Multilocus enzyme electrophoresis was used to examine genetic relationships among and between toxigenic and non-toxigenic isolates of Vibrio cholerae O1 obtained from patients and the environment in the US Gulf Coast and surrounding areas. A total of 23 toxigenic and 23 non-toxigenic strains were examined. All the toxigenic and 7 of the non-toxigenic strains had the same alleles at 16 enzyme loci, whereas the balance of the nontoxigenic strains had 9 distinct combinations of alleles. This study suggests that all of the toxigenic strains belong to a single clone, and that while some of the non-toxigenic isolates were related, most were of diverse origin.
Chen, F.; Evins, G. M.; Cook, W. L.; Almeida, R.; Hargrett-Bean, N.; Wachsmuth, K.
Multilocus enzyme electrophoresis was used to examine genetic relationships among and between toxigenic and non-toxigenic isolates of Vibrio cholerae O1 obtained from patients and the environment in the US Gulf Coast and surrounding areas. A total of 23 toxigenic and 23 non-toxigenic strains were examined. All the toxigenic and 7 of the non-toxigenic strains had the same alleles at 16 enzyme loci, whereas the balance of the nontoxigenic strains had 9 distinct combinations of alleles. This study suggests that all of the toxigenic strains belong to a single clone, and that while some of the non-toxigenic isolates were related, most were of diverse origin. PMID:1879486
Chen, F; Evins, G M; Cook, W L; Almeida, R; Hargrett-Bean, N; Wachsmuth, K
Nontoxigenic strains of Corynebacterium diphtheriae represent a potential reservoir for the emergence of toxigenic C. diphtheriae strains if they possessed functional diphtheria toxin repressor (dtxR) genes. We studied the predominant strain of nontoxigenic C. diphtheriae circulating in the United Kingdom to see if they possessed dtxR genes and ascertain whether they were functional. A total of 26 nontoxigenic C. diphtheriae
Aruni De Zoysa; Androulla Efstratiou; Peter M. Hawkey
We investigated the virulence properties of four Vibrio parahaemolyticus strains causing acute gastroenteritis following consumption of indigenous mussels in Italy. The isolated strains were cytotoxic and adhesive but, surprisingly, lacked tdh, trh, and type three secretion system 2 (T3SS2) genes. We emphasize that nontoxigenic V. parahaemolyticus can induce acute gastroenteritis, highlighting the need for more investigation of the pathogenicity of this microorganism.
Leoni, Francesca; Serra, Roberto; Serracca, Laura; Decastelli, Lucia; Rocchegiani, Elena; Masini, Laura; Canonico, Cristina; Talevi, Giulia; Carraturo, Antonio
A nonproteolytic, nontoxigenic Clostridium botulinum strain identified by conventional and molecular techniques as type B-, E-, or F-like (BEF-like) was isolated from a human postsurgical wound. All previous reports of such strains have been from environmental sources. Since toxin production is the main taxonomic denominator for C. botulinum, a new name is needed for nonproteolytic, nontoxigenic BEF-like clinical isolates.
Carlier, Jean-Philippe; K'ouas, Guylene; Lozniewski, Alain; Sirveaux, Francois; Cailloux, Philippe; Mory, Francine
A 54-year-old female with a prosthetic mitral valve presented with a 3-day history of dizziness, subjective fever, and chills. Blood cultures were positive for a pleomorphic Gram-positive rod. Initial phenotypic testing could only support the identification of a Corynebacterium species. Nucleic acid sequencing (16S rRNA) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) were conclusive for Corynebacterium diphtheriae. Definitive phenotypic testing classified the strain as nontoxigenic C. diphtheriae biotype Gravis. PMID:24006007
Clinton, Lani Kai; Bankowski, Matthew J; Shimasaki, Teppei; Sae-Ow, Wichit; Whelen, A Christian; O'Connor, Norman; Kim, Wesley; Young, Royden
Although infection by Corynebacterium diphtheriae is a model of extracellular mucosal pathogenesis, and diphtheria is one of the most worried diseases, this microorganism can be associated also with invasive infections such as endocarditis, septic arthritis, and osteomyelitis. Invasive infections are usually caused by non-toxigenic C. diphtheriae strains. Over the last years severe pharyngitis/tonsillitis associated with the isolation of non-toxigenic C. diphtheriae have been described. Penicillin treatment failure of these infections could only partially be explained by penicillin tolerance of the causing strain. Thus, we examined the in vitro ability of non-toxigenic C. diphtheriae throat clinical isolates to adhere to, and enter human respiratory epithelial cells. Trasmission and scanning electron microscopy demonstrated intracellular C. diphtheriae in laryngeal (HEp-2 cells) and pharyngeal (Detroit D562 cells) tissue culture. Live intracellular bacteria were detectable up to 48 h post-infection. Using a variety of compound that act on eukariotic cell structures, the internalization of C. diphtheriae seems to occur via a zipper-like mechanism. It is likely that internalization of C. diphtheriae can be involved in throat colonization contributing to bacterial eradication failure and asymptomatic carriage. PMID:15351033
Bertuccini, Lucia; Baldassarri, Lucilla; von Hunolstein, Christina
Two spontaneous phase variants of Bordetella avium were isolated at a frequency of 2 x 10(-4) by colony immunoblot assay of B. avium with antibody against B. avium dermonecrotic toxin. The two phase variants, designated GOBL309 and GOBL312, lack dermonecrotic toxin and four outer membrane proteins with molecular masses of 93, 48, 38, and 27 kDa but retain the ability to agglutinate guinea pig erythrocytes. The proteins which are not expressed by GOBL309 and GOBL312 correspond to five proteins which are phenotypically modulated in B. avium by growth in the presence of nicotinic acid or MgSO4. Growth of the phase variants in supplemented Stainer-Scholte media containing nicotinamide did not alter expression of these five proteins. Intranasal inoculation of the spontaneous phase variants into 3-day-old turkeys and reisolation of B. avium at 2 weeks postinoculation resulted in the recovery of B. avium which had the wild-type phenotype, colonized the turkey tracheas, and produced the four outer membrane proteins and dermonecrotic toxin. Hybridization of B. avium and B. avium-like chromosomal DNA with internal portions of the Bordetella pertussis virulence regulatory genes, bvgA and bvgS, revealed that B. avium and B. avium-like isolates contain 5.3- and 5.7-kb DNA fragments, respectively, which are homologous to bvgS. B. avium and B. avium-like chromosomal DNA failed to hybridize to B. pertussis bvgA. Images
Gentry-Weeks, C R; Provence, D L; Keith, J M; Curtiss, R
SUMMARY Stenotrophomonas maltophilia contains a novel chromosomally-encoded qnr gene named Smqnr that contributes to low intrinsic resistance to quinolone. We described Smqnr in 13 clinical isolates of S. maltophilia from two Brazilian hospitals, over a 2-year period. The strains were identified by API 20 NE (bioMérieux, France). Susceptibility by microdilution method to trimetroprim/sulfamethoxazole, ciprofloxacin, levofloxacin, minocycline, ceftazidime, chloramphenicol and ticarcillin/clavulanate was performed according to CLSI. PCR detection of Smqnr gene was carried out. The sequence of Smqnr was compared with those deposited in GenBank. Pulsed-field gel electrophoresis (PFGE) of all strains was performed. Thirteen Smqnr positives isolates were sequenced and three novel variants of Smqnr were identified. All 13 Smqnr isolates had distinguishable patterns by PFGE. This is the first report of Smqnr in S. maltophilia isolated in Brazil.
Gracia-Paez, Jorge Isaac; Ferraz, Juliana Rosa; Franca E Silva, Ivan Avelino; Rossi, Flavia; Levin, Anna Sara; Costa, Silvia Figueiredo
The genetic contribution to the pathogenesis of isolated single suture craniosynostosis is poorly understood. The role of mutations in genes known to be associated with syndromic synostosis appears to be limited. We present our findings of a candidate gene resequencing approach to identify rare variants associated with the most common forms of isolated craniosynostosis. Resequencing of the coding regions, splice junction sites, and 5? and 3? untranslated regions of 27 candidate genes in 186 cases of isolated nonsyndromic single suture synostosis revealed three novel and two rare sequence variants (R406H, R595H, N857S, P190S, M446V) in insulin-like growth factor I receptor (IGF1R) that are enriched relative to control samples. Mapping the resultant amino acid changes to the modeled homodimer protein structure suggests a structural basis for segregation between these and other disease-associated mutations found in IGF1R. These data suggest that IGF1R mutations may contribute to the risk and in some cases cause single suture craniosynostosis.
Cunningham, Michael L.; Horst, Jeremy A.; Rieder, Mark J.; Hing, Anne V.; Stanaway, Ian B.; Park, Sarah S.; Samudrala, Ram; Speltz, Matthew L.
Background Congenital malformations are present in approximately 2–3% of liveborn babies and 20% of stillborn fetuses. The mechanisms underlying the majority of sporadic and isolated congenital malformations are poorly understood, although it is hypothesized that the accumulation of rare genetic, genomic and epigenetic variants converge to deregulate developmental networks. Methodology/Principal Findings We selected samples from 95 fetuses with congenital malformations not ascribed to a specific syndrome (68 with isolated malformations, 27 with multiple malformations). Karyotyping and Multiplex Ligation-dependent Probe Amplification (MLPA) discarded recurrent genomic and cytogenetic rearrangements. DNA extracted from the affected tissue (46%) or from lung or liver (54%) was analyzed by molecular karyotyping. Validations and inheritance were obtained by MLPA. We identified 22 rare copy number variants (CNV) [>100 kb, either absent (n?=?7) or very uncommon (n?=?15, <1/2,000) in the control population] in 20/95 fetuses with congenital malformations (21%), including 11 deletions and 11 duplications. One of the 9 tested rearrangements was de novo while the remaining were inherited from a healthy parent. The highest frequency was observed in fetuses with heart hypoplasia (8/17, 62.5%), with two events previously related with the phenotype. Double events hitting candidate genes were detected in two samples with brain malformations. Globally, the burden of deletions was significantly higher in fetuses with malformations compared to controls. Conclusions/Significance Our data reveal a significant contribution of rare deletion-type CNV, mostly inherited but also de novo, to human congenital malformations, especially heart hypoplasia, and reinforce the hypothesis of a multifactorial etiology in most cases.
Serra-Juhe, Clara; Rodriguez-Santiago, Benjamin; Cusco, Ivon; Vendrell, Teresa; Camats, Nuria; Toran, Nuria; Perez-Jurado, Luis A.
Salmonella enterica serovar Typhimurium variant Copenhagen was isolated from 5 of 152 (3.3%) feral pigeons from the city of Ghent (Belgium) and from 26 pooled fecal samples from 114 pigeon lofts (22.8%). These isolates belonged to phage type (PT) 99. Seven of the pigeon isolates were further compared in vitro to five human variant Copenhagen isolates, 2 isolates of PT
Frank Pasmans; Filip Van Immerseel; Katleen Hermans; Marc Heyndrickx; Jean-Marc Collard; Richard Ducatelle; Freddy Haesebrouck
The aim of this study was to fortify the clinical importance and representation of toxigenic and non-toxigenic Clostridium difficile isolated from stool samples of hospitalized patients. This survey included 80 hospitalized patients with diarrhea and positive findings of Clostridium difficile in stool samples, and 100 hospitalized patients with formed stool as a control group. Bacteriological examination of a stool samples was conducted using standard microbiological methods. Stool sample were inoculated directly on nutrient media for bacterial cultivation (blood agar using 5% sheep blood, Endo agar, selective Salmonella Shigella agar, Selenite-F broth, CIN agar and Skirrow's medium), and to selective cycloserine-cefoxitin-fructose agar (CCFA) (Biomedics, Parg qe tehnicologico, Madrid, Spain) for isolation of Clostridium difficile. Clostridium difficile toxin was detected by ELISA-ridascreen Clostridium difficile Toxin A/B (R-Biopharm AG, Germany) and ColorPAC ToxinA test (Becton Dickinson, USA). Examination of stool specimens for the presence of parasites (causing diarrhea) was done using standard methods (conventional microscopy), commercial concentration test Paraprep S Gold kit (Dia Mondial, France) and RIDA(®)QUICK Cryptosporidium/Giardia Combi test (R-Biopharm AG, Germany). Examination of stool specimens for the presence of fungi (causing diarrhea) was performed by standard methods. All stool samples positive for Clostridium difficile were tested for Rota, Noro, Astro and Adeno viruses by ELISA - ridascreen (R-Biopharm AG, Germany). In this research we isolated 99 Clostridium difficile strains from 116 stool samples of 80 hospitalized patients with diarrhea. The 53 (66.25%) of patients with diarrhea were positive for toxins A and B, one (1.25%) were positive for only toxin B. Non-toxigenic Clostridium difficile isolated from samples of 26 (32.5%) patients. However, other pathogenic microorganisms of intestinal tract cultivated from samples of 16 patients. Examination of cultivated colonies revealed that most of cultivated species belonged to genera of Campylobacter spp., Salmonella spp., and Candida spp.. In control group, toxigenic Clostridium difficile cultivated from stool samples of two patients (2%) and non-toxigenic Clostridium difficile from samples of five patients (5%). This research confirmed clinical importance of toxigenic Clostridium difficile found in liquid stool samples of hospitalized patient, and the possibility of asymptomatic carriage in 2% of patients with formed stool. PMID:24031820
Predrag, Stojanovic; Branislava, Kocic; Miodrag, Stojanovic; Biljana, Miljkovic-Selimovic; Suzana, Tasic; Natasa, Miladinovic-Tasic; Tatjana, Babic
Bdellovibrios are Gram-negative bacteria that are characterized by predatory behavior. Although Bdellovibrios exhibit an obligatory parasitic life cycle, it is possible to isolate Bdellovibrio variants that no longer require host cells for their growth. In this study, a new method for isolating Bdellovibrio bacteriovorus host-independent (HI) variants was developed. Filtered B. bacteriovorus prey cells were cultured with E. coli diaminopimelic acid (DAP) auxotrophs as host cells. Thereafter, the lysate was plated on DAP minus media, allowing only HI colonies to develop. Using this method, we have isolated numerous HI variants and demonstrated that the emergence of HI variants may be occurring at a higher frequency than was previously suggested. PMID:19590595
Dashiff, Aliza; Kadouri, Daniel E
A parainfluenza 3 virus variant which failed to react with parainfluenza 3 virus-specific monoclonal anti-bodies from two commercial sources was isolated from a 14-month-old boy. Analysis of the coding region of the hemagglutinin-neuraminidase gene identified 36 nucleotide changes and 4 amino acid changes compared with a consensus sequence derived from strains isolated from 1957 through 1983. Two unique amino acid changes occurred at positions 174 and 283, which are close to identified epitopes in the hemagglutinin-neuraminidase protein. Ongoing viral surveillance to detect variants is important, particularly in regard to vaccine development.
Swierkosz, E M; Erdman, D D; Bonnot, T; Schneiderheinze, C; Waner, J L
Clostridium sporogenes PA 3679 is widely used as a nontoxigenic surrogate for proteolytic strains of Clostridium botulinum in the derivation and validation of thermal processes in food. Here we report the draft assembly and annotation of the C. sporogenes PA 3679 genome. Preliminary analysis demonstrates a high degree of relatedness between C. sporogenes PA 3679 and sequenced strains of proteolytic C. botulinum. PMID:22374960
Bradbury, Mark; Greenfield, Paul; Midgley, David; Li, Dongmei; Tran-Dinh, Nai; Vriesekoop, Frank; Brown, Janelle L
Isoelectric focusing on polyacrylamide gel in the absence of haem ligands represents a useful, convenient and rapid procedure to isolate silent Hb variants in their native forms, provided that they exhibit an abnormal Bohr effect. The amount of material which is eluted is sufficient for both a limited functional study and a structural determination using microscale high-performance liquid chromatography. This is exemplified by the isolation and the study of Hb Rainier. PMID:3793825
Rochette, J; Baudin, V; Bohn, B; Poyart, C; Wajcman, H
In this study we report the isolation and characterization of normal-sized and small-colony variants of Enterococcus faecalis from outbreaks of amyloid arthropathy in chickens. Postmortem examinations of 59 chickens revealed orange deposits in the knee joints, typical for amyloid arthropathy. Bacterial cultures from 102 joints and 43 spleens exhibited pure (n = 88) and mixed (n = 11) cultures of normal (n = 60) and pinpoint (n = 28) colonies of E. faecalis. Pulsed-field gel electrophoresis of 62 isolates demonstrated seven different band patterns with at most two band size variations, and multilocus sequence typing demonstrated two different sequence types, sharing six out of seven alleles, suggesting a close evolutionary relationship between isolates obtained from four outbreaks. In addition, all isolates were clonally related to an amyloid arthropathy reference strain from The Netherlands, previously shown to be globally dispersed. Initial investigation of the isolated small-colony variant phenotype revealed no difference in whole-cell protein profiling between normal and pinpoint colonies. However, the pinpoint colony isolates appeared to be more virulent in an in vivo challenge model in chickens than their normal-sized-colony counterparts. In addition, pinpoint morphology and associated slow growth were expressed without reversion after in vitro and in vivo passage, suggesting a genuine altered phenotype, and in some instances normal colonies converted to pinpoint morphology postinfection. In conclusion, small-colony variants of E. faecalis are described for the first time from veterinary clinical sources and in relation to amyloid arthropathy in chickens.
Petersen, Andreas; Chadfield, Mark S.; Christensen, Jens P.; Christensen, Henrik; Bisgaard, Magne
To save energy, space, and time, today's breweries make use of high-gravity brewing in which concentrated medium (wort) is fermented, resulting in a product with higher ethanol content. After fermentation, the product is diluted to obtain beer with the desired alcohol content. While economically desirable, the use of wort with an even higher sugar concentration is limited by the inability of brewer's yeast (Saccharomyces pastorianus) to efficiently ferment such concentrated medium. Here, we describe a successful strategy to obtain yeast variants with significantly improved fermentation capacity under high-gravity conditions. We isolated better-performing variants of the industrial lager strain CMBS33 by subjecting a pool of UV-induced variants to consecutive rounds of fermentation in very-high-gravity wort (>22° Plato). Two variants (GT336 and GT344) showing faster fermentation rates and/or more-complete attenuation as well as improved viability under high ethanol conditions were identified. The variants displayed the same advantages in a pilot-scale stirred fermenter under high-gravity conditions at 11°C. Microarray analysis identified several genes whose altered expression may be responsible for the superior performance of the variants. The role of some of these candidate genes was confirmed by genetic transformation. Our study shows that proper selection conditions allow the isolation of variants of commercial brewer's yeast with superior fermentation characteristics. Moreover, it is the first study to identify genes that affect fermentation performance under high-gravity conditions. The results are of interest to the beer and bioethanol industries, where the use of more-concentrated medium is economically advantageous.
Blieck, Lies; Toye, Geert; Dumortier, Francoise; Verstrepen, Kevin J.; Delvaux, Freddy R.; Thevelein, Johan M.; Van Dijck, Patrick
Isolated populations can empower the identification of rare variation associated with complex traits through next generation association studies, but the generalizability of such findings remains unknown. Here we genotype 1,267 individuals from a Greek population isolate on the Illumina HumanExome Beadchip, in search of functional coding variants associated with lipids traits. We find genome-wide significant evidence for association between R19X, a functional variant in APOC3, with increased high-density lipoprotein and decreased triglycerides levels. Approximately 3.8% of individuals are heterozygous for this cardioprotective variant, which was previously thought to be private to the Amish founder population. R19X is rare (<0.05% frequency) in outbred European populations. The increased frequency of R19X enables discovery of this lipid traits signal at genome-wide significance in a small sample size. This work exemplifies the value of isolated populations in successfully detecting transferable rare variant associations of high medical relevance.
Tachmazidou, Ioanna; Dedoussis, George; Southam, Lorraine; Farmaki, Aliki-Eleni; Ritchie, Graham R. S.; Xifara, Dionysia K.; Matchan, Angela; Hatzikotoulas, Konstantinos; Rayner, Nigel W.; Chen, Yuan; Pollin, Toni I.; O'Connell, Jeffrey R.; Yerges-Armstrong, Laura M.; Kiagiadaki, Chrysoula; Panoutsopoulou, Kalliope; Schwartzentruber, Jeremy; Moutsianas, Loukas; Tsafantakis, Emmanouil; Tyler-Smith, Chris; McVean, Gil; Xue, Yali; Zeggini, Eleftheria
Integrons are important genetic elements implicated in acquisition and expression of antimicrobial resistance genes. Gene cassettes of class 1 integrons may be differently expressed depending on the Pc promoter variant. Thirty-four Escherichia coli isolates (carrying 38 class 1 integrons), recovered from poultry meat in previous studies in Tunisia and selected by their specific traits, were further characterized in this study. Integron promoter variants and the pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) of isolates were determined. Three types of promoter variants were identified among the 38 class 1 integrons (PcW, PcH1, and PcS); the weak promoters were the most predominant. A high clonal diversity of the E. coli strains was demonstrated by PFGE or by MLST. Fifteen PFGE profiles were detected among 19 integron-positive isolates of phylogroup B2, and 12 different sequence types were identified by MLST among the remaining 15 isolates (ST48, ST88, ST101, ST117, ST155, ST189, ST351, ST359, ST410, ST641, ST665, and one new ST). These data reflect that the presence of integrons in these isolates is not due to the clonal dispersion but to dissemination of genetic structures carrying integrons in different clones. To the best of our knowledge, this is the first report examining the gene cassette promoter variants in class 1 integrons of E. coli isolates from poultry meat origin. The predominance of promoters implicated in weak expression/high excision activity of gene cassette arrays is of interest because they could theoretically enhance the capacity of integrons to adapt to antibiotic pressure. PMID:23988017
Soufi, Leila; Sáenz, Yolanda; Vinué, Laura; Abbassi, Mohamed Salah; Hammami, Salah; Torres, Carmen
NODAL and its signaling pathway are known to play a key role in specification and patterning of vertebrate embryos. Mutations in several genes encoding components of the NODAL signaling pathway have previously been implicated in the pathogenesis of human left–right (LR) patterning defects. Therefore, NODAL, a member of TGF-? superfamily of developmental regulators, is a strong candidate to be functionally involved in congenital LR axis patterning defects or heterotaxy. Here we have investigated whether variants in NODAL are present in patients with heterotaxy and/or isolated cardiovascular malformations (CVM) thought to be caused by abnormal heart tube looping. Analysis of a large cohort of cases (n = 269) affected with either classic heterotaxy or looping CVM revealed four different missense variants, one in-frame insertion/deletion and two conserved splice site variants in 14 unrelated subjects (14/269, 5.2%). Although similar with regard to other associated defects, individuals with the NODAL mutations had a significantly higher occurrence of pulmonary valve atresia (P = 0.001) compared with cases without a detectable NODAL mutation. Functional analyses demonstrate that the missense variant forms of NODAL exhibit significant impairment of signaling as measured by decreased Cripto (TDGF-1) co-receptor-mediated activation of artificial reporters. Expression of these NODAL proteins also led to reduced induction of Smad2 phosphorylation and impaired Smad2 nuclear import. Taken together, these results support a role for mutations and rare deleterious variants in NODAL as a cause for sporadic human LR patterning defects.
Mohapatra, Bhagyalaxmi; Casey, Brett; Li, Hua; Ho-Dawson, Trang; Smith, Liana; Fernbach, Susan D.; Molinari, Laura; Niesh, Stephen R.; Jefferies, John Lynn; Craigen, William J.; Towbin, Jeffrey A.; Belmont, John W.; Ware, Stephanie M.
Ornithobacterium rhinotracheale is a Gram-negative bacterium associated with respiratory diseases in many avian species, with worldwide distribution, and it causes significant economic loss to the poultry industry. In this study, the isolation and characterization of O. rhinotracheale small-colony variants (SCVs) are described for the first time. O. rhinotracheale isolates (n = 27) were recovered from tracheal samples (n = 321) collected from different avian species with clinical signs of respiratory disease. Of the 27 O. rhinotracheale isolates, 21 (77.8%) showed SCVs in their primary cultures. Five O. rhinotracheale SCV isolates showed high levels of stability and were chosen for further characterization with their wild-type (WT) isolates. Stable O. rhinotracheale SCVs were oxidase negative, while their WT isolates were positive. Growth curves for stable O. rhinotracheale SCVs indicated lower growth rates and longer lag phases than for their WT isolates. Furthermore, it was possible to increase the efficacy of the broth medium in supporting the growth of O. rhinotracheale WT isolates by supplementing it with 5% fetal bovine serum (FBS) and 2% IsoVitaleX Enrichment. Antibiotic susceptibility tests showed that O. rhinotracheale SCVs had higher MIC values than their WT isolates. This study suggests that successful antibiotic treatment of respiratory diseases associated with O. rhinotracheale must take into consideration the resistance patterns of O. rhinotracheale SCVs. Intracellular persistence in murine RAW 264.7 macrophages revealed that O. rhinotracheale SCV28 had higher survival rates than its WT isolate. Finally, small-colony variants may be important contributors to the pathogenesis of O. rhinotracheale.
Zahra, Mohammad; Ferreri, Miro; Alkasir, Rashad; Yin, Jinhua
Four ceftazidime-resistant isolates of a Klebsiella pneumoniae strain were collected from intensive care unit patients in Nijmegen, The Netherlands. These isolates had TEM-29 and SHV-14 ?-lactamases. SHV-14 is a novel variant, with two substitutions compared with the sequence of SHV-1: Ile8Phe and Arg43Ser. Its gene also had a silent C?T mutation at nucleotide 481. The SHV-14 enzyme had slightly higher Vmax rates than SHV-1 for oxyimino-aminothiazolyl cephalosporins, but this activity was insufficient for the enzyme to count as an extended-spectrum ?-lactamase.
Yuan, Meifang; Hall, Lucinda M. C.; Hoogkamp-Korstanje, Jaa; Livermore, David M.
Megacystis is frequently involved with chronic intestinal pseudoobstruction syndrome; however, isolated megacystis without intestinal obstruction is extremely rare. We present the case of a female patient with isolated congenital megacystis without severe intestinal obstruction. In this case, barium enema did not reveal any significant findings; however, histologic evaluation of her rectum showed hypoganglionosis of the submucous and myenteric plexuses. These findings indicate that this case may be a mild variant of chronic intestinal pseudoobstruction syndrome. The presence of megacystis should alert the physician to the possibility of chronic intestinal pseudoobstruction syndrome. PMID:22075369
Shimizu, Masaki; Nishio, Sayaka; Ueno, Kazuyuki; Yokoyama, Tadafumi; Sakai, Seisho; Nagaoki, Shuya; Sugimoto, Naotoshi; Ohta, Kazuhide; Miyamoto, Masatoshi; Yachie, Akihiro
We screened 650 isolates from historical collection of Vibrio cholerae O1 during the 7th cholera pandemic in China, by amplifying and sequencing the cholera toxin subunit gene ctxB. Ten isolates were identified as harboring three novel ctxB genotypes based on amino acid residue substitutions. Within them one isolate from a patient in 1964 was similar to the El Tor genotype, except for an 11 amino acid repeat sequence (LAGKREMAIIT) that was inserted after position 62. Six environmental isolates from different regions and years were identified as the Australia El Tor genotype, except at positions 36(T?A), 39(H?Y), and 55(K?N), while three environmental isolates were similar to genotype 5, except at position 24(Q?H). Sequencing of rstR, the marker gene for the CTX? allele typing, revealed that two isolates carried the rstR gene of the El Tor type, five carried the classical type rstR, while other isolates carried the rstR232 type. All 10 isolates contained the repeat in the toxin gene rtxC, an El Tor biotype-specific marker, and the El Tor toxin-coregulated pili subunit A gene tcpA, showing the El Tor traits of these isolates. Additionally, by phenotypic biotyping (susceptibility to polymyxin B, positive for chicken erythrocyte agglutination, and Voges-Proskauer test), all isolates except two were typical of the prototype El Tor isolate, while these two isolates had mixed classical phenotypes (hybrid biotype). Furthermore, pulsed-field gel electrophoresis analysis suggested that the new ctxB altered isolates possessed potential transmissibility and thatthey propagated in the local region(s). Taken together, these novel ctxB variants of V. cholerae O1 experienced complex hybrid and genetic exchange but belong to the El Tor lineage, and the pathogenic and epidemic potential of these lineages should be monitored. PMID:23954417
Zhang, Ping; Zhou, Haijian; Kan, Biao; Wang, Duochun
Staphylococcus aureus produces superantigens (SAgs) that bind and cross-link T cells and APCs, leading to activation and proliferation of immune cells. SAgs bind to variable regions of the ?-chains of T cell receptors (V?-TCRs), and each SAg binds a unique subset of V?-TCRs. This binding leads to massive cytokine production and can result in toxic shock syndrome (TSS). The most abundantly produced staphylococcal SAgs and the most common causes of staphylococcal TSS are TSS toxin-1 (TSST-1), and staphylococcal enterotoxins B and C (SEB and SEC, respectively). There are several characterized variants of humans SECs, designated SEC1-4, but only one variant of SEB has been described. Sequencing the seb genes from over 20 S. aureus isolates show there are at least five different alleles of seb, encoding forms of SEB with predicted amino acid substitutions outside of the predicted immune-cell binding regions of the SAgs. Examination of purified, variant SEBs indicates that these amino acid substitutions cause differences in proliferation of rabbit splenocytes in vitro. Additionally, the SEBs varied in lethality in a rabbit model of TSS. The SEBs were diverse in their abilities to cause proliferation of human peripheral blood mononuclear cells, and differed in their activation of subsets of T cells. A soluble, high-affinity V?-TCR, designed to neutralize the previously characterized variant of SEB (SEB1), was able to neutralize the variant SEBs, indicating that this high-affinity peptide may be useful in treating a variety of SEB-mediated illnesses.
Kohler, Petra L.; Greenwood, Seth D.; Nookala, Suba; Kotb, Malak; Kranz, David M.; Schlievert, Patrick M.
BACKGROUND: The nontoxigenic V. cholerae El Tor strains ferment sorbitol faster than the toxigenic strains, hence fast-fermenting and slow-fermenting strains are defined by sorbitol fermentation test. This test has been used for more than 40 years in cholera surveillance and strain analysis in China. Understanding of the mechanisms of sorbitol metabolism of the toxigenic and nontoxigenic strains may help to
Ruibai Wang; Hongzhi Zhang; Haiyan Qiu; Shouyi Gao; Biao Kan
Background Though multiple single nucleotide polymorphisms (SNPs) associated with type 2 diabetes have been identified, the genetic bases of isolated fasting hyperglycaemia (IFH) and isolated postprandial hyperglycaemia (IPH) were still unclear. In present study, we aimed to investigate the association of genome-wide association study-validated genetic variants and IFH or IPH in Han Chinese. Methods/Principal Findings We genotyped 27 validated SNPs in 6,663 unrelated individuals comprising 341 IFH, 865 IPH, 1,203 combined fasting hyperglycaemia and postprandial hyperglycaemia, and 4,254 normal glycaemic subjects of Han ancestry. The distributions of genotype frequencies of FTO, CDKAL1 and GCKR were significant different between individuals with IFH and those with IPH (SNP(ptrend): rs8050136(0.0024), rs9939609(0.0049), rs7756992(0.0122), rs780094(0.0037)). Risk allele of FTO specifically increased the risk of IFH (rs8050136: OR 1.403 [95% CI 1.125–1.750], p?=?0.0027; rs9939609: 1.398 [1.120–1.744], p?=?0.0030). G allele of CDKAL1 specifically increased the risk of IPH (1.217 [1.092–1.355], p?=?0.0004). G allele of GCKR increased the risk of IFH (1.167 [0.999–1.362], p?=?0.0513), but decreased the risk of IPH (0.891 [0.801–0.991], p?=?0.0331). In addition, TCF7L2 and KCNQ1 increased the risk of both IFH and IPH. When combined, each additional risk allele associated with IFH increased the risk for IFH by 1.246-fold (p<0.0001), while each additional risk allele associated with IPH increased the risk for IPH by 1.190-fold (p<0.0001). Conclusion/Significance Our results indicate that genotype distributions of variants from FTO, GCKR, CDKAL1 were different between IPH and IFH in Han Chinese. Variants of genes modulating insulin sensitivity (FTO, GCKR) contributed to the risk of IFH, while variants of genes related to beta cell function (CDKAL1) increase the risk of IPH.
Chen, Ying; Chen, Li; Zhao, Zhigang; Li, Qiang; Ge, Jiapu; Chen, Gang; Guo, Xiaohui; Lu, Juming; Weng, Jianping; Jia, Weiping; Ji, Linong; Xiao, Jianzhong; Shan, Zhongyan; Liu, Jie; Tian, Haoming; Ji, Qiuhe; Zhu, Dalong; Zhou, Zhiguang; Shan, Guangliang; Yang, Wenying
VP20621, spores of nontoxigenic Clostridium difficile (NTCD) strain M3, is protective against challenge with toxigenic strains in hamsters. Human administration and colonization may prevent primary C. difficile infection (CDI) or recurrent CDI. Healthy adult subjects 18 to 45 years old or ?60 years old received single or multiple doses of an oral suspension of VP20621 (104, 106, or 108 spores) or placebo. Group 4 (?60 years old) received oral vancomycin for 5 days, followed by 14 days of VP20621 or placebo. Subjects were monitored for safety and followed through day 28. Stool was cultured for C. difficile before, during, and after VP20621 administration. Isolates were tested for toxin by enzyme immunoassay, and VP20621 was confirmed by molecular typing. After single escalating doses, no subjects had C. difficile-positive stool cultures. VP20621 was found in the stool of all subjects given 108 spores twice a day. Following vancomycin administration, VP20621 was detected in the stool of all subjects given 104, 106, or 108 spores daily beginning on day 2 to 6. Recovered isolates were toxin negative and confirmed to be VP20621. There were no serious adverse events, and no subjects prematurely discontinued study drugs. Following vancomycin administration, 2 placebo subjects became colonized with toxigenic C. difficile and 3 placebo subjects became colonized with VP20621. Persistent colonization with VP20621 was detected in stools on days 21 to 28 in 44% of subjects. VP20621 was well tolerated and able to colonize the gastrointestinal tracts of subjects pretreated with vancomycin. Further study of VP20621 to prevent CDI in patients is warranted.
Villano, Stephen A.; Seiberling, Michael; Tatarowicz, Walter; Monnot-Chase, Elizabeth
In this study, we determined vt subtypes and evaluated verotoxicity in basal as well as induced conditions of verotoxin-producing Escherichia coli (VTEC) strains isolated from cattle and meat products. Most (87%) of the 186 isolates carried a vt2 gene. Moreover, the vt2 subtype, which is associated with serious disease, was present in 42% of our VTEC collection. The other vt subtypes detected were vt1, vt1d, vt2vha, vt2vhb, vt2O118, vt2d (mucus activatable), and vt2g. A total of 41 (22%) of the isolates possessed more than one vt subtype in its genome, and among them the most frequent combination was vt1/vt2, but we also observed multiple combinations among vt2 subtypes. Differences in verotoxicity titers were found among a selection of 54 isolates. Among isolates with a single vt2 variant, those carrying the vt2 subtype had high titers under both uninduced and induced conditions. However, the highest increase in cytotoxicity under mitomycin C treatment was detected among the strains carrying vt2vha or vt2hb variants. Notably, the isolates carrying the vt1 subtype showed a lesser increase than that of most of the vt2-positive VTEC strains. Furthermore, the presence of more than one vt gene variant in the same isolate was not reflected in higher titers, and generally the titers were lower than those for strains with only one gene variant. The main observation was that both basal and induced cytotoxic effects seemed to be associated with the type and number of vt variants more than with the serotype or origin of the isolate.
Kruger, Alejandra; Lucchesi, Paula M. A.; Parma, Alberto E.
Effective treatment of anthrax is hampered by our limited understanding of the pathophysiology of Bacillus anthracis infection. We used a genetically complete (pXO1+ pXO2+) virulent B. anthracis strain and four isogenic toxin-null mutants to determine the effects of the anthrax edema toxin (ET; edema factor [EF] plus protective antigen [PA]) and lethal toxin (LT; lethal factor [LF] plus PA) on the host innate response during systemic infection. Using the spleen as an indicator for host response, we found that intravenous inoculation of LT-deficient mutants into C57BL/6 mice significantly increased production of several cytokines over that observed after infection with the parent strain or an EF-deficient mutant. Bacteria producing one or both of the toxins were capable of inducing significant apoptosis of cells present in spleens, whereas apoptosis was greatly reduced in mice infected with nontoxigenic mutants. Mice infected with toxin-producing strains also showed increased splenic neutrophil recruitment compared to mice infected with nontoxigenic strains and neutrophil depletion prior to infection with toxin-producing strains, leading to decreased levels of apoptosis. Together, these studies indicate that anthrax LT suppresses cytokine secretion during infection, but both EF and LF play roles in inducing neutrophil recruitment and enhancing apoptosis. Interestingly, in the absence of LF the effect of EF-induced cell recruitment is further enhanced, perhaps because LF so effectively suppresses the secretion of chemokines.
Drysdale, Melissa; Olson, Gwyneth; Koehler, Theresa M.; Lipscomb, Mary F.; Lyons, C. Rick
Salmonella enterica serovar Typhimurium variant Copenhagen was isolated from 5 of 152 (3.3%) feral pigeons from the city of Ghent (Belgium) and from 26 pooled fecal samples from 114 pigeon lofts (22.8%). These isolates belonged to phage type (PT) 99. Seven of the pigeon isolates were further compared in vitro to five human variant Copenhagen isolates, 2 isolates of PT 208, 1 isolate each of PT 120 and U302, and a nontypeable isolate. No differences in invasiveness in human intestinal epithelial Caco-2 cells were found. The human strains, however, were able to multiply significantly more inside human THP-1 macrophages than the pigeon strains. After inoculation of mice with a pigeon PT 99 strain, high numbers of Salmonella bacteria were shed with the feces, the internal organs were heavily colonized, and the animals showed severe clinical symptoms resulting in death. In conclusion, the less-pronounced ability of the pigeon variant Copenhagen strains to multiply inside human macrophages than human strains as well as the lack of human PT 99 isolates during 2002, despite the relatively high frequency of this PT in the pigeon population, suggest these strains to be of low virulence to humans. However, the high virulence for mice of the tested strain implies that rodents may act as reservoirs. PMID:15131161
Pasmans, Frank; Van Immerseel, Filip; Hermans, Katleen; Heyndrickx, Marc; Collard, Jean-Marc; Ducatelle, Richard; Haesebrouck, Freddy
Kobuviruses comprise three species, the Aichivirus A, Aichivirus B, and Aichivirus C (porcine kobuvirus). Porcine kobuvirus is endemic to pig farms and is not restricted geographically but, rather, is distributed worldwide. The complete genomic sequences of four porcine kobuvirus strains isolated during a diarrhea outbreak in piglets in the Gansu province of China were determined. Two of these strains exhibited variations relative to the traditional strains. The potential 3C/3D cleavage sites of the variant strains were Q/C, which differed from the Q/S in the traditional porcine kobuvirus genome. A 90-nucleotide deletion in the 2B protein and a single nucleotide insertion in the 3'UTR were found in the variant strains. The VP1 regions of all four porcine kobuviruses in our study were highly variable (81%-86%). Ten common amino acid mutations were found specifically at certain positions within the VP1 region. Significant recombination sites were identified using SimPlot scans of whole genome sequences. Porcine kobuviruses were also detected in pig serum, indicating that the virus can escape the gastrointestinal tract and travel to the circulatory system. These findings suggest that mutations and recombination events may have contributed to the high level of genetic diversity of porcine kobuviruses and serve as a driving force in its evolution. PMID:24145960
Fan, Shengtao; Sun, Heting; Ying, Ying; Gao, Xiaolong; Wang, Zheng; Yu, Yicong; Li, Yuanguo; Wang, Tiecheng; Yu, Zhijun; Yang, Songtao; Zhao, Yongkun; Qin, Chuan; Gao, Yuwei; Xia, Xianzhu
Experiments were conducted to determine the potential for biological control of aflatoxin contamination of peanuts during storage. Florunner peanuts were treated in field plots by applying competitive, nontoxigenic strains of Aspergillus flavus and A. parasiticus, at 76 and 67 days after planting in 1998 and 1999, respectively. After harvest, half the peanuts from both treated and control plots were sprayed
Joe W Dorner; Richard J Cole
The healthy skin of two female domestic pigs (Sus scrofa domestica) was sampled with cotton-tipped swabs. Total genomic DNA was extracted from the samples and subjected to PCR with degenerate papillomavirus (PV)-specific primers. Similarity searches performed with blastn showed that partial E1 and L1 sequences of two novel PVs were amplified. Subsequently, the complete genomes of these Sus scrofa papillomaviruses (SsPVs) were amplified by long-template PCR, cloned and sequenced using a transposon insertion method. They contained the typical PV open reading frames (ORFs) E1, E2, E4, E6, L1 and L2, but the E7 ORF was absent in both viruses. Pairwise nucleotide sequence alignment of the L1 ORFs of the SsPVs showed 98.5 % similarity, classifying these viruses as SsPV type 1 'variants' (SsPV-1a and -1b). Based on a concatenated alignment of the E1, E2, L1 and L2 ORFs of SsPV-1 variants a and b, and 81 other human and animal PV type species, a neighbour-joining phylogenetic tree was constructed. This phylogenetic analysis showed that the SsPV-1a and -1b variants did not cluster with the other PVs of artiodactyls (cloven-hoofed) host species, but clustered on the edge of the genus Alphapapillomavirus, very near to the root of this genus. PMID:18796716
Stevens, Hans; Rector, Annabel; Van Der Kroght, Kees; Van Ranst, Marc
Two clinical strains of Klebsiella pneumoniae (K. pneumoniae) and one isolate of Escherichia coli (E. coli) were collected from two large general hospitals in China. Conjugation experiment, susceptibility testing, isoelectric focusing, PCR, and sequencing techniques as well as clone, expression, purification and kinetics were carried out to describe the characterization of the novel SHV-tpye enzyme. The analysis of plasmid profiling and pulsed-field gel electrophoresis of the novel enzyme were performed to investigate epidemiology. These isolates had CTX-M-14 and SHV-89 beta-lactamases. SHV-89 beta-lactamase of pI 7.6 is a novel variant with two substitutions compared with the sequence of SHV-1: Leu35Gln and Met129Val. Its gene also had two silent mutations at positions 369 and 774, respectively. The results of substrate profiles and MIC determinations showed the activity of the novel enzyme was insufficient for the enzyme to count as an extended-spectrum beta-lactamase (ESBL). The substrates of the enzyme were also characterized. Furthermore, the three novel SHV enzyme-producing strains were epidemiologically unrelated. The emergence of a novel SHV-type beta-lactamase is rarely described in other areas. This study illustrates the importance of molecular survelliance in tracking SHV-producing strains in large teaching hospitals and emphasizes the need for epidemiological monitoring. PMID:18587684
Li, Jia-Bin; Cheng, Jun; Wang, Qian; Chen, Yan; Ye, Ying; Zhang, Xue-Jun
BACKGROUND—Hirschsprung disease (HSCR), which may be sporadic or familial, occurs in 1:5000 live births and presents with functional intestinal obstruction secondary to aganglionosis of the hindgut. Germline mutations of the RET proto-oncogene are believed to account for up to 50% of familial cases and up to 30% of isolated cases in most series. However, these series are highly selected for the most obvious and severe cases and large familial aggregations. Population based studies indicate that germline RET mutations account for no more than 3% of isolated HSCR cases. Recently, we and others have noted that specific polymorphic sequence variants, notably A45A (exon 2), are over-represented in isolated HSCR.?PURPOSE—In order to determine if it is the variant per se, a combination thereof, or another locus in linkage disequilibrium which predisposes to HSCR, we looked for association of RET haplotype(s) and disease in HSCR cases compared to region matched controls.?METHODS—Seven loci across RET were typed and haplotypes formed for HSCR cases, their unaffected parents, and region matched controls. Haplotype and genotype frequencies and distributions were compared among these groups using the transmission disequilibrium test and standard case-control statistic.?RESULTS—Twelve unique haplotypes, labelled A-L, were obtained. The distributions of haplotypes between cases and controls (?112 =81.4, p<<0.0001) and between cases and non-transmitted parental haplotypes were significantly different (?211=53.1, p<0.0001). Genotypes comprising pairs of haplotypes were formed for cases and controls. There were 38 different genotypes among cases and controls combined. Inspection of the genotypes in these two groups showed that the genotype distribution between cases and controls was distinct (?372=93.8, p<<0.0001). For example, BB, BC, BD, and CD, all of which contain at least one allele with the polymorphic A45A, are prominently represented among HSCR cases, together accounting for >35% of the case genotypes, yet these four genotypes were not represented among the population matched normal controls. Conversely, AA, AG, DD, GG, and GJ, none of which contains A45A, are commonly represented in the controls, together accounting for 43% of the control genotypes, and yet they are never seen among the HSCR cases.?CONCLUSIONS—Our data suggest that genotypes comprising specific pairs of RET haplotypes are associated with predisposition to HSCR either in a simple autosomal recessive manner or in an additive, dose dependent fashion.???Keywords: transmission disequilibrium test; chromosome 10; polymorphisms
Borrego, S.; Ruiz, A.; Saez, M. E.; Gimm, O.; Gao, X.; Lopez-Alonso, M.; Hernandez, A.; Wright, F.; Antinolo, G.; Eng, C.
Methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered in Irish hospitals between 1971 and 2002 were characterized using multilocus sequence typing (MLST) (n = 130) and SCCmec typing (n = 172). Where atypical SCCmec typing results were obtained, PCR amplification of entire SCCmec elements, analysis of amplimer mobility, and nucleotide sequencing were undertaken. MLST revealed that 129/130 isolates had the same genotypes as internationally spread MRSA clones, including ST239, ST247, ST250, ST5, ST22, ST36, and ST8. A novel genotype, ST496, was identified in one isolate. Half of the isolates (86/172) had SCCmec type I, IA, II, III, or IV. The remaining 86 isolates harbored novel SCCmec variants in three distinct genetic backgrounds: (i) 74/86 had genotype ST8 and either one of five novel SCCmec II (IIA, IIB, IIC, IID, and IIE) or one of two novel SCCmec IV (IVE and IVF) variants; (ii) 3/86 had genotype ST239 and a novel SCCmec III variant; (iii) 9/86 had a novel SCCmec I variant associated with ST250. SCCmec IVE and IVF were similar to SCCmec IVc and IVb, respectively, but differed in the region downstream of mecA. The five SCCmec II variants were similar to SCCmec IVb in the region upstream of the ccr complex but otherwise were similar to SCCmec II, except for the following regions: SCCmec IIA and IID had a novel mec complex, A.4 (?mecI-IS1182-?mecI-mecR1-mecA-IS431mec); SCCmec IIC and IIE had a novel mec complex, A.3 (IS1182-?mecI-mecR1-mecA-IS431mec); SCCmec IID and IIE lacked pUB110; SCCmec IIC and IIE lacked a region of DNA between Tn554 and the mec complex; and SCCmec IIB lacked Tn554. This study has demonstrated a hitherto-undescribed degree of diversity within SCCmec.
Shore, Anna; Rossney, Angela S.; Keane, Conor T.; Enright, Mark C.; Coleman, David C.
Abstract Salmonella 4,,12:i:- is a variant of Salmonella Typhimurium, which lacks the expression of phase-2 flagellar antigen, generally associated with the deletion of the fljB gene. Additional mechanisms involving the fljAB operon (?fljA, fljB, and hin genes) lead to the lack of expression of phase-2 flagellar antigens also in Salmonella strains harboring the fljB gene. For 20 S. 4,,12:i:- strains, defined as "inconsistent" Salmonella Typhimurium variants since they had phenotypically behaved as monophasic, even though the fljB gene was conserved, the fljAB operon was characterized in order to explain the ineffective expression of the phase-2 flagellar antigen. The monophasic phenotype for a first group of strains (9) was likely due to the absence of the hin gene, leading to the inhibited switch between the expression of phase-1 and phase-2 flagellar genes. For a second group of strains (5), the monophasic phenotype could be attributed to nonconservative point mutations identified in fljA and hin genes, which could hamper the proper expression of invertase gene and the fljA, acting as repressor of the phase-1 flagellar gene. Finally, for a last group of inconsistent strains (6), a plausible reason for their monophasic phenotype was not found, since the genes involved in the expression of phase-2 flagellar antigen were fully conserved. Moreover, the collection of inconsistent Salmonella Typhimurium isolates investigated were characterized by distinct molecular profiles, as demonstrated by multiple-locus variable-number tandem repeat analysis, and phenotype variability, as demonstrated by phage-typing. This study highlights the usefulness of investigating the entire fljAB operon when a definitive identification of the monophasic or biphasic status of Salmonella Typhimurium strains is needed (for instance, in the context of epidemiological investigations aimed to identify the relatedness among strains). PMID:24666380
Barco, Lisa; Longo, Alessandra; Lettini, Antonia Anna; Cortini, Enzo; Saccardin, Cristina; Minorello, Claudio; Olsen, John Elmerdahl; Ricci, Antonia
Background & Aims The Helicobacter pylori toxin vacuolating cytotoxin (VacA) promotes gastric colonization and its presence (VacA+) is associated with more-severe disease. The exact mechanisms by which VacA contributes to infection are unclear. We previously found that limited exposure to VacA induces autophagy of gastric cells, which eliminates the toxin; we investigated whether autophagy serves as a defense mechanism against H pylori infection. Methods We investigated the effect of VacA on autophagy in human gastric epithelial cells (AGS) and primary gastric cells from mice. Expression of p62, a marker of autophagy, was also assessed in gastric tissues from patients infected with toxigenic (VacA+) or nontoxigenic strains. We analyzed the effect of VacA on autophagy in peripheral blood monocytes obtained from subjects with different genotypes of ATG16L1, which regulates autophagy. We performed genotyping for ATG16L1 in two cohorts of infected and uninfected subjects. Results Prolonged exposure of AGS and mouse gastric cells to VacA disrupted induction of autophagy in response to the toxin, because the cells lacked cathepsin-D in autophagosomes. Loss of autophagy resulted in the accumulation of p62 and reactive oxygen species. Gastric biopsies samples from patients infected with VacA+, but not nontoxigenic strains of H pylori, had increased levels of p62. Peripheral blood monocytes isolated from individuals with polymorphisms in ATG16L1 that increase susceptibility to Crohn's disease had reduced induction of autophagy in response to VacA+ compared to cells from individuals that did not have these polymorphisms. The presence of the ATG16L1 Crohn’s disease risk variant increased susceptibility to H pylori infection in 2 separate cohorts. Conclusions Autophagy protects against infection with H pylori; the toxin VacA disrupts autophagy to promote infection, which could contribute to inflammation and eventual carcinogenesis.
Raju, D; Hussey, S; Ang, M; Terebiznik, M.R.; Sibony, M; Galindo-Mata, E; Gupta, V; Blanke, S.R.; Delgado, A; Romero-Gallo, J; Ramjeet, M; Mascarenhas, H; Peek, R.M.; Correa, P; Streutker, C; Hold, G; Kunstmann, E; Yoshimori, T; Silverberg, M. S.; Girardin, S.E.; Philpott, D.J.; El Omar, E; Jones, N.L.
