Browsing by Subject "Mutation"

Now showing 1 - 20 of 137

Open Access
A Bayesian Approach to Inferring Rates of Selfing and Locus-Specific Mutation.
(Genetics, 2015-11) Redelings, Benjamin D; Kumagai, Seiji; Tatarenkov, Andrey; Wang, Liuyang; Sakai, Ann K; Weller, Stephen G; Culley, Theresa M; Avise, John C; Uyenoyama, Marcy K
We present a Bayesian method for characterizing the mating system of populations reproducing through a mixture of self-fertilization and random outcrossing. Our method uses patterns of genetic variation across the genome as a basis for inference about reproduction under pure hermaphroditism, gynodioecy, and a model developed to describe the self-fertilizing killifish Kryptolebias marmoratus. We extend the standard coalescence model to accommodate these mating systems, accounting explicitly for multilocus identity disequilibrium, inbreeding depression, and variation in fertility among mating types. We incorporate the Ewens sampling formula (ESF) under the infinite-alleles model of mutation to obtain a novel expression for the likelihood of mating system parameters. Our Markov chain Monte Carlo (MCMC) algorithm assigns locus-specific mutation rates, drawn from a common mutation rate distribution that is itself estimated from the data using a Dirichlet process prior model. Our sampler is designed to accommodate additional information, including observations pertaining to the sex ratio, the intensity of inbreeding depression, and other aspects of reproduction. It can provide joint posterior distributions for the population-wide proportion of uniparental individuals, locus-specific mutation rates, and the number of generations since the most recent outcrossing event for each sampled individual. Further, estimation of all basic parameters of a given model permits estimation of functions of those parameters, including the proportion of the gene pool contributed by each sex and relative effective numbers.
Open Access
A cell-based multiplex immunoassay platform using fluorescent protein-barcoded reporter cell lines.
(Communications biology, 2021-11) Song, Shengli; Manook, Miriam; Kwun, Jean; Jackson, Annette M; Knechtle, Stuart J; Kelsoe, Garnett
Multiplex immunoassays with acellular antigens are well-established based on solid-phase platforms such as the Luminex^® technology. Cell barcoding by amine-reactive fluorescent dyes enables analogous cell-based multiplex assays, but requires multiple labeling reactions and quality checks prior to every assay. Here we describe generation of stable, fluorescent protein-barcoded reporter cell lines suitable for multiplex screening of antibody to membrane proteins. The utility of this cell-based system, with the potential of a 256-plex cell panel, is demonstrated by flow cytometry deconvolution of barcoded cell panels expressing influenza A hemagglutinin trimers, or native human CCR2 or CCR5 multi-span proteins and their epitope-defining mutants. This platform will prove useful for characterizing immunity and discovering antibodies to membrane-associated proteins.
Open Access
A cerebellar learning model of vestibulo-ocular reflex adaptation in wild-type and mutant mice.
(The Journal of neuroscience : the official journal of the Society for Neuroscience, 2014-05) Clopath, Claudia; Badura, Aleksandra; De Zeeuw, Chris I; Brunel, Nicolas
Mechanisms of cerebellar motor learning are still poorly understood. The standard Marr-Albus-Ito theory posits that learning involves plasticity at the parallel fiber to Purkinje cell synapses under control of the climbing fiber input, which provides an error signal as in classical supervised learning paradigms. However, a growing body of evidence challenges this theory, in that additional sites of plasticity appear to contribute to motor adaptation. Here, we consider phase-reversal training of the vestibulo-ocular reflex (VOR), a simple form of motor learning for which a large body of experimental data is available in wild-type and mutant mice, in which the excitability of granule cells or inhibition of Purkinje cells was affected in a cell-specific fashion. We present novel electrophysiological recordings of Purkinje cell activity measured in naive wild-type mice subjected to this VOR adaptation task. We then introduce a minimal model that consists of learning at the parallel fibers to Purkinje cells with the help of the climbing fibers. Although the minimal model reproduces the behavior of the wild-type animals and is analytically tractable, it fails at reproducing the behavior of mutant mice and the electrophysiology data. Therefore, we build a detailed model involving plasticity at the parallel fibers to Purkinje cells' synapse guided by climbing fibers, feedforward inhibition of Purkinje cells, and plasticity at the mossy fiber to vestibular nuclei neuron synapse. The detailed model reproduces both the behavioral and electrophysiological data of both the wild-type and mutant mice and allows for experimentally testable predictions.
