scholarly journals Cloning of complete genome sets of six dsRNA viruses using an improved cloning method for large dsRNA genes

2002 ◽  
Vol 83 (9) ◽  
pp. 2215-2223 ◽  
Author(s):  
A. C. Potgieter ◽  
A. D. Steele ◽  
A. A. van Dijk
Keyword(s):  
Complete Genome ◽  
Vaccine Development ◽  
Sequence Data ◽  
Pcr Amplification ◽  
Cloning Method ◽  
Dsrna Genome ◽  
Stool Samples ◽  
Large Genes

Cloning full-length large (>3 kb) dsRNA genome segments from small amounts of dsRNA has thus far remained problematic. Here, a single-primer amplification sequence-independent dsRNA cloning procedure was perfected for large genes and tailored for routine use to clone complete genome sets or individual genes. Nine complete viral genome sets were amplified by PCR, namely those of two human rotaviruses, two African horsesickness viruses (AHSV), two equine encephalosis viruses (EEV), one bluetongue virus (BTV), one reovirus and bacteriophage Φ12. Of these amplified genomes, six complete genome sets were cloned for viruses with genes ranging in size from 0·8 to 6·8 kb. Rotavirus dsRNA was extracted directly from stool samples. Co-expressed EEV VP3 and VP7 assembled into core-like particles that have typical orbivirus capsomeres. This work presents the first EEV sequence data and establishes that EEV genes have the same conserved termini (5′ GUU and UAC 3′) and coding assignment as AHSV and BTV. To clone complete genome sets, one-tube reactions were developed for oligo-ligation, cDNA synthesis and PCR amplification. The method is simple and efficient compared to other methods. Complete genomes can be cloned from as little as 1 ng dsRNA and a considerably reduced number of PCR cycles (22–30 cycles compared to 30–35 of other methods). This progress with cloning large dsRNA genes is important for recombinant vaccine development and determination of the role of terminal sequences for replication and gene expression.

Frequent LOH of CYP2D6 in ER+ breast cancer determined by next-generation sequencing (NGS).

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 534-534
Author(s):  
Mark J. Ratain ◽  
James Sun ◽  
Yusuke Nakamura ◽  
Nancy J Cox ◽  
Tarek Sahmoud ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Sequence Data ◽  
Biological Significance ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Full Cohort ◽  
Next Generation Sequencing Ngs ◽  
Apparent Association

534 Background: The role of CYP2D6 genetic variation in predicting response to tamoxifen in ER+ breast cancer is a subject of ongoing debate. There has been great variability in approaches to both genotyping and phenotyping, and in particular many investigators have extracted DNA from breast cancer samples rather than peripheral blood. We hypothesized that CYP2D6 gene copy number alterations are common in ER+ breast cancer, affecting genotype results, and used NGS to characterize CYP2D6 in patients with ER+ disease. Methods: CYP2D6 sequencing was performed as part of a comprehensive NGS profile of cancer-related genes for 261 predominantly relapsed and metastatic ER+ breast cancer FFPE specimens. Sequence data were resolved into genotypes according to the * allele nomenclature. Tumor LOH was determined at CYP2D6, and its error impact on genotyping methods was estimated. To assess biological significance, the prevalence of CYP2D6 alleles and LOH in ER+ disease was compared against a control set of 99 ER- tumors. Results: CYP2D6 allele frequencies in our full cohort (ER+, 261; ER-, 99) were consistent with prior studies; 64.4%, 16.8%, 9.0% vs. 63.1%, 17.2%, 7.0% for *1/*2, *4, and *41 respectively, and 1%-2% for the rarer alleles *9, *10, and *5. The rate of CYP2D6 LOH was higher in ER+ disease (41% vs. 26%, p<0.01), with all excess arising from copy-loss (as opposed to copy-neutral) changes (22% vs. 7%, p<0.002). The estimated impact of LOH on germline genotype assessment from tumor was considerable; an assay sensitive at >20% minor allele frequency (e.g., Sanger sequencing) can misclassify >10% of heterozygotes, leading to significant Hardy-Weinberg disequilibrium (e.g., p=8.3x10-8 for *4). Interestingly, an enrichment of reduced or non-functional CYP2D6 alleles in ER+ samples was observed (61% vs. 47%, p<0.03). Conclusions: Our results demonstrate the distorting effect of extensive LOH on genotype assessment of CYP2D6 in breast cancer. Therefore, tumor DNA should not be routinely used for determination of germline 2D6 genotype, although it appears possible to use NGS. The apparent association between reduced function CYP2D6 alleles and ER+ breast cancer in our dataset requires further investigation.