A degenerated strain of Pleurotus eryngii KNR2312 was isolated from a commercial farm. Random amplified polymorphic DNA analysis performed on the genomic DNA of the normal and degenerated strains of this species revealed differences in the DNA banding pattern. A unique DNA fragment (1.7 kbp), which appeared only in the degenerated strain, was isolated and sequenced. Comparing this sequence with the KNR2312 genomic sequence showed that the sequence of the degenerated strain comprised three DNA regions that originated from nine distinct scaffolds of the genomic sequence, suggesting that chromosome-level changes had occurred in the degenerated strain. Using the unique sequence, three sets of PCR primers were designed that targeted the full length, the 5' half, and the 3' half of the DNA. The primer sets P2-1 and P2-2 yielded 1.76 and 0.97 kbp PCR products, respectively, only in the case of the degenerated strain, whereas P2-3 generated a 0.8 kbp product in both the normal and the degenerated strains because its target region was intact in the normal strain as well. In the case of the P2-1 and P2-2 sets, the priming regions of the forward and reverse primers were located at distinct genomic scaffolds in the normal strain. These two primer sets specifically detected the degenerate strain of KNR2312 isolated from various mushrooms including 10 different strains of P. eryngii, four strains of P. ostreatus, and 11 other wild mushrooms. PMID:24808734
Lee, Hyun-Jun; Kim, Sang-Woo; Ryu, Jae-San; Lee, Chang-Yun; Ro, Hyeon-Su
A degenerated strain of Pleurotus eryngii KNR2312 was isolated from a commercial farm. Random amplified polymorphic DNA analysis performed on the genomic DNA of the normal and degenerated strains of this species revealed differences in the DNA banding pattern. A unique DNA fragment (1.7 kbp), which appeared only in the degenerated strain, was isolated and sequenced. Comparing this sequence with the KNR2312 genomic sequence showed that the sequence of the degenerated strain comprised three DNA regions that originated from nine distinct scaffolds of the genomic sequence, suggesting that chromosome-level changes had occurred in the degenerated strain. Using the unique sequence, three sets of PCR primers were designed that targeted the full length, the 5' half, and the 3' half of the DNA. The primer sets P2-1 and P2-2 yielded 1.76 and 0.97 kbp PCR products, respectively, only in the case of the degenerated strain, whereas P2-3 generated a 0.8 kbp product in both the normal and the degenerated strains because its target region was intact in the normal strain as well. In the case of the P2-1 and P2-2 sets, the priming regions of the forward and reverse primers were located at distinct genomic scaffolds in the normal strain. These two primer sets specifically detected the degenerate strain of KNR2312 isolated from various mushrooms including 10 different strains of P. eryngii, four strains of P. ostreatus, and 11 other wild mushrooms.
Lee, Hyun-Jun; Kim, Sang-Woo; Ryu, Jae-San; Lee, Chang-Yun
We developed a method for rapidly generating thermostable enzyme variants. Our strategy is to introduce the gene coding for a given enzyme from a mesophilic organism into a thermophile, Bacillus stearothermophilus. Variants that retain the enzymatic activity at the higher growth temperatures of the thermophile are then selected. This strategy was applied to kanamycin nucleotidyltransferase, which confers resistance to the
Hans Liao; Tim McKenzie; Robert Hageman
Isolated clubfoot is a relatively common birth defect that affects approximately 4,000 newborns in the US each year. Calf muscles in the affected leg(s) are underdeveloped and remain small even after corrective treatment. This observation suggests that variants in genes that influence muscle development are priority candidate risk factors for clubfoot. This contention is further supported by the discovery that mutations in genes that encode components of the muscle contractile complex (MYH3, TPM2, TNNT3, TNNI2, and MYH8) cause congenital contractures, including clubfoot, in distal arthrogryposis (DA) syndromes. Interrogation of fifteen genes encoding proteins that control myofiber contractility in a cohort of both nonHispanic white (NHW) and Hispanic families, identified positive associations (p<0.05) with SNPs in twelve genes; only one was identified in a family-based validation dataset. Six SNPs in TNNC2 deviated from Hardy Weinberg Equilibrium (HWE) in mothers in our NHW discovery dataset. Relative risk and likelihood ratio tests showed evidence for a maternal genotypic effect with TNNC2/rs383112 and an inherited/child genotypic effect with two SNPs, TNNC2/rs4629 and rs383112. Associations with multiple SNPs in TPM1 were identified in the NHW discovery (rs4075583, p=0.01), family-based validation (rs1972041, p=0.000074) and case-control validation (rs12148828, p=0.04) datasets. Gene interactions were identified between multiple muscle contraction genes with many of the interactions involving at least one potential regulatory SNP. Collectively, our results suggest that variation in genes that encode contractile proteins of skeletal myofibers may play a role in the etiology of clubfoot.
Weymouth, Katelyn S.; Blanton, Susan H.; Bamshad, Michael J.; Beck, Anita E.; Alvarez, Christine; Richards, Steve; Gurnett, Christina A.; Dobbs, Matthew B.; Barnes, Douglas; Mitchell, Laura E.; Hecht, Jacqueline T.
Background Congenital malformations involving the Müllerian ducts are observed in around 5% of infertile women. Complete aplasia of the uterus, cervix, and upper vagina, also termed Müllerian aplasia or Mayer–Rokitansky–Kuster–Hauser (MRKH) syndrome, occurs with an incidence of around 1 in 4500 female births, and occurs in both isolated and syndromic forms. Previous reports have suggested that a proportion of cases, especially syndromic cases, are caused by variation in copy number at different genomic loci. Methods In order to obtain an overview of the contribution of copy number variation to both isolated and syndromic forms of Müllerian aplasia, copy number assays were performed in a series of 63 cases, of which 25 were syndromic and 38 isolated. Results A high incidence (9/63, 14%) of recurrent copy number variants in this cohort is reported here. These comprised four cases of microdeletion at 16p11.2, an autism susceptibility locus not previously associated with Müllerian aplasia, four cases of microdeletion at 17q12, and one case of a distal 22q11.2 microdeletion. Microdeletions at 16p11.2 and 17q12 were found in 4/38 (10.5%) cases with isolated Müllerian aplasia, and at 16p11.2, 17q12 and 22q11.2 (distal) in 5/25 cases (20%) with syndromic Müllerian aplasia. Conclusion The finding of microdeletion at 16p11.2 in 2/38 (5%) of isolated and 2/25 (8%) of syndromic cases suggests a significant contribution of this copy number variant alone to the pathogenesis of Müllerian aplasia. Overall, the high incidence of recurrent copy number variants in all forms of Müllerian aplasia has implications for the understanding of the aetiopathogenesis of the condition, and for genetic counselling in families affected by it.
Nik-Zainal, Serena; Strick, Reiner; Storer, Mekayla; Huang, Ni; Rad, Roland; Willatt, Lionel; Fitzgerald, Tomas; Martin, Vicki; Sandford, Richard; Carter, Nigel P; Janecke, Andreas R; Renner, Stefan P; Oppelt, Patricia G; Oppelt, Peter; Schulze, Christine; Brucker, Sara; Hurles, Matthew; Beckmann, Matthias W; Strissel, Pamela L; Shaw-Smith, Charles
The homotetrameric lactate dehydrogenase (LDH) isozymes, B2 prime and B2 double prime, from normal and variant rainbow trout livers have been purified to homogeneity by affinity chromatography using a Sepharose-linked oxamate ligand. The two isozymes have...
Y. H. J. Kao
A strategy to control the devastating late blight disease is providing potato cultivars with genes that are effective in resistance to a broad spectrum of Phytophthora infestans isolates. Thus far, most late blight resistance (R) genes that were introgressed in potato were quickly defeated. In contrast, the Rpi-blb1 gene originating from Solanum bulbocastanum has performed as an exclusive broad-spectrum R gene for many years. Recently, the RXLR effector family ipiO was identified to contain Avr-blb1. Monitoring the genetic diversity of the ipiO family in a large set of isolates of P. infestans and related species resulted in 16 ipiO variants in three distinct classes. Class I and class II but not class III ipiO variants induce cell death when coinfiltrated with Rpi-blb1 in Nicotiana benthamiana. Class I is highly diverse and is represented in all analyzed P. infestans isolates except two Mexican P. infestans isolates, and these were found virulent on Rpi-blb1 plants. In its C-terminal domain, IPI-O contains a W motif that is essential for triggering Rpi-blb1-mediated cell death and is under positive selection. This study shows that profiling the variation of Avr-blb1 within a P. infestans population is instrumental for predicting the effectiveness of Rpi-blb1-mediated resistance in potato. PMID:19888819
Champouret, Nicolas; Bouwmeester, Klaas; Rietman, Hendrik; van der Lee, Theo; Maliepaard, Chris; Heupink, Anika; van de Vondervoort, Peter J I; Jacobsen, Evert; Visser, Richard G F; van der Vossen, Edwin A G; Govers, Francine; Vleeshouwers, Vivianne G A A
Chinese hamster ovary cell populations were enriched for cells displaying low surface expression of the 140-kD integrin fibronectin receptor (FnR) by means of fluorescence-activated cell sorting using monoclonal anti-FnR antibodies. Selected cells were cloned by limiting dilution, and the resulting clones were screened for low cell surface FnR expression by ELISA. Two multiply sorted populations gave rise to variant clones possessing approximately 20 or 2% FnR expression, respectively, compared with wild-type cells. Growth rates of the "20%" and "2%" clones on serum-coated plastic dishes were similar to that of wild-type cells. Variant cells expressing 20% FnR could attach and spread on substrata coated with purified fibronectin, although somewhat more slowly than wild-type cells, while cells expressing 2% FnR could not attach or spread. Cells from all variant clones attached normally to vitronectin substrata, but some of the 2% clones displayed altered morphology on this type of substratum. Motility assays in blind well chambers showed a correlation of movement with level of expression of FnR. The number of cells migrating in response to fibronectin was greatly reduced compared with wild-type cells for the 20% FnR variant clones, while variant clones with 2% FnR showed virtually no migratory activity. Surface labeling with 125I and immunoaffinity purification of FnR showed reduced levels of intact FnR on the plasma membranes of variants with 20% FnR, while none was detected in variants expressing 2% FnR. Nevertheless, beta subunits were detected on the surfaces of all variant clones. Immunoblots of cell lysates from wild-type cells and from both types of variant clones showed substantial amounts of FnR beta chain as well as enhanced amounts of a pre-beta moiety in the variants. alpha chain was markedly reduced in the 20% variants and essentially absent in the 2% variants, indicating that failure to assemble intact FnR in these variants was due to deficiencies of alpha chain production. Dot blots of total mRNA from a representative clone expressing 20% FnR showed reduced levels of material hybridizing to an 0.97-kb hamster FnR alpha chain cDNA probe as compared with wild type, while mRNA from a representative clone expressing 2% FnR had no detectable hybridizable RNA; this seems to agree well with the results obtained by immunoblotting. Thus, the defect in the variant clones seems to be due to reduced levels of alpha chain mRNA leading to a deficit of mature FnR and consequent alterations in cell adhesion and motility on fibronectin substrata.
Glycoprotein E-negative (gE–) laboratory strains of bovine herpesvirus 1 (BHV-1) were recently introduced as novel marker vaccines, allowing serological discrimination between vaccinated and naturally infected animals on the basis of lack or presence of antibodies against gE epitopes. The applicability of this approach is based on the genetic stability of the gE. However, mutant field variants of BHV-1 with a
L. Egyed; C. Ros; S. Belák
Chinese hamster ovary cell populations were enriched for cells displaying low surface expres- sion of the 140-kD integrin fibronectin receptor (FnR) by means of fluorescence-activated cell sorting using monoclonal anti-FnR antibodies. Selected cells were cloned by limiting dilution, and the resulting clones were screened for low cell surface FnR expression by ELISA. Two multiply sorted populations gave rise to variant
Clara L. Schreiner; J. S. Bauer; Yuri N. Danilov; Saber Hussein; Melanie M. Sczekan; R. L. Juliano
Fowlpox virus (FPV) is one example of poultry viruses which undergoes recombination with Reticuloendotheliosis virus (REV). Trepidation had been raised, and it was well established on augmented pathogenicity of the FPV upon integration of the full intact REV. In this study, we therefore intended at assessing the integration of REV into FPV genome of the field isolates obtained in samples collected from different regions of Tanzania. DNA extraction of 85 samples (scabs) was performed, and FPV-specific PCR was done by the amplification of the highly conserved P4b gene. Evaluation of FPV-REV recombination was done to FPV-specific PCR positively identified samples by amplifying the env gene and REV long terminal repeats (5' LTR). A 578-bp PCR product was amplified from 43 samples. We are reporting for the first time in Tanzania the existence of variant stains of FPV integrated with REV in its genome as 65 % of FPV identified isolates were having full intact REV integration, 21 % had partial FPV-REV env gene integration and 5 % had partial 5' LTR integration. Despite of the fact that FPV-REV integrated stains prevailed, FPV-REV-free isolates (9 %) also existed. In view of the fact that full intact REV integration is connected with increased pathogenicity of FPV, its existence in the FPV genome of most field isolates could have played a role in increased endemic, sporadic and recurring outbreaks in selected areas in Tanzania. PMID:24557589
Mzula, Alexanda; Masola, Selemani N; Kasanga, Christopher J; Wambura, Philemon N
Sixteen variants of apolipoprotein A-I (ApoA-I) are associated with hereditary systemic amyloidoses, characterized by amyloid deposition in peripheral organs of patients. As these are heterozygous for the amyloidogenic variants, their isolation from plasma is impracticable and recombinant expression systems are needed. Here we report the expression of recombinant ApoA-I amyloidogenic variant Leu174 with Ser (L174S) in stably transfected Chinese hamster ovary-K1 cells. ApoA-I variant L174S was found to be efficiently secreted in the culture medium, from which it was isolated following a one-step purification procedure. Mass spectrometry analyses allowed the qualitative and quantitative definition of the amyloidogenic variant lipid content, which was found to consist of two saturated and two monounsaturated fatty acids. Interestingly, the same lipid species were found to be associated with the wild-type ApoA-I, expressed and isolated using the same cell system, with lower values of the lipid to protein molar ratios with respect to the amyloidogenic variant. A possible role of fatty acids in trafficking and secretion of apolipoproteins may be hypothesized. PMID:22295944
Monti, Daria Maria; Di Gaetano, Sonia; Del Giudice, Rita; Giangrande, Chiara; Amoresano, Angela; Monti, Maria; Arciello, Angela; Piccoli, Renata
Mitochondrial DNA (mtDNA) sequence variation can influence the penetrance of complex diseases and climatic adaptation. While studies in geographically defined human populations suggest that mtDNA mutations become fixed when they have conferred metabolic capabilities optimally suited for a specific environment, it has been challenging to definitively assign adaptive functions to specific mtDNA sequence variants in mammals. We investigated whether mtDNA genome variation functionally influences Caenorhabditis elegans wild isolates of distinct mtDNA lineages and geographic origins. We found that, relative to N2 (England) wild-type nematodes, CB4856 wild isolates from a warmer native climate (Hawaii) had a unique p.A12S amino acid substitution in the mtDNA-encoded COX1 core catalytic subunit of mitochondrial complex IV (CIV). Relative to N2, CB4856 worms grown at 20°C had significantly increased CIV enzyme activity, mitochondrial matrix oxidant burden, and sensitivity to oxidative stress but had significantly reduced lifespan and mitochondrial membrane potential. Interestingly, mitochondrial membrane potential was significantly increased in CB4856 grown at its native temperature of 25°C. A transmitochondrial cybrid worm strain, chpIR (M, CB4856>N2), was bred as homoplasmic for the CB4856 mtDNA genome in the N2 nuclear background. The cybrid strain also displayed significantly increased CIV activity, demonstrating that this difference results from the mtDNA-encoded p.A12S variant. However, chpIR (M, CB4856>N2) worms had significantly reduced median and maximal lifespan relative to CB4856, which may relate to their nuclear-mtDNA genome mismatch. Overall, these data suggest that C. elegans wild isolates of varying geographic origins may adapt to environmental challenges through mtDNA variation to modulate critical aspects of mitochondrial energy metabolism. PMID:24534730
Dingley, Stephen D; Polyak, Erzsebet; Ostrovsky, Julian; Srinivasan, Satish; Lee, Icksoo; Rosenfeld, Amy B; Tsukikawa, Mai; Xiao, Rui; Selak, Mary A; Coon, Joshua J; Hebert, Alexander S; Grimsrud, Paul A; Kwon, Young Joon; Pagliarini, David J; Gai, Xiaowu; Schurr, Theodore G; Hüttemann, Maik; Nakamaru-Ogiso, Eiko; Falk, Marni J
A slowly milk-coagulating variant (Fmc?) of Lactobacillus helveticus CRL 1062, designated S1, was isolated and characterized. Strain S1 possessed all the known essential components required to utilize casein as a nitrogen source, which include functional proteinase and peptidase activities as well as functional amino acid, di- and tripeptide, and oligopeptide transport systems. The amino acid requirements of strain S1 were similar to those of the parental strain. However, on a purine-free, chemically defined medium, the growth rate of the Fmc? strain was threefold lower than that of the wild-type strain. L. helveticus S1 was found to be defective in IMP dehydrogenase activity and therefore was deficient in the ability to synthesize XMP and GMP. This conclusion was further supported by the observation that the addition of guanine or xanthine to milk, a substrate poor in purine compounds, restored the Fmc+ phenotype of L. helveticus S1.
Hebert, Elvira M.; De Giori, Graciela S.; Raya, Raul R.
The genetic relationship among the three color variants (Red, Green, and Black) of the Japanese sea cucumber, S. japonicus, was investigated using 11 microsatellite markers. Genetic differentiation testing among the three sympatric color types showed the strong heterogeneity of Red (p<0.001), while no significant difference was observed between Green and Black (p=0.301 to 0.961). UPGMA trees constructed from 10 sample lots from 5 localities showed two distinct clusters, one from the Red types and the other from the Green and Black types. In addition, the sympatric Green and Black formed one subcluster with strong bootstrap support at each locality. These results indicate the separate species status of Red and the other color types, and also support the population identity of sympatric Green and Black. PMID:17043749
Kanno, Manami; Suyama, Yoshihisa; Li, Qi; Kijima, Akihiro
To validate the identification of Pasteurella multocida-like bacteria negative for acid formation from sucrose, including isolates from bite wounds caused by large cats, 17 strains were phenotypically and genotypically characterized. Phylogenetic analysis of partially sequenced rpoB and infB genes showed the monophyly of the strains characterized and the reference strains of P. multocida. The sucrose-negative strains formed two groups, one related to reference strains of P. multocida and the other related to a separate species-like group (taxon 45 of Bisgaard). DNA-DNA hybridization further documented the species-like nature of this group. Ribotyping showed the heterogeneity of all strains except four strains that shared the same ribotype and that were isolated from bovine lungs. Phylogenetic analysis by 16S rRNA sequence comparison showed the monophyly of the strains characterized and the reference strains of P. multocida. Two strains isolated from leopard bite wounds were related to the type strain of P. dagmatis; however, they represented a new taxon (taxon 46 of Bisgaard), in accordance with their distinct phenotypic and genotypic identifications. The present study documents that sucrose-negative strains of P. multocida-like bacteria belong to two genotypically distinct groups. The study further confirms the phenotypic heterogeneity of P. multocida strains and documents two new species-like taxa of Pasteurella related to P. multocida. Until diagnostic tools have been further elaborated, special care should be taken in the identification of Pasteurella-like bacteria isolated from bite wounds caused by large cats. The evidence of phenotypic and genotypic divergence calls for the further development of PCR tests and DNA sequencing to document doubtful isolates.
Christensen, Henrik; Bisgaard, Magne; Angen, ?ystein; Frederiksen, Wilhelm; Olsen, John Elmerdahl
To validate the identification of Pasteurella multocida-like bacteria negative for acid formation from sucrose, including isolates from bite wounds caused by large cats, 17 strains were phenotypically and genotypically characterized. Phylogenetic analysis of partially sequenced rpoB and infB genes showed the monophyly of the strains characterized and the reference strains of P. multocida. The sucrose-negative strains formed two groups, one related to reference strains of P. multocida and the other related to a separate species-like group (taxon 45 of Bisgaard). DNA-DNA hybridization further documented the species-like nature of this group. Ribotyping showed the heterogeneity of all strains except four strains that shared the same ribotype and that were isolated from bovine lungs. Phylogenetic analysis by 16S rRNA sequence comparison showed the monophyly of the strains characterized and the reference strains of P. multocida. Two strains isolated from leopard bite wounds were related to the type strain of P. dagmatis; however, they represented a new taxon (taxon 46 of Bisgaard), in accordance with their distinct phenotypic and genotypic identifications. The present study documents that sucrose-negative strains of P. multocida-like bacteria belong to two genotypically distinct groups. The study further confirms the phenotypic heterogeneity of P. multocida strains and documents two new species-like taxa of Pasteurella related to P. multocida. Until diagnostic tools have been further elaborated, special care should be taken in the identification of Pasteurella-like bacteria isolated from bite wounds caused by large cats. The evidence of phenotypic and genotypic divergence calls for the further development of PCR tests and DNA sequencing to document doubtful isolates. PMID:15634981
Christensen, Henrik; Bisgaard, Magne; Angen, Oystein; Frederiksen, Wilhelm; Olsen, John Elmerdahl
Tetralogy of Fallot (TOF), the most common severe congenital heart malformation, occurs sporadically, without other anomaly, and from unknown cause in 70% of cases. A genome-wide survey of 114 TOF patients and their unaffected parents identified 11 de novo copy number variants (CNVs) that were absent or extremely rare (<0.1%) in 2,265 controls. A second, independent TOF cohort (n = 398) was then examined for additional CNVs at these loci. In 1% (5/512, p = 0.0002, OR = 22.3) of non-syndromic sporadic TOF cases we identified CNVs at chromosome 1q21.1. Recurrent CNVs were also identified at 3p25.1, 7p21.3 and 22q11.2. CNVs in a single TOF case occurred at six loci, two that encode known (NOTCH1, JAG1) disease genes. Our data predicts that at least 10% (4.5–15.5, 95% CI) of sporadic, non-syndromic TOF reflects de novo CNVs and implicates mutations within these loci as etiologic in other cases of TOF.
Greenway, Steven C; Pereira, Alexandre C; Lin, Jennifer C; DePalma, Steven R; Israel, Samuel J; Mesquita, Sonia M; Ergul, Emel; Conta, Jessie R; Korn, Joshua M; McCarroll, Steven A; Gorham, Joshua M; Gabriel, Stacey; Altshuler, David A; de Lourdes Quintanilla-Dieck, Maria; Artunduaga, Maria Alexandra; Eavey, Roland D; Plenge, Robert M; Shadick, Nancy A; Weinblatt, Michael E; De Jager, Philip L; Hafler, David A; Breitbart, Roger E; Seidman, J G; Seidman, Christine E
In order to study the expression and function of laminin variants during chick embryonic development, we have generated monoclonal antibodies against chick heart laminin. One monoclonal antibody (mAb), called 9/F-10, could be used to purify chick laminin to homogeneity. By rotary shadowing, cross-shaped and T-shaped laminin particles as well as aggregates of two laminin molecules crosslinked via their short arms could be observed in this preparation. Purified chick laminin was very potent in mediating neurite growth by chick embryonic neurons. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reduced chick heart laminin revealed a complex pattern of polypeptides which are immunologically related to several mammalian laminin chains. The two mAbs, 9/F-10 and 3/E-8, recognize two different types of chick laminin subunits. By immunofluorescence, antibody 3/E-8 labels basement membranes, aortic smooth muscle, and mesenchyme of 6-day-old chick embryos. In contrast, staining by mAb 9/F-10 is confined to basement membranes. Therefore, the two antibodies seem to distinguish between two different chick laminin isoforms. PMID:1959563
Brubacher, D; Wehrle-Haller, B; Chiquet, M
Summary. Australian infectious bronchitis viruses (IBV) have undergone a separate evolution due to geographic isolation. Consequently,\\u000a changes occurring in Australian IBV illustrate, independently from other countries, types of variability that could occur\\u000a in emerging IBV strains. Previously, we have identified two distinct genetic groups of IBV, designated subgroups 1 and 2.\\u000a IBV strains of subgroup 1 have S1 and N proteins
J. Ignjatovic; G. Gould; S. Sapats
BackgroundCongenital malformations involving the Müllerian ducts are observed in around 5% of infertile women. Complete aplasia of the uterus, cervix, and upper vagina, also termed Müllerian aplasia or Mayer–Rokitansky–Kuster–Hauser (MRKH) syndrome, occurs with an incidence of around 1 in 4500 female births, and occurs in both isolated and syndromic forms. Previous reports have suggested that a proportion of cases, especially
Serena Nik-Zainal; Reiner Strick; Mekayla Storer; Ni Huang; Roland Rad; Lionel Willatt; Tomas Fitzgerald; Vicki Martin; Richard Sandford; Nigel P Carter; Andreas R Janecke; Stefan P Renner; Patricia G Oppelt; Peter Oppelt; Christine Schulze; Sara Brucker; Matthew Hurles; Matthias W Beckmann; Pamela L Strissel; Charles Shaw-Smith
Salmonella isolates harbour a range of resident prophages which can influence their virulence and ability to compete and survive in their environment. Phage gene profiling of a range of phage types of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) indicates a significant level of correlation of phage gene profile with phage type as well as correlation with genotypes determined by a combination of multi-locus variable-number tandem repeat (VNTR) typing and clustered regularly interspaced short palindromic repeats (CRISPR) typing. Variation in phage gene profiles appears to be partly linked to differences in composition of variants of known prophages. We therefore conducted a study of the distribution of variants of ST64B and Gifsy-1 prophages and coincidently the presence of Gifsy-3 prophage in a range of S. Typhimurium phage types and genotypes. We have discovered two variants of the DT104 variant of ST64B and at least two new variants of Gifsy-1 as well as variants of related phage genes. While there is definite correlation between phage type and the prophage profile based on ST64B and Gifsy-1 variants we find stronger correlation between the VNTR/CRISPR genotype and prophage profile. Further differentiation of some genotypes is obtained by addition of the distribution of Gifsy-3 and a sequence variant of the substituted SB26 gene from the DT104 variant of ST64B. To explain the correlation between genotype and prophage profile we propose that suites of resident prophages promote clonality possibly through superinfection exclusion systems. PMID:24475087
Hiley, Lester; Fang, Ning-Xia; Micalizzi, Gino R; Bates, John
Salmonella isolates harbour a range of resident prophages which can influence their virulence and ability to compete and survive in their environment. Phage gene profiling of a range of phage types of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) indicates a significant level of correlation of phage gene profile with phage type as well as correlation with genotypes determined by a combination of multi-locus variable-number tandem repeat (VNTR) typing and clustered regularly interspaced short palindromic repeats (CRISPR) typing. Variation in phage gene profiles appears to be partly linked to differences in composition of variants of known prophages. We therefore conducted a study of the distribution of variants of ST64B and Gifsy-1 prophages and coincidently the presence of Gifsy-3 prophage in a range of S. Typhimurium phage types and genotypes. We have discovered two variants of the DT104 variant of ST64B and at least two new variants of Gifsy-1 as well as variants of related phage genes. While there is definite correlation between phage type and the prophage profile based on ST64B and Gifsy-1 variants we find stronger correlation between the VNTR/CRISPR genotype and prophage profile. Further differentiation of some genotypes is obtained by addition of the distribution of Gifsy-3 and a sequence variant of the substituted SB26 gene from the DT104 variant of ST64B. To explain the correlation between genotype and prophage profile we propose that suites of resident prophages promote clonality possibly through superinfection exclusion systems.
Hiley, Lester; Fang, Ning-Xia; Micalizzi, Gino R.; Bates, John
A rapid enzyme immunoassay (EIA) was developed for the phenotypic detection of diphtheria toxin among clinical isolates of corynebacteria. The assay uses equine polyclonal antitoxin as the capture antibody and an alkaline phosphatase-labeled monoclonal antibody, specific for fragment A of the toxin molecule, as the detecting antibody. The assay is rapid, sensitive, and specific: a final result is available within 3 h of colony selection, and the limits of detection are 0.1 ng of pure diphtheria toxin/ml. Toxigenicity could be detected with isolates grown on a diverse range of culture media, including selective agars. Toxin detection using the EIA was compared to that with the Elek test and PCR detection of fragment A of the diphtheria toxin (tox) gene, using 245 isolates of corynebacteria. The results for the EIA were in complete concordance with those of the Elek test: 87 toxigenic and 158 nontoxigenic isolates. Ten of the phenotypically nontoxigenic strains were found to contain fragment A of the tox gene but did not express the toxin protein. These isolates were found to be nontoxigenic in the Vero cell tissue culture cytotoxicity assay and were therefore nontoxigenic for diagnostic purposes. The EIA is a simple rapid phenotypic test which provides a definitive result on toxigenicity within one working day.
Engler, Kathryn H.; Efstratiou, Androulla
Pepino mosaic virus (PepMV), a member of the genus Potexvirus, was first described in South America on pepino (Solanum muricatum A.). Only in recent years, it was reported to infect greenhouse-grown tomatoes. Genome nucleotide sequences from several European isolates showed extensive sequence identity (>99%). Recent genome nucleotide sequences from two US isolates (US1 and US2) however showed much greater sequence divergence from that of the European PepMV isolates. My interest in characterizing virus isolates from South America was due to an active commercial tomato seed production in Chile. Through genome sequence comparison and phylogenetic analyses, we may be able to understand the source of virus infection and control this devastating disease from further spreading into new tomato growing regions of the world. Complete genome nucleotide sequences from two PepMV variants (designated as Ch1 and Ch2) were determined from a virus isolate obtained from a commercial tomato seed lot produced in Chile. Using RT-PCR-based genome walking strategy, complete genome sequences from these two variants were determined. Excluding poly (A) tails, the genomes of PepMV Ch1 and Ch2 were 6414 and 6412 nucleotides (nt), respectively. Pairwise comparisons of PepMV Ch1 and Ch2 genomes with other PepMV isolates showed that the highest nucleotide sequence identity was with two US isolates, 98.7% between PepMV Ch1 and US1, and 90.7% between Ch2 and US2. Similar to PepMV US1 and US2, the two Chilean variants were the most divergent from one another (78% nt identity). These two Chilean PepMV variants also shared only 78-86% nucleotide sequence identity to that of five European isolates. The high level of nucleotide sequence identity between Chilean and US isolates suggests a common origin. Phylogenetic analyses with various gene products generated three distinct sequence clusters (or strains): US1 and Ch1 in the first group, US2 and Ch2 in the second, and the European tomato isolates in the third. Based on the host specificity, it was previously suggested that the original pepino isolate should be considered a distinct strain from that of the tomato isolates. PMID:16927118
Aim To study the association between genetic variants in myocilin and collagen type I alpha 1 genes and high myopia in an isolated island population. Methods A total of 944 examinees from the genetic epidemiology study conducted on the island of Kor?ula, Croatia, were included in the study. We selected 2 short nucleotide polymorphisms (SNP) available in our genome-wide scan set of SNPs that were previously associated with high myopia and used them to replicate previous claims of possible association. Results Nineteen cases of high myopia, defined as the refraction of ?-6.00 diopters, were identified and included in the analysis. We showed that rs2075555 in the COL1A1 gene was not associated with high myopia. In contrast, rs2421853 in the myocilin gene was significantly associated in both bivariate (P?=?0.006) and age- and sex-adjusted analysis (P?=?0.049). Conclusion Myocilin seems to be a very strong candidate for explaining some of the pathophysiological pathways leading to the development of both glaucoma and high myopia. As our finding was obtained in a relatively under-powered sample, further research and replication of these results is needed.
Vatavuk, Zoran; Skunca Herman, Jelena; Bencic, Goran; Andrijevic Derk, Biljana; Lacmanovic Loncar, Valentina; Petric Vickovic, Ivanka; Bucan, Kajo; Mandic, Kresimir; Mandic, Antonija; Skegro, Ivan; Pavicic Astalos, Jasna; Merc, Ivana; Martinovic, Miljenka; Kralj, Petra; Knezevic, Tamara; Barac-Juretic, Katja; Zgaga, Lina
Variants of Coconut cadang-cadang viroid have been identified in a plantation oil palm growing in Malaysia. Three size classes are described, comprising 297, 293, and 270 nt. Compared with the 296-nt form of coconut cadang-cadang viroid (CCCVd), all variants substituted C31 --> U in the pathogenicity domain and A175 --> C in the right-hand terminus. Other mutations and deletions accounted for the different sizes. These are the first sequences reported for variants of Coconut cadang-cadang viroid in a species other than coconut palm, and this is the first evidence that variants closely related to CCCVd occur outside the Philippines. PMID:16470341
Vadamalai, G; Hanold, D; Rezaian, M A; Randles, J W
The genomic DNA of four Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) variants isolated from Galleria mellonella, Spodoptera exigua, Spodoptera litura and Xestia c-nigrum was analyzed in comparison with the AcMNPV E2 strain. Restriction endonuclease analysis revealed a deletion and an insertion in collinear regions of the four variants. Polymerase chain reaction analysis indicated that, in the four variants, the deletion occurred in the region corresponding to AcMNPV C6 ORF86 (pnk/pnl). Also the insertion, with a length of approximately 1.1 kb, was commonly identified in the fragments corresponding to the PstI-J fragment (18.5 m.u.-21.2 m.u.) of AcMNPV E2. Sequencing analysis of the variant from S. litura showed that the insertion contains an additional open reading frame encoding 322 amino acids between homologues of AcMNPV ORF30 and ORF31 (the superoxide dismutase gene). This ORF has 82.8% amino acid identity to Bombyx mori NPV T3 ORF 22 (bro-a, one of the baculovirus repeated ORFs) and thus, it was named Splt-bro-a. Southern blot hybridization study indicated that the other three variants also contain Splt-bro-a homologue. In addition, the labeled Splt-bro-a gene weakly hybridized to the PstI-D fragment (99.0 m.u.-8.0 m.u.) of AcMNPV E2. This fragment contains AcMNPV ORF2, a member of bro family. The signal was also observed on the corresponding fragment of the four variants. This result suggested that two bro genes are present in the four variants, although AcMNPV E2 and C6 are known to contain a single bro gene. PMID:11129632
Yanase, T; Hashimoto, Y; Kawarabata, T
The objective of this study was to determine the prevalence of the plasmid-borne quinolone resistance genes qnrA, qnrB and qnrS in a collection of Enterobacteriaceae causing bacteraemia. The presence of the three genes was tested for using multiplex PCR in 306 clinical isolates. Plasmid analysis was performed using I-CeuI and S1 nuclease digestion and hybridization with specific probes for the qnr and 23S rRNA genes. Five strains were found to carry a qnr gene, one of which, qnrB16, a new variant of qnrB, was detected in a Citrobacter freundii isolate. The qnrB6 variant was found in two C. freundii isolates and in one Citrobacter werkmanii isolate. The qnrS2 gene was found in one Klebsiella pneumoniae isolate. The qnrA gene was not found in any of the isolates studied. The qnrS2 gene was located on a plasmid of c. 50 kb, whereas qnrB6 and qnrB16 were inserted in the chromosome between pspF and the orf2, which had previously been found in a complex integron. In the Hospital Clinic of Barcelona, Spain, the prevalence of qnrB was higher than that of qnrA and qnrS. The importance of the description of the new qnrB16 is emphasized. PMID:19392890
Sanchez-Cespedes, J; Marti, S; Soto, S M; Alba, V; Melción, C; Almela, M; Marco, F; Vila, J
Alternative splicing of the mu opioid receptor genes to create multiple mu receptor subtypes has been demonstrated in animals and humans. Previously, we identified a number of C-terminal variants in mice, rats and human, followed by several N-terminal variants associated with a new upstream exon in mice (exon 11). Behavioral studies in exon 11 knockout mice suggest an important role for the exon 11 variants in the analgesic actions of heroin and morphine-6beta-glucuronide, but not morphine or methadone. We now have identified a homologous human exon 11 and three similar human exon 11-associated variants, suggesting conservation of exon 11 and its associated variants across species. hMOR-1i has an additional 93 amino acids at the tip of the N-terminus but is otherwise identical to hMOR-1. When expressed in Chinese hamster ovary cells, the additional 93 amino acids in hMOR-1i had little effect on opioid binding, but significantly altered agonist-induced G-protein activation. hMOR-1G1 and hMOR-1G2 predicted six transmembrane domain variants, similar to those seen in mice. The regional expression of these exon 11-associated variants, as determined by RT-PCR, varied markedly, implying region-specific alternative splicing. The presence of exon 11-associated variants in humans raises questions regarding their potential role in heroin and morphine-6beta-glucuronide actions in people as they do in mice. PMID:19077058
Xu, Jin; Xu, Mingming; Hurd, Yasmin L; Pasternak, Gavril W; Pan, Ying-Xian
By using sequence analysis of Shiga toxin 1 (Stx 1) genes from human and ovine Stx-producing Escherichia coli (STEC) strains, we identified an Stx1 variant in STEC of human origin that was identical to the Stx1 variant from ovine STEC, but demonstrated only 97.1 and 96.6% amino acid sequence identity in its A and B subunits, respectively, to the Stx1 encoded by bacteriophage 933J. We designated this variant “Stx1c” and developed stxB1 restriction fragment length polymorphism and stx1c-specific PCR strategies to determine the frequency and distribution of stx1c among 212 STEC strains isolated from humans. stx1c was identified in 36 (17.0%) of 212 STEC strains, 19 of which originated from asymptomatic subjects and 16 of which were from patients with uncomplicated diarrhea. stx1c was most frequently (in 23 STEC strains [63.9%]) associated with stx2d, but 12 (33.3%) of the 36 STEC strains possessed stx1c only. A single STEC strain possessed stx1c together with stx2 and was isolated from a patient with hemolytic-uremic syndrome. All 36 stx1c-positive STEC strains were eae negative and belonged to 10 different serogroups, none of which was O157, O26, O103, O111, or O145. Stx1c was produced by all stx1c-containing STEC strains, but reacted weakly with a commercial immunoassay. We conclude that STEC strains harboring the stx1c variant account for a significant proportion of human STEC isolates. The procedures developed in this study now allow the determination of the frequency of STEC strains harboring stx1c among clinical STEC isolates and their association with human disease in prospective studies.
Zhang, Wenlan; Bielaszewska, Martina; Kuczius, Thorsten; Karch, Helge
Uric acid nephrolithiasis (UAN) is a common disease with an established genetic component that presents a complex mode of inheritance. While studying an ancient founder population in Talana, a village in Sardinia, we recently identified a susceptibility locus of ?2.5 cM for UAN on 10q21-q22 in a relatively small sample that was carefully selected through genealogical information. To refine the critical region and to identify the susceptibility gene, we extended our analysis to severely affected subjects from the same village. We confirm the involvement of this region in UAN through identical-by-descent sharing and autozygosity mapping, and we refine the critical region to an interval of ?67 kb associated with UAN by linkage-disequilibrium mapping. After inspecting the genomic sequences available in public databases, we determined that a novel gene overlaps this interval. This gene is divided into 15 exons, spanning a region of ?300 kb and generating at least four different proteins (407, 333, 462, and 216 amino acids). Interestingly, the last isoform was completely included in the 67-kb associated interval. Computer-assisted analysis of this isoform revealed at least one membrane-spanning domain and several N- and O-glycosylation consensus sites at N-termini, suggesting that it could be an integral membrane protein. Mutational analysis shows that a coding nucleotide variant (Ala62Thr), causing a missense in exon 12, is in strong association with UAN (P=.0051). Moreover, Ala62Thr modifies predicted protein secondary structure, suggesting that it may have a role in UAN etiology. The present study underscores the value of our small, genealogically well-characterized, isolated population as a model for the identification of susceptibility genes underlying complex diseases. Indeed, using a relatively small sample of affected and unaffected subjects, we identified a candidate gene for multifactorial UAN.
Gianfrancesco, Fernando; Esposito, Teresa; Ombra, Maria Neve; Forabosco, Paola; Maninchedda, Giuseppe; Fattorini, Mauro; Casula, Stefania; Vaccargiu, Simona; Casu, Giuseppina; Cardia, Francesco; Deiana, Ivo; Melis, Paola; Falchi, Mario; Pirastu, Mario
The aim of the present study was to investigate the epidemiological link of multidrug-resistant Klebsiella oxytoca isolates causing community-onset infections among patients attending our outpatient department and to investigate the underlying resistance mechanisms. The isolates were tested by agar dilution MICs, phenotypic carbapenemase testing, enterobacterial repetitive intergenic consensus-PCR, and pulsed-field gel electrophoresis (PFGE). PCR assays and nucleotide sequencing were employed for the identification of bla gene types and the mapping of the integron-containing metallo-?-lactamase (MBL) gene. During the study period (January 2005 to April 2007), nine broad-spectrum cephalosporin-resistant K. oxytoca clinical isolates were prospectively collected from separate outpatients with urinary tract infections. In all cases, the patients had been hospitalized or exposed to health care facilities during the preceding year. Molecular typing revealed that all isolates belonged to the same K. oxytoca clonal type, which contained five PFGE subtypes. A novel chromosomal OXY-2 ?-lactamase type variant (OXY-2-9) was detected in all isolates, but no mutations in the promoter region justifying blaOXY gene overproduction were detected. In addition, all isolates harbored the plasmidic CMY-31 (LAT-4) AmpC cephalosporinase, while three of them harbored VIM-1 MBL in a class 1 integron structure. This is the first study to present the dissemination in the community of multidrug-resistant K. oxytoca isolates causing extrahospital infections.
Tsakris, Athanassios; Poulou, Aggeliki; Markou, Fani; Pitiriga, Vassiliki; Piperaki, Evangelia-Theophano; Kristo, Ioulia; Pournaras, Spyros
We report the draft whole-genome sequences of two Vibrio cholerae O1 strains, the environmental toxigenic strain 2011EL-301 and the clinical nontoxigenic strain P-18785, both isolated in Russia. Some basic data comparing the two against the GenBank repository are provided. PMID:23969060
Kuleshov, Konstantin V; Vodop'ianov, Sergey O; Markelov, Mikhail L; Dedkov, Vladimir G; Kermanov, Anton V; Kruglikov, Vladimir D; Vodop'ianov, Alexey S; Pisanov, Ruslan V; Mazrukho, Alexey B; Shipulin, German A
Prevalence and genetic diversity of arginine catabolic mobile element (ACME) in clinical isolates of coagulase-negative staphylococci: identification of ACME type I variants in Staphylococcus epidermidis.
Arginine catabolic mobile element (ACME), a genomic island consisting of the arc and/or opp3 gene clusters found in staphylococcal species, is related to increased bacterial adaptability to hosts. Staphylococcus epidermidis is considered a major ACME reservoir; however, prevalence and genetic diversity of ACME in coagulase-negative staphylococci (CNS) have not yet been well characterized for clinical isolates in Japan. A total of 271 clinical isolates of CNS in a Japanese hospital were investigated for the presence and genotype of ACME and SCCmec. The prevalence of ACME-arcA was significantly higher (p<0.001) in S. epidermidis (45.8%) than in other CNS species (3.7%). ACME in S. epidermidis isolates (n=87) were differentiated into type I (n=33), variant forms of type I (?I, n=26) newly identified in this study, type II (n=6), and type ?II (n=19). ACME-type ?I, which were further classified into three subtypes, lacked some genetic components between the arc and opp3 clusters in archetypal type I, whereas the arc and opp3 clusters were intact. The arc cluster exhibited high sequence identity (95.8-100%) to that of type I ACME; in contrast, the opp3 cluster was highly diverse, and showed relatively lower identities (94.8-98.7%) to the identical regions in type I ACME. Twenty-one isolates of ?I ACME-carrying S. epidermidis possessed SCCmec IVa and belonged to ST5 (clonal complex 2). Phylogenetic analysis revealed that isolates harboring ACME ?I in this study clustered with previously reported S. epidermidis strains with other lineges, suggesting that S. epidermidis originally had some genetic variations in the opp3 cluster. In summary, ACME type ?I, a truncated variant of ACME-I, was first identified in S. epidermidis, and revealed to be prevalent in ST5 MRSE clinical isolates with SCCmec IVa. PMID:24113082
Onishi, Mayumi; Urushibara, Noriko; Kawaguchiya, Mitsuyo; Ghosh, Souvik; Shinagawa, Masaaki; Watanabe, Naoki; Kobayashi, Nobumichi
Background The development of anaemia in feline leukaemia virus (FeLV)-infected cats is associated with the emergence of a novel viral subgroup, FeLV-C. FeLV-C arises from the subgroup that is transmitted, FeLV-A, through alterations in the amino acid sequence of the receptor binding domain (RBD) of the envelope glycoprotein that result in a shift in the receptor usage and the cell tropism of the virus. The factors that influence the transition from subgroup A to subgroup C remain unclear, one possibility is that a selective pressure in the host drives the acquisition of mutations in the RBD, creating A/C intermediates with enhanced abilities to interact with the FeLV-C receptor, FLVCR. In order to understand further the emergence of FeLV-C in the infected cat, we examined primary isolates of FeLV-C for evidence of FeLV-A variants that bore mutations consistent with a gradual evolution from FeLV-A to FeLV-C. Results Within each isolate of FeLV-C, we identified variants that were ostensibly subgroup A by nucleic acid sequence comparisons, but which bore mutations in the RBD. One such mutation, N91D, was present in multiple isolates and when engineered into a molecular clone of the prototypic FeLV-A (Glasgow-1), enhanced replication was noted in feline cells. Expression of the N91D Env on murine leukaemia virus (MLV) pseudotypes enhanced viral entry mediated by the FeLV-A receptor THTR1 while soluble FeLV-A Env bearing the N91D mutation bound more efficiently to mouse or guinea pig cells bearing the FeLV-A and -C receptors. Long-term in vitro culture of variants bearing the N91D substitution in the presence of anti-FeLV gp70 antibodies did not result in the emergence of FeLV-C variants, suggesting that additional selective pressures in the infected cat may drive the subsequent evolution from subgroup A to subgroup C. Conclusions Our data support a model in which variants of FeLV-A, bearing subtle differences in the RBD of Env, may be predisposed towards enhanced replication in vivo and subsequent conversion to FeLV-C. The selection pressures in vivo that drive the emergence of FeLV-C in a proportion of infected cats remain to be established.
Thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus can be isolated from the airway secretions of patients suffering from cystic fibrosis (CF) and are implicated in persistent and treatment-resistant infections. These characteristics, as well as the variety of mutations in the thymidylate synthase-encoding thyA gene which are responsible for thymidine dependency, suggest that these morphological variants are hypermutable. To prove this hypothesis, we analyzed the mutator phenotype of different S. aureus phenotypes, in particular CF-derived TD-SCVs, CF-derived isolates with a normal phenotype (NCVs), and non-CF NCVs. The comparative analysis revealed that the CF isolates had significantly higher mutation rates than the non-CF isolates. The TD-SCVs, in turn, harbored significantly more strong hypermutators (mutation rate ? 10?7) than the CF and non-CF NCVs. In addition, antimicrobial resistance to non-beta-lactam antibiotics, including gentamicin, ciprofloxacin, erythromycin, fosfomycin, and rifampin, was significantly more prevalent in TD-SCVs than in CF and non-CF NCVs. Interestingly, macrolide resistance, which is usually mediated by mobile genetic elements, was conferred in half of the macrolide-resistant TD-SCVs by the point mutation A2058G or A2058T in the genes encoding the 23S rRNA. Sequence analysis of mutS and mutL, which are involved in DNA mismatch repair in gram-positive bacteria, revealed that in hypermutable CF isolates and especially in TD-SCVs, mutL was often truncated due to frameshift mutations. In conclusion, these data provide direct evidence that TD-SCVs are hypermutators. This hypermutability apparently favors the acquisition of antibiotic resistance and facilitates bacterial adaptation during long-term persistence.
Besier, Silke; Zander, Johannes; Kahl, Barbara C.; Kraiczy, Peter; Brade, Volker; Wichelhaus, Thomas A.
HCV variants are described. The variants include polynucleotides comprising non-naturally occurring HCV sequences and HCV variants that have a transfection efficiency and ability to survive subpassage greater than HCV that have wild-type polyprotein codin...
C. M. Rice K. J. Blight
Necrotoxigenic Escherichia coli (NTEC) are associated with intestinal and extraintestinal diseases in animals and human beings and produce Cytotoxic Necrotizing Factor 1 (CNF1) or 2 (CNF2). Fourty-three NTEC1, 42 NTEC2, and 32 CNF-negative isolates from cattle were tested by colony DNA hybridization, by plasmid DNA hybridization and by PCR assays for the presence of DNA sequences homologous to the operons coding for fimbrial (PAP/PRS, SFA/FIC, and F17) and afimbrial (AFA/Dr) adhesins of extraintestinal E. coli. Most NTEC1 isolates hybridized with the PAP probes and either the SFA probe (37%) or the AFA probes (49%). Most NTEC2 isolates, in contrast, hybridized with the F17 probe (45%), the AFA probes (19%), or the F17 and AFA probes (22%). A probe-positive plasmid was identified in each of the 19 NTEC2 isolates studied. They all hybridized with the CNF2 toxin probe (Vir plasmids) and most of them with the F17 (6 plasmids) or AFA (7 plasmids) probes. PCR amplification was obtained with 6 of the 11 NTEC isolates tested for the papGII/prsG genes; with all 5 NTEC isolates tested for the sfa and related operons; but with none of the 18 NTEC isolates tested for the afa and related operons. pap-, sfa-, and afa-related sequences are thus present in NTEC isolates from cattle in addition to f17-related operons and may code for adhesins corresponding to specific colonization factors. f17- and afa-related sequences can be located on the Vir plasmids along with the cnf2 gene. Existence of new variants of the AFA/Dr family is evident from the negative results of this family-specific PCR assay. Images Figure 1.
Mainil, J G; Jacquemin, E; Herault, F; Oswald, E
Phage type 99 of Salmonella enterica subsp. enterica serovar Typhimurium variant Copenhagen strains isolated from pigeons were examined for the presence of genotypic and phenotypic characteristics. The pulsed-field gel electrophoresis patterns obtained with XbaI and BlnI from 38 pigeon strains were compared with those obtained from 89 porcine, poultry, and human strains of variant Copenhagen. Identical patterns with XbaI and four closely related patterns with BlnI were obtained with the pigeon strains, whereas 16 XbaI patterns were found with the other strains. The XbaI patterns of the pigeon strains showed a low genetic similarity to the patterns of the porcine, poultry, and human strains and invariably showed a low-molecular-weight band that was absent in the majority of the other strains. The virulence genes shdA, spvR, pefA, sopE, and spvB were uniformly present in six pigeon isolates representing the genetic diversity found with BlnI. These six pigeon-derived strains were highly cytotoxic for pigeon macrophages compared to three porcine strains. After experimental infection of pigeons with a pigeon strain, clinical symptoms, fecal shedding, and colonization of internal organs were more pronounced than those after infection with a porcine strain. These data suggest that the phage type 99 strains used in this study are highly adapted to pigeons and should be classified as a host-restricted lineage of the serovar Typhimurium. PMID:14500532
Pasmans, Frank; Van Immerseel, Filip; Heyndrickx, Marc; Martel, An; Godard, Claudine; Wildemauwe, Christa; Ducatelle, Richard; Haesebrouck, Freddy
Transposon Tn558 integrated in the chromosomal radC gene was detected for the first time in Staphylococus pseudintermedius. It carried a novel fexA variant (fexAv) that confers only chloramphenicol resistance. The exporter FexAv exhibited two amino acid substitutions, Gly33Ala and Ala37Val, both of which seem to be important for substrate recognition. Site-directed mutagenesis that reverted the mutated base pairs to those present in the original fexA gene restored the chloramphenicol-plus-florfenicol resistance phenotype.