Open Access
A constitutively active mutant beta 2-adrenergic receptor is constitutively desensitized and phosphorylated.
(Proc Natl Acad Sci U S A, 1994-03-29) Pei, G; Samama, P; Lohse, M; Wang, M; Codina, J; Lefkowitz, RJ
The beta 2-adrenergic receptor (beta 2AR) can be constitutively activated by mutations in the third intracellular loop. Whereas the wild-type receptor exists predominantly in an inactive conformation (R) in the absence of agonist, the mutant receptor appears to spontaneously adopt an active conformation (R*). We now demonstrate that not only is the mutant beta 2AR constitutively active, it is also constitutively desensitized and down-regulated. To assess whether the mutant receptor can constitutively engage a known element of the cellular desensitization machinery, the receptor was purified and reconstituted into phospholipid vesicles. These preparations retained the essential properties of the constitutively active mutant receptor: agonist-independent activity [to stimulate guanine nucleotide-binding protein (Gs)-GTPase] and agonist-specific increase in binding affinity. Moreover, the purified mutant receptor, in the absence of agonist, was phosphorylated by recombinant beta AR-specific kinase (beta ARK) in a fashion comparable to the agonist-occupied wild-type receptor. Thus, the conformation of the mutated receptor is equivalent to the active conformation (R*), which stimulates Gs protein and is identical to the beta ARK substrate.
Open Access
A curative combination cancer therapy achieves high fractional cell killing through low cross-resistance and drug additivity.
(eLife, 2019-11) Palmer, Adam C; Chidley, Christopher; Sorger, Peter K
Curative cancer therapies are uncommon and nearly always involve multi-drug combinations developed by experimentation in humans; unfortunately, the mechanistic basis for the success of such combinations has rarely been investigated in detail, obscuring lessons learned. Here, we use isobologram analysis to score pharmacological interaction, and clone tracing and CRISPR screening to measure cross-resistance among the five drugs comprising R-CHOP, a combination therapy that frequently cures Diffuse Large B-Cell Lymphomas. We find that drugs in R-CHOP exhibit very low cross-resistance but not synergistic interaction: together they achieve a greater fractional kill according to the null hypothesis for both the Loewe dose-additivity model and the Bliss effect-independence model. These data provide direct evidence for the 50 year old hypothesis that a curative cancer therapy can be constructed on the basis of independently effective drugs having non-overlapping mechanisms of resistance, without synergistic interaction, which has immediate significance for the design of new drug combinations.
Open Access
A Genetic Mosaic Screen Reveals Ecdysone-Responsive Genes Regulating Drosophila Oogenesis.
(G3 (Bethesda, Md.), 2016-08) Ables, Elizabeth T; Hwang, Grace H; Finger, Danielle S; Hinnant, Taylor D; Drummond-Barbosa, Daniela
Multiple aspects of Drosophila oogenesis, including germline stem cell activity, germ cell differentiation, and follicle survival, are regulated by the steroid hormone ecdysone. While the transcriptional targets of ecdysone signaling during development have been studied extensively, targets in the ovary remain largely unknown. Early studies of salivary gland polytene chromosomes led to a model in which ecdysone stimulates a hierarchical transcriptional cascade, wherein a core group of ecdysone-sensitive transcription factors induce tissue-specific responses by activating secondary branches of transcriptional targets. More recently, genome-wide approaches have identified hundreds of putative ecdysone-responsive targets. Determining whether these putative targets represent bona fide targets in vivo, however, requires that they be tested via traditional mutant analysis in a cell-type specific fashion. To investigate the molecular mechanisms whereby ecdysone signaling regulates oogenesis, we used genetic mosaic analysis to screen putative ecdysone-responsive genes for novel roles in the control of the earliest steps of oogenesis. We identified a cohort of genes required for stem cell maintenance, stem and progenitor cell proliferation, and follicle encapsulation, growth, and survival. These genes encode transcription factors, chromatin modulators, and factors required for RNA transport, stability, and ribosome biogenesis, suggesting that ecdysone might control a wide range of molecular processes during oogenesis. Our results suggest that, although ecdysone target genes are known to have cell type-specific roles, many ecdysone response genes that control larval or pupal cell types at developmental transitions are used reiteratively in the adult ovary. These results provide novel insights into the molecular mechanisms by which ecdysone signaling controls oogenesis, laying new ground for future studies.
