Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens

We developed a conceptually new subtraction strategy for the detection and isolation of target DNA and/or RNA from complex nucleic acid mixtures, called Primer Extension Enrichment Reaction (PEER). PEER uses adapters and class IIS restriction enzymes to generate tagged oligonucleotides from dsDNA fragments derived from specimens containing an unknown target (‘tester’). Subtraction is achieved by selectively disabling these oligonucleotides by extension reaction using ddNTPs and a double stranded DNA template generated from a pool of normal specimens (‘driver’). Primers that do not acquire ddNTP are used to capture and amplify the unique target DNA from the original tester dsDNA. We successfully applied PEER to specimens containing known infectious agents (Hepatitis B Virus and Walrus Calicivirus) and demonstrated that it has higher efficiency than the best comparable technique. The strategy used for PEER is versatile and can be adapted for the identification of known and unknown pathogens and mutations, differential expression studies and other applications that allow the use of subtractive strategies

Prevalence of hepatitis B antiviral drug resistance variants in North American patients with chronic hepatitis B not receiving antiviral treatment

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138913/1/jvh12732.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138913/2/jvh12732_am.pd

Solution structure and dynamics of DNA duplexes containing the universal base analogues 5-nitroindole and 5-nitroindole 3-carboxamide

Universal bases hybridize with all other natural DNA or RNA bases, and have applications in PCR and sequencing. We have analysed by nuclear magnetic resonance spectroscopy the structure and dynamics of three DNA oligonucleotides containing the universal base analogues 5-nitroindole and 5-nitroindole-3-carboxamide. In all systems studied, both the 5-nitroindole nucleotide and the opposing nucleotide adopt a standard anti conformation and are fully stacked within the DNA duplex. The 5-nitroindole bases do not base pair with the nucleotide opposite them, but intercalate between this base and an adjacent Watson–Crick pair. In spite of their smooth accommodation within the DNA double-helix, the 5-nitroindole-containing duplexes exist as a dynamic mixture of two different stacking configurations exchanging fast on the chemical shift timescale. These configurations depend on the relative intercalating positions of the universal base and the opposing base, and their exchange implies nucleotide opening motions on the millisecond time range. The structure of these nitroindole-containing duplexes explains the mechanism by which these artificial moieties behave as universal bases

What factors determine the severity of hepatitis A‐related acute liver failure?

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87020/1/j.1365-2893.2010.01410.x.pd

Experimentelle Kaninchensyphilis und das Reticuloendothelialsystem

Bei experimenteller Kaninchensyphilis fuhrte der Verfasser seine Versuche aus, berucksichtigt auf die klinischen Befunde und die Wassermannsche Reaktion des mit Sp. pallida geimpften Kaninchens bei der Splenektomie, bei der Blockierung des Reticuloendothelialsystemes und bei der Einspritzung des Milzextractes. Die Resultate sind die folgenden: 1. Nachdem Spirochaeta pallida dem Kaninchen in den Hoden geimpft wurde, wurde die Blockierung mit Tusche ausgefuhrt. Im Verlaufe der Infektion zeigten sich lokale Befunde und Wassermannsche Reaktion die in jeder Hinsicht denen der Kontrollen glichen. 2. Beim Kaninchen, welches gleichzeizig entmilzt und mit Sp. pallida geimpft wurde, trat der Lokalaffekt etwas fruhzeizig auf und die Wassermannsche Reaktion war kurzdauernd positiv. 3. Falls die Impfung nach der Entmilzung ausgefuhrt war, stimmten die Resultate fast mit den obigen ein. 4. Wenn die Versuchstiere erst geimpft und dann entmilzt wurden, so wurde eine bemerkenswerte oedematose Anschwellung des Hodensacks und ein Kurzdauernder positive Ausfall der Wa. R. beobachtet. 5. Wenn die Entmilzung, Impfung und Blockierung gleichzeizig ausgefuhrt wurden, so zeigten sich die lokalen sowie metastatischen Befunde bei Versuchstieren wie ohne Entmilzung, doch war die positive Phase der Wa. R. bedeutend verkurzt und dabei ihre Grad niederwertig. 6. Erst geimpft und dann mit Milzextract wiederhohlt gespritzt, so verliefen die lokalen sowie metastatischen Befunde leichter und die Wa. R. brach fruhzeizig positiv aus und dauerte langer. (Autoreferat.