Gomez-Sanz, Elena; Kadlec, Kristina; Fessler, Andrea T.; Zarazaga, Myriam; Schwarz, Stefan
X-linked adrenoleukodystrophy (X-ALD) shows a wide range of phenotypic expression, but clinical presentation as an isolated lesion of the cerebellar white matter and dentate nuclei has not been reported. We report an unusual presentation of X-ALD only with an isolated lesion of the cerebellar white matter and dentate nuclei. The proband, a 37-year-old man presented with bladder incontinence, slurred speech, dysmetria in all limbs, difficulties in balancing, and gait ataxia. Brain magnetic resonance imaging showed an isolated signal change of white matter around the dentate nucleus in cerebellum. With high level of very long chain fatty acid, gene study showed a de novo mutation in exon 1 at nucleotide position c.277_296dup20 (p.Ala100Cysfs*10) of the adenosine triphosphate-binding cassette D1 gene. It is advised to consider X-ALD as a differential diagnosis in patients with isolated cerebellar degeneration symptoms. PMID:24954351
Kang, Joon Won; Lee, Sang Mi; Koo, Kyo Yeon; Lee, Young Mock; Nam, Hyo Suk; Quan, Zhejiu; Kang, Hoon Chul
X-linked adrenoleukodystrophy (X-ALD) shows a wide range of phenotypic expression, but clinical presentation as an isolated lesion of the cerebellar white matter and dentate nuclei has not been reported. We report an unusual presentation of X-ALD only with an isolated lesion of the cerebellar white matter and dentate nuclei. The proband, a 37-year-old man presented with bladder incontinence, slurred speech, dysmetria in all limbs, difficulties in balancing, and gait ataxia. Brain magnetic resonance imaging showed an isolated signal change of white matter around the dentate nucleus in cerebellum. With high level of very long chain fatty acid, gene study showed a de novo mutation in exon 1 at nucleotide position c.277_296dup20 (p.Ala100Cysfs*10) of the adenosine triphosphate-binding cassette D1 gene. It is advised to consider X-ALD as a differential diagnosis in patients with isolated cerebellar degeneration symptoms.
Kang, Joon Won; Lee, Sang Mi; Koo, Kyo Yeon; Lee, Young-Mock; Nam, Hyo Suk; Quan, Zhejiu
There is evidence for an association between structural variants in genes for lissencephaly, which are involved in neuronal migration, and prefrontal cognitive deficits in schizophrenia and bipolar patients. On the basis of these intriguing findings, we analyzed 16 markers located in the lissencephaly critical region (LCR in chromosome 17p13.3) in 124 schizophrenic, 56 bipolar, and 141 healthy individuals. All recruits were from a Spanish population isolate of Basque origin that is characterized by low genetic heterogeneity. In addition, we examined whether structural genomic variations in the LCR were associated with executive cognition. Twenty-three patients (12.8%), but none of the controls, showed structural variants (deletions and insertions) in either of two markers related with lissencephaly (D17S1566 on tumor suppressor gene TP53: tumor protein p53 and D17S22 on SMG6 gene: Smg-6 homolog, nonsense mediated mRNA decay factor- Caenorhabditis elegans). These patients performed significantly worse in the Wisconsin Card Sorting Test-Categories in comparison with patients without such variations in lissencephaly-related genes. The presence of structural variants was related to completed categories, and accounted for 10.7% of the variance (P=0.001). Finally, logistic regression showed that poor Wisconsin Card Sorting Test-Categories performance was the only predictor of belonging to the positive LCR variations group. These new findings provide further evidence for the association between some lissencephaly-related genes and both schizophrenia and bipolar disorder, and influence on frontal executive functioning. PMID:19018238
Tabarés-Seisdedos, Rafael; Mata, Ignacio; Escámez, Teresa; Vieta, Eduard; López-Ilundain, Jose M; Salazar, Jose; Selva, Gabriel; Balanzá, Vicente; Rubio, Cristina; Martínez-Arán, Anabel; Valdés-Sánchez, Lourdes; Geijo-Barrientos, Emilio; Martínez, Salvador
The heterogeneous environment of the lung of the cystic fibrosis (CF) patient gives rise to Pseudomonas aeruginosa small colony variants (SCVs) with increased antibiotic resistance, autoaggregative growth behavior, and an enhanced ability to form biofilms. In this study, oligonucleotide DNA microarrays were used to perform a genome-wide expression study of autoaggregative and highly adherent P. aeruginosa SCV 20265 isolated from a CF patient's lung in comparison with its clonal wild type and a revertant generated in vitro from the SCV population. Most strikingly, SCV 20265 showed a pronounced upregulation of the type III protein secretion system (TTSS) and the respective effector proteins. This differential expression was shown to be biologically meaningful, as SCV 20265 and other hyperpiliated and autoaggregative SCVs with increased TTSS expression were significantly more cytotoxic for macrophages in vitro and were more virulent in a mouse model of respiratory tract infection than the wild type. The observed cytotoxicity and virulence of SCV 20265 required exsA, an important transcriptional activator of the TTSS. Thus, the prevailing assumption that P. aeruginosa is subject to selection towards reduced cytotoxicity and attenuated virulence during chronic CF lung infection might not apply to all clonal variants.
von Gotz, Franz; Haussler, Susanne; Jordan, Doris; Saravanamuthu, Senthil Selvan; Wehmhoner, Dirk; Strussmann, Andre; Lauber, Joerg; Attree, Ina; Buer, Jan; Tummler, Burkhard; Steinmetz, Ivo
The spread of the blaNDM-1 gene is gaining worldwide attentions. This gene is usually carried by large plasmids and has been discovered in diverse bacteria since it was originally found in Klebsiella pneumoniae. Here we report the complete sequences of a blaNDM-1-bearing plasmid, pNDM-BJ01, and its variant, pNDM-BJ02, isolated from clinical Acinetobacter lwoffii strains. The plasmid pNDM-BJ01 is 47.3 kb in size and cannot be classified into any known plasmid incompatibility group, thus representing a novel plasmid with an unknown maintenance mechanism. This plasmid contains both a blaNDM-1 gene and a type IV secretion system (T4SS) gene cluster. The T4SS is assigned to the P-type T4SS group, which usually encode a short, rigid pilus, and the blaNDM-1 gene is located within a composite transposon flanked by two insertion elements of ISAba125. Plasmid pNDM-BJ02 is nearly identical to pNDM-BJ01 except that one copy of the ISAba125 element is missing, and it is therefore regarded as a variant of pNDM-BJ01. Sequence alignment indicated that this blaNDM-1-containing composite transposon, which can also be captured by other mobile elements, was probably a product of multiple recombination events and can move as a whole by transposition.
Hu, Hongyan; Hu, Yongfei; Pan, Yuanlong; Liang, Hui; Wang, Haiyan; Wang, Xiumei; Hao, Qinfang; Yang, Xiaoli; Yang, Xi; Xiao, Xue; Luan, Chunguang; Yang, Yi; Cui, Yujun; Yang, Ruifu; Gao, George F.
The bacteriocinogenic strain RJ16 isolated from goat cheese has been identified as Enterococcus faecium by species-specific PCR, DNA–rRNA hybridization and rDNA sequencing. Purified bacteriocin from strain RJ16 is a carboxypeptidase A-resistant peptide with a molecular mass (7125Da) very close to the cyclic peptide enterocin AS-48. Bacteriocin from strain RJ16 and AS-48 show identical antibacterial spectra, although the former is slightly
Hikmate Abriouel; Rosario Lucas; Nabil Ben Omar; Eva Valdivia; Mercedes Maqueda; Magdalena Martínez-Cañamero; Antonio Gálvez
CM-Sephadex C-25 column chromatography profile of Indian cobra (Naja naja) venom from eastern region showed a distinct and a dominant phospholipase peak, peak-10, while it was not seen in either southern or western venom samples. Peak-10 was subjected to CM-Sephadex C-25 and Sephadex G-50 column chromatography to isolate NN-X-PLA2. NN-X-PLA2 is a single chain protein with the relative molecular weight
R. Shashidharamurthy; K. Kemparaju
Summary The complications of malaria in pregnancy are caused by the massive sequestration of parasitized erythrocytes (PE) in the placenta. Placental isolates of Plasmodium falciparum are unusual in that they do not bind the primary microvasculature receptor CD36 but instead bind chondroitin sulphate A (CSA). Preg- nant mothers develop antibodies that recognize pla- cental variants worldwide, suggesting that a vaccine
Benoit Gamain; Joseph D. Smith; Marion Avril; Dror I. Baruch; Artur Scherf; Jurg Gysin; Louis H. Miller
Draft Genome Sequences of Two Genetic Variant Strains of Edwardsiella piscicida, JF1305 and RSB1309, Isolated from Olive Flounder (Paralichythys olivaceus) and Red Sea Bream (Pagrus major) Cultured in Japan, Respectively
Edwardsiella piscicida is a new species discovered within the group of organisms traditionally classified as Edwardsiella tarda. We present draft genome sequences of two variant strains of E. piscicida, JF1305 and RSB1309. Differences in protein-coding sequence between these isolates are associated with virulence, disease, and defense, suggesting differences in pathogenicity.
Oguro, Kazuki; Tamura, Kazuki; Yamane, Jin; Shimizu, Masato; Yamamoto, Takeshi; Ikawa, Takuya; Ohnishi, Kouhei; Oshima, Syun-ichirou
Because of the frequency of multiple antibiotic resistance, Staphylococcus species often represent a challenge in incisional infections of horses undergoing colic surgery. To investigate the evolution of antibiotic resistance patterns before and after preventative peri- and postoperative penicillin treatment, staphylococci were isolated from skin and wound samples at different times during hospitalization. Most staphylococci were normal skin commensals and belonged to the common coagulase-negative group. In some cases they turned out to be opportunistic pathogens present in wound infections. MICs were determined for 12 antibiotics, and antibiotic resistance genes were detected by microarray. At hospital admission, horses harbored staphylococci that were susceptible to antibiotics or resistant to one group of drugs, mainly due to the presence of new variants of the methicillin and macrolide resistance genes mecA and mph(C), respectively. After 3 days, the percentage of Staphylococcus isolates displaying antibiotic resistance, as well as the number of resistance genes per isolate, increased moderately in hospitalized horses without surgery or penicillin treatment but dramatically in hospitalized horses after colic surgery as well as penicillin treatment. Staphylococcus species displaying multiple resistance were found to harbor mainly genes conferring resistance to ?-lactams (mecA and blaZ), aminoglycosides [str and aac(6?)-Ie-aph(2?)-Ia], and trimethoprim [dfr(A) and dfr(D)]. Additional genes conferring resistance to macrolides [mph(C), erm(C), and erm(B)], tetracycline [tet(K) and tet(M)], chloramphenicol [cat(pC221) and cat(pC223)], and streptothricin (sat4) appeared in several strains. Hospitalization and preventive penicillin use were shown to act as selection agents for multidrug-resistant commensal staphylococcal flora.
Schnellmann, Christina; Gerber, Vinzenz; Rossano, Alexandra; Jaquier, Valentine; Panchaud, Yann; Doherr, Marcus G.; Thomann, Andreas; Straub, Reto; Perreten, Vincent
Venom of the Australian tiger snake, Notechis scutatus scutatus was fractionated by conventional ion-exchange chromatography. The fraction containing notexin, a well-known single-chain toxic phospholipase A2, was further purified by reverse-phase high-performance liquid chromatography. Two main components were isolated and the major one corresponded to notexin. The other component, designated as notechis Ns, was an isoform of notexin. Notechis Ns and notexin possessed similar in vitro esterase activity, in vitro neuromuscular activity and in vivo lethality. Amino acid composition and sequence of the Staphylococcus aureus V8-protease peptides demonstrated that primary structures of notechis Ns and notexin differed from each other by a single substitution amongst 119 amino acids: Lys----Arg at position 16. PMID:2155818
Chwetzoff, S; Mollier, P; Bouet, F; Rowan, E G; Harvey, A L; Ménez, A
In this study, the genetic organization of three novel genomic antibiotic resistance islands (AbaRs) in Acinetobacter baumannii isolates belonging to group of European clone II (EC II) comM integrated sequences of 18-, 21-, and 23-kb resistance islands were determined. These resistance islands carry the backbone of AbaR-type transposon structures, which are composed of the transposition module coding for potential transposition proteins and other genes coding for the intact universal stress protein (uspA), sulfate permease (sul), and proteins of unknown function. The antibiotic resistance genes strA, strB, tetB, and tetR and insertion sequence CR2 element were found to be inserted into the AbaR transposons. GenBank homology searches indicated that they are closely related to the AbaR sequences found integrated in comM in strains of EC II (A. baumannii strains 1656-2 and TCDC-AB0715) and AbaR4 integrated in another location of A. baumannii AB0057 (EC I). All of the AbaRs showed structural similarity to the previously described AbaR4 island and share a 12,008-bp backbone. AbaRs contain Tn1213, Tn2006, and the multiple fragments which could be derived from transposons Tn3, Tn10, Tn21, Tn1000, Tn5393, and Tn6020, the insertion sequences IS26, ISAba1, ISAba14, and ISCR2, and the class 1 integron. Moreover, chromosomal DNA was inserted into distinct regions of the AbaR backbone. Sequence analysis suggested that the AbaR-type transposons have evolved through insertions, deletions, and homologous recombination. AbaR islands, sharing the core structure similar to AbaR4, appeared to be distributed in isolates of EC I and EC II via integration into distinct genomic sites, i.e., pho and comM, respectively. PMID:22290980
Seputiene, Vaida; Povilonis, Justas; Suziedeliene, Edita
A total of 250 Corynebacterium diphtheriae isolates from clinical cases and carriers in Russia were assayed by PCR directed at the A subunit of the diphtheria toxin gene to distinguish toxigenic from nontoxigenic strains; 170 strains were positive as indicated by the presence of the 248-bp amplicon. The results of this PCR assay were in complete concordance with those of the standard immunoprecipitation assay (Elek), and the PCR assay is a useful tool for rapid identification in clinical laboratories. PMID:8576378
Mikhailovich, V M; Melnikov, V G; Mazurova, I K; Wachsmuth, I K; Wenger, J D; Wharton, M; Nakao, H; Popovic, T
A total of 250 Corynebacterium diphtheriae isolates from clinical cases and carriers in Russia were assayed by PCR directed at the A subunit of the diphtheria toxin gene to distinguish toxigenic from nontoxigenic strains; 170 strains were positive as indicated by the presence of the 248-bp amplicon. The results of this PCR assay were in complete concordance with those of the standard immunoprecipitation assay (Elek), and the PCR assay is a useful tool for rapid identification in clinical laboratories.
Mikhailovich, V M; Melnikov, V G; Mazurova, I K; Wachsmuth, I K; Wenger, J D; Wharton, M; Nakao, H; Popovic, T
CM-Sephadex C-25 column chromatography profile of Indian cobra (Naja naja) venom from eastern region showed a distinct and a dominant phospholipase peak, peak-10, while it was not seen in either southern or western venom samples. Peak-10 was subjected to CM-Sephadex C-25 and Sephadex G-50 column chromatography to isolate NN-X-PLA(2). NN-X-PLA(2) is a single chain protein with the relative molecular weight of 10kDa by SDS-PAGE. It was toxic to mice with an LD(50) value 0.098 mg/kg body weight (i.p.) and the mice exhibited acute neurotoxic symptoms. Upon indirect stimulation, it inhibited the twitching of frog's gastrocnemius muscle in a dose dependent manner. NN-X-PLA(2) was weakly anticoagulant and devoid of cytotoxicity, myotoxicity, hemorrhage, edema inducing, and directlytic activities and effects on platelet aggregation process. Upon chemical modification independently with p-bromophenacyl bromide and acetic anhydride, NN-X-PLA(2) lost both enzymatic and toxic properties. PMID:16574178
Shashidharamurthy, R; Kemparaju, K
Background Human height is a classical example of a polygenic quantitative trait. Recent large-scale genome-wide association studies (GWAS) have identified more than 200 height-associated loci, though these variants explain only 2?10% of overall variability of normal height. The objective of this study was to investigate the variance explained by these loci in a relatively isolated population of European descent with limited admixture and homogeneous genetic background from the Adriatic coast of Croatia. Methodology/Principal Findings In a sample of 1304 individuals from the island population of Hvar, Croatia, we performed genome-wide SNP typing and assessed the variance explained by genetic scores constructed from different panels of height-associated SNPs extracted from five published studies. The combined information of the 180 SNPs reported by Lango Allen el al. explained 7.94% of phenotypic variation in our sample. Genetic scores based on 20?50 SNPs reported by the remaining individual GWA studies explained 3?5% of height variance. These percentages of variance explained were within ranges comparable to the original studies and heterogeneity tests did not detect significant differences in effect size estimates between our study and the original reports, if the estimates were obtained from populations of European descent. Conclusions/Significance We have evaluated the portability of height-associated loci and the overall fitting of estimated effect sizes reported in large cohorts to an isolated population. We found proportions of explained height variability were comparable to multiple reference GWAS in cohorts of European descent. These results indicate similar genetic architecture and comparable effect sizes of height loci among populations of European descent.
Zhang, Ge; Karns, Rebekah; Sun, Guangyun; Indugula, Subba Rao; Cheng, Hong; Havas-Augustin, Dubravka; Novokmet, Natalija; Rudan, Dusko; Durakovic, Zijad; Missoni, Sasa; Chakraborty, Ranajit; Rudan, Pavao; Deka, Ranjan
After Clostridium botulinum type G organisms and toxin were identified in necropsy specimens in cases of unexplained death in adults and infants (O. Sonnabend, W. Sonnabend, R. Heinzle, T. Sigrist, R Dirnhofer, and U. Krech, J. Infect. Dis. 143:22-27, 1981), extensive research to detect C. botulinum type G in soil samples from Switzerland was done. A total of 41 specimens from virgin soil and from cultivated land were examined for the presence of C. botulinum type G and other toxin types. Because of the lack of the lipase marker in type G, the detection of C. botulinum type G was based on the demonstration of type G organisms in enrichment cultures by a type G-specific enzyme-linked immunosorbent assay to detect both the type G toxin and antigen; enrichment cultures in which type G toxin or antigen was identified by enzyme-linked immunosorbent assay were then tested by a type G-specific gel immunodiffusion agar procedure. This method not only isolated strains of type G but also strains of Clostridium subterminale, a nontoxigenic variant of C. botulinum type G. As a consequence of the observed cross-reactions caused by strains of C. subterminale within this test system, all isolates of type G had to be definitively confirmed by mouse bioassay. The sequential steps of these methods seem to be very useful for detecting C. botulinum type G organisms. C. botulinum type G strains were isolated in five soil samples from different locations in close association with cultivated land.(ABSTRACT TRUNCATED AT 250 WORDS) Images
Sonnabend, W F; Sonnabend, U P; Krech, T
Vibrio cholerae serogroup O139 was first identified in 1992 in India and Bangladesh, in association with major epidemics of cholera in both countries; cases were noted shortly thereafter in China. We characterized 211 V. cholerae O139 isolates that were isolated at multiple sites in China between 1993 and 2012 from patients (n = 92) and the environment (n = 119). Among clinical isolates, 88 (95.7%) of 92 were toxigenic, compared with 47 (39.5%) of 119 environmental isolates. Toxigenic isolates carried the El Tor CTX prophage and toxin-coregulated pilus A gene (tcpA), as well as the Vibrio seventh pandemic island I (VSP-I) and VSP-II. Among a subset of 42 toxigenic isolates screened by multilocus sequence typing (MLST), all were in the same sequence type as a clinical isolate (MO45) from the original Indian outbreak. Nontoxigenic isolates, in contrast, generally lacked VSP-I and -II, and fell within 13 additional sequence types in two clonal complexes distinct from the toxigenic isolates. In further pulsed-field gel electrophoresis (PFGE) (with NotI digestion) studies, toxigenic isolates formed 60 pulsotypes clustered in one group, while the nontoxigenic isolates formed 43 pulsotypes which clustered into 3 different groups. Our data suggest that toxigenic O139 isolates from widely divergent geographic locations, while showing some diversity, have maintained a relatively tight clonal structure across a 20-year time span. Nontoxigenic isolates, in contrast, exhibited greater diversity, with multiple clonal lineages, than did their toxigenic counterparts. PMID:24452176
Zhang, Ping; Zhou, Haijian; Diao, Baowei; Li, Fengjuan; Du, Pengcheng; Li, Jie; Kan, Biao; Morris, J Glenn; Wang, Duochun
Phage type 99 of Salmonella enterica subsp. enterica serovar Typhimurium variant Copenhagen strains iso- lated from pigeons were examined for the presence of genotypic and phenotypic characteristics. The pulsed- field gel electrophoresis patterns obtained with XbaI and BlnI from 38 pigeon strains were compared with those obtained from 89 porcine, poultry, and human strains of variant Copenhagen. Identical patterns with
Frank Pasmans; Filip Van Immerseel; Marc Heyndrickx; An Martel; Claudine Godard; Christa Wildemauwe; Richard Ducatelle; Freddy Haesebrouck
Since 2011, outbreaks caused by influenza A(H3N2) variant [A(H3N2)v] viruses have become a public health concern in the United States. The A(H3N2)v viruses share the A(H1N1)pdm09 M gene containing the marker of M2 blocker resistance, S31N, but do not contain any known molecular markers associated with resistance to neuraminidase (NA) inhibitors (NAIs). Using a fluorescent NA inhibition (NI) assay, the susceptibilities of recovered A(H3N2)v viruses (n=168) to FDA-approved (oseltamivir and zanamivir) and other (peramivir, laninamivir, and A-315675) NAIs were assessed. All A(H3N2)v viruses tested, with the exception of a single virus strain, A/Ohio/88/2012, isolated from an untreated patient, were susceptible to the NAIs tested. The A/Ohio/88/2012 virus contained two rare substitutions, S245N and S247P, in the NA and demonstrated reduced inhibition by oseltamivir (31-fold) and zanamivir (66-fold) in the NI assay. Using recombinant NA (recNA) proteins, S247P was shown to be responsible for the observed altered NAI susceptibility, in addition to an approximately 60% reduction in NA enzymatic activity. The S247P substitution has not been previously reported as a molecular marker of reduced susceptibility to the NAIs. Using cell culture assays, the investigational antiviral drugs nitazoxanide, favipiravir, and fludase were shown to inhibit the replication of A(H3N2)v viruses, including the virus with the S247P substitution in the NA. This report demonstrates the importance of continuous monitoring of susceptibility of zoonotic influenza viruses to available and investigational antiviral drugs. PMID:24449767
Sleeman, K; Mishin, V P; Guo, Z; Garten, R J; Balish, A; Fry, A M; Villanueva, J; Stevens, J; Gubareva, L V
Staphylococcus aureus produces a particular morphological variant called small colony variant (SCV) which is responsible for persistent subclinical infections in predisposed individuals and is usually resistant to aminoglycosides and cell wall active antibiotics. Infections by SCV of S. aureus are an upcoming problem due to difficulty in laboratory diagnosis and resistance to antimicrobial chemotherapy. We here report a case of infective endocarditis caused by SCV of Staphylococcus aureus in a pediatric patient.
Bhattacharyya, S; Roy, S; Mukhopadhyay, PK; Rit, K; Dey, JB; Ganguly, U; Ray, R
Several serious diseases are caused by biofilm-associated Staphylococcus aureus. Colonial variants occur in biofilms of other bacterial species, and S. aureus variants are frequently isolated from biofilm-associated infections. Thus, we studied the generation of variants with altered expression of virulence factors in S. aureus biofilms. We observed that the number of variants found in biofilms, as measured by hemolytic activity,
Jeremy M. Yarwood; Kara M. Paquette; Ilya B. Tikh; Esther M. Volper; E. Peter Greenberg
Aspergillus oryzae NRRL 35191 was isolated as an endophyte from coffee leaves and found to produce kojic acid (KA) in culture. When inoculated\\u000a into cacao seedlings (Theobroma cacao), A. oryzae grew endophytically and synthesized KA in planta. Cacao seedlings inoculated with A. oryzae produced higher levels of caffeine than non-inoculated ones. Aspergillus oryzae may be a useful endophyte to introduce
Fabio C. Chaves; Thomas J. Gianfagna; Madhu Aneja; Francisco Posada; Stephen W. Peterson; Fernando E. Vega
Revised description and classification of atypical isolates of Pasteurella multocida from bovine lungs based on genotypic characterization to include variants previously classified as biovar 2 of Pasteurella canis and Pasteurella avium.
Strains deviating in key phenotypic characters, mainly isolated from cases of bovine pneumonia in five European countries, were genotyped in order to examine their genotypic relationship with Pasteurella multocida. Twenty-two strains of Pasteurella avium biovar 2, including variants in indole, xylose and mannitol, 18 strains of Pasteurella canis biovar 2 and variants of this taxon, five strains of P. multocida subsp. septica showing variations in indole and ornithine decarboxylase, nine strains of P. multocida subsp. multocida showing variation in ornithine decarboxylase and mannitol, and type strains of the subspecies of P. multocida were included. Ribotyping was used to examine the relationship of the strains, and 13 types, each containing between one and 20 isolates, were observed. Identical ribotypes were observed in some cases for P. avium biovar 2 and either P. canis biovar 2 or P. multocida subsp. septica. ITS (16S-23S rRNA internal transcribed spacer) fragment-length profiling showed identity of the majority of strains (47 of 52), representing all four taxa, with only five divergent strains. A 16S rRNA sequence comparison of 11 strains representing the main ribotype clusters showed 99.9 % similarity to the type strain of P. multocida subsp. multocida, but only 97.4 % similarity was obtained to P. canis (biovar 1) and 93.7 % to P. avium (biovar 1). A species-specific PCR test for P. multocida gave a positive result with biovar 2 variants of P. avium and P. canis. DNA-DNA hybridizations between strains of P. multocida, biovar 2 variants of P. avium and P. canis, and P. multocida subsp. septica confirmed similarity at the species level. It is proposed, on the basis of genotypic similarity, that P. multocida be reclassified to include the biovar 2 variants of P. avium and P. canis and that the existence of the biovar 2 variants of P. avium and P. canis is highly questionable. It is concluded that the redefined P. multocida is genotypically homogeneous, although phenotypically diverse lineages exist with respect to ornithine decarboxylase, indole and mannitol, characters that have been regarded as essential for identification to the species level. A formal reclassification of the species is not possible, however, since too few strains have been found to vary in these key characters. Considering the phenotypic diversity of P. multocida, identification will have to depend partly on genotypic methods and the source host also seems important for safe diagnosis. PMID:15184562
Christensen, Henrik; Angen, Øystein; Olsen, John Elmerdahl; Bisgaard, Magne
Coffee, one of the most popular beverages in the world, contains many different physiologically active compounds with a potential impact on people’s health. Despite the recent attention given to the genetic basis of its consumption, very little has been done in understanding genes influencing coffee preference among different individuals. Given its markedly bitter taste, we decided to verify if bitter receptor genes (TAS2Rs) variants affect coffee liking. In this light, 4066 people from different parts of Europe and Central Asia filled in a field questionnaire on coffee liking. They have been consequently recruited and included in the study. Eighty-eight SNPs covering the 25 TAS2R genes were selected from the available imputed ones and used to run association analysis for coffee liking. A significant association was detected with three SNP: one synonymous and two functional variants (W35S and H212R) on the TAS2R43 gene. Both variants have been shown to greatly reduce in vitro protein activity. Surprisingly the wild type allele, which corresponds to the functional form of the protein, is associated to higher liking of coffee. Since the hTAS2R43 receptor is sensible to caffeine, we verified if the detected variants produced differences in caffeine bitter perception on a subsample of people coming from the FVG cohort. We found a significant association between differences in caffeine perception and the H212R variant but not with the W35S, which suggests that the effect of the TAS2R43 gene on coffee liking is mediated by caffeine and in particular by the H212R variant. No other significant association was found with other TAS2R genes. In conclusion, the present study opens new perspectives in the understanding of coffee liking. Further studies are needed to clarify the role of the TAS2R43 gene in coffee hedonics and to identify which other genes and pathways are involved in its genetics.
Pirastu, Nicola; Kooyman, Maarten; Traglia, Michela; Robino, Antonietta; Willems, Sara M.; Pistis, Giorgio; d'Adamo, Pio; Amin, Najaf; d'Eustacchio, Angela; Navarini, Luciano; Sala, Cinzia; Karssen, Lennart C.; van Duijn, Cornelia; Toniolo, Daniela; Gasparini, Paolo
Coffee, one of the most popular beverages in the world, contains many different physiologically active compounds with a potential impact on people's health. Despite the recent attention given to the genetic basis of its consumption, very little has been done in understanding genes influencing coffee preference among different individuals. Given its markedly bitter taste, we decided to verify if bitter receptor genes (TAS2Rs) variants affect coffee liking. In this light, 4066 people from different parts of Europe and Central Asia filled in a field questionnaire on coffee liking. They have been consequently recruited and included in the study. Eighty-eight SNPs covering the 25 TAS2R genes were selected from the available imputed ones and used to run association analysis for coffee liking. A significant association was detected with three SNP: one synonymous and two functional variants (W35S and H212R) on the TAS2R43 gene. Both variants have been shown to greatly reduce in vitro protein activity. Surprisingly the wild type allele, which corresponds to the functional form of the protein, is associated to higher liking of coffee. Since the hTAS2R43 receptor is sensible to caffeine, we verified if the detected variants produced differences in caffeine bitter perception on a subsample of people coming from the FVG cohort. We found a significant association between differences in caffeine perception and the H212R variant but not with the W35S, which suggests that the effect of the TAS2R43 gene on coffee liking is mediated by caffeine and in particular by the H212R variant. No other significant association was found with other TAS2R genes. In conclusion, the present study opens new perspectives in the understanding of coffee liking. Further studies are needed to clarify the role of the TAS2R43 gene in coffee hedonics and to identify which other genes and pathways are involved in its genetics. PMID:24647340
Pirastu, Nicola; Kooyman, Maarten; Traglia, Michela; Robino, Antonietta; Willems, Sara M; Pistis, Giorgio; d'Adamo, Pio; Amin, Najaf; d'Eustacchio, Angela; Navarini, Luciano; Sala, Cinzia; Karssen, Lennart C; van Duijn, Cornelia; Toniolo, Daniela; Gasparini, Paolo
We investigated the occurrence of multidrug resistance in 44 Enterobacter aerogenes and Klebsiella pneumoniae clinical isolates. Efflux was involved in resistance in E. aerogenes isolates more frequently than in K. pneumoniae isolates (100 versus 38% of isolates) and was associated with the expression of phenylalanine arginine ?-naphthylamide-susceptible active efflux. AcrA-TolC overproduction in E. aerogenes isolates was noted. An analysis of four E. aerogenes isolates for which cefepime MICs were high revealed no modification in porin expression but a new specific mutation in the AmpC ?-lactamase.
Tran, Que-Tien; Dupont, Myrielle; Lavigne, Jean-Philippe; Chevalier, Jacqueline; Pages, Jean-Marie; Sotto, Albert; Davin-Regli, Anne
The role of rare variants has become a focus in the search for association with complex traits. Imputation is a powerful and cost-efficient tool to access variants that have not been directly typed, but there are several challenges when imputing rare variants, most notably reference panel selection. Extensions to rare variant association tests to incorporate genotype uncertainty from imputation are discussed, as well as the use of imputed low frequency and rare variants in the study of population isolates.
Asimit, Jennifer L; Zeggini, Eleftheria
Chemical sanitizers are routinely used during commercial flume washing of fresh-cut leafy greens to minimize cross-contamination from the water. This study assessed the efficacy of five commercial sanitizer treatments against Escherichia coli O157:H7 on iceberg lettuce, in wash water, and on equipment during simulated commercial production in a pilot-scale processing line. Iceberg lettuce (5.4 kg) was inoculated to contain 10(6) CFU/g of a four-strain cocktail of nontoxigenic, green fluorescent protein-labeled, ampicillin-resistant E. coli O157:H7 and processed after 1 h of draining at ~22 °C. Lettuce was shredded using a commercial slicer, step-conveyed to a flume tank, washed for 90 s using six different treatments (water alone, 50 ppm of peroxyacetic acid, 50 ppm of mixed peracid, or 50 ppm of available chlorine either alone or acidified to pH 6.5 with citric acid [CA] or T-128), and then dried using a shaker table and centrifugal dryer. Various product (25-g) and water (50-ml) samples collected during processing along with equipment surface samples (100 cm(2)) from the flume tank, shaker table, and centrifugal dryer were homogenized in neutralizing buffer and plated on tryptic soy agar. During and after iceberg lettuce processing, none of the sanitizers were significantly more effective (P ? 0.05) than water alone at reducing E. coli O157:H7 populations on lettuce, with reductions ranging from 0.75 to 1.4 log CFU/g. Regardless of the sanitizer treatment used, the centrifugal dryer surfaces yielded E. coli O157:H7 populations of 3.49 to 4.98 log CFU/100 cm(2). Chlorine, chlorine plus CA, and chlorine plus T-128 were generally more effective (P ? 0.05) than the other treatments, with reductions of 3.79, 5.47, and 5.37 log CFU/ml after 90 s of processing, respectively. This indicates that chlorine-based sanitizers will likely prevent wash water containing low organic loads from becoming a vehicle for cross-contamination. PMID:24215685
Davidson, Gordon R; Buchholz, Annemarie L; Ryser, Elliot T
A total of 209 clinical isolates of Pseudomonas (193 Pseudomonas aeruginosa ,1 0P. putida ,4 P. stutzeri, and 2 P. fluorescens isolates) with reduced susceptibilities to imipenem and\\/or ceftazidime were subjected to PCR assays with primers specific for blaIMP-1, blaIMP-2, blaVIM-1, and blaVIM-2 and sequence analysis to identify the metallo-b-lactamases (MBLs) prevalent among these organisms in Taiwan; and 21 isolates
JING-JOU YAN; PO-REN HSUEH; WEN-CHIEN KO; KWEN-TAY LUH; SHU-HUEI TSAI; HSIU-MEI WU; JIUNN-JONG WU
Since 2009, erysipelas infection among pigs in Japan has been increasing. This study investigated the prevalence, and characteristics of Erysipelothrix rhusiopathiae isolates in Japan from 2008 to 2010 and assessed the efficacy of current commercial erysipelas vaccines. Based on polymorphisms in a 432-bp hypervariable region in the surface protective antigen A (spaA) gene, 34 isolates were classified into three groups: (i) Group 1 with methionine at position 203 (Met-203) and isoleucine at position 257 (Ile-257) (18 isolates of serotype 1a and one untypable isolate). (ii) Group 2 with Ile-257 (12 isolates of serotypes 1a, 1b, 2, 10 and 11), and (iii) Group 3 with alanine at position 195 (Ala-195) and Ile-257 (three isolates of serotype 1a). Isolates with Met-203 were highly pathogenic in mice and pigs, causing death in the pig and LD50 values of 0.45-1.45 CFU per mouse. One live and three inactivated commercial E. rhusiopathiae vaccines were evaluated for efficacy against a Met-203 isolate. Almost all mice and pigs that received vaccine survived, while non-vaccinated controls all died within 5 days of the challenge. This indicates that swine erysipelas vaccines might be still effective in protecting animals against the recently prevalent Met-203 isolates in Japan. PMID:24405986
Uchiyama, Mariko; Yamamoto, Kinya; Ochiai, Mariko; Yamamoto, Tsukasa; Hirano, Fumiya; Imamura, Saiki; Nagai, Hidetaka; Ohishi, Kouji; Horiuchi, Noriyuki; Kijima, Mayumi
The term "migrant variant" is not used in the headache classification of the International Headache Society (IHS), but it includes those forms of migraine that are not typical of migraine with or without aura. Headaches that do not quite fulfill all of the IHS criteria are termed "migrainous disorder." Migraine associated with auras arising from unusual sites includes basilar migraine, retinal migraine, and ophthalmoplegic migraine. Two of the chromosomal sites for hemiplegic migraine have been identified. Migraine aura may occur without headache and an aura may be prolonged. Migrainous infarct has occurred when the aura lasts more than 1 week or imaging studies are positive and other etiologies have been ruled out. If the migraine attack is prolonged beyond 3 days the term "status migrainousus" is applied. PMID:11252150
Hepatitis C virus (HCV), a single-stranded RNA virus of the family Flaviviridae, has a wide range of genetic heterogeneity: 6–11 genotypes (or 6 clades) have been known and each genotype comprises multiple subtypes. Here we report the entire nucleotide sequence of an HCV isolate from a patient in Moldova with chronic hepatitis (isolate name VAT96). The genetic organization of VAT96
Evgenyi I. Samokhvalov; Minako Hijikata; Rodica I. Gylka; Dmitri K. Lvov; Shunji Mishiro
It has long been assumed that cowpea golden mosaic disease (CGMD) in southern Asia is caused by a begomovirus distinct from\\u000a those causing disease in other legumes. The components of a begomovirus causing CGMD in western India were isolated, cloned\\u000a and sequenced. Analysis of the sequences shows the virus to be an isolate of Mungbean yellow mosaic India virus, but
P. John; P. N. Sivalingam; Q. M. I. Haq; N. Kumar; A. Mishra; R. W. Briddon; V. G. Malathi
A total of 514 Shiga toxin-producing Escherichia coli (STEC) isolates from diarrheic and healthy cattle in Spain were characterized in this study. PCR showed that 101 (20%) isolates carried stx1 genes, 278 (54%) possessed stx2 genes, and 135 (26%) possessed both stx1 and stx2. Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 326 (63%) and in 151 (29%)
M. Blanco; J. E. Blanco; A. Mora; G. Dahbi; M. P. Alonso; E. A. Gonzalez; M. I. Bernardez
Draft whole-genome sequencing of the Vibrio cholerae ?1 El Tor clinical strain L3226, isolated in Moscow in 2010, was carried out. Various mutations in the virulence-associated mobile elements were determined in its genome that differentiated this strain from the reference V. cholerae ?1 El Tor strain N16961. PMID:24874670
Smirnova, Nina I; Cherkasov, Alexander V; Krasnov, Yaroslav M; Agafonov, Dmitriy A; Kutyrev, Vladimir V
Draft whole-genome sequencing of the Vibrio cholerae ?1 El Tor clinical strain L3226, isolated in Moscow in 2010, was carried out. Various mutations in the virulence-associated mobile elements were determined in its genome that differentiated this strain from the reference V. cholerae ?1 El Tor strain N16961.
Smirnova, Nina I.; Krasnov, Yaroslav M.; Agafonov, Dmitriy A.; Kutyrev, Vladimir V.
Because of the frequency of multiple antibiotic resistance, Staphylococcus species often represent a challenge in incisional infections of horses undergoing colic surgery. To investigate the evolution of antibiotic resistance patterns before and after preventative peri- and postoperative penicillin treatment, staphylococci were isolated from skin and wound samples at different times during hospitalization. Most staphylococci were normal skin commensals and belonged
Christina Schnellmann; Vinzenz Gerber; Alexandra Rossano; Valentine Jaquier; Yann Panchaud; Marcus G. Doherr; Andreas Thomann; Reto Straub; Vincent Perreten
Because of variable degrees of phosphorylation, alternative splicing, and probable instability resulting from nonenzymatic deamidation, equine beta-casein presents a complex pattern by 2-dimensional electrophoresis that needs clarification. beta-Casein prepared from Haflinger mare's milk by hydrophobic interaction chromatography was fractionated by ion-exchange chromatography according to the degree of phosphorylation. Isoforms were identified by mass spectrometry; they corresponded to the full-length protein having 3 to 7 phosphate groups and to the splicing variant involving exon 5 and containing 4 to 7 phosphate groups. Investigations of nonenzymatic deamidation showed that beta-casein did not deamidate spontaneously in stored milk and during the different steps of chromatography, but deamidation could occur when 2-dimensional electrophoresis was performed, increasing the beta-casein pattern complexity. This phenomenon was strongly minimized when the first dimension step was carried out at 10 degrees C instead of at room temperature. Finally, spot attribution on 2-dimensional pattern of beta-casein was achieved by mixing each phosphorylation isoform in its native state with the whole beta-casein fraction. PMID:19447971
Matéos, A; Girardet, J-M; Mollé, D; Dary, A; Miclo, L; Gaillard, J-L
Variants of Hirschsprung disease are conditions that clinically resemble Hirschsprung disease, despite the presence of ganglion cells in rectal suction biopsies. The characterization and differentiation of various entities are mainly based on histologic, immunohistochemical, and electron microscopy findings of biopsies from patients with functional intestinal obstruction. Intestinal neuronal dysplasia is histologically characterized by hyperganglionosis, giant ganglia, and ectopic ganglion cells. In most intestinal neuronal dysplasia cases, conservative treatments such as laxatives and enema are sufficient. Some patients may require internal sphincter myectomy. Patients with the diagnosis of isolated hypoganglionosis show decreased numbers of nerve cells, decreased plexus area, as well as increased distance between ganglia in rectal biopsies, and resection of the affected segment has been the treatment of choice. The diagnosis of internal anal sphincter achalasia is based on abnormal rectal manometry findings, whereas rectal suction biopsies display presence of ganglion cells as well as normal acetylcholinesterase activity. Internal anal sphincter achalasia is either treated by internal sphincter myectomy or botulinum toxin injection. Megacystis microcolon intestinal hypoperistalsis is a rare condition, and the most severe form of functional intestinal obstruction in the newborn. Megacystis microcolon intestinal hypoperistalsis is characterized by massive abdominal distension caused by a largely dilated nonobstructed bladder, microcolon, and decreased or absent intestinal peristalsis. Although the outcome has improved in recent years, survivors have to be either maintained by total parenteral nutrition or have undergone multivisceral transplant. This review article summarizes the current knowledge of the aforementioned entities of variant HD. PMID:22985836
Puri, Prem; Gosemann, Jan-Hendrik
Enterobacter aerogenes resistant to cefepime (MIC, 32 g\\/ml) was isolated from a patient treated with cefepime for an infection caused by a strain of E. aerogenes overproducing its AmpC -lactamase (MIC of cefepime, 0.5 g\\/ml). The AmpC -lactamase of the resistant strain had an L-293-P amino acid substitution and a high kcat\\/Km ratio for cefepime. Both of these modifications were
G. Barnaud; Y. Benzerara; J. Gravisse; L. Raskine; M. J. Sanson-Le Pors; R. Labia; G. Arlet
Three types of cytolethal distending toxin (CDT), namely, CDT-I, CDT-II, and CDT-III, have been described in Escherichia coli. Using primers designed for the detection of sequences common to the cdtB genes, we analyzed by PCR a set of 21 CDT-producing E. coli strains of intestinal and extraintestinal origins isolated from human and different animal species in several European countries and
Istvan Toth; Frederique Herault; Lothar Beutin; Eric Oswald
A Gram-negative, rod-shaped, non-spore forming, non-motile and moderate halophilic bacteria designated as strain CMC-5 was\\u000a isolated from decomposing seaweeds by enrichment culture. The growth of strain CMC-5 was assessed in synthetic seawater-based\\u000a medium containing polysaccharide. The bacterium degraded and utilized agar, alginate, carrageenan, xylan, carboxymethyl cellulose\\u000a and chitin. The strain was characterized using a polyphasic approach for taxonomic identification. Cellular
RaviChand Jonnadula; Pankaj Verma; Yogesh S. Shouche; Sanjeev C. Ghadi
The enzyme S-adenosylmethionine-DNA (cytosine-5)-methyltransferase has been identified, first time for invertebrates, in embryos of the marine polychaete annelid worm Chaetopterus variopedatus. The molecule has been isolated from embryos at 15 h of development. It is a single peptide of about 200 kDa molecular weight, cross-reacting with antibodies against sea urchin DNA methyltransferase. The enzymatic properties of the molecule are similar
Rosanna del Gaudio; Rossella Di Giaimo; Nicoletta Potenza; Margherita Branno; Francesco Aniello; Giuseppe Geraci
[Persistent and recurrent skin and soft tissue infections by Staphylococcus aureus. Impact of the small colony-variant (SCV) phenotype and of Panton-Valentine leukocidin (PVL)-positive S. aureus isolates].
Staphylococcus aureus is one of the major pathogens causing chronic skin and soft tissue infections. Particularly isolates producing Panton-Valentine leukocidin (PVL) comprising methicillin-susceptible and community-associated methicillin-resistant S. aureus (CA-MRSA) have been associated with more aggressive and persistent or relapsing courses. Beyond classical resistance mechanisms, functional resistance as shown by the small colony-variant (SCV) phenotype could be also responsible for treatment failures, despite the administration of antibiotics tested in vitro as susceptible. Also this phenotype has been associated with chronic courses of infections often with multiple exacerbations. Due to their ability to persist intracellularly, SCVs are protected from host defense and antibiotic treatment if only extracellularly active agents are administered. Reduced growth, abnormal colony morphology and changes in the metabolism of the SCVs aggravate drastically their identification, differentiation and susceptibility testing. The diagnostic and therapeutic challenges of PVL-positive and SCV isolates necessitate close collaboration with microbiological and infectious disease specialists. PMID:24445941
Becker, K; Kriegeskorte, A; Sunderkötter, C; Löffler, B; von Eiff, C
Tn5801, originally detected in Staphylococcus aureus Mu50, is a Tn916 family element in which a unique int gene (int5801) replaces the int and xis genes in Tn916 (int916 and xis916). Among 62 tet(M)-positive tetracycline-resistant Streptococcus agalactiae isolates, 43 harbored Tn916, whereas 19 harbored a Tn5801-like element (Tn5801.Sag, ?20.6 kb). Tn5801.Sag was characterized (PCR mapping, partial sequencing, and chromosomal integration) and compared to other Tn5801-like elements. Similar to Tn5801 from S. aureus Mu50, tested in parallel, Tn5801.Sag was unable to undergo circularization and conjugal transfer.
Mingoia, Marina; Morici, Eleonora; Tili, Emily; Giovanetti, Eleonora; Montanari, Maria Pia
A Gram-negative, rod-shaped, non-spore forming, non-motile and moderate halophilic bacteria designated as strain CMC-5 was isolated from decomposing seaweeds by enrichment culture. The growth of strain CMC-5 was assessed in synthetic seawater-based medium containing polysaccharide. The bacterium degraded and utilized agar, alginate, carrageenan, xylan, carboxymethyl cellulose and chitin. The strain was characterized using a polyphasic approach for taxonomic identification. Cellular fatty acid analysis showed the presence of iso-C(15:0) as major fatty acid and significant amounts of iso-C(17:1x9c) and C(18:1x7c). Phylogenetic analysis based on 16S rDNA sequence indicated that strain CMC-5 is phylogenetically related to Microbulbifer genus and 99% similar to type strain Microbulbifer elongatus DSM6810T. However in contrast to Microbulbifer elongatus DSM6810T, strain CMC-5 is non-motile, utilizes glucose, galactose, inositol and xylan, does not utilize fructose and succinate nor does it produce H2S. Further growth of bacterial strain CMC-5 was observed when inoculated in seawater-based medium containing sterile pieces of Gracilaria corticata thalli. The bacterial growth was associated with release of reducing sugar in the broth suggesting its role in carbon recycling of polysaccharides from seaweeds in marine ecosystem. PMID:19701665
Jonnadula, RaviChand; Verma, Pankaj; Shouche, Yogesh S; Ghadi, Sanjeev C
Papillary thyroid carcinomas are the most common thyroid cancers and constitute more than 70% of thyroid malignancies. The\\u000a most common etiologic factor is radiation, but genetic susceptibility and other factors also contribute to the development\\u000a of papillary thyroid carcinoma. The most common variants include conventional, follicular variant and tall cell variant. However,\\u000a many other uncommon variants have been described including
Ricardo V. Lloyd; Darya Buehler; Elham Khanafshar
Summary Naturally occurring ‘color variant’ strains ofAureobasidium pullulans are distinguished from typical strains by their brilliant pigmentation, overproduction of secreted enzymes (xylanase), and low DNA relatedness. Color variants have not previously been examined for pullulan secretion. Among five independently isolated color variants, strains NRRL Y-12,974 and YB-4026 made the greatest amounts of pullulan from cornstarch, with conversion efficiencies of about
T. D. Leathers; G. W. Nofsinger; C. P. Kurtzman; R. J. Bothast
The paper tries to contribute to a better understanding of the behaviour of base isolated asymmetric structures. Numerous variants of originally symmetric four storey RC frame building isolated by a simple lead rubber bearing base isolation system with various distributions of isolators were considered as test examples. The symmetrical structural variant and appropriate LRB bearing properties were designed according to
Vojko Kilar; David Koren
Intracellular Activity of Antibiotics in a Model of Human THP-1 Macrophages Infected by a Staphylococcus aureus Small-Colony Variant Strain Isolated from a Cystic Fibrosis Patient: Pharmacodynamic Evaluation and Comparison with Isogenic Normal-Phenotype and Revertant Strains? †
Small-colony variant (SCV) strains of Staphylococcus aureus show reduced antibiotic susceptibility and intracellular persistence, potentially explaining therapeutic failures. The activities of oxacillin, fusidic acid, clindamycin, gentamicin, rifampin, vancomycin, linezolid, quinupristin-dalfopristin, daptomycin, tigecycline, moxifloxacin, telavancin, and oritavancin have been examined in THP-1 macrophages infected by a stable thymidine-dependent SCV strain in comparison with normal-phenotype and revertant isogenic strains isolated from the same cystic fibrosis patient. The SCV strain grew slowly extracellularly and intracellularly (1- and 0.2-log CFU increase in 24 h, respectively). In confocal and electron microscopy, SCV and the normal-phenotype bacteria remain confined in acid vacuoles. All antibiotics tested, except tigecycline, caused a net reduction in bacterial counts that was both time and concentration dependent. At an extracellular concentration corresponding to the maximum concentration in human serum (total drug), oritavancin caused a 2-log CFU reduction at 24 h; rifampin, moxifloxacin, and quinupristin-dalfopristin caused a similar reduction at 72 h; and all other antibiotics had only a static effect at 24 h and a 1-log CFU reduction at 72 h. In concentration dependence experiments, response to oritavancin was bimodal (two successive plateaus of ?0.4 and ?3.1 log CFU); tigecycline, moxifloxacin, and rifampin showed maximal effects of ?1.1 to ?1.7 log CFU; and the other antibiotics produced results of ?0.6 log CFU or less. Addition of thymidine restored intracellular growth of the SCV strain but did not modify the activity of antibiotics (except quinupristin-dalfopristin). All drugs (except tigecycline and oritavancin) showed higher intracellular activity against normal or revertant phenotypes than against SCV strains. The data may help rationalizing the design of further studies with intracellular SCV strains.
Nguyen, Hoang Anh; Denis, Olivier; Vergison, Anne; Theunis, Anne; Tulkens, Paul M.; Struelens, Marc J.; Van Bambeke, Francoise
Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants are often observed during routine biomanufacture or process changes and pose a challenge to demonstrating product comparability. To gain further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity, we isolated the major charge forms of a recombinant humanized IgG1 and compared their in vitro properties and in vivo PK. The mAb starting material had a pI range of 8.7–9.1 and was composed of about 20% acidic variants, 12% basic variants and 68% main peak. Cation exchange displacement chromatography was used to isolate the acidic, basic and main peak fractions for animal studies. Detailed analyses were performed on the isolated fractions to identify specific chemical modification contributing to the charge differences and were also characterized for purity and in vitro potency prior to being administered either subcutaneously (SC) or intravenously (IV) in rats. All isolated materials had similar potency and rat FcRn binding relative to the starting material. Following IV or SC administration (10 mg/kg) in rats, no difference in serum PK was observed, indicating that physiochemical modifications and pI differences among charge variants were not sufficient to result in PK changes. Thus, these results provided meaningful information for the comparative evaluation of charge-related heterogeneity of mAbs and suggested that charge variants of IgGs do not affect the in vitro potency, FcRn binding affinity or the PK properties in rats.
