results in the complete replacement of the host germ line with donor PGCs, was validated by the generation of maternal and maternal-zygotic mutants for the miles apart locus. This germ-line replacement technique provides a powerful tool for studying the maternal effects of zygotic lethal mutations. Furthermore, the ability to generate large clutches of purely mutant embryos will greatly facilitate embryological, genetic, genomic, and biochemical studies. PMID:12397179

The effect of polyandry on a distorter system with differential viabilities in the sexes.

PubMed

Manser, Andri; Lindholm, Anna K; König, Barbara; Bagheri, Homayoun C

2012-11-01

The presence of selfish genetic elements can have fatal consequences for populations that harbor them. In the well known t haplotype in wild house mice, large proportions of the population die from t/t recessive lethal effects. Due to strong advantages at the gamete level (drive), t haplotypes nevertheless occur at substantial frequencies. The stable presence of a lethal is not the only effect of the t. It also distorts the fate of mutations that differentially affect male and female survival and reproduction (such as in sexual conflict), by giving male selective effects a strong advantage over female selective effects. In a recent study, we proposed polyandry as a potential counterstrategy against t deleterious effects. Here, we show that (1) the efficiency of polyandry in reducing the t frequency strongly depends on the selective context and (2) polyandry helps to reduce male-biased leverage in sex dependent selection.

Social networks of educated nematodes

USDA-ARS?s Scientific Manuscript database

Entomopathogenic nematodes are obligate lethal parasitoids of insect larvae that navigate a chemically complex belowground environment while interacting with their insect hosts, plants, and each other. In this environment, prior exposure to volatile compounds appears to prime nematodes in a compound...

Review of insecticide resistance and behavioral avoidance of vectors of human diseases in Thailand

PubMed Central

2013-01-01

Physiological resistance and behavioral responses of mosquito vectors to insecticides are critical aspects of the chemical-based disease control equation. The complex interaction between lethal, sub-lethal and excitation/repellent ('excito-repellent’) properties of chemicals is typically overlooked in vector management and control programs. The development of “physiological” resistance, metabolic and/or target site modifications, to insecticides has been well documented in many insect groups and disease vectors around the world. In Thailand, resistance in many mosquito populations has developed to all three classes of insecticidal active ingredients currently used for vector control with a majority being synthetic-derived pyrethroids. Evidence of low-grade insecticide resistance requires immediate countermeasures to mitigate further intensification and spread of the genetic mechanisms responsible for resistance. This can take the form of rotation of a different class of chemical, addition of a synergist, mixtures of chemicals or concurrent mosaic application of different classes of chemicals. From the gathered evidence, the distribution and degree of physiological resistance has been restricted in specific areas of Thailand in spite of long-term use of chemicals to control insect pests and disease vectors throughout the country. Most surprisingly, there have been no reported cases of pyrethroid resistance in anopheline populations in the country from 2000 to 2011. The precise reasons for this are unclear but we assume that behavioral avoidance to insecticides may play a significant role in reducing the selection pressure and thus occurrence and spread of insecticide resistance. The review herein provides information regarding the status of physiological resistance and behavioral avoidance of the primary mosquito vectors of human diseases to insecticides in Thailand from 2000 to 2011. PMID:24294938

Loss of Nephrocystin-3 Function Can Cause Embryonic Lethality, Meckel-Gruber-like Syndrome, Situs Inversus, and Renal-Hepatic-Pancreatic Dysplasia

PubMed Central

Bergmann, Carsten; Fliegauf, Manfred; Brüchle, Nadina Ortiz; Frank, Valeska; Olbrich, Heike; Kirschner, Jan; Schermer, Bernhard; Schmedding, Ingolf; Kispert, Andreas; Kränzlin, Bettina; Nürnberg, Gudrun; Becker, Christian; Grimm, Tiemo; Girschick, Gundula; Lynch, Sally A.; Kelehan, Peter; Senderek, Jan; Neuhaus, Thomas J.; Stallmach, Thomas; Zentgraf, Hanswalter; Nürnberg, Peter; Gretz, Norbert; Lo, Cecilia; Lienkamp, Soeren; Schäfer, Tobias; Walz, Gerd; Benzing, Thomas; Zerres, Klaus; Omran, Heymut

2008-01-01

Many genetic diseases have been linked to the dysfunction of primary cilia, which occur nearly ubiquitously in the body and act as solitary cellular mechanosensory organelles. The list of clinical manifestations and affected tissues in cilia-related disorders (ciliopathies) such as nephronophthisis is broad and has been attributed to the wide expression pattern of ciliary proteins. However, little is known about the molecular mechanisms leading to this dramatic diversity of phenotypes. We recently reported hypomorphic NPHP3 mutations in children and young adults with isolated nephronophthisis and associated hepatic fibrosis or tapetoretinal degeneration. Here, we chose a combinatorial approach in mice and humans to define the phenotypic spectrum of NPHP3/Nphp3 mutations and the role of the nephrocystin-3 protein. We demonstrate that the pcy mutation generates a hypomorphic Nphp3 allele that is responsible for the cystic kidney disease phenotype, whereas complete loss of Nphp3 function results in situs inversus, congenital heart defects, and embryonic lethality in mice. In humans, we show that NPHP3 mutations can cause a broad clinical spectrum of early embryonic patterning defects comprising situs inversus, polydactyly, central nervous system malformations, structural heart defects, preauricular fistulas, and a wide range of congenital anomalies of the kidney and urinary tract (CAKUT). On the functional level, we show that nephrocystin-3 directly interacts with inversin and can inhibit like inversin canonical Wnt signaling, whereas nephrocystin-3 deficiency leads in Xenopus laevis to typical planar cell polarity defects, suggesting a role in the control of canonical and noncanonical (planar cell polarity) Wnt signaling. PMID:18371931

The Wright stuff: reimagining path analysis reveals novel components of the sex determination hierarchy in Drosophila melanogaster.

PubMed

Fear, Justin M; Arbeitman, Michelle N; Salomon, Matthew P; Dalton, Justin E; Tower, John; Nuzhdin, Sergey V; McIntyre, Lauren M

2015-09-04

The Drosophila sex determination hierarchy is a classic example of a transcriptional regulatory hierarchy, with sex-specific isoforms regulating morphology and behavior. We use a structural equation modeling approach, leveraging natural genetic variation from two studies on Drosophila female head tissues--DSPR collection (596 F1-hybrids from crosses between DSPR sub-populations) and CEGS population (75 F1-hybrids from crosses between DGRP/Winters lines to a reference strain w1118)--to expand understanding of the sex hierarchy gene regulatory network (GRN). This approach is completely generalizable to any natural population, including humans. We expanded the sex hierarchy GRN adding novel links among genes, including a link from fruitless (fru) to Sex-lethal (Sxl) identified in both populations. This link is further supported by the presence of fru binding sites in the Sxl locus. 754 candidate genes were added to the pathway, including the splicing factors male-specific lethal 2 and Rm62 as downstream targets of Sxl which are well-supported links in males. Independent studies of doublesex and transformer mutants support many additions, including evidence for a link between the sex hierarchy and metabolism, via Insulin-like receptor. The genes added in the CEGS population were enriched for genes with sex-biased splicing and components of the spliceosome. A common goal of molecular biologists is to expand understanding about regulatory interactions among genes. Using natural alleles we can not only identify novel relationships, but using supervised approaches can order genes into a regulatory hierarchy. Combining these results with independent large effect mutation studies, allows clear candidates for detailed molecular follow-up to emerge.

Effects of hypo-O-GlcNAcylation on Drosophila development.

PubMed

Mariappa, Daniel; Ferenbach, Andrew T; van Aalten, Daan M F

2018-05-11

Post-translational modification of serine/threonine residues in nucleocytoplasmic proteins with GlcNAc ( O -GlcNAcylation) is an essential regulatory mechanism in many cellular processes. In Drosophila , null mutants of the Polycomb gene O -GlcNAc transferase ( OGT ; also known as super sex combs ( sxc )) display homeotic phenotypes. To dissect the requirement for O -GlcNAc signaling in Drosophila development, we used CRISPR/Cas9 gene editing to generate rationally designed sxc catalytically hypomorphic or null point mutants. Of the fertile males derived from embryos injected with the CRISPR/Cas9 reagents, 25% produced progeny carrying precise point mutations with no detectable off-target effects. One of these mutants, the catalytically inactive sxc K872M , was recessive lethal, whereas a second mutant, the hypomorphic sxc H537A , was homozygous viable. We observed that reduced total protein O -GlcNAcylation in the sxc H537A mutant is associated with a wing vein phenotype and temperature-dependent lethality. Genetic interaction between sxc H537A and a null allele of Drosophila host cell factor ( dHcf ), encoding an extensively O -GlcNAcylated transcriptional coactivator, resulted in abnormal scutellar bristle numbers. A similar phenotype was also observed in sxc H537A flies lacking a copy of skuld ( skd ), a Mediator complex gene known to affect scutellar bristle formation. Interestingly, this phenotype was independent of OGT Polycomb function or dHcf downstream targets. In conclusion, the generation of the endogenous OGT hypomorphic mutant sxc H537A enabled us to identify pleiotropic effects of globally reduced protein O -GlcNAc during Drosophila development. The mutants generated and phenotypes observed in this study provide a platform for discovery of OGT substrates that are critical for Drosophila development. © 2018 Mariappa et al.

Tooth development in vitro in two teleost fish, the cichlid Hemichromis bimaculatus and the cyprinid Danio rerio.

PubMed

Van der Heyden, C; Allizard, F; Sire, J-Y; Huysseune, A

2005-09-01

A technique for organotypic in vitro culture with serum-free medium was tested for its appropriateness to mimic normal odontogenesis in the cichlid fish Hemichromis bimaculatus and the zebrafish Danio rerio. Serial semithin sections were observed by light microscopy to collect data on tooth patterning and transmission electron microscopy was used to compare cellular and extracellular features of tooth germs developing in vitro with the situation in vivo. Head explants of H. bimaculatus from 120 h post-fertilization (hPF) to 8.5 days post-fertilization (dPF) and of zebrafish from 45 hPF to 79 hPF and adults kept in culture for 3, 4 or 7 days revealed that tooth germs developed in vitro from explants in which the buccal or pharyngeal epithelium was apparently undifferentiated and, when present at the time of explantation, they continued their development up to a stage of attachment. In addition, the medium allowed the morphogenesis and cytodifferentiation of the tooth germs similar to that observed in vivo and the establishment of a dental pattern (place and order of tooth appearance and of attachment) that mimicked that in vivo. Organotypic culture in serum-free conditions thus provides us with the means of studying epithelial-mesenchymal interactions during tooth development in teleost fish and of analysing the genetic control of either mandibular or pharyngeal tooth development and replacement in these polyphyodont species. Importantly, it allows heads from embryonically lethal (zebrafish) mutants or from early lethal knockdown experiments to develop beyond the point at which the embryos normally die. Such organotypic culture in serum-free conditions could therefore become a powerful tool in developmental studies and open new perspectives for craniofacial research.

Functional diversity of non-lethal effects, chemical camouflage, and variation in fish avoidance in colonizing beetles.

PubMed

Resetarits, William J; Pintar, Matthew R

2016-12-01

Predators play an extremely important role in natural communities. In freshwater systems, fish can dominate sorting both at the colonization and post-colonization stage. Specifically, for many colonizing species, fish can have non-lethal, direct effects that exceed the lethal direct effects of predation. Functionally diverse fish species with a range of predatory capabilities have previously been observed to elicit functionally equivalent responses on oviposition in tree frogs. We tested this hypothesis of functional equivalence of non-lethal effects for four predatory fish species, using naturally colonizing populations of aquatic beetles. Among taxa other than mosquitoes, and with the exception of the chemically camouflaged pirate perch, Aphredoderus sayanus, we provide the first evidence of variation in colonization or oviposition responses to different fish species. Focusing on total abundance, Fundulus chrysotus, a gape-limited, surface-feeding fish, elicited unique responses among colonizing Hydrophilidae, with the exception of the smallest and most abundant taxa, Paracymus, while Dytiscidae responded similarly to all avoided fish. Neither family responded to A. sayanus. Analysis of species richness and multivariate characterization of the beetle assemblages for the four fish species and controls revealed additional variation among the three avoided species and confirmed that chemical camouflage in A. sayanus results in assemblages essentially identical to fishless controls. The origin of this variation in beetle responses to different fish is unknown, but may involve variation in cue sensitivity, different behavioral algorithms, or differential responses to species-specific fish cues. The identity of fish species occupying aquatic habitats is crucial to understanding community structure, as varying strengths of lethal and non-lethal effects, as well as their interaction, create complex landscapes of predator effects and challenge the notion of functional equivalence. © 2016 by the Ecological Society of America.

[Analytic methods for seed models with genotype x environment interactions].

PubMed

Zhu, J

1996-01-01

Genetic models with genotype effect (G) and genotype x environment interaction effect (GE) are proposed for analyzing generation means of seed quantitative traits in crops. The total genetic effect (G) is partitioned into seed direct genetic effect (G0), cytoplasm genetic of effect (C), and maternal plant genetic effect (Gm). Seed direct genetic effect (G0) can be further partitioned into direct additive (A) and direct dominance (D) genetic components. Maternal genetic effect (Gm) can also be partitioned into maternal additive (Am) and maternal dominance (Dm) genetic components. The total genotype x environment interaction effect (GE) can also be partitioned into direct genetic by environment interaction effect (G0E), cytoplasm genetic by environment interaction effect (CE), and maternal genetic by environment interaction effect (GmE). G0E can be partitioned into direct additive by environment interaction (AE) and direct dominance by environment interaction (DE) genetic components. GmE can also be partitioned into maternal additive by environment interaction (AmE) and maternal dominance by environment interaction (DmE) genetic components. Partitions of genetic components are listed for parent, F1, F2 and backcrosses. A set of parents, their reciprocal F1 and F2 seeds is applicable for efficient analysis of seed quantitative traits. MINQUE(0/1) method can be used for estimating variance and covariance components. Unbiased estimation for covariance components between two traits can also be obtained by the MINQUE(0/1) method. Random genetic effects in seed models are predictable by the Adjusted Unbiased Prediction (AUP) approach with MINQUE(0/1) method. The jackknife procedure is suggested for estimation of sampling variances of estimated variance and covariance components and of predicted genetic effects, which can be further used in a t-test for parameter. Unbiasedness and efficiency for estimating variance components and predicting genetic effects are tested by Monte Carlo simulations.

Redundant functions of I-BAR family members, IRSp53 and IRTKS, are essential for embryonic development

PubMed Central

Chou, Ai Mei; Sem, Kai Ping; Lam, Wei Jun; Ahmed, Sohail; Lim, Chin Yan

2017-01-01

The insulin receptor substrate of 53 kDa, IRSp53, is an adaptor protein that works with activated GTPases, Cdc42 and Rac, to modulate actin dynamics and generate membrane protrusions in response to cell signaling. Adult mice that lack IRSp53 fail to regulate synaptic plasticity and exhibit hippocampus-associated learning deficiencies. Here, we show that 60% of IRSp53 null embryos die at mid to late gestation, indicating a vital IRSp53 function in embryonic development. We find that IRSp53 KO embryos displayed pleiotropic phenotypes such as developmental delay, oligodactyly and subcutaneous edema, and died of severely impaired cardiac and placental development. We further show that double knockout of IRSp53 and its closest family member, IRTKS, resulted in exacerbated placental abnormalities, particularly in spongiotrophoblast differentiation and development, giving rise to complete embryonic lethality. Hence, our findings demonstrate a hitherto under-appreciated IRSp53 function in embryonic development, and further establish an essential genetic interaction between IRSp53 and IRTKS in placental formation. PMID:28067313

Mouse models for four types of Waardenburg syndrome.

PubMed

Tachibana, Masayoshi; Kobayashi, Yasuhito; Matsushima, Yoshibumi

2003-10-01

Waardenburg syndrome (WS) is an auditory-pigmentary syndrome caused by a deficiency of melanocytes and other neural crest-derived cells. Depending on a variety of symptoms associated with the auditory-pigmentary symptoms, WS is classified into four types: WS type 1 (WS1), WS2, WS3, and WS4. Six genes contributing to this syndrome--PAX3, SOX10, MITF, SLUG, EDN3 and EDNRB--have been cloned so far, all of them necessary for normal development of melanocytes. Mutant mice with coat color anomalies were helpful in identifying these genes, although the phenotypes of these mice did not necessarily perfectly match those of the four types of WS. Here we describe mice with mutations of murine homologs of WS genes and verify their suitability as models for WS with special interest in the cochlear disorder. The mice include splotch (Sp), microphthalmia (mi), Slugh-/-, WS4, JF1, lethal-spotting (ls), and Dominant megacolon (Dom). The influence of genetic background on the phenotypes of mice mutated in homologs of WS genes is also addressed. Finally, possible interactions among the six WS gene products are discussed.

Targeted Disruption of Orchestration between Stroma and Tumor Cells in Pancreatic Cancer: Molecular Basis and Therapeutic Implications

PubMed Central

Kong, Xiangyu; Li, Lei; Li, Zhaoshen; Xie, Keping

2012-01-01

Pancreatic cancer is one of the most lethal malignancies, with a prominent desmoplastic reaction as the defining hallmark of the disease. The past several decades have seen dramatic progress in understanding of pancreatic cancer pathogenesis, including the identification of precursor lesions, sequential transformation from normal pancreas to invasive pancreatic cancer and corresponding signature genetic events, and the biological impact of those alterations on malignant behaviors. However, the current therapeutic strategies for epithelial tumor cells, which have exhibited potent antitumor activity in cell culture and animal models, have failed to have significant effects in the clinic. The desmoplastic stroma surrounding pancreatic cancer cells, which accounts for about 90% of a tumor’s mass, clearly is not a passive scaffold for cancer cells but an active contributor to carcinogenesis. Improved understanding of the dynamic interaction between cancer cells and their stroma will be important to designing new, effective therapeutic strategies for pancreatic cancer. This review focuses on the origination of stromal molecular and cellular components in pancreatic tumors, their biological effects on pancreatic cancer cells, and the orchestration between these two components. PMID:22749856

Interallelic Complementation at the Suppressor of Forked Locus of Drosophila Reveals Complementation between Suppressor of Forked Proteins Mutated in Different Regions

PubMed Central

Simonelig, M.; Elliott, K.; Mitchelson, A.; O'Hare, K.

1996-01-01

The Su(f) protein of Drosophila melanogaster shares extensive homologies with proteins from yeast (RNA14) and man (77 kD subunit of cleavage stimulation factor) that are required for 3' end processing of mRNA. These homologies suggest that su(f) is involved in mRNA 3' end formation and that some aspects of this process are conserved throughout eukaryotes. We have investigated the genetic and molecular complexity of the su(f) locus. The su(f) gene is transcribed to produce three RNAs and could encode two proteins. Using constructs that contain different parts of the locus, we show that only the larger predicted gene product of 84 kD is required for the wild-type function of su(f). Some lethal alleles of su(f) complement to produce viable combinations. The structures of complementing and noncomplementing su(f) alleles indicate that 84-kD Su(f) proteins mutated in different domains can act in combination for partial su(f) function. Our results suggest protein-protein interaction between or within wild-type Su(f) molecules. PMID:8846900

Rare copy number variants in a population-based investigation of hypoplastic right heart syndrome.

PubMed

Dimopoulos, Aggeliki; Sicko, Robert J; Kay, Denise M; Rigler, Shannon L; Druschel, Charlotte M; Caggana, Michele; Browne, Marilyn L; Fan, Ruzong; Romitti, Paul A; Brody, Lawrence C; Mills, James L

2017-01-20

Hypoplastic right heart syndrome (HRHS) is a rare congenital defect characterized by underdevelopment of the right heart structures commonly accompanied by an atrial septal defect. Familial HRHS reports suggest genetic factor involvement. We examined the role of copy number variants (CNVs) in HRHS. We genotyped 32 HRHS cases identified from all New York State live births (1998-2005) using Illumina HumanOmni2.5 microarrays. CNVs were called with PennCNV and prioritized if they were ≥20 Kb, contained ≥10 SNPs and had minimal overlap with CNVs from in-house controls, the Database of Genomic Variants, HapMap3, and Childrens Hospital of Philadelphia database. We identified 28 CNVs in 17 cases; several encompassed genes important for right heart development. One case had a 2p16-2p23 duplication spanning LBH, a limb and heart development transcription factor. Lbh mis-expression results in right ventricular hypoplasia and pulmonary valve defects. This duplication also encompassed SOS1, a factor associated with pulmonary valve stenosis in Noonan syndrome. Sos1 -/- mice display thin and poorly trabeculated ventricles. In another case, we identified a 1.5 Mb deletion associated with Williams-Beuren syndrome, a disorder that includes valvular malformations. A third case had a 24 Kb deletion upstream of the TGFβ ligand ITGB8. Embryos genetically null for Itgb8, and its intracellular interactant Band 4.1B, display lethal cardiac phenotypes. To our knowledge, this is the first study of CNVs in HRHS. We identified several rare CNVs that overlap genes related to right ventricular wall and valve development, suggesting that genetics plays a role in HRHS and providing clues for further investigation. Birth Defects Research 109:16-26, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Rare Copy Number Variants in a Population Based Investigation of Hypoplastic Right Heart Syndrome

PubMed Central

Dimopoulos, Aggeliki; Sicko, Robert J.; Kay, Denise M.; Rigler, Shannon L.; Druschel, Charlotte M.; Caggana, Michele; Browne, Marilyn L.; Fan, Ruzong; Romitti, Paul A.; Brody, Lawrence C.; Mills, James L.

2016-01-01

Background Hypoplastic right heart syndrome (HRHS) is a rare congenital defect characterized by underdevelopment of the right heart structures commonly accompanied by an atrial septal defect. Familial HRHS reports suggest genetic factor involvement. We examined the role of copy number variants (CNVs) in HRHS. Methods We genotyped 32 HRHS cases identified from all New York State live births (1998–2005) using Illumina HumanOmni2.5 microarrays. CNVs were called with PennCNV and prioritized if they were ≥20Kb, contained ≥10 SNPs and had minimal overlap with CNVs from in-house controls, the Database of Genomic Variants, HapMap3 and CHOP database. Results We identified 28 CNVs in 17 cases; several encompassed genes important for right heart development. One case had a 2p16–2p23 duplication spanning LBH, a limb and heart development transcription factor. Lbh mis-expression results in right ventricular hypoplasia and pulmonary valve defects. This duplication also encompassed SOS1, a factor associated with pulmonary valve stenosis in Noonan syndrome. Sos1−/− mice display thin and poorly trabeculated ventricles. In another case, we identified a 1.5Mb deletion associated with Williams Beuren syndrome, a disorder that includes valvular malformations. A third case had a 24Kb deletion upstream of the TGFβ ligand ITGB8. Embryos genetically null for Itgb8, and its intracellular interactant Band 4.1B, display lethal cardiac phenotypes. Conclusions To our knowledge, this is the first study of CNVs in HRHS. We identified several rare CNVs that overlap genes related to right ventricular wall and valve development, suggesting that genetics plays a role in HRHS and providing clues for further investigation. PMID:28009100

Studying the Effect of Radiation in the Context of Deep Space Travel

NASA Technical Reports Server (NTRS)

Bhattacharya, Sharmila; Gilbert, Rachel R.; Lo, Rachel

2017-01-01

While it has been shown that decades of astronauts and cosmonauts can suffer from illnesses both during and after spaceflight, the underlying causes are still poorly understood, due in part to the fact that there are so many variables to consider when investigating the human immune system in a complex environment. Invertebrates have become popular models for studying human disease because they are cheap, highly amenable to experimental manipulation, and have innate immune systems with a high genetic similarity to humans. Fruit flies (Drosophila melanogaster) have been shown to experience a dramatic shift in immune gene expression following spaceflight, but are still able to fight off infections when exposed to bacteria. However, the common bacterial pathogen Serratia marcescens was shown to become more lethal to fruit flies after being cultured in space, suggesting that not only do we need to consider host changes in susceptibility, but also changes in the pathogen itself after spaceflight conditions. Being able to simulate spaceflight conditions in a controlled environment on the ground gives us the ability to not only evaluate the effects of microgravity on the host immune system, but also how the microorganisms that cause immune disorders are being affected by these drastic environmental shifts. In this study, I use a ground-based simulated microgravity environment to examine the genetic changes associated with increased S. marcescens virulence in order to understand how microgravity is affecting this pathogen, as well as how these genetic changes influence and interact with the host immune system. This study will provide us with more directed approaches to studying the effects of spaceflight on human beings, with the ultimate goal of being able to counteract immune dysfunction in future space exploration.

Effects of Simulated Microgravity on a Host-Pathogen System

NASA Technical Reports Server (NTRS)

Gilbert, Rachel; Lo, Rachel; Bhattacharya, Sharmila

2017-01-01

While it has been shown that decades of astronauts and cosmonauts can suffer from illnesses both during and after spaceflight, the underlying causes are still poorly understood, due in part to the fact that there are so many variables to consider when investigating the human immune system in a complex environment. Invertebrates have become popular models for studying human disease because they are cheap, highly amenable to experimental manipulation, and have innate immune systems with a high genetic similarity to humans. Fruit flies (Drosophila melanogaster) have been shown to experience a dramatic shift in immune gene expression following spaceflight, but are still able to fight off infections when exposed to bacteria. However, the common bacterial pathogen Serratia marcescens was shown to become more lethal to fruit flies after being cultured in space, suggesting that not only do we need to consider host changes in susceptibility, but also changes in the pathogen itself after spaceflight conditions. Being able to simulate spaceflight conditions in a controlled environment on the ground gives us the ability to not only evaluate the effects of microgravity on the host immune system, but also how the microorganisms that cause immune disorders are being affected by these drastic environmental shifts. In this study, I use a ground-based simulated microgravity environment to examine the genetic changes associated with increased S. marcescens virulence in order to understand how microgravity is affecting this pathogen, as well as how these genetic changes influence and interact with the host immune system. This study will provide us with more directed approaches to studying the effects of spaceflight on human beings, with the ultimate goal of being able to counteract immune dysfunction in future space exploration.

The genotoxic effects of the imidacloprid-based insecticide formulation Glacoxan Imida on Montevideo tree frog Hypsiboas pulchellus tadpoles (Anura, Hylidae).

PubMed

Pérez-Iglesias, J M; Ruiz de Arcaute, C; Nikoloff, N; Dury, L; Soloneski, S; Natale, G S; Larramendy, M L

2014-06-01

The neonicotinoid insecticide imidacloprid (IMI) affects the insect central nervous system and is successfully applied to control pests for a variety of agricultural crops. In the current study, acute toxicity and genotoxicity of the IMI-containing commercial formulation insecticide Glacoxan Imida (35 percent IMI) was evaluated on Hypsiboas pulchellus (Anura: Hylidae) tadpoles exposed under laboratory conditions. A lethal effect was evaluated as the end point for lethality, whereas micronucleus (MN) frequency and DNA single-strand breaks evaluated by the single cell gel electrophoresis (SCGE) assay were employed as end points for genotoxicity. Sublethal end points were assayed within the 12.5-37.5mg/L IMI concentration range. Experiments were performed on tadpoles at stage 36 (range, 35-37) according to the classification proposed by Gosner. Lethality studies revealed an LC50 96h value of 52.622mg/L IMI. Increased frequency of MNs was only observed when 25.0mg/L was assayed for 96h, whereas no other nuclear abnormalities were induced. Increase of the genetic damage index was observed at 48h of treatment within the 12.5-37.5mg/L concentration range, whereas an increased frequency of DNA damage was observed only in tadpoles treated with 37.5mg/L IMI for 96h. This study represents the first evidence of the acute lethal and genotoxic effects exerted by IMI on tadpoles of an amphibian species native to Argentina under laboratory conditions. Copyright © 2014 Elsevier Inc. All rights reserved.

Suppression of AKT phosphorylation restores rapamycin-based synthetic lethality in SMAD4-defective pancreatic cancer cells.

PubMed

Le Gendre, Onica; Sookdeo, Ayisha; Duliepre, Stephie-Anne; Utter, Matthew; Frias, Maria; Foster, David A

2013-05-01

mTOR has been implicated in survival signals for many human cancers. Rapamycin and TGF-β synergistically induce G1 cell-cycle arrest in several cell lines with intact TGF-β signaling pathway, which protects cells from the apoptotic effects of rapamycin during S-phase of the cell cycle. Thus, rapamycin is cytostatic in the presence of serum/TGF-β and cytotoxic in the absence of serum. However, if TGF-β signaling is defective, rapamycin induced apoptosis in both the presence and absence of serum/TGF-β in colon and breast cancer cell lines. Because genetic dysregulation of TGF-β signaling is commonly observed in pancreatic cancers-with defects in the Smad4 gene being most prevalent, we hypothesized that pancreatic cancers would display a synthetic lethality to rapamycin in the presence of serum/TGF-β. We report here that Smad4-deficient pancreatic cancer cells are killed by rapamycin in the absence of serum; however, in the presence of serum, we did not observe the predicted synthetic lethality with rapamycin. Rapamycin also induced elevated phosphorylation of the survival kinase Akt at Ser473. Suppression of rapamycin-induced Akt phosphorylation restored rapamycin sensitivity in Smad4-null, but not Smad4 wild-type pancreatic cancer cells. This study shows that the synthetic lethality to rapamycin in pancreatic cancers with defective TGF-β signaling is masked by rapamycin-induced increases in Akt phosphorylation. The implication is that a combination of approaches that suppress both Akt phosphorylation and mTOR could be effective in targeting pancreatic cancers with defective TGF-β signaling. ©2013 AACR.

From tails to toes: developing nonlethal tissue indicators of mercury exposure in five amphibian species.

PubMed

Pfleeger, Adam Z; Eagles-Smith, Collin A; Kowalski, Brandon M; Herring, Garth; Willacker, James J; Jackson, Allyson K; Pierce, John R

2016-04-01

Exposure to environmental contaminants has been implicated as a factor in global amphibian decline. Mercury (Hg) is a particularly widespread contaminant that biomagnifies in amphibians and can cause a suite of deleterious effects. However, monitoring contaminant exposure in amphibian tissues may conflict with conservation goals if lethal take is required. Thus, there is a need to develop non-lethal tissue sampling techniques to quantify contaminant exposure in amphibians. Some minimally invasive sampling techniques, such as toe-clipping, are common in population-genetic research, but it is unclear if these methods can adequately characterize contaminant exposure. We examined the relationships between mercury (Hg) concentrations in non-lethally sampled tissues and paired whole-bodies in five amphibian species. Specifically, we examined the utility of three different tail-clip sections from four salamander species and toe-clips from one anuran species. Both tail and toe-clips accurately predicted whole-body THg concentrations, but the relationships differed among species and the specific tail-clip section or toe that was used. Tail-clips comprised of the distal 0-2 cm segment performed the best across all salamander species, explaining between 82 and 92% of the variation in paired whole-body THg concentrations. Toe-clips were less effective predictors of frog THg concentrations, but THg concentrations in outer rear toes accounted for up to 79% of the variability in frog whole-body THg concentrations. These findings suggest non-lethal sampling of tails and toes has potential applications for monitoring contaminant exposure and risk in amphibians, but care must be taken to ensure consistent collection and interpretation of samples.

Genetic addiction: selfish gene's strategy for symbiosis in the genome.

PubMed

Mochizuki, Atsushi; Yahara, Koji; Kobayashi, Ichizo; Iwasa, Yoh

2006-02-01

The evolution and maintenance of the phenomenon of postsegregational host killing or genetic addiction are paradoxical. In this phenomenon, a gene complex, once established in a genome, programs death of a host cell that has eliminated it. The intact form of the gene complex would survive in other members of the host population. It is controversial as to why these genetic elements are maintained, due to the lethal effects of host killing, or perhaps some other properties are beneficial to the host. We analyzed their population dynamics by analytical methods and computer simulations. Genetic addiction turned out to be advantageous to the gene complex in the presence of a competitor genetic element. The advantage is, however, limited in a population without spatial structure, such as that in a well-mixed liquid culture. In contrast, in a structured habitat, such as the surface of a solid medium, the addiction gene complex can increase in frequency, irrespective of its initial density. Our demonstration that genomes can evolve through acquisition of addiction genes has implications for the general question of how a genome can evolve as a community of potentially selfish genes.

Pathways to genetic screening: Patient knowledges, Patient practices. Technical report of research progress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1994-08-29

This study is designed to clarify the integration of genetic knowledge into the lived experience of high-risk family members. A major focus is elucidation of the social and cultural barriers and bridges to the use of genetic information to increase reproductive options and improve the quality of family life. They study focuses on families at risk for cystic fibrosis and sickle cell disease. These two disease groups were selected because they are among the most common potentially lethal genetic diseases and because while each has a similar pattern of inheritance and raises similarly serious bio-medical challenges and issues of informationmore » management, they primarily affect different racial and ethno-cultural groups permitting a naturally occurring experiment. In the variable penetration and meaning of genetic medicine in two populations. We have conducted intensive interviews with more than 300 individuals in approximately 88 families. We have attempted to balance the effort equally between the two groups, but have found we are able to penetrate the family systems of the cystic fibrosis families more easily than the sickle cell families.« less

Social Network Analysis of Crowds

DTIC Science & Technology

2009-10-29

Response to non-lethal weapons fire depends on social relationships among crowd members – Pre-existing Personal Relationships – Ongoing Real Time...weapons and systems – Prior, existing social relationships – Real time social interactions – Formal/informal hierarchies 13-Feb-15 24UNCLASSIFIED

Temporal genetic population structure and interannual variation in migration behavior of Pacific Lamprey Entosphenus tridentatus

USGS Publications Warehouse

Clemens, Benjamin J.; Wyss, Lance A.; McCoun, Rebecca; Courter, Ian; Schwabe, Lawrence; Peery, Christopher; Schreck, Carl B.; Spice, Erin K.; Docker, Margaret F.