Open Access
A genetic screen reveals Arabidopsis stomatal and/or apoplastic defenses against Pseudomonas syringae pv. tomato DC3000.
(PLoS pathogens, 2011-10-06) Zeng, Weiqing; Brutus, Alexandre; Kremer, James M; Withers, John C; Gao, Xiaoli; Jones, A Daniel; He, Sheng Yang
Bacterial infection of plants often begins with colonization of the plant surface, followed by entry into the plant through wounds and natural openings (such as stomata), multiplication in the intercellular space (apoplast) of the infected tissues, and dissemination of bacteria to other plants. Historically, most studies assess bacterial infection based on final outcomes of disease and/or pathogen growth using whole infected tissues; few studies have genetically distinguished the contribution of different host cell types in response to an infection. The phytotoxin coronatine (COR) is produced by several pathovars of Pseudomonas syringae. COR-deficient mutants of P. s. tomato (Pst) DC3000 are severely compromised in virulence, especially when inoculated onto the plant surface. We report here a genetic screen to identify Arabidopsis mutants that could rescue the virulence of COR-deficient mutant bacteria. Among the susceptible to coronatine-deficient Pst DC3000 (scord) mutants were two that were defective in stomatal closure response, two that were defective in apoplast defense, and four that were defective in both stomatal and apoplast defense. Isolation of these three classes of mutants suggests that stomatal and apoplastic defenses are integrated in plants, but are genetically separable, and that COR is important for Pst DC3000 to overcome both stomatal guard cell- and apoplastic mesophyll cell-based defenses. Of the six mutants defective in bacterium-triggered stomatal closure, three are defective in salicylic acid (SA)-induced stomatal closure, but exhibit normal stomatal closure in response to abscisic acid (ABA), and scord7 is compromised in both SA- and ABA-induced stomatal closure. We have cloned SCORD3, which is required for salicylic acid (SA) biosynthesis, and SCORD5, which encodes an ATP-binding cassette (ABC) protein, AtGCN20/AtABCF3, predicted to be involved in stress-associated protein translation control. Identification of SCORD5 begins to implicate an important role of stress-associated protein translation in stomatal guard cell signaling in response to microbe-associated molecular patterns and bacterial infection.
Open Access
A kinesin motor in a force-producing conformation.
(BMC Struct Biol, 2010-07-05) Heuston, Elisabeth; Bronner, C Eric; Kull, F Jon; Endow, Sharyn A
BACKGROUND: Kinesin motors hydrolyze ATP to produce force and move along microtubules, converting chemical energy into work by a mechanism that is only poorly understood. Key transitions and intermediate states in the process are still structurally uncharacterized, and remain outstanding questions in the field. Perturbing the motor by introducing point mutations could stabilize transitional or unstable states, providing critical information about these rarer states. RESULTS: Here we show that mutation of a single residue in the kinesin-14 Ncd causes the motor to release ADP and hydrolyze ATP faster than wild type, but move more slowly along microtubules in gliding assays, uncoupling nucleotide hydrolysis from force generation. A crystal structure of the motor shows a large rotation of the stalk, a conformation representing a force-producing stroke of Ncd. Three C-terminal residues of Ncd, visible for the first time, interact with the central beta-sheet and dock onto the motor core, forming a structure resembling the kinesin-1 neck linker, which has been proposed to be the primary force-generating mechanical element of kinesin-1. CONCLUSIONS: Force generation by minus-end Ncd involves docking of the C-terminus, which forms a structure resembling the kinesin-1 neck linker. The mechanism by which the plus- and minus-end motors produce force to move to opposite ends of the microtubule appears to involve the same conformational changes, but distinct structural linkers. Unstable ADP binding may destabilize the motor-ADP state, triggering Ncd stalk rotation and C-terminus docking, producing a working stroke of the motor.