Goswami, Sirj; Hutchinson, Ryan; Kwong, Zephania W; Yang, Jihong; Wang, Xiangdan; Yao, Zhenling; Sreedhara, Alavattam; Cano, Tony; Tesar, Devin; Nijem, Ihsan; Allison, David E; Wong, Pin Yee; Kao, Yung-Hsiang; Quan, Cynthia; Joshi, Amita; Harris, Reed J; Motchnik, Paul
Cyprus, located at the eastern end of the Mediterranean region, has been a place of eastern and western civilizations, and the presence of various hemoglobin (Hb) variants can be considered a testimony to past colonizations of the island. In this study, we report the structural Hb variants identified in the Cypriot population (Greek Cypriots, Maronites, Armenians, and Latinos) during the thalassemia screening of 248,000 subjects carried out at the Thalassaemia Centre, Nicosia, Cyprus, over a period of 26 years. A sample population of 65,668 people was used to determine the frequency and localization of several of the variants identified in Cyprus. The localization of some of the variants in regions where the presence of foreign people was most prevalent provides important clues to the origin of the variants. Twelve structural variants have been identified by DNA sequencing, nine concerning the beta-globin gene and three concerning the alpha-globin gene. The most common beta-globin variants identified were Hb S (0.2%), Hb D-Punjab (0.02%), and Hb Lepore-Washington-Boston (Hb Lepore-WB) (0.03%); the most common alpha-globin variant was Hb Setif (0.1%). The presence of some of these variants is likely to be directly linked to the history of Cyprus, as archeological monuments have been found throughout the island which signify the presence for many years of the Greeks, Syrians, Persians, Arabs, Byzantines, Franks, Venetians, and Turks. PMID:19373583
Kyrri, Andreani R; Felekis, Xenia; Kalogerou, Eleni; Wild, Barbara J; Kythreotis, Loukas; Phylactides, Marios; Kleanthous, Marina
Amongst the human papillomaviruses (HPVs), the genus Alphapapillomavirus contains HPV types that are uniquely pathogenic. They can be classified into species and types based on genetic distances between viral genomes. Current circulating infectious HPVs constitute a set of viral genomes that have evolved with the rapid expansion of the human population. Viral variants were initially identified through restriction enzyme polymorphisms and more recently through sequence determination of viral fragments. Using partial sequence information, the history of variants, and the association of HPV variants with disease will be discussed with the main focus on the recent utilization of full genome sequence information for variant analyses. The use of multiple sequence alignments of complete viral genomes and phylogenetic analyses have begun to define variant lineages and sublineages using empirically defined differences of 1.0-10.0% and 0.5-1.0%, respectively. These studies provide the basis to define the genetics of HPV pathogenesis. PMID:23998342
Burk, Robert D; Harari, Ariana; Chen, Zigui
Sequencing of the human genome and introduction of clinical next-generation sequencing enable discovery of all DNA variants carried by an individual. Variants may be solely responsible for disease, may contribute to disease, or may have no influence on the development of disease. Interpreting the effect of these variants upon disease is a major challenge for medicine. Although the process is still evolving, certain methods are useful in discriminating the effect of variants upon phenotype. These methods have been employed to the greatest extent in Mendelian disorders where deleterious changes in one gene can cause disease. Here, we briefly review the relative merits of these methods, with emphasis on using a comprehensive approach modelled after the analysis of variants that causes cystic fibrosis. PMID:24343785
Sosnay, Patrick R; Cutting, Garry R
[PSI+] is an amyloid-based prion of Sup35p, a subunit of the translation termination factor. Prion “strains” or “variants” are amyloids with different conformations of a single protein sequence, conferring different phenotypes, but each relatively faithfully propagated. Wild Saccharomyces cerevisiae isolates have SUP35 alleles that fall into three groups, called reference, ?19, and E9, with limited transmissibility of [PSI+] between cells expressing these different polymorphs. Here we show that prion transmission pattern between different Sup35 polymorphs is prion variant-dependent. Passage of one prion variant from one Sup35 polymorph to another need not change the prion variant. Surprisingly, simple mitotic growth of a [PSI+] strain results in a spectrum of variant transmission properties among the progeny clones. Even cells that have grown for >150 generations continue to vary in transmission properties, suggesting that simple variant segregation is insufficient to explain the results. Rather, there appears to be continuous generation of a cloud of prion variants, with one or another becoming stochastically dominant, only to be succeeded by a different mixture. We find that among the rare wild isolates containing [PSI+], all indistinguishably “weak” [PSI+], are several different variants based on their transmission efficiencies to other Sup35 alleles. Most show some limitation of transmission, indicating that the evolved wild Sup35 alleles are effective in limiting the spread of [PSI+]. Notably, a “strong [PSI+]” can have any of several different transmission efficiency patterns, showing that “strong” versus “weak” is insufficient to indicate prion variant uniformity.
Bateman, David A.; Wickner, Reed B.
SUMMARY: Stable small-colony variants of Escherichia coli K-12 and several of its auxotrophs were isolated by treatment of cultures with copper sulphate solutions. These variants did not show any associated changes in nutritional, fermentative or serological characters. The small-colony forms showed normal recombining character in two instances, but in one variant the F - form showed a lower recombination rate
R. C. CLOWES; D. ROWLEY
Preharvest internalization of Escherichia coli O157:H7 into the roots of leafy greens is a food safety risk because the pathogen may be systemically transported to edible portions of the plant. In this study, both abiotic (degree of soil moisture) and biotic (E. coli O157:H7 exposure, presence of Shiga toxin genes, and type of leafy green) factors were examined to determine their potential effects on pathogen internalization into roots of leafy greens. Using field soil that should have an active indigenous microbial community, internalized populations in lettuce roots were 0.8 to 1.6 log CFU/g after exposure to soil containing E. coli O157:H7 at 5.6 to 6.1 log CFU/g. Internalization of E. coli O157:H7 into leafy green plant roots was higher when E. coli O157:H7 populations in soil were increased to 7 or 8 log CFU/g or when the soil was saturated with water. No differences were noted in the extent to which internalization of E. coli O157:H7 occurred in spinach, lettuce, or parsley roots; however, in saturated soil, maximum levels in parsley occurred later than did those in spinach or lettuce. Translocation of E. coli O157:H7 from roots to leaves was rare; therefore, decreases observed in root populations over time were likely the result of inactivation within the plant tissue. Shiga toxin-negative (nontoxigenic) E. coli O157:H7 isolates were more stable than were virulent isolates in soil, but the degree of internalization of E. coli O157:H7 into roots did not differ between isolate type. Therefore, these nontoxigenic isolates could be used as surrogates for virulent isolates in field trials involving internalization. PMID:24853507
Erickson, Marilyn C; Webb, Cathy C; Davey, Lindsey E; Payton, Alison S; Flitcroft, Ian D; Doyle, Michael P
Isolates of Listeria monocytogenes saved from outbreaks of listeriosis, cases of sporadic listeriosis, and similar events do not always belong to a solitary genetic variant. Variants of the same strain may have evolved from a unique clone, and plasmid loss or gain and phage-mediated genetic changes are suggested as the main mechanism. Some of these reports are summarized in this short communication. PMID:23988078
Tham, Wilhem; Lopez-Valladares, Gloria; Helmersson, Seved; Wennström, Stefan; Österlund, Anders; Danielsson-Tham, Marie-Louise
"Variants of Hirschsprung's disease" are conditions that clinically resemble Hirschsprung's disease (HD), despite the presence of ganglion cells in rectal suction biopsies. The diagnosis and management of these patients can be challenging. Specific histological, immunohistochemical and electron microscopic investigations are required to characterize this heterogeneous group of functional bowel disorders. Variants of HD include intestinal neuronal dysplasia, intestinal ganglioneuromatosis, isolated hypoganglionosis, immature ganglia, absence of the argyrophil plexus, internal anal sphincter achalasia and congenital smooth muscle cell disorders such as megacystis microcolon intestinal hypoperistalsis syndrome. This review article systematically classifies variants of HD based on current diagnostic criteria with an additional focus on pathogenesis, epidemiology, clinical presentation, management and outcome. PMID:23943250
Friedmacher, Florian; Puri, Prem
HBV replicates through reverse transcription of an RNA intermediate; the inherent lack of proofreading causes a high mutation frequency. Mutations in the precore and core promoter regions that abolish or reduce the production of hepatitis B e antigen occur most commonly. Patients with these HBV variants remain viremic and can develop progressive liver disease. Mutations in the core promoter region
Watcharasak Chotiyaputta; Anna S. F. Lok
Monoclonal antibodies (mAbs) have emerged as one of the most important classes of biotherapeutics, although development of these molecules is long and arduous. A production cell line must be established, and growth conditions for the cells and purification processes for the product must be optimized. Integration of the appropriate analytical strategies in these activities is the cornerstone of Quality by Design and in-process control approaches are encouraged by the Food and Drug Administration. We report here the development of a reversed phase-high performance liquid chromatography (RP-HPLC) method to follow the presence of a mAb product-related variant observed during the purification process development. The variant eluted as a later peak on RP-HPLC, compared with the mAb control (3.25 min and 2.85 min, respectively). We isolated this hydrophobic variant and further analyzed it by mass spectrometry. We identified the variant as a mAb with an incompletely processed leader sequence attached to the N-terminus of one of the two heavy chains.
Ambrogelly, Alexandre; Liu, Yan-Hui; Li, Hong; Mengisen, Selina; Yao, Bingyi; Xu, Wei; Cannon-Carlson, Susan
Writing task variants can increase test security in high-stakes essay assessments by substantially increasing the pool of available writing stimuli and by making the specific writing task less predictable. A given prompt (parent) may be used as the basis for one or more different variants. Six variant types based on argument essay prompts from a…
Bridgeman, Brent; Trapani, Catherine; Bivens-Tatum, Jennifer
The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.
Teter, Sarah; Ward, Connie; Cherry, Joel; Jones, Aubrey; Harris, Paul; Yi, Jung
The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.
Teter, Sarah (Davis, CA); Ward, Connie (Hamilton, MT); Cherry, Joel (Davis, CA); Jones, Aubrey (Davis, CA); Harris, Paul (Carnation, WA); Yi, Jung (Sacramento, CA)
BACKGROUND: We are conducting a genetic study of autism in the isolated population of the Central Valley of Costa Rica (CVCR). A novel Neuregulin 1 (NRG1) missense variant (exon 11 G>T) was recently associated with psychosis and schizophrenia (SCZ) in the same population isolate. METHODS: We genotyped the NRG1 exon 11 missense variant in 146 cases with autism, or autism
Lynne A McInnes; Leonid Ouchanov; Alisa Nakamine; Patricia Jimenez; Marcela Esquivel; Marietha Fallas; Silvia Monge; Pamela Bondy; Elina R Manghi
Keratins 8 and 18 (K8 and K18) are the cytoskeletal intermediate filament proteins of adult hepatocytes. Murine models indicate that mutations in the K8- and K18-encoding genes predispose to liver injury. Moreover, studies showed an association of K8/K18 variants with human acute and chronic liver diseases. However, only little is known about a putative association of K8/K18 variants with cryptogenic hepatitis, a frequent liver disease of enigmatic etiology, often necessitating liver transplantation. Therefore, we analyzed whether K8 variants associate with cryptogenic hepatitis in a German cohort of patients in comparison to control blood bank donors. Genomic DNA isolated from liver biopsies of cryptogenic hepatitis patients or peripheral blood of healthy donors was analyzed for K8 variants by PCR amplification and direct DNA sequencing. We identified 8 novel heterozygous or homozygous amino acid-altering K8 variants in 5 of 62 cryptogenic hepatitis patients (8.1 %) and in none of 67 controls (P?=?0.02). Previously described K8 variants p.G62C and p.R341H were found at similar incidence in cryptogenic hepatitis patients and controls. Hence, they were considered as polymorphisms not associated with liver disease progression. In conclusion, previously undescribed K8 variants associate with cryptogenic hepatitis in a German cohort of patients, possibly predisposing carriers to the development of liver disease. PMID:22419260
Zierden, Mario; Penner, Arndt-Hendrik; Montesinos-Rongen, Manuel; Weferling, Maren; Drebber, Uta; Stift, Judith; Fries, Jochen W U; Odenthal, Margarete; Rosenkranz, Stephan; Dienes, Hans-Peter
We report three cases of an unusual form of "reel syndrome" characterized by isolated, reeling dislodgement of a single lead in patients with dual-chamber or biventricular devices. One of these patients presented with worsening heart failure due to loss of left ventricular pacing and the others were detected incidentally during scheduled device checks. We suspect that a ratchet mechanism was probably responsible for this and that this type of dislodgement is not a twiddler variant. We propose a simple solution for prevention. PMID:18688700
Patel, Mehul B; Pandya, Khyati; Shah, Ashok J; Lojewski, Elizabeth; Castellani, Mark D; Thakur, Ranjan
Polymerase chain reaction (PCR) detected the presence of various genes associated with virulence in genome of strains V. cholerae eltor isolated in Turkmenistan territory during epidemic and epidemic-free perios. It was found that a complete set of virulence genes (ctxA+, tcpA+ and toxR+) contained strains isolated from patients, carriers and environment only in cholera epidemics. Strains isolated from the environment in the period free of epidemics did not contain ctxA and tcpA in 78.2% of cases, but 5.2% of the strains carried a complete set of virulence genes. There were also nontoxigenic strains containing genes tcpA and toxR. Such strains were isolated from the environment (16.6%) and vibrion carriers (42.9%). Isolated were also strains V.cholerae eltor carrying bacteriophage CTX phi with incomplete set of virulence genes and having genotype ctxA-, ace+ and zot+. Almost all the strains ctxA-, tcpA+ carry attRS1-site in genome. This shows that such strains may transform into toxigenic as a result of infection with bacteriophage CTX phi. PMID:12534264
Smirnova, N I; Kostromitina, E A; Cheldyshova, N B; Kutyrev, V V
A catalase-negative variant of Escherichia coli was isolated from the blood of a patient with acute leukemia who had been treated with various antibiotics and gentamicin. This small-colony variant grew almost as actively under anaerobic conditions as its large-colony revertant or E. coli NIHJ JC-2. The variant was resistant to gentamicin, in contrast with the revertant. Streptomycin and hemin stimulated growth of the variant slightly. With repeated subculturing the variant tended to increase slightly in colony size with coincident recovery of weak catalase production. The possibility that such a variant may have been induced by gentamicin was indicated. Images
Funada, H; Hattori, K I; Kosakai, N
Quantum measures play a key role in quantum interactions. In this paper, a variant simulation system – a variant double path model – is proposed that uses multiple variable logic functions and variant principle schemes applied to input\\/output relationships as a variant quaternion. Under this mechanism, a two-state system of quantum interactions can be simulated by the variant double path
Jeffrey Zheng; Chris Zheng
Quantum measures play a key role in quantum interactions. In this paper, a variant simulation system – a variant double path model – is proposed that uses multiple variable logic functions and variant principle schemes applied to input\\/output relationships as a variant quaternion. Under this mechanism, a two-state system of quantum interactions can be simulated by the variant double path
Jeffrey Zheng; Chris Zheng
A new variant of human galactokinase activity is described. This enzyme shows reduzed catalytic activity both in red and white blood cells, lower Km for ATPMg2–, and increased in vivo instability when compared to the normal enzyme. Thermo stability and pH optimum are not modified. We have labelled this enzyme the Urbino variant and have suggested the procedure to distinguish
Mauro Magnani; Luigi Cucchiarini; Marina Dachà; Giorgio Fornaini
Conventional actin has been implicated in various nuclear processes including chromatin remodeling, transcription, nuclear transport, and overall nuclear structure. Moreover, actin has been identified as a component of several chromatin remodeling complexes present in the nucleus. In animal cells, nuclear actin exists as a dynamic equilibrium of monomers and polymers. Actin binding proteins (ABPs) such as ADF/Cofilin and profilin play a role in actin import and export, respectively. However, very little is known about the localization and roles of nuclear actin in plants. In multicellular plants and animals, actin is comprised of an ancient and divergent family of protein variants. Here, we have investigated the presence and localization of two ancient subclasses of actin in isolated Arabidopsis nuclei. Although the subclass 1 variants ACT2 and ACT8 and subclass 2 variant ACT7 were found distributed throughout the nucleoplasm, ACT7 was often found more concentrated in nuclear speckles than subclass 1 variants. The nuclei from the act2-1/act8-2 double null mutant and the act7-5 null mutant lacked their corresponding actin variants. In addition, serial sectioning of several independent nuclei revealed that ACT7 was notably more abundant in the nucleolus than the subclass 1 actins. Profilin and ADF proteins were also found in significant levels in plant nuclei. The possible functions of differentially localized nuclear actin variants are discussed.
Kandasamy, Muthugapatti K.; McKinney, Elizabeth C.; Meagher, Richard B.
The present invention relates to variants of a parent cellobiohydrolase II. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.
This effort describes the use of time variant coherence causality based analysis to separate the effects of nonstationary time variant vibration excitation sources. A time variant coherence function using the Short Time Fourier Transform (STFT) is first discussed. The concept of a time variant coherent output power for source separation of systems with time variant transfer functions is developed. A parametric study is performed to examine the coherent output power separation capabilities with respect to the data processing parameters. The study guided the selection of the time-frequency processing parameters judged to provide a suitable compromise between the time event localization and output amplitude source separation. The time variant coherent output power is then applied to separate the effects of the two possible excitation sources on the prototype vibration isolation floor. The application was a subscale prototype isolation floor for a proposed vibration sensitive equipment site adjacent to a busy freight rail line. The moving train created time variant transmission paths. As there was a direct line of sight between the prototype floor and the rail line there was an airborne acoustic excitation path in addition to a ground path. The short time coherent output power was applied to separate prototype isolation floor vibration into respective components related to the two candidate sources. The analysis and discussion of the results focuses upon the interpretation and issues in such a complicated realistic environment. Ultimately the application was successful providing an explanation as to why the observed vibration isolation was degraded at higher frequencies.
Trethewey, Martin W.
Catalase and proteolytic activity of the culures and morphological variants of Bacillus mesentericus fuscus, Bac. mesentericus vulgatus were studied. The variants were obtained as a result of prolonged cultivation of the stock strains in the potato mash under the layer of vaseline oil. The level of catalase activity varies in different morphological variants of the same culture, changes with age and depends on the storage conditions. The catalase activity in the rough, smooth and papillar variants that were freshly isolated from the potato mash was 1.5=2.5 times lower than that in the variants long kept on the agar medium. The quantitative indexes of the proteolytic activity of different variants also varied. PMID:1208411
Vasilevskaya, I A; Kolchinskaya, I D; Sergeichuk, M G; Roy, A A
Background Heterochromatic variants of pericentromere of chromosome 9 are reported and discussed since decades concerning their detailed structure and clinical meaning. However, detailed studies are scarce. Thus, here we provide the largest ever done molecular cytogenetic research based on >300 chromosome 9 heteromorphism carriers. Results In this study, 334 carriers of heterochromatic variants of chromosome 9 were included, being 192 patients from Western Europe and the remainder from Easter-European origin. A 3-color-fluorescence in situ hybridization (FISH) probe-set directed against for 9p12 to 9q13~21.1 (9het-mix) and 8 different locus-specific probes were applied for their characterization. The 9het-mix enables the characterization of 21 of the yet known 24 chromosome 9 heteromorphic patterns. In this study, 17 different variants were detected including five yet unreported; the most frequent were pericentric inversions (49.4%) followed by 9qh-variants (23.9%), variants of 9ph (11.4%), cenh (8.2%), and dicentric- (3.8%) and duplication-variants (3.3%). For reasons of simplicity, a new short nomenclature for the yet reported 24 heteromorphic patterns of chromosome 9 is suggested. Six breakpoints involved in four of the 24 variants could be narrowed down using locus-specific probes. Conclusions Based on this largest study ever done in carriers of chromosome 9 heteromorphisms, three of the 24 detailed variants were more frequently observed in Western than in Eastern Europe. Besides, there is no clear evidence that infertility is linked to any of the 24 chromosome 9 heteromorphic variants.
Migraine is amongst the oldest of diseases known to mankind. Migraine is a heterogenous entity, usually characterised by periodic attacks of headache on one or both sides of the head. These may be accompanied by nausea, vomiting, increased sensitivity of the eyes to light (photophobia), increased sensitivity to sound (phonophobia), dizziness, blurred vision, cognitive disturbances, and other symptoms. Migraines are not always preceded by an aura and some migraines may not include headache. If migraine does not manifest itself in the form of headache but in some other form such as paroxysmal episodes of prolonged visual auras, atypical sensory, motor, or visual aura, confusion, dysarthria, focal neurologic deficits with or without a headache, it is labelled a Migraine Variant (MV). MV is therefore diagnosed by the history of paroxysmal symptoms with or without cephalgia and a prior history of migraine with aura, in the absence of other medical disorders that may contribute to the symptoms. Many of the MVs have been included and redefined in the revised edition of The International Classification of Headache Disorders (ICHD-II) 2004 classification. These include hemiplegic migraine, basilar migraine, childhood periodic syndromes, retinal migraine, complicated migraine and ophthalmoplegic migraine. Even though conditions such as vertiginous migraine, acute confusional migraine of childhood and nocturnal migraine are well recognized entities, they have not yet been included in IHCD-II, but will be discussed here in brief because they are relatively common conditions. PMID:21049701
Srinivasa, R; Kumar, Rahul
The present invention relates to variants of a parent beta-glucosidase, comprising a substitution at one or more positions corresponding to positions 142, 183, 266, and 703 of amino acids 1 to 842 of SEQ ID NO: 2 or corresponding to positions 142, 183, 266, and 705 of amino acids 1 to 844 of SEQ ID NO: 70, wherein the variant has beta-glucosidase activity. The present invention also relates to nucleotide sequences encoding the variant beta-glucosidases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.
Fidantsef, Ana (Davis, CA) [Davis, CA; Lamsa, Michael (Davis, CA) [Davis, CA; Clancy, Brian Gorre (Elk Grove, CA) [Elk Grove, CA
The present invention relates to variants of a parent beta-glucosidase, comprising a substitution at one or more positions corresponding to positions 142, 183, 266, and 703 of amino acids 1 to 842 of SEQ ID NO: 2 or corresponding to positions 142, 183, 266, and 705 of amino acids 1 to 844 of SEQ ID NO: 70, wherein the variant has beta-glucosidase activity. The present invention also relates to nucleotide sequences encoding the variant beta-glucosidases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.
Fidantsef, Ana (Davis, CA); Lamsa, Michael (Davis, CA); Gorre-Clancy, Brian (Elk Grove, CA)
Regulation of chromatin activity by covalent histone modifications has been long recognized. Histones that constitute the\\u000a nucleosome are encoded by large families of genes and display a strong degree of conservation. However, histone variants exist\\u000a and it is becoming clear that they play important roles in genome regulation. While most studies of the role of histone3 (H3)\\u000a variants in transcriptional
Mathieu Ingouff; Frédéric Berger
Most histones are assembled into nucleosomes behind the replication fork to package newly synthesized DNA, but some histones\\u000a are deposited independent of replication. Replication-independent histone variants of H3 and H2A have evolved to participate\\u000a in gene regulation, transcriptional elongation, chromosome segregation and DNA repair in almost all eukaryotes. Because histone\\u000a variants are deposited on a chromatinized template, they replace canonical
Four cases of variant angina are reported, in which total remission of anginal pain was documented during a follow-up of seven months, four years, five years, and 15 years, respectively. During this relatively long follow-up, the clinical course of the disease was apparently benign. The possibility of spontaneously and complete recovery may be postulated. The natural history of relatively benign forms a variant angina is poorly known and understood.
Girotti, A L; Rutitzky, B; Schmidberg, J; Crosatto, J; Rosenbaum, M B
Disease- and locus-specific variant databases have been a valuable resource to clinical and research geneticists. With the recent rapid developments in technologies, the number of DNA variants detected in a typical molecular genetics laboratory easily exceeds 1,000. To keep track of the growing inventory of DNA variants, many laboratories employ information technology to store the data as well as distributing the data and its associated information to clinicians and researchers via the Web. While it is a valuable resource, the hosting of a web-accessible database requires collaboration between bioinformaticians and biologists and careful planning to ensure its usability and availability. In this chapter, a series of tutorials on building a local DNA variant database out of a sample dataset will be provided. However, this tutorial will not include programming details on building a web interface and on constructing the web application necessary for web hosting. Instead, an introduction to the two commonly used methods for hosting web-accessible variant databases will be described. Apart from the tutorials, this chapter will also consider the resources and planning required for making a variant database project successful. PMID:18453092
Fung, David C Y
We sequenced 34 new peach latent mosaic viroid (PLMVd) variants isolated from nine different peach cultivars. This study provides the widest view of PLMVd diversity reported to date and includes the original characterization of North American variants, which cannot be differentiated from European sequences. PLMVd appears as a species in which each isolate is a complex mixture of RNAs. Analysis
M. Pelchat; D. Lévesque; J. Ouellet; S. Laurendeau; S. Lévesque; J. Lehoux; D. A. Thompson; K. C. Eastwell; L. J. Skrzeczkowski; J. P. Perreault
We isolated a novel hepatitis E virus (HEV-Au1) variant from a patient in Austria suffering from acute viral hepatitis, who had no known risk factors for acquiring hepatitis E. The clinical presentation and initial serological findings have been reported previously. In this paper we report the results of sequence and phylogenetic analysis of HEV products from viral RNA isolated from
Harald C. Worm; George G. Schlauder; Herbert Wurzer; Isa K. Mushahwar
Full-length genomes of 16 hepatitis C virus genotype 1 isolates representing subtypes 1c, 1d, 1e, 1g, 1h, 1i, 1j and 1k, and two new subtypes 1m and 1n, and four unclassified variants reveal ancestral relationships among subtypes.
We characterized the full-length genomes of 16 distinct hepatitis C virus genotype 1 (HCV-1) isolates. Among them, four represented the first full-length genomes for subtypes 1d (QC103), 1i (QC181), 1j (QC329) and 1k (QC82), and another four corresponded to subtypes 1c (QC165), 1g (QC78), 1h (QC156) and 1e (QC172). Both QC196 and QC87 were assigned into a new subtype 1m, and QC113 and QC74 into another new subtype 1n. The remaining four (QC60, QC316, QC152 and QC180) did not classify among the established subtypes and corresponded to four new lineages. Subtypes 1j, 1k, 1m, 1n and the unclassified isolate QC60 were identified in Haitian immigrants. In the updated HCV nomenclature of 2005, a total of 12 subtypes of HCV-1 were designated. Including the data from the present study, all but subtype 1f now have their full-length genomes defined. Further analysis of partial NS5B sequences available in GenBank denoted a total of 21 unclassified lineages, indicating the taxonomic complexity of HCV-1. Among them, six have had their full-length genomes characterized. Based on the available full-length genome sequences, a timescale phylogenetic tree was reconstructed which estimated important time points in the evolution of HCV-1. It revealed that subtype 1a diverged from its nearest relatives 135 years ago and subtype 1b diverged from its nearest relatives 112 years ago. When subtypes 1a, 1j, 1k, 1m, 1n and six close relatives (all but one from Haitian immigrants) were considered as a whole, the divergence time was 176 years ago. This diversification was concurrent with the time period when the transatlantic slave trade was active. When taking all the HCV-1 isolates as a single lineage, the divergence time was 326 years ago. This analysis suggested the existence of a recent common ancestor for subtype 1a and the Haitian variants; a co-origin for subtypes 1b, 1i and 1d was also implied. PMID:24718832
Lu, Ling; Li, Chunhua; Xu, Yan; Murphy, Donald G
Seventeen isolates of Bartonella henselae from the region of Freiburg, Germany, obtained from blood cultures of domestic cats, were examined for their genetic heterogeneity. On the basis of different DNA fingerprinting methods, including pulsed-field gel electrophoresis (PFGE), enterobacterial repetitive intergenic consensus (ERIC)-PCR, repetitive extragenic palindromic (REP) PCR, and arbitrarily primed (AP)-PCR, three different variants were identified among the isolates (variants I to III). Variant I included 6 strains, variant II included 10 strains, and variant III included only one strain. By all methods used, the isolates could be clearly distinguished from the type strain, Houston-1, which was designated variant IV. A previously published type-specific amplification of 16S rDNA differentiated two types of the B. henselae isolates (16S rRNA types 1 and 2). The majority of the isolates (16 of 17), including all variants I and II, were 16S rRNA type 2. Only one isolate (variant III) and the Houston-1 strain (variant IV) comprised the 16S rRNA type 1. Comparison of the 16S rDNA sequences from one representative strain from each of the three variants (I to III) confirmed the results obtained by 16S rRNA type-specific PCR. The sequences from variant I and variant II were identical, whereas the sequence of variant III differed in three positions. All methods applied in this study allowed subtyping of the isolates. PFGE and ERIC-PCR provided the highest discriminatory potential for subtyping B. henselae strains, whereas AP-PCR with the M13 primer showed a very clear differentiation between the four variants. Our results suggest that the genetic heterogeneity of B. henselae strains is high. The methods applied were found useful for typing B. henselae isolates, providing tools for epidemiological and clinical follow-up studies.
Sander, Anna; Ruess, Michael; Bereswill, Stefan; Schuppler, Markus; Steinbrueckner, Bernhard
Guillain-Barré syndrome (GBS) is a well-described condition involving the peripheral nervous system. The most well-known form of this disease is acute inflammatory demyelinating polyradiculoneuropathy. Among the different variants of GBS described in the literature, the sensory variant is scantily recognized. There has been a recent attempt to classify the sensory variants of the GBS and bring more objectivity to this diagnostic paradigm. We report a rare sensory variant of GBS presenting with isolated small nerve fiber involvement peripherally in the limbs and associated facial nerve palsy in a patient who had clinical and serological evidence of a preceding Mycoplasma pneumoniae infection. The symptoms resolved gradually with intravenous immunoglobulin therapy. This case adds to the growing literature of the rare form of acute small fiber neuropathy and GBS variants. PMID:24872212
Makonahalli, Rohitha; Seneviratne, Janaka; Seneviratne, Udaya
Embryonic development is regulated by both genetic and epigenetic mechanisms, with nearly all DNA-templated processes influenced by chromatin architecture. Sequence variations in histone proteins, core components of chromatin, provide a means to generate diversity in the chromatin structure, resulting in distinct and profound biological outcomes in the developing embryo. Emerging literature suggests that epigenetic contributions from histone variants play key roles in a number of developmental processes such as the initiation and maintenance of pericentric heterochromatin, X-inactivation, and germ cell differentiation. Here, we review the role of histone variants in the embryo with particular emphasis on early mammalian development.
Banaszynski, Laura A.; Allis, C. David; Lewis, Peter W.
. Reported here is a cluster of infections due to a nitrate-negative variant of Enterobacter sakazakii, which occurred among premature neonates at the Hadassah Hospital, Mount Scopus, Jerusalem, in December 1999–January 2000.\\u000a Pulsed-field gel electrophoresis showed cluster isolates to be identical but unrelated to previous systemic isolates recovered\\u000a in 1993 and 1998. The organism was not isolated from infant formula
C. Block; O. Peleg; N. Minster; B. Bar-Oz; A. Simhon; I. Arad; M. Shapiro
Virulent strains of Mycobacterium tuberculosis isolated from humans are divisible into five variants by using four tests: oxygen requirement (aerobic or microaerophilic), nitrate reductase activity, susceptibility to pyrazinamide (60 micrograms/ml) and susceptibility to thiophene-2-carboxylic acid hydrazide (5 micrograms/ml). The five variants are referred to as Classical human, Asian human, bovine, African I and African II. The relation of these variants to previously described types is discussed. This simple division has been shown to be useful in epidemiological studies.
Collins, C. H.; Yates, M. D.; Grange, J. M.
While federal regulations during the past 10 years have treated isolated wetlands as unconnected to aquatic resources protected by the Clean Water Act, they provide critical ecosystem services to society that extend well beyond their wetland boundaries. The authors offer well-documented examples from the scientific literature on some of the ecosystem services provided by isolated wetlands to society and other ecosystems.
Smith, Loren M.; Euliss, Ned H., Jr.; Haukos, David A.
Purpose: To identify sequence variants of the ataxia telangiectasia mutated (ATM) gene and establish their prevalence rate among American Indian (AI) as compared with non-AI cancer patients. Materials and Methods: DNA was isolated from blood samples collected from 100 AI and 100 non-AI cancer patients undergoing radiation therapy, and a blinded assessment of the ATM sequence was conducted. Quantitative PCR assessment of copy number for each exon was also performed. The main outcome measure was the prevalence of ATM variants in the two patient populations. Results: No statistically significant differences for total prevalence of ATM variants among AI and non-AI patients were found. Of the 25 variants identified, 5 variants had a prevalence of >2%, of which 4 occurred at a rate of >5% in one or both groups. The prevalence of these four variants could meaningfully be compared between the two groups. The only statistically significant difference among the groups was the c.4138C?>?T variant which is predicted not to affect protein function, seen in 8% of AI versus 0% of non-AI patients (P?=?0.007). No exonic copy number changes were found in these patients. Conclusion: This study is the first to determine the prevalence of ATM variants in AIs.
Petereit, Daniel G.; Hahn, L. Jennifer; Kanekar, Shalini; Boylan, Amy; Bentzen, S?ren M.; Ritter, Mark; Moser, Amy R.
The phenomenon of interference was exploited to isolate low-abundance noncytopathic human immunodeficiency virus type 1 (HIV-1) variants from a primary HIV-1 isolate from an asymptomatic HIV-1-seropositive hemophiliac. Successive rounds of virus infection of a cytolysis-susceptible CD4+ cell line and isolation of surviving cells resulted in selective amplification of an HIV-1 variant reduced in the ability to induce cytolysis. The presence of a PvuII polymorphism facilitated subsequent amplification and cloning of cytopathic and noncytopathic HIV-1 variants from the primary isolate. Cloned virus stocks from cytopathic and noncytopathic variants exhibited similar replication kinetics, infectivity, and syncytium induction in susceptible host cells. The noncytopathic HIV-1 variant was unable, however, to induce single-cell killing in susceptible host cells. Construction of viral hybrids in which regions of cytopathic and noncytopathic variants were exchanged indicated that determinants for the noncytopathic phenotype map to the envelope glycoprotein. Sequence analysis of the envelope coding regions indicated the absence of two highly conserved N-linked glycosylation sites in the noncytopathic HIV-1 variant, which accompanied differences in processing of precursor gp160 envelope glycoprotein. These results demonstrate that determinants for syncytium-independent single-cell killing are located within the envelope glycoprotein and suggest that single-cell killing is profoundly influenced by alterations in envelope sequence which affect posttranslational processing of HIV-1 envelope glycoprotein within the infected cell. Images
Stevenson, M; Haggerty, S; Lamonica, C; Mann, A M; Meier, C; Wasiak, A
In aquatic birds, influenza A viruses mainly replicate in the intestinal tract without significantly affecting the health of the host, but in mammals, they replicate in the respiratory tract and often cause disease. Occasionally, influenza viruses have been detected in stool samples of hospitalized patients and in rectal swabs of naturally or experimentally infected mammals. In this study, we compared the biological and molecular differences among four wild-type avian H1N1 influenza viruses and their corresponding fecal and lung isolates in DBA/2J and BALB/cJ mice. All fecal and lung isolates were more pathogenic than the original wild-type viruses, when inoculated into mice of both strains. The increased virulence was associated with the acquisition of genetic mutations. Most of the novel genotypes emerged as PB2E627K, HAF128V, HAF454L, or HAH300P variations, and double mutations frequently occurred in the same isolate. However, influenza virus strain- and host-specific differences were also observed in terms of selected variants. The avian H1N1 virus of shorebird origin appeared to be unique in its ability to rapidly adapt to BALB/cJ mice via the fecal route, compared to the adaptability of the H1N1 virus of mallard origin. Furthermore, a bimodal distribution in fecal shedding was observed in mice infected with the fecal isolates, while a normal distribution was observed after infection with the lung isolates or wild-type virus. Fecal isolates contained HA mutations that increased the activation pH of the HA protein. We conclude that influenza virus variants that emerge in fecal isolates in mammals might influence viral transmission, adaptation to mammals, and viral ecology or evolution.
Kocer, Zeynep A.; Obenauer, John; Zaraket, Hassan; Zhang, Jinghui; Rehg, Jerold E.; Russell, Charles J.
Disclosed are variants of Humicola grisea Cel7A (CBH1.1), H. jecorina CBH1 variant or S. thermophilium CBH1, nucleic acids encoding the same and methods for producing the same. The variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted.
Goedegebuur, Frits (Vlaardingen, NL); Gualfetti, Peter (San Francisco, CA); Mitchinson, Colin (Half Moon Bay, CA); Larenas, Edmund (Moss Beach, CA)
Disclosed are variants of Humicola grisea Cel7A (CBH1.1), H. jecorina CBH1 variant or S. thermophilium CBH1, nucleic acids encoding the same and methods for producing the same. The variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted.
Goedegebuur, Frits (Vlaardingen, NL); Gualfetti, Peter (San Francisco, CA); Mitchinson, Colin (Half Moon Bay, CA); Larenas, Edmund (Moss Beach, CA)
Disclosed are variants of Humicola grisea Cel7A (CBH1.1), H. jecorina CBH1 variant or S. thermophilium CBH1, nucleic acids encoding the same and methods for producing the same. The variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted.
Goedegeburr, Frits; Gualfetti, Peter; Mitchinson, Colin; Larenas, Edmund
Goedegebuur, Frits (Vlaardingen, NL); Gualfetti, Peter (San Francisco, CA); Mitchinson, Colin (Half Moon Bay, CA); Larenas, Edmund (Moss Beach, CA)
In this paper we deflne small scale variants of the AES. These variants inherit the design features of the AES and provide a suitable framework for comparing difierent cryptanalytic methods. In particular, we provide some preliminary results and insights when using ofi-the- shelf computational algebra techniques to solve the systems of equations arising from these small scale variants.
Carlos Cid; Sean Murphy; Matthew J. B. Robshaw
\\u000a Different types of genetic variants are present in the genome. Single nucleotide polymorphisms, or SNPs, are minor variations\\u000a in the genetic sequence that differ between members of a species or even between paired chromosomes in an individual. There\\u000a are common SNPs that occur in at least 1% of a population. These SNPs may be specific to an ethnic group or
Mounia Tannour-Louet; Dolores J. Lamb
The distribution of C3 variants in dermatitis herpetiformis, thyroid cancer, spinal muscular atrophy, multiple sclerosis and psoriasis was studied, and also the Bf phenotype distribution in thyroid cancer. In thyroid cancer there was a significant deficit of heterozygotes for the C3 locus, a possible decrease in the C3F allele frequency and a significant elevation of frequency of the BfF allele.
J. E. Bernal; S. S. Papiha; D. F. Roberts
Genome-wide association studies (GWAS) have identified >500 common variants associated with quantitative metabolic traits, but in aggregate such variants explain at most 20-30% of the heritable component of population variation in these traits. To further investigate the impact of genotypic variation on metabolic traits, we conducted re-sequencing studies in >6,000 members of a Finnish population cohort (The Northern Finland Birth Cohort of 1966 [NFBC]) and a type 2 diabetes case-control sample (The Finland-United States Investigation of NIDDM Genetics [FUSION] study). By sequencing the coding sequence and 5' and 3' untranslated regions of 78 genes at 17 GWAS loci associated with one or more of six metabolic traits (serum levels of fasting HDL-C, LDL-C, total cholesterol, triglycerides, plasma glucose, and insulin), and conducting both single-variant and gene-level association tests, we obtained a more complete understanding of phenotype-genotype associations at eight of these loci. At all eight of these loci, the identification of new associations provides significant evidence for multiple genetic signals to one or more phenotypes, and at two loci, in the genes ABCA1 and CETP, we found significant gene-level evidence of association to non-synonymous variants with MAF<1%. Additionally, two potentially deleterious variants that demonstrated significant associations (rs138726309, a missense variant in G6PC2, and rs28933094, a missense variant in LIPC) were considerably more common in these Finnish samples than in European reference populations, supporting our prior hypothesis that deleterious variants could attain high frequencies in this isolated population, likely due to the effects of population bottlenecks. Our results highlight the value of large, well-phenotyped samples for rare-variant association analysis, and the challenge of evaluating the phenotypic impact of such variants. PMID:24497850
Service, Susan K; Teslovich, Tanya M; Fuchsberger, Christian; Ramensky, Vasily; Yajnik, Pranav; Koboldt, Daniel C; Larson, David E; Zhang, Qunyuan; Lin, Ling; Welch, Ryan; Ding, Li; McLellan, Michael D; O'Laughlin, Michele; Fronick, Catrina; Fulton, Lucinda L; Magrini, Vincent; Swift, Amy; Elliott, Paul; Jarvelin, Marjo-Riitta; Kaakinen, Marika; McCarthy, Mark I; Peltonen, Leena; Pouta, Anneli; Bonnycastle, Lori L; Collins, Francis S; Narisu, Narisu; Stringham, Heather M; Tuomilehto, Jaakko; Ripatti, Samuli; Fulton, Robert S; Sabatti, Chiara; Wilson, Richard K; Boehnke, Michael; Freimer, Nelson B
Starting with an rII diploid isolate of T4 a procedure is reported for isolating diploid variants which generate segregants at a reduced frequency. The properties of one such variant, which exhibits a four-fold reduction in segregation frequency, is discussed in detail. It is shown: 1) this variant has acquired at least two extra mutations outside the rII region 2) these
P. van den Ende; N. Symonds
We examined the population structure and genetic variation of four genomic regions within and between 30 Citrus tristeza virus (CTV) isolates from Spain and California. Our analyses showed that most isolates contained a population of sequence variants, with one being predominant. Four isolates showed two major sequence variants in some genomic regions. The two major variants of three of these isolates showed very low nucleotide identity to each other but were very similar to those of other isolates, suggesting the possibility of mixed infections with two divergent isolates. Incongruencies of phylogenetic relationships in the different genomic regions and statistical analyses suggested that the genomes of some CTV sequence variants originated by recombination events between diverged sequence variants. No correlation was observed between geographic origin and nucleotide distance, and thus from a genetic view, the Spanish and Californian isolates analyzed here could be considered members of the same population.
Rubio, Luis; Ayllon, Maria Angeles; Kong, Ping; Fernandez, Andres; Polek, MaryLou; Guerri, Jose; Moreno, Pedro; Falk, Bryce W.
V-073, a small-molecule capsid inhibitor originally developed for nonpolio enterovirus indications is considerably more potent against polioviruses. All poliovirus isolates tested to date (n = 45), including wild, vaccine, vaccine-derived, and laboratory strains, are susceptible to the antiviral capsid inhibitor V-073. We grew poliovirus in the presence of V-073 to allow for the identification of variants with reduced susceptibility to the drug. Sequence analysis of 160 independent resistant variants (80 isolates of poliovirus type 1, 40 isolates each of types 2 and 3) established that V-073 resistance involved a single amino acid change in either of two virus capsid proteins, VP1 (67 of 160 [42%]) or VP3 (93 of 160 [58%]). In resistant variants with a VP1 change, the majority (53 of 67 [79%]) exhibited a substitution of isoleucine at position 194 (equivalent position 192 in type 3) with either methionine or phenylalanine. Of those with a VP3 change, alanine at position 24 was replaced with valine in all variants (n = 93). The resistance phenotype was relatively stable upon passage of viruses in cell culture in the absence of drug. Single-step growth studies showed no substantial differences between drug-resistant variants and the virus stocks from which they were derived, while the resistant viruses were generally more thermally labile than the corresponding drug-susceptible parental viruses. These studies provide a foundation from which to build a greater understanding of resistance to antiviral compound V-073.
Liu, Hong-Mei; Roberts, Jason A.; Moore, Deborah; Anderson, Barbara; Pallansch, Mark A.; Pevear, Daniel C.; Collett, Marc S.
Background As resequencing projects become more prevalent across a larger number of species, accurate variant identification will further elucidate the nature of genetic diversity and become increasingly relevant in genomic studies. However, the identification of larger genomic variants via DNA sequencing is limited by both the incomplete information provided by sequencing reads and the nature of the genome itself. Long-read sequencing technologies provide high-resolution access to structural variants often inaccessible to shorter reads. Results We present PBHoney, software that considers both intra-read discordance and soft-clipped tails of long reads (>10,000 bp) to identify structural variants. As a proof of concept, we identify four structural variants and two genomic features in a strain of Escherichia coli with PBHoney and validate them via de novo assembly. PBHoney is available for download at http://sourceforge.net/projects/pb-jelly/. Conclusions Implementing two variant-identification approaches that exploit the high mappability of long reads, PBHoney is demonstrated as being effective at detecting larger structural variants using whole-genome Pacific Biosciences RS II Continuous Long Reads. Furthermore, PBHoney is able to discover two genomic features: the existence of Rac-Phage in isolate; evidence of E. coli’s circular genome.
The poxviruses Warsaw Agricultural University 86 (WAU86) and 88-1 (WAU88-1) were isolated in 1986 to 1988 from separate outbreaks in laboratory mice in Poland and described as ectromelia virus isolates. The genome sequences of these poxviruses reveal that they are almost identical and represent a novel variant of the vaccinia virus Lister strain.
Mavian, Carla; Lopez-Bueno, Alberto
Yersinia pestis, the causative agent of plague, has been responsible for at least three pandemics. During the last pandemic, which started in Hong Kong in 1894, the microorganism colonized new, previously unscathed geographical areas where it has become well established. The aim of this longitudinal study was to investigate the genetic stability of Y. pestis strains introduced into a new environment just under a century ago and to follow the epidemiology of any new genetic variant detected. In the present study, 187 strains of Y. pestis isolated between 1939 and 1996 from different regions of Madagascar and responsible mainly for human cases of bubonic and pneumonic plague were studied. Our principal genotyping method was rRNA gene profiling (ribotyping), which has previously been shown to be an effective scheme for typing Y. pestis strains of different geographical origins. We report that all studied Y. pestis strains isolated in Madagascar before 1982 were of classical ribotype B, the ribotype attributed to the Y. pestis clone that spread around the world during the third pandemic. In 1982, 1983, and 1994, strains with new ribotypes, designated R, Q, and T, respectively, were isolated on the high-plateau region of the island. Analysis of other genotypic traits such as the NotI genomic restriction profiles and the EcoRV plasmid restriction profiles revealed that the new variants could also be distinguished by specific genomic and/or plasmid profiles. A follow-up of these new variants indicated that strains of ribotypes Q and R have become well established in their ecosystem and have a tendency to spread to new geographical areas and supplant the original classical strain.
Guiyoule, A; Rasoamanana, B; Buchrieser, C; Michel, P; Chanteau, S; Carniel, E
Carriage and acquisition rates of Clostridium difficile in hospitalized horses, including molecular characterization, multilocus sequence typing and antimicrobial susceptibility of bacterial isolates.
Clostridium difficile has been identified as a significant agent of diarrhoea and enterocolitis in both foals and adult horses. Hospitalization, antibiotic therapy or changes in diet may contribute to the development of C. difficile infection. Horses admitted to a care unit are therefore at greater risk of being colonized. The aim of this study was to investigate the carriage of C. difficile in hospitalized horses and the possible influence of some risk factors in colonization. During a seven-month period, faecal samples and data relating the clinical history of horses admitted to a veterinary teaching hospital were collected. C. difficile isolates were characterized through toxin profiles, cytotoxicity activity, PCR-ribotyping, antimicrobial resistance and multilocus sequence typing (MLST). Ten isolates were obtained with a total of seven different PCR-ribotypes, including PCR-ribotype 014. Five of them were identified as toxinogenic. A high resistance to gentamicin, clindamycin and ceftiofur was found. MLST revealed four different sequencing types (ST), which included ST11, ST26, ST2 and ST15, and phylogenetic analysis showed that most of the isolates clustered in the same lineage. Clinical history suggests that horses frequently harbour toxigenic and non-toxigenic C. difficile and that in most cases they are colonized regardless of the reason for hospitalization; the development of diarrhoea is more unusual. PMID:24894133
Rodriguez, C; Taminiau, B; Brévers, B; Avesani, V; Van Broeck, J; Leroux, A A; Amory, H; Delmée, M; Daube, G
Histone variants are key players in shaping chromatin structure, and, thus, in regulating fundamental cellular processes such as chromosome segregation and gene expression. Emerging evidence points towards a role for histone variants in contributing to tumor progression, and, recently, the first cancer-associated mutation in a histone variant-encoding gene was reported. In addition, genetic alterations of the histone chaperones that specifically regulate chromatin incorporation of histone variants are rapidly being uncovered in numerous cancers. Collectively, these findings implicate histone variants as potential drivers of cancer initiation and/or progression, and, therefore, targeting histone deposition or the chromatin remodeling machinery may be of therapeutic value. Here, we review the mammalian histone variants of the H2A and H3 families in their respective cellular functions, and their involvement in tumor biology.
Vardabasso, Chiara; Hasson, Dan; Ratnakumar, Kajan; Chung, Chi-Yeh; Duarte, Luis F.
Whereas phagocytic cells from normal individuals have the capacity to kill ingested bacteria and parasites, those from patients with several uncommon genetic deficiency diseases are known to be defective in bactericidal activity. Studies on neutrophils of these patients have revealed fundamental defects in their ability to reduce molecular oxygen and metabolize it to superoxide anion, hydrogen peroxide, and oxygen radicals. In the present experiments, we describe a clone of a continuous murine macrophage-like cell line, J774.16, that, upon appropriate stimulation, activates the hexose monophosphate shunt, and produces superoxide anion and hydrogen peroxide. With nitroblue tetrazolium to select against cells capable of being stimulated by phorbol myristate acetate to reduce the dye to polymer--formazan--which is toxic fot cells, we have selected for variants that are defective in oxygen metabolism. Four of these subclones have been characterized and found to be lacking in the ability (a) to generate superoxide anion, as measured by cytochrome c reduction; (b) to produce hydrogen peroxide, as measured by the ability to form complex I with cytochrome c peroxidase; and (c) to be stimulated to oxidize glucose via the hexose monophosphate shunt. These variants appear to represent a useful model for studying the molecular basis for macrophage cytocidal activity.
The established animal model for Lassa fever is based on the new world arenavirus Pichinde (PIC). Natural isolates of PIC virus are attenuated in guinea pigs, but serial guinea pig passage renders them extremely virulent in that host. We have compared the nucleotide sequences of the small RNA segments of two attenuated, low- passage variants of the PIC virus Munchique
LIHONG ZHANG; KATHLEEN MARRIOTT; JUDITH F. ARONSON
Helcococcus kunzii was isolated from a brain abscess in a diabetic patient with cholesteatoma and demonstrated satellitism around Staphylococcus aureus in culture. This is the first reported case of severe central nervous system infection due to H. kunzii and the first description of a satelliting phenotypic variant of this organism. PMID:24172152
Sridhar, Siddharth; Chan, Jasper F W; Yuen, Kwok-Yung
Helcococcus kunzii was isolated from a brain abscess in a diabetic patient with cholesteatoma and demonstrated satellitism around Staphylococcus aureus in culture. This is the first reported case of severe central nervous system infection due to H. kunzii and the first description of a satelliting phenotypic variant of this organism.
Sridhar, Siddharth; Chan, Jasper F. W.
In order to determine geographically related intratypic variation in human papillomavirus (HPV) type 16 and 18 isolates that could be associated with lesion development, data were analysed from an ongoing cohort study of the natural course of infection of HPVs and cervical neoplasia. Testing for HPVs was carried out by PCR and molecular variants of these HPVs were characterized by
Luisa L. Villa; Laura Sichero; Paula Rahal; Otavia Caballero; Alex Ferenczy; Tom Rohan; Eduardo L. Franco
Objective To explore the association of a functional germline variant in the 3?-UTR of KRAS with endometrial cancer risk, as well as the association of microRNA (miRNA) signatures and the KRAS-variant with clinical characteristics and survival outcomes in two prospective RTOG endometrial cancer trials. Methods/Materials The association of the KRAS-variant with endometrial cancer risk was evaluated by case-control analysis of 467 women with type 1 or 2 endometrial cancer and 582 age-matched controls. miRNA and DNA were isolated for expression profiling and genotyping from tumor specimens of 46 women with type 1 endometrial cancer enrolled in RTOG trials 9708 and 9905. miRNA expression levels and KRAS-variant genotype were correlated with patient and tumor characteristics, and survival outcomes were evaluated by variant allele type. Results The KRAS-variant was not significantly associated with overall endometrial cancer risk (14% controls and 17% type 1 cancers), although was enriched in type 2 endometrial cancers (24%, p?=?0.2). In the combined analysis of RTOG 9708/9905, miRNA expression differed by age, presence of lymphovascular invasion and KRAS-variant status. Overall survival rates at 3 years for patients with the variant and wild-type alleles were 100% and 77% (HR 0.3, p?=?0.24), respectively, favoring the variant. Conclusions The KRAS-variant may be a genetic marker of risk for type 2 endometrial cancers. In addition, tumor miRNA expression appears to be associated with patient age, lymphovascular invasion and the KRAS-variant, supporting the hypothesis that altered tumor biology can be measured by miRNA expression, and that the KRAS-variant likely impacts endometrial tumor biology.