2017-01-01

Studies using neutral loci suggest that Pacific lamprey, Entosphenus tridentatus, lack strong spatial genetic population structure. However, it is unknown whether temporal genetic population structure exists. We tested whether adult Pacific lamprey: (1) show temporal genetic population structure; and (2) migrate different distances between years. We non-lethally sampled lamprey for DNA in 2009 and 2010 and used eight microsatellite loci to test for genetic population structure. We used telemetry to record the migration behaviors of these fish. Lamprey were assignable to three moderately differentiated genetic clusters (FST = 0.16–0.24 for all pairwise comparisons): one cluster was composed of individuals from 2009, and the other two contained individuals from 2010. The FST value between years was 0.13 and between genetic clusters within 2010 was 0.20. A total of 372 (72.5%) fish were detected multiple times during their migrations. Most fish (69.9%) remained in the mainstem Willamette River; the remaining 30.1% migrated into tributaries. Eighty-two lamprey exhibited multiple back-and-forth movements among tributaries and the mainstem, which may indicate searching behaviors. All migration distances were significantly greater in 2010, when the amplitude of river discharge was greater. Our data suggest genetic structuring between and within years that may reflect different cohorts.

Genetic susceptibility to neuroblastoma: current knowledge and future directions.

PubMed

Ritenour, Laura E; Randall, Michael P; Bosse, Kristopher R; Diskin, Sharon J

2018-05-01

Neuroblastoma, a malignancy of the developing peripheral nervous system that affects infants and young children, is a complex genetic disease. Over the past two decades, significant progress has been made toward understanding the genetic determinants that predispose to this often lethal childhood cancer. Approximately 1-2% of neuroblastomas are inherited in an autosomal dominant fashion and a combination of co-morbidity and linkage studies has led to the identification of germline mutations in PHOX2B and ALK as the major genetic contributors to this familial neuroblastoma subset. The genetic basis of "sporadic" neuroblastoma is being studied through a large genome-wide association study (GWAS). These efforts have led to the discovery of many common susceptibility alleles, each with modest effect size, associated with the development and progression of sporadic neuroblastoma. More recently, next-generation sequencing efforts have expanded the list of potential neuroblastoma-predisposing mutations to include rare germline variants with a predicted larger effect size. The evolving characterization of neuroblastoma's genetic basis has led to a deeper understanding of the molecular events driving tumorigenesis, more precise risk stratification and prognostics and novel therapeutic strategies. This review details the contemporary understanding of neuroblastoma's genetic predisposition, including recent advances and discusses ongoing efforts to address gaps in our knowledge regarding this malignancy's complex genetic underpinnings.

NMDA antagonists exert distinct effects in experimental organophosphate or carbamate poisoning in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dekundy, Andrzej; Kaminski, Rafal M.; Zielinska, Elzbieta

2007-03-15

Organophosphate (OP) and carbamate acetylcholinesterase (AChE) inhibitors produce seizures and lethality in mammals. Anticonvulsant and neuroprotective properties of N-methyl-D-aspartate (NMDA) antagonists encourage the investigation of their effects in AChE inhibitor-induced poisonings. In the present study, the effects of dizocilpine (MK-801, 1 mg/kg) or 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP, 10 mg/kg), alone or combined with muscarinic antagonist atropine (1.8 mg/kg), on convulsant and lethal properties of an OP pesticide dichlorvos or a carbamate drug physostigmine, were studied in mice. Both dichlorvos and physostigmine induced dose-dependent seizure activity and lethality. Atropine did not prevent the occurrence of convulsions but decreased the lethal effects ofmore » both dichlorvos and physostigmine. MK-801 or CPP blocked or attenuated, respectively, dichlorvos-induced convulsions. Contrariwise, NMDA antagonists had no effect in physostigmine-induced seizures or lethality produced by dichlorvos or physostigmine. Concurrent pretreatment with atropine and either MK-801 or CPP blocked or alleviated seizures produced by dichlorvos, but not by physostigmine. Both MK-801 and CPP co-administered with atropine enhanced its antilethal effects in both dichlorvos and physostigmine poisoning. In both saline- and AChE inhibitor-treated mice, no interaction of the investigated antidotes with brain cholinesterase was found. The data indicate that both muscarinic ACh and NMDA receptor-mediated mechanisms contribute to the acute toxicity of AChE inhibitors, and NMDA receptors seem critical to OP-induced seizures.« less

Pathway-based discovery of genetic interactions in breast cancer

PubMed Central

Xu, Zack Z.; Boone, Charles; Lange, Carol A.

2017-01-01

Breast cancer is the second largest cause of cancer death among U.S. women and the leading cause of cancer death among women worldwide. Genome-wide association studies (GWAS) have identified several genetic variants associated with susceptibility to breast cancer, but these still explain less than half of the estimated genetic contribution to the disease. Combinations of variants (i.e. genetic interactions) may play an important role in breast cancer susceptibility. However, due to a lack of statistical power, the current tests for genetic interactions from GWAS data mainly leverage prior knowledge to focus on small sets of genes or SNPs that are known to have an association with breast cancer. Thus, many genetic interactions, particularly among novel variants, remain understudied. Reverse-genetic interaction screens in model organisms have shown that genetic interactions frequently cluster into highly structured motifs, where members of the same pathway share similar patterns of genetic interactions. Based on this key observation, we recently developed a method called BridGE to search for such structured motifs in genetic networks derived from GWAS studies and identify pathway-level genetic interactions in human populations. We applied BridGE to six independent breast cancer cohorts and identified significant pathway-level interactions in five cohorts. Joint analysis across all five cohorts revealed a high confidence consensus set of genetic interactions with support in multiple cohorts. The discovered interactions implicated the glutathione conjugation, vitamin D receptor, purine metabolism, mitotic prometaphase, and steroid hormone biosynthesis pathways as major modifiers of breast cancer risk. Notably, while many of the pathways identified by BridGE show clear relevance to breast cancer, variants in these pathways had not been previously discovered by traditional single variant association tests, or single pathway enrichment analysis that does not consider SNP-SNP interactions. PMID:28957314

A Haploid Genetic Screen Identifies Heparan Sulfate Proteoglycans Supporting Rift Valley Fever Virus Infection.

PubMed

Riblett, Amber M; Blomen, Vincent A; Jae, Lucas T; Altamura, Louis A; Doms, Robert W; Brummelkamp, Thijn R; Wojcechowskyj, Jason A

2016-02-01

Rift Valley fever virus (RVFV) causes recurrent insect-borne epizootics throughout the African continent, and infection of humans can lead to a lethal hemorrhagic fever syndrome. Deep mutagenesis of haploid human cells was used to identify host factors required for RVFV infection. This screen identified a suite of enzymes involved in glycosaminoglycan (GAG) biogenesis and transport, including several components of the cis-oligomeric Golgi (COG) complex, one of the central components of Golgi complex trafficking. In addition, disruption of PTAR1 led to RVFV resistance as well as reduced heparan sulfate surface levels, consistent with recent observations that PTAR1-deficient cells exhibit altered Golgi complex morphology and glycosylation defects. A variety of biochemical and genetic approaches were utilized to show that both pathogenic and attenuated RVFV strains require GAGs for efficient infection on some, but not all, cell types, with the block to infection being at the level of virion attachment. Examination of other members of the Bunyaviridae family for GAG-dependent infection suggested that the interaction with GAGs is not universal among bunyaviruses, indicating that these viruses, as well as RVFV on certain cell types, employ additional unidentified virion attachment factors and/or receptors. Rift Valley fever virus (RVFV) is an emerging pathogen that can cause severe disease in humans and animals. Epizootics among livestock populations lead to high mortality rates and can be economically devastating. Human epidemics of Rift Valley fever, often initiated by contact with infected animals, are characterized by a febrile disease that sometimes leads to encephalitis or hemorrhagic fever. The global burden of the pathogen is increasing because it has recently disseminated beyond Africa, which is of particular concern because the virus can be transmitted by widely distributed mosquito species. There are no FDA-licensed vaccines or antiviral agents with activity against RVFV, and details of its life cycle and interaction with host cells are not well characterized. We used the power of genetic screening in human cells and found that RVFV utilizes glycosaminoglycans to attach to host cells. This furthers our understanding of the virus and informs the development of antiviral therapeutics. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Network Hubs Buffer Environmental Variation in Saccharomyces cerevisiae

PubMed Central

Levy, Sasha F; Siegal, Mark L

2008-01-01

Regulatory and developmental systems produce phenotypes that are robust to environmental and genetic variation. A gene product that normally contributes to this robustness is termed a phenotypic capacitor. When a phenotypic capacitor fails, for example when challenged by a harsh environment or mutation, the system becomes less robust and thus produces greater phenotypic variation. A functional phenotypic capacitor provides a mechanism by which hidden polymorphism can accumulate, whereas its failure provides a mechanism by which evolutionary change might be promoted. The primary example to date of a phenotypic capacitor is Hsp90, a molecular chaperone that targets a large set of signal transduction proteins. In both Drosophila and Arabidopsis, compromised Hsp90 function results in pleiotropic phenotypic effects dependent on the underlying genotype. For some traits, Hsp90 also appears to buffer stochastic variation, yet the relationship between environmental and genetic buffering remains an important unresolved question. We previously used simulations of knockout mutations in transcriptional networks to predict that many gene products would act as phenotypic capacitors. To test this prediction, we use high-throughput morphological phenotyping of individual yeast cells from single-gene deletion strains to identify gene products that buffer environmental variation in Saccharomyces cerevisiae. We find more than 300 gene products that, when absent, increase morphological variation. Overrepresented among these capacitors are gene products that control chromosome organization and DNA integrity, RNA elongation, protein modification, cell cycle, and response to stimuli such as stress. Capacitors have a high number of synthetic-lethal interactions but knockouts of these genes do not tend to cause severe decreases in growth rate. Each capacitor can be classified based on whether or not it is encoded by a gene with a paralog in the genome. Capacitors with a duplicate are highly connected in the protein–protein interaction network and show considerable divergence in expression from their paralogs. In contrast, capacitors encoded by singleton genes are part of highly interconnected protein clusters whose other members also tend to affect phenotypic variability or fitness. These results suggest that buffering and release of variation is a widespread phenomenon that is caused by incomplete functional redundancy at multiple levels in the genetic architecture. PMID:18986213

The ADAR RNA editing enzyme controls neuronal excitability in Drosophila melanogaster

PubMed Central

Li, Xianghua; Overton, Ian M.; Baines, Richard A.; Keegan, Liam P.; O’Connell, Mary A.

2014-01-01

RNA editing by deamination of specific adenosine bases to inosines during pre-mRNA processing generates edited isoforms of proteins. Recoding RNA editing is more widespread in Drosophila than in vertebrates. Editing levels rise strongly at metamorphosis, and Adar5G1 null mutant flies lack editing events in hundreds of CNS transcripts; mutant flies have reduced viability, severely defective locomotion and age-dependent neurodegeneration. On the other hand, overexpressing an adult dADAR isoform with high enzymatic activity ubiquitously during larval and pupal stages is lethal. Advantage was taken of this to screen for genetic modifiers; Adar overexpression lethality is rescued by reduced dosage of the Rdl (Resistant to dieldrin), gene encoding a subunit of inhibitory GABA receptors. Reduced dosage of the Gad1 gene encoding the GABA synthetase also rescues Adar overexpression lethality. Drosophila Adar5G1 mutant phenotypes are ameliorated by feeding GABA modulators. We demonstrate that neuronal excitability is linked to dADAR expression levels in individual neurons; Adar-overexpressing larval motor neurons show reduced excitability whereas Adar5G1 null mutant or targeted Adar knockdown motor neurons exhibit increased excitability. GABA inhibitory signalling is impaired in human epileptic and autistic conditions, and vertebrate ADARs may have a relevant evolutionarily conserved control over neuronal excitability. PMID:24137011

Highly variable penetrance of abnormal phenotypes in embryonic lethal knockout mice

PubMed Central

Wilson, Robert; Geyer, Stefan H.; Reissig, Lukas; Rose, Julia; Szumska, Dorota; Hardman, Emily; Prin, Fabrice; McGuire, Christina; Ramirez-Solis, Ramiro; White, Jacqui; Galli, Antonella; Tudor, Catherine; Tuck, Elizabeth; Mazzeo, Cecilia Icoresi; Smith, James C.; Robertson, Elizabeth; Adams, David J.; Mohun, Timothy; Weninger, Wolfgang J.

2017-01-01

Background: Identifying genes that are essential for mouse embryonic development and survival through term is a powerful and unbiased way to discover possible genetic determinants of human developmental disorders. Characterising the changes in mouse embryos that result from ablation of lethal genes is a necessary first step towards uncovering their role in normal embryonic development and establishing any correlates amongst human congenital abnormalities. Methods: Here we present results gathered to date in the Deciphering the Mechanisms of Developmental Disorders (DMDD) programme, cataloguing the morphological defects identified from comprehensive imaging of 220 homozygous mutant and 114 wild type embryos from 42 lethal and subviable lines, analysed at E14.5. Results: Virtually all mutant embryos show multiple abnormal phenotypes and amongst the 42 lines these affect most organ systems. Within each mutant line, the phenotypes of individual embryos form distinct but overlapping sets. Subcutaneous edema, malformations of the heart or great vessels, abnormalities in forebrain morphology and the musculature of the eyes are all prevalent phenotypes, as is loss or abnormal size of the hypoglossal nerve. Conclusions: Overall, the most striking finding is that no matter how profound the malformation, each phenotype shows highly variable penetrance within a mutant line. These findings have challenging implications for efforts to identify human disease correlates. PMID:27996060

Delineating the requirements for spontaneous DNA damage resistance pathways in genome maintenance and viability in Saccharomyces cerevisiae.

PubMed

Morey, Natalie J; Doetsch, Paul W; Jinks-Robertson, Sue

2003-06-01

Cellular metabolic processes constantly generate reactive species that damage DNA. To counteract this relentless assault, cells have developed multiple pathways to resist damage. The base excision repair (BER) and nucleotide excision repair (NER) pathways remove damage whereas the recombination (REC) and postreplication repair (PRR) pathways bypass the damage, allowing deferred removal. Genetic studies in yeast indicate that these pathways can process a common spontaneous lesion(s), with mutational inactivation of any pathway increasing the burden on the remaining pathways. In this study, we examine the consequences of simultaneously compromising three or more of these pathways. Although the presence of a functional BER pathway alone is able to support haploid growth, retention of the NER, REC, or PRR pathway alone is not, indicating that BER is the key damage resistance pathway in yeast and may be responsible for the removal of the majority of either spontaneous DNA damage or specifically those lesions that are potentially lethal. In the diploid state, functional BER, NER, or REC alone can support growth, while PRR alone is insufficient for growth. In diploids, the presence of PRR alone may confer a lethal mutation load or, alternatively, PRR alone may be insufficient to deal with potentially lethal, replication-blocking lesions.

Population-level effects of fitness costs associated with repressible female-lethal transgene insertions in two pest insects

PubMed Central

Harvey-Samuel, Tim; Ant, Thomas; Gong, Hongfei; Morrison, Neil I; Alphey, Luke

2014-01-01

Genetic control strategies offer great potential for the sustainable and effective control of insect pests. These strategies involve the field release of transgenic insects with the aim of introducing engineered alleles into wild populations, either permanently or transiently. Their efficacy can therefore be reduced if transgene-associated fitness costs reduce the relative performance of released insects. We describe a method of measuring the fitness costs associated with transgenes by analyzing their evolutionary trajectories when placed in competition with wild-type alleles in replicated cage populations. Using this method, we estimated lifetime fitness costs associated with two repressible female-lethal transgenes in the diamondback moth and olive fly as being acceptable for field suppression programs. Furthermore, using these estimates of genotype-level fitness costs, we were able to project longer-term evolutionary trajectories for the transgenes investigated. Results from these projections demonstrate that although transgene-associated fitness costs will ultimately cause these transgenes to become extinct, even when engineered lethality is repressed, they may persist for varying periods of time before doing so. This implies that tetracycline-mediated transgene field persistence in these strains is unlikely and suggests that realistic estimates of transgene-associated fitness costs may be useful in trialing ‘uncoupled’ gene drive system components in the field. PMID:24944572

Population-level effects of fitness costs associated with repressible female-lethal transgene insertions in two pest insects.

PubMed

Harvey-Samuel, Tim; Ant, Thomas; Gong, Hongfei; Morrison, Neil I; Alphey, Luke

2014-05-01

Genetic control strategies offer great potential for the sustainable and effective control of insect pests. These strategies involve the field release of transgenic insects with the aim of introducing engineered alleles into wild populations, either permanently or transiently. Their efficacy can therefore be reduced if transgene-associated fitness costs reduce the relative performance of released insects. We describe a method of measuring the fitness costs associated with transgenes by analyzing their evolutionary trajectories when placed in competition with wild-type alleles in replicated cage populations. Using this method, we estimated lifetime fitness costs associated with two repressible female-lethal transgenes in the diamondback moth and olive fly as being acceptable for field suppression programs. Furthermore, using these estimates of genotype-level fitness costs, we were able to project longer-term evolutionary trajectories for the transgenes investigated. Results from these projections demonstrate that although transgene-associated fitness costs will ultimately cause these transgenes to become extinct, even when engineered lethality is repressed, they may persist for varying periods of time before doing so. This implies that tetracycline-mediated transgene field persistence in these strains is unlikely and suggests that realistic estimates of transgene-associated fitness costs may be useful in trialing 'uncoupled' gene drive system components in the field.

DNA replication error-induced extinction of diploid yeast.

PubMed

Herr, Alan J; Kennedy, Scott R; Knowels, Gary M; Schultz, Eric M; Preston, Bradley D

2014-03-01

Genetic defects in DNA polymerase accuracy, proofreading, or mismatch repair (MMR) induce mutator phenotypes that accelerate adaptation of microbes and tumor cells. Certain combinations of mutator alleles synergistically increase mutation rates to levels that drive extinction of haploid cells. The maximum tolerated mutation rate of diploid cells is unknown. Here, we define the threshold for replication error-induced extinction (EEX) of diploid Saccharomyces cerevisiae. Double-mutant pol3 alleles that carry mutations for defective DNA polymerase-δ proofreading (pol3-01) and accuracy (pol3-L612M or pol3-L612G) induce strong mutator phenotypes in heterozygous diploids (POL3/pol3-01,L612M or POL3/pol3-01,L612G). Both pol3-01,L612M and pol3-01,L612G alleles are lethal in the homozygous state; cells with pol3-01,L612M divide up to 10 times before arresting at random stages in the cell cycle. Antimutator eex mutations in the pol3 alleles suppress this lethality (pol3-01,L612M,eex or pol3-01,L612G,eex). MMR defects synergize with pol3-01,L612M,eex and pol3-01,L612G,eex alleles, increasing mutation rates and impairing growth. Conversely, inactivation of the Dun1 S-phase checkpoint kinase suppresses strong pol3-01,L612M,eex and pol3-01,L612G,eex mutator phenotypes as well as the lethal pol3-01,L612M phenotype. Our results reveal that the lethal error threshold in diploids is 10 times higher than in haploids and likely determined by homozygous inactivation of essential genes. Pronounced loss of fitness occurs at mutation rates well below the lethal threshold, suggesting that mutator-driven cancers may be susceptible to drugs that exacerbate replication errors.

The population genetics of human disease: The case of recessive, lethal mutations

PubMed Central

Gao, Ziyue; Baker, Zachary; Diesel, José Francisco; Simons, Yuval B.; Haque, Imran S.; Pickrell, Joseph; Przeworski, Molly

2017-01-01

Do the frequencies of disease mutations in human populations reflect a simple balance between mutation and purifying selection? What other factors shape the prevalence of disease mutations? To begin to answer these questions, we focused on one of the simplest cases: recessive mutations that alone cause lethal diseases or complete sterility. To this end, we generated a hand-curated set of 417 Mendelian mutations in 32 genes reported to cause a recessive, lethal Mendelian disease. We then considered analytic models of mutation-selection balance in infinite and finite populations of constant sizes and simulations of purifying selection in a more realistic demographic setting, and tested how well these models fit allele frequencies estimated from 33,370 individuals of European ancestry. In doing so, we distinguished between CpG transitions, which occur at a substantially elevated rate, and three other mutation types. Intriguingly, the observed frequency for CpG transitions is slightly higher than expectation but close, whereas the frequencies observed for the three other mutation types are an order of magnitude higher than expected, with a bigger deviation from expectation seen for less mutable types. This discrepancy is even larger when subtle fitness effects in heterozygotes or lethal compound heterozygotes are taken into account. In principle, higher than expected frequencies of disease mutations could be due to widespread errors in reporting causal variants, compensation by other mutations, or balancing selection. It is unclear why these factors would have a greater impact on disease mutations that occur at lower rates, however. We argue instead that the unexpectedly high frequency of disease mutations and the relationship to the mutation rate likely reflect an ascertainment bias: of all the mutations that cause recessive lethal diseases, those that by chance have reached higher frequencies are more likely to have been identified and thus to have been included in this study. Beyond the specific application, this study highlights the parameters likely to be important in shaping the frequencies of Mendelian disease alleles. PMID:28957316

Fine-tuning synthesis of Yersinia pestis LcrV from runaway-like replication balanced-lethal plasmid in a Salmonella enterica serovar typhimurium vaccine induces protection against a lethal Y. pestis challenge in mice.

PubMed

Torres-Escobar, Ascención; Juárez-Rodríguez, María Dolores; Gunn, Bronwyn M; Branger, Christine G; Tinge, Steven A; Curtiss, Roy

2010-06-01

A balanced-lethal plasmid expression system that switches from low-copy-number to runaway-like high-copy-number replication (pYA4534) was constructed for the regulated delayed in vivo synthesis of heterologous antigens by vaccine strains. This is an antibiotic resistance-free maintenance system containing the asdA gene (essential for peptidoglycan synthesis) as a selectable marker to complement the lethal chromosomal DeltaasdA allele in live recombinant attenuated Salmonella vaccines (RASVs) such as Salmonella enterica serovar Typhimurium strain chi9447. pYA4534 harbors two origins of replication, pSC101 and pUC (low and high copy numbers, respectively). The pUC replication origin is controlled by a genetic switch formed by the operator/promoter of the P22 cro gene (O/P(cro)) (P(R)), which is negatively regulated by an arabinose-inducible P22 c2 gene located on both the plasmid and the chromosome (araC P(BAD) c2). The absence of arabinose, which is unavailable in vivo, triggers replication to a high-copy-number plasmid state. To validate these vector attributes, the Yersinia pestis virulence antigen LcrV was used to develop a vaccine against plague. An lcrV sequence encoding amino acids 131 to 326 (LcrV196) was optimized for expression in Salmonella, flanked with nucleotide sequences encoding the signal peptide (SS) and the carboxy-terminal domain (CT) of beta-lactamase, and cloned into pYA4534 under the control of the P(trc) promoter to generate plasmid pYA4535. Our results indicate that the live Salmonella vaccine strain chi9447 harboring pYA4535 efficiently stimulated a mixed Th1/Th2 immune response that protected mice against lethal challenge with Y. pestis strain CO92 introduced through either the intranasal or subcutaneous route.

Fine-Tuning Synthesis of Yersinia pestis LcrV from Runaway-Like Replication Balanced-Lethal Plasmid in a Salmonella enterica Serovar Typhimurium Vaccine Induces Protection against a Lethal Y. pestis Challenge in Mice▿

PubMed Central

Torres-Escobar, Ascención; Juárez-Rodríguez, María Dolores; Gunn, Bronwyn M.; Branger, Christine G.; Tinge, Steven A.; Curtiss, Roy

2010-01-01

A balanced-lethal plasmid expression system that switches from low-copy-number to runaway-like high-copy-number replication (pYA4534) was constructed for the regulated delayed in vivo synthesis of heterologous antigens by vaccine strains. This is an antibiotic resistance-free maintenance system containing the asdA gene (essential for peptidoglycan synthesis) as a selectable marker to complement the lethal chromosomal ΔasdA allele in live recombinant attenuated Salmonella vaccines (RASVs) such as Salmonella enterica serovar Typhimurium strain χ9447. pYA4534 harbors two origins of replication, pSC101 and pUC (low and high copy numbers, respectively). The pUC replication origin is controlled by a genetic switch formed by the operator/promoter of the P22 cro gene (O/Pcro) (PR), which is negatively regulated by an arabinose-inducible P22 c2 gene located on both the plasmid and the chromosome (araC PBAD c2). The absence of arabinose, which is unavailable in vivo, triggers replication to a high-copy-number plasmid state. To validate these vector attributes, the Yersinia pestis virulence antigen LcrV was used to develop a vaccine against plague. An lcrV sequence encoding amino acids 131 to 326 (LcrV196) was optimized for expression in Salmonella, flanked with nucleotide sequences encoding the signal peptide (SS) and the carboxy-terminal domain (CT) of β-lactamase, and cloned into pYA4534 under the control of the Ptrc promoter to generate plasmid pYA4535. Our results indicate that the live Salmonella vaccine strain χ9447 harboring pYA4535 efficiently stimulated a mixed Th1/Th2 immune response that protected mice against lethal challenge with Y. pestis strain CO92 introduced through either the intranasal or subcutaneous route. PMID:20308296

Incorporating gene-environment interaction in testing for association with rare genetic variants.

PubMed

Chen, Han; Meigs, James B; Dupuis, Josée

2014-01-01

The incorporation of gene-environment interactions could improve the ability to detect genetic associations with complex traits. For common genetic variants, single-marker interaction tests and joint tests of genetic main effects and gene-environment interaction have been well-established and used to identify novel association loci for complex diseases and continuous traits. For rare genetic variants, however, single-marker tests are severely underpowered due to the low minor allele frequency, and only a few gene-environment interaction tests have been developed. We aimed at developing powerful and computationally efficient tests for gene-environment interaction with rare variants. In this paper, we propose interaction and joint tests for testing gene-environment interaction of rare genetic variants. Our approach is a generalization of existing gene-environment interaction tests for multiple genetic variants under certain conditions. We show in our simulation studies that our interaction and joint tests have correct type I errors, and that the joint test is a powerful approach for testing genetic association, allowing for gene-environment interaction. We also illustrate our approach in a real data example from the Framingham Heart Study. Our approach can be applied to both binary and continuous traits, it is powerful and computationally efficient.

Sex-lethal promotes nuclear retention of msl2 mRNA via interactions with the STAR protein HOW

PubMed Central

Graindorge, Antoine; Carré, Clément; Gebauer, Fátima

2013-01-01

Female-specific repression of male-specific-lethal-2 (msl2) mRNA in Drosophila melanogaster provides a paradigm for coordinated control of gene expression by RNA-binding complexes. Repression is orchestrated by Sex-lethal (SXL), which binds to the 5′ and 3′ untranslated regions (UTRs) of the mRNA and inhibits splicing in the nucleus and subsequent translation in the cytoplasm. Here we show that SXL ensures msl2 silencing by yet a third mechanism that involves inhibition of nucleocytoplasmic transport of msl2 mRNA. To identify SXL cofactors in msl2 regulation, we devised a two-step purification method termed GRAB (GST pull-down and RNA affinity binding) and identified Held-Out-Wings (HOW) as a component of the msl2 5′ UTR-associated complex. HOW directly interacts with SXL and binds to two sequence elements in the msl2 5′ UTR. Depletion of HOW reduces the capacity of SXL to repress the expression of msl2 reporters without affecting SXL-mediated regulation of splicing or translation. Instead, HOW is required for SXL to retain msl2 transcripts in the nucleus. Cooperation with SXL confers a sex-specific role to HOW. Our results uncover a novel function of SXL in nuclear mRNA retention and identify HOW as a mediator of this function. PMID:23788626

Morphogenesis Is Not Required for Candida albicans-Staphylococcus aureus Intra-Abdominal Infection-Mediated Dissemination and Lethal Sepsis

PubMed Central

Nash, Evelyn E.; Peters, Brian M.; Palmer, Glen E.; Fidel, Paul L.

2014-01-01

Intra-abdominal polymicrobial infections cause significant morbidity and mortality. An established experimental mouse model of Staphylococcus aureus-Candida albicans intra-abdominal infection results in ∼60% mortality within 48 h postinoculation, concomitant with amplified local inflammatory responses, while monomicrobial infections are avirulent. The purpose of this study was to characterize early local and systemic innate responses during coinfection and determine the role of C. albicans morphogenesis in lethality, a trait involved in virulence and physical interaction with S. aureus. Local and systemic proinflammatory cytokines were significantly elevated during coinfection at early time points (4 to 12 h) compared to those in monoinfection. In contrast, microbial burdens in the organs and peritoneal lavage fluid were similar between mono- and coinfected animals through 24 h, as was peritoneal neutrophil infiltration. After optimizing the model for 100% mortality within 48 h, using 3.5 × 107 C. albicans (5× increase), coinfection with C. albicans yeast-locked or hypha-locked mutants showed similar mortality, dissemination, and local and systemic inflammation to the isogenic control. However, coinfection with the yeast-locked C. albicans mutant given intravenously (i.v.) and S. aureus given intraperitoneally (i.p.) failed to induce mortality. These results suggest a unique intra-abdominal interaction between the host and C. albicans-S. aureus that results in strong inflammatory responses, dissemination, and lethal sepsis, independent of C. albicans morphogenesis. PMID:24891104

Genetic variation in insecticide tolerance in a population of southern leopard frogs (Rana sphenocephala): Implications for amphibian conservation

USGS Publications Warehouse

Bridges, C.M.; Semlitsch, R.D.

2001-01-01

Currently, conservation efforts are devoted to determining the extent and the causes of the decline of many amphibian species worldwide. Human impacts frequently degrade amphibian habitat and have been implicated in many declines. Because genetic variance is critical in determining the persistence of a species in a changing environment, we examined the amount of genetic variability present in a single population for tolerance to an environmental stressor. We examined the amount of genetic variability among full- and half-sib families in a single population of southern leopard frogs (Rana sphenocephala) with respect to their tolerance to lethal concentrations of the agricultural chemical, carbaryl. Analysis of time-to-death data indicated significant differences among full-sib families and suggests a large amount of variability present in the responses to this environmental stressor. Significant differences in responses among half-sib families indicated that there is additive genetic variance. These data suggest that this population may have the ability to adapt to environmental stressors. It is possible that declines of amphibian populations in the western United States may be attributed to low genetic variability resulting from limited migration among populations and small population sizes.

A Case Based Approach to Clinical Genetics of Thoracic Aortic Aneurysm/Dissection

PubMed Central

Giusti, Betti; Nistri, Stefano; Sticchi, Elena; De Cario, Rosina; Abbate, Rosanna; Gensini, Gian Franco; Pepe, Guglielmina

2016-01-01

Thoracic aortic aneurysm/dissection (TAAD) is a potential lethal condition with a rising incidence. This condition may occur sporadically; nevertheless, it displays familial clustering in >20% of the cases. Family history confers a six- to twentyfold increased risk of TAAD and has to be considered in the identification and evaluation of patients needing an adequate clinical follow-up. Familial TAAD recognizes a number of potential etiologies with a significant genetic heterogeneity, in either syndromic or nonsyndromic forms of the manifestation. The clinical impact and the management of patients with TAAD differ according to the syndromic and nonsyndromic forms of the manifestation. The clinical management of TAAD patients varies, depending on the different forms. Starting from the description of patient history, in this paper, we summarized the state of the art concerning assessment of clinical/genetic profile and therapeutic management of TAAD patients. PMID:27314043

Wild worm embryogenesis harbors ubiquitous polygenic modifier variation

PubMed Central

Paaby, Annalise B; White, Amelia G; Riccardi, David D; Gunsalus, Kristin C; Piano, Fabio; Rockman, Matthew V

2015-01-01

Embryogenesis is an essential and stereotypic process that nevertheless evolves among species. Its essentiality may favor the accumulation of cryptic genetic variation (CGV) that has no effect in the wild-type but that enhances or suppresses the effects of rare disruptions to gene function. Here, we adapted a classical modifier screen to interrogate the alleles segregating in natural populations of Caenorhabditis elegans: we induced gene knockdowns and used quantitative genetic methodology to examine how segregating variants modify the penetrance of embryonic lethality. Each perturbation revealed CGV, indicating that wild-type genomes harbor myriad genetic modifiers that may have little effect individually but which in aggregate can dramatically influence penetrance. Phenotypes were mediated by many modifiers, indicating high polygenicity, but the alleles tend to act very specifically, indicating low pleiotropy. Our findings demonstrate the extent of conditional functionality in complex trait architecture. DOI: http://dx.doi.org/10.7554/eLife.09178.001 PMID:26297805

A maternal-effect selfish genetic element in Caenorhabditis elegans.

