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991.
Soumya Raychaudhuri Robert M. Plenge Elizabeth J. Rossin Aylwin C. Y. Ng International Schizophrenia Consortium Shaun M. Purcell Pamela Sklar Edward M. Scolnick Ramnik J. Xavier David Altshuler Mark J. Daly 《PLoS genetics》2009,5(6)
Translating a set of disease regions into insight about pathogenic mechanisms requires not only the ability to identify the key disease genes within them, but also the biological relationships among those key genes. Here we describe a statistical method, Gene Relationships Among Implicated Loci (GRAIL), that takes a list of disease regions and automatically assesses the degree of relatedness of implicated genes using 250,000 PubMed abstracts. We first evaluated GRAIL by assessing its ability to identify subsets of highly related genes in common pathways from validated lipid and height SNP associations from recent genome-wide studies. We then tested GRAIL, by assessing its ability to separate true disease regions from many false positive disease regions in two separate practical applications in human genetics. First, we took 74 nominally associated Crohn''s disease SNPs and applied GRAIL to identify a subset of 13 SNPs with highly related genes. Of these, ten convincingly validated in follow-up genotyping; genotyping results for the remaining three were inconclusive. Next, we applied GRAIL to 165 rare deletion events seen in schizophrenia cases (less than one-third of which are contributing to disease risk). We demonstrate that GRAIL is able to identify a subset of 16 deletions containing highly related genes; many of these genes are expressed in the central nervous system and play a role in neuronal synapses. GRAIL offers a statistically robust approach to identifying functionally related genes from across multiple disease regions—that likely represent key disease pathways. An online version of this method is available for public use (http://www.broad.mit.edu/mpg/grail/). 相似文献
992.
F. Joseph Pollock Pamela J. Morris Bette L. Willis David G. Bourne 《Applied and environmental microbiology》2010,76(15):5282-5286
A real-time quantitative PCR-based detection assay targeting the dnaJ gene (encoding heat shock protein 40) of the coral pathogen Vibrio coralliilyticus was developed. The assay is sensitive, detecting as little as 1 CFU per ml in seawater and 104 CFU per cm2 of coral tissue. Moreover, inhibition by DNA and cells derived from bacteria other than V. coralliilyticus was minimal. This assay represents a novel approach to coral disease diagnosis that will advance the field of coral disease research.Vibrio coralliilyticus has recently emerged as a coral pathogen of concern on reefs throughout the Indo-Pacific. It was first implicated as the etiological agent responsible for bleaching and tissue lysis of the coral Pocillopora damicornis on Zanzibar reefs (2). More recently, V. coralliilyticus has been identified as the causative agent of white syndrome (WS) outbreaks on several Pacific reefs (14). WS is a collective term describing coral diseases characterized by a spreading band of tissue loss exposing white skeleton on Indo-Pacific scleractinian corals (16). V. coralliilyticus is an emerging model pathogen for understanding the mechanisms linking bacterial infection and coral disease (13) and therefore provides an ideal model for the development of diagnostic assays to detect coral disease. Current coral disease diagnostic methods, which are based primarily upon field-based observations of macroscopic disease signs, often detect disease only at the latest stages of infection, when control measures are least effective. The development of diagnostic tools targeting pathogens underlying coral disease pathologies may provide early indications of infection, aid the identification of disease vectors and reservoirs, and assist managers in developing strategies to prevent the spread of coral disease outbreaks. In this paper, we describe the development and validation of a TaqMan-based real-time quantitative PCR (qPCR) assay that targets a segment of the V. coralliilyticus heat shock protein 40-encoding gene (dnaJ).Nucleotide sequences of the dnaJ gene were retrieved from relevant Vibrio species, including V. coralliilyticus (LMG 20984), using the National Center for Biotechnology Information''s (NCBI) Entrez Nucleotide Database search tool (http://www.ncbi.nlm.nih.gov/). Gene sequences of strains not available in public databases (V. coralliilyticus strains LMG 21348, LMG 21349, LMG 21350, LMG 10953, LMG 20538, LMG 23696, LMG 23691, LMG 23693, LMG 23692, and LMG 23694) were obtained through extraction of total DNA using a Promega Wizard Prep DNA Purification Kit (Promega, Sydney, Australia), PCR amplification, and sequencing using primers and thermal cycling parameters described by Nhung et al. (8). A 128-bp region (nucleotides 363 to 490) containing high concentrations of single nucleotide polymorphisms (SNPs), which were conserved within V. coralliilyticus strains but differed from non-V. coralliilyticus strains, was identified, and oligonucleotide primers Vc_dnaJ_F1 (5′-CGG TTC GYG GTG TTT CAA AA-3′) and Vc_dnaJ_R1 (5′-AAC CTG ACC ATG ACC GTG ACA-3′) and a