Uduman, Mohamed; Winter, Kathryn; Boeke, Marta; Greven, Kathryn M.; King, Stephanie; Burke, Thomas W.; Underhill, Kelly; Kim, Harold; Boulware, Raleigh J.; Yu, Herbert; Parkash, Vinita; Lu, Lingeng; Gaffney, David; Dicker, Adam P.; Weidhaas, Joanne
Exome sequencing studies in complex diseases are challenged by the allelic heterogeneity, large number and modest effect sizes of associated variants on disease risk and the presence of large numbers of neutral variants, even in phenotypically relevant genes. Isolated populations with recent bottlenecks offer advantages for studying rare variants in complex diseases as they have deleterious variants that are present at higher frequencies as well as a substantial reduction in rare neutral variation. To explore the potential of the Finnish founder population for studying low-frequency (0.5-5%) variants in complex diseases, we compared exome sequence data on 3,000 Finns to the same number of non-Finnish Europeans and discovered that, despite having fewer variable sites overall, the average Finn has more low-frequency loss-of-function variants and complete gene knockouts. We then used several well-characterized Finnish population cohorts to study the phenotypic effects of 83 enriched loss-of-function variants across 60 phenotypes in 36,262 Finns. Using a deep set of quantitative traits collected on these cohorts, we show 5 associations (p<5×10-8) including splice variants in LPA that lowered plasma lipoprotein(a) levels (P?=?1.5×10-117). Through accessing the national medical records of these participants, we evaluate the LPA finding via Mendelian randomization and confirm that these splice variants confer protection from cardiovascular disease (OR?=?0.84, P?=?3×10-4), demonstrating for the first time the correlation between very low levels of LPA in humans with potential therapeutic implications for cardiovascular diseases. More generally, this study articulates substantial advantages for studying the role of rare variation in complex phenotypes in founder populations like the Finns and by combining a unique population genetic history with data from large population cohorts and centralized research access to National Health Registers. PMID:25078778
Lim, Elaine T; Würtz, Peter; Havulinna, Aki S; Palta, Priit; Tukiainen, Taru; Rehnström, Karola; Esko, Tõnu; Mägi, Reedik; Inouye, Michael; Lappalainen, Tuuli; Chan, Yingleong; Salem, Rany M; Lek, Monkol; Flannick, Jason; Sim, Xueling; Manning, Alisa; Ladenvall, Claes; Bumpstead, Suzannah; Hämäläinen, Eija; Aalto, Kristiina; Maksimow, Mikael; Salmi, Marko; Blankenberg, Stefan; Ardissino, Diego; Shah, Svati; Horne, Benjamin; McPherson, Ruth; Hovingh, Gerald K; Reilly, Muredach P; Watkins, Hugh; Goel, Anuj; Farrall, Martin; Girelli, Domenico; Reiner, Alex P; Stitziel, Nathan O; Kathiresan, Sekar; Gabriel, Stacey; Barrett, Jeffrey C; Lehtimäki, Terho; Laakso, Markku; Groop, Leif; Kaprio, Jaakko; Perola, Markus; McCarthy, Mark I; Boehnke, Michael; Altshuler, David M; Lindgren, Cecilia M; Hirschhorn, Joel N; Metspalu, Andres; Freimer, Nelson B; Zeller, Tanja; Jalkanen, Sirpa; Koskinen, Seppo; Raitakari, Olli; Durbin, Richard; MacArthur, Daniel G; Salomaa, Veikko; Ripatti, Samuli; Daly, Mark J; Palotie, Aarno
Summary Differences in the clinical pathology of mammalian prion diseases reflect distinct heritable conformations of aggregated PrP proteins, called prion strains. Here, using the yeast [PSI+] prion, we examine the de novo establishment of prion strains (called variants in yeast). The [PSI+] prion protein, Sup35, is efficiently induced to take on numerous prion variant conformations following transient overexpression of Sup35 in the presence of another prion, e.g. [PIN+]. One hypothesis is that the first [PSI+] prion seed to arise in a cell causes propagation of only that seed’s variant, but that different variants could be initiated in different cells. However, we now show that even within a single cell, Sup35 retains the potential to fold into more than one variant type. When individual cells segregating different [PSI+] variants were followed in pedigrees, establishment of a single variant phenotype generally occurred in daughters, granddaughters or great granddaughters—but in 5% of the pedigrees cells continued to segregate multiple variants indefinitely. The data is consistent with the idea that many newly formed prions go through a maturation phase before they reach a single specific variant conformation. These findings may be relevant to mammalian PrP prion strain establishment and adaptation.
Sharma, Jaya; Liebman, Susan W.
Increasing evidence suggests that rare and generally deleterious genetic variants might have strong impact on disease risks of not only Mendelian disease, but also many common diseases. However, identifying such rare variants remains to be challenging, and novel statistical methods and bioinformatic software must be developed. Hence, we have to extensively evaluate various methods under reasonable genetic models. While there are abundant genomic data, they are not most helpful for the evaluation of the methods because the disease mechanism is unknown. Thus, it is imperative that we simulate genomic data that mimic the real data containing rare variants and that enable us to impose a known disease penetrance model. Although resampling simulation methods have shown their advantages in computational efficiency and in preserving important properties such as linkage disequilibrium (LD) and allele frequency, they still have limitations as we demonstrated. We propose an algorithm that combines a regression-based imputation with resampling to simulate genetic data with both rare and common variants. Logistic regression model was employed to fit the relationship between a rare variant and its nearby common variants in the 1000 Genomes Project data and then applied to the real data to fill in one rare variant at a time using the fitted logistic model based on common variants. Individuals then were simulated using the real data with imputed rare variants. We compared our method with existing simulators and demonstrated that our method performed well in retaining the real sample properties, such as LD and minor allele frequency, qualitatively.
Xu, Yaji; Wu, Yinghua; Song, Chi; Zhang, Heping
An outbreak of cholera struck Bihar, an Indian state, in August 2008 following a massive flood. Here we report the phenotypic and genotypic characteristics of Vibrio cholerae strains isolated from patients with diarrhea. Rectal swabs were obtained from patients with diarrhea who were admitted to medical camps or the hospital, and the strains were biochemically and serologically characterized. V. cholerae was isolated from 21 (65.6%) of 32 rectal swabs. Serological studies revealed that all the 21 isolates belonged to V. cholerae O1 Ogawa. Mismatch amplification mutation assay (MAMA)-PCR showed that the isolates belonged to El Tor variant group, and pulsed-field gel electrophoresis (PFGE) proved that these isolates were of a different lineage than the conventional El Tor variant strains. These isolates were resistant to several drugs, including ampicillin, streptomycin, tetracycline, nalidixic acid, and furazolidone. The uniqueness of the current report arises from the fact that records of cholera in Bihar are availiable for the early 1960s but not for the next 4 decades. Moreover, the present study is the first to report a cholera outbreak in Bihar that was caused by an El Tor variant strain. PMID:24858614
Koley, Hemanta; Ray, Nivedita; Chowdhury, Goutam; Barman, Soumik; Mitra, Soma; Ramamurthy, T; Mukhopadhyay, Asish K; Sarkar, B L; Katyal, Rakesh; Das, Pradeep; Panda, Samiran; Ghosh, Subrata
Size exclusion chromatography (SEC) is the most commonly used method to separate and quantify monoclonal antibody (mAb) size variants. MAb-A is an IgG1 subtype humanized monoclonal antibody recombinantly produced in Chinese hamster ovary (CHO) cells. SEC analysis of MAb-A resolved a peak, named Peak 1, which elutes between monomer and dimer peaks. MAb-A lots produced from different clones and production scales all have 0.2–0.3% of SEC Peak 1. Electron spray ionization—time of flight mass spectrometry (ESI-TOF MS), microfluidics capillary electrophoresis and sodium dodecyl sulfate-PAGE (SDS PAGE) results demonstrated that SEC Peak 1 contains two structural variants: MAb-A with one extra light chain (2H3L) and MAb-A with two extra light chains (2H4L). The C-terminal Cys of the extra light chain in Peak 1 variants is either a free thiol, capped by glutathione, cysteine, or another light chain. Both electrophoresis and LC/MS analyses of non-reduced and reduced samples suggested that the extra light chains are linked to the MAb-A light chain through disulfide bonds. Isolated SEC Peak 1 fraction had a potency of 50% relative to MAb-A reference material. The 50% potency loss may result from the reduced accessibility to the antigen-binding site caused by the extra light chain(s)’ steric hindrance.
Lu, Connie; Liu, Dandan; Liu, Hongbin; Motchnik, Paul
Heritability estimates for body mass index (BMI) variation are high. For mothers and their offspring higher BMI correlations have been described than for fathers. Variation(s) in the exclusively maternally inherited mitochondrial DNA (mtDNA) might contribute to this parental effect. Thirty-two to 40 mtDNA single nucleotide polymorphisms (SNPs) were available from genome-wide association study SNP arrays (Affymetrix 6.0). For discovery, we analyzed association in a case-control (CC) sample of 1,158 extremely obese children and adolescents and 435 lean adult controls. For independent confirmation, 7,014 population-based adults were analyzed as CC sample of n = 1,697 obese cases (BMI ? 30 kg/m2) and n = 2,373 normal weight and lean controls (BMI<25 kg/m2). SNPs were analyzed as single SNPs and haplogroups determined by HaploGrep. Fisher's two-sided exact test was used for association testing. Moreover, the D-loop was re-sequenced (Sanger) in 192 extremely obese children and adolescents and 192 lean adult controls. Association testing of detected variants was performed using Fisher's two-sided exact test. For discovery, nominal association with obesity was found for the frequent allele G of m.8994G/A (rs28358887, p = 0.002) located in ATP6. Haplogroup W was nominally overrepresented in the controls (p = 0.039). These findings could not be confirmed independently. For two of the 252 identified D-loop variants nominal association was detected (m.16292C/T, p = 0.007, m.16189T/C, p = 0.048). Only eight controls carried the m.16292T allele, five of whom belonged to haplogroup W that was initially enriched among these controls. m.16189T/C might create an uninterrupted poly-C tract located near a regulatory element involved in replication of mtDNA. Though follow-up of some D-loop variants still is conceivable, our hypothesis of a contribution of variation in the exclusively maternally inherited mtDNA to the observed larger correlations for BMI between mothers and their offspring could not be substantiated by the findings of the present study. PMID:24788344
Knoll, Nadja; Jarick, Ivonne; Volckmar, Anna-Lena; Klingenspor, Martin; Illig, Thomas; Grallert, Harald; Gieger, Christian; Wichmann, Heinz-Erich; Peters, Annette; Wiegand, Susanna; Biebermann, Heike; Fischer-Posovszky, Pamela; Wabitsch, Martin; Völzke, Henry; Nauck, Matthias; Teumer, Alexander; Rosskopf, Dieter; Rimmbach, Christian; Schreiber, Stefan; Jacobs, Gunnar; Lieb, Wolfgang; Franke, Andre; Hebebrand, Johannes; Hinney, Anke
Heritability estimates for body mass index (BMI) variation are high. For mothers and their offspring higher BMI correlations have been described than for fathers. Variation(s) in the exclusively maternally inherited mitochondrial DNA (mtDNA) might contribute to this parental effect. Thirty-two to 40 mtDNA single nucleotide polymorphisms (SNPs) were available from genome-wide association study SNP arrays (Affymetrix 6.0). For discovery, we analyzed association in a case-control (CC) sample of 1,158 extremely obese children and adolescents and 435 lean adult controls. For independent confirmation, 7,014 population-based adults were analyzed as CC sample of n?=?1,697 obese cases (BMI?30 kg/m2) and n?=?2,373 normal weight and lean controls (BMI<25 kg/m2). SNPs were analyzed as single SNPs and haplogroups determined by HaploGrep. Fisher's two-sided exact test was used for association testing. Moreover, the D-loop was re-sequenced (Sanger) in 192 extremely obese children and adolescents and 192 lean adult controls. Association testing of detected variants was performed using Fisher's two-sided exact test. For discovery, nominal association with obesity was found for the frequent allele G of m.8994G/A (rs28358887, p?=?0.002) located in ATP6. Haplogroup W was nominally overrepresented in the controls (p?=?0.039). These findings could not be confirmed independently. For two of the 252 identified D-loop variants nominal association was detected (m.16292C/T, p?=?0.007, m.16189T/C, p?=?0.048). Only eight controls carried the m.16292T allele, five of whom belonged to haplogroup W that was initially enriched among these controls. m.16189T/C might create an uninterrupted poly-C tract located near a regulatory element involved in replication of mtDNA. Though follow-up of some D-loop variants still is conceivable, our hypothesis of a contribution of variation in the exclusively maternally inherited mtDNA to the observed larger correlations for BMI between mothers and their offspring could not be substantiated by the findings of the present study.
Knoll, Nadja; Jarick, Ivonne; Volckmar, Anna-Lena; Klingenspor, Martin; Illig, Thomas; Grallert, Harald; Gieger, Christian; Wichmann, Heinz-Erich; Peters, Annette; Wiegand, Susanna; Biebermann, Heike; Fischer-Posovszky, Pamela; Wabitsch, Martin; Volzke, Henry; Nauck, Matthias; Teumer, Alexander; Rosskopf, Dieter; Rimmbach, Christian; Schreiber, Stefan; Jacobs, Gunnar; Lieb, Wolfgang; Franke, Andre; Hebebrand, Johannes; Hinney, Anke
Next-generation sequencing technology will soon allow sequencing the whole genome of large groups of individuals, and thus will make directly testing rare variants possible. Currently, most of existing methods for rare variant association studies are essentially testing the effect of a weighted combination of variants with different weighting schemes. Performance of these methods depends on the weights being used and no optimal weights are available. By putting large weights on rare variants and small weights on common variants, these methods target at rare variants only, although increasing evidence shows that complex diseases are caused by both common and rare variants. In this paper, we analytically derive optimal weights under a certain criterion. Based on the optimal weights, we propose a Variable Weight Test for testing the effect of an Optimally Weighted combination of variants (VW-TOW). VW-TOW aims to test the effects of both rare and common variants. VW-TOW is applicable to both quantitative and qualitative traits, allows covariates, can control for population stratification, and is robust to directions of effects of causal variants. Extensive simulation studies and application to the Genetic Analysis Workshop 17 (GAW17) data show that VW-TOW is more powerful than existing ones either for testing effects of both rare and common variants or for testing effects of rare variants only. PMID:22714994
Sha, Qiuying; Wang, Xuexia; Wang, Xinli; Zhang, Shuanglin
A novel variant strain of porcine epidemic diarrhea virus (PEDV) emerged on pig farms in South Korea during late 2013. Genomic DNA isolated from a K14JB01 strain identified in a diarrheal pig showed high sequence similarity to PEDV strains prevailing in the United States in 2013. This is the first study to identify the complete genome sequence of a novel variant PEDV in South Korea.
Cho, Yoon-Young; Lim, Seong-In; Kim, Yong Kwan; Song, Jae-Young; Lee, Joong-Bok
It is widely believed that both common and rare variants contribute to the risks of common diseases or complex traits and the cumulative effects of multiple rare variants can explain a significant proportion of trait variances. Advances in high-throughput DNA sequencing technologies allow us to genotype rare causal variants and investigate the effects of such rare variants on complex traits. We developed an adaptive ridge regression method to analyze the collective effects of multiple variants in the same gene or the same functional unit. Our model focuses on continuous trait and incorporates covariate factors to remove potential confounding effects. The proposed method estimates and tests multiple rare variants collectively but does not depend on the assumption of same direction of each rare variant effect. Compared with the Bayesian hierarchical generalized linear model approach, the state-of-the-art method of rare variant detection, the proposed new method is easy to implement, yet it has higher statistical power. Application of the new method is demonstrated using the well-known data from the Dallas Heart Study.
Zhan, Haimao; Xu, Shizhong
It is widely believed that both common and rare variants contribute to the risks of common diseases or complex traits and the cumulative effects of multiple rare variants can explain a significant proportion of trait variances. Advances in high-throughput DNA sequencing technologies allow us to genotype rare causal variants and investigate the effects of such rare variants on complex traits. We developed an adaptive ridge regression method to analyze the collective effects of multiple variants in the same gene or the same functional unit. Our model focuses on continuous trait and incorporates covariate factors to remove potential confounding effects. The proposed method estimates and tests multiple rare variants collectively but does not depend on the assumption of same direction of each rare variant effect. Compared with the Bayesian hierarchical generalized linear model approach, the state-of-the-art method of rare variant detection, the proposed new method is easy to implement, yet it has higher statistical power. Application of the new method is demonstrated using the well-known data from the Dallas Heart Study. PMID:22952918
Zhan, Haimao; Xu, Shizhong
This note discusses two integral variants of the input-to-state stability (ISS) property, which represent nonlinear generalizations of L, stability, in much the same way that ISS generalizes L, stability. Both variants are equivalent to ISS for linear systems. For general nonlinear systems, it is shown that one of the new properties is strictly weaker than ISS, while the other one
Eduardo D. Sontag
A recent result shows that a variant of communicating distri buted H system with two components can generate recursive enumerable languages. In this variant the filters are a finite union of sets whose number depends upon the simulated system. We prove here that it is possible to obtain the same result usi ng filters testing the presence of couples of
Pierluigi Frisco; Claudio Zandron
The prion protein-encoding gene (prnp) strongly influences the susceptibility of small ruminants to transmissible spongiform encephalopathies (TSEs). Hence, selective breeding programs have been implemented to increase sheep resistance to scrapie. For goats, epidemiological and experimental studies have provided some association between certain polymorphisms of the cellular prion protein (PrP(C)) and resistance to TSEs. Among them, the Q/K polymorphism at PrP(C) codon 222 (Q/K222) yielded the most promising results. In this work, we investigated the individual effects of the K222-PrP(C) variant on the resistance/susceptibility of goats to TSEs. For that purpose, we generated two transgenic mouse lines, expressing either the Q222 (wild type) or K222 variant of goat PrP(C). Both mouse lines were challenged intracerebrally with a panel of TSE isolates. Transgenic mice expressing the wild-type (Q222) allele were fully susceptible to infection with all tested isolates, whereas transgenic mice expressing similar levels of the K222 allele were resistant to all goat scrapie and cattle BSE isolates but not to goat BSE isolates. Finally, heterozygous K/Q222 mice displayed a reduced susceptibility to the tested panel of scrapie isolates. These results demonstrate a highly protective effect of the K222 variant against a broad panel of different prion isolates and further reinforce the argument supporting the use of this variant in breeding programs to control TSEs in goat herds. Importance: The objective of this study was to determine the role of the K222 variant of the prion protein (PrP) in the susceptibility/resistance of goats to transmissible spongiform encephalopathies (TSEs). Results showed that transgenic mice expressing the goat K222-PrP polymorphic variant are resistant to scrapie and bovine spongiform encephalopathy (BSE) agents. This protective effect was also observed in heterozygous Q/K222 animals. Therefore, the single amino acid exchange from Q to K at codon 222 of the cellular prion protein provides resistance against TSEs. All the results presented here support the view that the K222 polymorphic variant is a good candidate for selective breeding programs to control and eradicate scrapie in goat herds. PMID:24352451
Aguilar-Calvo, Patricia; Espinosa, Juan Carlos; Pintado, Belén; Gutiérrez-Adán, Alfonso; Alamillo, Elia; Miranda, Alberto; Prieto, Irene; Bossers, Alex; Andreoletti, Olivier; Torres, Juan María
The allelic architecture of complex traits is likely to be underpinned by a combination of multiple common frequency and rare variants. Targeted genotyping arrays and next-generation sequencing technologies at the whole-genome sequencing (WGS) and whole-exome scales (WES) are increasingly employed to access sequence variation across the full minor allele frequency (MAF) spectrum. Different study design strategies that make use of diverse technologies, imputation and sample selection approaches are an active target of development and evaluation efforts. Initial insights into the contribution of rare variants in common diseases and medically relevant quantitative traits point to low-frequency and rare alleles acting either independently or in aggregate and in several cases alongside common variants. Studies conducted in population isolates have been successful in detecting rare variant associations with complex phenotypes. Statistical methodologies that enable the joint analysis of rare variants across regions of the genome continue to evolve with current efforts focusing on incorporating information such as functional annotation, and on the meta-analysis of these burden tests. In addition, population stratification, defining genome-wide statistical significance thresholds and the design of appropriate replication experiments constitute important considerations for the powerful analysis and interpretation of rare variant association studies. Progress in addressing these emerging challenges and the accrual of sufficiently large data sets are poised to help the field of complex trait genetics enter a promising era of discovery.
Panoutsopoulou, Kalliope; Tachmazidou, Ioanna; Zeggini, Eleftheria
Isolated protoplasts from thalli of Porphyra haitanensis and Porphyra yezoensis were treated with colchicine or irradiated by ultraviolet (UV). Several types of color variants were observed among the protoplast offspring. After treatment with colchicine: (1) 0.04 0.09% of red type variants in P. haitanensis were obtained; (2) The rate of red type variants and the variegated chimeral thalli composed of red type and wild type of sectors were 0.31 1.11% in P. yezoensis. After irradiation with UV: (1) 3.5 10.5% of red type variants in P. yezoensis were obtained: (2) 0.5 2.0% of red type variants and the variegated chimeral thalli composed of red type and wild type of sectors were obtained in P. haitanensis. Colchicine and UV’s mutangenic effects on P. yezoensis protoplasts were stronger than those on P. haitanensis protoplasts. The most efficient concentration of colchicine was 0.05%. The optimal length of UV-radiation was 1/2 min (radiation distance 5 cm). The red type variants induced by colchicine treatment grew faster than the wild type thalli. The clones of vegetative propagation from protoplasts of red type variants were still red type thalli. The red type variants will be good materials for genetic studies and improvement of Porphyra strains.
Rabies viruses isolated from different animal species in various parts of the world were in the past considered to be antigenically closely related. Only when the antibodies produced in animals immunized with whole virions or viral components were assayed by the plaque reduction method, were some minor differences detected in the antigenic composition of various rabies strains (1). On the
T. J. WIKTOR; H. KOPROWSKI
Legumes inoculated with tryptophan catabolic variants (tan variants) of wild-type bradyrhizobia are characterized by an enhanced capacity to fix atmospheric nitrogen as compared to parent strains. Responses of the symbiotic system to the variants include ...
Primary progressive aphasia (PPA) is a language predominant neurodegenerative disorder that has three recognized variants: nonfluent/agrammatic, semantic, and logopenic. This report describes a 60-year-old man who presented with a progressive decline in verbal output that does not fit the currently accepted PPA subtypes. The patient exhibited a paucity of verbal output and impaired phonemic fluency with minimal associated language, cognitive, or behavioral deficits. Focal cortical thinning/hypometabolism of the left superior frontal region and a cerebrospinal fluid profile not consistent with Alzheimer's disease pathology were identified. This case of isolated progressive dynamic aphasia extends the current boundaries of PPA diagnostic variants. PMID:23168447
Perez, David L; Dickerson, Bradford C; McGinnis, Scott M; Sapolsky, Daisy; Johnson, Keith; Searl, Meghan; Daffner, Kirk R
Primary progressive aphasia (PPA) is a language predominant neurodegenerative disorder that has three recognized variants: nonfluent/agrammatic, semantic, and logopenic. This report describes a 60-year-old man who presented with a progressive decline in verbal output that does not fit the currently accepted PPA subtypes. The patient exhibited a paucity of verbal output and impaired phonemic fluency with minimal associated language, cognitive, or behavioral deficits. Focal cortical thinning/hypometabolism of the left superior frontal region and a cerebrospinal fluid profile not consistent with Alzheimer’s disease pathology were identified. This case of isolated progressive dynamic aphasia extends the current boundaries of PPA diagnostic variants.
Perez, David L.; Dickerson, Bradford C.; McGinnis, Scott M.; Sapolsky, Daisy; Johnson, Keith; Searl, Meghan; Daffner, Kirk R.
In the USA, vancomycin-resistant Enterococcus faecium (VREF) is endemic in hospitals, despite lack of carriage among healthy individuals. In Europe, however, hospital outbreaks are rare, but VREF carriage among healthy individuals and livestock is common. We used amplified fragment-length polymorphism analysis to genotype 120 VREF isolates associated with hospital outbreaks and 45 non-epidemic isolates from the USA, Europe, and Australia. We also looked for the esp virulence gene in these isolates and in 98 VREF from animals. A specific E. faecium subpopulation genetically distinct from non-epidemic VREF isolates was found to be the cause of the hospital epidemics in all three continents. This subpopulation contained a variant of the esp gene that was absent in all non-epidemic and animal isolates. Identification of the variant esp gene will be important in guiding infection-control strategies, and the Esp protein could be a new target for antibacterial therapy. PMID:11265956
Willems, R J; Homan, W; Top, J; van Santen-Verheuvel, M; Tribe, D; Manzioros, X; Gaillard, C; Vandenbroucke-Grauls, C M; Mascini, E M; van Kregten, E; van Embden, J D; Bonten, M J
Although Bouveret’s syndrome, i.e. gastric outlet obstruction by a large gallstone impacted in the proximal duodenum secondary to a cholecystoduodenal fistula, is rare, its pathogenesis and clinical features are well characterized. However, existence of variant forms of the syndrome are not well known, and as far as we know, only two cases of variant Bouveret’s syndrome have been described in the English-language literature. We present a case of another new variant of Bouveret’s syndrome in a 54-year-old Korean woman.
Park, Seong-Heum; Lee, Sang-Woo; Song, Tae-Jin
This report describes the characterization by whole-genome sequencing of four PVY isolates with unique combinations of molecular and symptomatic characteristics. Three of these four isolates were of type PVY(N:O) (ID-1, OR-1, PN10A), including one of "type B", which contains an extra recombination event in the 5'UTR/P1 cistron; the other (NE-11) represents a novel PVY molecular genotype, previously misclassified as a PVY(NA-NTN) isolate. The full genome sequence of this latter isolate is unique inasmuch as it is nearly identical to that of PVY(N) isolates for the first 2,000 nucleotides (nts), after which it very strongly resembles PVY(NA-NTN) isolates for the next 600 nts. For the final 7,000 nts of its genome, NE-11 shares intermediate identity with these other two previously reported classes of PVY(N) genomes, except for a portion of the capsid protein region in which it resembles neither. Recombination in each of the four isolates was verified by a suite of recombination detection programs. PN10A represents the first complete sequence of a PVY strain variant of the class reported as PVY(N)-W (or PVY(N:O)) type B. Specific PCR assays for two unique regions of NE-11 are presented that will allow the identification of this strain variant by other researchers. PMID:18193154
Lorenzen, Jim; Nolte, Phil; Martin, Darren; Pasche, Julie S; Gudmestad, Neil C
Viewgraphs on vibration isolation are presented. Techniques to control and isolate centrifuge disturbances were identified. Topics covered include: disturbance sources in the microgravity environment; microgravity assessment criteria; life sciences centrifuge; flight support equipment for launch; active vibration isolation system; active balancing system; and fuzzy logic control.
The chicken anemia virus (CAV), is a known member of the genus Gyrovirus and was first isolated from chickens in Japan in 1979. Some reports have also demonstrated that CAV can be identified in human stool specimens. In this study, a variant of CAV was detected using PCR with CAV-based primers in fecal samples of stray cats. The genome of CAV variant was sequenced and the results suggest that it could be a recombinant viral strain from parental CAV strains JQ690762 and AF311900. Recombination is an important evolutionary mechanism that contributes to genetic diversification. These findings indicate that CAV variant might have originated from CAV-infected chickens. The epidemiology and pathogenesis of this novel virus remains to be elucidated. This study underscores the importance of CAV surveillance and it presents the first evidence suggesting the possibility of CAV homologous recombination in cat. PMID:24689034
Zhang, Xinheng; Liu, Yuanjia; Ji, Jun; Chen, Feng; Sun, Baoli; Xue, Chunyi; Ma, Jingyun; Bi, Yingzuo; Xie, Qingmei
The aim of this communication was to describe the detection of the coexistence of HIV-1 variant with dipeptide insertion between codons 69 and 70 of reverse transcriptase. These variants were isolated from a 16-year-old male patient, undergoing treatment in the city of Marilia, SP, Southeastern Brazil. After confirmation of treatment failure, resistance to antiretroviral drugs testing was performed and two variants with the insertions of the aminoacids Ser-Gly/Ser-Ala at codon 69 of reverse transcriptase were detected, besides the T69S mutation. These insertions have low prevalence, have not been reported in situations of coexistence in Brazil and are related to multidrug resistance, which makes this epidemiological finding relevant. PMID:24346670
Tanikawa, Aline Aki; Barreto, Sarita Fiorelli Dias; Grotto, Rejane Maria Tommasini; Pardini, Maria Inês de Moura Campos
An unstable hemoglobin variant termed Hb Louisville, was found in four members of a Caucasian family, who were suffering from a mild hemolytic anemia. The variant showed a decreased stability upon warming at 65°C and an increased tendency to dissociate in the presence of sulfhydryl group-blocking agents. The structural abnormality was identified as a replacement of phenylalanyl residue in position 42 (CD1) by a leucyl residue. Substitution of this phenylalanyl residue, which participates in the contact with heme, by a nonpolar leucyl residue has apparently less severe consequences than a replacement of the same residue by a polar seryl residue as in Hb Hammersmith. Oxygen equilibrium studies of total hemolysate from one Hb Louisville heterozygote indicated a decreased oxygen affinity, a marked decrease in heme-heme interaction, and a normal Bohr effect. Studies with isolated Hb Louisville were not made because it was not possible to separate the variant from normal Hb A. Images
Keeling, Marie M.; Ogden, Lynn L.; Wrightstone, Ruth N.; Wilson, J. B.; Reynolds, Cecelia A.; Kitchens, Janice L.; Huisman, T. H. J.
One hundred and four scrapie positive and 77 negative goats from 34 Greek mixed flocks were analysed by prion protein gene sequencing and 17 caprine scrapie isolates from 11 flocks were submitted to molecular isolate typing. For the first time, the protective S146 variant was reported in Greece, while the protective K222 variant was detected in negative but also in five scrapie positive goats from heavily infected flocks. By immunoblotting six isolates, including two goat flockmates carrying the K222 variant, showed molecular features slightly different from all other Greek and Italian isolates co-analysed, possibly suggesting the presence of different scrapie strains in Greece.
Synopsis Guillain-Barré syndrome (GBS) is characterized by rapidly evolving ascending weakness, mild sensory loss and hypo- or areflexia, progressing to a nadir over up to four weeks. Cerebrospinal fluid evaluation demonstrates albuminocytologic dissociation in 90% of cases. Acute inflammatory demyelinating polyneuropathy (AIDP) was the first to be recognized over a century ago and is the most common form of GBS. In AIDP, the immune attack is directed at peripheral nerve myelin with secondary by-stander axon loss. Axonal motor and sensorimotor variants have been described in the last 3 decades and are mediated by molecular mimicry targeting peripheral nerve motor axons. Besides the Miller-Fisher syndrome (MFS) and descending weakness, other rare phenotypic variants have been recently described with pure sensory variant, restricted autonomic manifestations and the pharyngeal-cervical-brachial pattern. It is important to recognize GBS and its variants due to the availability of equally effective therapies in the form of plasmapheresis and intravenous immunoglobulins.
Barohn, Richard J.
Technologic advances in human immunodeficiency virus type 1 (HIV-1) sequencing have revolutionized the study of antiretroviral drug resistance and are increasingly moving from the laboratory to clinical practice. These techniques are able to detect HIV-1 drug resistance mutations present at low frequencies not detectable by current HIV-1 genotyping assays. For a number of commonly used antiretroviral medications, such as nonnucleoside reverse transcriptase inhibitors, the detection of these drug-resistant minority variants significantly increases the risk of treatment failure. The level of evidence, however, is insufficient to determine the impact of HIV-1 minority variants for several other classes of antiretroviral medications. Clinicians should be aware of the novel technologies that are moving into routine clinical use and the clinical implications of HIV-1 minority variants. Additional studies are needed to determine the optimal platform for clinical application of these new technologies and to provide guidance to clinicians on the type and frequency of clinically important HIV-1 minority variants.
Li, Jonathan Z.; Kuritzkes, Daniel R.
A catalog of human papillomavirus (HPV) type 16 (HPV-16) E6 and L1 signature nucleotides was used to develop PCR-based oligonucleotide probe systems capable of distinguishing HPV-16 class and subclass variants. Twenty-three E6-specific oligonucleotide probes targeting 13 variant nucleotide positions and 12 L1-specific oligonucleotide probes targeting 6 variant nucleotide positions were used to characterize HPV-16-containing cervicovaginal lavage specimens. Nucleotide positions that could be distinguished included E6 nucleotides 109, 131, 132, 143, 145, 178, 183, 286, 289, 335, 350, 403, and 532 and L1 nucleotides 6695, 6721, 6803, 6854, 6862, and 6994. Combined hybridization patterns were assigned on the basis of the predicted HPV-16 class, subclass, or minor class variants described previously (T. Yamada, C. M. Wheeler, A. L. Halpern, A.-C. M. Stewart, A. Hildesheim, and S.A. Jenison, J. Virol. 69:7743-7753, 1995). The major HPV-16 variant lineages detected included European prototype-like (E-P), Asian (As), Asian-American (AA), and African (Af1 and Af2) lineages. In addition, E-G131, an E-class variant, and AA-G183, an AA-class variant, were also identified. For each clinical specimen, DNA hybridization results were compared to nucleotide sequence determinations. Targeted L1 and E6 marker nucleotides covaried within all HPV-16 variant isolates examined. These hybridization-based methods result in minimal misclassification error, are amenable to targeting additional lineage-specific nucleotide positions, and should facilitate the large-scale, low-cost analysis of HPV-16 variants in epidemiologic investigations. Specifically, these methods will facilitate epidemiologic studies of HPV-16 transmission and natural history, as well as studies of associations between HPV variants, host immune responses, and cervical neoplasia.
Wheeler, C M; Yamada, T; Hildesheim, A; Jenison, S A
. Variant B1 is a rare type of GM2 gangliosidosis. Clinically, it shows a wide spectrum of forms ranging from infantile to\\u000a juvenile. We report the first magnetic resonance imaging (MRI) findings from three patients affected by GM2 gangliosidosis\\u000a variant B1, two presenting with the infantile form and one with the juvenile form. The MRI appearances of the two patients
Salvatore Grosso; Maria Angela Farnetani; Rosario Berardi; Maria Margollicci; Paolo Galluzzi; Rossella Vivarelli; Guido Morgese; Paolo Ballestri
Of 18 members of a Fiji Indian family investigated, eight of the 12 males and two of the six females had an electrophoretically slow-type bisalbuminemia (alloalbuminemia). The albumin was characterized by the hiterto unique ratio of the two bands (Al A 35%: variant 65%), and by dye-binding studies and electrophoretic mobility in different media. The data suggest that this is a new variant, which we propose to call albumin Vancouver (Al Va). PMID:709819
Frohlich, J; Kozier, J; Campbell, D J; Curnow, J V; Tárnoky, A L
Obligately alkalophilic Bacillus firmus RAB cannot grow well on media containing less than 5 mM Na+. However, variant strains can be isolated on plates containing 2 to 3 mM Na+. These variants are observed only rarely in cultures that are plated before being subjected to repeated transfers in liquid medium. Cultures which have been transferred several times produce variants at an apparent frequency of 2 X 10(-4). Most of these variants are unstable, generating parental types at the high frequency of 10%; however, stable variants can be isolated. These strains grow better than the parental strain at very high pH values in the presence of 5 mM Na+ and have enhanced activity of the Na+ -H+ antiporter that has been implicated in pH homeostasis. By contrast, Na+ -coupled solute uptake is indistinguishable from that of the parental strain, and no obvious changes in the respiratory chain components are apparent in reduced versus oxidized difference spectra. The membranes of the variants show a marked enhancement, on sodium dodecyl sulfate-polyacrylamide gradient electrophoresis, in one polypeptide band with a molecular weight in the range of 90,000. The findings are discussed from the point of view of genetic mechanisms that might confer adaptability to even more extreme environments than usual and in view of earlier models relating the Na+ -translocating activities of the alkalophiles. Images
Krulwich, T A; Guffanti, A A; Fong, M Y; Falk, L; Hicks, D B
Tick-borne encephalitis virus (TBEV) is a zoonotic agent that causes acute central nervous system (CNS) disease in humans. We previously suggested that immune response in addition to CNS infection contribute to mouse mortality following TBEV infection. However, we did not examine the influence of virus variants in the previous study. Therefore, in this study, we investigated the biological and pathologic potentials of the variant clones in the TBEV Oshima strain. We isolated eight variant clones from the stock virus of the Oshima 5-10. These variants exhibited different plaque morphologies in BHK cells and pathogenic potentials in mice. Full sequences of viral genomes revealed that each of the variant clones except one had specific combinations of nucleotide and amino acid changes at certain positions different from the parent strain. We also showed that an amino acid substitution of Glu122?Gly in the E protein could have affected virus infection and replication in vivo, as well as the attenuated pathogenicity in mice. These data confirm the presence of virus variants or quasispecies from the parent strain. Further elucidation of the effect of each variant clone on immune responses such as the T-cell response is an important priority in the development of an effective vaccine and treatment strategies for tick-borne encephalitis.
Luat, Le Xuan; Tun, Mya Myat Ngwe; Buerano, Corazon C.; Aoki, Kotaro; Morita, Kouichi; Hayasaka, Daisuke
G-protein-coupled receptors (GPCRs) play essential roles in various physiological processes, and are widely targeted by pharmaceutical drugs. Despite their importance, studying GPCRs has been problematic due to difficulties in isolating large quantities of these membrane proteins in forms that retain their ligand binding capabilities. Creating water-soluble variants of GPCRs by mutating the exterior, transmembrane residues provides a potential method to overcome these difficulties. Here we present the first study involving the computational design, expression and characterization of water-soluble variant of a human GPCR, the human mu opioid receptor (MUR), which is involved in pain and addiction. An atomistic structure of the transmembrane domain was built using comparative (homology) modeling and known GPCR structures. This structure was highly similar to the subsequently determined structure of the murine receptor and was used to computationally design 53 mutations of exterior residues in the transmembrane region, yielding a variant intended to be soluble in aqueous media. The designed variant expressed in high yield in Escherichia coli and was water soluble. The variant shared structural and functionally related features with the native human MUR, including helical secondary structure and comparable affinity for the antagonist naltrexone (Kd ?=?65 nM). The roles of cholesterol and disulfide bonds on the stability of the receptor variant were also investigated. This study exemplifies the potential of the computational approach to produce water-soluble variants of GPCRs amenable for structural and functionally related characterization in aqueous solution.
Matsunaga, Felipe; Cui, Xu; Selling, Bernard; Saven, Jeffery G.; Liu, Renyu
Restriction endonucleases (REases) with 8-base specificity are rare specimens in nature. NotI from Nocardia otitidis-caviarum (recognition sequence 5?-GCGGCCGC-3?) has been cloned, thus allowing for mutagenesis and screening for enzymes with altered 8-base recognition and cleavage activity. Variants possessing altered specificity have been isolated by the application of two genetic methods. In step 1, variant E156K was isolated by its ability to induce DNA-damage in an indicator strain expressing M.EagI (to protect 5?-NCGGCCGN-3? sites). In step 2, the E156K allele was mutagenized with the objective of increasing enzyme activity towards the alternative substrate site: 5?-GCTGCCGC-3?. In this procedure, clones of interest were selected by their ability to eliminate a conditionally toxic substrate vector and induce the SOS response. Thus, specific DNA cleavage was linked to cell survival. The secondary substitutions M91V, F157C and V348M were each found to have a positive effect on specific activity when paired with E156K. For example, variant M91V/E156K cleaves 5?-GCTGCCGC-3? with a specific activity of 8.2 × 104 U/mg, a 32-fold increase over variant E156K. A comprehensive analysis indicates that the cleavage specificity of M91V/E156K is relaxed to a small set of 8 bp substrates while retaining activity towards the NotI sequence.
Outbreaks of infectious coryza have been reported in vaccinated flocks in different countries, indicating that new serotype(s) of Haemophilus paragallinarum may have evolved. Several field isolates from vaccinated flocks in the US, Ecuador, Argentina and Zimbabwe were examined and, apart from one serotype C strain, all were typed as serotype B. An inactivated commercial trivalent vaccine, containing serotypes A, B and C, protected against challenge with the serotype C isolate but protection against challenge with serotype B isolates was weaker, suggesting that they might represent a new variant immunotype. An experimental tetravalent oil adjuvant vaccine, containing one of the serotype B isolates, appeared immunogenic against all isolates after one vaccination. Its efficacy and safety were further tested in layer chickens housed under field conditions. Chickens were vaccinated at 8 and 16 weeks of age while controls were unvaccinated. Vaccinates and controls were challenged with type A, B, C and variant type B at 25, 45 or 65 weeks of age. There was good protection (P<0.05) against all four immunotypes after all challenges. No systemic reactions were observed and local reactions were similar to those found with the commercial trivalent vaccine. The tetravalent vaccine may therefore be a good choice for control of new field isolates. PMID:12850915
Jacobs, Anton A C; van den Berg, Karin; Malo, Aris
Experimental study of the role of disorder in protein function is challenging. It has been proposed that proteins utilize disordered regions in the adaptive recognition of their various binding partners. However apart from a few exceptions, defining the importance of disorder in promiscuous binding interactions has proven to be difficult. In this paper, we have utilized a genetic selection that links protein stability to antibiotic resistance to isolate variants of the newly discovered chaperone Spy that show an up to 7 fold improved chaperone activity against a variety of substrates. These “Super Spy” variants show tighter binding to client proteins and are generally more unstable than is wild type Spy and show increases in apparent flexibility. We establish a good relationship between the degree of their instability and the improvement they show in their chaperone activity. Our results provide evidence for the importance of disorder and flexibility in chaperone function. DOI: http://dx.doi.org/10.7554/eLife.01584.001
Quan, Shu; Wang, Lili; Petrotchenko, Evgeniy V; Makepeace, Karl AT; Horowitz, Scott; Yang, Jianyi; Zhang, Yang; Borchers, Christoph H; Bardwell, James CA
Experimental study of the role of disorder in protein function is challenging. It has been proposed that proteins utilize disordered regions in the adaptive recognition of their various binding partners. However apart from a few exceptions, defining the importance of disorder in promiscuous binding interactions has proven to be difficult. In this paper, we have utilized a genetic selection that links protein stability to antibiotic resistance to isolate variants of the newly discovered chaperone Spy that show an up to 7 fold improved chaperone activity against a variety of substrates. These "Super Spy" variants show tighter binding to client proteins and are generally more unstable than is wild type Spy and show increases in apparent flexibility. We establish a good relationship between the degree of their instability and the improvement they show in their chaperone activity. Our results provide evidence for the importance of disorder and flexibility in chaperone function. DOI: http://dx.doi.org/10.7554/eLife.01584.001. PMID:24497545
Quan, Shu; Wang, Lili; Petrotchenko, Evgeniy V; Makepeace, Karl At; Horowitz, Scott; Yang, Jianyi; Zhang, Yang; Borchers, Christoph H; Bardwell, James Ca
Light beam transmits heartbeat signal from electrodes on patient to electrocardiograph without exposing patient to possible severe electrical shock. System provides complete isolation between patient and EKG instrumentation.
Palmer, E.; Rasquin, J. R.; Smith, H. E.
Eight erythrocyte enzymes were examine for thermostability in an unselected sample of 100 newborn infants. Three thermolabile variants, one each of lactate dehydrogenase, glucosephosphate isomerase, and glucose-6-phosphate dehydrogenase, were identified, none of which was detectable as a variant by standard electrophoretic techniques. All were inherited. This frequency of 3.8 heritable thermostability variants per 1000 determinations is to be compared with a frequency of electrophoretically detectable variants of 1.1 per 1000 determinations, a frequency of 2.4 enzyme-deficiency variants per 1000 determinations, and a frequency of individuals with rare enzyme deficiency or electrophoretic or thermostability (or both) variants at these loci is 8.4 per 1000 determinations. A similar distribution and frequency is seen when the comparison is limited to the seven loci studied by all techniques. it is clear that not all of the electrophoretic and thermostability variants present in the population are detected by the techniques used in this study. Accordingly, it is estimated that the true frequency of carriers of a rare variant for each of these enzyme-coding loci averages greater than 10/1000. Some implications of these frequencies for human disease are discussed.
Mohrenweiser, H.W.; Neel, J.V.
The diversity of the cytotoxin-associated gene (cagA )o fHelicobacter pylori was analyzed in 45 isolates obtained from nine countries. We examined variation in the 5* end of the cagA open reading frame as determined by PCR and sequencing. Phylogenetic analysis revealed the existence of at least two distinct types of cagA. One variant (cagA1) was found exclusively in strains from
LEEN-JAN VAN DOORN; C EU FIGUEIREDO; RICARDO SANNA; MARTIN J. BLASER; WIM G. V. QUINT
Four typical manifestations of the occipital vertebra are described from both an anatomic and a radiologic point of view; the basilar process, the condylus tertius, the paracondylar process, and the isolated prebasioccipital arch. The clinical importance of the described variants is discussed. ImagesFigure 1aFigure 1bFigure 1p86-bFigure 2Figure 2Figure 3Figure 3Figure 4p91-bFigure 4
Prescher, Andreas; Brors, Dominik; Adam, Gerhard
Porcine kobuvirus, an emerging virus, may be the underlying etiological cause of a large-scale outbreak of diarrhea in suckling piglets in China that started in 2010. We report the complete genome sequence of the porcine kobuvirus variant CH/HNXX-4/2012 with a 30-amino-acid deletion in its 2B-coding region that was isolated in this outbreak. This will help the phenotypic variation and evolutionary characteristics of porcine kobuvirus to be understood.
Cao, Weijun; Zhang, Keshan; Jin, Ye; Lv, Lv; Yang, Fan
Two isolates of haemophilic bacteria originally isolated in the 1980s from chickens were re-examined. The addition of a 10% sterile filtrate from an overnight culture of Staphylococcus epidermidis allowed growth of both isolates in solid and liquid media that were otherwise not capable of supporting the growth of these isolates. Using the modified media, genotypic and serotypic studies were performed, which confirmed both isolates to be Avibacterium paragallinarum, with one isolate being serovar A and the other serovar C. The unusual growth requirements of these two isolates reinforces the need for careful interpretation by diagnostic laboratories examining chickens showing signs of upper respiratory tract disease. PMID:21696378
Blackall, P J; Christensen, H; Bisgaard, M
The silver-haired bat variant of rabies virus (SHBRV) has been identified as the etiological agent of a number of recent human rabies cases in the United States that are unusual in not having been associated with any known history of conventional exposure. Comparison of the different biological and biochemical properties of isolates of this virus with those of a coyote
Kinjiro Morimoto; Menal Patel; Susanne Corisdeo; D. Craig Hooper; Zhen Fang Fu; Charles E. Rupprecht; Hilary Koprowski; Bernhard Dietzschold
Purpose The gene encoding nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) was recently found to be mutated in a subset of patients with Leber congenital amaurosis (LCA) with macular atrophy. The aim of this study was to determine the occurrence and frequency of NMNAT1 mutations and associated phenotypes in different types of inherited retinal dystrophies. Methods DNA samples of 161 patients with LCA without genetic diagnosis were analyzed for variants in NMNAT1 using Sanger sequencing. Variants in exon 5 of NMNAT1, which harbors the majority of the previously identified mutations, were screened in 532 additional patients with retinal dystrophies. This cohort encompassed 108 persons with isolated or autosomal recessive cone-rod dystrophy (CRD), 271 with isolated or autosomal recessive retinitis pigmentosa (RP), and 49 with autosomal dominant RP, as well as 104 persons with LCA in whom the causative mutation was previously identified. Results Compound heterozygous alterations were found in six patients with LCA and in one person with early-onset RP. All except one carried the common p.E257K variant on one allele. Macular atrophy was absent in one patient, who carried this variant in combination with a truncating mutation on the other allele. The p.E257K alteration was also found in a heterozygous state in five individuals with LCA and one with RP while no mutation was detected on the other allele. Two individuals with LCA carried other NMNAT1 variants in a heterozygous state, whereas no NMNAT1 variants in exon 5 were identified in individuals with CRD. The p.E257K variant was found to be enriched in a heterozygous state in individuals with LCA (0.94%) compared to Caucasian controls (0.18%), although the difference was statistically insignificant (p=0.12). Conclusions Although macular atrophy can occur in LCA and CRD, no NMNAT1 mutations were found in the latter cohort. NMNAT1 variants were also not found in a large group of patients with sporadic or autosomal recessive RP. The enrichment of p.E257K in a heterozygous state in patients with LCA versus controls suggests that this allele could act as a modifier in other genetic subtypes of LCA.
Siemiatkowska, Anna M.; van den Born, L. Ingeborgh; van Genderen, Maria M.; Bertelsen, Mette; Zobor, Ditta; Rohrschneider, Klaus; van Huet, Ramon A.C.; Nurohmah, Siska; Klevering, B. Jeroen; Kohl, Susanne; Faradz, Sultana M.H.; Rosenberg, Thomas; den Hollander, Anneke I.; Collin, Rob W.J.
Background Missense pharmacogenomic (PGx) variants refer to amino acid substitutions that potentially affect the pharmacokinetic (PK) or pharmacodynamic (PD) response to drug therapies. The PGx variants, as compared to disease-associated variants, have not been investigated as deeply. The ability to computationally predict future PGx variants is desirable; however, it is not clear what data sets should be used or what features are beneficial to this end. Hence we carried out a comparative characterization of PGx variants with annotated neutral and disease variants from UniProt, to test the predictive power of sequence conservation and structural information in discriminating these three groups. Results 126 PGx variants of high quality from PharmGKB were selected and two data sets were created: one set contained 416 variants with structural and sequence information, and, the other set contained 1,265 variants with sequence information only. In terms of sequence conservation, PGx variants are more conserved than neutral variants and much less conserved than disease variants. A weighted random forest was used to strike a more balanced classification for PGx variants. Generally structural features are helpful in discriminating PGx variant from the other two groups, but still classification of PGx from neutral polymorphisms is much less effective than between disease and neutral variants. Conclusions We found that PGx variants are much more similar to neutral variants than to disease variants in the feature space consisting of residue conservation, neighboring residue conservation, number of neighbors, and protein solvent accessibility. Such similarity poses great difficulty in the classification of PGx variants and polymorphisms.