Open Access
A mutation in TNNC1-encoded cardiac troponin C, TNNC1-A31S, predisposes to hypertrophic cardiomyopathy and ventricular fibrillation.
(The Journal of biological chemistry, 2012-09) Parvatiyar, MS; Landstrom, AP; Figueiredo-Freitas, C; Potter, JD; Ackerman, MJ; Pinto, JR
Defined as clinically unexplained hypertrophy of the left ventricle, hypertrophic cardiomyopathy (HCM) is traditionally understood as a disease of the cardiac sarcomere. Mutations in TNNC1-encoded cardiac troponin C (cTnC) are a relatively rare cause of HCM. Here, we report clinical and functional characterization of a novel TNNC1 mutation, A31S, identified in a pediatric HCM proband with multiple episodes of ventricular fibrillation and aborted sudden cardiac death. Diagnosed at age 5, the proband is family history-negative for HCM or sudden cardiac death, suggesting a de novo mutation. TnC-extracted cardiac skinned fibers were reconstituted with the cTnC-A31S mutant, which increased Ca(2+) sensitivity with no effect on the maximal contractile force generation. Reconstituted actomyosin ATPase assays with 50% cTnC-A31S:50% cTnC-WT demonstrated Ca(2+) sensitivity that was intermediate between 100% cTnC-A31S and 100% cTnC-WT, whereas the mutant increased the activation of the actomyosin ATPase without affecting the inhibitory qualities of the ATPase. The secondary structure of the cTnC mutant was evaluated by circular dichroism, which did not indicate global changes in structure. Fluorescence studies demonstrated increased Ca(2+) affinity in isolated cTnC, the troponin complex, thin filament, and to a lesser degree, thin filament with myosin subfragment 1. These results suggest that this mutation has a direct effect on the Ca(2+) sensitivity of the myofilament, which may alter Ca(2+) handling and contribute to the arrhythmogenesis observed in the proband. In summary, we report a novel mutation in the TNNC1 gene that is associated with HCM pathogenesis and may predispose to the pathogenesis of a fatal arrhythmogenic subtype of HCM.
Open Access
A novel mutation of the ACADM gene (c.145C>G) associated with the common c.985A>G mutation on the other ACADM allele causes mild MCAD deficiency: a case report.
(Orphanet J Rare Dis, 2010-10-05) Dessein, Anne-Frédérique; Fontaine, Monique; Andresen, Brage S; Gregersen, Niels; Brivet, Michèle; Rabier, Daniel; Napuri-Gouel, Silvia; Dobbelaere, Dries; Mention-Mulliez, Karine; Martin-Ponthieu, Annie; Briand, Gilbert; Millington, David S; Vianey-Saban, Christine; Wanders, Ronald JA; Vamecq, Joseph
A female patient, with normal familial history, developed at the age of 30 months an episode of diarrhoea, vomiting and lethargy which resolved spontaneously. At the age of 3 years, the patient re-iterated vomiting, was sub-febrile and hypoglycemic, fell into coma, developed seizures and sequels involving right hemi-body. Urinary excretion of hexanoylglycine and suberylglycine was low during this metabolic decompensation. A study of pre- and post-prandial blood glucose and ketones over a period of 24 hours showed a normal glycaemic cycle but a failure to form ketones after 12 hours fasting, suggesting a mitochondrial β-oxidation defect. Total blood carnitine was lowered with unesterified carnitine being half of the lowest control value. A diagnosis of mild MCAD deficiency (MCADD) was based on rates of 1-14C-octanoate and 9, 10-3H-myristate oxidation and of octanoyl-CoA dehydrogenase being reduced to 25% of control values. Other mitochondrial fatty acid oxidation proteins were functionally normal. De novo acylcarnitine synthesis in whole blood samples incubated with deuterated palmitate was also typical of MCADD. Genetic studies showed that the patient was compound heterozygous with a sequence variation in both of the two ACADM alleles; one had the common c.985A>G mutation and the other had a novel c.145C>G mutation. This is the first report for the ACADM gene c.145C>G mutation: it is located in exon 3 and causes a replacement of glutamine to glutamate at position 24 of the mature protein (Q24E). Associated with heterozygosity for c.985A>G mutation, this mutation is responsible for a mild MCADD phenotype along with a clinical story corroborating the emerging literature view that patients with genotypes representing mild MCADD (high residual enzyme activity and low urinary levels of glycine conjugates), similar to some of the mild MCADDs detected by MS/MS newborn screening, may be at risk for disease presentation.