TaqMan probe, Vc_dnaJ_TMP (5′-6-FAM-CAG TGG CGC GAA G-MGBNFQ-3′; 6-FAM is 6-carboxyfluorescein and MGBNFQ is molecular groove binding nonfluorescent quencher), were designed to target this region. The qPCR assay was optimized and validated using DNA extracted from V. coralliilyticus isolates, nontarget Vibrio species, and other bacterial species grown in marine broth (MB) (Table (Table1),1), under the following optimal conditions: 1× TaqMan buffer A, 0.5 U of AmpliTaq Gold DNA polymerase, 200 μM deoxynucleotide triphosphates (with 400 μM dUTP replacing deoxythymidine triphosphate), 0.2 U of AmpErase uracil N-glycosylase (UNG), 3 mM MgCl2, 0.6 μM each primer, 0.2 μM fluorophore-labeled TaqMan, 1 μl of template, and sterile MilliQ water for a total reaction volume to 20 μl. All assays were conducted on a RotoGene 300 (Corbett Research, Sydney, Australia) real-time analyzer with the following cycling parameters: 50°C for 120 s (UNG activation) and 95°C for 10 min (AmpliTaq Gold DNA polymerase activation), followed by 40 cycles of 95°C for 15 s (denaturation) and 60°C for 60 s (annealing/extension). During the annealing/extension phase of each thermal cycle, fluorescence was measured in the FAM channel (470-nm excitation and 510-nm detection).
Open in a separate windowaOrigin, host organism, and dnaJ gene sequence accession numbers are shown for V. coralliilyticus strains.bStrain designations beginning with LMG were derived from the Belgian Coordinated Collections of Microorganisms, ATCC strains are from the American Type Culture Collection, DSM strains are from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH culture collection, AIMS strains are from the Australian Institute of Marine Science culture collection, and C1 and C2 were provided by Pamela Morris.c†, amplification in one of three reactions; ††, amplification in two of three reactions; NA, no amplification.dIsolated from seawater above coral.The qPCR assay specifically detected 12 out of 13 isolated V. coralliilyticus strains tested in this study (Table (Table1).1). The exception was one Caribbean strain (C2), which failed to give specific amplification despite repeated attempts. Positive detection of the target gene segment was determined by the increase in fluorescent signal beyond the fluorescence threshold value (normalized fluorescence, 0.010) at a specific cycle, referred to as the threshold cycle (CT). Specific detection was further confirmed by gel electrophoresis, which revealed a PCR product of the correct theoretical size (128 bp) (data not shown), and DNA sequencing, which confirmed the target amplified product to be a segment of the dnaJ gene. No amplification with the assay was detected for 13 other closely related Vibrio strains, including the closely related Vibrio neptunius and two non-Vibrio species (Table (Table1).1). A total of five other Vibrio strains and one non-Vibrio strain (Shewanella sp.) exhibited CT values less than the cutoff of 32 cycles. However, CT values for these strains (mean ± standard error of the mean [SEM], 27.96 ± 2.40) were all much higher than those for V. coralliilyticus strains (12.30 ± 1.52), and no amplicons were evident in post-qPCR gel electrophoresis (data not shown).The detection limit for purified V. coralliilyticus genomic DNA was 0.1 pg of DNA, determined by performing 10-fold serial dilutions (100 ng to 0.01 pg per reaction), followed by qPCR amplification. Similarly, qPCR assays of serial dilutions of V. coralliilyticus (LMG 23696) cells cultured overnight in MB (108 CFU ml−1 to extinction) were able to detect as few as 104 CFU (Fig. (Fig.1).1). Standard curves revealed a strong linear negative correlation between CT values and both DNA and cell concentrations of V. coralliilyticus over several orders of magnitude, with r2 values of 0.998 and 0.953 for DNA and cells, respectively (Fig. (Fig.11).Open in a separate windowFIG. 1.Standard curves delineating threshold (CT) values of fluorescence for indicators of pathogen presence: (A) concentration of V. coralliilyticus DNA and (B) number of V. coralliilyticus cells in pure culture. Error bars indicate standard error of the mean for three replicate qPCRs.Little interference of the qPCR assay was observed when purified V. coralliilyticus (LMG 23696) DNA (10 ng) was combined with 10-fold serial dilutions (0.01 to 100 ng per reaction) of non-V. coralliilyticus DNA (i.e., Vibrio campbellii [ATCC 25920T]). Over the entire range of nontarget DNA concentrations tested, the resulting CT values (mean ± SEM, 17.76 ± 0.53) were not significantly different from those of a control treatment containing 10 ng of V. coralliilyticus DNA and no nonspecific DNA (16.75 ± 0.18; analysis of variance [ANOVA], P = 0.51) (Table (Table2).2). Detection of V. coralliilyticus (LMG 23696) bacterial cells (104, 105, 106, 107, or 108 CFU per ml) in a background of non-V. coralliilyticus cells (i.e., V. campbellii [ATCC 25920T] at 0, 10, 104, or 107 CFU per ml) showed little reduction in assay sensitivity (see Fig. S1 in the supplemental material). For example, when V. coralliilyticus was seeded at 107 cells with similarly high concentrations of nontarget cells, little inhibition of the assay was observed.