Background: Porokeratosis, a well recognized disorder of keratinization, is known to have several clinical variants. This report describes a rare variant characterized by verrucous plaques. Methods: An adult male presented with a slowly progressive verrucous plaque on the gluteal region that was resistant to conventional therapy. Careful inspection revealed a keratotic ridge at the plaque border leading to the diagnosis. Results: Histopathology showed the presence of multiple cornoid lamellae confirming the diagnosis of porokeratosis ptychotropica. Conclusions: Porokeratosis ptychotropica is a rare variant of porokeratosis with fewer than 25 cases described in the literature. This report is to highlight the importance of considering this particular entity in the diagnosis of genitogluteal plaques, especially those not responding to conventional modalities. PMID:24945649
D'souza, Paschal; Dhali, Tapan Kumar; Arora, Shikha; Gupta, Himanshu; Khanna, Urmi
Motivation: Structural variants, including duplications, insertions, deletions and inversions of large blocks of DNA sequence, are an important contributor to human genome variation. Measuring structural variants in a genome sequence is typically more challenging than measuring single nucleotide changes. Current approaches for structural variant identification, including paired-end DNA sequencing/mapping and array comparative genomic hybridization (aCGH), do not identify the boundaries of variants precisely. Consequently, most reported human structural variants are poorly defined and not readily compared across different studies and measurement techniques. Results: We introduce Geometric Analysis of Structural Variants (GASV), a geometric approach for identification, classification and comparison of structural variants. This approach represents the uncertainty in measurement of a structural variant as a polygon in the plane, and identifies measurements supporting the same variant by computing intersections of polygons. We derive a computational geometry algorithm to efficiently identify all such intersections. We apply GASV to sequencing data from nine individual human genomes and several cancer genomes. We obtain better localization of the boundaries of structural variants, distinguish genetic from putative somatic structural variants in cancer genomes, and integrate aCGH and paired-end sequencing measurements of structural variants. This work presents the first general framework for comparing structural variants across multiple samples and measurement techniques, and will be useful for studies of both genetic structural variants and somatic rearrangements in cancer. Availability: http://cs.brown.edu/people/braphael/software.html Contact: firstname.lastname@example.org
Sindi, Suzanne; Helman, Elena; Bashir, Ali; Raphael, Benjamin J.
At least three variants of avian pox virus are present in Hawai‘i - Fowlpox from domestic poultry and a group of genetically distinct viruses that cluster within two clades (Pox Variant 1 and Pox Variant 2) that are most similar to Canarypox based on DNA sequence of the virus 4b core protein gene. We tested whether Hawai‘i ‘Amakihi can be protected from wild virus isolates with an attenuated live Canarypox vaccine that is closely related to isolates that cluster within clade 1 (Pox Variant 1) based on sequence of the attenuated Canarypox virus 4b core protein. Thirty-one (31) Hawai`i ‘Amakihi (Hemignathus virens) with no prior physical evidence of pox infection were collected on Mauna Kea from xeric, high elevation habitats with low pox prevalence and randomly divided into two groups. One group of 16 was vaccinated with Poximmune C® while the other group received a sham vaccination with virus diluent. Four of 15 (27%) vaccinated birds developed potentially life-threatening disseminated lesions or lesions of unusually long duration, while one bird never developed a vaccine-associated lesion or "take". After vaccine-associated lesions healed, vaccinated birds were randomly divided into three groups of five and challenged with either a wild isolate of Fowlpox, a Hawai`i `Amakihi isolate of a Canarypox-like virus from clade 1 (Pox Variant 1) or a Hawai`i `Amakihi isolate of a Canarypox-like virus from clade 2 (Pox Variant 2). Similarly, three random groups of five unvaccinated ‘Amakihi were challenged with the same virus isolates. Vaccinated and unvaccinated ‘Amakihi challenged with Fowlpox had transient infections with no clinical signs of infection. Mortality in vaccinated ‘Amakihi that were challenged with Pox Variant 1 and Pox Variant 2 ranged from 0% (0/5) for Pox Variant 1 to 60% (3/5) for Pox Variant 2. Mortality in unvaccinated ‘Amakihi ranged from 40% (2/5) for Pox Variant 1 to 100% (5/5) for Pox Variant 2. While the vaccine provided some protection against Pox Variant 1, serious side effects and low efficacy against Pox Variant 2 make it risky to use in captive or wild honeycreepers.
Atkinson, Carter T.; Wiegand, Kimberly C.; Triglia, Dennis; Jarvi, Susan I.
Eukaryotic gene regulation involves a balance between packaging of the genome into nucleosomes and enabling access to regulatory proteins and RNA polymerase. Nucleosomes are integral components of gene regulation that restrict access to both regulatory sequences and the underlying template. Whereas canonical histones package the newly replicated genome, they can be replaced with histone variants that alter nucleosome structure, stability, dynamics, and, ultimately, DNA accessibility. Here we consider how histone variants and their interacting partners are involved in transcriptional regulation through the creation of unique chromatin states.
Weber, Christopher M.; Henikoff, Steven
Eukaryotic gene regulation involves a balance between packaging of the genome into nucleosomes and enabling access to regulatory proteins and RNA polymerase. Nucleosomes are integral components of gene regulation that restrict access to both regulatory sequences and the underlying template. Whereas canonical histones package the newly replicated genome, they can be replaced with histone variants that alter nucleosome structure, stability, dynamics, and, ultimately, DNA accessibility. Here we consider how histone variants and their interacting partners are involved in transcriptional regulation through the creation of unique chromatin states. PMID:24696452
Weber, Christopher M; Henikoff, Steven
DNA shuffling facilitated the evolution of a human immunodeficiency virus type 1 (HIV-1) variant with enhanced replication in pig-tailed macaque peripheral blood mononuclear cells (pt mPBMC). This variant consists exclusively of HIV-1-derived sequences with the exception of simian immunodeficiency virus (SIV) nef. Sequences spanning the gag-protease-reverse transcriptase (gag-pro-RT) region from several HIV-1 isolates were shuffled and cloned into a parental
Katja Pekrun; Riri Shibata; Tatsuhiko Igarashi; Margaret Reed; Liana Sheppard; Philip A. Patten; Willem P. C. Stemmer; Malcolm A. Martin; Nay-Wei Soong
Background Meningococci produce a penta-acylated instead of hexa-acylated lipid A when their lpxL1 gene is inactivated. Meningococcal strains with such lipid A endotoxin variants have been found previously in adult meningitis patients, where they caused less blood coagulopathy because of decreased TLR4 activation. Methods A cohort of 448 isolates from patients with invasive meningococcal disease in the Netherlands were screened for the ability to induce IL-6 in monocytic cell Mono Mac 6 cells. The lpxL1 gene was sequenced of isolates, which show poor capacity to induce IL-6.. Clinical characteristics of patients were retrieved from hospital records. Results Of 448 patients, 29 (6.5%) were infected with meningococci expressing a lipid A variant strain. Lipid A variation was not associated with a specific serogroup or genotype. Infections with lipid A variants were associated with older age (19.3 vs. 5.9 (median) years, p?=?0.007) and higher prevalence of underlying comorbidities (39% vs. 17%; p?=?0.004) compared to wild-type strains. Patients infected with lipid A variant strains had less severe infections like meningitis or shock (OR 0.23; 95%CI 0.09–0.58) and were less often admitted to intensive care (OR 0.21; 95%CI 0.07–0.60) compared to wild-type strains, independent of age, underlying comorbidities or strain characteristics. Conclusions In adults with meningococcal disease lipid A variation is rather common. Infection with penta-acylated lipid A variant meningococci is associated with a less severe disease course.
Bogaert, Debby; Schipper, Kim; Groenwold, Rolf H. H.; Hamstra, Hendrik Jan; Westerhuis, Brenda M.; van de Beek, Diederik; van der Ley, Peter; Sanders, Elisabeth A. M.; van der Ende, Arie
Background Breastfeeding is a leading cause of infant HIV-1 infection in the developing world, yet only a minority of infants exposed to HIV-1 via breastfeeding become infected. As a genetic bottleneck severely restricts the number of postnatally-transmitted variants, genetic or phenotypic properties of the virus Envelope (Env) could be important for the establishment of infant infection. We examined the efficiency of virologic functions required for initiation of infection in the gastrointestinal tract and the neutralization sensitivity of HIV-1 Env variants isolated from milk of three postnatally-transmitting mothers (n=13 viruses), five clinically-matched nontransmitting mothers (n=16 viruses), and seven postnatally-infected infants (n = 7 postnatally-transmitted/founder (T/F) viruses). Results There was no difference in the efficiency of epithelial cell interactions between Env virus variants from the breast milk of transmitting and nontransmitting mothers. Moreover, there was similar efficiency of DC-mediated trans-infection, CCR5-usage, target cell fusion, and infectivity between HIV-1 Env-pseudoviruses from nontransmitting mothers and postnatal T/F viruses. Milk Env-pseudoviruses were generally sensitive to neutralization by autologous maternal plasma and resistant to breast milk neutralization. Infant T/F Env-pseudoviruses were equally sensitive to neutralization by broadly-neutralizing monoclonal and polyclonal antibodies as compared to nontransmitted breast milk Env variants. Conclusion Postnatally-T/F Env variants do not appear to possess a superior ability to interact with and cross a mucosal barrier or an exceptional resistance to neutralization that define their capability to initiate infection across the infant gastrointestinal tract in the setting of preexisting maternal antibodies.
Biofuel cell is an energy conversion device of the next generation which enables use of safer and higher energy-density fuels such as glucose. We have been developing a biofuel cell that comprises the three enzymes: glucose dehydrogenase (GDH) and diaphorase (DI) on anode, and bilirubin oxidase (BOD) on cathode. In this work, we have developed a DI variant suitable for our biofuel cell by using directed molecular evolution method. A gene library of DI variants was constructed by using error-prone PCR and the variant proteins were expressed in an Escherichia coli system. 8000 isolated variants have been screened with activity against 2-amino-1,4-naphthoquinone (ANQ), and 10 of them have been qualified which were then purified and examined their activities against ANQ. A highest activity was observed in G122D variant of which glycine residue at position 122 is substituted to aspartate. Enzymatic kinetic analyses show that KM for ANQ in G122D is 1/3 of that in wild type (G122D: 356 ?M, wild type: 1.08 mM), whereas kcat and KM for NADH is almost the same, clearly showing that G122D mutation has given DI an improvement in enzymatic activity at lower ANQ concentration. The effect of this mutation was considered electrochemically in solution and in immobilized layer. The results show that G122D variant DI gave a higher current at lower ANQ concentration in solution, as well as in immobilized condition where GDH is co-immobilized within. PMID:20739172
Sugiyama, Taiki; Goto, Yoshio; Matsumoto, Ryuhei; Sakai, Hideki; Tokita, Yuichi; Hatazawa, Tsuyonobu
Hepatitis C virus (HCV) is a positive-strand enveloped RNA virus that shows diverse viral populations even in one individual. Though Sanger sequencing has been used to determine viral sequences, deep sequencing technologies are much faster and can perform large-scale sequencing. We demonstrate the successful use of Illumina deep sequencing technology and subsequent analyses to determine the genetic variants and amino acid substitutions in both treatment-naïve (patient 1) and treatment-experienced (patient 7) isolates from HCV-infected patients. As a result, almost the full nucleotide sequence of HCV was detectable for patients 1 and 7. The reads were mapped to the HCV reference sequence. The coverage was 99.8% and the average depth was 69.5× for patient 7, with values of 99.4% (coverage) and 51.1× (average depth) for patient 1. In patient 7, amino acid (aa) 70 in the core region showed arginine, with methionine at aa 91, by Sanger sequencing. Major variants showed the same amino acid sequence, but minor variants were detectable in 18% (6/34 sequences) of sequences, with replacement of methionine by leucine at aa 91. In NS3, 8 amino acid positions showed mixed variants (T72T/I, K213K/R, G237G/S, P264P/S/A, S297S/A, A358A/T, S457S/C, and I615I/M) in patient 7. In patient 1, 3 amino acid positions showed mixed variants (L14L/F/V, S61S/A, and I586T/I). In conclusion, deep sequencing technologies are powerful tools for obtaining more profound insight into the dynamics of variants in the HCV quasispecies in human serum.
Ninomiya, Masashi; Funayama, Ryo; Nagashima, Takeshi; Nishida, Yuichiro; Kondo, Yasuteru; Inoue, Jun; Kakazu, Eiji; Kimura, Osamu; Nakayama, Keiko; Shimosegawa, Tooru
Background The inhibitor of apoptosis (IAP) protein Survivin and its splice variants are differentially expressed in breast cancer tissues. Our previous work showed Survivin is released from tumor cells via small membrane-bound vesicles called exosomes. We, therefore, hypothesize that analysis of serum exosomal Survivin and its splice variants may provide a novel biomarker for early diagnosis of breast cancer. Methods We collected sera from forty breast cancer patients and ten control patients who were disease free for 5 years after treatment. In addition, twenty-three paired breast cancer tumor tissues from those same 40 patients were analyzed for splice variants. Serum levels of Survivin were analyzed using ELISA and exosomes were isolated from this serum using the commercially available ExoQuick kit, with subsequent Western blots and immunohistochemistry performed. Results Survivin levels were significantly higher in all the breast cancer samples compared to controls (p?0.05) with exosome amounts significantly higher in cancer patient sera compared to controls (p?0.01). While Survivin and Survivin-?Ex3 splice variant expression and localization was identical in serum exosomes, differential expression of Survivin-2B protein existed in the exosomes. Similarly, Survivin and Survivin-?Ex3 proteins were the predominant forms detected in all of the breast cancer tissues evaluated in this study, whereas a more variable expression of Survivin-2B level was found at different cancer stages. Conclusion In this study we show for the first time that like Survivin, the Survivin splice variants are also exosomally packaged in the breast cancer patients’ sera, mimicking the survivin splice variant pattern that we also report in breast cancer tissues. Differential expression of exosomal-Survivin, particularly Survivin-2B, may serve as a diagnostic and/or prognostic marker, a “liquid biopsy” if you will, in early breast cancer patients. Furthermore, a more thorough understanding of the role of this prominent antiapoptotic pathway could lead to the development of potential therapeutics for breast cancer patients.
To investigate the distribution of Human papillomavirus (HPV)-31 A, B and C variants as well as the common amino acid polymorphisms in Chinese women, all 14 HPV-31 positive cervical exfoliated cell specimens identified from a descriptive study including ?2700 women from Northern China were analyzed. HPV-31 positive specimens were identified by Mass Spectrometry and the fragments of partial Long Control Region, E6 and E7 were amplified and directly sequenced or cloned into vector and then sequenced to confirm the variant information. HPV-31 prevalence in Northern Chinese female population was 0.52%. Six different sequences represented all 14 isolates, and these isolates were subsequently classified into variant lineage A (9), B (0) and C (5) by phylogenetic analysis. Five common amino acid polymorphism sites (2 in E6 and 3 in E7) and a novel non-synonymous mutation were detected in the current study. Our investigation suggested that HPV-31 was much less detected in Chinese women population than that in western countries. A and C variants were commonly detected while B variants were rarely detected in this population.
Xi, Longfu; Li, Jingjing; Liu, Fangfang; Liu, Ying; Pan, Yaqi; Ning, Tao; Guo, Chuanhai; Xu, Ruiping; Zhang, Lixin; Cai, Hong; Ke, Yang
Purpose: Increased prevalence of cavum septi pellucidi (CSP) in schizophrenic patients in comparison to healthy subjects was reported previously. Our purpose was to evaluate the prevalence of variants of the septum pellucidum in healthy subjects in three different age groups. Methods: 151 healthy subjects, including 46 children (age 6 ± 4 years), 72 young adults (age 31 ± 8 years)
Christine M. Born; Eva M. Meisenzahl; Thomas Frodl; Thomas Pfluger; Maximilian Reiser; H. J. Möller; Gerda L. Leinsinger
The disclosure provides a method for preparing an active exoglucanase in a heterologous host of eukaryotic origin. The method includes mutagenesis to reduce glycosylation of the exoglucanase when expressed in a heterologous host. It is further disclosed a method to produce variant cellobiohydrolase that is stable at high temperature through mutagenesis.
Adney, William S. (Golden, CO) [Golden, CO; Decker, Stephen R. (Berthoud, CO) [Berthoud, CO; Mc Carter, Suzanne (San Carlos, CA) [San Carlos, CA; Baker, John O. (Golden, CO) [Golden, CO; Nieves, Raphael (Lakewood, CO) [Lakewood, CO; Himmel, Michael E. (Littleton, CO) [Littleton, CO; Vinzant, Todd B. (Golden, CO) [Golden, CO
Array-based genome-wide segmental aneuploidy screening detects both de novo and inherited copy number variations (CNVs). In sporadic patients de novo CNVs are interpreted as potentially pathogenic. However, a deletion, transmitted from a healthy parent, may be pathogenic if it overlaps with a mutated second allele inherited from the other healthy parent. To detect such events, we performed multiplex enrichment and next-generation sequencing of the entire coding sequence of all genes within unique hemizygous deletion regions in 20 patients (1.53?Mb capture footprint). Out of the detected 703 non-synonymous single-nucleotide variants (SNVs), 8 represented variants being unmasked by a hemizygous deletion. Although evaluation of inheritance patterns, Grantham matrix scores, evolutionary conservation and bioinformatic predictions did not consistently indicate pathogenicity of these variants, no definitive conclusions can be drawn without functional validation. However, in one patient with severe mental retardation, lack of speech, microcephaly, cheilognathopalatoschisis and bilateral hearing loss, we discovered a second smaller deletion, inherited from the other healthy parent, resulting in loss of both alleles of the highly conserved heat shock factor binding protein 1 (HSBP1) gene. Conceivably, inherited deletions may unmask rare pathogenic variants that may exert a phenotypic impact through a recessive mode of gene action.
Hochstenbach, Ron; Poot, Martin; Nijman, Isaac J; Renkens, Ivo; Duran, Karen J; van'T Slot, Ruben; van Binsbergen, Ellen; van der Zwaag, Bert; Vogel, Maartje J; Terhal, Paulien A; Ploos van Amstel, Hans Kristian; Kloosterman, Wigard P; Cuppen, Edwin
Several variants of dermatofibroma have been described. They are essentially distinguished by their clinical and histopathological features. To review the mainfeaturesof these variants, a retrospective study of skin biopsies and tissue excisions of dermatofibromasperformed in the dermatology and venereology service at the Hospital Garcia de Orta between May 2007 and April 2012 was carried out. During that period, 192 dermatofibromas were diagnosed in 181 patients, the lesions being more common in women. Median age of the study population was 48 years. The most common lesion site was the limbs (74% of patients). The histopathological types found were common fibrous histiocytoma (80%) and the aneurysmal (5.7%),hemosiderotic (5.7%), epithelioid (2.6%), cellular (2.1%), lipidized (2.1%), atrophic (1.0) and clear cell (0.5%) variants. Based on these findings, this review focuses on the clinical and histological features of the various variants of dermatofibroma in terms of their clinical presentation, distinct histopathological features, differential diagnosis and prognosis. PMID:24937822
Alves, João Vítor Pina; Matos, Diogo Miguel; Barreiros, Hugo Frederico; Bártolo, Elvira Augusta Felgueira Leonardo Fernandes
Several variants of dermatofibroma have been described. They are essentially distinguished by their clinical and histopathological features. To review the mainfeaturesof these variants, a retrospective study of skin biopsies and tissue excisions of dermatofibromasperformed in the dermatology and venereology service at the Hospital Garcia de Orta between May 2007 and April 2012 was carried out. During that period, 192 dermatofibromas were diagnosed in 181 patients, the lesions being more common in women. Median age of the study population was 48 years. The most common lesion site was the limbs (74% of patients). The histopathological types found were common fibrous histiocytoma (80%) and the aneurysmal (5.7%),hemosiderotic (5.7%), epithelioid (2.6%), cellular (2.1%), lipidized (2.1%), atrophic (1.0) and clear cell (0.5%) variants. Based on these findings, this review focuses on the clinical and histological features of the various variants of dermatofibroma in terms of their clinical presentation, distinct histopathological features, differential diagnosis and prognosis.
Alves, Joao Vitor Pina; Matos, Diogo Miguel; Barreiros, Hugo Frederico; Bartolo, Elvira Augusta Felgueira Leonardo Fernandes
Green fluorescent protein (GFP) has become an important tool in cell biology and is widely used as a reporter for imaging intracellular proteins and structures in live cells. Recently, spectral variants of GFP with red- and blue-shifted fluorescence emissions have been characterized, opening the possibility of double labelling with two different-coloured GFP fusion proteins. This article reviews recent advances in
Jan Ellenberg; Jennifer Lippincott-Schwartz; John F. Presley
Based on tape recorded conversations of 28 informants in 18 Louisiana communities, this study investigated regional phonological variants in Louisiana speech. On the basis of settlement history and previous dialect studies, four regions are defined: northern Louisiana, the Florida Parishes, French Louisiana, and New Orleans. The informants are all…
Rubrecht, August Weston
The present invention concerns polypeptides comprising a variant Fc region. More particularly, the present invention concerns Fc region-containing polypeptides that have altered effector function as a consequence of one or more amino acid modifications in the Fc region thereof.
UK NHS diagnostic service sequence analysis of genes generally examines and reports on variations within a designated region 5? and 3? of each exon, typically 30?bp up and downstream. However, because of the degenerate nature of the splice sites, intronic variants outside the AG and GT dinucleotides of the acceptor and donor splice sites (ASS and DSS) are most often classified as being of unknown clinical significance, unless there is some functional evidence of their pathogenicity. It is now becoming clear that mutations deep within introns can also interfere with normal processing of pre-mRNA and result in pathogenic effects on the mature transcript. In diagnostic laboratories, these deep intronic variants most often fall outside of the regions analysed and so are rarely reported. With the likelihood that next generation sequencing will identify more of these unclassified variants, it will become important to perform additional studies to determine the pathogenicity of such sequence anomalies. Here, we analyse variants detected in either COL2A1 or COL11A1 in patients with Stickler syndrome. These have been analysed both in silico and functionally using either RNA isolated from the patient's cells or, more commonly, minigenes as splicing reporters. We show that deep intronic mutations are not a rare occurrence, including one variant that results in multiple transcripts, where both de novo donor and ASS are created by the mutation. Another variant produces transcripts that result in either haploinsufficiency or a dominant negative effect, potentially modifying the disease phenotype.
Richards, Allan J; McNinch, Annie; Whittaker, Joanne; Treacy, Becky; Oakhill, Kim; Poulson, Arabella; Snead, Martin P
Variants of the mouse embryo fibroblast X melanoma hybrid clone 100A have been isolated by a procedure that selects against cells that are able to grow in medium containing low concentrations of serum plus insulin. Three variant clones derived from this selection were found to have a much higher serum requirement than the parental clone 100A cells, as evidenced by a very low rate of DNA synthesis and growth in medium containing low concentrations of serum. Two of the variants had approximately double the number of chromosomes as the parental cell line, while one had approximately the same number of chromosomes as the parental cells. One of the variants was very strongly reverted by 5- azacytidine but not by ethyl methanesulfonate, suggesting that it reverted by a nonmutational mechanism such as a stable change in DNA methylation. Analysis of the growth requirements in hormone- supplemented serum-free media of the 100A parent, the INS 471 variant, and revertants of the variant indicated that the variant had a specific deficiency in its growth response to platelet-derived growth factor (PDGF). PDGF dose-response curves obtained with the variant cells were shifted approximately an order of magnitude toward higher PDGF concentrations relative to PDGF dose-response curves obtained with the parental 100A cells. This quantitative increase in PDGF requirement of the INS 471 variant appears to explain the increased serum requirement of this variant. Equilibrium binding experiments performed with 125I- PDGF suggest that the variant does not have a decreased number of PDGF receptors.
The study of human serum albumin variants is reviewed with reference to albumin Kashmir, a typical variant. Its published instances are listed and its position in this field of investigations is indicated. PMID:1289226
Tárnoky, A L; Vickers, M F; Savva, D
Abnormal differentiation of the renal stem/progenitor pool into kidney tissue can lead to renal hypodysplasia (RHD), but the underlying causes of RHD are not well understood. In this multicenter study, we identified 20 Israeli pedigrees with isolated familial, nonsyndromic RHD and screened for mutations in candidate genes involved in kidney development, including PAX2, HNF1B, EYA1, SIX1, SIX2, SALL1, GDNF, WNT4, and WT1. In addition to previously reported RHD-causing genes, we found that two affected brothers were heterozygous for a missense variant in the WNT4 gene. Functional analysis of this variant revealed both antagonistic and agonistic canonical WNT stimuli, dependent on cell type. In HEK293 cells, WNT4 inhibited WNT3A induced canonical activation, and the WNT4 variant significantly enhanced this inhibition of the canonical WNT pathway. In contrast, in primary cultures of human fetal kidney cells, which maintain WNT activation and more closely represent WNT signaling in renal progenitors during nephrogenesis, this mutation caused significant loss of function, resulting in diminished canonical WNT/?-catenin signaling. In conclusion, heterozygous WNT4 variants are likely to play a causative role in renal hypodysplasia.
Vivante, Asaf; Mark-Danieli, Michal; Davidovits, Miriam; Harari-Steinberg, Orit; Omer, Dorit; Gnatek, Yehudit; Cleper, Roxana; Landau, Daniel; Kovalski, Yael; Weissman, Irit; Eisenstein, Israel; Soudack, Michalle; Wolf, Haike Reznik; Issler, Naomi; Lotan, Danny; Anikster, Yair
Abnormal differentiation of the renal stem/progenitor pool into kidney tissue can lead to renal hypodysplasia (RHD), but the underlying causes of RHD are not well understood. In this multicenter study, we identified 20 Israeli pedigrees with isolated familial, nonsyndromic RHD and screened for mutations in candidate genes involved in kidney development, including PAX2, HNF1B, EYA1, SIX1, SIX2, SALL1, GDNF, WNT4, and WT1. In addition to previously reported RHD-causing genes, we found that two affected brothers were heterozygous for a missense variant in the WNT4 gene. Functional analysis of this variant revealed both antagonistic and agonistic canonical WNT stimuli, dependent on cell type. In HEK293 cells, WNT4 inhibited WNT3A induced canonical activation, and the WNT4 variant significantly enhanced this inhibition of the canonical WNT pathway. In contrast, in primary cultures of human fetal kidney cells, which maintain WNT activation and more closely represent WNT signaling in renal progenitors during nephrogenesis, this mutation caused significant loss of function, resulting in diminished canonical WNT/?-catenin signaling. In conclusion, heterozygous WNT4 variants are likely to play a causative role in renal hypodysplasia. PMID:23520208
Vivante, Asaf; Mark-Danieli, Michal; Davidovits, Miriam; Harari-Steinberg, Orit; Omer, Dorit; Gnatek, Yehudit; Cleper, Roxana; Landau, Daniel; Kovalski, Yael; Weissman, Irit; Eisenstein, Israel; Soudack, Michalle; Wolf, Haike Reznik; Issler, Naomi; Lotan, Danny; Anikster, Yair; Dekel, Benjamin
Summary The capacity of isotype variants of BR55-2, an anti-tumor monoclonal antibody directed against Y oligosaccharide, to mediate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in the human system was evaluated using freshly isolated peripheral blood mononuclear cells, lymphocytes, monocytes and complement. The ADCC activities of the BR55-2 IgG3 isotype and its switch variants (IgG1, IgG2b, and IgG2a) with
Dieter Scholz; Michael Lubeck; Hans Loibner; Joan McDonaldlSmith; Yasuhiko Kimoto; Hilary Koprowski; Zeoon Steplewski
The culture of toxigenic Clostridium difficile from stool specimens is still seen as the gold standard for the laboratory diagnosis of C. difficile infection (CDI). bioMérieux have released ChromID Cdiff chromogenic agar (CDIF) for the isolation and identification of C. difficile in 24 h. In this study, we compared CDIF to pre-reduced cycloserine-cefoxitin-fructose agar with sodium taurocholate (TCCFA) in the examination of glutamate dehydrogenase-positive faecal specimens that were either GeneOhm positive or negative, using direct culture or culture following alcohol shock. Direct culture on CDIF had a sensitivity of 100 % and recovery of 94 % while for TCCFA these were 87 % and 82 %, respectively. For GeneOhm-positive alcohol-shocked faecal samples, sensitivity and recovery on CDIF was similar to direct culture while on TCCFA they were about 10 % higher. For direct culture, there was a significant difference between growth on CDIF at 24 h and TCCFA at 48 h (P = 0.001) and between the two media at 48 h (P<0.001). A total of 142 strains of C. difficile were recovered in pure culture from all GeneOhm-positive samples used in this study and 11 (7.7 %) of these were A(-)B(-)CDT(-) and may represent mixed infections of toxigenic and non-toxigenic C. difficile. The most dominant ribotype was UK 014 (14.7 %) followed by 002 (11.9 %) and 020 (11.9 %), and 36 % of toxigenic isolates, including an A(-)B(+)CDT(-) strain, could not be assigned a UK ribotype. CDIF outperformed pre-reduced TCCFA by negating the need for alcohol shock treatment and by giving a time saving of 24 h in the isolation of C. difficile. CDIF plates were also more selective than TCCFA and C. difficile colonies were easy to identify and subculture prior to strain typing. PMID:23579394
Carson, Kerry C; Boseiwaqa, Lusiana V; Thean, Sara K; Foster, Niki F; Riley, Thomas V
HPV16 accounts for 50–70% of cervical cancer cases worldwide. Characterization of HPV16 variants previously indicated that they differ in risks for viral persistence, progression to cervical precancer and malignant cancer. The aim of this study was to examine the association of severity of disease with HPV16 variants identified in specimens (n?=?281) obtained from a Cervical Pathology and Colposcopy outpatient clinic in the University Hospital of Espírito Santo State, Southeastern Brazil, from April 2010 to November 2011. All cytologic and histologic diagnoses were determined prior to definitive treatment. The DNA was isolated using QIAamp DNA Mini Kit and HPV was detected by amplification with PGMY09/11 primers and positive samples were genotyped by RFLP analyses and reverse line blot. The genomes of the HPV16 positive samples were sequenced, from which variant lineages were determined. Chi2 statistics was performed to test the association of HPV16 variants between case and control groups. The prevalence of HR-HPV types in
Freitas, Luciana Bueno; Chen, Zigui; Muqui, Elaine Freire; Boldrini, Neide Aparecida Tosato; Miranda, Angelica Espinosa; Spano, Liliana Cruz; Burk, Robert D.
Tells benefits of approaching the study of literature through a study of folk tale variants; suggests a plan for helping children analyze and compare folk tale variants. Includes a model of a discussion based on three versions of "Jack and the Beanstalk," and lists and annotates variant editions of nine familiar folk tales. (ET)
Western, Linda E.
Solid variant of aneurysmal bone cyst is a variant of aneurysmal bone cyst in which the predominant histology is that of the solid material of a cystic aneurysmal bone cyst. In this article, we present a patient with solid variant of aneurysmal bone cyst of the hamate and discuss the differential diagnosis and current treatment for this lesion. PMID:21113696
Mavrogenis, Andreas F; Skarpidi, Evangelia; Papagelopoulos, Panayiotis J
The limitations of genome-wide association (GWA) studies that focus on the phenotypic influence of common genetic variants have motivated human geneticists to consider the contribution of rare variants to phenotypic expression. The increasing availability of high-throughput sequencing technologies has enabled studies of rare variants but these methods will not be sufficient for their success as appropriate analytical methods are also
Vikas Bansal; Ondrej Libiger; Ali Torkamani; Nicholas J. Schork
Perinatal infection with variants of hepatitis B virus occurs despite combined immunoprophylaxis with hepatitis B immunoglobulin and currently licensed plasma-derived and recombinant yeast hepatitis B vaccines. Several variants have been detected during a large study of infants born to carrier mothers in Singapore. The most frequent variant was a virus in which a single amino acid substitution Gly to Arg
Chong-Jin Oon; Gek-Keow Lim; Zhao Ye; Kee-Tai Goh; Kim-Leong Tan; Sui-Lan Yo; Elaine Hopes; Tim J. Harrison; Arie J. Zuckerman
We have studied the diversity of B. henselae circulating in patients, reservoir hosts and vectors in Spain. In total, we have fully characterized 53 clinical samples from 46 patients, as well as 78 B. henselae isolates obtained from 35 cats from La Rioja and Catalonia (northeastern Spain), four positive cat blood samples from which no isolates were obtained, and three positive fleas by Multiple Locus Sequence Typing and Multiple Locus Variable Number Tandem Repeats Analysis. This study represents the largest series of human cases characterized with these methods, with 10 different sequence types and 41 MLVA profiles. Two of the sequence types and 35 of the profiles were not described previously. Most of the B. henselae variants belonged to ST5. Also, we have identified a common profile (72) which is well distributed in Spain and was found to persist over time. Indeed, this profile seems to be the origin from which most of the variants identified in this study have been generated. In addition, ST5, ST6 and ST9 were found associated with felines, whereas ST1, ST5 and ST8 were the most frequent sequence types found infecting humans. Interestingly, some of the feline associated variants never found on patients were located in a separate clade, which could represent a group of strains less pathogenic for humans. PMID:23874563
Gil, Horacio; Escudero, Raquel; Pons, Inmaculada; Rodríguez-Vargas, Manuela; García-Esteban, Coral; Rodríguez-Moreno, Isabel; García-Amil, Cristina; Lobo, Bruno; Valcárcel, Félix; Pérez, Azucena; Jiménez, Santos; Jado, Isabel; Juste, Ramón; Segura, Ferrán; Anda, Pedro
The 18S rRNA gene is fundamental to cellular and organismal protein synthesis and because of its stable persistence through generations it is also used in phylogenetic analysis among taxa. Sequence variation in this gene within a single species is rare, but it has been observed in few metazoan organisms. More frequently it has mostly been reported in the non-transcribed spacer region. Here, we have identified two sequence variants within the near full coding region of 18S rRNA gene from a single reniform nematode (RN) Rotylenchulus reniformis labeled as reniform nematode variant 1 (RN_VAR1) and variant 2 (RN_VAR2). All sequences from three of the four isolates had both RN variants in their sequences; however, isolate 13B had only RN variant 2 sequence. Specific variable base sites (96 or 5.5%) were found within the 18S rRNA gene that can clearly distinguish the two 18S rDNA variants of RN, in 11 (25.0%) and 33 (75.0%) of the 44 RN clones, for RN_VAR1 and RN_VAR2, respectively. Neighbor-joining trees show that the RN_VAR1 is very similar to the previously existing R. reniformis sequence in GenBank, while the RN_VAR2 sequence is more divergent. This is the first report of the identification of two major variants of the 18S rRNA gene in the same single RN, and documents the specific base variation between the two variants, and hypothesizes on simultaneous co-existence of these two variants for this gene.
Nyaku, Seloame T.; Sripathi, Venkateswara R.; Kantety, Ramesh V.; Gu, Yong Q.; Lawrence, Kathy; Sharma, Govind C.
Over 90% of the human listeriosis cases are caused by Listeria monocytogenes serotypes 1/2a, 1/2b and 4b strains. As an alternative to antigen-antibody based serotyping, a PCR-based method for serogrouping has been developed and validated. In this communication, we report an in-depth analysis of five 4b variant strains, four clinical isolates from Australia and one environmental isolate from USA. Although these five strains were serotype 4b by classical serotyping method, the serogrouping PCR profiles of these strains show the presence of a 1/2a-3a specific amplicon in addition to the standard 4b-4d-4e specific amplicons. These strains were further analyzed by pulsed field gel electrophoresis, binary gene typing, multi-locus variable-number-tandem-repeat analysis and a high density pan-genomic Listeria microarray. Using these sub-typing results, the clinical isolates were grouped into two distinct genomic groups- one of which could be part of an unidentified outbreak. The microarray results when compared with our database of other 4b outbreak isolates indicated that the serotype 4b variant strains represent very different genotypic profiles than the known reported 4b outbreak strains representing major epidemic clones. The acquisition of serotype 1/2a gene clusters by the 4b variant strains appears to be independent in origin, spanning large areas of geographical and temporal space and may indicate predisposition of some 4b strains towards accepting DNA from related organisms. PMID:24586485
Laksanalamai, Pongpan; Huang, Bixing; Sabo, Jonathan; Burall, Laurel S; Zhao, Shaohua; Bates, John; Datta, Atin R
Over 90% of the human listeriosis cases are caused by Listeria monocytogenes serotypes 1/2a, 1/2b and 4b strains. As an alternative to antigen-antibody based serotyping, a PCR-based method for serogrouping has been developed and validated. In this communication, we report an in-depth analysis of five 4b variant strains, four clinical isolates from Australia and one environmental isolate from USA. Although these five strains were serotype 4b by classical serotyping method, the serogrouping PCR profiles of these strains show the presence of a 1/2a-3a specific amplicon in addition to the standard 4b-4d-4e specific amplicons. These strains were further analyzed by pulsed field gel electrophoresis, binary gene typing, multi-locus variable-number-tandem-repeat analysis and a high density pan-genomic Listeria microarray. Using these sub-typing results, the clinical isolates were grouped into two distinct genomic groups- one of which could be part of an unidentified outbreak. The microarray results when compared with our database of other 4b outbreak isolates indicated that the serotype 4b variant strains represent very different genotypic profiles than the known reported 4b outbreak strains representing major epidemic clones. The acquisition of serotype 1/2a gene clusters by the 4b variant strains appears to be independent in origin, spanning large areas of geographical and temporal space and may indicate predisposition of some 4b strains towards accepting DNA from related organisms.
Laksanalamai, Pongpan; Huang, Bixing; Sabo, Jonathan; Burall, Laurel S.; Zhao, Shaohua; Bates, John; Datta, Atin R.
Lobular carcinoma is a special type of breast cancer that shows distinct clinical presentation, morphologic and molecular features, and clinical behavior, and its incidence is rising in recent years. Infiltrating lobular carcinoma (ILC) and its precursor lesions may result in diagnostic difficulties, particularly in the screening settings and their management may be problematic. Variants of lobular carcinoma, such as the pleomorphic variant, although not common, exist and some show differences in behavior warranting their recognition in view of requirements for different management strategies. Here we present a review of lobular carcinomas with particular attention to lobular in situ lesions, epidemiology, subtypes, diagnosis, molecular pathology, and grading of ILC in addition to the clinical behavior, response to therapy, and outcome of patients with ILC. PMID:20306830
Rakha, Emad A; Ellis, Ian O
This review aims to provide a snapshot of the actual state of knowledge on genetic variants of nuclear receptors (NR) involved in regulating important aspects of liver metabolism. It recapitulates recent evidence for the application of NR in genetic diagnosis of monogenic (“Mendelian”) liver disease and their use in clinical diagnosis. Genetic analysis of multifactorial liver diseases such as viral hepatitis or fatty liver disease identifies key players in disease predisposition and progression. Evidence from these analyses points towards a role of NR polymorphisms in common diseases, linking regulatory networks to complex and variable phenotypes. The new insights into NR variants also offer perspectives and cautionary advice for their use as handles towards diagnosis and treatment.
Mullenbach, Roman; Weber, Susanne N.; Lammert, Frank
Clinical and neuropathological studies of a case of AB variant GM2-gangliosidosis have been presented. The patient was a 14 months old black female infant who had “black cherry spot” in the retinas. The total activities of ß-galactosidase and N-acetyl-ß-hexosaminidase, as well as the proportion of hexosaminidase A and B components in her serum and leukocytes were normal when the assays
Cecile M. Baecque; Kinuko Suzuki; Isabelle Rapin; Anne B. Johnson; Doris L. Whethers; Kunihiko Suzuki
Abstract 1. The possibility that infectious bronchitis virus (IBV) variants isolated from broilers with enteric and respiratory problems have a different tropism and pathological outcome from those IBV strains causing classical respiratory disease was investigated. 2. IBV variants were isolated from broiler flocks with enteric and respiratory problems in two regions of Brazil. The USP-10 isolate, of enteric origin, was inoculated via the oral oroculonasal routes into IBV-antibody-free broilers and specific pathogen-free (SPF) chickens to determine tissue tropism and pathogenicity and compared with an IBV variant (USP-50) isolated from chickens showing signs of respiratory disease only. 3. Both USP-10 and USP-50 strains caused similar pathological patterns by either route of inoculation. Both variants were detected in respiratory and non-respiratory tissues, including the kidney, intestine and testis. 4. Broilers were more susceptible to infection than SPF chickens, and seroconversion was detected in all of the chicks. PMID:24678626
Chacón, J L; Assayag, M S; Revolledo, L; Astolfi-Ferreira, C S; Vejarano, M P; Jones, R C; Piantino Ferreira, A J
Thiopeptides are a family of highly modified peptide metabolites, characterized by a macrocycle bearing a central piperidine/dehydropiperidine/pyridine ring, multiple thiazole rings, and several dehydrated amino acid residues. Thiopeptides have useful antibacterial, antimalarial, and anticancer properties but have not been adapted for human clinical applications, owing in part to their poor water solubility. In 2009, it was revealed that the thiopeptide scaffold is derived from a ribosomally synthesized precursor peptide subjected to extensive posttranslational modifications. Shortly thereafter, three groups developed two types of in vivo strategies to generate thiopeptide variants: precursor peptide mutagenesis and gene inactivation. The thiopeptide analogs and biosynthetic intermediates obtained from these studies provide much-needed insight into the biosynthetic process for these complicated metabolites. Furthermore, the in vivo production of variants can be employed to interrogate thiopeptide structure-activity relationships and may be useful to address the bioavailability issues plaguing these otherwise promising lead molecules. This chapter discusses the in vivo systems developed to generate thiopeptide variants. PMID:23034221
Zhang, Feifei; Kelly, Wendy L
The goal of the research described in this dissertation is to be able to model propagation of light through shift -variant optics. Shift-variant optical elements have a point spread function which is a function of the transverse coordinates. This shift-variance can be caused by aberration or by the first order properties of the optical system. In this work the latter is emphasized. Specifically, this dissertation discusses propagation through lenses and prisms and between tilted planes or a plane and a spherical surface. Extension to other types of shift-variant optical elements is possible. Two methods for performing the propagation are described. One, the beam division model, divides the beam into isoplanatic patches, separately propagates the patches and recombines them on the observation surface. The second method, the mapping model, maps the beam into a space in which the propagation is shift-invariant, propagates and then maps back into real space. Experimental verification of these methods is demonstrated by means of the Talbot effect. The setup consists of a collimated laser beam passing through a Ronchi ruling of about ten cycles per millimeter. With no intervening optics, Talbot images of the ruling are formed which are parallel to the wavefronts. When a prism at minimum deviation is placed in the outgoing beam, it causes the Talbot images to be tilted with respect to the wavefronts. If a stigmatic unit magnification telescope replaces the prism, the Talbot images are formed on surfaces congruent to the Petzval surface.
Eckhardt, Stephen Karl
The new isolated circovirus variant PCV-2 is discussed to be the etiological agent of a new emerging swine disease with a variable morbidity and high lethality, postweaning multisystemic wasting syndrome (PMWS). PMWS has been diagnosed in North America and West Europe. Clinical signs include dyspnea, loss of weight, lymph node enlargement and lymphocyte depletion in lymphoid tissues. This report describes
Annette Mankertz; Mariano Domingo; Josep M Folch; Pierre LeCann; André Jestin; Joaquim Segalés; Barbara Chmielewicz; Juan Plana-Durán; Dirk Soike
Human serum contains, in addition to the "classical" 7.5-kDa insulin-like growth factors (IGFs) I and II, small amounts of larger IGF-II. A 10-kDa IGF-II was isolated by gel filtration, immunoaffinity chromatography, and reversed-phase HPLC. Upon amino acid sequence determination, a substitution of Cys-Gly-Asp for Ser-33 was found as well as a COOH-terminal extension of 21 residues (E peptide). These sequence differences suggest that 10-kDa IGF-II is a precursor of a variant IGF-II. Since the substitution is not located at a known intron/exon hinge region, the finding of this variant IGF-II is evidence for the presence of more than one gene for IGF-II.
Zumstein, P P; Luthi, C; Humbel, R E
Two isolates of Neodiprion sertifer (Geoffr.) nuclear polyhedrosis virus from Britain and North America were compared using three biochemical techniques. Alkaline protease assays of polyhedra revealed the presence of endogenous enzyme activity in the British isolate but not in the North American isolate. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of virus particle structural polypeptides revealed only minor differences, with the exception that the North American virus was contaminated with polyhedral protein. The restriction endonucleases SalI, HindIII, and HpaII were used as a definitive method of distinguishing the two variants, with all endonucleases achieving this to a greater or lesser extent. The possible significance of all of these observations is discussed in terms of their possible influence on the registration and field application of this virus. PMID:16345930
Brown, D A
In this chapter we aim to provide an overview of DNA variant databases, commonly known as Locus-Specific Databases (LSDBs), or Gene-Disease Specific Databases (GDSDBs), but the term variant database will be used for simplicity. We restrict this overview to germ-line variants, particularly as related to Mendelian diseases, which are diseases caused by a variant in a single gene. Common difficulties associated with variant databases and some proposed solutions are reviewed. Finally, systems where technical solutions have been implemented are discussed. This work will be useful for anyone wishing to establish their own variant database, or to learn about the global picture of variant databases, and the technical challenges to be overcome. PMID:24870141
Plazzer, John-Paul; Macrae, Finlay
The most lethal organophosphorus nerve agents (NA), like sarin, soman, agent-VX and Russian-VX, share a methylphosphonate moiety. Pseudomonas diminuta phosphotriesterase (PTE) catalyses the hydrolysis of methylphosphonate NA analogues with a catalytic efficiency orders of magnitude lower than that towards the pesticide paraoxon. With a view to obtaining PTE variants that more readily accept methylphosphonate NA, ~75,000 PTE variants of the substrate-binding residues Gly-60, Ile-106, Leu-303 and Ser-308 were screened with fluorogenic analogues of the NA Russian-VX and cyclosarin. Seven new PTE variants were isolated, purified and their k(cat)/K(M) determined against five phosphotriesters and five methylphosphonate analogues of sarin, cyclosarin, soman, agent-VX and Russian-VX. The novel PTE variants exhibited as much as a 10-fold increase in activity towards the methylphosphonate compounds--many reaching a k(cat)/K(M) of 10? M?¹ s?¹--and as much as a 29,000-fold decrease in their phosphotriesterase activity. The mutations found in two of the variants, SS0.5 (G60V/I106L/S308G) and SS4.5 (G60V/I106A/S308G), were modelled into a high-resolution structure of PTE-wild type and docked with analogues of cyclosarin and Russian-VX using Autodock 4.2. The kinetic data and docking simulations suggest that the increase in activity towards the methylphosphonates and the loss of function against the phosphotriesters were due to an alteration of the shape and hydrophobicity of the binding pocket that hinders the productive binding of non-chiral racemic phosphotriesters, yet allows the binding of the highly asymmetric methylphosphonates. PMID:21037279
Briseño-Roa, Luis; Timperley, Christopher M; Griffiths, Andrew D; Fersht, Alan R
Neuroblastoma (NB) is the most commonly occurring solid tumor in children. The disease usually arises in the adrenal medulla, and it is characterized by a remarkable heterogeneity in its progression. Most NB patients with an advanced disease have massive bone marrow infiltration at diagnosis. Lung metastasis represents a widely disseminated stage and is typically considered to be a terminal event. Much like other malignancies, NB progression is a complex, multistep process. The expression, function, and significance of the various factors involved in NB progression must be studied in relevant in vivo and in vitro models. Currently, models consisting of metastatic and nonmetastatic cell variants of the same genetic background exist for several types of cancer; however, none exists for NB. In the present study, we describe the generation of a NB metastasis model. SH-SY5Y and MHH-NB-11 NB cells were inoculated orthotopically into the adrenal glands of athymic nude mice. Neuroblastoma cells metastasizing to the lungs were isolated from mice bearing adrenal tumors. Lung metastatic variants were generated by repeated cycles of in vivo passage. Characterization of these variants included cellular morphology and immunophenotyping in vitro, aggressiveness in vivo, and various biologic parameters in vitro. The NB metastatic variant in each model displayed unique properties, and both metastatic variants demonstrated a metastatic phenotype in vivo. These reproducible models of human NB metastasis will serve as an unlimited source of transcriptomic and proteomic material. Such models can facilitate future studies on NB metastasis and the identification of novel NB biomarkers and targets for therapy.
Nevo, Ido; Sagi-Assif, Orit; Botzer, Liat Edry; Amar, Dana; Maman, Shelly; Kariv, Naam; Leider-Trejo, Leonor E; Savelyeva, Larissa; Schwab, Manfred; Yron, Ilana; Witz, Isaac P
Three liver-specific growth media, respectively free of arginine (Arg-), tyrosine (Tyr-) and glucose (G-), have been used to characterize cells of the rat H4IIEC3, human HepG2 and mouse BW hepatoma lines. Cells of clone FaO, a derivative of line H4IIEC3, freely grew in Tyr- and G- media, and gave rise to stable variants in Arg- conditions. Cells of line HepG2 and clone BWTG3, a derivative of line BW, degenerated in all three media. Arg and tyr variants were however derived from HepG2 cells; their genesis appeared to be pathway specific, illustrating the complexity of the regulatory loops that are implicated in the control of the differentiated state. No variant was ever obtained with BWTG3 cells, demonstrating the stability of their deficiency in the post-natal hepatic functions that are involved in Arg-, Tyr- and G- selections. Variant clones of HepG2 and FaO cells that have been isolated in Arg- medium were characterized in details for liver-specific urea-cycle enzyme activities and mRNA. These variants were shown to be controlled at the mRNA level, most likely at transcription. Isolation of stable FaO and HepG2 variant clones as well as the converse demonstration of the stable deficiency of BWTG3 cells in post-natal hepatic functions were aimed at expression cloning. Our results are thus discussed in terms of transfection with full-length cDNA expression libraries and cloning of regulatory genes that could activate or extinguish liver specific genes. PMID:1322334
Armbruster, L; Cavard, C; Briand, P; Bertolotti, R
Naturally occurring NAD-independent variants of Haemophilus paragallinarum, which have been isolates from poultry showing clinical signs of infectious coryza, were used to determine their virulence using a newly developed challenge model for infectious coryza. It was established that the NAD-independent isolates belonging to a particular serogroup, were less virulent when compared to the virulence of the NAD-dependent isolates from the same serogroup. It was shown that the virulence of the NAD-independent isolates belonging to serogroup C and serogroup A were very similar to each other. This differs to the results obtained with NAD-dependent isolates reported on previously, in which the serogroup C isolates were found to be more virulent then the serogroup A isolates. PMID:12234004
Bragg, R R
Attention deficit\\/hyperactivity disorder (ADHD) is a common, highly heritable, neuropsychiatric disorder among children. Linkage studies in isolated populations have proved powerful to detect variants for complex diseases, such as ADHD. We performed a genome-wide linkage scan for ADHD in nine patients from a genetically isolated population in the Netherlands, who were linked to each other within 10 generations through multiple
Najaf Amin; Yuri S Aulchenko; Marieke C Dekker; Robert F Ferdinand; Alwin van Spreeken; Alfons H Temmink; Frank C Verhulst; Ben A Oostra; Cornelia M van Duijn; CM van Duijn
The preserved speech variant is the milder form of Rett syndrome: affected girls show the same stages of this condition and by the second half of the first decade are making slow progress in manual and verbal abilities. They walk without help, and may be able to make simple drawings and write a few words. Most of them can speak in sentences. Autistic behavior can often be observed. We previously described several cases in the pre-molecular era and subsequently reported a survey of 12 cases with MECP2 mutations. Seventeen new patients with the preserved speech variant and a proven MECP2 mutation have been clinically evaluated. Additional clinical data of our previously described cases are reported. These 29 preserved speech variant cases were compared with 129 classic Rett patients using a clinical severity score system including 22 different signs. There was both statistical and clinical evidence of the existence of this variant. On the basis of their abilities these girls can be distinguished as low-, intermediate- and high-functioning. Girls of the last two groups show a greater homogeneity: they speak in sentences, use their hands more easily, have normal somatic features, mild neurovegetative abnormalities, with autistic behavior in 76%, epilepsy in 30%, while girls of the first group are closer to classic Rett syndrome. The majority of patients carries either missense mutations (especially the p.R133C change) or late truncating mutations in the MECP2 gene. These results confirm the existence of this variant of Rett syndrome (Zappella variant), a clear example of progress of manual and verbal abilities, and not of a "preserved speech" and suggest corresponding diagnostic criteria. PMID:18562141
Renieri, A; Mari, F; Mencarelli, M A; Scala, E; Ariani, F; Longo, I; Meloni, I; Cevenini, G; Pini, G; Hayek, G; Zappella, M
Coxsackievirus A24 variant is, together with enterovirus 70 and adenoviruses, the major etiological agent involved in acute hemorrhagic conjunctivitis outbreaks worldwide. However, the standard virus isolation method followed by serotyping or VP1 region sequencing is time-consuming. A rapid method for the detection of coxsackievirus A24 variant from conjunctival swab specimens would be useful in the context of explosive and extensive outbreaks. A one-step real-time RT-PCR assay based on TaqMan technology was thus developed and assessed on 36 conjunctival swabs from outbreaks of conjunctivitis in Morocco in 2004 due to a coxsackievirus A24 variant and in Corsica in 2006 due to adenovirus type 3, and 83 virus strains including 41 coxsackievirus A24 variant collected in French Guiana and Guadeloupe in 2003, in the Democratic Republic of the Congo in 2003, in Morocco in 2004 and 42 other virus species genetically close or known to be responsible for conjunctivitis. All the conjunctival swabs from coxsackievirus A24 variant related outbreak and the 41 coxsackievirus A24 variant strains were tested positive by the RT-PCR assay within 4h. This novel single-tube real-time RT-PCR assay is sensitive and specific, and consists in a reliable and faster alternative to the viral culture for recent and future acute hemorrhagic conjunctivitis outbreaks caused by coxsackievirus A24 variant. PMID:17328967
Lévêque, Nicolas; Lahlou Amine, Idriss; Tcheng, Remy; Falcon, Delphine; Rivat, Nathalie; Dussart, Philippe; Muyembe, Jean-Jacques; Chomel, Jean-Jacques; Norder, Helene; Eugene, Maxime; Lina, Bruno
Whole-exome or gene targeted resequencing in hundreds to thousands of individuals has shown that the majority of genetic variants are at low frequency in human populations. Rare variants are enriched for functional mutations and are expected to explain an important fraction of the genetic etiology of human disease, therefore having a potential medical interest. In this work, we analyze the whole-exome sequences of French-Canadian individuals, a founder population with a unique demographic history that includes an original population bottleneck less than 20 generations ago, followed by a demographic explosion, and the whole exomes of French individuals sampled from France. We show that in less than 20 generations of genetic isolation from the French population, the genetic pool of French-Canadians shows reduced levels of diversity, higher homozygosity, and an excess of rare variants with low variant sharing with Europeans. Furthermore, the French-Canadian population contains a larger proportion of putatively damaging functional variants, which could partially explain the increased incidence of genetic disease in the province. Our results highlight the impact of population demography on genetic fitness and the contribution of rare variants to the human genetic variation landscape, emphasizing the need for deep cataloguing of genetic variants by resequencing worldwide human populations in order to truly assess disease risk.