PubMed

Ben-David, Eyal; Burga, Alejandro; Kruglyak, Leonid

2017-06-09

Selfish genetic elements spread in natural populations and have an important role in genome evolution. We discovered a selfish element causing embryonic lethality in crosses between wild strains of the nematode Caenorhabditis elegans The element is made up of sup-35 , a maternal-effect toxin that kills developing embryos, and pha-1 , its zygotically expressed antidote. pha-1 has long been considered essential for pharynx development on the basis of its mutant phenotype, but this phenotype arises from a loss of suppression of sup-35 toxicity. Inactive copies of the sup-35/pha-1 element show high sequence divergence from active copies, and phylogenetic reconstruction suggests that they represent ancestral stages in the evolution of the element. Our results suggest that other essential genes identified by genetic screens may turn out to be components of selfish elements. Copyright © 2017, American Association for the Advancement of Science.

Spontaneous Generation of Infectious Prion Disease in Transgenic Mice

PubMed Central

Castilla, Joaquín; Pintado, Belén; Gutiérrez-Adan, Alfonso; Andréoletti, Olivier; Aguilar-Calvo, Patricia; Arroba, Ana-Isabel; Parra-Arrondo, Beatriz; Ferrer, Isidro; Manzanares, Jorge; Espinosa, Juan-Carlos

2013-01-01

We generated transgenic mice expressing bovine cellular prion protein (PrPC) with a leucine substitution at codon 113 (113L). This protein is homologous to human protein with mutation 102L, and its genetic link with Gerstmann–Sträussler–Scheinker syndrome has been established. This mutation in bovine PrPC causes a fully penetrant, lethal, spongiform encephalopathy. This genetic disease was transmitted by intracerebral inoculation of brain homogenate from ill mice expressing mutant bovine PrP to mice expressing wild-type bovine PrP, which indicated de novo generation of infectious prions. Our findings demonstrate that a single amino acid change in the PrPC sequence can induce spontaneous generation of an infectious prion disease that differs from all others identified in hosts expressing the same PrPC sequence. These observations support the view that a variety of infectious prion strains might spontaneously emerge in hosts displaying random genetic PrPC mutations. PMID:24274622

Development of a genetic system for the archaeal virus Sulfolobus turreted icosahedral virus (STIV).

PubMed

Wirth, Jennifer Fulton; Snyder, Jamie C; Hochstein, Rebecca A; Ortmann, Alice C; Willits, Deborah A; Douglas, Trevor; Young, Mark J

2011-06-20

Our understanding of archaeal viruses has been limited by the lack of genetic systems for examining viral function. We describe the construction of an infectious clone for the archaeal virus Sulfolobus turreted icosahedral virus (STIV). STIV was isolated from a high temperature (82°C) acidic (pH 2.2) hot spring in Yellowstone National Park and replicates in the archaeal model organism Sulfolobus solfataricus (Rice et al., 2004). While STIV is one of most studied archaeal viruses, little is known about its replication cycle. The development of an STIV infectious clone allows for directed gene disruptions and detailed genetic analysis of the virus. The utility of the STIV infectious clone was demonstrated by gene disruption of STIV open reading frame (ORF) B116 which resulted in crippled virus replication, while disruption of ORFs A197, C381 and B345 was lethal for virus replication. Copyright © 2011. Published by Elsevier Inc.

Wolbachia Protein TomO Targets nanos mRNA and Restores Germ Stem Cells in Drosophila Sex-lethal Mutants.

PubMed

Ote, Manabu; Ueyama, Morio; Yamamoto, Daisuke

2016-09-12

Wolbachia, endosymbiotic bacteria prevalent in invertebrates, manipulate their hosts in a variety of ways: they induce cytoplasmic incompatibility, male lethality, male-to-female transformation, and parthenogenesis. However, little is known about the molecular basis for host manipulation by these bacteria. In Drosophila melanogaster, Wolbachia infection makes otherwise sterile Sex-lethal (Sxl) mutant females capable of producing mature eggs. Through a functional genomic screen for Wolbachia genes with growth-inhibitory effects when expressed in cultured Drosophila cells, we identified the gene WD1278 encoding a novel protein we call toxic manipulator of oogenesis (TomO), which phenocopies some of the Wolbachia effects in Sxl mutant D. melanogaster females. We demonstrate that TomO enhances the maintenance of germ stem cells (GSCs) by elevating Nanos (Nos) expression via its interaction with nos mRNA, ultimately leading to the restoration of germ cell production in Sxl mutant females that are otherwise without GSCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

The non-pathogenic Australian rabbit calicivirus RCV-A1 provides temporal and partial cross protection to lethal Rabbit Haemorrhagic Disease Virus infection which is not dependent on antibody titres

PubMed Central

2013-01-01

The endemic non-pathogenic Australian rabbit calicivirus RCV-A1 is known to provide some cross protection to lethal infection with the closely related Rabbit Haemorrhagic Disease Virus (RHDV). Despite its obvious negative impacts on viral biocontrol of introduced European rabbits in Australia, little is known about the extent and mechanisms of this cross protection. In this study 46 rabbits from a colony naturally infected with RCV-A1 were exposed to RHDV. Survival rates and survival times did not correlate with titres of serum antibodies specific to RCV-A1 or cross reacting to RHDV, but were instead influenced by the time between infection with the two viruses, demonstrating for the first time that the cross protection to lethal RHDV infection is transient. These findings are an important step towards a better understanding of the complex interactions of co-occurring pathogenic and non-pathogenic lagoviruses. PMID:23834204

Caffeine: cognitive and physical performance enhancer or psychoactive drug?

PubMed

Cappelletti, Simone; Piacentino, Daria; Daria, Piacentino; Sani, Gabriele; Aromatario, Mariarosaria

2015-01-01

Caffeine use is increasing worldwide. The underlying motivations are mainly concentration and memory enhancement and physical performance improvement. Coffee and caffeine-containing products affect the cardiovascular system, with their positive inotropic and chronotropic effects, and the central nervous system, with their locomotor activity stimulation and anxiogenic-like effects. Thus, it is of interest to examine whether these effects could be detrimental for health. Furthermore, caffeine abuse and dependence are becoming more and more common and can lead to caffeine intoxication, which puts individuals at risk for premature and unnatural death. The present review summarizes the main findings concerning caffeine's mechanisms of action (focusing on adenosine antagonism, intracellular calcium mobilization, and phosphodiesterases inhibition), use, abuse, dependence, intoxication, and lethal effects. It also suggests that the concepts of toxic and lethal doses are relative, since doses below the toxic and/or lethal range may play a causal role in intoxication or death. This could be due to caffeine's interaction with other substances or to the individuals' preexisting metabolism alterations or diseases.

Caffeine: Cognitive and Physical Performance Enhancer or Psychoactive Drug?

PubMed Central

Cappelletti, Simone; Daria, Piacentino; Sani, Gabriele; Aromatario, Mariarosaria

2015-01-01

Caffeine use is increasing worldwide. The underlying motivations are mainly concentration and memory enhancement and physical performance improvement. Coffee and caffeine-containing products affect the cardiovascular system, with their positive inotropic and chronotropic effects, and the central nervous system, with their locomotor activity stimulation and anxiogenic-like effects. Thus, it is of interest to examine whether these effects could be detrimental for health. Furthermore, caffeine abuse and dependence are becoming more and more common and can lead to caffeine intoxication, which puts individuals at risk for premature and unnatural death. The present review summarizes the main findings concerning caffeine’s mechanisms of action (focusing on adenosine antagonism, intracellular calcium mobilization, and phosphodiesterases inhibition), use, abuse, dependence, intoxication, and lethal effects. It also suggests that the concepts of toxic and lethal doses are relative, since doses below the toxic and/or lethal range may play a causal role in intoxication or death. This could be due to caffeine’s interaction with other substances or to the individuals' preexisting metabolism alterations or diseases. PMID:26074744

A mutually exclusive stem–loop arrangement in roX2 RNA is essential for X-chromosome regulation in Drosophila

PubMed Central

Ilik, Ibrahim Avsar; Maticzka, Daniel; Georgiev, Plamen; Gutierrez, Noel Marie; Backofen, Rolf; Akhtar, Asifa

2017-01-01

The X chromosome provides an ideal model system to study the contribution of RNA–protein interactions in epigenetic regulation. In male flies, roX long noncoding RNAs (lncRNAs) harbor several redundant domains to interact with the ubiquitin ligase male-specific lethal 2 (MSL2) and the RNA helicase Maleless (MLE) for X-chromosomal regulation. However, how these interactions provide the mechanics of spreading remains unknown. By using the uvCLAP (UV cross-linking and affinity purification) methodology, which provides unprecedented information about RNA secondary structures in vivo, we identified the minimal functional unit of roX2 RNA. By using wild-type and various MLE mutant derivatives, including a catalytically inactive MLE derivative, MLEGET, we show that the minimal roX RNA contains two mutually exclusive stem–loops that exist in a peculiar structural arrangement: When one stem–loop is unwound by MLE, an alternate structure can form, likely trapping MLE in this perpetually structured region. We show that this functional unit is necessary for dosage compensation, as mutations that disrupt this formation lead to male lethality. Thus, we propose that roX2 lncRNA contains an MLE-dependent affinity switch to enable reversible interactions of the MSL complex to allow dosage compensation of the X chromosome. PMID:29066499

Inhibition of N-type Ca2+ channels ameliorates an imbalance in cardiac autonomic nerve activity and prevents lethal arrhythmias in mice with heart failure.

PubMed

Yamada, Yuko; Kinoshita, Hideyuki; Kuwahara, Koichiro; Nakagawa, Yasuaki; Kuwabara, Yoshihiro; Minami, Takeya; Yamada, Chinatsu; Shibata, Junko; Nakao, Kazuhiro; Cho, Kosai; Arai, Yuji; Yasuno, Shinji; Nishikimi, Toshio; Ueshima, Kenji; Kamakura, Shiro; Nishida, Motohiro; Kiyonaka, Shigeki; Mori, Yasuo; Kimura, Takeshi; Kangawa, Kenji; Nakao, Kazuwa

2014-10-01

Dysregulation of autonomic nervous system activity can trigger ventricular arrhythmias and sudden death in patients with heart failure. N-type Ca(2+) channels (NCCs) play an important role in sympathetic nervous system activation by regulating the calcium entry that triggers release of neurotransmitters from peripheral sympathetic nerve terminals. We have investigated the ability of NCC blockade to prevent lethal arrhythmias associated with heart failure. We compared the effects of cilnidipine, a dual N- and L-type Ca(2+) channel blocker, with those of nitrendipine, a selective L-type Ca(2+) channel blocker, in transgenic mice expressing a cardiac-specific, dominant-negative form of neuron-restrictive silencer factor (dnNRSF-Tg). In this mouse model of dilated cardiomyopathy leading to sudden arrhythmic death, cardiac structure and function did not significantly differ among the control, cilnidipine, and nitrendipine groups. However, cilnidipine dramatically reduced arrhythmias in dnNRSF-Tg mice, significantly improving their survival rate and correcting the imbalance between cardiac sympathetic and parasympathetic nervous system activity. A β-blocker, bisoprolol, showed similar effects in these mice. Genetic titration of NCCs, achieved by crossing dnNRSF-Tg mice with mice lacking CACNA1B, which encodes the α1 subunit of NCCs, improved the survival rate. With restoration of cardiac autonomic balance, dnNRSF-Tg;CACNA1B(+/-) mice showed fewer malignant arrhythmias than dnNRSF-Tg;CACNA1B(+/+) mice. Both pharmacological blockade of NCCs and their genetic titration improved cardiac autonomic balance and prevented lethal arrhythmias in a mouse model of dilated cardiomyopathy and sudden arrhythmic death. Our findings suggest that NCC blockade is a potentially useful approach to preventing sudden death in patients with heart failure. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.

Quantitative maps of genetic interactions in yeast - comparative evaluation and integrative analysis.

PubMed

Lindén, Rolf O; Eronen, Ville-Pekka; Aittokallio, Tero

2011-03-24

High-throughput genetic screening approaches have enabled systematic means to study how interactions among gene mutations contribute to quantitative fitness phenotypes, with the aim of providing insights into the functional wiring diagrams of genetic interaction networks on a global scale. However, it is poorly known how well these quantitative interaction measurements agree across the screening approaches, which hinders their integrated use toward improving the coverage and quality of the genetic interaction maps in yeast and other organisms. Using large-scale data matrices from epistatic miniarray profiling (E-MAP), genetic interaction mapping (GIM), and synthetic genetic array (SGA) approaches, we carried out here a systematic comparative evaluation among these quantitative maps of genetic interactions in yeast. The relatively low association between the original interaction measurements or their customized scores could be improved using a matrix-based modelling framework, which enables the use of single- and double-mutant fitness estimates and measurements, respectively, when scoring genetic interactions. Toward an integrative analysis, we show how the detections from the different screening approaches can be combined to suggest novel positive and negative interactions which are complementary to those obtained using any single screening approach alone. The matrix approximation procedure has been made available to support the design and analysis of the future screening studies. We have shown here that even if the correlation between the currently available quantitative genetic interaction maps in yeast is relatively low, their comparability can be improved by means of our computational matrix approximation procedure, which will enable integrative analysis and detection of a wider spectrum of genetic interactions using data from the complementary screening approaches.

A genome-wide RNAi screen identifies novel targets of neratinib resistance leading to identification of potential drug resistant genetic markers.

PubMed

Seyhan, Attila A; Varadarajan, Usha; Choe, Sung; Liu, Wei; Ryan, Terence E

2012-04-01

Neratinib (HKI-272) is a small molecule tyrosine kinase inhibitor of the ErbB receptor family currently in Phase III clinical trials. Despite its efficacy, the mechanism of potential cellular resistance to neratinib and genes involved with it remains unknown. We have used a pool-based lentiviral genome-wide functional RNAi screen combined with a lethal dose of neratinib to discover chemoresistant interactions with neratinib. Our screen has identified a collection of genes whose inhibition by RNAi led to neratinib resistance including genes involved in oncogenesis (e.g. RAB33A, RAB6A and BCL2L14), transcription factors (e.g. FOXP4, TFEC, ZNF), cellular ion transport (e.g. CLIC3, TRAPPC2P1, P2RX2), protein ubiquitination (e.g. UBL5), cell cycle (e.g. CCNF), and genes known to interact with breast cancer-associated genes (e.g. CCNF, FOXP4, TFEC, several ZNF factors, GNA13, IGFBP1, PMEPA1, SOX5, RAB33A, RAB6A, FXR1, DDO, TFEC, OLFM2). The identification of novel mediators of cellular resistance to neratinib could lead to the identification of new or neoadjuvant drug targets. Their use as patient or treatment selection biomarkers could make the application of anti-ErbB therapeutics more clinically effective.

Toxoplasma gondii Relies on Both Host and Parasite Isoprenoids and Can Be Rendered Sensitive to Atorvastatin

PubMed Central

Li, Zhu-Hong; Ramakrishnan, Srinivasan; Striepen, Boris; Moreno, Silvia N. J.

2013-01-01

Intracellular pathogens have complex metabolic interactions with their host cells to ensure a steady supply of energy and anabolic building blocks for rapid growth. Here we use the obligate intracellular parasite Toxoplasma gondii to probe this interaction for isoprenoids, abundant lipidic compounds essential to many cellular processes including signaling, trafficking, energy metabolism, and protein translation. Synthesis of precursors for isoprenoids in Apicomplexa occurs in the apicoplast and is essential. To synthesize longer isoprenoids from these precursors, T. gondii expresses a bifunctional farnesyl diphosphate/geranylgeranyl diphosphate synthase (TgFPPS). In this work we construct and characterize T. gondii null mutants for this enzyme. Surprisingly, these mutants have only a mild growth phenotype and an isoprenoid composition similar to wild type parasites. However, when extracellular, the loss of the enzyme becomes phenotypically apparent. This strongly suggests that intracellular parasite salvage FPP and/or geranylgeranyl diphosphate (GGPP) from the host. We test this hypothesis using inhibitors of host cell isoprenoid synthesis. Mammals use the mevalonate pathway, which is susceptible to statins. We document strong synergy between statin treatment and pharmacological or genetic interference with the parasite isoprenoid pathway. Mice can be cured with atorvastatin (Lipitor) from a lethal infection with the TgFPPs mutant. We propose a double-hit strategy combining inhibitors of host and parasite pathways as a novel therapeutic approach against Apicomplexan parasites. PMID:24146616

Toxoplasma gondii relies on both host and parasite isoprenoids and can be rendered sensitive to atorvastatin.

PubMed

Li, Zhu-Hong; Ramakrishnan, Srinivasan; Striepen, Boris; Moreno, Silvia N J

2013-01-01

Intracellular pathogens have complex metabolic interactions with their host cells to ensure a steady supply of energy and anabolic building blocks for rapid growth. Here we use the obligate intracellular parasite Toxoplasma gondii to probe this interaction for isoprenoids, abundant lipidic compounds essential to many cellular processes including signaling, trafficking, energy metabolism, and protein translation. Synthesis of precursors for isoprenoids in Apicomplexa occurs in the apicoplast and is essential. To synthesize longer isoprenoids from these precursors, T. gondii expresses a bifunctional farnesyl diphosphate/geranylgeranyl diphosphate synthase (TgFPPS). In this work we construct and characterize T. gondii null mutants for this enzyme. Surprisingly, these mutants have only a mild growth phenotype and an isoprenoid composition similar to wild type parasites. However, when extracellular, the loss of the enzyme becomes phenotypically apparent. This strongly suggests that intracellular parasite salvage FPP and/or geranylgeranyl diphosphate (GGPP) from the host. We test this hypothesis using inhibitors of host cell isoprenoid synthesis. Mammals use the mevalonate pathway, which is susceptible to statins. We document strong synergy between statin treatment and pharmacological or genetic interference with the parasite isoprenoid pathway. Mice can be cured with atorvastatin (Lipitor) from a lethal infection with the TgFPPs mutant. We propose a double-hit strategy combining inhibitors of host and parasite pathways as a novel therapeutic approach against Apicomplexan parasites.

Hip3 interacts with the HIRA proteins Hip1 and Slm9 and is required for transcriptional silencing and accurate chromosome segregation.

PubMed

Greenall, Amanda; Williams, Emma S; Martin, Katherine A; Palmer, Jeremy M; Gray, Joe; Liu, Cong; Whitehall, Simon K

2006-03-31

The fission yeast HIRA proteins Hip1 and Slm9 are members of an evolutionarily conserved family of histone chaperones that are implicated in nucleosome assembly. Here we have used single-step affinity purification and mass spectrometry to identify factors that interact with both Hip1 and Slm9. This analysis identified Hip3, a previously uncharacterized 187-kDa protein, with similarity to S. cerevisiae Hir3. Consistent with this, cells disrupted for hip3+ exhibit a range of growth defects that are similar to those associated with loss of Hip1 and Slm9. These include temperature sensitivity, a cell cycle delay, and synthetic lethality with cdc25-22. Furthermore, genetic analysis also indicates that disruption of hip3+ is epistatic with mutation of hip1+ and slm9+. Mutation of hip3+ alleviates transcriptional silencing at several heterochromatic loci, including in the outer (otr) centromeric repeats, indicating that Hip3 is required for the integrity of pericentric heterochromatin. As a result, loss of Hip3 function leads to high levels of minichromosome loss and an increased frequency of lagging chromosomes during mitosis. Importantly, the function of Hip1, Slm9, and Hip3 is not restricted to constitutive heterochromatic loci, since these proteins also repress the expression of a number of genes, including the Tf2 retrotransposons.

Membrane-localized extra-large G proteins and Gbg of the heterotrimeric G proteins form functional complexes engaged in plant immunity in Arabidopsis.

PubMed

Maruta, Natsumi; Trusov, Yuri; Brenya, Eric; Parekh, Urvi; Botella, José Ramón

2015-03-01

In animals, heterotrimeric G proteins, comprising Ga, Gb, and Gg subunits, are molecular switches whose function tightly depends on Ga and Gbg interaction. Intriguingly, in Arabidopsis (Arabidopsis thaliana), multiple defense responses involve Gbg, but not Ga. We report here that the Gbg dimer directly partners with extra-large G proteins (XLGs) to mediate plant immunity. Arabidopsis mutants deficient in XLGs, Gb, and Gg are similarly compromised in several pathogen defense responses, including disease development and production of reactive oxygen species. Genetic analysis of double, triple, and quadruple mutants confirmed that XLGs and Gbg functionally interact in the same defense signaling pathways. In addition, mutations in XLG2 suppressed the seedling lethal and cell death phenotypes of BRASSINOSTEROID INSENSITIVE1-associated receptor kinase1-interacting receptor-like kinase1 mutants in an identical way as reported for Arabidopsis Gb-deficient mutants. Yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescent complementation assays revealed that XLG2 physically interacts with all three possible Gbg dimers at the plasma membrane. Phylogenetic analysis indicated a close relationship between XLGs and plant Ga subunits, placing the divergence point at the dawn of land plant evolution. Based on these findings, we conclude that XLGs form functional complexes with Gbg dimers, although the mechanism of action of these complexes, including activation/deactivation, must be radically different form the one used by the canonical Ga subunit and are not likely to share the same receptors. Accordingly, XLGs expand the repertoire of heterotrimeric G proteins in plants and reveal a higher level of diversity in heterotrimeric G protein signaling.

Knockdown of genes in the Toll pathway reveals new lethal RNA interference targets for insect pest control.

PubMed

Bingsohn, L; Knorr, E; Billion, A; Narva, K E; Vilcinskas, A

2017-02-01

RNA interference (RNAi) is a promising alternative strategy for ecologically friendly pest management. However, the identification of RNAi candidate genes is challenging owing to the absence of laboratory strains and the seasonality of most pest species. Tribolium castaneum is a well-established model, with a strong and robust RNAi response, which can be used as a high-throughput screening platform to identify potential RNAi target genes. Recently, the cactus gene was identified as a sensitive RNAi target for pest control. To explore whether the spectrum of promising RNAi targets can be expanded beyond those found by random large-scale screening, to encompass others identified using targeted knowledge-based approaches, we constructed a Cactus interaction network. We tested nine genes in this network and found that the delivery of double-stranded RNA corresponding to fusilli and cactin showed lethal effects. The silencing of cactin resulted in 100% lethality at every developmental stage from the larva to the adult. The knockdown of pelle, Dorsal-related immunity factor and short gastrulation reduced or even prevented egg hatching in the next generation. The combination of such targets with lethal and parental RNAi effects can now be tested against different pest species in field studies. © 2016 The Royal Entomological Society.

Scaling laws and universality for the strength of genetic interactions in yeast

NASA Astrophysics Data System (ADS)

Velenich, Andrea; Dai, Mingjie; Gore, Jeff

2012-02-01

Genetic interactions provide a window to the organization of the thousands of biochemical reactions in living cells. If two mutations affect unrelated cellular functions, the fitness effects of their combination can be easily predicted from the two separate fitness effects. However, because of interactions, for some pairs of mutations their combined fitness effect deviates from the naive prediction. We study genetic interactions in yeast cells by analyzing a publicly available database containing experimental growth rates of 5 million double mutants. We show that the characteristic strength of genetic interactions has a simple power law dependence on the fitness effects of the two interacting mutations and that the probability distribution of genetic interactions is a universal function. We further argue that the strength of genetic interactions depends only on the fitness effects of the interacting mutations and not on their biological origin in terms of single point mutations, entire gene knockouts or even more complicated physiological perturbations. Finally, we discuss the implications of the power law scaling of genetic interactions on the ruggedness of fitness landscapes and the consequent evolutionary dynamics.

Global Mapping of the Yeast Genetic Interaction Network

NASA Astrophysics Data System (ADS)

Tong, Amy Hin Yan; Lesage, Guillaume; Bader, Gary D.; Ding, Huiming; Xu, Hong; Xin, Xiaofeng; Young, James; Berriz, Gabriel F.; Brost, Renee L.; Chang, Michael; Chen, YiQun; Cheng, Xin; Chua, Gordon; Friesen, Helena; Goldberg, Debra S.; Haynes, Jennifer; Humphries, Christine; He, Grace; Hussein, Shamiza; Ke, Lizhu; Krogan, Nevan; Li, Zhijian; Levinson, Joshua N.; Lu, Hong; Ménard, Patrice; Munyana, Christella; Parsons, Ainslie B.; Ryan, Owen; Tonikian, Raffi; Roberts, Tania; Sdicu, Anne-Marie; Shapiro, Jesse; Sheikh, Bilal; Suter, Bernhard; Wong, Sharyl L.; Zhang, Lan V.; Zhu, Hongwei; Burd, Christopher G.; Munro, Sean; Sander, Chris; Rine, Jasper; Greenblatt, Jack; Peter, Matthias; Bretscher, Anthony; Bell, Graham; Roth, Frederick P.; Brown, Grant W.; Andrews, Brenda; Bussey, Howard; Boone, Charles

2004-02-01

A genetic interaction network containing ~1000 genes and ~4000 interactions was mapped by crossing mutations in 132 different query genes into a set of ~4700 viable gene yeast deletion mutants and scoring the double mutant progeny for fitness defects. Network connectivity was predictive of function because interactions often occurred among functionally related genes, and similar patterns of interactions tended to identify components of the same pathway. The genetic network exhibited dense local neighborhoods; therefore, the position of a gene on a partially mapped network is predictive of other genetic interactions. Because digenic interactions are common in yeast, similar networks may underlie the complex genetics associated with inherited phenotypes in other organisms.

The Identification of a Legionella pneumophila Toxin with in vivo Lethality

DTIC Science & Technology

1980-01-01

It lacked hydroxy fatty a similar extract, obtained from a newly acids , was minimally inhibited by treat- discovered, but genetically unrelated, ment...applied to the Molecular Weight Estimation column depending upon the individual acid supernatant preparation. There A Sephadex G-50 column (1.5 x...Table 1). Although 0.5 ml of the first peak readi- One-milligram amounts of protein ly kills mice within 18 hours, as much as from Legionella acid

Cytokine Autoantibody Screening in the Swedish Addison Registry Identifies Patients With Undiagnosed APS1.

PubMed

Eriksson, Daniel; Dalin, Frida; Eriksson, Gabriel Nordling; Landegren, Nils; Bianchi, Matteo; Hallgren, Åsa; Dahlqvist, Per; Wahlberg, Jeanette; Ekwall, Olov; Winqvist, Ola; Catrina, Sergiu-Bogdan; Rönnelid, Johan; Hulting, Anna-Lena; Lindblad-Toh, Kerstin; Alimohammadi, Mohammad; Husebye, Eystein S; Knappskog, Per Morten; Rosengren Pielberg, Gerli; Bensing, Sophie; Kämpe, Olle

2018-01-01

Autoimmune polyendocrine syndrome type 1 (APS1) is a monogenic disorder that features autoimmune Addison disease as a major component. Although APS1 accounts for only a small fraction of all patients with Addison disease, early identification of these individuals is vital to prevent the potentially lethal complications of APS1. To determine whether available serological and genetic markers are valuable screening tools for the identification of APS1 among patients diagnosed with Addison disease. We systematically screened 677 patients with Addison disease enrolled in the Swedish Addison Registry for autoantibodies against interleukin-22 and interferon-α4. Autoantibody-positive patients were investigated for clinical manifestations of APS1, additional APS1-specific autoantibodies, and DNA sequence and copy number variations of AIRE. In total, 17 patients (2.5%) displayed autoantibodies against interleukin-22 and/or interferon-α4, of which nine were known APS1 cases. Four patients previously undiagnosed with APS1 fulfilled clinical, genetic, and serological criteria. Hence, we identified four patients with undiagnosed APS1 with this screening procedure. We propose that patients with Addison disease should be routinely screened for cytokine autoantibodies. Clinical or serological support for APS1 should warrant DNA sequencing and copy number analysis of AIRE to enable early diagnosis and prevention of lethal complications. Copyright © 2017 Endocrine Society

Protruding Domain of Capsid Protein Is Necessary and Sufficient To Determine Murine Norovirus Replication and Pathogenesis In Vivo

PubMed Central

Strong, David W.; Thackray, Larissa B.; Smith, Tom J.

2012-01-01

Human noroviruses (HuNoVs) are the major cause of epidemic, nonbacterial gastroenteritis worldwide. Due to the lack of a tractable model system and the inability to grow HuNoVs in cell culture, factors required for the norovirus (NoV) life cycle and pathogenesis in the host remain largely unknown. The discovery of murine norovirus (MNV) and the development of reverse-genetics systems for this virus provide an opportunity to study these aspects of NoV infection in vitro and in vivo. Previous studies identified a single amino acid at residue 296 in the protruding (P) domain of the capsid protein that is responsible for determining the virulence of the MNV clone MNV1.CW1 in 12956/SvEv background STAT1-deficient (STAT1−/−) mice. In this report, we identified and characterized another determinant of lethality in the P domain that is necessary and sufficient to determine the replication and pathogenesis of the MNV clones MNV1.CW3 and CR6.STL1 in C57BL/6 background STAT1−/− mice. Furthermore, we describe how the role of residue 296 in MNV virulence differs between STAT1−/− mouse strains. We also describe potential interactions between subdomains of the P domain, as well as between other virus elements, which facilitate recovery of MNV using a reverse-genetics system. PMID:22258242

Mapping DNA damage-dependent genetic interactions in yeast via party mating and barcode fusion genetics.

PubMed

Díaz-Mejía, J Javier; Celaj, Albi; Mellor, Joseph C; Coté, Atina; Balint, Attila; Ho, Brandon; Bansal, Pritpal; Shaeri, Fatemeh; Gebbia, Marinella; Weile, Jochen; Verby, Marta; Karkhanina, Anna; Zhang, YiFan; Wong, Cassandra; Rich, Justin; Prendergast, D'Arcy; Gupta, Gaurav; Öztürk, Sedide; Durocher, Daniel; Brown, Grant W; Roth, Frederick P

2018-05-28

Condition-dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State-of-the-art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double-mutant strains, does not scale readily to multi-condition studies. Here, we describe barcode fusion genetics to map genetic interactions (BFG-GI), by which double-mutant strains generated via en masse "party" mating can also be monitored en masse for growth to detect genetic interactions. By using site-specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG-GI enables multiplexed quantitative tracking of double mutants via next-generation sequencing. We applied BFG-GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4NQO), bleomycin, zeocin, and three other DNA-damaging environments. BFG-GI recapitulated known genetic interactions and yielded new condition-dependent genetic interactions. We validated and further explored a subnetwork of condition-dependent genetic interactions involving MAG1 , SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to an increase in the activation of the checkpoint protein kinase Rad53. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

rigor mortis encodes a novel nuclear receptor interacting protein required for ecdysone signaling during Drosophila larval development.

PubMed

Gates, Julie; Lam, Geanette; Ortiz, José A; Losson, Régine; Thummel, Carl S

2004-01-01

Pulses of the steroid hormone ecdysone trigger the major developmental transitions in Drosophila, including molting and puparium formation. The ecdysone signal is transduced by the EcR/USP nuclear receptor heterodimer that binds to specific response elements in the genome and directly regulates target gene transcription. We describe a novel nuclear receptor interacting protein encoded by rigor mortis (rig) that is required for ecdysone responses during larval development. rig mutants display defects in molting, delayed larval development, larval lethality, duplicated mouth parts, and defects in puparium formation--phenotypes that resemble those seen in EcR, usp, E75A and betaFTZ-F1 mutants. Although the expression of these nuclear receptor genes is essentially normal in rig mutant larvae, the ecdysone-triggered switch in E74 isoform expression is defective. rig encodes a protein with multiple WD-40 repeats and an LXXLL motif, sequences that act as specific protein-protein interaction domains. Consistent with the presence of these elements and the lethal phenotypes of rig mutants, Rig protein interacts with several Drosophila nuclear receptors in GST pull-down experiments, including EcR, USP, DHR3, SVP and betaFTZ-F1. The ligand binding domain of betaFTZ-F1 is sufficient for this interaction, which can occur in an AF-2-independent manner. Antibody stains reveal that Rig protein is present in the brain and imaginal discs of second and third instar larvae, where it is restricted to the cytoplasm. In larval salivary gland and midgut cells, however, Rig shuttles between the cytoplasm and nucleus in a spatially and temporally regulated manner, at times that correlate with the major lethal phase of rig mutants and major switches in ecdysone-regulated gene expression. Taken together, these data indicate that rig exerts essential functions during larval development through gene-specific effects on ecdysone-regulated transcription, most likely as a cofactor for one or more nuclear receptors. Furthermore, the dynamic intracellular redistribution of Rig protein suggests that it may act to refine spatial and temporal responses to ecdysone during development.

Skin microbes on frogs prevent morbidity and mortality caused by a lethal skin fungus.

PubMed

Harris, Reid N; Brucker, Robert M; Walke, Jenifer B; Becker, Matthew H; Schwantes, Christian R; Flaherty, Devon C; Lam, Brianna A; Woodhams, Douglas C; Briggs, Cheryl J; Vredenburg, Vance T; Minbiole, Kevin P C

2009-07-01

Emerging infectious diseases threaten human and wildlife populations. Altered ecological interactions between mutualistic microbes and hosts can result in disease, but an understanding of interactions between host, microbes and disease-causing organisms may lead to management strategies to affect disease outcomes. Many amphibian species in relatively pristine habitats are experiencing dramatic population declines and extinctions due to the skin disease chytridiomycosis, which is caused by the chytrid fungus Batrachochytrium dendrobatidis. Using a randomized, replicated experiment, we show that adding an antifungal bacterial species, Janthinobacterium lividum, found on several species of amphibians to the skins of the frog Rana muscosa prevented morbidity and mortality caused by the pathogen. The bacterial species produces the anti-chytrid metabolite violacein, which was found in much higher concentrations on frog skins in the treatments where J. lividum was added. Our results show that cutaneous microbes are a part of amphibians' innate immune system, the microbial community structure on frog skins is a determinant of disease outcome and altering microbial interactions on frog skins can prevent a lethal disease outcome. A bioaugmentation strategy may be an effective management tool to control chytridiomycosis in amphibian survival assurance colonies and in nature.