Open in a separate windowaV. coralliilyticus (LMG 23696) DNA (10 ng) free of nontarget DNA and cells served as positive controls.bA qPCR mixture containing no bacterial DNA served as a no-template, or negative, control (NTC).The assay''s detection limit in seawater was tested by inoculating 10-fold serial dilutions of V. coralliilyticus (LMG 23696) cultures (grown overnight in MB medium, pelleted at 14,000 rpm for 10 min, and washed twice with sterile phosphate-buffered saline [PBS]) into 1 liter of seawater (equivalent final concentrations were 106 to 1 CFU ml−1). The entire volume of V. coralliilyticus-seeded seawater was filtered through a Sterivex-GP filter (Millipore), and DNA was extracted using the method described by Schauer et al. (11). The lowest detection limit for V. coralliilyticus cells seeded into seawater was 1 CFU ml−1 (Fig. (Fig.2),2), with no detection in a 1-liter volume of an unseeded seawater negative control. Standard curves revealed a strong correlation between CT values and the concentrations of V. coralliilyticus bacteria seeded into the seawater over several orders of magnitude (r2 of 0.968) (Fig. (Fig.22).Open in a separate windowFIG. 2.Standard curves showing CT values of the fluorescent signal versus the number of V. coralliilyticus cells per ml seawater (▿), and cells per cm2 of M. aequituberculata tissue, with (○) or without (·) enrichment. Each dot represents an independent experiment. Error bars indicate standard error of the mean for three replicate qPCR runs.The detection limit in seeded coral tissue homogenate was determined by seeding 10-fold dilutions (1010 to 103 CFU ml−1) of pelleted, PBS-washed and resuspended (in 10 ml of sterile PBS) V. coralliilyticus cells onto healthy fragments (∼10 cm2) of the coral Montipora aequituberculata collected from Nelly Bay (Magnetic Island, Australia). Corals were collected in March 2009 and maintained in holding tanks supplied with flowthrough ambient seawater. Resuspended cells were inoculated onto M. aequituberculata fragments, each contained in an individual 3.8-liter plastic bag, allowed to sit at room temperature for 30 min, and then air brushed with compressed air until only white skeleton remained. One-milliliter aliquots of the resulting slurry (PBS, bacteria, and coral tissue) was vortexed for 10 min at 14,000 rpm, and DNA was extracted using a PowerPlant DNA Isolation Kit (Mo Bio, Carlsbad, CA). The lowest detection limits for V. coralliilyticus cells seeded onto coral fragments was 104 CFU per cm2 of coral tissue (Fig. (Fig.2).2). Again, standard curves revealed a strong correlation between CT values and the concentrations of seeded bacteria over several orders of magnitude (r2 of 0.981) (Fig. (Fig.2).2). When a 1-ml aliquot of the slurry was also inoculated into 25 ml of MB and enriched for 6 h at 28°C (with shaking at 170 rpm), the detection limit increased by 1 order of magnitude, to 103 CFU of V. coralliilyticus per cm2 of coral tissue (Fig. (Fig.2).2). The slope of the standard curve reveals some inhibition, particularly at the highest V. coralliilyticus concentrations, which could result from lower replication rates in the cultures with the highest bacterial densities (i.e., 109 CFU). However, since this effect is most pronounced only at the highest bacterial concentrations, the detection limit is still valid. In all trials, unseeded coral fragments and enrichment cultures derived from uninoculated coral fragments served as negative controls.The current study describes the first assay developed to detect and quantify a coral pathogen using a real-time quantitative PCR (qPCR) approach. While previous studies have utilized antibodies or fluorescent in situ hybridization (FISH) to detect coral pathogens (1, 6), the combination of high sensitivity and specificity, low contamination risk, and ease and speed of performance (5) make qPCR technology an ideal choice for rapid pathogen