Open Access
A plant genetic network for preventing dysbiosis in the phyllosphere.
(Nature, 2020-04-08) Chen, Tao; Nomura, Kinya; Wang, Xiaolin; Sohrabi, Reza; Xu, Jin; Yao, Lingya; Paasch, Bradley C; Ma, Li; Kremer, James; Cheng, Yuti; Zhang, Li; Wang, Nian; Wang, Ertao; Xin, Xiu-Fang; He, Sheng Yang
The aboveground parts of terrestrial plants, collectively called the phyllosphere, have a key role in the global balance of atmospheric carbon dioxide and oxygen. The phyllosphere represents one of the most abundant habitats for microbiota colonization. Whether and how plants control phyllosphere microbiota to ensure plant health is not well understood. Here we show that the Arabidopsis quadruple mutant (min7 fls2 efr cerk1; hereafter, mfec)1, simultaneously defective in pattern-triggered immunity and the MIN7 vesicle-trafficking pathway, or a constitutively activated cell death1 (cad1) mutant, carrying a S205F mutation in a membrane-attack-complex/perforin (MACPF)-domain protein, harbour altered endophytic phyllosphere microbiota and display leaf-tissue damage associated with dysbiosis. The Shannon diversity index and the relative abundance of Firmicutes were markedly reduced, whereas Proteobacteria were enriched in the mfec and cad1S205F mutants, bearing cross-kingdom resemblance to some aspects of the dysbiosis that occurs in human inflammatory bowel disease. Bacterial community transplantation experiments demonstrated a causal role of a properly assembled leaf bacterial community in phyllosphere health. Pattern-triggered immune signalling, MIN7 and CAD1 are found in major land plant lineages and are probably key components of a genetic network through which terrestrial plants control the level and nurture the diversity of endophytic phyllosphere microbiota for survival and health in a microorganism-rich environment.
Open Access
ADAP2 Is an Interferon Stimulated Gene That Restricts RNA Virus Entry.
(PLoS pathogens, 2015-09-15) Shu, Qian; Lennemann, Nicholas J; Sarkar, Saumendra N; Sadovsky, Yoel; Coyne, Carolyn B
Interferon stimulated genes (ISGs) target viruses at various stages of their infectious life cycles, including at the earliest stage of viral entry. Here we identify ArfGAP with dual pleckstrin homology (PH) domains 2 (ADAP2) as a gene upregulated by type I IFN treatment in a STAT1-dependent manner. ADAP2 functions as a GTPase-activating protein (GAP) for Arf6 and binds to phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) and PI(3,4)P2. We show that overexpression of ADAP2 suppresses dengue virus (DENV) and vesicular stomatitis virus (VSV) infection in an Arf6 GAP activity-dependent manner, while exerting no effect on coxsackievirus B (CVB) or Sendai virus (SeV) replication. We further show that ADAP2 expression induces macropinocytosis and that ADAP2 strongly associates with actin-enriched membrane ruffles and with Rab8a- and LAMP1-, but not EEA1- or Rab7-, positive vesicles. Utilizing two techniques--light-sensitive neutral red (NR)-containing DENV and fluorescence assays for virus internalization--we show that ADAP2 primarily restricts DENV infection at the stage of virion entry and/or intracellular trafficking and that incoming DENV and VSV particles associate with ADAP2 during their entry. Taken together, this study identifies ADAP2 as an ISG that exerts antiviral effects against RNA viruses by altering Arf6-mediated trafficking to disrupt viral entry.
Open Access
Afatinib induces apoptosis in NSCLC without EGFR mutation through Elk-1-mediated suppression of CIP2A.