Hussin, Julie; Idaghdour, Youssef; Bruat, Vanessa; de Maillard, Thibault; Grenier, Jean-Cristophe; Gbeha, Elias; Hamdan, Fadi F.; Girard, Simon; Spinella, Jean-Francois; Lariviere, Mathieu; Saillour, Virginie; Healy, Jasmine; Fernandez, Isabel; Sinnett, Daniel; Michaud, Jacques L.; Rouleau, Guy A.; Haddad, Elie; Le Deist, Francoise; Awadalla, Philip
Whole-exome or gene targeted resequencing in hundreds to thousands of individuals has shown that the majority of genetic variants are at low frequency in human populations. Rare variants are enriched for functional mutations and are expected to explain an important fraction of the genetic etiology of human disease, therefore having a potential medical interest. In this work, we analyze the whole-exome sequences of French-Canadian individuals, a founder population with a unique demographic history that includes an original population bottleneck less than 20 generations ago, followed by a demographic explosion, and the whole exomes of French individuals sampled from France. We show that in less than 20 generations of genetic isolation from the French population, the genetic pool of French-Canadians shows reduced levels of diversity, higher homozygosity, and an excess of rare variants with low variant sharing with Europeans. Furthermore, the French-Canadian population contains a larger proportion of putatively damaging functional variants, which could partially explain the increased incidence of genetic disease in the province. Our results highlight the impact of population demography on genetic fitness and the contribution of rare variants to the human genetic variation landscape, emphasizing the need for deep cataloguing of genetic variants by resequencing worldwide human populations in order to truly assess disease risk. PMID:24086152
Casals, Ferran; Hodgkinson, Alan; Hussin, Julie; Idaghdour, Youssef; Bruat, Vanessa; de Maillard, Thibault; Grenier, Jean-Christophe; Grenier, Jean-Cristophe; Gbeha, Elias; Hamdan, Fadi F; Girard, Simon; Spinella, Jean-François; Larivière, Mathieu; Saillour, Virginie; Healy, Jasmine; Fernández, Isabel; Sinnett, Daniel; Michaud, Jacques L; Rouleau, Guy A; Haddad, Elie; Le Deist, Françoise; Awadalla, Philip
One-step real-time PCR using one set of primers and four probes was developed for differentiation of F18 variants (F18 common, F18ab, F18ac, F18new variant) of enterotoxigenic (ETEC) and Shiga toxin-producing (STEC) Escherichia coli from piglets with diarrhoea and oedema disease. The limits of detection for F18common, F18ab, F18ac, and F18new variant were 10(7), 10(7), 10(5) and 10(7)colony forming units/g faeces, respectively. Of 94 Korean isolates of E. coli encoding F18, 70 were F18ac (43 STEC/ETEC, 4 STEC and 23 ETEC), 15 were F18ab (all STEC) and nine were F18new variant (1 STEC/ETEC, 7 STEC, 1 ETEC). PMID:23992871
Byun, Jae-Won; Jung, Byeong Yeal; Kim, Ha-Young; Fairbrother, John M; Lee, Myoung-Heon; Lee, Wan-Kyu
One of several splice variants of CD44 expressed in metastasizing cell lines of rat tumors has been shown to confer metastatic potential to the non-metastatic variant of a rat pancreatic carcinoma line (U. Günthert et al., Cell, 65: 13-24, 1991). The variant-specific rat CD44 sequences were used to detect RNA expression in human cell lines: in carcinoma lines from lung, breast and colon; and in keratinocyte lines. By polymerase chain reaction amplification, complementary DNAs encoding human homologues were isolated and sequenced. The largest splice variant has been found in a large cell lung carcinoma line and in keratinocyte cell lines. It carries at least 5 additional domains (exons) encoding a total of 338 amino acids in the membrane-proximal extracellular region of the standard CD44. Various alternative splice products have been detected in other human tumor cell lines. The distribution of CD44 splice variants is consistent with the speculation that they fulfill functions in only a few restricted differentiation pathways and that in tumor cells these pathways have been reactivated. PMID:1717145
Hofmann, M; Rudy, W; Zöller, M; Tölg, C; Ponta, H; Herrlich, P; Günthert, U
During September 2010, an outbreak of acute hemorrhagic conjunctivitis reemerged in Jiangsu, three years after the nationwide epidemic in China in 2007. In total, 2409 cases were reported, 2118 of which were reported in September; 79.8% of those affected were students or teachers, with a median age of 16 years. To identify and demonstrate the genetic characteristics of the etiological agent, 52 conjunctival swabs were randomly collected from four different cities. After detection and isolation, 43 patients were positive for coxsackievirus A24 variant according to PCR and 20 according to culture isolation. Neither adenovirus nor EV70 was detected. A phylogenetic study of the complete 3Cpro and VP1 regions showed that the Jiangsu isolates clustered into a new lineage, GIV-C5, with two uniform amino-acid mutations that distinguished them from all previous strains. Another new cluster, GIV-C4, formed by Indian isolates from 2007 and Brazilian isolates from 2009, was also identified in this study. Interestingly, our isolates shared greatest homology with the GIV-C4 strains, not with the isolates that were responsible for the nationwide acute hemorrhagic conjunctivitis epidemic in China in 2007. Although all our isolates were closely related, they could be differentiated into two subclusters within GIV-C5. In conclusion, our study suggests that a new cluster of coxsackievirus A24 variant that had already evolved into diverse strains was associated with the acute hemorrhagic conjunctivitis outbreaks in Jiangsu in September 2010. These viruses might have originated from the virus isolated in India in 2007, rather than from the epidemic strains isolated in China in 2007.
Wu, Bin; Qi, Xian; Xu, Ke; Ji, Hong; Zhu, Yefei; Tang, Fenyang; Zhou, Minghao
The acquired carbapenem-hydrolyzing oxacillinase (OXA) OXA-143 has thus far been detected only in Acinetobacter baumannii isolates from Brazil. The aim of this study was to characterize three OXA-143 variants: OXA-231 and OXA-253 from carbapenem-resistant A. baumannii isolates and OXA-255 in a carbapenem-susceptible Acinetobacter pittii isolate originating from Brazil, Honduras, and the United States, respectively. The 5' rapid amplification of cDNA ends (RACE) technique identified the same transcription initiation site for all blaOXA-143-like genes and revealed differences in the putative promoter regions. However, all cloned OXA-143 variants conferred carbapenem resistance on A. baumannii ATCC 17978 and OXA-255 conferred carbapenem resistance on A. pittii SH024, which was correlated with blaOXA-255 gene expression. This is the first description of OXA-143-like outside A. baumannii. Detection of OXA-143-like in the United States and Honduras indicates its dissemination through the American continent. PMID:24566181
Zander, Esther; Bonnin, Rémy A; Seifert, Harald; Higgins, Paul G
Borrelia afzelii is one of two most important pathogens of the ixodes tick borrelioses (ITB) in Russia and neighboring countries. This pathogen circulates in various ecosystems and has a wide range of reservoir hosts and transmitters. The results of studies of genetic heterogeneity of the spirochaetae B. afzelii are considered. A total of 139 primary isolates were studied. The isolates were isolated from three species of Ixodes ticks at different stages of development and obtained from the laboratory of infection transmitters, Gamaleya Scientific Research Institute of Epidemiology and Microbiology, Russran Academy of Medical Sciences, Moscow. The transmitters and reservoir hosts of borrelias were caught in natural foci of Russia (from Kaliningrad region irk the west to south Sakhalin in the east), Czechia, Lithuania, Estonia, Ukraine, and Moldavia. Analysis of genotype sequence similarity obtained by sequencing of the rrf(SS)-rrl(23S) spacer demonstrated that the B. afzelii genospecies incorporated no less than 10 genetic variants of spirochaetae, most variants being geographically widespread. PMID:16173394
Fadeeva, I A; Nefedova, V V; Korenberg, E I; Gorelova, N B
Identification of causal rare variants that are associated with complex traits poses a central challenge on genome-wide association studies. However, most current research focuses only on testing the global association whether the rare variants in a given genomic region are collectively associated with the trait. Although some recent work, e.g., the Bayesian risk index method, have tried to address this problem, it is unclear whether the causal rare variants can be consistently identified by them in the small--large- situation. We develop a new Bayesian method, the so-called Bayesian Rare Variant Detector (BRVD), to tackle this problem. The new method simultaneously addresses two issues: (i) (Global association test) Are there any of the variants associated with the disease, and (ii) (Causal variant detection) Which variants, if any, are driving the association. The BRVD ensures the causal rare variants to be consistently identified in the small--large- situation by imposing some appropriate prior distributions on the model and model specific parameters. The numerical results indicate that the BRVD is more powerful for testing the global association than the existing methods, such as the combined multivariate and collapsing test, weighted sum statistic test, RARECOVER, sequence kernel association test, and Bayesian risk index, and also more powerful for identification of causal rare variants than the Bayesian risk index method. The BRVD has also been successfully applied to the Early-Onset Myocardial Infarction (EOMI) Exome Sequence Data. It identified a few causal rare variants that have been verified in the literature.
Liang, Faming; Xiong, Momiao
The nutritionally variant streptococci (NVS) are a fastidious group of bacteria first recognized in the early 1960s in the blood cultures of patients with subacute bacterial endocarditis. Since that time, the NVS have been implicated in 5 to 6% of all cases of human bacterial endocarditis. The NVS possess membrane-associated amphipathic molecules different from those described for other streptococci. Unitl recently, chemical characterization of these new amphipathic polymers was hampered by unsuccessful attempts at isolating large quantities of these molecules in a form free from other bacterial components. Presently, stationary-phase-culture supernatants provide an optimum source of crude material for amphiphile purification procedures. Hydrophobic-interaction chromatography in conjunction with immunoaffinity chromatography yields an NVS serotype I amphiphile preparation free of contaminants, as determined by immunoelectrophoretic and chemical analyses. Tandem crossed immunoelectrophoresis of the purified extracellular NVS amphiphile demonstrated that it is immunologically similar to the intracellular amphiphile. Finally, this amphiphile serves as the NVS serotype I antigen. Images
George, M; van de Rijn, I
Background: The Bosley-Salih-Alorainy syndrome (BSAS) variant of the congenital human HOXA1 syndrome results from autosomal recessive truncating HOXA1 mutations. We describe the currently recognized spectrum of ocular motility, inner ear malformations, cerebrovascular anomalies, and cognitive function. Methods: We examined nine affected individuals from five consanguineous Saudi Arabian families, all of whom harbored the same I75-I76insG homozygous mutation in the HOXA1 gene. Patients underwent complete neurologic, neuro-ophthalmologic, orthoptic, and neuropsychological examinations. Six individuals had CT, and six had MRI of the head. Results: All nine individuals had bilateral Duane retraction syndrome (DRS) type 3, but extent of abduction and adduction varied between eyes and individuals. Eight patients were deaf with the common cavity deformity of the inner ear, while one patient had normal hearing and skull base development. Six had delayed motor milestones, and two had cognitive and behavioral abnormalities meeting Diagnostic and Statistical Manual of Mental Disorders-IV criteria for autism spectrum disorder. MRI of the orbits, extraocular muscles, brainstem, and supratentorial brain appeared normal. All six appropriately studied patients had cerebrovascular malformations ranging from unilateral internal carotid artery hypoplasia to bilateral agenesis. Conclusions: This report extends the Bosley-Salih-Alorainy syndrome phenotype and documents the clinical variability resulting from identical HOXA1 mutations within an isolated ethnic population. Similarities between this syndrome and thalidomide embryopathy suggest that the teratogenic effects of early thalidomide exposure in humans may be due to interaction with the HOX cascade.
Bosley, T.M.; Salih, M.A.; Alorainy, I.A.; Oystreck, D.T.; Nester, M.; Abu-Amero, K.K.; Tischfield, M.A.; Engle, E.C.
Recent discoveries of surface proteins involved in tumor metastasis formation have revived an old hypothesis that tumor cells may acquire, and use for their metastatic spreading, properties which lymphoid cells had developed to defend the organism against foreign antigens. Splice variants of CD44 and integrins are expressed on metastasizing tumor cells and also on leukocytes at defined stages of their differentiation. Expression and function appear to be essential not only for the generation of an immune response but also for the establishment of metastatic tumor colonies. PMID:7691067
Herrlich, P; Zöller, M; Pals, S T; Ponta, H
Some patients with dystonic movements and postures not known to be caused by environmental or degenerative disorders can be segregated from classical-appearing idiopathic torsion dystonia on the basis of distinctive clinical and pharmacologic features. Many of them should be considered within the family of dystonia, as clinical variants of idiopathic torsion dystonia, while others are better classified as being part of other families of dyskinesias. In the former group are paradoxical dystonia, myoclonic dystonia, diurnal dystonia, and dopa-responsive dystonia. The latter group consists of dystonic tics and the various entities comprising paroxysmal dystonia, namely kinesigenic, nonkinesigenic and hypnogenic dystonia. PMID:2666583
Some patients with dystonic movements and postures not known to be caused by environmental or degenerative disorders can be segregated from classical-appearing idiopathic torsion dystonia on the basis of distinctive clinical and pharmacologic features. Many of them should be considered within the family of dystonia, as clinical variants of idiopathic torsion dystonia, while others are better classified as being part of other families of dyskinesias. In the former group are paradoxical dystonia, myoclonic dystonia, diurnal dystonia, and dopa-responsive dystonia. The latter group consists of dystonic tics and the various entities comprising paroxysmal dystonia, namely kinesigenic, nonkinesigenic and hypnogenic dystonia.
Four elderly women had intense fears of falling when there was no visible support at hand or on seeing space cues while driving. Two patients had cervical spondylosis. The mean age at onset of the fear was 54--thirty years later than that for agoraphobia. Fear of public places and of heights was not prominent, nor was depersonalisation or depression. These "space phobias" might be a hitherto unrecognised syndrome or an unusual variant of agoraphobia. The visuospatial reflexes involved might illuminate the pathogenesis of certain fears.
Marks, I; Bebbington, P
The mandibular canal transmits the inferior alveolar artery, vein and the inferior alveolar nerve. From an embryological perspective, there might be three inferior dental nerves innervating three groups of mandibular teeth. During rapid prenatal growth and remodeling in the ramus region there is spread of intramembranous ossification that eventually forms the mandibular canal. Occurrence of bifid/trifid mandibular canals in some patients is secondary to incomplete fusion of these three nerves. Various types of bifid mandibular canals have been classified according to anatomical location and configuration. This case report highlights an unusual variant of the mandibular canal. PMID:18197857
Wadhwani, P; Mathur, R M; Kohli, M; Sahu, R
Summary\\u000a Background: Genetic polymorphism of serum transferrin (Tf) was studied in order to differentiate between protein genetic variants and\\u000a congenital disorders of glycosylation (CDG), further focusing on unusual findings.\\u000a \\u000a \\u000a \\u000a Methods: Screening of Tf hypoglycosylation was carried out by isoelectric focusing with direct immunofixation and Coomassie blue staining\\u000a in 100 healthy controls and a group of 1247 patients with various symptoms and diagnoses.
E. Marklová; Z. Albahri; H. Vaní?ek; P. D?dek; M. Vališ; M. Kopá?ová; V. Vávrová
In Nigeria, quinolones and ?-lactam antibiotics are widely used to treat bacterial infections. This study aimed to identify the prevalence of resistance to these drugs and to determine the mechanisms of resistance to these agents. In total, 134 non-duplicate, Gram-negative enteric isolates of 13 species from different hospitals were investigated for susceptibility to a panel of antibiotics, carriage of plasmid-mediated quinolone and ?-lactam resistance genes, production of extended-spectrum ?-lactamases (ESBLs), and mutations within topoisomerase genes. The level of resistance to all antibiotics tested was extremely high, with minimum inhibitory concentrations for 90% of the organisms (MIC(90) values) of ? 256 ?g/mL for all drugs. Of the 134 isolates, 92 had mutations within the quinolone resistance-determining region (QRDR) of gyrA or within gyrA and parC. In addition, the plasmid-mediated quinolone resistance genes qnrA, qnrB, aac(6')-Ib-cr and qepA were identified. The qnrD allele, which has previously only been found in Salmonella isolates from China, was identified in two Proteus isolates and one Pseudomonas isolate. Of the 134 isolates, 23 (17.2%) carried aac(6')-Ib-cr, 11 (8.2%) carried a qnr variant and 5 (3.7%) were positive for qepA. Twenty-eight isolates (20.9%) produced ESBL variants, with a CTX-M variant being carried by 25 isolates (18.7%). In addition, six isolates (4.5%) carried ampC variants [ACT-1 (1 isolate), DHA-1 (4 isolates) and CMY-2 (1 isolate)]. This study demonstrates a very high level of multidrug resistance amongst Gram-negative enteric bacilli isolated from different sites from patients in Nigerian hospitals as well as the presence of a variety of plasmid-associated resistance genes, including some identified from Africa for the first time. PMID:21074376
Ogbolu, D O; Daini, O A; Ogunledun, A; Alli, A O; Webber, M A
Background Isoniazid (INH) is a highly effective antibiotic central for the treatment of Mycobacterium tuberculosis (MTB). INH-resistant MTB clinical isolates are frequently mutated in the katG gene and the inhA promoter region, but 10 to 37% of INH-resistant clinical isolates have no detectable alterations in currently known gene targets associated with INH-resistance. We aimed to identify novel genes associated with INH-resistance in these latter isolates. Methodology/Principal Findings INH-resistant clinical isolates of MTB were pre-screened for mutations in the katG, inhA, kasA and ndh genes and the regulatory regions of inhA and ahpC. Twelve INH-resistant isolates with no mutations, and 17 INH-susceptible MTB isolates were subjected to whole genome sequencing. Phylogenetically related variants and synonymous mutations were excluded and further analysis revealed mutations in 60 genes and 4 intergenic regions associated with INH-resistance. Sanger sequencing verification of 45 genes confirmed that mutations in 40 genes were observed only in INH-resistant isolates and not in INH-susceptible isolates. The ratios of non-synonymous to synonymous mutations (dN/dS ratio) for the INH-resistance associated mutations identified in this study were 1.234 for INH-resistant and 0.654 for INH-susceptible isolates, strongly suggesting that these mutations are indeed associated with INH-resistance. Conclusion The discovery of novel targets associated with INH-resistance described in this study may potentially be important for the development of improved molecular detection strategies.
Chan, Maurice K. L.; Ong, Danny C. T.; Tongyoo, Pumipat; Wong, Sin-Yew; Lee, Ann S. G.
Cissus quadrangularis L. variant II belonging to the family Vitaceae was screened for its activity Hellcobacter pylori (Hp) human isolates. Flowering and vegetative period samples were analyzed. Aqueous (hot and cold) and solvent extracts (acetone, chloroform and methanol) were screened. Among them chloroform was observed to recover bioactive principles with low MIC and MLC. MIC and MLC was 40 ?g/ml for flowering period. Whereas for vegetative period MIC was 40 ?g/ml and MLC was 40 ?g/ml respectively. Extracts from samples collected during flowering period were better than that of vegetative period. The results confirm the traditional use of the plant in PUD. PMID:22557114
Austin, Anoop; Jegadeesan, M; Gowrishankar, R
Pseudomonas aeruginosa ED-1, isolated from a pulmonary brush of a patient hospitalized in a suburb of Paris, France, was resistant to ceftazidime and of intermediate susceptibility to ureidopenicillins and to cefotaxime. Cloning and expression of the b-lactamase gene content of this isolate in Escherichia coli DH10B identified a novel OXA-10 variant, OXA-28, with a pI value of 8.1 and a
LAURENT POIREL; DELPHINE GIRLICH; THIERRY NAAS; PATRICE NORDMANN
Recognition of a spoken word phonological variant--schwa vowel deletion (e.g., corporate --> corp'rate)--was investigated in vowel detection (absent/present) and syllable number judgment (two or three syllables) tasks. Variant frequency corpus analyses (Patterson, LoCasto, & Connine, 2003) were used to select words with either high or low schwa vowel deletion rates. Speech continua were created for each word in which schwa vowel length was manipulated (unambiguous schwa-present and schwa-absent endpoints, along with intermediate ambiguous tokens). Matched control nonwords were created with identical schwa vowel continua and surrounding segments. The low-deletion-rate words showed more three-syllable judgments than did the high-deletion-rate words. Matched control nonwords did not differ as a function of deletion rate. Experiments 2 and 3 showed a lexical decision reaction time advantage for more frequent surface forms, as compared with infrequent ones, for schwa-deleted (Experiment 2) and schwa-present (Experiment 3) stimuli. The results are discussed in terms of representations of variant forms of words based on variant frequency. PMID:18459250
Connine, Cynthia M; Ranbom, Larissa J; Patterson, David J
Two colonial variants of Staphylococcus epidermidis were isolated from the valvular tissue of a patient with native valve endocarditis. In addition to differing in colonial morphology, the two variants differed in hemolysis on blood-containing media, in adherence capacity, and in the expression of certain enzymes. Under suitable conditions, both variants were themselves capable of phenotypic variation, although they differed in the rate at which variants were generated. The variants yielded identical profiles on restriction endonuclease analysis of plasmid DNA and pulsed-field gel electrophoresis of whole-cell DNA. This report suggests a possible role for phenotypic variation in coagulase-negative staphylococcal virulence. Congo red agar would be an excellent medium for studying the contribution of variation to the virulence of these organisms. Images
Deighton, M; Pearson, S; Capstick, J; Spelman, D; Borland, R
BackgroundSmall colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch.Methodology\\/Principal FindingsOne SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence
Qing Wei; Saeed Tarighi; Andreas Dötsch; Susanne Häussler; Mathias Müsken; Victoria J. Wright; Miguel Cámara; Paul Williams; Steven Haenen; Bart Boerjan; Annelies Bogaerts; Evy Vierstraete; Peter Verleyen; Liliane Schoofs; Ronnie Willaert; Valérie N. De Groote; Jan Michiels; Ken Vercammen; Aurélie Crabbé; Pierre Cornelis
Two nisin-resistant variants of a strain of Listeria innocua were isolated after growth in the presence of 500 and 4000 IU ml?1 of nisin A showed increased cell wall hydrophobicity, resistance to phage attack and three different cell wall-acting antibiotics, as well as to the peptidoglycan hydrolytic enzymes lysozyme and mutanolysin, as compared to the parental strain. Transmission electron microscopy
Sophie Maisnier-Patin; Jean Richard
We characterized 7 highly pathogenic avian influenza A(H5N1) viruses isolated from poultry in China during 2009–2012 and found that they belong to clade 2.3.4 but do not fit within the 3 defined subclades. Antigenic drift in subtype H5N1 variants may reduce the efficacy of vaccines designed to control these viruses in poultry.
Gu, Min; Zhao, Guo; Zhao, Kunkun; Zhong, Lei; Huang, Junqing; Wan, Hongquan; Wang, Xiaoquan; Liu, Wenbo; Liu, Huimou; Peng, Daxin
Background Aspergillus flavus is intensively studied for its role in infecting crop plants and contaminating produce with aflatoxin, but its role as a human pathogen is less well understood. In parts of the Middle East and India, A. flavus surpasses A. fumigatus as a cause of invasive aspergillosis and is a significant cause of cutaneous, sinus, nasal and nail infections. Methods A collection of 45 clinical and 10 environmental A. flavus isolates from Iran were analysed using Variable-Number Tandem-Repeat (VNTR) markers with MICROSAT and goeBURST to determine their genetic diversity and their relatedness to clinical and environmental A. flavus isolates from Australia. Phylogeny was assessed using partial ?-tubulin and calmodulin gene sequencing, and mating type was determined by PCR. Antifungal susceptibility testing was performed on selected isolates using a reference microbroth dilution method. Results There was considerable diversity in the A. flavus collection, with no segregation on goeBURST networks according to source or geographic location. Three Iranian isolates, two from sinus infections and one from a paranasal infection grouped with Aspergillus minisclerotigenes, and all produced B and G aflatoxin. Phylogenic analysis using partial ?-tubulin and calmodulin sequencing confirmed two of these as A. minisclerotigenes, while the third could not be differentiated from A. flavus and related species within Aspergillus section flavi. Based on epidemiological cut-off values, the A. minisclerotigens and A. flavus isolates tested were susceptible to commonly used antifungal drugs. Conclusions This is the first report of human infection due to A. minisclerotigenes, and it raises the possiblity that other species within Aspergillus section flavi may also cause clinical disease. Clinical isolates of A. flavus from Iran are not distinct from Australian isolates, indicating local environmental, climatic or host features, rather than fungal features, govern the high incidence of A. flavus infection in this region. The results of this study have important implications for biological control strategies that aim to reduce aflatoxin by the introduction of non-toxigenic strains, as potentially any strain of A. flavus, and closely related species like A. minisclerotigenes, might be capable of human infection.
The aerial parts of Cissus quadrangularis L.Variant I and II are being used therapeutically for various ailments in indigenous system of medicine. Detailed pharmacognostical studies on the aerial parts were made. Variant I and II were analysed for their physiochemical, microscopical, fluorescent, qualitative and quantitative phytochemical, TLC and HPTLC characteristics. Quantitative variations were noted among seasonal samples and between variants and the results are presented. PMID:22557140
Austin, Anoop; Kannan, R; Jegadeesan, M
The interaction between Hfq and RNA is central to multiple regulatory processes. Using site-directed mutagenesis, we have found a missense mutation in Hfq (V43R) which strongly affects2 the RNA binding capacity of the Hfq protein and its ability to stimulate poly(A) tail elongation by poly(A)-polymerase in vitro. In vivo, overexpression of this Hfq variant fails to stimulate rpoS–lacZ expression and does not restore a normal growth rate in hfq null mutant. Cells in which the wild-type gene has been replaced by the hfqV43R allele exhibit a phenotype intermediate between those of the wild-type and of the hfq minus or null strains. This missense mutation derepresses Hfq synthesis. However, not all Hfq functions are affected by this mutation. For example, HfqV43R represses OppA synthesis as strongly as the wild-type protein. The dominant negative effect of the V43R mutation over the wild-type allele suggests that hexamers containing variant and genuine subunits are presumably not functional. Finally, molecular dynamics studies indicate that the V43R substitution mainly changes the position of the K56 and Y55 side chains involved in the Hfq–RNA interaction but has probably no effect on the folding and the oligomerization of the protein.
Ziolkowska, Katarzyna; Derreumaux, Philippe; Folichon, Marc; Pellegrini, Olivier; Regnier, Philippe; Boni, Irina V.; Hajnsdorf, Eliane
We report the isolation of thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus from unusual infection sites of patients with chronic soft tissue infection, tympanitis, bronchitis, peritonitis, and septicemia. Furthermore, we provide evidence that the essential growth factor for TD-SCVs, i.e., thymidine, and its metabolite dTMP are present in various human specimens.
Besier, Silke; Zander, Johannes; Siegel, Ekkehard; Saum, Stephan H.; Hunfeld, Klaus-Peter; Ehrhart, Annabelle; Brade, Volker; Wichelhaus, Thomas A.
Forty isolates from 97 raw milk samples (heated to 80 C for 10 min and stored at 4 to 7 C for 3 to 4 weeks) were sporeforming, aerobic, gram-positive or gram-variable, rod-shaped bacteria. Fifteen isolates that were identified had characteristics similar to species of Bacillus, except that they had lower growth temperature ranges, were gram-variable, and were somewhat different in sugar fermentations. Four isolates grew well within 2 weeks at 0 C, but they grew faster at 20 to 25 C. These psychrophilic sporeforming bacteria, the importance of which is discussed, are considered to be variant strains of mesophilic bacilli adapted to low temperatures.
Shehata, T. E.; Collins, E. B.
Curli are adhesive fimbriae of Enterobactericaeae and are involved in surface attachment, cell aggregation, and biofilm formation. We reported previously that curli-producing (C+) variants of E. coli O157:H7 (EcO157) were much more acid sensitive than their corresponding curli-deficient (C?) variants; however, this difference was not linked to the curli fimbriae per se. Here, we investigated the underlying molecular basis of this phenotypic divergence. We identified large deletions in the rcsB gene of C+ variants isolated from the 1993 U.S. hamburger-associated outbreak strains. rcsB encodes the response regulator of the RcsCDB two-component signal transduction system, which regulates curli biogenesis negatively but acid resistance positively. Further comparison of stress fitness revealed that C+ variants were also significantly more sensitive to heat shock but were resistant to osmotic stress and oxidative damage, similar to C? variants. Transcriptomics analysis uncovered a large number of differentially expressed genes between the curli variants, characterized by enhanced expression in C+ variants of genes related to biofilm formation, virulence, catabolic activity, and nutrient uptake but marked decreases in transcription of genes related to various types of stress resistance. Supplying C+ variants with a functional rcsB restored resistance to heat shock and acid challenge in cells but blocked curli production, confirming that inactivation of RcsB in C+ variants was the basis of fitness segregation within the EcO157 population. This study provides an example of how genome instability of EcO157 promotes intrapopulation diversification, generating subpopulations carrying an array of distinct phenotypes that may confer the pathogen with survival advantages in diverse environments.
Parker, Craig T.; Louie, Jacqueline W.; Huynh, Steven; Fagerquist, Clifton K.; Mandrell, Robert E.
Although genome-wide association studies have been successful in detecting associations with common variants, there is currently an increasing interest in identifying low frequency and rare variants associated with complex traits. Next-generation sequencing technologies make it feasible to survey the full spectrum of genetic variation in coding regions or the entire genome. Due to the low frequency of rare variants, coupled with allelic heterogeneity, however, the association analysis for rare variants is challenging and traditional methods are ineffective. Recently a battery of new statistical methods has been proposed for identifying rare variants associated with complex traits. These methods test for associations by aggregating multiple rare variants across a gene or a genomic region, or a group of variants in the genome. In this Unit, we describe key concepts for rare variant association for complex traits, survey some of the recent methods and discuss their statistical power under various scenarios, and provide practical guidance on analyzing next-generation sequencing data for identifying rare variants associated with complex traits.
Li, Bingshan; Liu, Dajiang J.; Leal, Suzanne M.
Coxsackie A viruses belong to the enteroviruses, the isolation of which from infectious materials and further cultivation are possible only when laboratory animals are infected. The authors could adapt the strains of 17 of 23 serotypes of these viruses to RD cell culture. The strains of 8 serotypes were additionally adapted to Vero cell culture. The cultural variants of Coxsackle A viruses were used to prepare immune sera. The Bacterial and Viral Agents Enterprise, M. P. Chumakov Institute of Poliomyelitis and Virus Encephalitides, Russian Academy of Medical Sciences, has set up the production of bacterial and viral drugs based on the cultural variants of 5 Coxsackie A virus serotypes. The cultural variants of 14 Coxsackie A virus serotypes were used to carry out a virus neutralization test. Examination of more than 600 children from Moscow and the Moscow Region showed the wide circulation of individual Coxsackie A virus serotypes. It also demonstrated a drastic reduction in Coxsackie A-7 virus circulation in the past 50 years. PMID:22834148
Se?bil', V B; Malyshkina, L P; Gracheva, L A; Kozlov, V G
Parvovirus B19 (B19), currently the only accepted member of the Erythrovirus genus, is the only parvovirus known to be pathogenic in humans. Recently a viral sequence, tentatively termed V9 which showed 11% variability from the published B19 sequences, was described from a patient with aplastic crisis. To search for additional parvovirus variants, we used the new NS1/7.5EC PCR assay whose primers were designed from a conserved region of the B19/V9 sequence and encompasses an MfeI restriction enzyme site that would allow differentiation between B19- and V9-like sequences. Screening of 225 serum and bone marrow samples and 62 plasma pools identified one new atypical parvovirus sequence, A6, from an anemic HIV-positive patient. A6 exhibited 88% similarity to B19 and 92% to V9, compared to >98% correspondence between reported B19 isolates. Based on the genome similarity to B19, an RT-PCR for A6 capsid transcripts was developed and used to test for A6 infectivity of UT7/Epo/S1 cells. Despite high viral titers, A6 viral transcripts were not detected. Thus, although the prevalence of B19 variants probably is low, the true clinical significance remains unknown. Current PCR analyses are unlikely to detect novel variants without the design of specific primers to the A6/V9/B19 common sequences. PMID:12359439
Nguyen, Quang Tri; Wong, Susan; Heegaard, Erik D; Brown, Kevin E
Extensive alternative splicing and RNA editing have been documented for the transcript of DmNaV (formerly para), the sole sodium channel gene in Drosophila melanogaster. However, the functional consequences of these post-transcriptional modifications are not well understood. In this study we isolated 64 full-length DmNaV cDNA clones from D. melanogaster adults. Based on the usage of 11 alternative exons, 64 clones could be grouped into 29 splice types. When expressed in Xenopus oocytes, 33 DmNaV variants generated sodium currents large enough for functional characterization. Among these variants, DmNaV5-1 and DmNaV7-1 channels activated at the most hyperpolarizing potentials, whereas DmNaV1-6 and DmNaV19 channels activated at the most depolarizing membrane potentials. We identified an A-to-I editing event in DmNaV5-1 that is responsible for its uniquely low-voltage-dependent activation. The wide range of voltage dependence of gating properties exhibited by DmNaV variants represents a rich resource for future studies to determine the role of DmNaV in regulating sodium channel gating, pharmacology, and neuronal excitability in insects.
Olson, Rachel O'Donnell; Liu, Zhiqi; Nomura, Yoshiko; Song, Weizhong; Dong, Ke
The genomic clone, RRNpss1, representing the short ribosomal DNA length variant in Pisum sativum L. cv. Alaska, has been isolated and the 2859 bp intergenic spacer, along with the 25S rRNA 3' border and 18S rRNA 5' border, has been sequenced. The intergenic spacer contains nine tandem repeats, approximately 180 bp in length, which show greater than 80% sequence homology to each other. The RNA polymerase I transcription start site and a processing site, located 776 bp and 536 bp upstream of the 5' end of 18S rRNA, respectively, have been determined using S1 analysis. The region surrounding the +1 site shows strong homology between the positions -6 to +10 to the rDNA sites of initiation in radish, maize, and wheat. The sequence CATGCAAA is located 19 bp upstream of the site of initiation, and appears once within each subrepeat and twice more between the end of the subrepeat array and the site of initiation. A previously characterized HpaII site which shows developmental regulation of methylation is located 31 bp downstream of the site of initiation. Using RFLP linkage analysis, the short rDNA length variant of cv. Alaska is assigned to Chromosome 4 where it is genetically independent of the long rDNA length variant which is putatively assigned to Chromosome 7. Images
Piller, K J; Baerson, S R; Polans, N O; Kaufman, L S
Calcium (Ca2+) influx is a fundamental intracellular signal that is required to initiate and sustain T lymphocyte activation. Dihydropyridine-sensitive, L-type Ca2+ channels appear to play a significant role in Ca2+ mobilization during T cell activation, but very little is known about the molecular structure of these channels in T lymphocytes. Here we identify two novel splice variants of the Ca(V)1.4 (alpha1F) L-type Ca2+ channel that are expressed in human T lymphocytes, and also demonstrate expression of the Ca(V)1.4 protein in the human Jurkat T cell leukemia line and human peripheral blood T lymphocytes (PBTs). The carboxy-termini of both Ca(V)1.4 splice isoforms contain unique exon usages distinct from the Ca(V)1.4 channel isolated from human retina that may render these channel variants insensitive to changes in membrane depolarization. Additional evidence of the importance of these new splice variants comes from the demonstration that the mRNA expression of the Ca(V)1.4 splice isoforms is regulated by TCR-induced activation in Jurkat T cells, and to a lesser extent in human PBTs. Overall these results provide the first evidence that structurally unique L-type Ca2+ channels exist in T lymphocytes, which can contribute to a Ca2+ influx during T lymphocyte activation. PMID:15899519
Kotturi, Maya F; Jefferies, Wilfred A
A lipopolysaccharide O-side chain-producing phenotypic variant was isolated from a uridine 5'-diphosphogalactose 4-epimeraseless Escherichia coli J5 rough mutant strain by fluorescence-activated cell sorting with a monoclonal antibody (MAb) specific for the O-side chain of E. coli O111:B4 smooth parent lipopolysaccharide. The variant (J5-2) was recognized by both core- and O-side chain-specific MAbs, while the "original" rough mutant (J5-1) and smooth parent strains stained only with core- and O-side chain-specific MAb, respectively. J5-2 produced complete and incomplete (Rc chemotype) core and O polysaccharide in the presence of galactose. Three other E. coli J5 variants were either phenotypically similar to J5-1 (J5-UK) or distinct from J5-1 and J5-2 (J5-A, -B). The latter phenotype had a lower-molecular-weight core, compared with J5-1 and J5-2, and distinct MAb specificities. Various J5 phenotypes also differed in galactokinase levels, the ability to use galactose, and susceptibility to core-specific MAb binding on solid media. The J5-2 strain showed reciprocal changes in O-side chain and core expression during log-phase growth. E. coli J5 thus undergoes spontaneous alterations in lipopolysaccharide phenotype. PMID:1527415
Evans, M E; Pollack, M; Koles, N L; Hardegen, N J; Panopoulos, D
A total of 20 Vibrio cholerae isolates were recovered for investigation from a cholera outbreak in Kelantan, Malaysia, that occurred between November and December 2009. All isolates were biochemically characterized as V. cholerae serogroup O1 Ogawa of the El Tor biotype. They were found to be resistant to multiple antibiotics, including tetracycline, erythromycin, sulfamethoxazole-trimethoprim, streptomycin, penicillin G, and polymyxin B, with 35% of the isolates being resistant to ampicillin. All isolates were sensitive to ciprofloxacin, norfloxacin, chloramphenicol, gentamicin, and kanamycin. Multiplex PCR analysis confirmed the biochemical identification and revealed the presence of virulence genes, viz., ace, zot, and ctxA, in all of the isolates. Interestingly, the sequencing of the ctxB gene showed that the outbreak strain harbored the classical cholera toxin gene and therefore belongs to the newly assigned El Tor variant biotype. Clonal analysis by pulsed-field gel electrophoresis demonstrated that a single clone of a V. cholerae strain was responsible for this outbreak. Thus, we present the first molecular evidence that the toxigenic V. cholerae O1 El Tor variant has invaded Malaysia, highlighting the need for continuous monitoring to facilitate early interventions against any potential epidemic by this biotype. PMID:20826646
Ang, Geik Yong; Yu, Choo Yee; Balqis, Kamarudin; Elina, Husni Tan; Azura, Hussin; Hani, Mat Hussin; Yean, Chan Yean
The protozoan Trichomonas vaginalis a sexually transmitted protozoan parasite causes vaginitis, urethritis and cervicitis in humans. The present study highlights phenotypic 'variant' forms of trophozoites isolated from patients suffering from cervical neoplasia condition. The growth curve of 10 isolates i.e., four non-cervical neoplasia (NCN) isolates (NCN1-NCN4) and six cervical neoplasia (CN) isolates (CN1-CN6) showed two distinct and different in vitro growth profiles. The parasite count and growth rates were significantly higher in trophozoites from CN isolates in cultures of day 2 up to day 8 (p<0.05, Mann-Whitney test). The average generation time was 1.84±0.40 and 3.38±0.55h for NCN and CN isolates respectively. The nucleus of trophozoites in CN isolates using acridine orange and DAPI showed more intense staining revealing higher nuclear content. The FITC-labeled Concanavalin A stained stronger green fluorescence with surface of trophozoites in CN isolates showing more rough and creased surface with numerous deep micropores. Transmission electron microscopy studies revealed that there was higher numbers of vacuoles and hydrogenosomes in these forms. The study mounted staining techniques, growth profiles, morphology, morphometry studies using scanning and transmission electron microscopy and confirms that the trophozoites from cervical neoplasia proliferates at a higher rate, shows higher FITC-labeled Concanavalin A binding with rough and creased surface implying that these are virulent forms which can aggravate or exacerbate cervical neoplasia conditions. The large numbers of hyrogenosomes and vacuoles implies that these forms are active and implicates a possible role in such conditions. PMID:22525014
Yusof, Afzan M; Kumar, Suresh
Microorganisms from the oral flora were examined for the production of bacteriolytic substances. Among human viridans group streptococci, only one group of strains with thiol-dependent properties was shown to secrete enzymes with bacteriolytic activity on heat-killed cells of Micrococcus luteus on double-layer nutrient agar plates. By morphology, culture requirements, and biochemical properties, they were found to conform to descriptions of nutritionally variant streptococci (NVS). Bacteriolytic activity was shown to be a constant property of all of the human oral NVS isolated and a property of some reference strains of NVS from clinical sources. No other known species of viridans group streptococci demonstrated bacteriolytic activity. Analysis of bacteriolytic activity could be a useful tool for both the isolation and identification of this fastidious group of microorganisms. Images
Pompei, R; Caredda, E; Piras, V; Serra, C; Pintus, L
Pichinde virus (PV) strain AN 3739 was determined to be sensitive to natural killer (NK) cells in vivo by enhanced replication in NK-cell-depleted mice. An NK-sensitive subclone (PV-NKs1) was serially passed in mice whose NK cells had previously been activated by an interferon inducer, and two plaque isolates were shown to be resistant to NK cells but not to interferon. Inoculation of severe-combined-immunodeficient mice with PV-NKs1 led to a persistent infection resulting in an NK-resistant viral population. This is the first demonstration of the isolation of viral "NK-escape" variants, as defined by the ability of the virus to replicate in vivo.
Vargas-Cortes, M; O'Donnell, C L; Maciaszek, J W; Welsh, R M
Epigenetic changes related to human disease cannot be fully addressed by studies of cells from cultures or from other mammals. We isolated human fat cells from subcutaneous abdominal fat tissue of female subjects and extracted histones from either purified nuclei or intact cells. Direct acid extraction of whole adipocytes was more efficient, yielding about 100 µg of protein with histone content of 60% –70% from 10 mL of fat cells. Differential proteolysis of the protein extracts by trypsin or ArgC-protease followed by nanoLC/MS/MS with alternating CID/ETD peptide sequencing identified 19 histone variants. Four variants were found at the protein level for the first time; particularly HIST2H4B was identified besides the only H4 isoform earlier known to be expressed in humans. Three of the found H2A potentially organize small nucleosomes in transcriptionally active chromatin, while two H2AFY variants inactivate X chromosome in female cells. HIST1H2BA and three of the identified H1 variants had earlier been described only as oocyte or testis specific histones. H2AFX and H2AFY revealed differential and variable N-terminal processing. Out of 78 histone modifications by acetylation/trimethylation, methylation, dimethylation, phosphorylation and ubiquitination, identified from six subjects, 68 were found for the first time. Only 23 of these modifications were detected in two or more subjects, while all the others were individual specific. The direct acid extraction of adipocytes allows for personal epigenetic analyses of human fat tissue, for profiling of histone modifications related to obesity, diabetes and metabolic syndrome, as well as for selection of individual medical treatments.
Jufvas, Asa; Stralfors, Peter; Vener, Alexander V.
Isolation of polyadenilated mRNA from human immortalized bronchial epithelial cell line BEP2D revealed the presence of multiple isoforms of RNA coded by the CHRNA9 gene for ?9 nicotinic acetylcholine receptor (nAChR). BEP2D cells were homozygous for the rs10009228 polymorphism encoding for N442S amino acid substitution, and also contained mRNA coding for several truncated isoforms of ?9 protein. To elucidate the biologic significance of the naturally occurring variants of ?9 nAChR, we compared the biologic effects of overexpression of full-length ?9 N442 and S442 proteins, and the truncated ?9 variant occurring due to a loss of the exon 4 sequence that causes frame shift and early termination of the translation. These as well as control vector were overexpressed in the BEP2D cells that were used in the assays of proliferation rate, spontaneous vs. tobacco nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced cellular transformation, and tumorigenicity in cell culture and mice. Overexpression of the S442 variant significantly increased cellular proliferation, and spontaneous and NNK-induced transformation. The N442 variant significantly decreased cellular transformation, without affecting proliferation rate. Overexpression of the truncated ?9 significantly decreased proliferation and suppressed cellular transformation. These results suggested that ?9 nAChR plays important roles in regulation of bronchial cell growth by endogenous acetylcholine and exogenous nicotine, and susceptibility to NNK-induced carcinogenic transformation. The biologic activities of ?9 nAChR may be regulated at the splicing level, and genetic polymorphisms in CHRNA9 affecting protein levels, amino acid sequence and RNA splicing may influence the risk for lung cancer.
Chikova, Anna; Grando, Sergei A.
Rare mutations in PARK loci genes cause Parkinson's disease (PD) in some families and isolated populations. We investigated the association of common variants in PARK loci and related genes with PD susceptibility and age at onset in an outbred population. A total of 1,103 PD cases from the upper Midwest, USA, were individually matched to unaffected siblings (n = 654) or unrelated controls (n = 449) from the same region. Using a sequencing approach in 25 cases and 25 controls, single nucleotide polymorphisms (SNPs) in species-conserved regions of PARK loci and related genes were detected. We selected additional tag SNPs from the HapMap. We genotyped a total of 235 SNPs and two variable number tandem repeats in the ATP13A2, DJ1, LRRK1, LRRK2, MAPT, Omi/HtrA2, PARK2, PINK1, SNCA, SNCB, SNCG, SPR, and UCHL1 genes in all 2,206 subjects. Case-control analyses were performed to study association with PD susceptibility, while cases-only analyses were used to study association with age at onset. Only MAPT SNP rs2435200 was associated with PD susceptibility after correction for multiple testing (OR = 0.74, 95% CI = 0.64-0.86, uncorrected P < 0.0001, log additive model); however, 16 additional MAPT variants, seven SNCA variants, and one LRRK2, PARK2, and UCHL1 variants each had significant uncorrected P-values. There were no significant associations for age at onset after correction for multiple testing. Our results confirm the association of MAPT and SNCA genes with PD susceptibility but show limited association of other PARK loci and related genes with PD. PMID:21412835
Chung, Sun Ju; Armasu, Sebastian M; Biernacka, Joanna M; Lesnick, Timothy G; Rider, David N; Lincoln, Sarah J; Ortolaza, Alexandra I; Farrer, Matthew J; Cunningham, Julie M; Rocca, Walter A; Maraganore, Demetrius M
The Thermal Analysis Variant of the COMOC (computational continuum mechanics) computer system solves problems involving transient heat conduction and convection in stationary continua spanning arbitrarily irregular two-dimensional and axisymmetric solution domains. COMOC is based upon a finite element solution algorithm for the energy equation, and solves for the transient nodal temperature distribution using a highly stable and automatic explicit integration procedure. COMOC is extensively user-oriented, requires minimal input, and no a priori knowledge concerning the stability character of the differential equation system. It can readily output computed data in user-specified format fields, that geometrically resemble the solution domain discretization (for rapid engineering evaluation). Complete information is provided for applying COMOC to a specific problem.
Bauer, A. M.; Baker, A. J.
We report on a child with microcephaly, small facial and body size, and immune deficiency. The phenotype is consistent with Nijmegen breakage syndrome (NBS), with additional clinical manifestations and laboratory findings not reported heretofore. Most investigations, including the results of radiation-resistant DNA synthesis, concurred with the diagnosis of NBS. Cytogenetic analysis documented abnormalities in virtually all cells examined. Along with the high frequency of breaks and rearrangements of chromosomes 7 and 14, we found breakage and monosomies involving numerous other chromosomes. Because of some variation in the clinical presentation and some unusual cytogenetic findings, we suggest that our patient may represent a new variant of Nijmegen breakage syndrome. 34 refs., 3 figs., 7 tabs.
Der Kaloustian, V.M.; Booth, A.; Elliott, A.M. [Montreal Children`s Hospital and McGill Univ., Montreal (Canada)] [and others] [Montreal Children`s Hospital and McGill Univ., Montreal (Canada); and others
Vascular anomalies are frequently encountered in abdomen. But they are usually asymptomatic and diagnosed accidently during angiography or surgery leading into severe complications. Thus knowledge of angioarchitecture in abdomen, whether normal or variant, is considered prerequisite for successful, uncomplicated surgeries and interventional radiology. This case report describes one of such varying branching pattern of celiac trunk and superior mesenteric artery. During routine abdominal dissection, gastroduodenal artery was seen arising from celiac trunk along with its usual three branches. Common hepatic artery continued as left hepatic artery after giving rise the right gastric artery and a tortuous replaced right hepatic artery arose from superior mesenteric artery. An unusually long cystic artery arose from left hepatic artery and gave rise to 2-3 small anastomotic branches towards hepatic flexor of colon, in addition to its normal gallbladder supply. Awareness of such variations would certainly be helpful in upper abdominal surgeries.