detection in complex hosts, such as corals. The assay developed is highly sensitive for V. coralliilyticus, detecting as few as 1 CFU ml−1 of seawater and 104 CFU cm−2 of coral tissue (103 CFU cm−2 of coral tissue with a 6-h enrichment). These detection limits are likely to be within biologically relevant pathogen concentrations. For example, antibodies for specific detection of the coral bleaching pathogen Vibrio shiloi showed that bacterial densities reached 8.4 × 108 cells cm−3 1 month prior to maximum visual bleaching signs on the coral Oculina patagonica (6). Each seeded seawater and coral (enriched and nonenriched) dilution assay was performed in triplicate. The linearity of the resulting standard curves indicates consistent extraction efficiencies over V. coralliilyticus concentrations spanning 6 orders of magnitude (Fig. (Fig.2)2) and provides strong support for the robustness of the assay. In addition, the presence of competing, non-V. coralliilyticus bacterial cells and DNA had a minimal impact on the detection of V. coralliilyticus. This is an important consideration for accurate detection within the complex coral holobiont, where the target organism is present within a matrix of other microbial and host cells.V. coralliilyticus, like V. shiloi (10), is becoming a model pathogen for the study of coral disease. Recent research efforts have characterized the organism''s genome (W. R. Johnson et al., submitted for publication), proteome (N. E. Kimes et al., submitted for publication), resistome (15), and metabolome (4) and enhanced our understanding of the genetic (7, 9) and physiological (7, 13) basis of its virulence. Before effective management response plans can be formulated, however, continuing research on the genetic and cellular aspects of V. coralliilyticus must be complemented with knowledge of the epidemiology of this pathogen, including information on its distribution, incidence of infection, and rates of transmission throughout populations. The V. coralliilyticus-specific qPCR assay developed in this study will provide important insights into the dynamics of pathogen invasion and spread within populations (6) while also aiding in the identification of disease vectors and reservoirs (12). These capabilities will play an important role in advancing the field of coral disease research and effective management of coral reefs worldwide. 相似文献
TABLE 1.
Species, strain, and threshold cycle for all bacterial strains testedaSpecies | Strainb | Origin | Host organism | CT ± SEMc | dnaJ gene sequence accession no. | Reference |
---|---|---|---|---|---|---|
Vibrio coralliilyticus | LMG 23696 | Nelly Bay, Magnetic Island, Australia | Montipora aequituberculata | 12.43 ± 0.20 | HM215570 | 14 |
LMG 23691 | Majuro Atoll, Republic of Marshall Islands | Acropora cytherea | 14.07 ± 1.33 | HM215571 | 14 | |
LMG 23693 | Nikko Bay, Palau | Pachyseris speciosa | 10.83 ± 2.76 | HM215572 | 14 | |
LMG 23692 | Nikko Bay, Palau | Pachyseris speciosa | 9.40 ± 0.36 | HM215573 | 14 | |
LMG 23694 | Nikko Bay, Palau | Pachyseris speciosad | 12.54 ± 0.24 | HM215574 | 14 | |
LMG 20984T | Indian Ocean, Zanzibar, Tanzania | Pocillopora damicornis | 12.80 ± 0.71 | HM215575 | 2 | |
LMG 21348 | Red Sea, Eilat, Israel | Pocillopora damicornis | 13.81 ± 0.49 | HM215576 | 3 | |
LMG 21349 | Red Sea, Eilat, | Pocillopora damicornis | 12.98 ± 0.94 | HM215577 | 3 | |
LMG 21350 | Red Sea, Eilat, | Pocillopora damicornis | 11.49 ± 0.19 | HM215578 | 3 | |
LMG 10953 | Kent, United Kingdom | Crassostrea gigas (oyster) larvae | 10.53 ± 0.40 | HM215579 | 3 | |
LMG 20538 | Atlantic Ocean, Florianópolis, Brazil | Nodipecten nodosus (bivalve) larvae | 12.13 ± 0.50 | HM215580 | 3 | |
C1 | Caribbean Sea, La Parguera, Puerto Rico | Pseudopterogorgia americana | 14.53 ± 0.28 | HM215568 | 15 | |
C2 | Caribbean Sea, La Parguera, Puerto Rico | Pseudopterogorgia americana | NA | HM215569 | 15 | |