A global interaction network maps a wiring diagram of cellular function

PubMed Central

Costanzo, Michael; VanderSluis, Benjamin; Koch, Elizabeth N.; Baryshnikova, Anastasia; Pons, Carles; Tan, Guihong; Wang, Wen; Usaj, Matej; Hanchard, Julia; Lee, Susan D.; Pelechano, Vicent; Styles, Erin B.; Billmann, Maximilian; van Leeuwen, Jolanda; van Dyk, Nydia; Lin, Zhen-Yuan; Kuzmin, Elena; Nelson, Justin; Piotrowski, Jeff S.; Srikumar, Tharan; Bahr, Sondra; Chen, Yiqun; Deshpande, Raamesh; Kurat, Christoph F.; Li, Sheena C.; Li, Zhijian; Usaj, Mojca Mattiazzi; Okada, Hiroki; Pascoe, Natasha; Luis, Bryan-Joseph San; Sharifpoor, Sara; Shuteriqi, Emira; Simpkins, Scott W.; Snider, Jamie; Suresh, Harsha Garadi; Tan, Yizhao; Zhu, Hongwei; Malod-Dognin, Noel; Janjic, Vuk; Przulj, Natasa; Troyanskaya, Olga G.; Stagljar, Igor; Xia, Tian; Ohya, Yoshikazu; Gingras, Anne-Claude; Raught, Brian; Boutros, Michael; Steinmetz, Lars M.; Moore, Claire L.; Rosebrock, Adam P.; Caudy, Amy A.; Myers, Chad L.; Andrews, Brenda; Boone, Charles

2017-01-01

We generated a global genetic interaction network for Saccharomyces cerevisiae, constructing over 23 million double mutants, identifying ~550,000 negative and ~350,000 positive genetic interactions. This comprehensive network maps genetic interactions for essential gene pairs, highlighting essential genes as densely connected hubs. Genetic interaction profiles enabled assembly of a hierarchical model of cell function, including modules corresponding to protein complexes and pathways, biological processes, and cellular compartments. Negative interactions connected functionally related genes, mapped core bioprocesses, and identified pleiotropic genes, whereas positive interactions often mapped general regulatory connections among gene pairs, rather than shared functionality. The global network illustrates how coherent sets of genetic interactions connect protein complex and pathway modules to map a functional wiring diagram of the cell. PMID:27708008

EINVis: a visualization tool for analyzing and exploring genetic interactions in large-scale association studies.

PubMed

Wu, Yubao; Zhu, Xiaofeng; Chen, Jian; Zhang, Xiang

2013-11-01

Epistasis (gene-gene interaction) detection in large-scale genetic association studies has recently drawn extensive research interests as many complex traits are likely caused by the joint effect of multiple genetic factors. The large number of possible interactions poses both statistical and computational challenges. A variety of approaches have been developed to address the analytical challenges in epistatic interaction detection. These methods usually output the identified genetic interactions and store them in flat file formats. It is highly desirable to develop an effective visualization tool to further investigate the detected interactions and unravel hidden interaction patterns. We have developed EINVis, a novel visualization tool that is specifically designed to analyze and explore genetic interactions. EINVis displays interactions among genetic markers as a network. It utilizes a circular layout (specially, a tree ring view) to simultaneously visualize the hierarchical interactions between single nucleotide polymorphisms (SNPs), genes, and chromosomes, and the network structure formed by these interactions. Using EINVis, the user can distinguish marginal effects from interactions, track interactions involving more than two markers, visualize interactions at different levels, and detect proxy SNPs based on linkage disequilibrium. EINVis is an effective and user-friendly free visualization tool for analyzing and exploring genetic interactions. It is publicly available with detailed documentation and online tutorial on the web at http://filer.case.edu/yxw407/einvis/. © 2013 WILEY PERIODICALS, INC.

The genetic landscape of a physical interaction

PubMed Central

Diss, Guillaume

2018-01-01

A key question in human genetics and evolutionary biology is how mutations in different genes combine to alter phenotypes. Efforts to systematically map genetic interactions have mostly made use of gene deletions. However, most genetic variation consists of point mutations of diverse and difficult to predict effects. Here, by developing a new sequencing-based protein interaction assay – deepPCA – we quantified the effects of >120,000 pairs of point mutations on the formation of the AP-1 transcription factor complex between the products of the FOS and JUN proto-oncogenes. Genetic interactions are abundant both in cis (within one protein) and trans (between the two molecules) and consist of two classes – interactions driven by thermodynamics that can be predicted using a three-parameter global model, and structural interactions between proximally located residues. These results reveal how physical interactions generate quantitatively predictable genetic interactions. PMID:29638215

Evaluation of the Immune Responses to and Cross-Protective Efficacy of Eurasian H7 Avian Influenza Viruses

PubMed Central

Kwon, Hyeok-Il; Kim, Young-Il; Park, Su-Jin; Song, Min-Suk; Kim, Eun-Ha; Kim, Se Mi; Si, Young-Jae; Lee, In-Won; Song, Byung-Min; Lee, Youn-Jeong; Yun, Seok Joong; Kim, Wun-Jae

2017-01-01

ABSTRACT Due to increasing concerns about human infection by various H7 influenza viruses, including recent H7N9 viruses, we evaluated the genetic relationships and cross-protective efficacies of three different Eurasian H7 avian influenza viruses. Phylogenic and molecular analyses revealed that recent Eurasian H7 viruses can be separated into two different lineages, with relatively high amino acid identities within groups (94.8 to 98.8%) and low amino acid identities between groups (90.3 to 92.6%). In vivo immunization with representatives of each group revealed that while group-specific cross-reactivity was induced, cross-reactive hemagglutination inhibition (HI) titers were approximately 4-fold lower against heterologous group viruses than against homologous group viruses. Moreover, the group I (RgW109/06) vaccine protected 100% of immunized mice from various group I viruses, while only 20 to 40% of immunized mice survived lethal challenge with heterologous group II viruses and exhibited high viral titers in the lung. Moreover, while the group II (RgW478/14) vaccine also protected mice from lethal challenge with group II viruses, it failed to elicit cross-protection against group I viruses. However, it is noteworthy that vaccination with RgAnhui1/13, a virus of a sublineage of group I, cross-protected immunized mice against lethal challenge with both group I and II viruses and significantly attenuated lung viral titers. Interestingly, immune sera from RgAnhui1/13-vaccinated mice showed a broad neutralizing spectrum rather than the group-specific pattern observed with the other viruses. These results suggest that the recent human-infective H7N9 strain may be a candidate broad cross-protective vaccine for Eurasian H7 viruses. IMPORTANCE Genetic and phylogenic analyses have demonstrated that the Eurasian H7 viruses can be separated into at least two different lineages, both of which contain human-infective fatal H7 viruses, including the recent novel H7N9 viruses isolated in China since 2013. Due to the increasing concerns regarding the global public health risk posed by H7 viruses, we evaluated the genetic relationships between Eurasian H7 avian influenza viruses and the cross-protective efficacies of three different H7 viruses: W109/06 (group I), W478/14 (group II), and Anhui1/13 (a sublineage of group I). While each vaccine induced group-specific antibody responses and cross-protective efficacy, only Anhui1/13 was able to cross-protect immunized hosts against lethal challenge across groups. In fact, the Anhui1/13 virus induced not only cross-protection but also broad serum neutralizing antibody responses against both groups of viruses. This suggests that Anhui1/13-like H7N9 viruses may be viable vaccine candidates for broad protection against Eurasian H7 viruses. PMID:28331080

Evaluation of the Immune Responses to and Cross-Protective Efficacy of Eurasian H7 Avian Influenza Viruses.

PubMed

Kwon, Hyeok-Il; Kim, Young-Il; Park, Su-Jin; Song, Min-Suk; Kim, Eun-Ha; Kim, Se Mi; Si, Young-Jae; Lee, In-Won; Song, Byung-Min; Lee, Youn-Jeong; Yun, Seok Joong; Kim, Wun-Jae; Choi, Young Ki

2017-06-01

Due to increasing concerns about human infection by various H7 influenza viruses, including recent H7N9 viruses, we evaluated the genetic relationships and cross-protective efficacies of three different Eurasian H7 avian influenza viruses. Phylogenic and molecular analyses revealed that recent Eurasian H7 viruses can be separated into two different lineages, with relatively high amino acid identities within groups (94.8 to 98.8%) and low amino acid identities between groups (90.3 to 92.6%). In vivo immunization with representatives of each group revealed that while group-specific cross-reactivity was induced, cross-reactive hemagglutination inhibition (HI) titers were approximately 4-fold lower against heterologous group viruses than against homologous group viruses. Moreover, the group I (RgW109/06) vaccine protected 100% of immunized mice from various group I viruses, while only 20 to 40% of immunized mice survived lethal challenge with heterologous group II viruses and exhibited high viral titers in the lung. Moreover, while the group II (RgW478/14) vaccine also protected mice from lethal challenge with group II viruses, it failed to elicit cross-protection against group I viruses. However, it is noteworthy that vaccination with RgAnhui1/13, a virus of a sublineage of group I, cross-protected immunized mice against lethal challenge with both group I and II viruses and significantly attenuated lung viral titers. Interestingly, immune sera from RgAnhui1/13-vaccinated mice showed a broad neutralizing spectrum rather than the group-specific pattern observed with the other viruses. These results suggest that the recent human-infective H7N9 strain may be a candidate broad cross-protective vaccine for Eurasian H7 viruses. IMPORTANCE Genetic and phylogenic analyses have demonstrated that the Eurasian H7 viruses can be separated into at least two different lineages, both of which contain human-infective fatal H7 viruses, including the recent novel H7N9 viruses isolated in China since 2013. Due to the increasing concerns regarding the global public health risk posed by H7 viruses, we evaluated the genetic relationships between Eurasian H7 avian influenza viruses and the cross-protective efficacies of three different H7 viruses: W109/06 (group I), W478/14 (group II), and Anhui1/13 (a sublineage of group I). While each vaccine induced group-specific antibody responses and cross-protective efficacy, only Anhui1/13 was able to cross-protect immunized hosts against lethal challenge across groups. In fact, the Anhui1/13 virus induced not only cross-protection but also broad serum neutralizing antibody responses against both groups of viruses. This suggests that Anhui1/13-like H7N9 viruses may be viable vaccine candidates for broad protection against Eurasian H7 viruses. Copyright © 2017 American Society for Microbiology.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

PubMed Central

Bernut, Audrey; Dupont, Christian; Sahuquet, Alain; Herrmann, Jean-Louis; Lutfalla, Georges; Kremer, Laurent

2015-01-01

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions. The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model. In this respect, the present manuscript describes the use of zebrafish as an in vivo model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate. PMID:26382225

Heat shock modulates the subcellular localization, stability, and activity of HIPK2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Upadhyay, Mamta; Bhadauriya, Pratibha; Ganesh, Subramaniam, E-mail: sganesh@iitk.ac.in

2016-04-15

The homeodomain-interacting protein kinase-2 (HIPK2) is a highly conserved serine/threonine kinase and is involved in transcriptional regulation. HIPK2 is a highly unstable protein, and is kept at a low level under normal physiological conditions. However, exposure of cells to physiological stress – such as hypoxia, oxidative stress, or UV damage – is known to stabilize HIPK2, leading to the HIPK2-dependent activation of p53 and the cell death pathway. Therefore HIPK2 is also known as a stress kinase and as a stress-activated pro-apoptotic factor. We demonstrate here that exposure of cells to heat shock results in the stabilization of HIPK2 andmore » the stabilization is mediated via K63-linked ubiquitination. Intriguingly, a sub-lethal heat shock (42 °C, 1 h) results in the cytoplasmic localization of HIPK2, while a lethal heat shock (45 °C, 1 h) results in its nuclear localization. Cells exposed to the lethal heat shock showed significantly higher levels of the p53 activity than those exposed to the sub-lethal thermal stress, suggesting that both the level and the nuclear localization are essential for the pro-apoptotic activity of HIPK2 and that the lethal heat shock could retain the HIPK2 in the nucleus to promote the cell death. Taken together our study underscores the importance of HIPK2 in stress mediated cell death, and that the HIPK2 is a generic stress kinase that gets activated by diverse set of physiological stressors.« less

Lack of a peroxiredoxin suppresses the lethality of cells devoid of electron donors by channelling electrons to oxidized ribonucleotide reductase.

PubMed

Boronat, Susanna; Domènech, Alba; Carmona, Mercè; García-Santamarina, Sarela; Bañó, M Carmen; Ayté, José; Hidalgo, Elena

2017-06-01

The thioredoxin and glutaredoxin pathways are responsible of recycling several enzymes which undergo intramolecular disulfide bond formation as part of their catalytic cycles such as the peroxide scavengers peroxiredoxins or the enzyme ribonucleotide reductase (RNR). RNR, the rate-limiting enzyme of deoxyribonucleotide synthesis, is an essential enzyme relying on these electron flow cascades for recycling. RNR is tightly regulated in a cell cycle-dependent manner at different levels, but little is known about the participation of electron donors in such regulation. Here, we show that cytosolic thioredoxins Trx1 and Trx3 are the primary electron donors for RNR in fission yeast. Unexpectedly, trx1 transcript and Trx1 protein levels are up-regulated in a G1-to-S phase-dependent manner, indicating that the supply of electron donors is also cell cycle-regulated. Indeed, genetic depletion of thioredoxins triggers a DNA replication checkpoint ruled by Rad3 and Cds1, with the final goal of up-regulating transcription of S phase genes and constitutive RNR synthesis. Regarding the thioredoxin and glutaredoxin cascades, one combination of gene deletions is synthetic lethal in fission yeast: cells lacking both thioredoxin reductase and cytosolic dithiol glutaredoxin. We have isolated a suppressor of this lethal phenotype: a mutation at the Tpx1-coding gene, leading to a frame shift and a loss-of-function of Tpx1, the main client of electron donors. We propose that in a mutant strain compromised in reducing equivalents, the absence of an abundant and competitive substrate such as the peroxiredoxin Tpx1 has been selected as a lethality suppressor to favor RNR function at the expense of the non-essential peroxide scavenging function, to allow DNA synthesis and cell growth.

From tails to toes: developing nonlethal tissue indicators of mercury exposure in five amphibian species

USGS Publications Warehouse

Pfleeger, Adam Z.; Eagles-Smith, Collin A.; Kowalski, Brandon M.; Herring, Garth; Willacker, James J.; Jackson, Allyson K.; Pierce, John

2016-01-01

Exposure to environmental contaminants has been implicated as a factor in global amphibian decline. Mercury (Hg) is a particularly widespread contaminant that biomagnifies in amphibians and can cause a suite of deleterious effects. However, monitoring contaminant exposure in amphibian tissues may conflict with conservation goals if lethal take is required. Thus, there is a need to develop non-lethal tissue sampling techniques to quantify contaminant exposure in amphibians. Some minimally invasive sampling techniques, such as toe-clipping, are common in population-genetic research, but it is unclear if these methods can adequately characterize contaminant exposure. We examined the relationships between mercury (Hg) concentrations in non-lethally sampled tissues and paired whole-bodies in five amphibian species. Specifically, we examined the utility of three different tail-clip sections from four salamander species and toe-clips from one anuran species. Both tail and toe-clips accurately predicted whole-body THg concentrations, but the relationships differed among species and the specific tail-clip section or toe that was used. Tail-clips comprised of the distal 0–2 cm segment performed the best across all salamander species, explaining between 82 and 92 % of the variation in paired whole-body THg concentrations. Toe-clips were less effective predictors of frog THg concentrations, but THg concentrations in outer rear toes accounted for up to 79 % of the variability in frog whole-body THg concentrations. These findings suggest non-lethal sampling of tails and toes has potential applications for monitoring contaminant exposure and risk in amphibians, but care must be taken to ensure consistent collection and interpretation of samples.

What Are Reasons for the Large Gender Differences in the Lethality of Suicidal Acts? An Epidemiological Analysis in Four European Countries

PubMed Central

Heinrichs, Katherina; Székely, András; Tóth, Mónika Ditta; Coyne, James; Quintão, Sónia; Arensman, Ella; Coffey, Claire; Maxwell, Margaret; Värnik, Airi; van Audenhove, Chantal; McDaid, David; Sarchiapone, Marco; Schmidtke, Armin; Genz, Axel; Gusmão, Ricardo; Hegerl, Ulrich

2015-01-01

Background In Europe, men have lower rates of attempted suicide compared to women and at the same time a higher rate of completed suicides, indicating major gender differences in lethality of suicidal behaviour. The aim of this study was to analyse the extent to which these gender differences in lethality can be explained by factors such as choice of more lethal methods or lethality differences within the same suicide method or age. In addition, we explored gender differences in the intentionality of suicide attempts. Methods and Findings Methods. Design: Epidemiological study using a combination of self-report and official data. Setting: Mental health care services in four European countries: Germany, Hungary, Ireland, and Portugal. Data basis: Completed suicides derived from official statistics for each country (767 acts, 74.4% male) and assessed suicide attempts excluding habitual intentional self-harm (8,175 acts, 43.2% male). Main Outcome Measures and Data Analysis. We collected data on suicidal acts in eight regions of four European countries participating in the EU-funded “OSPI-Europe”-project (www.ospi-europe.com). We calculated method-specific lethality using the number of completed suicides per method * 100 / (number of completed suicides per method + number of attempted suicides per method). We tested gender differences in the distribution of suicidal acts for significance by using the χ2-test for two-by-two tables. We assessed the effect sizes with phi coefficients (φ). We identified predictors of lethality with a binary logistic regression analysis. Poisson regression analysis examined the contribution of choice of methods and method-specific lethality to gender differences in the lethality of suicidal acts. Findings Main Results Suicidal acts (fatal and non-fatal) were 3.4 times more lethal in men than in women (lethality 13.91% (regarding 4106 suicidal acts) versus 4.05% (regarding 4836 suicidal acts)), the difference being significant for the methods hanging, jumping, moving objects, sharp objects and poisoning by substances other than drugs. Median age at time of suicidal behaviour (35–44 years) did not differ between males and females. The overall gender difference in lethality of suicidal behaviour was explained by males choosing more lethal suicide methods (odds ratio (OR) = 2.03; 95% CI = 1.65 to 2.50; p < 0.000001) and additionally, but to a lesser degree, by a higher lethality of suicidal acts for males even within the same method (OR = 1.64; 95% CI = 1.32 to 2.02; p = 0.000005). Results of a regression analysis revealed neither age nor country differences were significant predictors for gender differences in the lethality of suicidal acts. The proportion of serious suicide attempts among all non-fatal suicidal acts with known intentionality (NFSAi) was significantly higher in men (57.1%; 1,207 of 2,115 NFSAi) than in women (48.6%; 1,508 of 3,100 NFSAi) (χ2 = 35.74; p < 0.000001). Main limitations of the study Due to restrictive data security regulations to ensure anonymity in Ireland, specific ages could not be provided because of the relatively low absolute numbers of suicide in the Irish intervention and control region. Therefore, analyses of the interaction between gender and age could only be conducted for three of the four countries. Attempted suicides were assessed for patients presenting to emergency departments or treated in hospitals. An unknown rate of attempted suicides remained undetected. This may have caused an overestimation of the lethality of certain methods. Moreover, the detection of attempted suicides and the registration of completed suicides might have differed across the four countries. Some suicides might be hidden and misclassified as undetermined deaths. Conclusions Men more often used highly lethal methods in suicidal behaviour, but there was also a higher method-specific lethality which together explained the large gender differences in the lethality of suicidal acts. Gender differences in the lethality of suicidal acts were fairly consistent across all four European countries examined. Males and females did not differ in age at time of suicidal behaviour. Suicide attempts by males were rated as being more serious independent of the method used, with the exceptions of attempted hanging, suggesting gender differences in intentionality associated with suicidal behaviour. These findings contribute to understanding of the spectrum of reasons for gender differences in the lethality of suicidal behaviour and should inform the development of gender specific strategies for suicide prevention. PMID:26147965

Genetic background effects in quantitative genetics: gene-by-system interactions.

PubMed

Sardi, Maria; Gasch, Audrey P

2018-04-11

Proper cell function depends on networks of proteins that interact physically and functionally to carry out physiological processes. Thus, it seems logical that the impact of sequence variation in one protein could be significantly influenced by genetic variants at other loci in a genome. Nonetheless, the importance of such genetic interactions, known as epistasis, in explaining phenotypic variation remains a matter of debate in genetics. Recent work from our lab revealed that genes implicated from an association study of toxin tolerance in Saccharomyces cerevisiae show extensive interactions with the genetic background: most implicated genes, regardless of allele, are important for toxin tolerance in only one of two tested strains. The prevalence of background effects in our study adds to other reports of widespread genetic-background interactions in model organisms. We suggest that these effects represent many-way interactions with myriad features of the cellular system that vary across classes of individuals. Such gene-by-system interactions may influence diverse traits and require new modeling approaches to accurately represent genotype-phenotype relationships across individuals.

Identification of Genes and Genetic Variants Associated with Poor Treatment Response in Patients with Prostate Cancer

DTIC Science & Technology

2014-12-01

Tsuchiya N, Narita S, Cao GW, Slavov C, Mitev V, Chanock S, Gronberg H, Haiman CA, Kraft P, Easton DF, Eeles RA. (2013). A meta - analysis of genome...S, Cao GW, Slavov C, Mitev V, Chanock S, Gronberg H, Haiman CA, Kraft P, Easton DF, Eeles RA. (2013). A meta - analysis of genome- wide association...ABSTRACT We have created an informative set of high risk lethal prostate cancer pedigrees and recurrent prostate cancer cases. Linkage analysis has

A Functional Genomics Approach to Identify Novel Breast Cancer Gene Targets in Yeast

DTIC Science & Technology

2005-05-01

Chaleff DT, Valent B, Fink GR. Mutations affecting Ty-mediated expression of the HIS4 gene of Saccharomyces cerevisiae. Genetics 1984; 107(2): 179-97... mutations , and are synthetically lethal with rotl mutations ROX3 YBL093C Repressor Of hypoXic genes : RNA polymerase I1 holcenzyme component 3,3 SSS...mitochondrial gene products; mutation causes an elevated rate of mitochondrial turnover; 3 MOD after 60 generations, MOD on NaCI YNDI YER005W Yeast Nucleoside

Integration of Genomic, Biologic, and Chemical Approaches to Target p53 Loss and Gain-of-Function in Triple Negative Breast Cancer

DTIC Science & Technology

2014-09-01

in this renewal: p53 triple negative breast cancer subtypes gene expression somatic cell genetics CRISPR /Cas 3. OVERALL PROJECT SUMMARY...to the efficacy of the synthetic lethality screen. In addition, we have optimized the use of CRISPR /Cas, a novel somatic cell recombination...completing this stage of the research within the upcoming Year 2 of the award period. Figure 1. CRISPR /Cas-mediated in vitro somatic cell

[Spontaneous and induced sterility by ethyl methanesulfonate (EMS) in Nigella damascena L].

PubMed

Gilot-Delhalle, J

1976-01-01

EMS-induced sterility could be very partially due to chromosomal aberrations appearing during male and female meiosis or even to mechanical abnormalities of the double fertilization. The sterility could be also related to diplontic origin (lethal factors or small deficiencies appearing in homozygous state on account of self-pollinization). Owing to our histological and genetical data, a female gametophytic origin could be mainly ascribed to EMS induced sterility. It could arise from a damage of the feeding function of the nucellus.

Mutation of Breast Cancer Cell Genomic DNA by APOBEC3B

DTIC Science & Technology

2013-09-01

lethal prostate cancers. Proc. Natl Acad. Sci. USA 108, 17087–17092 (2011). 5. Parsons, D. W. et al. The genetic landscape of the childhood cancer...7. Stransky, N. et al. The mutational landscape of head and neck squamous cell carcinoma. Science 333, 1157–1160 (2011). 8. Nik-Zainal, S. et al...Mutational processes molding the genomes of 21 breast cancers. Cell 149, 979–993 (2012). 9. Stephens, P. J. et al. The landscape of cancer genes and

Fear and loathing in the benthos: Responses of aquatic insect larvae to the pesticide imidacloprid in the presence of chemical signals of predation risk.

PubMed

Pestana, João L T; Loureiro, Susana; Baird, Donald J; Soares, Amadeu M V M

2009-06-28

The influence of interactions between pesticide exposure and perceived predation risk on the lethal and sub-lethal responses of two aquatic insects was investigated using the pesticide imidacloprid, and a combination of predator-release kairomones from trout and alarm substances from conspecifics. Laboratory experiments examined feeding and respiration rates of the caddisfly Sericostoma vittatum as well as the growth, emergence and respiration rates of the midge Chironomus riparius, exposed to sub-lethal concentrations of imidacloprid. The effects of the two stressors on burrowing behaviour of both species were also assessed. The results show significant effects of environmentally relevant concentrations of imidacloprid on all endpoints studied. Perceived predation risk also elicited sub-lethal effects in C. riparius and S. vittatum, the latter species being less responsive to predation cues. The effects of simultaneous exposure to both types of stressors were assessed using two different approaches: analysis of variance and conceptual models [concentration addition (CA) and independent action (IA)] normally used for the evaluation of contaminant mixture exposure. Both statistical approaches showed no significant interactions on responses in simultaneous exposures in the majority of parameters assessed with only a signification deviation from the reference CA and IA models being found for C. riparius respiration data contrary to the ANOVA results. Exposure to imidacloprid also compromised antipredator behavioural responses of both insect species, with potential negative consequences in terms of mortality from predation in the field. The results obtained demonstrate that natural and anthropogenic stressors can be treated within the same framework providing compatible data for modelling. For an improved interpretation of ecological effects it will be important to expand the mechanistic study of effects of combined exposure to pesticides and perceived predation risk by measuring different endpoints over a wider range of pesticide concentrations.

Preparation and characterization of cobalt-substituted anthrax lethal factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saebel, Crystal E.; Carbone, Ryan; Dabous, John R.

2011-12-09

Highlights: Black-Right-Pointing-Pointer Cobalt-substituted anthrax lethal factor (CoLF) is highly active. Black-Right-Pointing-Pointer CoLF can be prepared by bio-assimilation and direct exchange. Black-Right-Pointing-Pointer Lethal factor binds cobalt tightly. Black-Right-Pointing-Pointer The electronic spectrum of CoLF reveals penta-coordination. Black-Right-Pointing-Pointer Interaction of CoLF with thioglycolic acid follows a 2-step mechanism. -- Abstract: Anthrax lethal factor (LF) is a zinc-dependent endopeptidase involved in the cleavage of mitogen-activated protein kinase kinases near their N-termini. The current report concerns the preparation of cobalt-substituted LF (CoLF) and its characterization by electronic spectroscopy. Two strategies to produce CoLF were explored, including (i) a bio-assimilation approach involving the cultivation of LF-expressingmore » Bacillus megaterium cells in the presence of CoCl{sub 2}, and (ii) direct exchange by treatment of zinc-LF with CoCl{sub 2}. Independent of the method employed, the protein was found to contain one Co{sup 2+} per LF molecule, and was shown to be twice as active as its native zinc counterpart. The electronic spectrum of CoLF suggests the Co{sup 2+} ion to be five-coordinate, an observation similar to that reported for other Co{sup 2+}-substituted gluzincins, but distinct from that documented for the crystal structure of native LF. Furthermore, spectroscopic studies following the exposure of CoLF to thioglycolic acid (TGA) revealed a sequential mechanism of metal removal from LF, which likely involves the formation of an enzyme: Co{sup 2+}:TGA ternary complex prior to demetallation of the active site. CoLF reported herein constitutes the first spectroscopic probe of LF's active site, which may be utilized in future studies to gain further insight into the enzyme's mechanism and inhibitor interactions.« less

Real time observation of the ultrasound stimulated disintegration of optically trapped microbubbles in proximity to biological cells

NASA Astrophysics Data System (ADS)

Prentice, Paul; MacDonald, Michael P.; Cuschieri, Alfred; Dholakia, Kishan; Campbell, Paul

2005-08-01

Cells that are exposed to varying amounts of ultrasonic energy in the presence of ultrasound contrast agent (UCA) may undergo either permanent cell membrane damage (lethal sonoporation), or a transient enhancement of membrane permeability (reversible or non lethal sonoporation). The merits of each mode are clear; lethal sonoporation constitutes a significant tumour therapy weapon, whilst its less intrusive counterpart, reversible sonoporation, represents an effective non-invasive targeted drug delivery technique. Our working hypothesis for understanding this problem was that the root cause and effect in sonoporation involves the interaction of individual cells with single microbubbles, and to that end we devised an experiment that facilitates video rate observation of this specific scenario under well defined optical control. Specifically, we have constructed an innovative hybridization apparatus involving holographic optical trapping of single and multiple UCA microbubbles, together with the facility to irradiate with MHz pulsed ultrasound energy in the presence cancerous cells. This approach allows the isolation of a target microbubble from a resident population and the relocation to a [controllable] predetermined position relative to a cell within a monolayer. Frame extraction from standard framing rate video microscopy demonstrates the individuality of single microbubble-cell interactions. We describe a fluorescence microscopy protocol that will allow future study of the potential to deliver molecular species to cells, the dependence of the delivery on the initial microbubble-cell distance and to determine the targeted cell survival.

Clostridium perfringens epsilon toxin induces permanent neuronal degeneration and behavioral changes.

PubMed

Morris, Winston E; Goldstein, Jorge; Redondo, Leandro M; Cangelosi, Adriana; Geoghegan, Patricia; Brocco, Marcela; Loidl, Fabián C; Fernandez-Miyakawa, Mariano E

2017-05-01

Clostridium perfringens epsilon toxin (ETX), the most potent toxin produced by this bacteria, plays a key role in the pathogenesis of enterotoxaemia in ruminants, causing brain edema and encephalomalacia. Studies of animals suffering from ETX intoxication describe severe neurological disorders that are thought to be the result of vasogenic brain edemas and indirect neuronal toxicity, killing oligodendrocytes but not astrocytes, microglia, or neurons in vitro. In this study, by means of intravenous and intracerebroventricular delivery of sub-lethal concentrations of ETX, the histological and ultrastructural changes of the brain were studied in rats and mice. Histological analysis showed degenerative changes in neurons from the cortex, hippocampus, striatum and hypothalamus. Ultrastructurally, necrotic neurons and apoptotic cells were observed in these same areas, among axons with accumulation of neurofilaments and demyelination as well as synaptic stripping. Lesions observed in the brain after sub-lethal exposure to ETX, result in permanent behavioral changes in animals surviving ETX exposure, as observed individually in several animals and assessed in the Inclined Plane Test and the Wire Hang Test. Pharmacological studies showed that dexamethasone and reserpine but not ketamine or riluzole were able to reduce the brain lesions and the lethality of ETX. Cytotoxicity was not observed upon neuronal primary cultures in vitro. Therefore, we hypothesize that ETX can affect the brain of animals independently of death, producing changes on neurons or glia as the result of complex interactions, independently of ETX-BBB interactions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Eliminating Legionella by inhibiting BCL-XL to induce macrophage apoptosis.

PubMed

Speir, Mary; Lawlor, Kate E; Glaser, Stefan P; Abraham, Gilu; Chow, Seong; Vogrin, Adam; Schulze, Keith E; Schuelein, Ralf; O'Reilly, Lorraine A; Mason, Kylie; Hartland, Elizabeth L; Lithgow, Trevor; Strasser, Andreas; Lessene, Guillaume; Huang, David C S; Vince, James E; Naderer, Thomas

2016-02-24

Human pathogenic Legionella replicate in alveolar macrophages and cause a potentially lethal form of pneumonia known as Legionnaires' disease(1). Here, we have identified a host-directed therapeutic approach to eliminate intracellular Legionella infections. We demonstrate that the genetic deletion, or pharmacological inhibition, of the host cell pro-survival protein BCL-XL induces intrinsic apoptosis of macrophages infected with virulent Legionella strains, thereby abrogating Legionella replication. BCL-XL is essential for the survival of Legionella-infected macrophages due to bacterial inhibition of host-cell protein synthesis, resulting in reduced levels of the short-lived, related BCL-2 pro-survival family member, MCL-1. Consequently, a single dose of a BCL-XL-targeted BH3-mimetic therapy, or myeloid cell-restricted deletion of BCL-XL, limits Legionella replication and prevents lethal lung infections in mice. These results indicate that repurposing BH3-mimetic compounds, originally developed to induce cancer cell apoptosis, may have efficacy in treating Legionnaires' and other diseases caused by intracellular microbes.

The Bold and the Beautiful: a Neurotoxicity Comparison of New World Coral Snakes in the Micruroides and Micrurus Genera and Relative Neutralization by Antivenom.