scholarly journals The impact of DNA polymerase and number of rounds of amplification in PCR on 16S rRNA gene sequence data

2019 ◽  
Author(s):  
Marc A Sze ◽  
Patrick D Schloss
Keyword(s):  
16S Rrna ◽  
Microbial Communities ◽  
Error Rate ◽  
Sequence Data ◽  
Pcr Amplification ◽  
Sequencing Error ◽  
Mock Community ◽  
Sequencing Error Rate ◽  
Stool Samples ◽  
Human Stool

AbstractPCR amplification of 16S rRNA genes is a critical, yet under appreciated step in the generation of sequence data to describe the taxonomic composition of microbial communities. Numerous factors in the design of PCR can impact the sequencing error rate, the abundance of chimeric sequences, and the degree to which the fragments in the product represent their abundance in the original sample (i.e. bias). We compared the performance of high fidelity polymerases and varying number of rounds of amplification when amplifying a mock community and human stool samples. Although it was impossible to derive specific recommendations, we did observe general trends. Namely, using a polymerase with the highest possible fidelity and minimizing the number of rounds of PCR reduced the sequencing error rate, fraction of chimeric sequences, and bias. Evidence of bias at the sequence level was subtle and could not be ascribed to the fragments’ fraction of bases that were guanines or cytosines. When analyzing mock community data, the amount that the community deviated from the expected composition increased with rounds of PCR. This bias was inconsistent for human stool samples. Overall the results underscore the difficulty of comparing sequence data that are generated by different PCR protocols. However, the results indicate that the variation in human stool samples is generally larger than that introduced by the choice of polymerase or number of rounds of PCR.ImportanceA steep decline in sequencing costs drove an explosion in studies characterizing microbial communities from diverse environments. Although a significant amount of effort has gone into understanding the error profiles of DNA sequencers, little has been done to understand the downstream effects of the PCR amplification protocol. We quantified the effects of the choice of polymerase and number of PCR cycles on the quality of downstream data. We found that these choices can have a profound impact on the way that a microbial community is represented in the sequence data. The effects are relatively small compared to the variation in human stool samples, however, care should be taken to use polymerases with the highest possible fidelity and to minimize the number of rounds of PCR. These results also underscore that it is not possible to directly compare sequence data generated under different PCR conditions.

scholarly journals Multiple novel astrovirus species in human stool

2009 ◽  
Vol 90 (12) ◽  
pp. 2965-2972 ◽  
Author(s):  
A. Kapoor ◽  
L. Li ◽  
J. Victoria ◽  
B. Oderinde ◽  
C. Mason ◽  
...  
Keyword(s):  
Developing Countries ◽  
Genetic Distance ◽  
Complete Genome ◽  
Rt Pcr ◽  
Rna Sequences ◽  
Human Astroviruses ◽  
Human Astrovirus ◽  
Stool Samples ◽  
Human Stool