Vibrio alginolyticus | ATCC 17749 | 33.74 ± 0.33 | ||||
Vibio brasiliensis | DSM 17184 | 37.84† | ||||
Vibrio calviensis | DSM 14347 | 27.06 ± 0.52 | ||||
Vibrio campbellii | ATCC 25920T | 39.10† | ||||
Enterovibrio campbellii | LMG 21363 | 37.33 ± 2.41 | ||||
Alliivibrio fischeri | DSM 507 | 31.36 ± 1.42 | ||||
Vibrio fortis | DSM 19133 | NA | ||||
Vibrio furnissii | DSM 19622 | NA | ||||
Vibrio harveyi | DSM 19623 | NA | ||||
Vibrio natriegens | ATCC 14048 | 28.56 ± 0.60 | ||||
Vibrio neptunius | LMG 20536 | NA | ||||
Vibrio ordalii | ATCC 33509 | 25.56 ± 0.41 | ||||
Vibrio parahaemolyticus | ATCC 17802 | NA | ||||
Vibrio proteolyticus | ATCC 15338 | 30.00 ± 0.89†† | ||||
Vibrio rotiferianus | LMG 21460 | NA | ||||
Vibrio splendidus | ATCC 33125 | 32.31 ± 0.82 | ||||
Vibrio tubiashii | ATCC 19109 | NA | ||||
Vibrio xuii | LMG 21346 | NA | ||||
Escherichia coli | ATCC 25922 | NA | ||||
Psychrobacter sp. | AIMS 1618 | NA | ||||
Shewanella sp. | AIMS C041 | 25.34 ± 0.45 |
TABLE 2.
Effect of nontarget bacterial DNA on the detection of 10 ng of purified V. coralliilyticus DNAAmt of nontarget DNA (ng) | CT (mean ± SEM) |
---|---|
100 | 16.97 ± 0.33 |
10 | 16.9 ± 0.08 |
1 | 16.74 ± 0.10 |
0.1 | 17 ± 0.09 |
0.01 | 16.37 ± 0.43 |
0a | 16.75 ± 0.18 |
NTCb | 35.04 ± 0.02 |
993.
? Premise of the study: Microsatellites were isolated from two species of the genus Haageocereus (H. tenuis and H. pseudomelanostele) to be applied in studies of genetic diversity and population structure. ? Methods and Results: Five loci were employed in a preliminary study of genetic diversity and population differentiation in two rare (H. tenuis and H. repens) and two widespread (H. acranthus and H. pseudomelanostele) species, yielding between one and 44 alleles per locus. All five loci were polymorphic, with overall levels of observed and expected heterozygosities ranging from 0.478 to 0.871 and from 0.564 to 0.956, respectively. Three additional loci were scored in H. pseudomelanostele. These eight plus the remaining 11 loci were amplified from putative parents of three hybrids involving Haageocereus and Espostoa. ? Conclusions: These markers will facilitate analysis of genetic diversity, hybridization, and population differentiation throughout Haageocereus and Espostoa. 相似文献
994.
995.
Pamela E. Hoppe Johnnie Chau Kelly A. Flanagan April R. Reedy Lawrence A. Schriefer 《Genetics》2010,184(1):79-90
Mutations in the unc-82 locus of Caenorhabditis elegans were previously identified by screening for disrupted muscle cytoskeleton in otherwise apparently normal mutagenized animals. Here we demonstrate that the locus encodes a serine/threonine kinase orthologous to human ARK5/SNARK (NUAK1/NUAK2) and related to the PAR-1 and SNF1/AMP-Activated kinase (AMPK) families. The predicted 1600-amino-acid polypeptide contains an N-terminal catalytic domain and noncomplex repetitive sequence in the remainder of the molecule. Phenotypic analyses indicate that unc-82 is required for maintaining the organization of myosin filaments and internal components of the M-line during cell-shape changes. Mutants exhibit normal patterning of cytoskeletal elements during early embryogenesis. Defects in localization of thick filament and M-line components arise during embryonic elongation and become progressively more severe as development proceeds. The phenotype is independent of contractile activity, consistent with unc-82 mutations preventing proper cytoskeletal reorganization during growth, rather than undermining structural integrity of the M-line. This is the first report establishing a role for the UNC-82/ARK5/SNARK kinases in normal development. We propose that activation of UNC-82 kinase during cell elongation regulates thick filament attachment or growth, perhaps through phosphorylation of myosin and paramyosin. We speculate that regulation of myosin is an ancestral characteristic of kinases in this region of the kinome.THE contractile apparatus