(Oncotarget, 2015-02) Chao, Ting-Ting; Wang, Cheng-Yi; Chen, Yen-Lin; Lai, Chih-Cheng; Chang, Fang-Yu; Tsai, Yi-Ting; Chao, Chung-Hao H; Shiau, Chung-Wai; Huang, Yuh-Chin T; Yu, Chong-Jen; Chen, Kuen-Feng
Afatinib has anti-tumor effect in non-small cell lung carcinoma (NSCLC) with epidermal growth factor receptor (EGFR) mutation. We found afatinib can also induce apoptosis in NSCLC cells without EGFR mutation through CIP2A pathway. Four NSCLC cell lines (H358 H441 H460 and A549) were treated with afatinib to determine their sensitivity to afatinib-induced cell death and apoptosis. The effects of CIP2A on afatinib-induced apoptosis were confirmed by overexpression and knockdown of CIP2A expression in the sensitive and resistant cells, respectively. Reduction of Elk-1 binding to the CIP2A promoter and suppression of CIP2A transcription were analyzed. In vivo efficacy of afatinib against H358 and H460 xenografts tumors were also determined in nude mice. Afatinib induced significant cell death and apoptosis in H358 and H441 cells, but not in H460 or A549 cells. The apoptotic effect of afatinib in sensitive cells was associated with downregulation of CIP2A, promotion of PP2A activity and decrease in AKT phosphorylation. Afatinib suppressed CIP2A at the gene transcription level by reducing the promoter binding activity of Elk-1. Clinical samples showed that higher CIP2A expression predicted a poor prognosis and Elk-1 and CIP2A expressions were highly correlated. In conclusion, afatinib induces apoptosis in NSCLC without EGFR mutations through Elk-1/CIP2A/PP2A/AKT pathway.
Open Access
Amino acid-level signal-to-noise analysis of incidentally identified variants in genes associated with long QT syndrome during pediatric whole exome sequencing reflects background genetic noise.
(Heart rhythm, 2018-07) Landstrom, Andrew P; Fernandez, Ernesto; Rosenfeld, Jill A; Yang, Yaping; Dailey-Schwartz, Andrew L; Miyake, Christina Y; Allen, Hugh D; Penny, Daniel J; Kim, Jeffrey J
BACKGROUND:Due to rapid expansion of clinical genetic testing, an increasing number of genetic variants of undetermined significance and unclear diagnostic value are being identified in children. Variants found in genes associated with heritable channelopathies, such as long QT syndrome (LQTS), are particularly difficult to interpret given the risk of sudden cardiac death associated with pathologic mutations. OBJECTIVE:The purpose of this study was to determine whether variants in LQTS-associated genes from whole exome sequencing (WES) represent disease-associated biomarkers or background genetic "noise." METHODS:WES variants from Baylor Genetics Laboratories were obtained for 17 LQTS-associated genes. Rare variants from healthy controls were obtained from the GnomAD database. LQTS case variants were extracted from the literature. Amino acid-level mapping and signal-to-noise calculations were conducted. Clinical history and diagnostic studies were analyzed for WES subjects evaluated at our institution. RESULTS:Variants in LQTS case-associated genes were present in 38.3% of 7244 WES probands. There was a similar frequency of variants in the WES and healthy cohorts for LQTS1-3 (11.2% and 12.9%, respectively) and LQTS4-17 (27.1% and 38.4%, respectively). WES variants preferentially localized to amino acids altered in control individuals compared to cases. Based on amino acid-level analysis, WES-identified variants are indistinguishable from healthy background variation, whereas LQTS1 and 2 case-identified variants localized to clear pathologic "hotspots." No individuals who underwent clinical evaluation had clinical suspicion for LQTS. CONCLUSION:The prevalence of incidentally identified LQTS-associated variants is ∼38% among WES tests. These variants most likely represent benign healthy background genetic variation rather than disease-associated mutations.
Open Access
An immunoglobulin C kappa-reactive single chain antibody fusion protein induces tolerance through receptor editing in a normal polyclonal immune system.