Diarrhoea remains a significant cause of morbidity and mortality in developing countries where numerous cases remain without identified aetiology. Astroviruses are a recently identified cause of animal gastroenteritis which currently includes two species suspected of causing human diarrhoea. Using pan-astrovirus RT-PCR, we analysed human stool samples from different continents for astrovirus-related RNA sequences. We identified variants of the two known human astrovirus species plus, based on genetic distance criteria, three novel astrovirus species all distantly related to mink and ovine astroviruses, which we provisionally named HMOAstV species A–C. The complete genome of species A displayed all the conserved characteristics of mammalian astroviruses. Each of the now three groups of astroviruses found in human stool (HAstV, AstV-MLB and HMOAstV) were more closely related to animal astroviruses than to each other, indicating that human astroviruses may periodically emerge from zoonotic transmissions. Based on the pathogenic impact of their closest phylogenetic relatives in animals, further investigations of the role of HMOAstV, so far detected in Nigeria, Nepal and Pakistan, in human gastroenteritis are warranted.

scholarly journals The Impact of DNA Polymerase and Number of Rounds of Amplification in PCR on 16S rRNA Gene Sequence Data

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Marc A. Sze ◽  
Patrick D. Schloss
Keyword(s):  
16S Rrna ◽  
Microbial Communities ◽  
Error Rate ◽  
Sequence Data ◽  
Pcr Amplification ◽  
Sequencing Error ◽  
Mock Community ◽  
Sequencing Error Rate ◽  
Stool Samples ◽  
Human Stool

ABSTRACTPCR amplification of 16S rRNA genes is a critical yet underappreciated step in the generation of sequence data to describe the taxonomic composition of microbial communities. Numerous factors in the design of PCR can impact the sequencing error rate, the abundance of chimeric sequences, and the degree to which the fragments in the product represent their abundance in the original sample (i.e., bias). We compared the performance of high fidelity polymerases and various numbers of rounds of amplification when amplifying a mock community and human stool samples. Although it was impossible to derive specific recommendations, we did observe general trends. Namely, using a polymerase with the highest possible fidelity and minimizing the number of rounds of PCR reduced the sequencing error rate, fraction of chimeric sequences, and bias. Evidence of bias at the sequence level was subtle and could not be ascribed to the fragments’ fraction of bases that were guanines or cytosines. When analyzing mock community data, the amount that the community deviated from the expected composition increased with the number of rounds of PCR. This bias was inconsistent for human stool samples. Overall, the results underscore the difficulty of comparing sequence data that are generated by different PCR protocols. However, the results indicate that the variation in human stool samples is generally larger than that introduced by the choice of polymerase or number of rounds of PCR.IMPORTANCEA steep decline in sequencing costs drove an explosion in studies characterizing microbial communities from diverse environments. Although a significant amount of effort has gone into understanding the error profiles of DNA sequencers, little has been done to understand the downstream effects of the PCR amplification protocol. We quantified the effects of the choice of polymerase and number of PCR cycles on the quality of downstream data. We found that these choices can have a profound impact on the way that a microbial community is represented in the sequence data. The effects are relatively small compared to the variation in human stool samples; however, care should be taken to use polymerases with the highest possible fidelity and to minimize the number of rounds of PCR. These results also underscore that it is not possible to directly compare sequence data generated under different PCR conditions.

scholarly journals Determination of Geohelminthiasis and Its Association with Allergic Sensitization among Selected Children in Batangas, Philippines

2016 ◽  
Vol 9 (2) ◽  
pp. 33 ◽  
Author(s):  
Carina R. Magbojos ◽  
Shereen O. Chua ◽  
Charlene R. Alegria ◽  
Faustina C. Macalalad ◽  
Anne Camille R. Malayba ◽  
...  
Keyword(s):  
Allergic Sensitization ◽  
International Study ◽  
Allergic Diseases ◽  
Cross Reactivity ◽  
Total Ige ◽  
Stool Samples ◽  
Serum Total Ige ◽  
Level Of Significance