of striated muscle is a highly ordered cytoskeletal structure (Figure 1) composed of actin and myosin filaments, the filament anchoring structures, and a host of regulatory proteins. During Caenorhabditis elegans embryogenesis, the body-wall muscle cells polarize and assemble their cytoskeletons in response to contact with the epidermal cells, to which they attach through focal-adhesion-like structures. The epidermal cells respond in a similar fashion and assemble attachment structures and fibrous organelles at the sites of muscle-cell contact (reviewed in Moerman and Williams 2006). The coordination of the cytoskeletons of the two tissue types provides the physical attachment that transmits the force of muscle-cell contraction to the epidermis and its secreted cuticle and allows the worm to locomote through its environment. The patterning of the contractile apparatus occurs through integrin-mediated signaling at the plasma membrane where muscle cells contact the epidermis. The assembly of more interior (membrane-distal) components of the contractile apparatus follows and requires the membrane-proximal events (Hresko et al. 1994). Failure to assemble functional epidermal–muscle-cell contacts or failure to make contractile muscle cells prevents elongation of the embryo from an egg shape into a long tube. Many genes required for these early patterning events, as well as those essential for muscle contraction, have been identified by screening for embryonic lethal mutations that produce the Pat phenotype (paralyzed, arrested elongation at two-fold) (Williams and Waterston 1994).Open in a separate windowFigure 1.—unc-82 mutants show dramatic defects in localization of thick-filament and M-line components, but normal patterning of membrane and dense-body proteins. A diagram of the sarcomere (top) is highlighted to indicate those components affected in unc-82 mutants. Structures represented include the actin filaments anchored to the dense body (the Z-line analog) and myosin-containing thick filaments associated with the M-line. The components represented in white exhibit abnormal staining patterns in unc-82 mutants; those represented in gray are relatively unaffected. (A–N) Adult fragments from wild-type (left column) and unc-82 mutant worms (right column) were stained with antibodies specific for components of the contractile apparatus. Thick-filament proteins myosin A (A and B) and paramyosin (C and D) are grossly mislocalized in unc-82 mutants, as is the M-line component UNC-89/obscurin (E and F). White arrows (B, D, F, and H) indicate abnormal accumulations of thick-filament and M-line proteins, and asterisks (A and K) mark a cell border. Actin staining (G and H) is mildly disrupted, but does not appear in large clumps. The distribution of α-actinin, vinculin, and integrin (I–N) (organized lines of puncta, solid arrows) is similar in mutant and wild type. Antibodies: myosin A, 5.6; paramyosin, 5.23; UNC-89, EU30; actin, C4; α-actinin, MH35; vinculin, MH24; integrin MH25. Bar, 10 μm.However, proteins that act subsequent to the early patterning events or are not essential for contraction would not have been identified in the Pat screens. Mutations in the unc-82 gene were isolated by screening apparently normal animals for muscle-cell disorganization using polarized light microscopy (Waterston et al. 1980). Animals homozygous for unc-82 mutations exhibit patchy, bright birefringence rather than the uniform bright bands of signal that mark the areas of organized myosin-containing thick filaments in wild-type worms. To define the mechanisms underlying filament organization within the contractile apparatus, we undertook molecular and phenotypic analyses of unc-82 mutants. Our data suggest that UNC-82 is a kinase, orthologous to human ARK5 and SNARK, that is required specifically for myosin filament reorganization during cellular elongation in normal development. 相似文献
996.