(J Exp Med, 2005-03-07) Ait-Azzouzene, Djemel; Verkoczy, Laurent; Peters, Jorieke; Gavin, Amanda; Skog, Patrick; Vela, José Luis; Nemazee, David
Understanding immune tolerance mechanisms is a major goal of immunology research, but mechanistic studies have generally required the use of mouse models carrying untargeted or targeted antigen receptor transgenes, which distort lymphocyte development and therefore preclude analysis of a truly normal immune system. Here we demonstrate an advance in in vivo analysis of immune tolerance that overcomes these shortcomings. We show that custom superantigens generated by single chain antibody technology permit the study of tolerance in a normal, polyclonal immune system. In the present study we generated a membrane-tethered anti-Igkappa-reactive single chain antibody chimeric gene and expressed it as a transgene in mice. B cell tolerance was directly characterized in the transgenic mice and in radiation bone marrow chimeras in which ligand-bearing mice served as recipients of nontransgenic cells. We find that the ubiquitously expressed, Igkappa-reactive ligand induces efficient B cell tolerance primarily or exclusively by receptor editing. We also demonstrate the unique advantages of our model in the genetic and cellular analysis of immune tolerance.
Open Access
Ancestral population genomics: the coalescent hidden Markov model approach.
(Genetics, 2009-09) Dutheil, Julien Y; Ganapathy, Ganesh; Hobolth, Asger; Mailund, Thomas; Uyenoyama, Marcy K; Schierup, Mikkel H
With incomplete lineage sorting (ILS), the genealogy of closely related species differs along their genomes. The amount of ILS depends on population parameters such as the ancestral effective population sizes and the recombination rate, but also on the number of generations between speciation events. We use a hidden Markov model parameterized according to coalescent theory to infer the genealogy along a four-species genome alignment of closely related species and estimate population parameters. We analyze a basic, panmictic demographic model and study its properties using an extensive set of coalescent simulations. We assess the effect of the model assumptions and demonstrate that the Markov property provides a good approximation to the ancestral recombination graph. Using a too restricted set of possible genealogies, necessary to reduce the computational load, can bias parameter estimates. We propose a simple correction for this bias and suggest directions for future extensions of the model. We show that the patterns of ILS along a sequence alignment can be recovered efficiently together with the ancestral recombination rate. Finally, we introduce an extension of the basic model that allows for mutation rate heterogeneity and reanalyze human-chimpanzee-gorilla-orangutan alignments, using the new models. We expect that this framework will prove useful for population genomics and provide exciting insights into genome evolution.
Open Access
Angiopoietin receptor TEK mutations underlie primary congenital glaucoma with variable expressivity.
(The Journal of clinical investigation, 2016-07) Souma, Tomokazu; Tompson, Stuart W; Thomson, Benjamin R; Siggs, Owen M; Kizhatil, Krishnakumar; Yamaguchi, Shinji; Feng, Liang; Limviphuvadh, Vachiranee; Whisenhunt, Kristina N; Maurer-Stroh, Sebastian; Yanovitch, Tammy L; Kalaydjieva, Luba; Azmanov, Dimitar N; Finzi, Simone; Mauri, Lucia; Javadiyan, Shahrbanou; Souzeau, Emmanuelle; Zhou, Tiger; Hewitt, Alex W; Kloss, Bethany; Burdon, Kathryn P; Mackey, David A; Allen, Keri F; Ruddle, Jonathan B; Lim, Sing-Hui; Rozen, Steve; Tran-Viet, Khanh-Nhat; Liu, Xiaorong; John, Simon; Wiggs, Janey L; Pasutto, Francesca; Craig, Jamie E; Jin, Jing; Quaggin, Susan E; Young, Terri L
Primary congenital glaucoma (PCG) is a devastating eye disease and an important cause of childhood blindness worldwide. In PCG, defects in the anterior chamber aqueous humor outflow structures of the eye result in elevated intraocular pressure (IOP); however, the genes and molecular mechanisms involved in the etiology of these defects have not been fully characterized. Previously, we observed PCG-like phenotypes in transgenic mice that lack functional angiopoietin-TEK signaling. Herein, we identified rare TEK variants in 10 of 189 unrelated PCG families and demonstrated that each mutation results in haploinsufficiency due to protein loss of function. Multiple cellular mechanisms were responsible for the loss of protein function resulting from individual TEK variants, including an absence of normal protein production, protein aggregate formation, enhanced proteasomal degradation, altered subcellular localization, and reduced responsiveness to ligand stimulation. Further, in mice, hemizygosity for Tek led to the formation of severely hypomorphic Schlemm's canal and trabecular meshwork, as well as elevated IOP, demonstrating that anterior chamber vascular development is sensitive to Tek gene dosage and the resulting decrease in angiopoietin-TEK signaling. Collectively, these results identify TEK mutations in patients with PCG that likely underlie disease and are transmitted in an autosomal dominant pattern with variable expressivity.