PubMed

Yang, Daryl C; Dobson, James; Cochran, Chip; Dashevsky, Daniel; Arbuckle, Kevin; Benard, Melisa; Boyer, Leslie; Alagón, Alejandro; Hendrikx, Iwan; Hodgson, Wayne C; Fry, Bryan G

2017-10-01

Coral snake envenomations are well characterized to be lethally neurotoxic. Despite this, few multispecies, neurotoxicity and antivenom efficacy comparisons have been undertaken and only for the Micrurus genus; Micruroides has remained entirely uninvestigated. As the USA's supplier of antivenom has currently stopped production, alternative sources need to be explored. The Mexican manufacturer Bioclon uses species genetically related to USA species, thus we investigated the efficacy against Micrurus fulvius (eastern coral snake), the main species responsible for lethal envenomations in the USA as well as additional species from the Americas. The use of Coralmyn® coral snake antivenom was effective in neutralizing the neurotoxic effects exhibited by the venom of M. fulvius but was ineffective against the venoms of Micrurus tener, Micrurus spixii, Micrurus pyrrhocryptus, and Micruroides euryxanthus. Our results suggest that the Mexican antivenom may be clinically useful for the treatment of M. fulvius in the USA but may be of only limited efficacy against the other species studied.

ATM and MET kinases are synthetic lethal with non-genotoxic activation of p53

PubMed Central

Sullivan, Kelly D.; Padilla-Just, Nuria; Henry, Ryan E.; Porter, Christopher C.; Kim, Jihye; Tentler, John J.; Eckhardt, S. Gail; Tan, Aik Choon; DeGregori, James; Espinosa, Joaquín M.

2012-01-01

The p53 tumor suppressor orchestrates alternative stress responses including cell cycle arrest and apoptosis, but the mechanisms defining cell fate upon p53 activation are poorly understood. Several small molecule activators of p53 have been developed, including Nutlin-3, but their therapeutic potential is limited by the fact that they induce reversible cell cycle arrest in most cancer cell types. We report here the results of a ‘Synthetic Lethal with Nutlin-3’ genome-wide shRNA screen, which revealed that the ATM and MET kinases govern cell fate choice upon p53 activation. Genetic or pharmacological interference with ATM or MET activity converts the cellular response from cell cycle arrest into apoptosis in diverse cancer cell types without affecting expression of key p53 target genes. ATM and MET inhibitors enable Nutlin-3 to kill tumor spheroids. These results identify novel pathways controlling the cellular response to p53 activation and aid in the design of p53-based therapies. PMID:22660439

Lethal Respiratory Disease Associated with Human Rhinovirus C in Wild Chimpanzees, Uganda, 2013.

PubMed

Scully, Erik J; Basnet, Sarmi; Wrangham, Richard W; Muller, Martin N; Otali, Emily; Hyeroba, David; Grindle, Kristine A; Pappas, Tressa E; Thompson, Melissa Emery; Machanda, Zarin; Watters, Kelly E; Palmenberg, Ann C; Gern, James E; Goldberg, Tony L

2018-02-01

We describe a lethal respiratory outbreak among wild chimpanzees in Uganda in 2013 for which molecular and epidemiologic analyses implicate human rhinovirus C as the cause. Postmortem samples from an infant chimpanzee yielded near-complete genome sequences throughout the respiratory tract; other pathogens were absent. Epidemiologic modeling estimated the basic reproductive number (R 0 ) for the epidemic as 1.83, consistent with the common cold in humans. Genotyping of 41 chimpanzees and examination of 24 published chimpanzee genomes from subspecies across Africa showed universal homozygosity for the cadherin-related family member 3 CDHR3-Y 529 allele, which increases risk for rhinovirus C infection and asthma in human children. These results indicate that chimpanzees exhibit a species-wide genetic susceptibility to rhinovirus C and that this virus, heretofore considered a uniquely human pathogen, can cross primate species barriers and threatens wild apes. We advocate engineering interventions and prevention strategies for rhinovirus infections for both humans and wild apes.

Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.

PubMed

Kayagaki, Nobuhiko; Stowe, Irma B; Lee, Bettina L; O'Rourke, Karen; Anderson, Keith; Warming, Søren; Cuellar, Trinna; Haley, Benjamin; Roose-Girma, Merone; Phung, Qui T; Liu, Peter S; Lill, Jennie R; Li, Hong; Wu, Jiansheng; Kummerfeld, Sarah; Zhang, Juan; Lee, Wyne P; Snipas, Scott J; Salvesen, Guy S; Morris, Lucy X; Fitzgerald, Linda; Zhang, Yafei; Bertram, Edward M; Goodnow, Christopher C; Dixit, Vishva M

2015-10-29

Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1β processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1β maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd(-/-) mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1β secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd(-/-) mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.

The Protective Effect of Dietary Arthrospira (Spirulina) maxima Against Mutagenicity Induced by Benzo[alpha]pyrene in Mice

PubMed Central

Garduño-Siciliano, Leticia; Martínez-Galero, Elizdath; Mojica-Villegas, Angélica; Pages, Nicole; Gutiérrez-Salmeán, Gabriela

2014-01-01

Abstract Benzo[alpha]pyrene (B[α]P) was used to test the possible antimutagenic effects of Arthrospira (Spirulina) maxima (SP) on male and female mice. SP was orally administered at 0, 200, 400, or 800 mg/kg of body weight to animals of both sexes for 2 weeks before starting the B[α]P (intraperitoneal injection) at 125 mg/kg of body weight for 5 consecutive days. For the male dominant lethal test, each male was caged with two untreated females per week for 3 weeks. For the female dominant lethal test, each female was caged for 1 week with one untreated male. All the females were evaluated 13–15 days after mating for incidence of pregnancy, total corpora lutea, total implants and pre- and postimplant losses. SP protected from B[α]P-induced pre- and postimplant losses in the male dominant lethal test, and from B[α]P-induced postimplantation losses in treated females. Moreover, SP treatment significantly reduced the detrimental effect of B[α]P on the quality of mouse semen. Our results illustrate the protective effects of SP in relation to B[α]P-induced genetic damage to germ cells. We conclude that SP, owing mainly to the presence of phycocyanin, could be of potential clinical interest in cancer treatment or prevention of relapse. PMID:24787733

Deviation of the typical AAA substrate-threading pore prevents fatal protein degradation in yeast Cdc48.

PubMed

Esaki, Masatoshi; Islam, Md Tanvir; Tani, Naoki; Ogura, Teru

2017-07-14

Yeast Cdc48 is a well-conserved, essential chaperone of ATPases associated with diverse cellular activity (AAA) proteins, which recognizes substrate proteins and modulates their conformations to carry out many cellular processes. However, the fundamental mechanisms underlying the diverse pivotal roles of Cdc48 remain unknown. Almost all AAA proteins form a ring-shaped structure with a conserved aromatic amino acid residue that is essential for proper function. The threading mechanism hypothesis suggests that this residue guides the intrusion of substrate proteins into a narrow pore of the AAA ring, thereby becoming unfolded. By contrast, the aromatic residue in one of the two AAA rings of Cdc48 has been eliminated through evolution. Here, we show that artificial retrieval of this aromatic residue in Cdc48 is lethal, and essential features to support the threading mechanism are required to exhibit the lethal phenotype. In particular, genetic and biochemical analyses of the Cdc48 lethal mutant strongly suggested that when in complex with the 20S proteasome, essential proteins are abnormally forced to thread through the Cdc48 pore to become degraded, which was not detected in wild-type Cdc48. Thus, the widely applicable threading model is less effective for wild-type Cdc48; rather, Cdc48 might function predominantly through an as-yet-undetermined mechanism.

Fine Structure Genetic and Physical Map of the Gene 3 to 10 Region of the Bacteriophage P22 Chromosome

PubMed Central

Casjens, S.; Eppler, K.; Sampson, L.; Parr, R.; Wyckoff, E.

1991-01-01

The mechanism by which dsDNA is packaged by viruses is not yet understood in any system. Bacteriophage P22 has been a productive system in which to study the molecular genetics of virus particle assembly and DNA packaging. Only five phage encoded proteins, the products of genes 3, 2, 1, 8 and 5, are required for packaging the virus chromosome inside the coat protein shell. We report here the construction of a detailed genetic and physical map of these genes, the neighboring gene 4 and a portion of gene 10, in which 289 conditional lethal amber, opal, temperature sensitive and cold sensitive mutations are mapped into 44 small (several hundred base pair) intervals of known sequence. Knowledge of missense mutant phenotypes and information on the location of these mutations allows us to begin the assignment of partial protein functions to portions of these genes. The map and mapping strains will be of use in the further genetic dissection of the P22 DNA packaging and prohead assembly processes. PMID:2029965

Canine and Feline Models of Human Genetic Diseases and Their Contributions to Advancing Clinical Therapies
.

PubMed

Gurda, Brittney L; Bradbury, Allison M; Vite, Charles H

2017-09-01

For many lethal or debilitating genetic disorders in patients there are no satisfactory therapies. Several barriers exist that hinder the developments of effective therapies including the limited availability of clinically relevant animal models that faithfully recapitulate human genetic disease. In 1974, the Referral Center for Animal Models of Human Genetic Disease (RCAM) was established by Dr. Donald F. Patterson and continued by Dr. Mark E. Haskins at the University of Pennsylvania with the mission to discover, understand, treat, and maintain breeding colonies of naturally occurring hereditary disorders in dogs and cats that are orthologous to those found in human patients. Although non-human primates, sheep, and pig models are also available within the medical community, naturally occurring diseases are rarely identified in non-human primates, and the vast behavioral, clinicopathological, physiological, and anatomical knowledge available regarding dogs and cats far surpasses what is available in ovine and porcine species. The canine and feline models that are maintained at RCAM are presented here with a focus on preclinical therapy data. Clinical studies that have been generated from preclinical work in these models are also presented.

Functional Analysis With a Barcoder Yeast Gene Overexpression System

PubMed Central

Douglas, Alison C.; Smith, Andrew M.; Sharifpoor, Sara; Yan, Zhun; Durbic, Tanja; Heisler, Lawrence E.; Lee, Anna Y.; Ryan, Owen; Göttert, Hendrikje; Surendra, Anu; van Dyk, Dewald; Giaever, Guri; Boone, Charles; Nislow, Corey; Andrews, Brenda J.

2012-01-01

Systematic analysis of gene overexpression phenotypes provides an insight into gene function, enzyme targets, and biological pathways. Here, we describe a novel functional genomics platform that enables a highly parallel and systematic assessment of overexpression phenotypes in pooled cultures. First, we constructed a genome-level collection of ~5100 yeast barcoder strains, each of which carries a unique barcode, enabling pooled fitness assays with a barcode microarray or sequencing readout. Second, we constructed a yeast open reading frame (ORF) galactose-induced overexpression array by generating a genome-wide set of yeast transformants, each of which carries an individual plasmid-born and sequence-verified ORF derived from the Saccharomyces cerevisiae full-length EXpression-ready (FLEX) collection. We combined these collections genetically using synthetic genetic array methodology, generating ~5100 strains, each of which is barcoded and overexpresses a specific ORF, a set we termed “barFLEX.” Additional synthetic genetic array allows the barFLEX collection to be moved into different genetic backgrounds. As a proof-of-principle, we describe the properties of the barFLEX overexpression collection and its application in synthetic dosage lethality studies under different environmental conditions. PMID:23050238

RanBP2 modulates Cox11 and hexokinase I activities and haploinsufficiency of RanBP2 causes deficits in glucose metabolism.

PubMed

Aslanukov, Azamat; Bhowmick, Reshma; Guruju, Mallikarjuna; Oswald, John; Raz, Dorit; Bush, Ronald A; Sieving, Paul A; Lu, Xinrong; Bock, Cheryl B; Ferreira, Paulo A

2006-10-01

The Ran-binding protein 2 (RanBP2) is a large multimodular and pleiotropic protein. Several molecular partners with distinct functions interacting specifically with selective modules of RanBP2 have been identified. Yet, the significance of these interactions with RanBP2 and the genetic and physiological role(s) of RanBP2 in a whole-animal model remain elusive. Here, we report the identification of two novel partners of RanBP2 and a novel physiological role of RanBP2 in a mouse model. RanBP2 associates in vitro and in vivo and colocalizes with the mitochondrial metallochaperone, Cox11, and the pacemaker of glycolysis, hexokinase type I (HKI) via its leucine-rich domain. The leucine-rich domain of RanBP2 also exhibits strong chaperone activity toward intermediate and mature folding species of Cox11 supporting a chaperone role of RanBP2 in the cytosol during Cox11 biogenesis. Cox11 partially colocalizes with HKI, thus supporting additional and distinct roles in cell function. Cox11 is a strong inhibitor of HKI, and RanBP2 suppresses the inhibitory activity of Cox11 over HKI. To probe the physiological role of RanBP2 and its role in HKI function, a mouse model harboring a genetically disrupted RanBP2 locus was generated. RanBP2(-/-) are embryonically lethal, and haploinsufficiency of RanBP2 in an inbred strain causes a pronounced decrease of HKI and ATP levels selectively in the central nervous system. Inbred RanBP2(+/-) mice also exhibit deficits in growth rates and glucose catabolism without impairment of glucose uptake and gluconeogenesis. These phenotypes are accompanied by a decrease in the electrophysiological responses of photosensory and postreceptoral neurons. Hence, RanBP2 and its partners emerge as critical modulators of neuronal HKI, glucose catabolism, energy homeostasis, and targets for metabolic, aging disorders and allied neuropathies.

Nutritional pyhsiology of the eastern spruce budworm, Choristoneura fumiferana, infected with Nosema fumiferanae, and interactions with dietary nitrogen

Treesearch

Leah S. Bauer; G.L. Nordin

1988-01-01

Female eastern spurce budworm larvae, Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae), inoculated with a medium lethal spore dosage of the microsporidium. Nosema fumiferanae (Thomson) exhibited significant reductions in a consumptive index (CI), nitrogen consumptive index (NCI), relative growth rate (RGR), and gross(...

Detrimental effects of an autosomal selfish genetic element on sperm competitiveness in house mice

PubMed Central

Sutter, Andreas; Lindholm, Anna K.

2015-01-01

Female multiple mating (polyandry) is widespread across many animal taxa and indirect genetic benefits are a major evolutionary force favouring polyandry. An incentive for polyandry arises when multiple mating leads to sperm competition that disadvantages sperm from genetically inferior mates. A reduction in genetic quality is associated with costly selfish genetic elements (SGEs), and studies in invertebrates have shown that males bearing sex ratio distorting SGEs are worse sperm competitors than wild-type males. We used a vertebrate model species to test whether females can avoid an autosomal SGE, the t haplotype, through polyandry. The t haplotype in house mice exhibits strong drive in t heterozygous males by affecting spermatogenesis and is associated with homozygous in utero lethality. We used controlled matings to test the effect of the t haplotype on sperm competitiveness. Regardless of mating order, t heterozygous males sired only 11% of zygotes when competing against wild-type males, suggesting a very strong effect of the t haplotype on sperm quality. We provide, to our knowledge, the first substantial evidence that polyandry ameliorates the harmful effects of an autosomal SGE arising through genetic incompatibility. We discuss potential mechanisms in our study species and the broader implications for the benefits of polyandry. PMID:26136452

Genetic correction of β-thalassemia patient-specific iPS cells and its use in improving hemoglobin production in irradiated SCID mice.

PubMed

Wang, Yixuan; Zheng, Chen-Guang; Jiang, Yonghua; Zhang, Jiqin; Chen, Jiayu; Yao, Chao; Zhao, Qingguo; Liu, Sheng; Chen, Ke; Du, Juan; Yang, Ze; Gao, Shaorong

2012-04-01

The generation of induced pluripotent stem cells (iPSCs) from differentiated somatic cells by over-expression of several transcription factors has the potential to cure many genetic and degenerative diseases currently recalcitrant to traditional clinical approaches. One such genetic disease is β-thalassemia major (Cooley's anemia). This disease is caused by either a point mutation or the deletion of several nucleotides in the β-globin gene, and it threatens the lives of millions of people in China. In the present study, we successfully generated iPSCs from fibroblasts collected from a 2-year-old patient who was diagnosed with a homozygous 41/42 deletion in his β-globin gene. More importantly, we successfully corrected this genetic mutation in the β-thalassemia iPSCs by homologous recombination. Furthermore, transplantation of the genetically corrected iPSCs-derived hematopoietic progenitors into sub-lethally irradiated immune deficient SCID mice showed improved hemoglobin production compared with the uncorrected iPSCs. Moreover, the generation of human β-globin could be detected in the mice transplanted with corrected iPSCs-derived hematopietic progenitors. Our study provides strong evidence that iPSCs generated from a patient with a genetic disease can be corrected by homologous recombination and that the corrected iPSCs have potential clinical uses.

ATM inhibition induces synthetic lethality and enhances sensitivity of PTEN-deficient breast cancer cells to cisplatin.

PubMed

Li, Ke; Yan, Huaying; Guo, Wenhao; Tang, Mei; Zhao, Xinyu; Tong, Aiping; Peng, Yong; Li, Qintong; Yuan, Zhu

2018-05-01

PTEN deficiency often causes defects in DNA damage repair. Currently, effective therapies for breast cancer are lacking. ATM is an attractive target for cancer treatment. Previous studies suggested a synthetic lethality between PTEN and PARP. However, the synthetically lethal interaction between PTEN and ATM in breast cancer has not been reported. Moreover, the mechanism remains elusive. Here, using KU-60019, an ATM kinase inhibitor, we investigated ATM inhibition as a synthetically lethal strategy to target breast cancer cells with PTEN defects. We found that KU-60019 preferentially sensitizes PTEN-deficient MDA-MB-468 breast cancer cells to cisplatin, though it also slightly enhances sensitivity of PTEN wild-type breast cancer cells. The increased cytotoxic sensitivity is associated with apoptosis, as evidenced by flow cytometry and PARP cleavage. Additionally, the increase of DNA damage accumulation due to the decreased capability of DNA repair, as indicated by γ-H2AX and Rad51 foci, also contributed to this selective cytotoxicity. Mechanistically, compared with PTEN wild-type MDA-MB-231 cells, PTEN-deficient MDA-MB-468 cells have lower level of Rad51, higher ATM kinase activity, and display the elevated level of DNA damage. Moreover, these differences could be further enlarged by cisplatin. Our findings suggest that ATM is a promising target for PTEN-defective breast cancer. Copyright © 2018 Elsevier Inc. All rights reserved.

Radiosensitizing effects of neem (Azadirachta indica) oil.

PubMed

Kumar, Ashok; Rao, A R; Kimura, H

2002-02-01

Radiosensitization by neem oil was studied using Balbc/3T3 cells and SCID cells. Neem oil enhanced the radiosensitivity of the cells when applied both during and after x-irradiation under aerobic conditions. Neem oil completely inhibited the repair of sublethal damage and potentially lethal damage repair in Balbc/3T3 cells. The cytofluorimeter data show that neem oil treatment before and after x-irradiation reduced the G(2) + M phase, thus inhibiting the expression of the radiation induced arrest of cells in the G(2) phase of the cell cycle. However, SCIK cells (derived from the SCID mouse), deficient in DSB repair, treated with neem oil did not show any enhancement in the radiosensitivity. There was no effect of neem oil on SLD repair or its inhibition in SCIK cells. These results suggest that neem oil enhanced the radiosensitivity of cells by interacting with residual damage after x-irradiation, thereby converting the sublethal damage or potentially lethal damage into lethal damage, inhibiting the double-strand break repair or reducing the G(2) phase of the cell cycle. Copyright 2002 John Wiley & Sons, Ltd.

Inviability of a DNA2 deletion mutant is due to the DNA damage checkpoint.

PubMed

Budd, Martin E; Antoshechkin, Igor A; Reis, Clara; Wold, Barbara J; Campbell, Judith L

2011-05-15

Dna2 is a dual polarity exo/endonuclease, and 5' to 3' DNA helicase involved in Okazaki Fragment Processing (OFP) and Double-Strand Break (DSB) Repair. In yeast, DNA2 is an essential gene, as expected for a DNA replication protein. Suppression of the lethality of dna2Δ mutants has been found to occur by two mechanisms: overexpression of RAD27 (scFEN1) , encoding a 5' to 3' exo/endo nuclease that processes Okazaki fragments (OFs) for ligation, or deletion of PIF1, a 5' to 3' helicase involved in mitochondrial recombination, telomerase inhibition and OFP. Mapping of a novel, spontaneously arising suppressor of dna2Δ now reveals that mutation of rad9 and double mutation of rad9 mrc1 can also suppress the lethality of dna2Δ mutants. Interaction of dna2Δ and DNA damage checkpoint mutations provides insight as to why dna2Δ is lethal but rad27Δ is not, even though evidence shows that Rad27 (ScFEN1) processes most of the Okazaki fragments, while Dna2 processes only a subset.

Purification and biophysical characterization of the core protease domain of anthrax lethal factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gkazonis, Petros V.; Dalkas, Georgios A.; Chasapis, Christos T.

2010-06-04

Anthrax lethal toxin (LeTx) stands for the major virulence factor of the anthrax disease. It comprises a 90 kDa highly specific metalloprotease, the anthrax lethal factor (LF). LF possesses a catalytic Zn{sup 2+} binding site and is highly specific against MAPK kinases, thus representing the most potent native biomolecule to alter and inactivate MKK [MAPK (mitogen-activated protein kinase) kinases] signalling pathways. Given the importance of the interaction between LF and substrate for the development of anti-anthrax agents as well as the potential treatment of nascent tumours, the analysis of the structure and dynamic properties of the LF catalytic site aremore » essential to elucidate its enzymatic properties. Here we report the recombinant expression and purification of a C-terminal part of LF (LF{sub 672-776}) that harbours the enzyme's core protease domain. The biophysical characterization and backbone assignments ({sup 1}H, {sup 13}C, {sup 15}N) of the polypeptide revealed a stable, well folded structure even in the absence of Zn{sup 2+}, suitable for high resolution structural analysis by NMR.« less

Genetic evolution of pancreatic cancer: lessons learnt from the pancreatic cancer genome sequencing project

PubMed Central

Iacobuzio-Donahue, Christine A

2012-01-01

Pancreatic cancer is a disease caused by the accumulation of genetic alterations in specific genes. Elucidation of the human genome sequence, in conjunction with technical advances in the ability to perform whole exome sequencing, have provided new insight into the mutational spectra characteristic of this lethal tumour type. Most recently, exomic sequencing has been used to clarify the clonal evolution of pancreatic cancer as well as provide time estimates of pancreatic carcinogenesis, indicating that a long window of opportunity may exist for early detection of this disease while in the curative stage. Moving forward, these mutational analyses indicate potential targets for personalised diagnostic and therapeutic intervention as well as the optimal timing for intervention based on the natural history of pancreatic carcinogenesis and progression. PMID:21749982

Monitoring genetic damage to ecosystems from hazardous waste

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anderson, S.L.

1992-03-01

Applications of ecological toxicity testing to hazardous waste management have increased dramatically over the last few years, resulting in a greater awareness of the need for improved biomonitoring techniques. Our laboratory is developing advanced techniques to assess the genotoxic effects of environmental contamination on ecosystems. We have developed a novel mutagenesis assay using the nematode Caenorhabditis elegans, which is potentially applicable for multimedia studies in soil, sediment, and water. In addition, we are conducting validation studies of a previously developed anaphase aberration test that utilizes sea urchin embryos. Other related efforts include field validation studies of the new tests, evaluationmore » of their potential ecological relevance, and analysis of their sensitivity relative to that of existing toxicity tests that assess only lethal effects, rather than genetic damage.« less

Hydrogen peroxide induced by the fungicide prothioconazole triggers deoxynivalenol (DON) production by Fusarium graminearum

PubMed Central

2010-01-01

Background Fusarium head blight is a very important disease of small grain cereals with F. graminearum as one of the most important causal agents. It not only causes reduction in yield and quality but from a human and animal healthcare point of view, it produces mycotoxins such as deoxynivalenol (DON) which can accumulate to toxic levels. Little is known about external triggers influencing DON production. Results In the present work, a combined in vivo/in vitro approach was used to test the effect of sub lethal fungicide treatments on DON production. Using a dilution series of prothioconazole, azoxystrobin and prothioconazole + fluoxastrobin, we demonstrated that sub lethal doses of prothioconazole coincide with an increase in DON production 48 h after fungicide treatment. In an artificial infection trial using wheat plants, the in vitro results of increased DON levels upon sub lethal prothioconazole application were confirmed illustrating the significance of these results from a practical point of view. In addition, further in vitro experiments revealed a timely hyperinduction of H2O2 production as fast as 4 h after amending cultures with prothioconazole. When applying H2O2 directly to germinating conidia, a similar induction of DON-production by F. graminearum was observed. The effect of sub lethal prothioconazole concentrations on DON production completely disappeared when applying catalase together with the fungicide. Conclusions These cumulative results suggest that H2O2 induced by sub lethal doses of the triazole fungicide prothioconazole acts as a trigger of DON biosynthesis. In a broader framework, this work clearly shows that DON production by the plant pathogen F. graminearum is the result of the interaction of fungal genomics and external environmental triggers. PMID:20398299

Genetic and forensic implications in epilepsy and cardiac arrhythmias: a case series.

PubMed

Partemi, Sara; Vidal, Monica Coll; Striano, Pasquale; Campuzano, Oscar; Allegue, Catarina; Pezzella, Marianna; Elia, Maurizio; Parisi, Pasquale; Belcastro, Vincenzo; Casellato, Susanna; Giordano, Lucio; Mastrangelo, Massimo; Pietrafusa, Nicola; Striano, Salvatore; Zara, Federico; Bianchi, Amedeo; Buti, Daniela; La Neve, Angela; Tassinari, Carlo Alberto; Oliva, Antonio; Brugada, Ramon

2015-05-01

Epilepsy affects approximately 3% of the world's population, and sudden death is a significant cause of death in this population. Sudden unexpected death in epilepsy (SUDEP) accounts for up to 17% of all these cases, which increases the rate of sudden death by 24-fold as compared to the general population. The underlying mechanisms are still not elucidated, but recent studies suggest the possibility that a common genetic channelopathy might contribute to both epilepsy and cardiac disease to increase the incidence of death via a lethal cardiac arrhythmia. We performed genetic testing in a large cohort of individuals with epilepsy and cardiac conduction disorders in order to identify genetic mutations that could play a role in the mechanism of sudden death. Putative pathogenic disease-causing mutations in genes encoding cardiac ion channel were detected in 24% of unrelated individuals with epilepsy. Segregation analysis through genetic screening of the available family members and functional studies are crucial tasks to understand and to prove the possible pathogenicity of the variant, but in our cohort, only two families were available. Despite further research should be performed to clarify the mechanism of coexistence of both clinical conditions, genetic analysis, applied also in post-mortem setting, could be very useful to identify genetic factors that predispose epileptic patients to sudden death, helping to prevent sudden death in patients with epilepsy.

GENETIC EFFECTS OF SPACEFLIGHT FACTORS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Glembotskii, Ya.L.; Parfenov, G.P.

1962-12-01

With the object of investigating effects of spaceflight factors on heredity, Drosophila melanogaster was carried on the second, fourth, and fifth orbital spaceships and on Vostok-1 and Vostok-2. Four different spaceflight effects were investigated. Nondisjunction of chromosomes was investigated by exposing unfertilized white-eyed Drosophila females on Vostoks 1 and 2 and mating them on their return with red-eyed males. Primary nondisjunction of chromosomes resulted in the appearance of four times as many unusual genotypes (XXY females and XO males) among the progeny of the exposed group as among offspring of the controls. However, the increase in nondisjunction cannot be ascribedmore » to radiation effects. Induced crossovers were investigated by exposing heterozygotic males (having normal phenotypes but three recessive genes in the second chromosome) on the fifth orbital spaceship and on Vostoks 1 and 2. Upon return they were mated with homozygotic females displaying the three recessive characteristics (black body, cinnabar eyes, and vestigial wings). Drosophila carried in the fifth orbital spaceship with no protection against low-frequency vibrations showed crossover incidence of 0.50 450 deg C in a 0.12%, compared to an incidence of 0.05 450 deg C in a 0.05% or none at all on Vostok spaceships, where the insect containers were cushioned against vibration. Dominant lethal mutations were investigated by exposing two strains of Drosophila melanogaster (D- 18 with a high rate of spontaneous lethal mutations, and D-32 with a low rate for the same mutations) of the five spacecraft. The number of dominant lethal mutations was found to increase somewhat in all groups exposed to space flight. Sex-linked recessive lethal mutations were investigated by exposing young males of the D-18 and D-32 strains of Drosophila melanogaster on all five vehicles. Exposure on the second and fourth orbital spaceships and on Vostok-1 resulted in statistically significant numbers of sex-linked recessive lethal mutations for spermatozoa and spermatids of both strains. However, no increase in mutations was observed following exposure on the fifth orbital spaceship and on Vostok-2. (TCO)« less

MKLN1 splicing defect in dogs with lethal acrodermatitis

PubMed Central

Bauer, Anina; McEwan, Neil A.; Cadieu, Edouard; André, Catherine; Roosje, Petra; Mellersh, Cathryn; Casal, Margret L.

2018-01-01

Lethal acrodermatitis (LAD) is a genodermatosis with monogenic autosomal recessive inheritance in Bull Terriers and Miniature Bull Terriers. The LAD phenotype is characterized by poor growth, immune deficiency, and skin lesions, especially at the paws. Utilizing a combination of genome wide association study and haplotype analysis, we mapped the LAD locus to a critical interval of ~1.11 Mb on chromosome 14. Whole genome sequencing of an LAD affected dog revealed a splice region variant in the MKLN1 gene that was not present in 191 control genomes (chr14:5,731,405T>G or MKLN1:c.400+3A>C). This variant showed perfect association in a larger combined Bull Terrier/Miniature Bull Terrier cohort of 46 cases and 294 controls. The variant was absent from 462 genetically diverse control dogs of 62 other dog breeds. RT-PCR analysis of skin RNA from an affected and a control dog demonstrated skipping of exon 4 in the MKLN1 transcripts of the LAD affected dog, which leads to a shift in the MKLN1 reading frame. MKLN1 encodes the widely expressed intracellular protein muskelin 1, for which diverse functions in cell adhesion, morphology, spreading, and intracellular transport processes are discussed. While the pathogenesis of LAD remains unclear, our data facilitate genetic testing of Bull Terriers and Miniature Bull Terriers to prevent the unintentional production of LAD affected dogs. This study may provide a starting point to further clarify the elusive physiological role of muskelin 1 in vivo. PMID:29565995

Allelic asymmetry of the Lethal hybrid rescue (Lhr) gene expression in the hybrid between Drosophila melanogaster and D. simulans: confirmation by using genetic variations of D. melanogaster.

PubMed

Shirata, Mika; Araye, Quenta; Maehara, Kazunori; Enya, Sora; Takano-Shimizu, Toshiyuki; Sawamura, Kyoichi

2014-02-01

In the cross between Drosophila melanogaster females and D. simulans males, hybrid males die at the late larval stage, and the sibling females also die at later stages at high temperatures. Removing the D. simulans allele of the Lethal hybrid rescue gene (Lhr (sim) ) improves the hybrid incompatibility phenotypes. However, the loss-of-function mutation of Lhr (sim) (Lhr (sim0) ) does not rescue the hybrid males in crosses with several D. melanogaster strains. We first describe the genetic factor possessed by the D. melanogaster strains. It has been suggested that removing the D. melanogaster allele of Lhr (Lhr (mel) ), that is Lhr (mel0) , does not have the hybrid male rescue effect, contrasting to Lhr (sim0) . Because the expression level of the Lhr gene is known to be Lhr (sim) > Lhr (mel) in the hybrid, Lhr (mel0) may not lead to enough of a reduction in total Lhr expression. Then, there is a possibility that the D. melanogaster factor changes the expression level to Lhr (sim) < Lhr (mel) . But in fact, the expression level was Lhr (sim) > Lhr (mel) in the hybrid irrespectively of the presence of the factor. At last, we showed that Lhr (mel0) slightly improves the viability of hybrid females, which was not realized previously. All of the present results are consistent with the allelic asymmetry model of the Lhr gene expression in the hybrid.

Genetic interaction analysis of point mutations enables interrogation of gene function at a residue-level resolution

PubMed Central

Braberg, Hannes; Moehle, Erica A.; Shales, Michael; Guthrie, Christine; Krogan, Nevan J.

2014-01-01

We have achieved a residue-level resolution of genetic interaction mapping – a technique that measures how the function of one gene is affected by the alteration of a second gene – by analyzing point mutations. Here, we describe how to interpret point mutant genetic interactions, and outline key applications for the approach, including interrogation of protein interaction interfaces and active sites, and examination of post-translational modifications. Genetic interaction analysis has proven effective for characterizing cellular processes; however, to date, systematic high-throughput genetic interaction screens have relied on gene deletions or knockdowns, which limits the resolution of gene function analysis and poses problems for multifunctional genes. Our point mutant approach addresses these issues, and further provides a tool for in vivo structure-function analysis that complements traditional biophysical methods. We also discuss the potential for genetic interaction mapping of point mutations in human cells and its application to personalized medicine. PMID:24842270

Sequential structures provide insights into the fidelity of RNA replication.