<p>Several studies have explored the cross-reactivity between geohelminthiasis and allergy which share the same immune response in humans by triggering the increase in serum IgE level. Yet, the role of soil-transmitted intestinal parasitism as a protective or contributing factor for allergy remains inconclusive. In this study, the association of geohelminthiasis with allergic sensitization was investigated in a cohort of 50 children aged 5 to 12 years old residing in the areas of Batangas, Philippines where parasitism is prevalent. Stool samples of the subjects were qualitatively examined for the presence of helminth infection through modified Kato-Katz technique. They were also assessed for allergic diseases using the modified International Study of Asthma and Allergies in Childhood Phase I questionnaire. Fecalysis results were correlated with their serum total IgE (tIgE) levels and screening test results for allergic sensitization to common aeroallergens and food allergens determined through ImmunoCAP<sup>®</sup> system. Of the participants, 78% were under 10 years old while 22% were aged 10 years old and above. Mixed infection of <em>Ascaris lumbricoides </em>and<em> Trichuris trichiura </em>were found to be the most common (40%) followed by trichuriasis (34%) and ascariasis (26%). Atopic allergy to inhalants is prevalent in children under 10 years old (74.36%) whereas, some of them (5.13%) are fX2 positive which confirms the participants’ allergy to seafood. It was revealed that the occurrence of geohelminthic infection and allergy may affect any child regardless of age and serum total IgE level does not considerably vary with age, geohelminthiasis and presence of allergy. Hence, geohelminthiasis does not play contributory nor inhibitory role in allergic sensitization as established by a 0.05 level of significance (p≤0.05).<strong></strong></p>

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

1987 ◽  
Vol 26 (01) ◽  
pp. 1-6 ◽  
Author(s):  
S. Selvaraj ◽  
M. R. Suresh ◽  
G. McLean ◽  
D. Willans ◽  
C. Turner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
T Antigen ◽  
Human Carcinomas ◽  
Animal Tumor ◽  
Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

On The Mechanism Of Heparin-Induced Potentiation Of Platelet Aggregation

1981 ◽  
Author(s):  
M Yamamoto ◽  
K Watanabe ◽  
Y Ando ◽  
H Iri ◽  
N Fujiyama ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Coagulation Factors ◽  
Marked Enhancement ◽  
Matrix Bound ◽  
Induced Aggregation ◽  
Aggregation Of Platelets ◽  
Plasma Heparin

It has been suggested that heparin caused potentiation of aggregation induced by ADP or epinephrine. The exact mechanism of heparin-induced platelet activation, however, remained unknown. In this paper, we have investigated the role of anti-thrombin III ( AT ) in heparin-induced platelet activation using purified AT and AT depleted plasma. When ADP or epinephrine was added to citrated PRP one minute after addition of heparin ( 1 u/ml, porcine intestinal mucosal heparin, Sigma Co. USA ), marked enhancement of platelet aggregation was observed, compared with the degree of aggregation in the absence of heparin. However, in platelet suspensions prepared in modified Tyrode’s solution, heparin exhibited no potentiating effect on platelet aggregation induced by epinephrine or ADP. Potentiation of epinephrine- or ADP-induced platelet aggregation by heparin was demonstrated when purified AT was added to platelet suspensions at a concentration of 20 μg/ml. AT depleted plasma, which was prepared by immunosorption using matrix-bound antibodies to AT, retained no AT, while determination of α1-antitrypsinα2- macroglobulin and fibrinogen in AT depleted plasma produced values which corresponded to those of the original plasma when dilution factor was taken into account. The activities of coagulation factors were also comparable to those of the original plasma. Heparin exhibited potentiating effect on ADP- or epinephrine-induced aggregation of platelets in original plasma, but no effect in AT depleted plasma. When purified AT was added back to AT depleted plasma at a concentration of 20 μg/ml, potentiation of platelet aggregation by heparin was clearly demonstrated.Our results suggest that effect of heparin on platelet aggregation is also mediated by anti-thrombin III.