Evaluation of Intra-Host Variants of the Entire Hepatitis B Virus Genome

Genetic analysis of hepatitis B virus (HBV) frequently involves study of intra-host variants, identification of which is commonly achieved using short regions of the HBV genome. However, the use of short sequences significantly limits evaluation of genetic relatedness among HBV strains. Although analysis of HBV complete genomes using genetic cloning has been developed, its application is highly labor intensive and practiced only infrequently. We describe here a novel approach to whole genome (WG) HBV quasispecies analysis based on end-point, limiting-dilution real-time PCR (EPLD-PCR) for amplification of single HBV genome variants, and their subsequent sequencing. EPLD-PCR was used to analyze WG quasispecies from serum samples of patients (n = 38) infected with HBV genotypes A, B, C, D, E and G. Phylogenetic analysis of the EPLD-isolated HBV-WG quasispecies showed the presence of mixed genotypes, recombinant variants and sub-populations of the virus. A critical observation was that HBV-WG consensus sequences obtained by direct sequencing of PCR fragments without EPLD are genetically close, but not always identical to the major HBV variants in the intra-host population, thus indicating that consensus sequences should be judiciously used in genetic analysis. Sequence-based studies of HBV WG quasispecies should afford a more accurate assessment of HBV evolution in various clinical and epidemiological settings

Transparency of reporting in molecular diagnostics

The major advances made over the past few years in molecular and cell biology are providing a progressively more detailed understanding of the molecular pathways that control normal processes and become dysregulated in disease. This has resulted in the documentation of numerous genetic, epigenetic, transcriptomic, proteomic and metabolomic biomarkers that promise earlier disease detection, more accurate patient stratification and better prognosis. Furthermore, molecular fingerprinting of diseases can be predictive of drug response and so assist with specific targeting of drugs against disease-associated molecules and function

Epidemic History and Evolutionary Dynamics of Hepatitis B Virus Infection in Two Remote Communities in Rural Nigeria

BACKGROUND: In Nigeria, hepatitis B virus (HBV) infection has reached hyperendemic levels and its nature and origin have been described as a puzzle. In this study, we investigated the molecular epidemiology and epidemic history of HBV infection in two semi-isolated rural communities in North/Central Nigeria. It was expected that only a few, if any, HBV strains could have been introduced and effectively transmitted among these residents, reflecting limited contacts of these communities with the general population in the country. METHODS AND FINDINGS: Despite remoteness and isolation, approximately 11% of the entire population in these communities was HBV-DNA seropositive. Analyses of the S-gene sequences obtained from 55 HBV-seropositive individuals showed the circulation of 37 distinct HBV variants. These HBV isolates belong predominantly to genotype E (HBV/E) (n=53, 96.4%), with only 2 classified as sub-genotype A3 (HBV/A3). Phylogenetic analysis showed extensive intermixing between HBV/E variants identified in these communities and different countries in Africa. Quasispecies analysis of 22 HBV/E strains using end-point limiting-dilution real-time PCR, sequencing and median joining networks showed extensive intra-host heterogeneity and inter-host variant sharing. To investigate events that resulted in such remarkable HBV/E diversity, HBV full-size genome sequences were obtained from 47 HBV/E infected persons and P gene was subjected to Bayesian coalescent analysis. The time to the most recent common ancestor (tMRCA) for these HBV/E variants was estimated to be year 1952 (95% highest posterior density (95% HPD): 1927-1970). Using additional HBV/E sequences from other African countries, the tMRCA was estimated to be year 1948 (95% HPD: 1924-1966), indicating that HBV/E in these remote communities has a similar time of origin with multiple HBV/E variants broadly circulating in West/Central Africa. Phylogenetic analysis and statistical neutrality tests suggested rapid HBV/E population expansion. Additionally, skyline plot analysis showed an increase in the size of the HBV/E-infected population over the last approximately 30-40 years. CONCLUSIONS: Our data suggest a massive introduction and relatively recent HBV/E expansion in the human population in Africa. Collectively, these data show a significant shift in the HBV/E epidemic dynamics in Africa over the last century

Characteristics of Adults in the Hepatitis B Research Network in North America Reflect Their Country of Origin and Hepatitis B Virus Genotype

Chronic hepatitis B virus (HBV) infection is an important cause of cirrhosis and hepatocellular carcinoma worldwide; populations that migrate to the US and Canada might be disproportionately affected. The Hepatitis B Research Network (HBRN) is a cooperative network of investigators from the United States and Canada, created to facilitate clinical, therapeutic, and translational research in adults and children with hepatitis B. We describe the structure of the network and baseline characteristics of adults with hepatitis B enrolled in the network