PubMed

Ferrer-Orta, Cristina; Arias, Armando; Pérez-Luque, Rosa; Escarmís, Cristina; Domingo, Esteban; Verdaguer, Nuria

2007-05-29

RNA virus replication is an error-prone event caused by the low fidelity of viral RNA-dependent RNA polymerases. Replication fidelity can be decreased further by the use of mutagenic ribonucleoside analogs to a point where viral genetic information can no longer be maintained. For foot-and-mouth disease virus, the antiviral analogs ribavirin and 5-fluorouracil have been shown to be mutagenic, contributing to virus extinction through lethal mutagenesis. Here, we report the x-ray structure of four elongation complexes of foot-and-mouth disease virus polymerase 3D obtained in presence of natural substrates, ATP and UTP, or mutagenic nucleotides, ribavirin triphosphate and 5-fluorouridine triphosphate with different RNAs as template-primer molecules. The ability of these complexes to synthesize RNA in crystals allowed us to capture different successive replication events and to define the critical amino acids involved in (i) the recognition and positioning of the incoming nucleotide or analog; (ii) the positioning of the acceptor base of the template strand; and (iii) the positioning of the 3'-OH group of the primer nucleotide during RNA replication. The structures identify key interactions involved in viral RNA replication and provide insights into the molecular basis of the low fidelity of viral RNA polymerases.

Human skin pigmentation, migration and disease susceptibility

PubMed Central

Jablonski, Nina G.; Chaplin, George

2012-01-01

Human skin pigmentation evolved as a compromise between the conflicting physiological demands of protection against the deleterious effects of ultraviolet radiation (UVR) and photosynthesis of UVB-dependent vitamin D3. Living under high UVR near the equator, ancestral Homo sapiens had skin rich in protective eumelanin. Dispersals outside of the tropics were associated with positive selection for depigmentation to maximize cutaneous biosynthesis of pre-vitamin D3 under low and highly seasonal UVB conditions. In recent centuries, migrations and high-speed transportation have brought many people into UVR regimes different from those experienced by their ancestors and, accordingly, exposed them to new disease risks. These have been increased by urbanization and changes in diet and lifestyle. Three examples—nutritional rickets, multiple sclerosis (MS) and cutaneous malignant melanoma (CMM)—are chosen to illustrate the serious health effects of mismatches between skin pigmentation and UVR. The aetiology of MS in particular provides insight into complex and contingent interactions of genetic and environmental factors necessary to trigger lethal disease states. Low UVB levels and vitamin D deficiencies produced by changes in location and lifestyle pose some of the most serious disease risks of the twenty-first century. PMID:22312045

Unexpected Function of the Glucanosyltransferase Gas1 in the DNA Damage Response Linked to Histone H3 Acetyltransferases in Saccharomyces cerevisiae

PubMed Central

Eustice, Moriah; Pillus, Lorraine

2014-01-01

Chromatin organization and structure are crucial for transcriptional regulation, DNA replication, and damage repair. Although initially characterized in remodeling cell wall glucans, the β-1,3-glucanosyltransferase Gas1 was recently discovered to regulate transcriptional silencing in a manner separable from its activity at the cell wall. However, the function of Gas1 in modulating chromatin remains largely unexplored. Our genetic characterization revealed that GAS1 had critical interactions with genes encoding the histone H3 lysine acetyltransferases Gcn5 and Sas3. Specifically, whereas the gas1gcn5 double mutant was synthetically lethal, deletion of both GAS1 and SAS3 restored silencing in Saccharomyces cerevisiae. The loss of GAS1 also led to broad DNA damage sensitivity with reduced Rad53 phosphorylation and defective cell cycle checkpoint activation following exposure to select genotoxins. Deletion of SAS3 in the gas1 background restored both Rad53 phosphorylation and checkpoint activation following exposure to genotoxins that trigger the DNA replication checkpoint. Our analysis thus uncovers previously unsuspected functions for both Gas1 and Sas3 in DNA damage response and cell cycle regulation. PMID:24532730

Menin determines K-RAS proliferative outputs in endocrine cells

PubMed Central

Chamberlain, Chester E.; Scheel, David W.; McGlynn, Kathleen; Kim, Hail; Miyatsuka, Takeshi; Wang, Juehu; Nguyen, Vinh; Zhao, Shuhong; Mavropoulos, Anastasia; Abraham, Aswin G.; O’Neill, Eric; Ku, Gregory M.; Cobb, Melanie H.; Martin, Gail R.; German, Michael S.

2014-01-01

Endocrine cell proliferation fluctuates dramatically in response to signals that communicate hormone demand. The genetic alterations that override these controls in endocrine tumors often are not associated with oncogenes common to other tumor types, suggesting that unique pathways govern endocrine proliferation. Within the pancreas, for example, activating mutations of the prototypical oncogene KRAS drive proliferation in all pancreatic ductal adenocarcimomas but are never found in pancreatic endocrine tumors. Therefore, we asked how cellular context impacts K-RAS signaling. We found that K-RAS paradoxically suppressed, rather than promoted, growth in pancreatic endocrine cells. Inhibition of proliferation by K-RAS depended on antiproliferative RAS effector RASSF1A and blockade of the RAS-activated proproliferative RAF/MAPK pathway by tumor suppressor menin. Consistent with this model, a glucagon-like peptide 1 (GLP1) agonist, which stimulates ERK1/2 phosphorylation, did not affect endocrine cell proliferation by itself, but synergistically enhanced proliferation when combined with a menin inhibitor. In contrast, inhibition of MAPK signaling created a synthetic lethal interaction in the setting of menin loss. These insights suggest potential strategies both for regenerating pancreatic β cells for people with diabetes and for targeting menin-sensitive endocrine tumors. PMID:25133424

The p53–Mdm2 feedback loop protects against DNA damage by inhibiting p53 activity but is dispensable for p53 stability, development, and longevity

PubMed Central

Pant, Vinod; Xiong, Shunbin; Jackson, James G.; Post, Sean M.; Abbas, Hussein A.; Quintás-Cardama, Alfonso; Hamir, Amirali N.; Lozano, Guillermina

2013-01-01

The p53–Mdm2 feedback loop is perceived to be critical for regulating stress-induced p53 activity and levels. However, this has never been tested in vivo. Using a genetically engineered mouse with mutated p53 response elements in the Mdm2 P2 promoter, we show that feedback loop-deficient Mdm2P2/P2 mice are viable and aphenotypic and age normally. p53 degradation kinetics after DNA damage in radiosensitive tissues remains similar to wild-type controls. Nonetheless, DNA damage response is elevated in Mdm2P2/P2 mice. Enhanced p53-dependent apoptosis sensitizes hematopoietic stem cells (HSCs), causing drastic myeloablation and lethality. These results suggest that while basal Mdm2 levels are sufficient to regulate p53 in most tissues under homeostatic conditions, the p53–Mdm2 feedback loop is critical for regulating p53 activity and sustaining HSC function after DNA damage. Therefore, transient disruption of p53–Mdm2 interaction could be explored as a potential adjuvant/therapeutic strategy for targeting stem cells in hematological malignancies. PMID:23973961

Intrathymic lymphopoiesis: stromal cell-associated proliferation of T cells is independent of lymphocyte genotype.

PubMed

Kyewski, B A; Travis, M; Kaplan, H S

1984-09-01

We analyzed the genetic restriction of direct cell-cell interactions between thymocytes and a) cortical epithelial cells, b) macrophages, and c) medullary dendritic cells in the mouse thymus. Thymectomized (C3H X C57BL/Ka)F1 hybrid mice were doubly grafted with P1 and P2 neonatal thymus grafts, were lethally irradiated, and were reconstituted with a mixture of P1 and P2 bone marrow cells which differed in the Thy-1 locus. The contributions of both parental inocula to the composition of the free and stromal cell-associated T cell compartments were analyzed separately in thymic grafts of each parental strain. The lymphoid composition in both compartments essentially reflected the peripheral T cell-chimerism in the host. The development of lymphostromal complexes was not restricted by the genotype of the partner cells. Statistical analysis of the distributions of P1 and P2 T cells among free thymocytes and within individual lymphostromal complexes, however, suggests that the T cells of an individual complex are the progeny of oligoclonal proliferation. Thus, both epithelial cells and bone marrow-derived stromal cells seem to be involved in different stages of intrathymic lymphopoiesis.

The Case for Adopting the "Species Complex" Nomenclature for the Etiologic Agents of Cryptococcosis.

PubMed

Kwon-Chung, Kyung J; Bennett, John E; Wickes, Brian L; Meyer, Wieland; Cuomo, Christina A; Wollenburg, Kurt R; Bicanic, Tihana A; Castañeda, Elizabeth; Chang, Yun C; Chen, Jianghan; Cogliati, Massimo; Dromer, Françoise; Ellis, David; Filler, Scott G; Fisher, Matthew C; Harrison, Thomas S; Holland, Steven M; Kohno, Shigeru; Kronstad, James W; Lazera, Marcia; Levitz, Stuart M; Lionakis, Michail S; May, Robin C; Ngamskulrongroj, Popchai; Pappas, Peter G; Perfect, John R; Rickerts, Volker; Sorrell, Tania C; Walsh, Thomas J; Williamson, Peter R; Xu, Jianping; Zelazny, Adrian M; Casadevall, Arturo

2017-01-01

Cryptococcosis is a potentially lethal disease of humans/animals caused by Cryptococcus neoformans and Cryptococcus gattii . Distinction between the two species is based on phenotypic and genotypic characteristics. Recently, it was proposed that C. neoformans be divided into two species and C. gattii into five species based on a phylogenetic analysis of 115 isolates. While this proposal adds to the knowledge about the genetic diversity and population structure of cryptococcosis agents, the published genotypes of 2,606 strains have already revealed more genetic diversity than is encompassed by seven species. Naming every clade as a separate species at this juncture will lead to continuing nomenclatural instability. In the absence of biological differences between clades and no consensus about how DNA sequence alone can delineate a species, we recommend using " Cryptococcus neoformans species complex" and " C. gattii species complex" as a practical intermediate step, rather than creating more species. This strategy recognizes genetic diversity without creating confusion.

Cardiac Channelopathies and Sudden Death: Recent Clinical and Genetic Advances.

PubMed

Fernández-Falgueras, Anna; Sarquella-Brugada, Georgia; Brugada, Josep; Brugada, Ramon; Campuzano, Oscar

2017-01-29

Sudden cardiac death poses a unique challenge to clinicians because it may be the only symptom of an inherited heart condition. Indeed, inherited heart diseases can cause sudden cardiac death in older and younger individuals. Two groups of familial diseases are responsible for sudden cardiac death: cardiomyopathies (mainly hypertrophic cardiomyopathy, dilated cardiomyopathy, and arrhythmogenic cardiomyopathy) and channelopathies (mainly long QT syndrome, Brugada syndrome, short QT syndrome, and catecholaminergic polymorphic ventricular tachycardia). This review focuses on cardiac channelopathies, which are characterized by lethal arrhythmias in the structurally normal heart, incomplete penetrance, and variable expressivity. Arrhythmias in these diseases result from pathogenic variants in genes encoding cardiac ion channels or associated proteins. Due to a lack of gross structural changes in the heart, channelopathies are often considered as potential causes of death in otherwise unexplained forensic autopsies. The asymptomatic nature of channelopathies is cause for concern in family members who may be carrying genetic risk factors, making the identification of these genetic factors of significant clinical importance.

Synthetic biology approaches to biological containment: pre-emptively tackling potential risks

PubMed Central

Krüger, Antje; Csibra, Eszter; Gianni, Edoardo

2016-01-01

Biocontainment comprises any strategy applied to ensure that harmful organisms are confined to controlled laboratory conditions and not allowed to escape into the environment. Genetically engineered microorganisms (GEMs), regardless of the nature of the modification and how it was established, have potential human or ecological impact if accidentally leaked or voluntarily released into a natural setting. Although all evidence to date is that GEMs are unable to compete in the environment, the power of synthetic biology to rewrite life requires a pre-emptive strategy to tackle possible unknown risks. Physical containment barriers have proven effective but a number of strategies have been developed to further strengthen biocontainment. Research on complex genetic circuits, lethal genes, alternative nucleic acids, genome recoding and synthetic auxotrophies aim to design more effective routes towards biocontainment. Here, we describe recent advances in synthetic biology that contribute to the ongoing efforts to develop new and improved genetic, semantic, metabolic and mechanistic plans for the containment of GEMs. PMID:27903826

[Impacts of malathion on population genetic structure of Oxya chinensis].

PubMed

Lu, Fu-Ping; Li, Cui-Lan; Duan, Yi-Hao; Guo, Ya-Ping; Ma, En-Bo

2004-09-01

Allozyme electrophoresis was employed to compare the difference in mortality among the genotypes at two polymorphic loci of Pgm and Me of grasshopper Oxya chinensis individuals acutely exposed to 1.5g/L malathion which resulted in 56% mortality in 24 hours. The selective lethal effects were observed among the genotypes at Pgm locus but not at Me locus. It is noted that the genotype Pgm-ab experienced the highest mortality (80%), whereas Pgm-bb and Pgm-bc were 49%, lower than the average. The chi(2) tests showed significant difference in morality between Pgm-bb and Pgm-cc. After exposure the allele frequency of Pgm-b showed a notable increase among surviving individuals. The cluster analysis based on Roger's genetic distance indicated that the acute exposure to malathion can cause differentiation in genetic composition at population level in Oxya chinensis. Because malathion is commonly used as the insecticide for grasshopper control, the data obtained in this study suggest that the similar genotype-mortality effects may occur in crop fields.

Synthetic biology approaches to biological containment: pre-emptively tackling potential risks.

PubMed

Torres, Leticia; Krüger, Antje; Csibra, Eszter; Gianni, Edoardo; Pinheiro, Vitor B

2016-11-30

Biocontainment comprises any strategy applied to ensure that harmful organisms are confined to controlled laboratory conditions and not allowed to escape into the environment. Genetically engineered microorganisms (GEMs), regardless of the nature of the modification and how it was established, have potential human or ecological impact if accidentally leaked or voluntarily released into a natural setting. Although all evidence to date is that GEMs are unable to compete in the environment, the power of synthetic biology to rewrite life requires a pre-emptive strategy to tackle possible unknown risks. Physical containment barriers have proven effective but a number of strategies have been developed to further strengthen biocontainment. Research on complex genetic circuits, lethal genes, alternative nucleic acids, genome recoding and synthetic auxotrophies aim to design more effective routes towards biocontainment. Here, we describe recent advances in synthetic biology that contribute to the ongoing efforts to develop new and improved genetic, semantic, metabolic and mechanistic plans for the containment of GEMs. © 2016 The Author(s).

On ballistic parameters of less lethal projectiles influencing the severity of thoracic blunt impacts.

PubMed

Pavier, Julien; Langlet, André; Eches, Nicolas; Jacquet, Jean-François

2015-01-01

The development and safety certification of less lethal projectiles require an understanding of the influence of projectile parameters on projectile-chest interaction and on the resulting terminal effect. Several energy-based criteria have been developed for chest injury assessment. Many studies consider kinetic energy (KE) or energy density as the only projectile parameter influencing terminal effect. In a common KE range (100-160 J), analysis of the firing tests of two 40 mm projectiles of different masses on animal surrogates has been made in order to investigate the severity of the injuries in the thoracic region. Experimental results have shown that KE and calibre are not sufficient to discriminate between the two projectiles as regards their injury potential. Parameters, such as momentum, shape and impedance, influence the projectile-chest interaction and terminal effect. A simplified finite element model of projectile-structure interaction confirms the experimental tendencies. Within the range of ballistic parameters used, it has been demonstrated that maximum thoracic deflection is a useful parameter to predict the skeletal level of injury, and it largely depends on the projectile pre-impact momentum. However, numerical simulations show that these results are merely valid for the experimental conditions used and cannot be generalised. Nevertheless, the transmitted impulse seems to be a more general factor governing the thorax deflection.

Lethal control of an apex predator has unintended cascading effects on forest mammal assemblages.

PubMed

Colman, N J; Gordon, C E; Crowther, M S; Letnic, M

2014-05-07

Disruption to species-interaction networks caused by irruptions of herbivores and mesopredators following extirpation of apex predators is a global driver of ecosystem reorganization and biodiversity loss. Most studies of apex predators' ecological roles focus on effects arising from their interactions with herbivores or mesopredators in isolation, but rarely consider how the effects of herbivores and mesopredators interact. Here, we provide evidence that multiple cascade pathways induced by lethal control of an apex predator, the dingo, drive unintended shifts in forest ecosystem structure. We compared mammal assemblages and understorey structure at seven sites in southern Australia. Each site comprised an area where dingoes were poisoned and an area without control. The effects of dingo control on mammals scaled with body size. Activity of herbivorous macropods, arboreal mammals and a mesopredator, the red fox, were greater, but understorey vegetation sparser and abundances of small mammals lower, where dingoes were controlled. Structural equation modelling suggested that both predation by foxes and depletion of understorey vegetation by macropods were related to small mammal decline at poisoned sites. Our study suggests that apex predators' suppressive effects on herbivores and mesopredators occur simultaneously and should be considered in tandem in order to appreciate the extent of apex predators' indirect effects.

Lethal control of an apex predator has unintended cascading effects on forest mammal assemblages

PubMed Central

Colman, N. J.; Gordon, C. E.; Crowther, M. S.; Letnic, M.

2014-01-01

Disruption to species-interaction networks caused by irruptions of herbivores and mesopredators following extirpation of apex predators is a global driver of ecosystem reorganization and biodiversity loss. Most studies of apex predators' ecological roles focus on effects arising from their interactions with herbivores or mesopredators in isolation, but rarely consider how the effects of herbivores and mesopredators interact. Here, we provide evidence that multiple cascade pathways induced by lethal control of an apex predator, the dingo, drive unintended shifts in forest ecosystem structure. We compared mammal assemblages and understorey structure at seven sites in southern Australia. Each site comprised an area where dingoes were poisoned and an area without control. The effects of dingo control on mammals scaled with body size. Activity of herbivorous macropods, arboreal mammals and a mesopredator, the red fox, were greater, but understorey vegetation sparser and abundances of small mammals lower, where dingoes were controlled. Structural equation modelling suggested that both predation by foxes and depletion of understorey vegetation by macropods were related to small mammal decline at poisoned sites. Our study suggests that apex predators’ suppressive effects on herbivores and mesopredators occur simultaneously and should be considered in tandem in order to appreciate the extent of apex predators’ indirect effects. PMID:24619441

Are High-Lethality Suicide Attempters With Bipolar Disorder a Distinct Phenotype?

PubMed Central

Oquendo, Maria A.; Carballo, Juan Jose; Rajouria, Namita; Currier, Dianne; Tin, Adrienne; Merville, Jessica; Galfalvy, Hanga C.; Sher, Leo; Grunebaum, Michael F.; Burke, Ainsley K.; Mann, J. John

2013-01-01

Because Bipolar Disorder (BD) individuals making highly lethal suicide attempts have greater injury burden and risk for suicide, early identification is critical. BD patients were classified as high- or low-lethality attempters. High-lethality attempts required inpatient medical treatment. Mixed effects logistic regression models and permutation analyses examined correlations between lethality, number, and order of attempts. High-lethality attempters reported greater suicidal intent and more previous attempts. Multiple attempters showed no pattern of incremental lethality increase with subsequent attempts, but individuals with early high-lethality attempts more often made high-lethality attempts later. A subset of high-lethality attempters make only high-lethality attempts. However, presence of previous low-lethality attempts does not indicate that risk for more lethal, possibly successful, attempts is reduced. PMID:19590998

Mate choice for genetic compatibility in the house mouse

PubMed Central

Lindholm, Anna K; Musolf, Kerstin; Weidt, Andrea; König, Barbara

2013-01-01

In house mice, genetic compatibility is influenced by the t haplotype, a driving selfish genetic element with a recessive lethal allele, imposing fundamental costs on mate choice decisions. Here, we evaluate the cost of genetic incompatibility and its implication for mate choice in a wild house mice population. In laboratory reared mice, we detected no fertility (number of embryos) or fecundity (ability to conceive) costs of the t, and yet we found a high cost of genetic incompatibility: heterozygote crosses produced 40% smaller birth litter sizes because of prenatal mortality. Surprisingly, transmission of t in crosses using +/t males was influenced by female genotype, consistent with postcopulatory female choice for + sperm in +/t females. Analysis of paternity patterns in a wild population of house mice showed that +/t females were more likely than +/+ females to have offspring sired by +/+ males, and unlike +/+ females, paternity of their offspring was not influenced by +/t male frequency, further supporting mate choice for genetic compatibility. As the major histocompatibility complex (MHC) is physically linked to the t, we investigated whether females could potentially use variation at the MHC to identify male genotype at the sperm or individual level. A unique MHC haplotype is linked to the t haplotype. This MHC haplotype could allow the recognition of t and enable pre- and postcopulatory mate choice for genetic compatibility. Alternatively, the MHC itself could be the target of mate choice for genetic compatibility. We predict that mate choice for genetic compatibility will be difficult to find in many systems, as only weak fertilization biases were found despite an exceptionally high cost of genetic incompatibility. PMID:23762510

A functional cancer genomics screen identifies a druggable synthetic lethal interaction between MSH3 and PRKDC.

PubMed

Dietlein, Felix; Thelen, Lisa; Jokic, Mladen; Jachimowicz, Ron D; Ivan, Laura; Knittel, Gero; Leeser, Uschi; van Oers, Johanna; Edelmann, Winfried; Heukamp, Lukas C; Reinhardt, H Christian

2014-05-01

Here, we use a large-scale cell line-based approach to identify cancer cell-specific mutations that are associated with DNA-dependent protein kinase catalytic subunit (DNA-PKcs) dependence. For this purpose, we profiled the mutational landscape across 1,319 cancer-associated genes of 67 distinct cell lines and identified numerous genes involved in homologous recombination-mediated DNA repair, including BRCA1, BRCA2, ATM, PAXIP, and RAD50, as being associated with non-oncogene addiction to DNA-PKcs. Mutations in the mismatch repair gene MSH3, which have been reported to occur recurrently in numerous human cancer entities, emerged as the most significant predictors of DNA-PKcs addiction. Concordantly, DNA-PKcs inhibition robustly induced apoptosis in MSH3-mutant cell lines in vitro and displayed remarkable single-agent efficacy against MSH3-mutant tumors in vivo. Thus, we here identify a therapeutically actionable synthetic lethal interaction between MSH3 and the non-homologous end joining kinase DNA-PKcs. Our observations recommend DNA-PKcs inhibition as a therapeutic concept for the treatment of human cancers displaying homologous recombination defects.

Reconstruction and Validation of a Genome-Scale Metabolic Model for the Filamentous Fungus Neurospora crassa Using FARM

PubMed Central

Hood, Heather M.; Ocasio, Linda R.; Sachs, Matthew S.; Galagan, James E.

2013-01-01

The filamentous fungus Neurospora crassa played a central role in the development of twentieth-century genetics, biochemistry and molecular biology, and continues to serve as a model organism for eukaryotic biology. Here, we have reconstructed a genome-scale model of its metabolism. This model consists of 836 metabolic genes, 257 pathways, 6 cellular compartments, and is supported by extensive manual curation of 491 literature citations. To aid our reconstruction, we developed three optimization-based algorithms, which together comprise Fast Automated Reconstruction of Metabolism (FARM). These algorithms are: LInear MEtabolite Dilution Flux Balance Analysis (limed-FBA), which predicts flux while linearly accounting for metabolite dilution; One-step functional Pruning (OnePrune), which removes blocked reactions with a single compact linear program; and Consistent Reproduction Of growth/no-growth Phenotype (CROP), which reconciles differences between in silico and experimental gene essentiality faster than previous approaches. Against an independent test set of more than 300 essential/non-essential genes that were not used to train the model, the model displays 93% sensitivity and specificity. We also used the model to simulate the biochemical genetics experiments originally performed on Neurospora by comprehensively predicting nutrient rescue of essential genes and synthetic lethal interactions, and we provide detailed pathway-based mechanistic explanations of our predictions. Our model provides a reliable computational framework for the integration and interpretation of ongoing experimental efforts in Neurospora, and we anticipate that our methods will substantially reduce the manual effort required to develop high-quality genome-scale metabolic models for other organisms. PMID:23935467

Linking Yeast Gcn5p Catalytic Function and Gene Regulation Using a Quantitative, Graded Dominant Mutant Approach

PubMed Central

Lanza, Amanda M.; Blazeck, John J.; Crook, Nathan C.; Alper, Hal S.

2012-01-01

Establishing causative links between protein functional domains and global gene regulation is critical for advancements in genetics, biotechnology, disease treatment, and systems biology. This task is challenging for multifunctional proteins when relying on traditional approaches such as gene deletions since they remove all domains simultaneously. Here, we describe a novel approach to extract quantitative, causative links by modulating the expression of a dominant mutant allele to create a function-specific competitive inhibition. Using the yeast histone acetyltransferase Gcn5p as a case study, we demonstrate the utility of this approach and (1) find evidence that Gcn5p is more involved in cell-wide gene repression, instead of the accepted gene activation associated with HATs, (2) identify previously unknown gene targets and interactions for Gcn5p-based acetylation, (3) quantify the strength of some Gcn5p-DNA associations, (4) demonstrate that this approach can be used to correctly identify canonical chromatin modifications, (5) establish the role of acetyltransferase activity on synthetic lethal interactions, and (6) identify new functional classes of genes regulated by Gcn5p acetyltransferase activity—all six of these major conclusions were unattainable by using standard gene knockout studies alone. We recommend that a graded dominant mutant approach be utilized in conjunction with a traditional knockout to study multifunctional proteins and generate higher-resolution data that more accurately probes protein domain function and influence. PMID:22558379

Synthesis of mannosylinositol phosphorylceramides is involved in maintenance of cell integrity of yeast Saccharomyces cerevisiae.

PubMed

Morimoto, Yuji; Tani, Motohiro

2015-02-01

Complex sphingolipids play important roles in many physiologically important events in yeast Saccharomyces cerevisiae. In this study, we screened yeast mutant strains showing a synthetic lethal interaction with loss of mannosylinositol phosphorylceramide (MIPC) synthesis and found that a specific group of glycosyltransferases involved in the synthesis of mannan-type N-glycans is essential for the growth of cells lacking MIPC synthases (Sur1 and Csh1). The genetic interaction was also confirmed by repression of MNN2, which encodes alpha-1,2-mannosyltransferase that synthesizes mannan-type N-glycans, by a tetracycline-regulatable system. MNN2-repressed sur1Δ csh1Δ cells exhibited high sensitivity to zymolyase treatment, and caffeine and sodium dodecyl sulfate (SDS) strongly inhibited the growth of sur1Δ csh1Δ cells, suggesting impairment of cell integrity due to the loss of MIPC synthesis. The phosphorylated form of Slt2, a mitogen-activated protein (MAP) kinase activated by impaired cell integrity, increased in sur1Δ csh1Δ cells, and this increase was dramatically enhanced by the repression of Mnn2. Moreover, the growth defect of MNN2-repressed sur1Δ csh1Δ cells was enhanced by the deletion of SLT2 or RLM1 encoding a downstream target of Slt2. These results indicated that loss of MIPC synthesis causes impairment of cell integrity, and this effect is enhanced by impaired synthesis of mannan-type N-glycans. © 2014 John Wiley & Sons Ltd.

An integrated approach to characterize genetic interaction networks in yeast metabolism

PubMed Central

Szappanos, Balázs; Kovács, Károly; Szamecz, Béla; Honti, Frantisek; Costanzo, Michael; Baryshnikova, Anastasia; Gelius-Dietrich, Gabriel; Lercher, Martin J.; Jelasity, Márk; Myers, Chad L.; Andrews, Brenda J.; Boone, Charles; Oliver, Stephen G.; Pál, Csaba; Papp, Balázs

2011-01-01

Intense experimental and theoretical efforts have been made to globally map genetic interactions, yet we still do not understand how gene-gene interactions arise from the operation of biomolecular networks. To bridge the gap between empirical and computational studies, we: i) quantitatively measure genetic interactions between ~185,000 metabolic gene pairs in Saccharomyces cerevisiae, ii) superpose the data on a detailed systems biology model of metabolism, and iii) introduce a machine-learning method to reconcile empirical interaction data with model predictions. We systematically investigate the relative impacts of functional modularity and metabolic flux coupling on the distribution of negative and positive genetic interactions. We also provide a mechanistic explanation for the link between the degree of genetic interaction, pleiotropy, and gene dispensability. Last, we demonstrate the feasibility of automated metabolic model refinement by correcting misannotations in NAD biosynthesis and confirming them by in vivo experiments. PMID:21623372

Environmental confounding in gene-environment interaction studies.

PubMed

Vanderweele, Tyler J; Ko, Yi-An; Mukherjee, Bhramar

2013-07-01

We show that, in the presence of uncontrolled environmental confounding, joint tests for the presence of a main genetic effect and gene-environment interaction will be biased if the genetic and environmental factors are correlated, even if there is no effect of either the genetic factor or the environmental factor on the disease. When environmental confounding is ignored, such tests will in fact reject the joint null of no genetic effect with a probability that tends to 1 as the sample size increases. This problem with the joint test vanishes under gene-environment independence, but it still persists if estimating the gene-environment interaction parameter itself is of interest. Uncontrolled environmental confounding will bias estimates of gene-environment interaction parameters even under gene-environment independence, but it will not do so if the unmeasured confounding variable itself does not interact with the genetic factor. Under gene-environment independence, if the interaction parameter without controlling for the environmental confounder is nonzero, then there is gene-environment interaction either between the genetic factor and the environmental factor of interest or between the genetic factor and the unmeasured environmental confounder. We evaluate several recently proposed joint tests in a simulation study and discuss the implications of these results for the conduct of gene-environment interaction studies.

KEAP1-dependent synthetic lethality induced by AKT and TXNRD1 inhibitors in lung cancer

PubMed Central

Dai, Bingbing; Yoo, Suk-Yuong; Bartholomeusz, Geoffrey; Graham, Ryan A.; Majidi, Mourad; Yan, Shaoyu; Meng, Jieru; Ji, Lin; Coombes, Kevin; Minna, John D.; Fang, Bingliang; Roth, Jack A.

2013-01-01

Intrinsic resistance to agents targeting phosphatidylinositol-3-kinase (PI3K)/AKT pathway is one of the major challenges in cancer treatment with such agents. The objective of this study is to identify the genes or pathways that can be targeted to overcome the resistance of non-small cell lung cancer to the AKT inhibitor, MK2206, which is currently being evaluated in phase I and II clinical trials. Using a genome-wide small interfering RNA (siRNA) library screening and biological characterization we identified that inhibition of Thioredoxin Reductase-1 (TXNRD1), one of the key anti-oxidant enzymes, with siRNAs or its inhibitor, Auranofin, sensitized non-small cell lung cancer cells to MK2206 treatment in vitro and in vivo. We found that simultaneous inhibition of TXNRD1 and AKT pathways induced robust reactive oxygen species (ROS) production, which was involved in c-Jun N-terminal Kinase (JNK, MAPK8) activation and cell apoptosis. Furthermore we found that the synthetic lethality interaction between the TXNRD1 and AKT pathways occurred through the KEAP1/NRF2 cellular antioxidant pathway. Lastly, we found that synthetic lethality induced by TXNRD1 and AKT inhibitors relied on wild type KEAP1 function. Our study indicates that targeting the interaction between AKT and TXNRD1 antioxidant pathways with MK2206 and Auranofin, a FDA approved drug, is a rational strategy to treat lung cancer and that KEAP1 mutation status may offer a predicative biomarker for such combination approaches. PMID:23824739

Mapping the epitopes of a neutralizing antibody fragment directed against the lethal factor of Bacillus anthracis and cross-reacting with the homologous edema factor.

PubMed

Thullier, Philippe; Avril, Arnaud; Mathieu, Jacques; Behrens, Christian K; Pellequer, Jean-Luc; Pelat, Thibaut

2013-01-01

The lethal toxin (LT) of Bacillus anthracis, composed of the protective antigen (PA) and the lethal factor (LF), plays an essential role in anthrax pathogenesis. PA also interacts with the edema factor (EF, 20% identity with LF) to form the edema toxin (ET), which has a lesser role in anthrax pathogenesis. The first recombinant antibody fragment directed against LF was scFv 2LF; it neutralizes LT by blocking the interaction between PA and LF. Here, we report that scFv 2LF cross-reacts with EF and cross-neutralizes ET, and we present an in silico method taking advantage of this cross-reactivity to map the epitope of scFv 2LF on both LF and EF. This method identified five epitope candidates on LF, constituted of a total of 32 residues, which were tested experimentally by mutating the residues to alanine. This combined approach precisely identified the epitope of scFv 2LF on LF as five residues (H229, R230, Q234, L235 and Y236), of which three were missed by the consensus epitope candidate identified by pre-existing in silico methods. The homolog of this epitope on EF (H253, R254, E258, L259 and Y260) was experimentally confirmed to constitute the epitope of scFv 2LF on EF. Other inhibitors, including synthetic molecules, could be used to target these epitopes for therapeutic purposes. The in silico method presented here may be of more general interest.

Targeting synthetic lethality between the SRC kinase and the EPHB6 receptor may benefit cancer treatment.