Peng Xiao Jun Zhao L. Jean Patterson Egidio Brocca-Cofano David Venzon Pamela A. Kozlowski Rachmat Hidajat Thorsten Demberg Marjorie Robert-Guroff 《Journal of virology》2010,84(14):7161-7173
We have shown that following priming with replicating adenovirus type 5 host range mutant (Ad5hr)-human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) recombinants, boosting with gp140 envelope protein enhances acute-phase protection against intravenous simian/human immunodeficiency virus (SHIV)89.6P challenge compared to results with priming and no boosting or boosting with an HIV polypeptide representing the CD4 binding site of gp120. We retrospectively analyzed antibodies in sera and rectal secretions from these same macaques, investigating the hypothesis that vaccine-elicited nonneutralizing antibodies contributed to the better protection. Compared to other immunized groups or controls, the gp140-boosted group exhibited significantly greater antibody activities mediating antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell-mediated viral inhibition (ADCVI) in sera and transcytosis inhibition in rectal secretions. ADCC and ADCVI activities were directly correlated with antibody avidity, suggesting the importance of antibody maturation for functionality. Both ADCVI and percent ADCC killing prechallenge were significantly correlated with reduced acute viremia. The latter, as well as postchallenge ADCVI and ADCC, was also significantly correlated with reduced chronic viremia. We have previously demonstrated induction by the prime/boost regimen of mucosal antibodies that inhibit transcytosis of SIV across an intact epithelial cell layer. Here, antibody in rectal secretions was significantly correlated with transcytosis inhibition. Importantly, the transcytosis specific activity (percent inhibition/total secretory IgA and IgG) was strongly correlated with reduced chronic viremia, suggesting that mucosal antibody may help control cell-to-cell viral spread during the course of infection. Overall, the replicating Ad5hr-HIV/SIV priming/gp140 protein boosting approach elicited strong systemic and mucosal antibodies with multiple functional activities associated with control of both acute and chronic viremia.A major goal of human immunodeficiency virus (HIV) vaccine development is the elicitation of protective antibodies capable of neutralizing the diversity of isolates in the worldwide pandemic (6, 61). Indeed, passively administered neutralizing antibodies have been shown to protect against pathogenic HIV/simian immunodeficiency virus (SIV) challenge in rhesus macaque models (4, 44, 45, 57). However, the extent to which other antibody-mediated protective mechanisms impact HIV/SIV infection is still unclear. Whether these alternate biologic activities would augment vaccine-induced protection has not been definitively established.In HIV-infected individuals, as in SIV- or simian/human immunodeficiency virus (SHIV)-infected rhesus macaques, systemic nonneutralizing antibodies appear early during acute infection, often preceding a neutralizing antibody response (21, 55). Although neutralizing antibody activity is critical for sterilizing immunity, recent studies suggest that antibodies may contribute to protection by other functional activities, such as antibody-dependent cellular cytotoxicity (ADCC) (20, 29), antibody-dependent cell-mediated viral inhibition (ADCVI) (22, 23), and transcytosis inhibition (19, 35, 59). Antibodies in secretions may directly block viral entry into intestinal and endocervical tissues by inhibiting transcytosis across epithelium, whereas local or serum-derived antibodies that mediate ADCC or ADCVI may exert protective effects by eliminating small foci of infected cells during the brief window of time that exists between transmission of virus across an epithelial cell barrier to the lamina propria and subsequent systemic spread (32). In support of this notion, mutation of the Fc portion of the broadly neutralizing monoclonal antibody, IgGb12, thereby preventing interaction with the FcγR on effector cells, rendered the antibody less able to mediate protection upon subsequent passive transfer and challenge of rhesus macaques (34). Thus, neutralizing antibodies themselves may mediate protection by additional functional activities.ADCC bridges innate and adaptive immunity. Mechanistically, it involves FcγR-bearing effector cells, such as NK cells, macrophages, neutrophils, and γδ T cells, and antibodies specific for antigens expressed on the surface of target cells. Upon interaction of these three components, the target cells are killed. Since the effector cells are not major histocompatibility complex restricted, ADCC is broadly applicable to diverse populations. Because the antibody specificity need not be restricted to neutralizing epitopes, ADCC may increase the breadth of antibody reactivity. In fact, we have shown that an HIV clade B immunization regimen elicited antibodies that mediated ADCC across several HIV clades (28). Antibodies that mediate ADCC have been shown to arise early in infection, before neutralizing antibodies (55, 60). They are present in the majority of infected individuals, and they have been associated with slow disease progression following both HIV and SIV infection (5, 8).ADCVI is closely related to ADCC, also requiring antibody that forms a bridge between an infected target cell and an FcγR-bearing effector cell (24). However, ADCVI is a broader activity not restricted solely to target cell lysis but, rather, encompassing several mechanisms by which viral replication following infection of target cells is inhibited. Thus, it