Open Access
Anti-AIDS agents 81. Design, synthesis, and structure-activity relationship study of betulinic acid and moronic acid derivatives as potent HIV maturation inhibitors.
(J Med Chem, 2010-04-22) Qian, Keduo; Kuo, Reen-Yun; Chen, Chin-Ho; Huang, Li; Morris-Natschke, Susan L; Lee, Kuo-Hsiung
In our continuing study of triterpene derivatives as potent anti-HIV agents, different C-3 conformationally restricted betulinic acid (BA, 1) derivatives were designed and synthesized in order to explore the conformational space of the C-3 pharmacophore. 3-O-Monomethylsuccinyl-betulinic acid (MSB) analogues were also designed to better understand the contribution of the C-3' dimethyl group of bevirimat (2), the first-in-class HIV maturation inhibitor, which is currently in phase IIb clinical trials. In addition, another triterpene skeleton, moronic acid (MA, 3), was also employed to study the influence of the backbone and the C-3 modification toward the anti-HIV activity of this compound class. This study enabled us to better understand the structure-activity relationships (SAR) of triterpene-derived anti-HIV agents and led to the design and synthesis of compound 12 (EC(50): 0.0006 microM), which displayed slightly better activity than 2 as a HIV-1 maturation inhibitor.
Open Access
Anti-phospholipid human monoclonal antibodies inhibit CCR5-tropic HIV-1 and induce beta-chemokines.
(J Exp Med, 2010-04-12) Moody, MA; Liao, HX; Alam, SM; Scearce, RM; Plonk, MK; Kozink, DM; Drinker, MS; Zhang, R; Xia, SM; Sutherland, LL; Tomaras, GD; Giles, IP; Kappes, JC; Ochsenbauer Jambor, C; Edmonds, TG; Soares, M; Barbero, G; Forthal, DN; Landucci, G; Chang, C; King, SW; Kavlie, A; Denny, TN; Hwang, KK; Chen, PP; Thorpe, PE; Montefiori, DC; Haynes, BF
Traditional antibody-mediated neutralization of HIV-1 infection is thought to result from the binding of antibodies to virions, thus preventing virus entry. However, antibodies that broadly neutralize HIV-1 are rare and are not induced by current vaccines. We report that four human anti-phospholipid monoclonal antibodies (mAbs) (PGN632, P1, IS4, and CL1) inhibit HIV-1 CCR5-tropic (R5) primary isolate infection of peripheral blood mononuclear cells (PBMCs) with 80% inhibitory concentrations of <0.02 to approximately 10 microg/ml. Anti-phospholipid mAbs inhibited PBMC HIV-1 infection in vitro by mechanisms involving binding to monocytes and triggering the release of MIP-1alpha and MIP-1beta. The release of these beta-chemokines explains both the specificity for R5 HIV-1 and the activity of these mAbs in PBMC cultures containing both primary lymphocytes and monocytes.
Open Access
ARFGAP1 plays a central role in coupling COPI cargo sorting with vesicle formation.
(J Cell Biol, 2005-01-17) Lee, SY; Yang, JS; Hong, W; Premont, RT; Hsu, VW
Examining how key components of coat protein I (COPI) transport participate in cargo sorting, we find that, instead of ADP ribosylation factor 1 (ARF1), its GTPase-activating protein (GAP) plays a direct role in promoting the binding of cargo proteins by coatomer (the core COPI complex). Activated ARF1 binds selectively to SNARE cargo proteins, with this binding likely to represent at least a mechanism by which activated ARF1 is stabilized on Golgi membrane to propagate its effector functions. We also find that the GAP catalytic activity plays a critical role in the formation of COPI vesicles from Golgi membrane, in contrast to the prevailing view that this activity antagonizes vesicle formation. Together, these findings indicate that GAP plays a central role in coupling cargo sorting and vesicle formation, with implications for simplifying models to describe how these two processes are coupled during COPI transport.