PubMed

Paul, James M; Toosi, Behzad; Vizeacoumar, Frederick S; Bhanumathy, Kalpana Kalyanasundaram; Li, Yue; Gerger, Courtney; El Zawily, Amr; Freywald, Tanya; Anderson, Deborah H; Mousseau, Darrell; Kanthan, Rani; Zhang, Zhaolei; Vizeacoumar, Franco J; Freywald, Andrew

2016-08-02

Application of tumor genome sequencing has identified numerous loss-of-function alterations in cancer cells. While these alterations are difficult to target using direct interventions, they may be attacked with the help of the synthetic lethality (SL) approach. In this approach, inhibition of one gene causes lethality only when another gene is also completely or partially inactivated. The EPHB6 receptor tyrosine kinase has been shown to have anti-malignant properties and to be downregulated in multiple cancers, which makes it a very attractive target for SL applications. In our work, we used a genome-wide SL screen combined with expression and interaction network analyses, and identified the SRC kinase as a SL partner of EPHB6 in triple-negative breast cancer (TNBC) cells. Our experiments also reveal that this SL interaction can be targeted by small molecule SRC inhibitors, SU6656 and KX2-391, and can be used to improve elimination of human TNBC tumors in a xenograft model. Our observations are of potential practical importance, since TNBC is an aggressive heterogeneous malignancy with a very high rate of patient mortality due to the lack of targeted therapies, and our work indicates that FDA-approved SRC inhibitors may potentially be used in a personalized manner for treating patients with EPHB6-deficient TNBC. Our findings are also of a general interest, as EPHB6 is downregulated in multiple malignancies and our data serve as a proof of principle that EPHB6 deficiency may be targeted by small molecule inhibitors in the SL approach.

Plastidial NAD-Dependent Malate Dehydrogenase: A Moonlighting Protein Involved in Early Chloroplast Development Through its Interaction with an FtsH12-FtsHi Protease Complex.

PubMed

Schreier, Tina B; Antoine, Cléry; Schläfli, Michael; Galbier, Florian; Stadler, Martha; Demarsy, Emilie; Albertini, Daniele; Maier, Benjamin A; Kessler, Felix; Hörtensteiner, Stefan; Zeeman, Samuel C; Kötting, Oliver

2018-06-22

Malate dehydrogenases (MDH) convert malate to oxaloacetate using NAD(H) or NADP(H) as a cofactor. Arabidopsis thaliana mutants lacking plastidial NAD-dependent MDH (pdnad-mdh) are embryo-lethal, and constitutive silencing (miR-mdh-1) causes a pale, dwarfed phenotype. The reason for these severe phenotypes is unknown. Here, we rescued the embryo lethality of pdnad-mdh via embryo-specific expression of pdNAD-MDH. Rescued seedlings developed white leaves with aberrant chloroplasts and failed to reproduce. Inducible silencing of pdNAD-MDH at the rosette stage also resulted in white newly emerging leaves. These data suggest that pdNAD-MDH is important for early plastid development, which is consistent with the reductions in major plastidial galactolipid, carotenoid and protochlorophyllide levels in miR-mdh-1 seedlings. Surprisingly, the targeting of other NAD-dependent MDH isoforms to the plastid did not complement the embryo lethality of pdnad-mdh, while expression of enzymatically inactive pdNAD-MDH did. These complemented plants grew indistinguishably from the wild type. Both active and inactive forms of pdNAD-MDH interact with a heteromeric AAA-ATPase complex at the inner membrane of the chloroplast envelope. Silencing the expression of FtsH12, a key member of this complex, resulted in a phenotype that strongly resembles miR-mdh-1. We propose that pdNAD-MDH is essential for chloroplast development due to its moonlighting role in stabilizing FtsH12, distinct from its enzymatic function. © 2018 American Society of Plant Biologists. All rights reserved.

APOBEC3G-Induced Hypermutation of Human Immunodeficiency Virus Type-1 Is Typically a Discrete “All or Nothing” Phenomenon

PubMed Central

Armitage, Andrew E.; Deforche, Koen; Chang, Chih-hao; Wee, Edmund; Kramer, Beatrice; Welch, John J.; Gerstoft, Jan; Fugger, Lars; McMichael, Andrew; Rambaut, Andrew; Iversen, Astrid K. N.

2012-01-01

The rapid evolution of Human Immunodeficiency Virus (HIV-1) allows studies of ongoing host–pathogen interactions. One key selective host factor is APOBEC3G (hA3G) that can cause extensive and inactivating Guanosine-to-Adenosine (G-to-A) mutation on HIV plus-strand DNA (termed hypermutation). HIV can inhibit this innate anti-viral defense through binding of the viral protein Vif to hA3G, but binding efficiency varies and hypermutation frequencies fluctuate in patients. A pivotal question is whether hA3G-induced G-to-A mutation is always lethal to the virus or if it may occur at sub-lethal frequencies that could increase viral diversification. We show in vitro that limiting-levels of hA3G-activity (i.e. when only a single hA3G-unit is likely to act on HIV) produce hypermutation frequencies similar to those in patients and demonstrate in silico that potentially non-lethal G-to-A mutation rates are ∼10-fold lower than the lowest observed hypermutation levels in vitro and in vivo. Our results suggest that even a single incorporated hA3G-unit is likely to cause extensive and inactivating levels of HIV hypermutation and that hypermutation therefore is typically a discrete “all or nothing” phenomenon. Thus, therapeutic measures that inhibit the interaction between Vif and hA3G will likely not increase virus diversification but expand the fraction of hypermutated proviruses within the infected host. PMID:22457633

Control of cellular morphogenesis by the Ip12/Bem2 GTPase-activating protein: possible role of protein phosphorylation

PubMed Central

1994-01-01

The IPL2 gene is known to be required for normal polarized cell growth in the budding yeast Saccharomyces cerevisiae. We now show that IPL2 is identical to the previously identified BEM2 gene. bem2 mutants are defective in bud site selection at 26 degrees C and localized cell surface growth and organization of the actin cytoskeleton at 37 degrees C. BEM2 encodes a protein with a COOH-terminal domain homologous to sequences found in several GTPase-activating proteins, including human Bcr. The GTPase-activating protein-domain from the Bem2 protein (Bem2p) or human Bcr can functionally substitute for Bem2p. The Rho1 and Rho2 GTPases are the likely in vivo targets of Bem2p because bem2 mutant phenotypes can be partially suppressed by increasing the gene dosage of RHO1 or RHO2. CDC55 encodes the putative regulatory B subunit of protein phosphatase 2A, and mutations in BEM2 have previously been identified as suppressors of the cdc55-1 mutation. We show here that mutations in the previously identified GRR1 gene can suppress bem2 mutations. grr1 and cdc55 mutants are both elongated in shape and cold- sensitive for growth, and cells lacking both GRR1 and CDC55 exhibit a synthetic lethal phenotype. bem2 mutant phenotypes also can be suppressed by the SSD1-vl (also known as SRK1) mutation, which was shown previously to suppress mutations in the protein phosphatase- encoding SIT4 gene. Cells lacking both BEM2 and SIT4 exhibit a synthetic lethal phenotype even in the presence of the SSD1-v1 suppressor. These genetic interactions together suggest that protein phosphorylation and dephosphorylation play an important role in the BEM2-mediated process of polarized cell growth. PMID:7962097

Control of cellular morphogenesis by the Ip12/Bem2 GTPase-activating protein: possible role of protein phosphorylation.

PubMed

Kim, Y J; Francisco, L; Chen, G C; Marcotte, E; Chan, C S

1994-12-01

The IPL2 gene is known to be required for normal polarized cell growth in the budding yeast Saccharomyces cerevisiae. We now show that IPL2 is identical to the previously identified BEM2 gene. bem2 mutants are defective in bud site selection at 26 degrees C and localized cell surface growth and organization of the actin cytoskeleton at 37 degrees C. BEM2 encodes a protein with a COOH-terminal domain homologous to sequences found in several GTPase-activating proteins, including human Bcr. The GTPase-activating protein-domain from the Bem2 protein (Bem2p) or human Bcr can functionally substitute for Bem2p. The Rho1 and Rho2 GTPases are the likely in vivo targets of Bem2p because bem2 mutant phenotypes can be partially suppressed by increasing the gene dosage of RHO1 or RHO2. CDC55 encodes the putative regulatory B subunit of protein phosphatase 2A, and mutations in BEM2 have previously been identified as suppressors of the cdc55-1 mutation. We show here that mutations in the previously identified GRR1 gene can suppress bem2 mutations. grr1 and cdc55 mutants are both elongated in shape and cold-sensitive for growth, and cells lacking both GRR1 and CDC55 exhibit a synthetic lethal phenotype. bem2 mutant phenotypes also can be suppressed by the SSD1-vl (also known as SRK1) mutation, which was shown previously to suppress mutations in the protein phosphatase-encoding SIT4 gene. Cells lacking both BEM2 and SIT4 exhibit a synthetic lethal phenotype even in the presence of the SSD1-v1 suppressor. These genetic interactions together suggest that protein phosphorylation and dephosphorylation play an important role in the BEM2-mediated process of polarized cell growth.

Maintenance of genetic diversity through plant-herbivore interactions

PubMed Central

Gloss, Andrew D.; Dittrich, Anna C. Nelson; Goldman-Huertas, Benjamin; Whiteman, Noah K.

2013-01-01

Identifying the factors governing the maintenance of genetic variation is a central challenge in evolutionary biology. New genomic data, methods and conceptual advances provide increasing evidence that balancing selection, mediated by antagonistic species interactions, maintains functionally-important genetic variation within species and natural populations. Because diverse interactions between plants and herbivorous insects dominate terrestrial communities, they provide excellent systems to address this hypothesis. Population genomic studies of Arabidopsis thaliana and its relatives suggest spatial variation in herbivory maintains adaptive genetic variation controlling defense phenotypes, both within and among populations. Conversely, inter-species variation in plant defenses promotes adaptive genetic variation in herbivores. Emerging genomic model herbivores of Arabidopsis could illuminate how genetic variation in herbivores and plants interact simultaneously. PMID:23834766

God and Genes in the Caring Professions: Clinician and Clergy Perceptions of Religion and Genetics

PubMed Central

Bartlett, Virginia L; Johnson, Rolanda L

2013-01-01

Little is known about how care providers’ perceptions of religion and genetics affect interactions with patients/parishioners. This study investigates clinicians’ and clergy’s perceptions of and experiences with religion and genetics in their clinical and pastoral interactions. An exploratory qualitative study designed to elicit care providers’ descriptions of experiences with religion and genetics in clinical or pastoral interactions. Thirteen focus groups were conducted with members of the caring professions: physicians, nurses, and genetics counselors (clinicians), ministers and chaplains (clergy). Preliminary analysis of qualitative data is presented here. Preliminary analysis highlights four positions in professional perceptions of the relationship between science and faith. Further, differences among professional perceptions appear to influence perceptions of needed or available resources for interactions with religion and genetics. Clinicians’ and clergy’s perceptions of how religion and genetics relate are not defined solely by professional affiliation. These non-role-defined perceptions may affect clinical and pastoral interactions, especially regarding resources for patients and parishioners. PMID:19170091

Myc requires RhoA/SRF to reprogram glutamine metabolism.

PubMed

Haikala, Heidi M; Marques, Elsa; Turunen, Mikko; Klefström, Juha

2018-05-04

RhoA regulates actin cytoskeleton but recent evidence suggest a role for this conserved Rho GTPase also in other cellular processes, including transcriptional control of cell proliferation and survival. Interestingy, loss of RhoA is synthetic lethal with oncogenic Myc, a master transcription factor that turns on anabolic metabolism to promote cell growth in many cancers. We show evidence indicating that the synthetic lethal interaction between RhoA loss and Myc arises from deficiency in glutamine utilization, resulting from impaired co-regulation of glutaminase expression and anaplerosis by Myc and RhoA - serum response factor (SRF) pathway. The results suggest metabolic coordination between Myc and RhoA/SRF in sustaining cancer cell viability and indicate RhoA/SRF as a potential vulnerability in cancer cells for therapeutic targeting.

Toxic and hormetic-like effects of three components of citrus essential oils on adult Mediterranean fruit flies (Ceratitis capitata)

PubMed Central

Papanastasiou, Stella A.; Bali, Eleftheria-Maria D.; Ioannou, Charalampos S.; Papachristos, Dimitrios P.; Zarpas, Kostas D.

2017-01-01

Plant essential oils (EOs) and a wide range of their individual components are involved in a variety of biological interactions with insect pests including stimulatory, deterrent, toxic and even hormetic effects. Both the beneficial and toxic properties of citrus EOs on the Mediterranean fruit fly (medfly) have been experimentally evidenced over the last years. However, no information is available regarding the toxic or beneficial effects of the major components of citrus EOs via contact with the adults of the Mediterranean fruit fly. In the present study, we explored the toxicity of limonene, linalool and α-pinene (3 of the main compounds of citrus EOs) against adult medflies and identified the effects of sub-lethal doses of limonene on fitness traits in a relaxed [full diet (yeast and sugar)] and in a stressful (sugar only) feeding environment. Our results demonstrate that all three compounds inferred high toxicity to adult medflies regardless of the diet, with males being more sensitive than females. Sub-lethal doses of limonene (LD20) enhanced the lifespan of adult medflies when they were deprived of protein. Fecundity was positively affected when females were exposed to limonene sub-lethal doses. Therefore, limonene, a major constituent of citrus EOs, induces high mortality at increased doses and positive effects on life history traits of medfly adults through contact at low sub-lethal doses. A hormetic-like effect of limonene to adult medflies and its possible underlying mechanisms are discussed. PMID:28520791

Promises, pitfalls and practicalities of prenatal whole exome sequencing.

PubMed

Best, Sunayna; Wou, Karen; Vora, Neeta; Van der Veyver, Ignatia B; Wapner, Ronald; Chitty, Lyn S

2018-01-01

Prenatal genetic diagnosis provides information for pregnancy and perinatal decision-making and management. In several small series, prenatal whole exome sequencing (WES) approaches have identified genetic diagnoses when conventional tests (karyotype and microarray) were not diagnostic. Here, we review published prenatal WES studies and recent conference abstracts. Thirty-one studies were identified, with diagnostic rates in series of five or more fetuses varying between 6.2% and 80%. Differences in inclusion criteria and trio versus singleton approaches to sequencing largely account for the wide range of diagnostic rates. The data suggest that diagnostic yields will be greater in fetuses with multiple anomalies or in cases preselected following genetic review. Beyond its ability to improve diagnostic rates, we explore the potential of WES to improve understanding of prenatal presentations of genetic disorders and lethal fetal syndromes. We discuss prenatal phenotyping limitations, counselling challenges regarding variants of uncertain significance, incidental and secondary findings, and technical problems in WES. We review the practical, ethical, social and economic issues that must be considered before prenatal WES could become part of routine testing. Finally, we reflect upon the potential future of prenatal genetic diagnosis, including a move towards whole genome sequencing and non-invasive whole exome and whole genome testing. © 2017 John Wiley & Sons, Ltd. © 2017 John Wiley & Sons, Ltd.

In utero transplantation of adult bone marrow decreases perinatal lethality and rescues the bone phenotype in the knockin murine model for classical, dominant osteogenesis imperfecta

PubMed Central

Panaroni, Cristina; Gioia, Roberta; Lupi, Anna; Besio, Roberta; Goldstein, Steven A.; Kreider, Jaclynn; Leikin, Sergey; Vera, Juan Carlos; Mertz, Edward L.; Perilli, Egon; Baruffaldi, Fabio; Villa, Isabella; Farina, Aurora; Casasco, Marco; Cetta, Giuseppe; Rossi, Antonio; Frattini, Annalisa; Marini, Joan C.; Vezzoni, Paolo

2009-01-01

Autosomal dominant osteogenesis imperfecta (OI) caused by glycine substitutions in type I collagen is a paradigmatic disorder for stem cell therapy. Bone marrow transplantation in OI children has produced a low engraftment rate, but surprisingly encouraging symptomatic improvements. In utero transplantation (IUT) may hold even more promise. However, systematic studies of both methods have so far been limited to a recessive mouse model. In this study, we evaluated intrauterine transplantation of adult bone marrow into heterozygous BrtlIV mice. Brtl is a knockin mouse with a classical glycine substitution in type I collagen [α1(I)-Gly349Cys], dominant trait transmission, and a phenotype resembling moderately severe and lethal OI. Adult bone marrow donor cells from enhanced green fluorescent protein (eGFP) transgenic mice engrafted in hematopoietic and nonhematopoietic tissues differentiated to trabecular and cortical bone cells and synthesized up to 20% of all type I collagen in the host bone. The transplantation eliminated the perinatal lethality of heterozygous BrtlIV mice. At 2 months of age, femora of treated Brtl mice had significant improvement in geometric parameters (P < .05) versus untreated Brtl mice, and their mechanical properties attained wild-type values. Our results suggest that the engrafted cells form bone with higher efficiency than the endogenous cells, supporting IUT as a promising approach for the treatment of genetic bone diseases. PMID:19414862

Proteomic Characterization of Inbreeding-Related Cold Sensitivity in Drosophila melanogaster

PubMed Central

Beck, Hans C.; Petersen, Jørgen; Gagalova, Kristina Kirilova; Loeschcke, Volker

2013-01-01

Inbreeding depression is a widespread phenomenon of central importance to agriculture, medicine, conservation biology and evolutionary biology. Although the population genetic principles of inbreeding depression are well understood, we know little about its functional genomic causes. To provide insight into the molecular interplay between intrinsic stress responses, inbreeding depression and temperature tolerance, we performed a proteomic characterization of a well-defined conditional inbreeding effect in a single line of Drosophila melanogaster, which suffers from extreme cold sensitivity and lethality. We identified 48 differentially expressed proteins in a conditional lethal line as compared to two control lines. These proteins were enriched for proteins involved in hexose metabolism, in particular pyruvate metabolism, and many were found to be associated with lipid particles. These processes can be linked to known cold tolerance mechanisms, such as the production of cryoprotectants, membrane remodeling and the build-up of energy reserves. We checked mRNA-expression of seven genes with large differential protein expression. Although protein expression poorly correlated with gene expression, we found a single gene (CG18067) that, after cold shock, was upregulated in the conditional lethal line both at the mRNA and protein level. Expression of CG18067 also increased in control flies after cold shock, and has previously been linked to cold exposure and chill coma recovery time. Many differentially expressed proteins in our study appear to be involved in cold tolerance in non-inbred individuals. This suggest the conditional inbreeding effect to be caused by misregulation of physiological cold tolerance mechanisms. PMID:23658762

1H NMR-based metabolomics reveals interactive effects between the carrier solvent methanol and a pharmaceutical mixture in an amphibian developmental bioassay with Limnodynastes peronii.

PubMed

Melvin, Steven D; Jones, Oliver A H; Carroll, Anthony R; Leusch, Frederic D L

2018-05-01

Organic carrier solvents are used in aquatic toxicity testing to improve chemical solubility and facilitate the exploration of dose-response relationships. Both water- and solvent-control groups are normally included in these scenarios to ensure that the solvent itself has no effect on the test organism, but this fails to consider possible interactive effects between carrier solvents and contaminants of interest. We explored this topic by exposing Limnodynastes peronii tadpoles to a mixture of common water-soluble pharmaceuticals (diclofenac, metformin and valproic acid) in the presence and absence of the carrier solvent methanol, according to standard developmental bioassay methodology. Nuclear Magnetic Resonance (NMR) spectroscopy was applied as a platform for untargeted metabolomics, to compare broad sub-lethal hepatotoxicity in solvent- and solvent-free exposure scenarios. Considerable interactive effects were identified between the pharmaceutical mixture and a typical dose of methanol (0.003%). Specifically, pronounced differences were observed between the solvent- and solvent-free exposure groups for leucine, acetate, glutamine, citrate, glycogen, tyrosine, arginine, purine nucleotides and an unidentified metabolite at 6.53 ppm. Various other metabolites exhibited similar disparity related to the use of carrier solvent, but the interactions were non-significant. These results raise important questions about the use of carrier solvents for chemical exposures in aquatic ecotoxicology, and particularly for studies interested in sub-lethal mechanistic information and/or biomarker discovery. Copyright © 2018 Elsevier Ltd. All rights reserved.

Interactions between immunotoxicants and parasite stress: Implications for host health.

PubMed

Booton, Ross D; Yamaguchi, Ryo; Marshall, James A R; Childs, Dylan Z; Iwasa, Yoh

2018-05-14

Many organisms face a wide variety of biotic and abiotic stressors which reduce individual survival, interacting to further reduce fitness. Here we studied the effects of two such interacting stressors: immunotoxicant exposure and parasite infection. We model the dynamics of a within-host infection and the associated immune response of an individual. We consider both the indirect sub-lethal effects on immunosuppression and the direct effects on health and mortality of individuals exposed to toxicants. We demonstrate that sub-lethal exposure to toxicants can promote infection through the suppression of the immune system. This happens through the depletion of the immune response which causes rapid proliferation in parasite load. We predict that the within-host parasite density is maximised by an intermediate toxicant exposure, rather than continuing to increase with toxicant exposure. In addition, high toxicant exposure can alter cellular regulation and cause the breakdown of normal healthy tissue, from which we infer higher mortality risk of the host. We classify this breakdown into three phases of increasing toxicant stress, and demonstrate the range of conditions under which toxicant exposure causes failure at the within-host level. These phases are determined by the relationship between the immunity status, overall cellular health and the level of toxicant exposure. We discuss the implications of our model in the context of individual bee health. Our model provides an assessment of how pesticide stress and infection interact to cause the breakdown of the within-host dynamics of individual bees. Copyright © 2018 Elsevier Ltd. All rights reserved.

Modeling the effects of temperature, sodium chloride, and green tea and their interactions on the thermal inactivation of Listeria monocytogenes in turkey.

PubMed

Juneja, Vijay K; Garcia-Dávila, Jimena; Lopez-Romero, Julio Cesar; Pena-Ramos, Etna Aida; Camou, Juan Pedro; Valenzuela-Melendres, Martin

2014-10-01

The interactive effects of heating temperature (55 to 65°C), sodium chloride (NaCl; 0 to 2%), and green tea 60% polyphenol extract (GTPE; 0 to 3%) on the heat resistance of a five-strain mixture of Listeria monocytogenes in ground turkey were determined. Thermal death times were quantified in bags that were submerged in a circulating water bath set at 55, 57, 60, 63, and 65°C. The recovery medium was tryptic soy agar supplemented with 0.6% yeast extract and 1% sodium pyruvate. D-values were analyzed by second-order response surface regression for temperature, NaCl, and GTPE. The data indicated that all three factors interacted to affect the inactivation of the pathogen. The D-values for turkey with no NaCl or GTPE at 55, 57, 60, 63, and 65°C were 36.3, 20.8, 13.2, 4.1, and 2.9 min, respectively. Although NaCl exhibited a concentration-dependent protective effect against heat lethality on L. monocytogenes in turkey, addition of GTPE rendered the pathogen more sensitive to the lethal effect of heat. GTPE levels up to 1.5% interacted with NaCl and reduced the protective effect of NaCl on heat resistance of the pathogen. Food processors can use the predictive model to design an appropriate heat treatment that would inactivate L. monocytogenes in cooked turkey products without adversely affecting the quality of the product.

Was skin cancer a selective force for black pigmentation in early hominin evolution?

PubMed Central

Greaves, Mel

2014-01-01

Melanin provides a crucial filter for solar UV radiation and its genetically determined variation influences both skin pigmentation and risk of cancer. Genetic evidence suggests that the acquisition of a highly stable melanocortin 1 receptor allele promoting black pigmentation arose around the time of savannah colonization by hominins at some 1–2 Ma. The adaptive significance of dark skin is generally believed to be protection from UV damage but the pathologies that might have had a deleterious impact on survival and/or reproductive fitness, though much debated, are uncertain. Here, I suggest that data on age-associated cancer incidence and lethality in albinos living at low latitudes in both Africa and Central America support the contention that skin cancer could have provided a potent selective force for the emergence of black skin in early hominins. PMID:24573849

Molecular pathogenesis of hepatocellular carcinoma and impact of therapeutic advances

PubMed Central

Dhanasekaran, Renumathy; Bandoh, Salome; Roberts, Lewis R.

2016-01-01

Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality and has an increasing incidence worldwide. HCC can be induced by multiple etiologies, is influenced by many risk factors, and has a complex pathogenesis. Furthermore, HCCs exhibit substantial heterogeneity, which compounds the difficulties in developing effective therapies against this highly lethal cancer. With advances in cancer biology and molecular and genetic profiling, a number of different mechanisms involved in the development and progression of HCC have been identified. Despite the advances in this area, the molecular pathogenesis of hepatocellular carcinoma is still not completely understood. This review aims to elaborate our current understanding of the most relevant genetic alterations and molecular pathways involved in the development and progression of HCC, and anticipate the potential impact of future advances on therapeutic drug development. PMID:27239288

A lethal murine infection model for dengue virus 3 in AG129 mice deficient in type I and II interferon receptors leads to systemic disease.

PubMed

Sarathy, Vanessa V; White, Mellodee; Li, Li; Gorder, Summer R; Pyles, Richard B; Campbell, Gerald A; Milligan, Gregg N; Bourne, Nigel; Barrett, Alan D T

2015-01-15

The mosquito-borne disease dengue (DEN) is caused by four serologically and genetically related viruses, termed DENV-1 to DENV-4. Infection with one DENV usually leads to acute illness and results in lifelong homotypic immunity, but individuals remain susceptible to infection by the other three DENVs. The lack of a small-animal model that mimics systemic DEN disease without neurovirulence has been an obstacle, but DENV-2 models that resemble human disease have been recently developed in AG129 mice (deficient in interferon alpha/beta and interferon gamma receptor signaling). However, comparable DENV-1, -3, and -4 models have not been developed. We utilized a non-mouse-adapted DENV-3 Thai human isolate to develop a lethal infection model in AG129 mice. Intraperitoneal inoculation of six to eight-week-old animals with strain C0360/94 led to rapid, fatal disease. Lethal C0360/94 infection resulted in physical signs of illness, high viral loads in the spleen, liver, and large intestine, histological changes in the liver and spleen tissues, and increased serum cytokine levels. Importantly, the animals developed vascular leakage, thrombocytopenia, and leukopenia. Overall, we have developed a lethal DENV-3 murine infection model, with no evidence of neurotropic disease based on a non-mouse-adapted human isolate, which can be used to investigate DEN pathogenesis and to evaluate candidate vaccines and antivirals. This suggests that murine models utilizing non-mouse-adapted isolates can be obtained for all four DENVs. Dengue (DEN) is a mosquito-borne disease caused by four DENV serotypes (DENV-1, -2, -3, and -4) that have no treatments or vaccines. Primary infection with one DENV usually leads to acute illness followed by lifelong homotypic immunity, but susceptibility to infection by the other three DENVs remains. Therefore, a vaccine needs to protect from all four DENVs simultaneously. To date a suitable animal model to mimic systemic human illness exists only for DENV-2 in immunocompromised mice using passaged viruses; however, models are still needed for the remaining serotypes. This study describes establishment of a lethal systemic DENV-3 infection model with a human isolate in immunocompromised mice and is the first report of lethal infection by a nonadapted clinical DENV isolate without evidence of neurological disease. Our DENV-3 model provides a relevant platform to test DEN vaccines and antivirals. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Genetic, chromosomal, and syndromic causes of neural tube defects.

PubMed

Seidahmed, Mohammed Z; Abdelbasit, Omer B; Shaheed, Meeralebbae M; Alhussein, Khalid A; Miqdad, Abeer M; Samadi, Abdulmohsen S; Khalil, Mohammed I; Al-Mardawi, Elham; Salih, Mustafa A

2014-12-01

To ascertain the incidence, and describe the various forms of neural tube defects (NTDs) due to genetic, chromosomal, and syndromic causes. We carried out a retrospective analysis of data retrieved from the medical records of newborn infants admitted to the Neonatal Intensive Care Unit with NTDs and their mothers spanning 14 years (1996-2009) at the Security Forces Hospital, Riyadh, Saudi Arabia. The cases were ascertained by a perinatologist, neonatologist, geneticist, radiologist, and neurologist. The literature was reviewed via a MEDLINE search. Only liveborn babies were included. Permission from the Educational Committee at the Security Forces Hospital was obtained prior to the collection of data. Out of 103 infants with NTDs admitted during this period, 20 (19.4%) were found to have an underlying genetic syndromic, chromosomal and/or other anomalies. There were 5 cases of Meckel-Gruber syndrome, 2 Joubert syndrome, one Waardenburg syndrome, one Walker-Warburg syndrome, 2 chromosomal disorders, 2 caudal regression, one amniotic band disruption sequence, one associated with omphalocele, one with diaphragmatic hernia, and 4 with multiple congenital anomalies. There is a high rate of underlying genetic syndromic and/or chromosomal causes of NTDs in the Saudi Arabian population due to the high consanguinity rate. Identification of such association can lead to more accurate provisions of genetic counseling to the family including preimplantation genetic diagnosis or early termination of pregnancies associated with lethal conditions.

Genetic, chromosomal, and syndromic causes of neural tube defects

PubMed Central

Seidahmed, Mohammed Z.; Abdelbasit, Omer B.; Shaheed, Meeralebbae M.; Alhussein, Khalid A.; Miqdad, Abeer M.; Samadi, Abdulmohsen S.; Khalil, Mohammed I.; Al-Mardawi, Elham; Salih, Mustafa A.

2014-01-01

Objective: To ascertain the incidence, and describe the various forms of neural tube defects (NTDs) due to genetic, chromosomal, and syndromic causes. Methods: We carried out a retrospective analysis of data retrieved from the medical records of newborn infants admitted to the Neonatal Intensive Care Unit with NTDs and their mothers spanning 14 years (1996-2009) at the Security Forces Hospital, Riyadh, Saudi Arabia. The cases were ascertained by a perinatologist, neonatologist, geneticist, radiologist, and neurologist. The literature was reviewed via a MEDLINE search. Only liveborn babies were included. Permission from the Educational Committee at the Security Forces Hospital was obtained prior to the collection of data. Results: Out of 103 infants with NTDs admitted during this period, 20 (19.4%) were found to have an underlying genetic syndromic, chromosomal and/or other anomalies. There were 5 cases of Meckel-Gruber syndrome, 2 Joubert syndrome, one Waardenburg syndrome, one Walker-Warburg syndrome, 2 chromosomal disorders, 2 caudal regression, one amniotic band disruption sequence, one associated with omphalocele, one with diaphragmatic hernia, and 4 with multiple congenital anomalies. Conclusions: There is a high rate of underlying genetic syndromic and/or chromosomal causes of NTDs in the Saudi Arabian population due to the high consanguinity rate. Identification of such association can lead to more accurate provisions of genetic counseling to the family including preimplantation genetic diagnosis or early termination of pregnancies associated with lethal conditions. PMID:25551112

Collective Genetic Interaction Effects and the Role of Antigen Presenting Cells in Autoimmune Diseases

DTIC Science & Technology

2017-01-12

RESEARCH ARTICLE Collective Genetic Interaction Effects and the Role of Antigen-Presenting Cells in Autoimmune Diseases Hyung Jun Woo*, Chenggang Yu...autoimmunity. Genetic predispositions center around the major histocompatibility complex (MHC) class II loci involved in antigen presentation, the key...helper and regulatory T cells showing strong dis- ease-associated interactions with B cells. Our results provide direct genetic evidence point- ing to

Anatomy and genetic diversity of two populations of Schinus terebinthifolius (Anacardiaceae) from the Tibagi River basin in Paraná, Brazil.

PubMed

Ruas, E A; Ruas, C F; Medri, P S; Medri, C; Medri, M E; Bianchini, E; Pimenta, J A; Rodrigues, L A; Ruas, P M

2011-03-29

Knowledge of the effects of flooding on plant survival is relevant for the efficiency of management and conservation programs. Schinus terebinthifolius is a tree of economic and ecological importance that is common in northeast Brazil. Flooding tolerance and genetic variation were investigated in two riparian populations of S. terebinthifolius distributed along two different ecological regions of the Tibagi River basin. Flooding tolerance was evaluated through the investigation of young plants, submitted to different flooding intensities to examine the morphological and anatomical responses to this stress. The growth rate of S. terebinthifolius was not affected by flooding, but total submersion proved to be lethal for 100% of the plants. Morphological alterations such as hypertrophied lenticels were observed in both populations and lenticel openings were significantly higher in plants from one population. Genetic analysis using DNA samples obtained from both populations showed a moderate degree of genetic variation between populations (13.7%); most of the variation was found within populations (86.3%). These results show that for conservation purposes and management of degraded areas, both populations should be preserved and could be used in programs that intend to recompose riparian forests.

Core signaling pathways in human pancreatic cancers revealed by global genomic analyses.

PubMed

Jones, Siân; Zhang, Xiaosong; Parsons, D Williams; Lin, Jimmy Cheng-Ho; Leary, Rebecca J; Angenendt, Philipp; Mankoo, Parminder; Carter, Hannah; Kamiyama, Hirohiko; Jimeno, Antonio; Hong, Seung-Mo; Fu, Baojin; Lin, Ming-Tseh; Calhoun, Eric S; Kamiyama, Mihoko; Walter, Kimberly; Nikolskaya, Tatiana; Nikolsky, Yuri; Hartigan, James; Smith, Douglas R; Hidalgo, Manuel; Leach, Steven D; Klein, Alison P; Jaffee, Elizabeth M; Goggins, Michael; Maitra, Anirban; Iacobuzio-Donahue, Christine; Eshleman, James R; Kern, Scott E; Hruban, Ralph H; Karchin, Rachel; Papadopoulos, Nickolas; Parmigiani, Giovanni; Vogelstein, Bert; Velculescu, Victor E; Kinzler, Kenneth W

2008-09-26

There are currently few therapeutic options for patients with pancreatic cancer, and new insights into the pathogenesis of this lethal disease are urgently needed. Toward this end, we performed a comprehensive genetic analysis of 24 pancreatic cancers. We first determined the sequences of 23,219 transcripts, representing 20,661 protein-coding genes, in these samples. Then, we searched for homozygous deletions and amplifications in the tumor DNA by using microarrays containing probes for approximately 10(6) single-nucleotide polymorphisms. We found that pancreatic cancers contain an average of 63 genetic alterations, the majority of which are point mutations. These alterations defined a core set of 12 cellular signaling pathways and processes that were each genetically altered in 67 to 100% of the tumors. Analysis of these tumors' transcriptomes with next-generation sequencing-by-synthesis technologies provided independent evidence for the importance of these pathways and processes. Our data indicate that genetically altered core pathways and regulatory processes only become evident once the coding regions of the genome are analyzed in depth. Dysregulation of these core pathways and processes through mutation can explain the major features of pancreatic tumorigenesis.