ІНТЕРПРЕТАЦІЯ КАТЕГОРІЇ РОЗВИТКУ В СУЧАСНІЙ ПСИХОЛОГІЇ

1970 ◽  
Vol 8 (1) ◽  
pp. 22-30
Author(s):  
Анжеліка Шамне
Keyword(s):  
Cultural Context ◽  
Developmental Psychology ◽  
Structure And Dynamics ◽  
Modern Psychology ◽  
Development Site ◽  
Approaches To Study ◽  
Methodological Approaches ◽  
Self Motion

У статті розглянуто сучасні підходи до інтерпретації категорії розвитку, розкрито теоретичні  та методологічні підходи до вивчення категорії розвитку у сучасній психології, визначено її психологічний  зміст,   моделі,   структуру   та   динаміку.   Категорія   розвитку   розглядається   як   епіцентр   наукової  проблематики у психології та як поняття інтегративного типу. Розвиток проаналізовано як категорію,  явище і проблему психології розвитку в різних аспектах аналізу. Розглянуто місце розвитку в системі  споріднених психологічних понять. У статті також аналізуються психологічні аспекти теоретичних та  методологічних  постнекласичних  тенденцій  вивчення  природи,  характеру  та  визначення  психічного  розвитку. Постнекласична парадигма та плюралістична методологія пізнання визначають розмитість  дисциплінарної мови  та  врахування  ролі  соціокультурного  контексту  при  вивченні  психологічних явищ.  Важливими тенденціями сучасного теоретико-методологічного стану психологічних досліджень розвитку  також є визнання неефективності моністичного підходу до його вивчення, взаємозв'язок теоретичних ідей  та   спроби   створення   метатеоретичних   схем,   постнекласичне   розуміння   розвитку   як   принципово  незавершеного   процесу   саморуху,   актуалізація   антропологічного   діапазону   проблем   та   посилення  спрямованості на роль культурного контексту в дослідженні розвитку людини.  The article deals with the modern approaches to the interpretation of the category of development, reveals  the theoretical and methodological approaches to study of development in modern psychology, its psychological  content, patterns, structure and dynamics. Category of development is viewed as an epicenter of scientific issues in  modern  psychology  and  the  concept  of  the  integrative  type.  Category  of  development  is  considered  as  the  phenomenon  and  the  problem  of  developmental  psychology  in  various  aspects  of  the  analysis.  Analyzed  the  development site in the related psychological concepts. The article analyzes the psychological aspects of theoretical  and methodological postnonclassical contemporary trends in the study of nature, character, and determination of  mental  development.  Postnonclassical  paradigm  and  pluralistic  methodology  of  knowledge  determine  the  disciplinary blurring and increase of the role of the analysis of socio-cultural context in the study of psychological  phenomenon. The important tendencies of modern theoretical and methodological state of psychological researches  of development are facts of inefficiency of the monistic approach to its study, interconnection of theoretical ideas  and   attempts   of  creating   metatheoretical   schemes,   postnonclassical   understanding   of   development   as   a  fundamentally  uncompleted  process  of  self-motion,  actualization  of  anthropological  range  of  problems  and  strengthening of focus on the role of cultural context in research of human development.   

Role of the family focused technologies in the clinical course of pregnancy at women of high obstetric risk

2016 ◽  
pp. 64-66
Author(s):  
S.Yu. Vdovichenko ◽  
Keyword(s):  
Comparison Group ◽  
Traditional Approach ◽  
Married Couple ◽  
Premature Births ◽  
Individual Support ◽  
The Family ◽  
Risk Patients ◽  
Individual Preparation

The objective: to show a role of the family focused technologies in depression of frequency of pathology of pregnancy at women of high obstetric risk. Patients and methods. For determination of efficiency of prophylaxis of pathology of pregnancy on the basis of use of the family focused technologies complex clinical-psychological and laboratory and tool examination of 300 women with factors of obstetric risk which were divided into two groups was conducted. In the main group – 182 women with motivation on partner labors to which provided training on system of individual preparation of married couple to labors. The comparison group consisted of 118 women who were not in prenatal training and had individual support in childbirth, with the traditional approach to pain management. Results. Use of the family focused technologies during pregnancy allows to reduce significantly the frequency of the main complications of pregnancy, especially not incubation and premature births. Conclusion. In our opinion, the technique is simple, available and can widely be used in practical health care at women with high obstetric risk. Key words: obstetric risk, the family focused technologies, prophylaxis.

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