may include ADCC activity but also involve noncytotoxic mechanisms of virus control, such as the secretion of inhibitory chemokines or FcγR-mediated phagocytosis of immune complexes (24, 25).Most HIV infections occur via a mucosal route, including cervicovaginal and rectal tissues (39, 52). Several nonmutually exclusive mechanisms for HIV-1 transmission across mucosal epithelia have been proposed (13, 56). Transcytosis of infectious virus across polarized columnar epithelial cells following contact of virally infected cells with apical epithelial cell surfaces is one mechanism for mucosal HIV entry (12). Rather than fusion and infection, interactions between the viral envelope proteins and epithelial surface molecules, such as glycosphingolipid galactosyl-ceramide (GalCer) (13, 47), an important component of endocytotic “raft” membrane microdomains, lead to transcytosis of the virus across the epithelial barrier and its trapping by submucosal dendritic cells which disseminate it to their target CD4+ T cells. Studies have shown that mucosal immunoglobulin A (IgA) antibody, a major component of the mucosal immune response, could block mucosal HIV-1 entry via transcytosis in vitro (2, 19). Therefore, mucosal antibodies blocking adherence of virus to epithelial cells and preventing HIV-1 transcytosis across the epithelial barrier and subsequent CD4+ T cell infection may afford additional protection against HIV/SIV infection.We have been pursuing a replicating adenovirus (Ad)-HIV/SIV prime/protein subunit boost AIDS vaccine approach (30, 51), which has elicited strong, durable protection against HIV, SIV, and SHIV challenges (11, 18, 41, 42, 50). An underlying goal of these studies has been elucidation of immune responses that correlate with protective efficacy. Recently, we studied the contribution of novel protein boosts to immunogenicity and protective efficacy in a SHIV89.6P model (49). Immunized rhesus macaques were primed with Ad type 5 host range mutant (Ad5hr)-HIV89.6Pgp140, -SIV239gag, and -SIV239nef recombinants. One group was not boosted, one was boosted with HIV89.6P gp140ΔCFI protein (gp140 envelope with deletions in the cleavage site, fusion peptide, and part of the interspace between the two heptad repeats) (40), and one was boosted with a novel HIV-1 polypeptide “peptomer” representing the CD4 binding site of the envelope (54). The best protection was seen in the gp140-boosted group, with significant reductions in both acute and chronic viremia. Although Env-specific antibody and cellular responses were readily detected, none directly correlated with the better protection. Furthermore, neutralizing antibodies against SHIV89.6P did not develop until 4 weeks postchallenge. Therefore, we hypothesized that vaccine-elicited nonneutralizing anti-Env antibodies might have contributed to the better control of acute and/or chronic viremia in the gp140 group. Here, we report retrospective evaluations of sera and rectal secretions from macaques in this comparative study for serum binding antibody avidity, an important characteristic of functional antibodies (38, 58), and nonneutralizing activities of systemic and mucosal antibodies, including ADCC, ADCVI, and transcytosis inhibition. 相似文献
997.
998.
Anh P. Truong Gergley Tóth Gary D. Probst Jennifer M. Sealy Simeon Bowers David W.G. Wone Darren Dressen Roy K. Hom Andrei W. Konradi Hing L. Sham Jing Wu Brian T. Peterson Lany Ruslim Michael P. Bova Dora Kholodenko Ruth N. Motter Frédérique Bard Pamela Santiago Huifang Ni David Chian John-Michael Sauer 《Bioorganic & medicinal chemistry letters》2010,20(21):6231-6236
In this Letter, we describe our efforts to design HEA BACE-1 inhibitors that are highly permeable coupled with negligible levels of permeability-glycoprotein activity. These efforts culminate in producing 16 which lowers Αβ by 28% and 32% in the cortex and CSF, respectively, in the preclinical wild type Hartley guinea pig animal model when dosed orally at 30 mpk BID for 2.5 days. 相似文献
999.
Jutta Wanner Lijing Chen Rémy C. Lemoine Rama Kondru Andreas Jekle Gabrielle Heilek André deRosier Changhua Ji Pamela W. Berry David M. Rotstein 《Bioorganic & medicinal chemistry letters》2010,20(22):6802-6807
Replacement of a secondary amide with a piperidine or azetidine moiety in a series of CCR5 antagonists led to the discovery of compounds with increased intrinsic permeability. This effort led to the identification of a potent CCR5 antagonist which exhibited an improved in vivo pharmacokinetic profile. 相似文献
1000.
Pamela J. Schofield William F. Loftus Robert M. Kobza Mark I. Cook Daniel H. Slone 《Biological invasions》2010,12(8):2441-2457
The cold tolerance of two non-native cichlids (Hemichromis letourneuxi and Cichlasoma urophthalmus) that are established in south Florida was tested in the field and laboratory. In the laboratory, fishes were acclimated
to two temperatures (24 and 28°C), and three salinities (0, 10, and 35 ppt). Two endpoints were identified: loss of equilibrium
(11.5–13.7°C for C. urophthalmus; 10.8–12.5°C for H. letourneuxi), and death (9.5–11.1°C for C. urophthalmus; 9.1–13.3°C for H. letourneuxi). In the field, fishes were caged in several aquatic habitats during two winter cold snaps. Temperatures were lowest (4.0°C)
in the shallow marsh, where no fish survived, and warmest in canals and solution-holes. Canals and ditches as shallow as 50 cm
provided thermal refuges for these tropical fishes. Because of the effect on survival of different habitat types, simple predictions
of ultimate geographic expansion by non-native fishes using latitude and thermal isoclines are insufficient for freshwater
fishes. 相似文献