Anand, Mamta; Gupta, Smrity
A case of Prinzmetal variant angina with transient complete atrioventricular block and syncopal episodes following an anteroseptal myocardial infarction is described. The syncopal attacks were not prevented by demand cardiac pacing and were presumably caused by transient severe ischaemia of the left ventricle, with a consequent reduction in cardiac output. The left ventriculogram showed a large anterior dyskinetic area corresponding to the high grade proximal obstruction in the left anterior descending artery demonstrated by coronary angiography. All other coronary vessels appeared free of disease and it is suggested that the anginal episodes were caused by transient proximal segmental spasm of the right coronary artery. The anginal episodes were successfully prevented by a regimen of two-hourly coronary arterial vasodilator therapy. Images
Harper, R; Peter, T; Hunt, D
We report five patients with hyperlexia who presented for evaluation in the Learning Disabilities Clinic at the Medical College of Georgia over a two year period. Analysis of the data from the neurologic and neuropsychologic evaluations indicates that the primary and essential cognitive deficit in these children is a disorder in speech and language involving a severe deficit in their ability to comprehend language whether it be spoken or written, as opposed to a dyslexia syndrome involving only recognition and/or comprehension of written language. This finding is in contrast to the Nosology of Disorders of Higher Cerebral Function proposed by the Child Neurology Society which views hyperlexia as a variant of the language disorder subtype of dyslexia. PMID:2468342
Cohen, M; Campbell, R; Gelardo, M
Background Designing an ideal vaccine against HIV-1 has been difficult due to enormous genetic variability as a result of high replication rate and lack of proofreading activity of reverse transcriptase leading to emergence of genetic variants and recombinants. Tat transactivates HIV-1 LTR, resulting in a remarkable increase in viral gene expression, and plays a vital role in pathogenesis. The aim of this study was to characterize the genetic variations of Tat exon-1 from HIV-1 infected patients from North India. Methods Genomic DNA was isolated from PBMCs and Tat exon-1 was PCR amplified with specific primers followed by cloning, sequencing and sequence analyses using bioinformatic tools for predicting HIV-1 subtypes, recombination events, conservation of domains and phosphorylation sites, and LTR transactivation by luciferase assay. Results Phylogenetic analysis of Tat exon-1 variants (n?=?120) revealed sequence similarity with South African Tat C sequences and distinct geographical relationships were observed for B/C recombinants. Bootscan analysis of our variants showed 90% homology to Tat C and 10% to B/C recombinants with a precise breakpoint. Natural substitutions were observed with high allelic frequencies which may be beneficial for virus. High amino acid conservation was observed in Tat among Anti Retroviral Therapy (ART) recipients. Barring few changes, most of the functional domains, predicted motifs and phosphorylation sites were well conserved in most of Tat variants. dN/dS analysis revealed purifying selection, implying the importance of functional conservation of Tat exon-1. Our Indian Tat C variants and B/C recombinants showed differential LTR transactivation. Conclusions The possible role of Tat exon-1 variants in shaping the current HIV-1 epidemic in North India was highlighted. Natural substitutions across conserved functional domains were observed and provided evidence for the emergence of B/C recombinants within the ORF of Tat exon-1. These events are likely to have implications for viral pathogenesis and vaccine formulations.
Ronsard, Larance; Lata, Sneh; Singh, Jyotsna; Ramachandran, Vishnampettai G.; Das, Shukla; Banerjea, Akhil C.
Summary Background Normal protein C (PC) plasma levels range widely in the general population. Factors influencing normal PC levels are thought to influence the risk of venous thrombosis. Little is known about the underlying genetic variants. Objectives We performed a genome scan of normal PC levels to identify genes that regulate normal PC levels. Patients/Methods We performed a variance components linkage analysis for normal PC levels in 275 individuals from a single, large family. We then sequenced candidate genes under the identified linkage peak in eight family members: four with high and four with low, but normal PC levels. For variants showing a difference in carriers between those with high and low PC levels, we re-evaluated linkage in the 275 family members conditional on the measured genotype effect. Genotype-specific mean PC levels were determined using likelihood analysis. Findings were replicated in the Leiden Thrombophilia Study (LETS). Results We identified a quantitative trait locus at chromosome 5q14.1 affecting normal PC plasma level variability. Next-generation sequencing of 113 candidate genes under the linkage peak revealed four SNPs in BHMT2, ACOT12, SSBP2 and XRCC4, which significantly increased PC levels in our thrombophilic family, but not in LETS. Conclusions We identified four genes at chromosome 5q14.1 that might influence normal PC levels. BHMT2 seems the most likely candidate to regulate PC levels via homocysteine, a competitive inhibitor to thrombin. Failure to replicate our findings in LETS might be due to differences between the studies in genetic background and linkage disequilibrium patterns.
Vossen, C.Y.; Koeleman, B.P.; Hasstedt, S.J.; Nijman, I.J.; Renkens, I.J.; Callas, P.W.; Rosendaal, F.R.; Bovill, E.G.
Human ribonuclease inhibitor (hRI) is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type ribonucleases. hRI has 32 cysteine residues. The oxidation of these cysteine residues to form disulfide bonds is a rapid, cooperative process that inactivates hRI. The most proximal cysteine residues in native hRI are two pairs that are adjacent in sequence: Cys94 and Cys95, and Cys328 and Cys329. A cystine formed from such adjacent cysteine residues would likely contain a perturbing cis peptide bond within its eight-membered ring, which would disrupt the structure of hRI and could facilitate further oxidation. We find that replacing Cys328 and Cys329 with alanine residues has little effect on the affinity of hRI for bovine pancreatic ribonuclease A (RNase A), but increases its resistance to oxidation by 10- to 15-fold. Similar effects are observed for the single variants, C328A hRI and C329A hRI, suggesting that oxidation resistance arises from the inability to form a Cys328-Cys329 disulfide bond. Replacing Cys94 and Cys95 with alanine residues increases oxidation resistance to a lesser extent, and decreases the affinity of hRI for RNase A. The C328A, C329A, and C328A/C329A variants are likely to be more useful than wild-type hRI for inhibiting pancreatic-type ribonucleases in vitro and in vivo. We conclude that replacing adjacent cysteine residues can confer oxidation resistance in a protein.
Kim, B. M.; Schultz, L. W.; Raines, R. T.
We demonstrate that the alternatively spliced variable (V) region of fibronectin (FN) is required for secretion of FN dimers during biosynthesis. Alternative splicing of the V segment of the rat FN transcript generates three subunit variants (V120, V95, V0) that differ by the inclusion or omission of an additional 120 or 95 amino acids. We are exploring the functions of this segment by expressing variant cDNAs in normal and transformed fibroblasts. Like FN itself, the cDNA-encoded polypeptides (deminectins [DNs]) containing the V120 or V95 segment are efficiently secreted as disulfide-bonded homodimers. However, few homodimers of DNs lacking this region, V0 DNs, are secreted. V0 homodimers do form inside the cell, as demonstrated by biosynthetic analyses of dimer formation and secretion using pulse-chase and time course experiments, but these dimers seldom reach the cell surface and are probably degraded intracellularly. Coexpression of V0 and V120 subunits results in intracellular formation of three types of dimers, V0-V0, V0-V120, and V120-V120, but only the V120-containing dimers are secreted. This selective retention of V0 homodimers indicates that the V region is required for formation and secretion of native FN dimers. In an analogous in vivo situation, we show that plasma FN also lacks V0- V0 dimers and consists of V0-V+ and V+-V+ combinations. Dissection of V region sequences by deletion mapping localizes the major site involved in DN dimer secretion to an 18-amino acid segment within V95. In addition, high levels of dimer secretion can be restored by insertion of V into a heterologous site 10 kD COOH terminal to its normal location. We discuss the potential role of intracellular protein- protein interactions in FN dimer formation.
The major virulence factor of Shiga toxin producing E. coli, is Shiga toxin (Stx), an AB5 toxin that consists of a ribosomal RNA-cleaving A-subunit surrounded by a pentamer of receptor-binding B subunits. The two major isoforms, Stx1 and Stx2, and Stx2 variants (Stx2a-h) significantly differ in toxicity. The exact reason for this toxicity difference is unknown, however different receptor binding preferences are speculated to play a role. Previous studies used enzyme linked immunosorbent assay (ELISA) to study binding of Stx1 and Stx2a toxoids to glycolipid receptors. Here, we studied binding of holotoxin and B-subunits of Stx1, Stx2a, Stx2b, Stx2c and Stx2d to glycolipid receptors globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4) in the presence of cell membrane components such as phosphatidylcholine (PC), cholesterol (Ch) and other neutral glycolipids. In the absence of PC and Ch, holotoxins of Stx2 variants bound to mixtures of Gb3 with other glycolipids but not to Gb3 or Gb4 alone. Binding of all Stx holotoxins significantly increased in the presence of PC and Ch. Previously, Stx2a has been shown to form a less stable B-pentamer compared to Stx1. However, its effect on glycolipid receptor binding is unknown. In this study, we showed that even in the absence of the A-subunit, the B-subunits of both Stx1 and Stx2a were able to bind to the glycolipids and the more stable B-pentamer formed by Stx1 bound better than the less stable pentamer of Stx2a. B-subunit mutant of Stx1 L41Q, which shows similar stability as Stx2a B-subunits, lacked glycolipid binding, suggesting that pentamerization is more critical for binding of Stx1 than Stx2a.
Karve, Sayali S.; Weiss, Alison A.
Abstract A typing procedure based on polymorphism of the coagulase gene (coa) was used to discriminate Staphylococcus aureus isolated from Minas Gerais dairy cows with mastitis. Amplification of the gene from the 64 S. aureus isolates produced 27 different polymerase chain reaction (PCR) products; 60 isolates showed only 1 amplicon, and 4 showed 2 amplicons. The isolates were grouped into 49 types by analyzing the restriction fragment length polymorphism (RFLP) of the coa gene; the 10 most common types accounted for 39% of the isolates. The results demonstrate that many variants of the coa gene are present in the studied region, although only a few predominate.
Motivation: In sequencing studies of common diseases and quantitative traits, power to test rare and low frequency variants individually is weak. To improve power, a common approach is to combine statistical evidence from several genetic variants in a region. Major challenges are how to do the combining and which statistical framework to use. General approaches for testing association between rare variants and quantitative traits include aggregating genotypes and trait values, referred to as ‘collapsing’, or using a score-based variance component test. However, little attention has been paid to alternative models tailored for protein truncating variants. Recent studies have highlighted the important role that protein truncating variants, commonly referred to as ‘loss of function’ variants, may have on disease susceptibility and quantitative levels of biomarkers. We propose a Bayesian modelling framework for the analysis of protein truncating variants and quantitative traits. Results: Our simulation results show that our models have an advantage over the commonly used methods. We apply our models to sequence and exome-array data and discover strong evidence of association between low plasma triglyceride levels and protein truncating variants at APOC3 (Apolipoprotein C3). Availability: Software is available from http://www.well.ox.ac.uk/~rivas/mamba Contact: email@example.com
Rivas, Manuel A.; Pirinen, Matti; Neville, Matthew J.; Gaulton, Kyle J.; Moutsianas, Loukas; Lindgren, Cecilia M.; Karpe, Fredrik; McCarthy, Mark I.; Donnelly, Peter
Hemimandibular hypertrophy and its variants result from unilateral excessive growth of the mandible and involve both the body and ramus of mandible. This causes facial asymmetry and in turn accompanying psychological problems. In this report we discuss use of imaging in diagnosis of these lesions and investigate the different variants.
Mohan, Ravi Prakash Sasankoti; Verma, Sankalp; Singh, Udita; Agarwal, Neha
Disclosed are a number of homologs and variants of Hypocrea jecorina Cel7A (formerly Trichoderma reesei cellobiohydrolase I or CBH1), nucleic acids encoding the same and methods for producing the same. The homologs and variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted and/or deleted.
Goedegebuur, Frits (Rozenlaan, NL); Gualfetti, Peter (San Francisco, CA); Mitchinson, Colin (Half Moon Bay, CA); Neefe, Paulien (Zoetermeer, NL)
Advances in high-throughput sequencing technologies have brought us into the individual genome era. Projects such as the 1000 Genomes Project have led the individual genome sequencing to become more and more popular. How to visualize, analyse and annotate individual genomes with knowledge bases to support genome studies and personalized healthcare is still a big challenge. The Personal Genome Browser (PGB) is developed to provide comprehensive functional annotation and visualization for individual genomes based on the genetic-molecular-phenotypic model. Investigators can easily view individual genetic variants, such as single nucleotide variants (SNVs), INDELs and structural variations (SVs), as well as genomic features and phenotypes associated to the individual genetic variants. The PGB especially highlights potential functional variants using the PGB built-in method or SIFT/PolyPhen2 scores. Moreover, the functional risks of genes could be evaluated by scanning individual genetic variants on the whole genome, a chromosome, or a cytoband based on functional implications of the variants. Investigators can then navigate to high risk genes on the scanned individual genome. The PGB accepts Variant Call Format (VCF) and Genetic Variation Format (GVF) files as the input. The functional annotation of input individual genome variants can be visualized in real time by well-defined symbols and shapes. The PGB is available at http://www.pgbrowser.org/. PMID:24799434
Juan, Liran; Teng, Mingxiang; Zang, Tianyi; Hao, Yafeng; Wang, Zhenxing; Yan, Chengwu; Liu, Yongzhuang; Li, Jie; Zhang, Tianjiao; Wang, Yadong
Inactivating germline mutations in DNA mismatch repair (MMR) genes are diagnostic for Lynch syndrome. However, the clinical significance of missense variants is uncertain. A threshold level of compromised MLH1 expression, correlating with greater protein instability and MMR functional defect, has been identified to help classify the pathogenicity of missense variants. PMID:23532885
You, Y Nancy; Vilar, Eduardo
Here, we give a historical overview of the search for genetic variants that influence the susceptibility of an individual to a chronic disease, from RA Fisher's seminal work to the current excitement of whole-genome association studies (WGAS). We then discuss the concepts behind the identification of common variants as disease causal factors and contrast them to the basic ideas that
Walter Bodmer; Carolina Bonilla
It is well established that invasive urothelial carcinoma, involving the urinary bladder and renal pelvis, has marked propensity for divergent differentiation. In recent years, several ‘variant’ morphologies have been described and most have been recognized in the 2004 World Health Organization Classification. These histological variants of urothelial carcinoma have clinical significance at various levels, including diagnostic, that is, awareness of
Mahul B Amin
Approximately 20 % of individuals with Parkinson's disease (PD) report a positive family history. Yet, a large portion of causal and disease-modifying variants is still unknown. We used exome sequencing in two affected individuals from a family with late-onset PD to identify 15 potentially causal variants. Segregation analysis and frequency assessment in 862 PD cases and 1,014 ethnically matched controls highlighted variants in EEF1D and LRRK1 as the best candidates. Mutation screening of the coding regions of these genes in 862 cases and 1,014 controls revealed several novel non-synonymous variants in both genes in cases and controls. An in silico multi-model bioinformatics analysis was used to prioritize identified variants in LRRK1 for functional follow-up. However, protein expression, subcellular localization, and cell viability were not affected by the identified variants. Although it has yet to be proven conclusively that variants in LRRK1 are indeed causative of PD, our data strengthen a possible role for LRRK1 in addition to LRRK2 in the genetic underpinnings of PD but, at the same time, highlight the difficulties encountered in the study of rare variants identified by next-generation sequencing in diseases with autosomal dominant or complex patterns of inheritance. PMID:24241507
Schulte, Eva C; Ellwanger, Daniel C; Dihanich, Sybille; Manzoni, Claudia; Stangl, Katrin; Schormair, Barbara; Graf, Elisabeth; Eck, Sebastian; Mollenhauer, Brit; Haubenberger, Dietrich; Pirker, Walter; Zimprich, Alexander; Brücke, Thomas; Lichtner, Peter; Peters, Annette; Gieger, Christian; Trenkwalder, Claudia; Mewes, Hans-Werner; Meitinger, Thomas; Lewis, Patrick A; Klünemann, Hans H; Winkelmann, Juliane
This paper describes a technique for recording representations of space-variant optical systems as volume holograms. Due to the space-variant nature of the system, the input plane is spatially sampled in the recording step. The transfer-function holograms...
L. M. Deen J. F. Walkup M. O. Hagler
Next generation sequencing has dramatically increased our ability to localize disease-causing variants by providing base-pair level information at costs increasingly feasible for the large sample sizes required to detect complex-trait associations. Yet, identification of causal variants within an established region of association remains a challenge. Counter-intuitively, certain factors that increase power to detect an associated region can decrease power to localize the causal variant. First, combining GWAS with imputation or low coverage sequencing to achieve the large sample sizes required for high power can have the unintended effect of producing differential genotyping error among SNPs. This tends to bias the relative evidence for association toward better genotyped SNPs. Second, re-use of GWAS data for fine-mapping exploits previous findings to ensure genome-wide significance in GWAS-associated regions. However, using GWAS findings to inform fine-mapping analysis can bias evidence away from the causal SNP toward the tag SNP and SNPs in high LD with the tag. Together these factors can reduce power to localize the causal SNP by more than half. Other strategies commonly employed to increase power to detect association, namely increasing sample size and using higher density genotyping arrays, can, in certain common scenarios, actually exacerbate these effects and further decrease power to localize causal variants. We develop a re-ranking procedure that accounts for these adverse effects and substantially improves the accuracy of causal SNP identification, often doubling the probability that the causal SNP is top-ranked. Application to the NCI BPC3 aggressive prostate cancer GWAS with imputation meta-analysis identified a new top SNP at 2 of 3 associated loci and several additional possible causal SNPs at these loci that may have otherwise been overlooked. This method is simple to implement using R scripts provided on the author's website.
Faye, Laura L.; Machiela, Mitchell J.; Kraft, Peter; Bull, Shelley B.; Sun, Lei
Low-frequency coding DNA sequence variants in the proprotein convertase subtilisin/kexin type 9 gene (PCSK9) lower plasma low-density lipoprotein cholesterol (LDL-C), protect against risk of coronary heart disease (CHD), and have prompted the development of a new class of therapeutics. It is uncertain whether the PCSK9 example represents a paradigm or an isolated exception. We used the "Exome Array" to genotype >200,000 low-frequency and rare coding sequence variants across the genome in 56,538 individuals (42,208 European ancestry [EA] and 14,330 African ancestry [AA]) and tested these variants for association with LDL-C, high-density lipoprotein cholesterol (HDL-C), and triglycerides. Although we did not identify new genes associated with LDL-C, we did identify four low-frequency (frequencies between 0.1% and 2%) variants (ANGPTL8 rs145464906 [c.361C>T; p.Gln121*], PAFAH1B2 rs186808413 [c.482C>T; p.Ser161Leu], COL18A1 rs114139997 [c.331G>A; p.Gly111Arg], and PCSK7 rs142953140 [c.1511G>A; p.Arg504His]) with large effects on HDL-C and/or triglycerides. None of these four variants was associated with risk for CHD, suggesting that examples of low-frequency coding variants with robust effects on both lipids and CHD will be limited. PMID:24507774
Peloso, Gina M; Auer, Paul L; Bis, Joshua C; Voorman, Arend; Morrison, Alanna C; Stitziel, Nathan O; Brody, Jennifer A; Khetarpal, Sumeet A; Crosby, Jacy R; Fornage, Myriam; Isaacs, Aaron; Jakobsdottir, Johanna; Feitosa, Mary F; Davies, Gail; Huffman, Jennifer E; Manichaikul, Ani; Davis, Brian; Lohman, Kurt; Joon, Aron Y; Smith, Albert V; Grove, Megan L; Zanoni, Paolo; Redon, Valeska; Demissie, Serkalem; Lawson, Kim; Peters, Ulrike; Carlson, Christopher; Jackson, Rebecca D; Ryckman, Kelli K; Mackey, Rachel H; Robinson, Jennifer G; Siscovick, David S; Schreiner, Pamela J; Mychaleckyj, Josyf C; Pankow, James S; Hofman, Albert; Uitterlinden, Andre G; Harris, Tamara B; Taylor, Kent D; Stafford, Jeanette M; Reynolds, Lindsay M; Marioni, Riccardo E; Dehghan, Abbas; Franco, Oscar H; Patel, Aniruddh P; Lu, Yingchang; Hindy, George; Gottesman, Omri; Bottinger, Erwin P; Melander, Olle; Orho-Melander, Marju; Loos, Ruth J F; Duga, Stefano; Merlini, Piera Angelica; Farrall, Martin; Goel, Anuj; Asselta, Rosanna; Girelli, Domenico; Martinelli, Nicola; Shah, Svati H; Kraus, William E; Li, Mingyao; Rader, Daniel J; Reilly, Muredach P; McPherson, Ruth; Watkins, Hugh; Ardissino, Diego; Zhang, Qunyuan; Wang, Judy; Tsai, Michael Y; Taylor, Herman A; Correa, Adolfo; Griswold, Michael E; Lange, Leslie A; Starr, John M; Rudan, Igor; Eiriksdottir, Gudny; Launer, Lenore J; Ordovas, Jose M; Levy, Daniel; Chen, Y-D Ida; Reiner, Alexander P; Hayward, Caroline; Polasek, Ozren; Deary, Ian J; Borecki, Ingrid B; Liu, Yongmei; Gudnason, Vilmundur; Wilson, James G; van Duijn, Cornelia M; Kooperberg, Charles; Rich, Stephen S; Psaty, Bruce M; Rotter, Jerome I; O'Donnell, Christopher J; Rice, Kenneth; Boerwinkle, Eric; Kathiresan, Sekar; Cupples, L Adrienne
Technological advances make it possible to use high-throughput sequencing as a primary discovery tool of medical genetics, specifically for assaying rare variation. Still this approach faces the analytic challenge that the influence of very rare variants can only be evaluated effectively as a group. A further complication is that any given rare variant could have no effect, could increase risk, or could be protective. We propose here the C-alpha test statistic as a novel approach for testing for the presence of this mixture of effects across a set of rare variants. Unlike existing burden tests, C-alpha, by testing the variance rather than the mean, maintains consistent power when the target set contains both risk and protective variants. Through simulations and analysis of case/control data, we demonstrate good power relative to existing methods that assess the burden of rare variants in individuals.
Voight, Benjamin F.; Altshuler, David; Devlin, Bernie; Orho-Melander, Marju; Kathiresan, Sekar; Purcell, Shaun M.; Roeder, Kathryn; Daly, Mark J.
Alternative mRNA splicing in the region encoding the C-terminus of nuclear receptors results in receptor variants lacking the entire ligand-binding domain (LBD), or a part of it, and instead contain a sequence of splice variant-specific C-terminal amino acids. A total of thirteen such splice variants have been shown to occur in vertebrates, and at least nine occur in humans. None of these receptor variants appear to be able to bind endogenous ligands and to induce transcription on promoters containing the response element for the respective canonical receptor variant. Interestingly, ten of these C-terminal splice variants have been shown to display dominant-negative activity on the transactivational properties of their canonical equivalent. Research on most of these splice variants has been limited, and the dominant-negative effect of these receptor variants has only been demonstrated in reporter assays in vitro, using transiently transfected receptors and reporter constructs. Therefore, the in vivo function and relevance of most C-terminal splice variants remains unclear. By reviewing the literature on the human glucocorticoid receptor ?-isoform (hGR?), we show that the dominant-negative effect of hGR? is well established using more physiologically relevant readouts. The hGR ?-isoform may alter gene transcription independent from the canonical receptor and increased hGR? levels correlate with glucocorticoid resistance and the occurrence of several immune-related diseases. Thus, available data suggests that C-terminal splice variants of nuclear receptors act as dominant-negative inhibitors of receptor-mediated signaling in vivo, and that aberrant expression of these isoforms may be involved in the pathogenesis of a variety of diseases.
van der Vaart, Michiel; Schaaf, Marcel J.M.
GATA binding protein 3 (GATA-3) is a novel immunohistochemical marker for urothelial carcinoma (UC); however, few studies have investigated GATA-3's role as a marker for UC variants. We used immunohistochemistry to assess GATA-3 expression in different UC variants, including micropapillary (n = 46), sarcomatoid (n = 43), small cell carcinoma (n = 22), and plasmacytoid (n = 16) variants, and we also compared GATA-3 expression in conventional bladder UC (n = 103) to that in squamous cell carcinoma (n = 14). GATA-3 expression was present in 70% (72/103) of conventional bladder UCs and highly concordant between matched primary and metastatic UCs. The GATA-3 expression levels of the micropapillary variants (57%; 26/46) and plasmacytoid variants (44%; 7/16) were not significantly different from that of conventional UC. However, the GATA-3 expression levels of the sarcomatoid variants (16%; 7/43) and small cell carcinoma variants (5%; 1/22), which only weakly expressed the protein, were significantly lower than that of conventional UC (P < .001). Only 7% of squamous cell carcinomas (1/14) expressed GATA-3, and it was also significantly lower than that of conventional UC (P < .001). GATA-3 expression was not significantly associated with tumor stage or patients' clinical outcomes. In conclusion, GATA-3 expression differed among UC variants. GATA-3 is a useful marker for confirming the urothelial origin of micropapillary and plasmacytoid UC variants but not that of sarcomatoid or small cell carcinoma variants. GATA-3 can also be used in differentiating UC from squamous cell carcinoma. PMID:24745616
Liang, Yu; Heitzman, Joseph; Kamat, Ashish M; Dinney, Colin P; Czerniak, Bogdan; Guo, Charles C
To demonstrate vertical transmission of hepatitis C virus (HCV) from an HCV-infected, non-human immunodeficiency virus type 1-infected mother to her infant and to assess the distribution of viral species in the mother and infant, the hypervariable region of the gene encoding the putative envelope glycoprotein E2 (E2HV) was sequenced in three mothers and one mother-infant pair. The data indicate that (i) quasi-species distributions of HCV E2HV variants were found in all four mothers, (ii) a single predominant HCV E2HV variant was found in the infant of a mother shown to have nine predominant E2HV variants, and (iii) the infant's E2HV variant was highly related to, but not identical with, the nine variants identified in the mother at the time of birth. These findings indicate that HCV is transmitted from mother to infant and raise the possibility that the transmission occurs in utero.
Weiner, A J; Thaler, M M; Crawford, K; Ching, K; Kansopon, J; Chien, D Y; Hall, J E; Hu, F; Houghton, M
Recent studies indicate that defective activity of complement factor H (FH) is associated with several human diseases, suggesting that pure FH may be used for therapy. Here, we describe a simple method to isolate human FH, based on the specific interaction between FH and the hypervariable region (HVR) of certain Streptococcus pyogenes M proteins. Special interest was focused on the FH polymorphism Y402H, which is associated with the common eye disease age-related macular degeneration (AMD) and has also been implicated in the binding to M protein. Using a fusion protein containing two copies of the M5-HVR, we found that the Y402 and H402 variants of FH could be efficiently purified by single-step affinity chromatography from human serum containing the corresponding protein. Different M proteins vary in their binding properties, and the M6 and M5 proteins, but not the M18 protein, showed selective binding of the FH Y402 variant. Accordingly, chromatography on a fusion protein derived from the M6-HVR allowed enrichment of the Y402 protein from serum containing both variants. Thus, the exquisite binding specificity of a bacterial protein can be exploited to develop a simple and robust procedure to purify FH and to enrich for the FH variant that protects against AMD.
Nilsson, O. Rickard; Lannergard, Jonas; Morgan, B. Paul; Lindahl, Gunnar; Gustafsson, Mattias C. U.
Background Lamivudine (LAM) is associated with the highest known rate of resistance mutations among nucleotide analogs used to treat chronic hepatitis B virus (HBV) infection. Despite this, LAM continues in widespread use, especially in combination therapies. The primary LAM resistance mutation (rtM204V/I) occurs in the YMDD motif of HBV polymerase. The aim of this study was to characterize Brazilian HBV isolates from acute and chronic cases by direct sequencing, and to identify HBV quasispecies in the YMDD motif using a pyrosequencing method capable of detecting single-nucleotide polymorphisms. HBV DNA from serum samples of 20 individuals with acute HBV infection and 44 with chronic infection undergoing antiviral therapies containing LAM were analyzed by direct sequencing and pyrosequencing methods. Results Phylogenic analyses of direct-sequenced isolates showed the expected genotypes (A, D and F) for the Brazilian population in both acute and chronic infections. However, within genotype A isolates, subgenotype A2 was more frequently detected in acute cases than in chronic cases (P?=?0.012). As expected, none of the individuals with acute hepatitis B had LAM-resistant isolates as a dominant virus population, whether detected by direct sequencing or pyrosequencing. However, pyrosequencing analyses showed that 45% of isolates (9/20) had minor subpopulations (4-17%) of LAM-resistant isolates. Among chronic patients undergoing LAM treatment, YMDD mutants were frequently found as a dominant virus population. In cases where wild-type virus was the dominant population, subpopulations of YMDD variants were usually found, demonstrating the complexity of HBV quasispecies. Conclusions YMDD variants were frequently detected as a minor population in acute HBV infection. The occurrence of pre-existing variants may lead to a high frequency of resistant mutants during antiviral therapy in the chronic phase. In chronic infection, detection of YMDD variants before virological or biochemical breakthrough might contribute to making better therapy choices and thus improving treatment outcome.
Adenovirus (Ad) vectors for gene therapy are made replication defective by deletion of E1 region genes. For isolation, propagation, and large-scale production of such vectors, E1 functions are supplied in trans from a stable cell line. Virtually all Ad vectors used for clinical studies are produced in the 293 cell, a human embryonic kidney cell line expressing E1 functions from an integrated segment of the left end of the Ad type 5 (Ad5) genome. Replication-competent vector variants that have regained E1 sequences have been observed within populations of Ad vectors grown on 293 cells. These replication-competent variants presumably result from recombination between vector and 293 cell Ad5 sequences. We have developed Ad2-based vectors and have characterized at the molecular level examples of replication-competent variants. All such variants analyzed are Ad2-Ad5 chimeras in which the 293 cell Ad5 E1 sequences have become incorporated into the viral genome by legitimate recombination events. A map of Ad5 sequences within the 293 cell genome developed in parallel is consistent with the proposed recombination events. To provide a convenient vector production system that circumvents the generation of replication-competent variants, we have modified the Ad2 vector backbone by deleting or rearranging the protein IX coding region normally present downstream from the E1 region such that the frequency of recombination between vector and 293 cell Ad5 sequences is greatly reduced. Twelve serial passages of an Ad2 vector lacking the protein IX gene were carried out without generating replication-competent variants. In the course of producing and testing more than 30 large-scale preparations of vectors lacking the protein IX gene or having a rearranged protein IX gene, only three examples of replication-competent variants were observed. Use of these genome modifications allows use of conventional 293 cells for production of large-scale preparations of Ad-based vectors lacking replication-competent variants.
Hehir, K M; Armentano, D; Cardoza, L M; Choquette, T L; Berthelette, P B; White, G A; Couture, L A; Everton, M B; Keegan, J; Martin, J M; Pratt, D A; Smith, M P; Smith, A E; Wadsworth, S C
NECTAR (Non-synonymous Enriched Coding muTation ARchive; http://nectarmutation.org) is a database and web application to annotate disease-related and functionally important amino acids in human proteins. A number of tools are available to facilitate the interpretation of DNA variants identified in diagnostic or research sequencing. These typically identify previous reports of DNA variation at a given genomic location, predict its effects on transcript and protein sequence and may predict downstream functional consequences. Previous reports and functional annotations are typically linked by the genomic location of the variant observed. NECTAR collates disease-causing variants and functionally important amino acid residues from a number of sources. Importantly, rather than simply linking annotations by a shared genomic location, NECTAR annotates variants of interest with details of previously reported variation affecting the same codon. This provides a much richer data set for the interpretation of a novel DNA variant. NECTAR also identifies functionally equivalent amino acid residues in evolutionarily related proteins (paralogues) and, where appropriate, transfers annotations between them. As well as accessing these data through a web interface, users can upload batches of variants in variant call format (VCF) for annotation on-the-fly. The database is freely available to download from the ftp site: ftp://ftp.nectarmutation.org. PMID:24297257
Gong, Sungsam; Ware, James S; Walsh, Roddy; Cook, Stuart A
Addition of N-linked glycosylation sites has been shown to increase serum half-life and decrease clearance for proteins such as recombinant erythropoietin (EPO). However, factor IX (FIX) variants with additional N-linked glycans ("HG" variants) that were expressed in HKB11 cells showed increased clearance in rat in vivo pharmacokinetic studies relative to FIX variants with no additional glycans. Variants with multiple additional glycans were the most rapidly cleared. A rat hepatocyte clearance assay was developed to measure intrinsic clearance of these FIX variants in vitro. The rank order of clearance of the variants was the same both in vivo and in the in vitro hepatocyte assay. In the in vitro assay, heparin, galactose, and asialo-orosomucoid inhibited clearance of a FIX HG variant by hepatocytes, and asialo-FIX was rapidly cleared, suggesting roles for the asialoglycoprotein receptor (ASGPR) and cell surface proteoglycans in FIX clearance. Thus the in vitro hepatocyte intrinsic clearance assay is both useful and predictive for identifying rapidly cleared recombinant proteins and for helping to identify receptors involved in clearance of proteins by the liver. PMID:24036269
Blasko, Eric; Brooks, Alan R; Ho, Elena; Wu, James M; Zhao, Xiao-Yan; Subramanyam, Babu
Interactions between mitochondrial deoxyribonucleic acid (mtDNA) variants and the risk of developing breast cancer were investigated using DNA samples collected from non-Jewish European American breast cancer patients and ethnically age-matched female controls. Logistic regression was used to evaluate two-way interactions between 17 mtDNA variants. To control for multiple testing, empirical P values were calculated using permutation. Odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were calculated to measure the contribution of variants in modifying the risk of developing breast cancer. A highly significant interaction was identified between variants 12308G and 10398G (empirical P value = 0.0028), with results suggesting these variants increase the risk of a woman developing breast cancer (OR = 3.03; 95% CI 1.53–6.11). Nominal significant P values were also observed for interactions between mtDNA variants 709A and 16189C; 4216C and 10398G; 4216C and 16189C; 10398G and 16159C; 13368A and 16189C; and 14766T and 16519C. However, after adjusting for multiple testing, the P values did not remain significant. Although it is important to elucidate the main effect of mtDNA variants on the risk of developing breast cancer, understanding gene × gene interactions will give a greater knowledge of disease etiology and aid in interpreting a woman's risk of developing breast cancer.
Covarrubias, Daniel; Bai, Ren-Kui; Wong, Lee-Jun C.; Leal, Suzanne M.
Background Variant discovery for rare genetic diseases using Illumina genome or exome sequencing involves screening of up to millions of variants to find only the one or few causative variant(s). Sequencing or alignment errors create "false positive" variants, which are often retained in the variant screening process. Methods to remove false positive variants often retain many false positive variants. This report presents VarBin, a method to prioritize variants based on a false positive variant likelihood prediction. Methods VarBin uses the Genome Analysis Toolkit variant calling software to calculate the variant-to-wild type genotype likelihood ratio at each variant change and position divided by read depth. The resulting Phred-scaled, likelihood-ratio by depth (PLRD) was used to segregate variants into 4 Bins with Bin 1 variants most likely true and Bin 4 most likely false positive. PLRD values were calculated for a proband of interest and 41 additional Illumina HiSeq, exome and whole genome samples (proband's family or unrelated samples). At variant sites without apparent sequencing or alignment error, wild type/non-variant calls cluster near -3 PLRD and variant calls typically cluster above 10 PLRD. Sites with systematic variant calling problems (evident by variant quality scores and biases as well as displayed on the iGV viewer) tend to have higher and more variable wild type/non-variant PLRD values. Depending on the separation of a proband's variant PLRD value from the cluster of wild type/non-variant PLRD values for background samples at the same variant change and position, the VarBin method's classification is assigned to each proband variant (Bin 1 to Bin 4). Results To assess VarBin performance, Sanger sequencing was performed on 98 variants in the proband and background samples. True variants were confirmed in 97% of Bin 1 variants, 30% of Bin 2, and 0% of Bin 3/Bin 4. Conclusions These data indicate that VarBin correctly classifies the majority of true variants as Bin 1 and Bin 3/4 contained only false positive variants. The "uncertain" Bin 2 contained both true and false positive variants. Future work will further differentiate the variants in Bin 2.
Recently, many statistical methods have been proposed to test for associations between rare genetic variants and complex traits. Most of these methods test for association by aggregating genetic variations within a predefined region, such as a gene. Although there is evidence that "aggregate" tests are more powerful than the single marker test, these tests generally ignore neutral variants and therefore are unable to identify specific variants driving the association with phenotype. We propose a novel aggregate rare-variant test that explicitly models a fraction of variants as neutral, tests associations at the gene-level, and infers the rare-variants driving the association. Simulations show that in the practical scenario where there are many variants within a given region of the genome with only a fraction causal our approach has greater power compared to other popular tests such as the Sequence Kernel Association Test (SKAT), the Weighted Sum Statistic (WSS), and the collapsing method of Morris and Zeggini (MZ). Our algorithm leverages a fast variational Bayes approximate inference methodology to scale to exome-wide analyses, a significant computational advantage over exact inference model selection methodologies. To demonstrate the efficacy of our methodology we test for associations between von Willebrand Factor (VWF) levels and VWF missense rare-variants imputed from the National Heart, Lung, and Blood Institute's Exome Sequencing project into 2,487 African Americans within the VWF gene. Our method suggests that a relatively small fraction (~10%) of the imputed rare missense variants within VWF are strongly associated with lower VWF levels in African Americans. PMID:24482836
Logsdon, Benjamin A; Dai, James Y; Auer, Paul L; Johnsen, Jill M; Ganesh, Santhi K; Smith, Nicholas L; Wilson, James G; Tracy, Russell P; Lange, Leslie A; Jiao, Shuo; Rich, Stephen S; Lettre, Guillaume; Carlson, Christopher S; Jackson, Rebecca D; O'Donnell, Christopher J; Wurfel, Mark M; Nickerson, Deborah A; Tang, Hua; Reiner, Alexander P; Kooperberg, Charles
Advances in genome sequencing are providing unprecedented resolution of rare and private variants. However, methods which assess the effect of these variants have relied predominantly on information within coding sequences. Assessing their impact in non-coding sequences remains a significant contemporary challenge. In this review, we highlight the role of regulatory variation as causative agents and modifiers of monogenic disorders. We further discuss how advances in functional genomics are now providing new opportunity to assess the impact of rare non-coding variants and their role in disease.
Li, Xin; Montgomery, Stephen B.
Voltage-gated sodium channels are the target sites of both DDT and pyrethroid insecticides. The importance of alternative splicing as a key mechanism governing the structural and functional diversity of sodium channels and the resulting development of insecticide and acaricide resistance is widely recognized, as shown by the extensive research on characterizing alternative splicing and variants of sodium channels in medically and agriculturally important insect species. Here we present the first comparative study of multiple variants of the sodium channel transcripts in the mosquito Culex quinquefasciatus. The variants were classified into two categories, CxNa-L and CxNa-S based on their distinguishing sequence sizes of ~6.5 kb and ~4.0 kb, respectively, and generated via major extensive alternative splicing with minor small deletions/ insertions in susceptible S-Lab, low resistant HAmCqG0, and highly resistant HAmCqG8 Culex strains. Four alternative Cx-Na-L splice variants were identified, including three full length variants with three optional exons (2, 5, and 21i) and one with in-frame-stop codons. Large, multi-exon-alternative splices were identified in the CxNa-S category. All CxNa-S splicing variants in the S-Lab and HAmCqG0 strains contained in-frame stop codons, suggesting that any resulting proteins would be truncated. The ~1000 to ~3000-fold lower expression of these splice variants with stop codons compared with the CxNa-L splicing variants may support the lower importance of these variants in S-Lab and HAmCqG0. Interestingly, two alternative splicing variants of CxNa-S in HAmCqG8 included entire ORFs but lacked exons 5 to18 and these two variants had much higher expression levels in HAmCqG8 than in S-Lab and HAmCqG0. These results provide a functional basis for further characterizing how alternative splicing of a voltage-gated sodium channel contributes to diversity in neuronal signaling in mosquitoes in response to pyrethroids, and possibly indicates the role of these variants in the development of pyrethroid resistance.
He, Lin; Li, Ting; Zhang, Lee; Liu, Nannan
Recent advances in sequencing technology have presented both opportunities and challenges, with limited statistical power to detect a single causal rare variant with practical sample sizes. To overcome this, the contributors to Group 1 of Genetic Analysis Workshop 17 sought to develop methods to detect the combined signal of multiple causal rare variants in a biologically meaningful way. The contributors used genes, genome location proximity, or genetic pathways as the basic unit in combining the information from multiple variants. Weaknesses of the exome sequence data and the relative strengths and weaknesses of the five approaches are discussed.
Ye, Kenny Q.; Engelman, Corinne D.
Shigella spp. are the causative agent of shigellosis with Shigella flexneri serotype 2a being the most prevalent in developing countries. Epidemiological surveillance in China found that a new serotype of S. flexneri appeared in 2001 and replaced serotype 2a in 2003 as the most prevalent serotype in Henan Province. The new serotype also became the dominant serotype in 7 of the 10 other provinces under surveillance in China by 2007. The serotype was identified as a variant of serotype X. It differs from serotype X by agglutination to the monovalent anti-IV type antiserum and the group antigen-specific monoclonal antibody MASF IV-I. Genome sequencing of a serotype X variant isolate, 2002017, showed that it acquired a Shigella serotype conversion island, also as an SfX bacteriophage, containing gtr genes for type X-specific glucosylation. Multilocus sequence typing of 15 genes from 37 serotype X variant isolates and 69 isolates of eight other serotypes, 1a, 2a, 2b, 3a, 4a, 5b, X, and Y, found that all belong to a new sequence type (ST), ST91. Pulsed-field gel electrophoresis revealed 154 pulse types with 655 S. flexneri isolates analyzed and identified 57 serotype switching events. The data suggest that S. flexneri epidemics in China have been caused by a single epidemic clone, ST91, with frequent serotype switching to evade infection-induced immunity to serotypes to which the population was exposed previously. The clone has also acquired resistance to multiple antibiotics. These findings underscore the challenges to the current vaccine development and control strategies for shigellosis.
Ye, Changyun; Lan, Ruiting; Xia, Shengli; Zhang, Jin; Sun, Qiangzheng; Zhang, Shaomin; Jing, Huaiqi; Wang, Lei; Li, Zhenjun; Zhou, Zhemin; Zhao, Ailan; Cui, Zhigang; Cao, Jingjing; Jin, Dong; Huang, Lili; Wang, Yiting; Luo, Xia; Bai, Xuemei; Wang, Yan; Wang, Ping; Xu, Qiang; Xu, Jianguo
During the present investigation a total of forty Indian animal isolates were screened by single strand conformation polymorphism\\u000a (SSCP) collected from sheep, goat, cattle and buffalo. The result of the study indicated that nuclear variants of Echinococcus granulosus were present in both small and large ruminants. SSCP phenotypes of AgB, intron of actin II and Hbx-2 have been deduced. Presence
D. Bhattacharya; D. Pan; A. K. Bera; A. Konar; S. K. Das
Meta-analysis of genome-wide association studies (GWASs) has led to the discoveries of many common variants associated with complex human diseases. There is a growing recognition that identifying “causal” rare variants also requires large-scale meta-analysis. The fact that association tests with rare variants are performed at the gene level rather than at the variant level poses unprecedented challenges in the meta-analysis. First, different studies may adopt different gene-level tests, so the results are not compatible. Second, gene-level tests require multivariate statistics (i.e., components of the test statistic and their covariance matrix), which are difficult to obtain. To overcome these challenges, we propose to perform gene-level tests for rare variants by combining the results of single-variant analysis (i.e., p values of association tests and effect estimates) from participating studies. This simple strategy is possible because of an insight that multivariate statistics can be recovered from single-variant statistics, together with the correlation matrix of the single-variant test statistics, which can be estimated from one of the participating studies or from a publicly available database. We show both theoretically and numerically that the proposed meta-analysis approach provides accurate control of the type I error and is as powerful as joint analysis of individual participant data. This approach accommodates any disease phenotype and any study design and produces all commonly used gene-level tests. An application to the GWAS summary results of the Genetic Investigation of ANthropometric Traits (GIANT) consortium reveals rare and low-frequency variants associated with human height. The relevant software is freely available.
Hu, Yi-Juan; Berndt, Sonja I.; Gustafsson, Stefan; Ganna, Andrea; Berndt, Sonja I.; Gustafsson, Stefan; Mägi, Reedik; Ganna, Andrea; Wheeler, Eleanor; Feitosa, Mary F.; Justice, Anne E.; Monda, Keri L.; Croteau-Chonka, Damien C.; Day, Felix R.; Esko, Tõnu; Fall, Tove; Ferreira, Teresa; Gentilini, Davide; Jackson, Anne U.; Luan, Jian’an; Randall, Joshua C.; Vedantam, Sailaja; Willer, Cristen J.; Winkler, Thomas W.; Wood, Andrew R.; Workalemahu, Tsegaselassie; Hu, Yi-Juan; Lee, Sang Hong; Liang, Liming; Lin, Dan-Yu; Min, Josine L.; Neale, Benjamin M.; Thorleifsson, Gudmar; Yang, Jian; Albrecht, Eva; Amin, Najaf; Bragg-Gresham, Jennifer L.; Cadby, Gemma; den Heijer, Martin; Eklund, Niina; Fischer, Krista; Goel, Anuj; Hottenga, Jouke-Jan; Huffman, Jennifer E.; Jarick, Ivonne; Johansson, Åsa; Johnson, Toby; Kanoni, Stavroula; Kleber, Marcus E.; König, Inke R.; Kristiansson, Kati; Kutalik, Zoltán; Lamina, Claudia; Lecoeur, Cecile; Li, Guo; Mangino, Massimo; McArdle, Wendy L.; Medina-Gomez, Carolina; Müller-Nurasyid, Martina; Ngwa, Julius S.; Nolte, Ilja M.; Paternoster, Lavinia; Pechlivanis, Sonali; Perola, Markus; Peters, Marjolein J.; Preuss, Michael; Rose, Lynda M.; Shi, Jianxin; Shungin, Dmitry; Smith, Albert Vernon; Strawbridge, Rona J.; Surakka, Ida; Teumer, Alexander; Trip, Mieke D.; Tyrer, Jonathan; Van Vliet-Ostaptchouk, Jana V.; Vandenput, Liesbeth; Waite, Lindsay L.; Zhao, Jing Hua; Absher, Devin; Asselbergs, Folkert W.; Atalay, Mustafa; Attwood, Antony P.; Balmforth, Anthony J.; Basart, Hanneke; Beilby, John; Bonnycastle, Lori L.; Brambilla, Paolo; Bruinenberg, Marcel; Campbell, Harry; Chasman, Daniel I.; Chines, Peter S.; Collins, Francis S.; Connell, John M.; Cookson, William; de Faire, Ulf; de Vegt, Femmie; Dei, Mariano; Dimitriou, Maria; Edkins, Sarah; Estrada, Karol; Evans, David M.; Farrall, Martin; Ferrario, Marco M.; Ferrières, Jean; Franke, Lude; Frau, Francesca; Gejman, Pablo V.; Grallert, Harald; Grönberg, Henrik; Gudnason, Vilmundur; Hall, Alistair S.; Hall, Per; Hartikainen, Anna-Liisa; Hayward, Caroline; Heard-Costa, Nancy L.; Heath, Andrew C.; Hebebrand, Johannes; Homuth, Georg; Hu, Frank B.; Hunt, Sarah E.; Hyppönen, Elina; Iribarren, Carlos; Jacobs, Kevin B.; Jansson, John-Olov; Jula, Antti; Kähönen, Mika; Kathiresan, Sekar; Kee, Frank; Khaw, Kay-Tee; Kivimaki, Mika; Koenig, Wolfgang; Kraja, Aldi T.; Kumari, Meena; Kuulasmaa, Kari; Kuusisto, Johanna; Laitinen, Jaana H.; Lakka, Timo A.; Langenberg, Claudia; Launer, Lenore J.; Lind, Lars; Lindström, Jaana; Liu, Jianjun; Liuzzi, Antonio; Lokki, Marja-Liisa; Lorentzon, Mattias; Madden, Pamela A.; Magnusson, Patrik K.; Manunta, Paolo; Marek, Diana; März, Winfried; Leach, Irene Mateo; McKnight, Barbara; Medland, Sarah E.; Mihailov, Evelin; Milani, Lili; Montgomery, Grant W.; Mooser, Vincent; Mühleisen, Thomas W.; Munroe, Patricia B.; Musk, Arthur W.; Narisu, Narisu; Navis, Gerjan; Nicholson, George; Nohr, Ellen A.; Ong, Ken K.; Oostra, Ben A.; Palmer, Colin N.A.; Palotie, Aarno; Peden, John F.; Pedersen, Nancy; Peters, Annette; Polasek, Ozren; Pouta, Anneli; Pramstaller, Peter P.; Prokopenko, Inga; Pütter, Carolin; Radhakrishnan, Aparna; Raitakari, Olli; Rendon, Augusto; Rivadeneira, Fernando; Rudan, Igor; Saaristo, Timo E.; Sambrook, Jennifer G.; Sanders, Alan R.; Sanna, Serena; Saramies, Jouko; Schipf, Sabine; Schreiber, Stefan; Schunkert, Heribert; Shin, So-Youn; Signorini, Stefano; Sinisalo, Juha; Skrobek, Boris; Soranzo, Nicole; Stan?áková, Alena; Stark, Klaus; Stephens, Jonathan C.; Stirrups, Kathleen; Stolk, Ronald P.; Stumvoll, Michael; Swift, Amy J.; Theodoraki, Eirini V.; Thorand, Barbara; Tregouet, David-Alexandre; Tremoli, Elena; Van der Klauw, Melanie M.; van Meurs, Joyce B.J.; Vermeulen, Sita H.; Viikari, Jorma; Virtamo, Jarmo; Vitart, Veronique; Waeber, Gérard; Wang, Zhaoming; Widén, Elisabeth; Wild, Sarah H.; Willemsen, Gonneke; Winkelmann, Bernhard R.; Witteman, Jacqueline C.M.; Wolffenbuttel, Bruce H.R.; Wong, Andrew; Wright, Alan F.
The inducing capacity of cefpirome (HR 810) and the ability of the compound to select for stable derepressed mutants was determined and compared with those of cefodizime (HR 221), cefotaxime, ceftazidime and cefamandol. Variations in both characteristics between and within species was observed. Overall, cefodizime showed the lowest, cefamandol the highest inducing capacity. Antibiotic resistant variants were isolated from all strains tested at a frequency of around 10(-9). A stable increased enzyme production was found in Pseudomonas aeruginosa after exposure to ceftazidime as well as in the resistant mutants from Enterobacter cloacae after selection with cefpirome, ceftazidime, cefotaxime and cefamandol. In the other resistant mutants the resistance was probably due to changes in permeability. All resistant variants remained relatively susceptible to cefpirome. PMID:3072153
Stobberingh, E E; Houben, A W
The disclosure provides proteins that can be used to determine the redox status of an environment (such as the environment within a cell or subcellular compartment). These proteins are green fluorescent protein (GFP) variants (also referred to as redox se...
G. T. Hanson S. J. Remington
The present paper describes the structure of MmtDB-a specialized database designed to collect Metazoa mitochondrial DNA variants. Priority in the data collection is given to the Metazoa species for which a large amount of variants is available, as it is the case for human variants. Starting from the sequences available in the Nucleotide Sequence Databases, the redundant sequences are removed and new sequences from other sources are added. Value-added information are associated to each variant sequence, e.g. analysed region, experimental method, tissue and cell lines, population data, sex, age, family code and information about the variation events (nucleotide position, involved gene, restriction site's gain or loss). Cross-references are introduced to the EMBL Data Library, as well as an internal cross-referencing among MmtDB entries according to their tissual, heteroplasmic, familiar and aplotypical correlation. MmtDB can be accessed through the World Wide Web at URL [see text].
Calo, D; De Pascali, A; Sasanelli, D; Tanzariello, F; Tommaseo Ponzetta, M; Saccone, C; Attimonelli, M
The frequency of enzyme deficiency variants, defined as alleles whose products are either absent or almost devoid of normal activity in erythrocytes, was determined for nine erythrocyte enzymes in some 675 newborn infants and in approximately 200 adults. Examples of this type of genetic abnormality, which in the homozygous condition are often associated with significant health consequences, were detected for seven of the nine enzymes studied. Fifteen inherited enzyme deficiency variants in 1809 determinations from adults were identified. Seven of the deficiency variants involved triosephosphate isomerase, a frequency of 0.01 in the newborn population. The average frequency of 2.4/1000 is 2 to 3 times the frequency observed for rare electrophoretic variants of erythrocyte enzymes in this same population.