Canine and Feline Models of Human Genetic Diseases and Their Contributions to Advancing Clinical Therapies


PubMed Central

Gurda, Brittney L.; Bradbury, Allison M.; Vite, Charles H.

2017-01-01

For many lethal or debilitating genetic disorders in patients there are no satisfactory therapies. Several barriers exist that hinder the developments of effective therapies including the limited availability of clinically relevant animal models that faithfully recapitulate human genetic disease. In 1974, the Referral Center for Animal Models of Human Genetic Disease (RCAM) was established by Dr. Donald F. Patterson and continued by Dr. Mark E. Haskins at the University of Pennsylvania with the mission to discover, understand, treat, and maintain breeding colonies of naturally occurring hereditary disorders in dogs and cats that are orthologous to those found in human patients. Although non-human primates, sheep, and pig models are also available within the medical community, naturally occurring diseases are rarely identified in non-human primates, and the vast behavioral, clinicopathological, physiological, and anatomical knowledge available regarding dogs and cats far surpasses what is available in ovine and porcine species. The canine and feline models that are maintained at RCAM are presented here with a focus on preclinical therapy data. Clinical studies that have been generated from preclinical work in these models are also presented. PMID:28955181

Aggression-impulsivity, mental pain, and communication difficulties in medically serious and medically non-serious suicide attempters.

PubMed

Gvion, Yari; Horresh, Netta; Levi-Belz, Yossi; Fischel, Tsvi; Treves, Ilan; Weiser, Mark; David, Haim Shem; Stein-Reizer, Orit; Apter, Alan

2014-01-01

Unbearable mental pain, depression, and hopelessness have been associated with suicidal behavior in general, while difficulties with social communication and loneliness have been associated with highly lethal suicide attempts in particular. The literature also links aggression and impulsivity with suicidal behavior but raises questions about their influence on the lethality and outcome of the suicide attempt. To evaluate the relative effects of aggression and impulsivity on the lethality of suicide attempts we hypothesized that impulsivity and aggression differentiate between suicide attempters and non-attempters and between medically serious and medically non-serious suicide attempters. The study group included 196 participants divided into four groups: 43 medically serious suicide attempters; 49 medically non-serious suicide attempters, 47 psychiatric patients who had never attempted suicide; and 57 healthy control subjects. Data on sociodemographic parameters, clinical history, and details of the suicide attempts were collected. Participants completed a battery of instruments for assessment of aggression-impulsivity, mental pain, and communication difficulties. The medically serious and medically non-serious suicide attempters scored significantly higher than both control groups on mental pain, depression, and hopelessness (p<.001 for all) and on anger-in, anger-out, violence, and impulsivity (p<.05 for all), with no significant difference between the two suicide attempter groups. Medically serious suicide attempters had significantly lower self-disclosure (p<.05) and more schizoid tendencies (p<.001) than the other three groups and significantly more feelings of loneliness than the medically non-serious suicide attempters and nonsuicidal psychiatric patients (p<.05). Analysis of aggression-impulsivity, mental pain, and communication variables with suicide lethality yielded significant correlations for self-disclosure, schizoid tendency, and loneliness. The interaction between mental pain and schizoid traits explained some of the variance in suicide lethality, over and above the contribution of each component alone. Aggression-impulsivity and mental pain are risk factors for suicide attempts. However, only difficulties in communication differentiate medically serious from medically non-serious suicide attempters. The combination of unbearable mental pain and difficulties in communication has a magnifying effect on the risk of lethal suicidal behavior. © 2014.

Gene-Gene and Gene-Environment Interactions in Ulcerative Colitis

PubMed Central

Wang, Ming-Hsi; Fiocchi, Claudio; Zhu, Xiaofeng; Ripke, Stephan; Kamboh, M. Ilyas; Rebert, Nancy; Duerr, Richard H.; Achkar, Jean-Paul

2014-01-01

Genome-wide association studies (GWAS) have identified at least 133 ulcerative colitis (UC) associated loci. The role of genetic factors in clinical practice is not clearly defined. The relevance of genetic variants to disease pathogenesis is still uncertain because of not characterized gene-gene and gene-environment interactions. We examined the predictive value of combining the 133 UC risk loci with genetic interactions in an ongoing inflammatory bowel disease (IBD) GWAS. The Wellcome Trust Case-Control Consortium (WTCCC) IBD GWAS was used as a replication cohort. We applied logic regression (LR), a novel adaptive regression methodology, to search for high order interactions. Exploratory genotype correlations with UC sub-phenotypes (extent of disease, need of surgery, age of onset, extra-intestinal manifestations and primary sclerosing cholangitis (PSC)) were conducted. The combination of 133 UC loci yielded good UC risk predictability (area under the curve [AUC] of 0.86). A higher cumulative allele score predicted higher UC risk. Through LR, several lines of evidence for genetic interactions were identified and successfully replicated in the WTCCC cohort. The genetic interactions combined with the gene-smoking interaction significantly improved predictability in the model (AUC, from 0.86 to 0.89, P=3.26E-05). Explained UC variance increased from 37% to 42% after adding the interaction terms. A within case analysis found suggested genetic association with PSC. Our study demonstrates that the LR methodology allows the identification and replication of high order genetic interactions in UC GWAS datasets. UC risk can be predicted by a 133 loci and improved by adding gene-gene and gene-environment interactions. PMID:24241240

An Allele of Sequoia Dominantly Enhances a Trio Mutant Phenotype to Influence Drosophila Larval Behavior

PubMed Central

Liebl, Eric C.

2013-01-01

The transition of Drosophila third instar larvae from feeding, photo-phobic foragers to non-feeding, photo-neutral wanderers is a classic behavioral switch that precedes pupariation. The neuronal network responsible for this behavior has recently begun to be defined. Previous genetic analyses have identified signaling components for food and light sensory inputs and neuropeptide hormonal outputs as being critical for the forager to wanderer transition. Trio is a Rho-Guanine Nucleotide Exchange Factor integrated into a variety of signaling networks including those governing axon pathfinding in early development. Sequoia is a pan-neuronally expressed zinc-finger transcription factor that governs dendrite and axon outgrowth. Using pre-pupal lethality as an endpoint, we have screened for dominant second-site enhancers of a weakly lethal trio mutant background. In these screens, an allele of sequoia has been identified. While these mutants have no obvious disruption of embryonic central nervous system architecture and survive to third instar larvae similar to controls, they retain forager behavior and thus fail to pupariate at high frequency. PMID:24376789

An allele of sequoia dominantly enhances a trio mutant phenotype to influence Drosophila larval behavior.

PubMed

Dean, Kathryn E; Fields, April; Geer, Marcus J; King, Eric C; Lynch, Brian T; Manohar, Rohan R; McCall, Julianne R; Palozola, Katherine C; Zhang, Yan; Liebl, Eric C

2013-01-01

The transition of Drosophila third instar larvae from feeding, photo-phobic foragers to non-feeding, photo-neutral wanderers is a classic behavioral switch that precedes pupariation. The neuronal network responsible for this behavior has recently begun to be defined. Previous genetic analyses have identified signaling components for food and light sensory inputs and neuropeptide hormonal outputs as being critical for the forager to wanderer transition. Trio is a Rho-Guanine Nucleotide Exchange Factor integrated into a variety of signaling networks including those governing axon pathfinding in early development. Sequoia is a pan-neuronally expressed zinc-finger transcription factor that governs dendrite and axon outgrowth. Using pre-pupal lethality as an endpoint, we have screened for dominant second-site enhancers of a weakly lethal trio mutant background. In these screens, an allele of sequoia has been identified. While these mutants have no obvious disruption of embryonic central nervous system architecture and survive to third instar larvae similar to controls, they retain forager behavior and thus fail to pupariate at high frequency.

PDE5 inhibitors enhance the lethality of [pemetrexed + sorafenib

PubMed Central

Booth, Laurence; Roberts, Jane L.; Poklepovic, Andrew; Dent, Paul

2017-01-01

The combination of pemetrexed and sorafenib has significant clinical activity against a wide variety of tumor types in patients and the present studies were performed to determine whether sildenafil enhances the killing potential of [pemetrexed + sorafenib]. In multiple genetically diverse lung cancer cell lines, sildenafil enhanced the lethality of [pemetrexed + sorafenib]. The three-drug combination reduced the activities of AKT, mTOR and STAT transcription factors; increased the activities of eIF2α and ULK-1; lowered the expression of MCL-1, BCL-XL, thioredoxin and SOD2; and increased the expression of Beclin1. Enhanced cell killing by sildenafil was blocked by inhibition of death receptor signaling and autophagosome formation. Enforced activation of STAT3 and AKT or inhibition of JNK significantly reduced cell killing. The enhanced cell killing caused by sildenafil was more reliant on increased PKG signaling than on the generation of nitric oxide. In vivo sildenafil enhanced the anti-tumor properties of [pemetrexed + sorafenib]. Based on our data we argue that additional clinical studies combining pemetrexed, sorafenib and sildenafil are warranted. PMID:28088782

Lethal genes surviving by mosaicism: a possible explanation for sporadic birth defects involving the skin.

PubMed

Happle, R

1987-04-01

A genetic concept is advanced to explain the origin of several sporadic syndromes characterized by a mosaic distribution of skin defects. It is postulated that these disorders are due to the action of a lethal gene surviving by mosaicism. The presence of the mutation in the zygote will lead to death of the embryo at an early stage of development. Cells bearing the mutation can survive only in a mosaic state, in close proximity with normal cells. The mosaic may arise either from a gametic half chromatid mutation or from an early somatic mutation. This concept of origin is proposed to apply to the Schimmelpenning-Feuerstein-Mims syndrome, the McCune-Albright syndrome, the Klippel-Trenaunay syndrome, the Sturge-Weber syndrome, and neurocutaneous melanosis. Moreover, this etiologic hypothesis may apply to two other birth defects that have recently been delineated, the Proteus syndrome (partial gigantism of hands or feet, hemihypertrophy, macrocephaly, linear papillomatous epidermal nevus, subcutaneous hemangiomas and lipomas, accelerated growth, and visceral anomalies), and the Delleman-Oorthuys syndrome (orbital cyst, porencephaly, periorbital appendages, and focal aplasia of the skin.

Rewiring the severe acute respiratory syndrome coronavirus (SARS-CoV) transcription circuit: Engineering a recombination-resistant genome

NASA Astrophysics Data System (ADS)

Yount, Boyd; Roberts, Rhonda S.; Lindesmith, Lisa; Baric, Ralph S.

2006-08-01

Live virus vaccines provide significant protection against many detrimental human and animal diseases, but reversion to virulence by mutation and recombination has reduced appeal. Using severe acute respiratory syndrome coronavirus as a model, we engineered a different transcription regulatory circuit and isolated recombinant viruses. The transcription network allowed for efficient expression of the viral transcripts and proteins, and the recombinant viruses replicated to WT levels. Recombinant genomes were then constructed that contained mixtures of the WT and mutant regulatory circuits, reflecting recombinant viruses that might occur in nature. Although viable viruses could readily be isolated from WT and recombinant genomes containing homogeneous transcription circuits, chimeras that contained mixed regulatory networks were invariantly lethal, because viable chimeric viruses were not isolated. Mechanistically, mixed regulatory circuits promoted inefficient subgenomic transcription from inappropriate start sites, resulting in truncated ORFs and effectively minimize viral structural protein expression. Engineering regulatory transcription circuits of intercommunicating alleles successfully introduces genetic traps into a viral genome that are lethal in RNA recombinant progeny viruses. regulation | systems biology | vaccine design

Two O-linked N-acetylglucosamine transferase genes of Arabidopsis thaliana L. Heynh. have overlapping functions necessary for gamete and seed development.

PubMed Central

Hartweck, Lynn M; Scott, Cheryl L; Olszewski, Neil E

2002-01-01

The Arabidopsis SECRET AGENT (SEC) and SPINDLY (SPY) proteins are similar to animal O-linked N-acetylglucosamine transferases (OGTs). OGTs catalyze the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to Ser/Thr residues of proteins. In animals, O-GlcNAcylation has been shown to affect protein activity, stability, and/or localization. SEC protein expressed in Escherichia coli had autocatalytic OGT activity. To determine the function of SEC in plants, two tDNA insertional mutants were identified and analyzed. Although sec mutant plants did not exhibit obvious phenotypes, sec and spy mutations had a synthetic lethal interaction. This lethality was incompletely penetrant in gametes and completely penetrant postfertilization. The rate of both female and male sec spy gamete transmission was higher in plants heterozygous for both mutations than in plants heterozygous for sec and homozygous for spy. Double-mutant embryos aborted at various stages of development and no double-mutant seedlings were obtained. These results indicate that OGT activity is required during gametogenesis and embryogenesis with lethality occurring when parentally derived SEC, SPY, and/or O-GlcNAcylated proteins become limiting. PMID:12136030

Glucose-6-phosphate dehydrogenase and Trypanothione reductase interaction protects Leishmania donovani from metalloid mediated oxidative stress.

PubMed

Ghosh, Ayan Kumar; Saini, Savita; Das, Sushmita; Mandal, Abhishek; Sardar, Abul Hasan; Ansari, Md Yousuf; Abhishek, Kumar; Kumar, Ajay; Singh, Ruby; Verma, Sudha; Equbal, Asif; Ali, Vahab; Das, Pradeep

2017-05-01

Exploration of metabolons as viable drug target is rare in kinetoplastid biology. Here we present a novel protein-protein interaction among Glucose-6-phosphate dehydrogenase (LdG6PDH) and Trypanothione reductase (LdTryR) of Leishmania donovani displaying interconnection between central glucose metabolism and thiol metabolism of this parasite. Digitonin fractionation patterns observed through immunoblotting indicated localisation of both LdG6PDH and LdTryR in cytosol. In-silico and in-vitro interaction observed by size exclusion chromatography, co-purification, pull-down assay and spectrofluorimetric analysis revealed LdG6PDH and LdTryR physically interact with each other in a NADPH dependent manner. Coupled enzymatic assay displayed that NADPH generation was severely impaired by addition of Sb III , As III and Te IV extraneously, which hint towards metalloid driven structural changes of the interacting proteins. Co-purification patterns and pull-down assays also depicted that metalloids (Sb III , As III and Te IV ) hinder the in-vitro interaction of these two enzymes. Surprisingly, metalloids at sub-lethal concentrations induced the in-vivo interaction of LdG6PDH and LdTryR, as analyzed by pull-down assays and fluorescence microscopy signifying protection against metalloid mediated ROS. Inhibition of LdTryR by thioridazine in LdG6PDH -/- parasites resulted in metalloid induced apoptotic death of the parasites due to abrupt fall in reduced thiol content, disrupted NADPH/NADP + homeostasis and lethal oxidative stress. Interestingly, clinical isolates of L.donovani resistant to SAG exhibited enhanced interaction between LdG6PDH and LdTryR and showed cross resistivity towards As III and Te IV . Thus, our findings propose the metabolon of LdG6PDH and LdTryR as an alternate therapeutic target and provide mechanistic insight about metalloid resistance in Visceral Leishmaniasis. Copyright © 2017. Published by Elsevier Inc.

Cardiac Med1 deletion promotes early lethality, cardiac remodeling, and transcriptional reprogramming

PubMed Central

Spitler, Kathryn M.; Ponce, Jessica M.; Oudit, Gavin Y.; Hall, Duane D.

2017-01-01

The mediator complex, a multisubunit nuclear complex, plays an integral role in regulating gene expression by acting as a bridge between transcription factors and RNA polymerase II. Genetic deletion of mediator subunit 1 (Med1) results in embryonic lethality, due in large part to impaired cardiac development. We first established that Med1 is dynamically expressed in cardiac development and disease, with marked upregulation of Med1 in both human and murine failing hearts. To determine if Med1 deficiency protects against cardiac stress, we generated two cardiac-specific Med1 knockout mouse models in which Med1 is conditionally deleted (Med1cKO mice) or inducibly deleted in adult mice (Med1cKO-MCM mice). In both models, cardiac deletion of Med1 resulted in early lethality accompanied by pronounced changes in cardiac function, including left ventricular dilation, decreased ejection fraction, and pathological structural remodeling. We next defined how Med1 deficiency alters the cardiac transcriptional profile using RNA-sequencing analysis. Med1cKO mice demonstrated significant dysregulation of genes related to cardiac metabolism, in particular genes that are coordinated by the transcription factors Pgc1α, Pparα, and Errα. Consistent with the roles of these transcription factors in regulation of mitochondrial genes, we observed significant alterations in mitochondrial size, mitochondrial gene expression, complex activity, and electron transport chain expression under Med1 deficiency. Taken together, these data identify Med1 as an important regulator of vital cardiac gene expression and maintenance of normal heart function. NEW & NOTEWORTHY Disruption of transcriptional gene expression is a hallmark of dilated cardiomyopathy; however, its etiology is not well understood. Cardiac-specific deletion of the transcriptional coactivator mediator subunit 1 (Med1) results in dilated cardiomyopathy, decreased cardiac function, and lethality. Med1 deletion disrupted cardiac mitochondrial and metabolic gene expression patterns. PMID:28159809

Cloning and Characterization of the Scalloped Region of Drosophila Melanogaster

PubMed Central

Campbell, S. D.; Duttaroy, A.; Katzen, A. L.; Chovnick, A.

1991-01-01

Viable mutants of the scalloped gene (sd) of Drosophila melanogaster exhibit defects that can include gapping of the wing margin and ectopic bristle formation on the wing. Lethal sd alleles characterized in the present study now implicate this gene in a genetic function essential for normal development. In order to further characterize the developmental role of this gene, we have undertaken to clone and characterize the region where sd maps. A P[ry(+)] transposon insertion at 13F associated with sd([ry+2216]) served as the starting point for a 42-kb chromosomal walk. Molecular lesions associated with viable and lethal sd alleles were characterized by genomic hybridization analysis as a means of defining the extent of the gene. DNA rearrangements associated with 11 viable sd alleles map to a 2-kb interval which appears to be a ``hot spot'' for P element activity. Four of five recessive lethal sd mutations were mapped by denaturing gradient gel electrophoresis to a region 12-14 kb away from the region of viable lesions. In a sd(+) genotype, at least two structurally related and developmentally regulated transcripts hybridize to the genomic region where several sd lethal alleles have been localized. A viable mutation, sd(58), used for comparison in the transcript analysis, makes at least two slightly smaller transcripts that also hybridize to this region. Preliminary analysis of cDNA clones has identified three structurally related transcripts that hybridize to this genomic region. The 5' end of these transcripts extends into the 2-kb genomic region wherein DNA rearrangements were seen in the P element rearrangements. We favor the view that the transcripts represented by these cDNA clones are products of the sd gene. If this is true, the sd gene would include genomic sequences extending over at least 14 kb of the described chromosomal walk, and would appear to be subject to alternative splicing. PMID:1706292

Genetic Risk by Experience Interaction for Childhood Internalizing Problems: Converging Evidence across Multiple Methods

ERIC Educational Resources Information Center

Vendlinski, Matthew K.; Lemery-Chalfant, Kathryn; Essex, Marilyn J.; Goldsmith, H. Hill

2011-01-01

Background: Identifying how genetic risk interacts with experience to predict psychopathology is an important step toward understanding the etiology of mental health problems. Few studies have examined genetic risk by experience interaction (GxE) in the development of childhood psychopathology. Methods: We used both co-twin and parent mental…

The role of PARP inhibition in triple-negative breast cancer: Unraveling the wide spectrum of synthetic lethality.

PubMed

Papadimitriou, Marios; Mountzios, Giannis; Papadimitriou, Christos A

2018-05-02

Triple-negative breast cancer (TNBC) accounts for approximately 15-20% of all breast cancers and is characterized by a lack of immunohistochemical expression of estrogen receptors (ER), progesterone receptors (PR) and HER2. TNBC is associated with poor long-term outcomes compared with other breast cancer subtypes. Many of these tumors are also basal-like cancers which are characterized by an aggressive biological behavior with a distant recurrence peak observed early at 3 years following diagnosis. Furthermore, metastatic TNBC bears a dismal prognosis with an average survival of 12 months. Although the prevalence of genetic alterations among women with TNBC differs significantly by ethnicity, race and age, BRCA mutations (including both germline mutations and somatic genetic aberrations) are found in up to 20-25% of unselected patients and especially in those of the basal-like immunophenotype. Therefore, defects in the DNA repair pathway could represent a promising therapeutic target for this subgroup of TNBC patients. Poly(ADP-ribose) polymerase (PARP) inhibitors exploit this deficiency through synthetic lethality and have emerged as promising anticancer therapies, especially in BRCA1 or BRCA2 mutation carriers. Several PARP inhibitors are currently being evaluated in the adjuvant, neo-adjuvant, and metastatic setting for the treatment of breast cancer patients with a deficient homologous recombination pathway. In this article, we review the major molecular characteristics of TNBC, the mechanisms of homologous recombination, and the role of PARP inhibition as an emerging therapeutic strategy. Copyright © 2018 Elsevier Ltd. All rights reserved.

Parallel universes of Black Six biology.

PubMed

Kraev, Alexander

2014-07-19

Creation of lethal and synthetic lethal mutations in an experimental organism is a cornerstone of genetic dissection of gene function, and is related to the concept of an essential gene. Common inbred mouse strains carry background mutations, which can act as genetic modifiers, interfering with the assignment of gene essentiality. The inbred strain C57BL/6J, commonly known as "Black Six", stands out, as it carries a spontaneous homozygous deletion in the nicotinamide nucleotide transhydrogenase (Nnt) gene [GenBank: AH009385.2], resulting in impairment of steroidogenic mitochondria of the adrenal gland, and a multitude of indirect modifier effects, coming from alteration of glucocorticoid-regulated processes. Over time, the popular strain has been used, by means of gene targeting technology, to assign "essential" and "redundant" qualifiers to numerous genes, thus creating an internally consistent "parallel universe" of knowledge. It is unrealistic to suggest phasing-out of this strain, given the scope of shared resources built around it, however, continuing on the road of "strain-unawareness" will result in profound waste of effort, particularly where translational research is concerned. The review analyzes the historical roots of this phenomenon and proposes that building of "parallel universes" should be urgently made visible to a critical reader by obligatory use of unambiguous and persistent tags in publications and databases, such as hypertext links, pointing to a vendor's strain description web page, or to a digital object identifier (d.o.i.) of the original publication, so that any research done exclusively in C57BL/6J, could be easily identified. This article was reviewed by Dr. Neil Smalheiser and Dr. Miguel Andrade-Navarro.

Genetic characterization of the role of the Cip/Kip family of proteins as cyclin-dependent kinase inhibitors and assembly factors.

PubMed

Cerqueira, Antonio; Martín, Alberto; Symonds, Catherine E; Odajima, Junko; Dubus, Pierre; Barbacid, Mariano; Santamaría, David

2014-04-01

The Cip/Kip family, namely, p21(Cip1), p27(Kip1), and p57(Kip2), are stoichiometric cyclin-dependent kinase inhibitors (CKIs). Paradoxically, they have been proposed to also act as positive regulators of Cdk4/6-cyclin D by stabilizing these heterodimers. Loss of p21(Cip1) and p27(Kip1) reduces Cdk4/6-cyclin D complexes, although with limited phenotypic consequences compared to the embryonic lethality of Cdk4/6 or triple cyclin D deficiency. This milder phenotype was attributed to Cdk2 compensatory mechanisms. To address this controversy using a genetic approach, we generated Cdk2(-/-) p21(-/-) p27(-/-) mice. Triple-knockout mouse embryonic fibroblasts (MEFs) displayed minimal levels of D-type cyclins and Cdk4/6-cyclin D complexes. p57(Kip2) downregulation in the absence of p21(Cip1) and p27(Kip1) aggravated this phenotype, yet MEFs lacking all Cip/Kip proteins exhibited increased retinoblastoma phosphorylation, together with enhanced proliferation and transformation capacity. In vivo, Cdk2 ablation induced partial perinatal lethality in p21(-/-) p27(-/-) mice, suggesting partial Cdk2-dependent compensation. However, Cdk2(-/-) p21(-/-) p27(-/-) survivors displayed all phenotypes described for p27(-/-) mice, including organomegalia and pituitary tumors. Thus, Cip/Kip deficiency does not impair interphasic Cdk activity even in the absence of Cdk2, suggesting that their Cdk-cyclin assembly function is dispensable for homeostatic control in most cell types.

Jmjd5 functions as a regulator of p53 signaling during mouse embryogenesis.

PubMed

Ishimura, Akihiko; Terashima, Minoru; Tange, Shoichiro; Suzuki, Takeshi

2016-03-01

Genetic studies have shown that aberrant activation of p53 signaling leads to embryonic lethality. Maintenance of a fine balance of the p53 protein level is critical for normal development. Previously, we have reported that Jmjd5, a member of the Jumonji C (JmjC) family, regulates embryonic cell proliferation through the control of Cdkn1a expression. Since Cdkn1a is the representative p53-regulated gene, we have examined whether the expression of other p53 target genes is coincidentally upregulated with Cdkn1a in Jmjd5-deficient embryos. The expression of a subset of p53-regulated genes was increased in both Jmjd5 hypomorphic mouse embryonic fibroblasts (MEFs) and Jmjd5-deficient embryos at embryonic day 8.25 without the induced expression of Trp53. Intercrossing of Jmjd5-deficient mice with Trp53 knockout mice showed that the growth defect of Jmjd5 mutant cells was significantly recovered under a Trp53 null genetic background. Chromatin immunoprecipitation analysis in Jmjd5 hypomorphic MEFs indicated the increased recruitment of p53 at several p53 target gene loci, such as Cdkn1a, Pmaip1, and Mdm2. These results suggest that Jmjd5 is involved in the transcriptional regulation of a subset of p53-regulated genes, possibly through the control of p53 recruitment at the gene loci. In Jmjd5-deficient embryos, the enhanced recruitment of p53 might result in the abnormal activation of p53 signaling leading to embryonic lethality.

Global Nav1.7 Knockout Mice Recapitulate the Phenotype of Human Congenital Indifference to Pain

PubMed Central

Gingras, Jacinthe; Smith, Sarah; Matson, David J.; Johnson, Danielle; Nye, Kim; Couture, Lauren; Feric, Elma; Yin, Ruoyuan; Moyer, Bryan D.; Peterson, Matthew L.; Rottman, James B.; Beiler, Rudolph J.; Malmberg, Annika B.; McDonough, Stefan I.

2014-01-01

Clinical genetic studies have shown that loss of Nav1.7 function leads to the complete loss of acute pain perception. The global deletion is reported lethal in mice, however, and studies of mice with promoter-specific deletions of Nav1.7 have suggested that the role of Nav1.7 in pain transduction depends on the precise form of pain. We developed genetic and animal husbandry strategies that overcame the neonatal-lethal phenotype and enabled construction of a global Nav1.7 knockout mouse. Knockouts were anatomically normal, reached adulthood, and had phenotype wholly analogous to human congenital indifference to pain (CIP): compared to littermates, knockouts showed no defects in mechanical sensitivity or overall movement yet were completely insensitive to painful tactile, thermal, and chemical stimuli and were anosmic. Knockouts also showed no painful behaviors resulting from peripheral injection of nonselective sodium channel activators, did not develop complete Freund’s adjuvant-induced thermal hyperalgesia, and were insensitive to intra-dermal histamine injection. Tetrodotoxin-sensitive sodium current recorded from cell bodies of isolated sensory neurons and the mechanically-evoked spiking of C-fibers in a skin-nerve preparation each were reduced but not eliminated in tissue from knockouts compared to littermates. Results support a role for Nav1.7 that is conserved between rodents and humans and suggest several possibly translatable biomarkers for the study of Nav1.7-targeted therapeutics. Results further suggest that Nav1.7 may retain its key role in persistent as well as acute forms of pain. PMID:25188265

Analysis of genetic effects of nuclear-cytoplasmic interaction on quantitative traits: genetic model for diploid plants.

PubMed

Han, Lide; Yang, Jian; Zhu, Jun

2007-06-01

A genetic model was proposed for simultaneously analyzing genetic effects of nuclear, cytoplasm, and nuclear-cytoplasmic interaction (NCI) as well as their genotype by environment (GE) interaction for quantitative traits of diploid plants. In the model, the NCI effects were further partitioned into additive and dominance nuclear-cytoplasmic interaction components. Mixed linear model approaches were used for statistical analysis. On the basis of diallel cross designs, Monte Carlo simulations showed that the genetic model was robust for estimating variance components under several situations without specific effects. Random genetic effects were predicted by an adjusted unbiased prediction (AUP) method. Data on four quantitative traits (boll number, lint percentage, fiber length, and micronaire) in Upland cotton (Gossypium hirsutum L.) were analyzed as a worked example to show the effectiveness of the model.

Novel Burkholderia mallei Virulence Factors Linked to Specific Host-Pathogen Protein Interactions*

PubMed Central

Memišević, Vesna; Zavaljevski, Nela; Pieper, Rembert; Rajagopala, Seesandra V.; Kwon, Keehwan; Townsend, Katherine; Yu, Chenggang; Yu, Xueping; DeShazer, David; Reifman, Jaques; Wallqvist, Anders

2013-01-01

Burkholderia mallei is an infectious intracellular pathogen whose virulence and resistance to antibiotics makes it a potential bioterrorism agent. Given its genetic origin as a commensal soil organism, it is equipped with an extensive and varied set of adapted mechanisms to cope with and modulate host-cell environments. One essential virulence mechanism constitutes the specialized secretion systems that are designed to penetrate host-cell membranes and insert pathogen proteins directly into the host cell's cytosol. However, the secretion systems' proteins and, in particular, their host targets are largely uncharacterized. Here, we used a combined in silico, in vitro, and in vivo approach to identify B. mallei proteins required for pathogenicity. We used bioinformatics tools, including orthology detection and ab initio predictions of secretion system proteins, as well as published experimental Burkholderia data to initially select a small number of proteins as putative virulence factors. We then used yeast two-hybrid assays against normalized whole human and whole murine proteome libraries to detect and identify interactions among each of these bacterial proteins and host proteins. Analysis of such interactions provided both verification of known virulence factors and identification of three new putative virulence proteins. We successfully created insertion mutants for each of these three proteins using the virulent B. mallei ATCC 23344 strain. We exposed BALB/c mice to mutant strains and the wild-type strain in an aerosol challenge model using lethal B. mallei doses. In each set of experiments, mice exposed to mutant strains survived for the 21-day duration of the experiment, whereas mice exposed to the wild-type strain rapidly died. Given their in vivo role in pathogenicity, and based on the yeast two-hybrid interaction data, these results point to the importance of these pathogen proteins in modulating host ubiquitination pathways, phagosomal escape, and actin-cytoskeleton rearrangement processes. PMID:23800426

TheCellMap.org: A Web-Accessible Database for Visualizing and Mining the Global Yeast Genetic Interaction Network

PubMed Central

Usaj, Matej; Tan, Yizhao; Wang, Wen; VanderSluis, Benjamin; Zou, Albert; Myers, Chad L.; Costanzo, Michael; Andrews, Brenda; Boone, Charles

2017-01-01

Providing access to quantitative genomic data is key to ensure large-scale data validation and promote new discoveries. TheCellMap.org serves as a central repository for storing and analyzing quantitative genetic interaction data produced by genome-scale Synthetic Genetic Array (SGA) experiments with the budding yeast Saccharomyces cerevisiae. In particular, TheCellMap.org allows users to easily access, visualize, explore, and functionally annotate genetic interactions, or to extract and reorganize subnetworks, using data-driven network layouts in an intuitive and interactive manner. PMID:28325812

TheCellMap.org: A Web-Accessible Database for Visualizing and Mining the Global Yeast Genetic Interaction Network.

PubMed

Usaj, Matej; Tan, Yizhao; Wang, Wen; VanderSluis, Benjamin; Zou, Albert; Myers, Chad L; Costanzo, Michael; Andrews, Brenda; Boone, Charles

2017-05-05

Providing access to quantitative genomic data is key to ensure large-scale data validation and promote new discoveries. TheCellMap.org serves as a central repository for storing and analyzing quantitative genetic interaction data produced by genome-scale Synthetic Genetic Array (SGA) experiments with the budding yeast Saccharomyces cerevisiae In particular, TheCellMap.org allows users to easily access, visualize, explore, and functionally annotate genetic interactions, or to extract and reorganize subnetworks, using data-driven network layouts in an intuitive and interactive manner. Copyright © 2017 Usaj et al.