Chromatin immunoprecipitation followed by sequencing the precipitated DNA (ChIP-Seq) is the state-of-the-art method to study protein-DNA interactions. ChIP-Seq allows identification of binding sites of proteins across the entire genome in an unbiased manner. One of the major limitations of currently available ChIP-Seq protocols is the necessity to isolate sufficient amounts of immune precipitated DNA for subsequent sequencing. The NARG 2013/14 study evaluated library preparation alternatives starting from one and two orders of magnitude DNA less than standard protocols. Library preparation kits from seven different commercial providers were utilized in this project. Some of these kits were intended for low input while other kits were used outside of specifications of the manufacturers. Aliquots of the same preparation of ChIPed DNA were processed using the standard protocol for 10 ng of input DNA and the evaluated library preparation alternatives for 1 ng and 100 pg of input DNA. Each library type was prepared at two different ABRF Member labs and sequencing was done on a single Illumina HiSeq flow cell.
The results of low input compared to the standard protocol will be presented. The NARG 2013/14 study provides information how ChIP-Seq can be performed from 100 times less DNA than by standard methods with minimal compromising quality of results.
1992 witnessed the publication of the first method claiming to allow the generation of an expression profile from a single cell. Since then, about one new and/or improved method for quantitative single cell transcriptomics has appeared in a high impact journal per year, the latest only a few months ago. None of these approaches have been widely accepted in the scientific community. Here we offer several explanations why the extraction of biologically relevant information from the data generated by these published single cell expression profiling methods was hampered, namely the following: 1) missing information if the described method generates data representative of the interrogated biological samples; 2) false positive rates of over 60%; and 3) false negative rates of over 50%.
In addition to the technical issues mentioned above, careful evaluation of results from measurements of individuals (like cells) is hindered by a more philosophical reason, namely, if the analytical technique involves the destruction of the interrogated individual, independent evaluation of the results by alternative analytical methods is no longer possible. Here we present two strategies to overcome this dilemma: 1) if different degrees of heterogeneity of analyzed parameters (e.g. levels of gene expression) are observed within a population of individuals, these measurements can be validated in an independent sample of individuals of the same population; and 2) if an identical copy of an individual is available, one copy of this individual can be destroyed while the second can be used for validation purposes.
Austria is part of the classical area of central Europe to which alveolar echinococcosis (AE) is endemic. Annual incidences in Austria were 2.4 and 2.8 cases/100,000 population during 1991–2000 and 2001–2010, respectively. Hence, the registration of 13 new AE patients in 2011 was unexpected. Increasing fox populations and past AE underreporting might have caused this increase.
Alveolar echinococcosis; incidence; Austria; helminth; parasites; Echinococcus multilocularis; tapeworm
Leishmania guyanensis; Leishmania; Viannia; Phlebotomus; Lutzomyia; Sandfly; leishmaniasis; parasites; Austria
Uveitis is an autoimmune disease of the eye that refers to any of a number of intraocular inflammatory conditions. Because it is a rare disease, uveitis is often overlooked, and the possible associations between uveitis and extra-ocular disease manifestations are not well known. The aim of this study was to characterize uveitis in a large sample of patients and to evaluate the relationship between uveitis and systemic diseases.
The present study is a cross-sectional study of a cohort of patients with uveitis. Records from consecutive uveitis patients who were seen by the Uveitis Service in the Department of Ophthalmology at the Medical University of Vienna between 1995 and 2009 were selected from the clinical databases. The cases were classified according to the Standardization of Uveitis Nomenclature Study Group criteria for Uveitis.
Data were available for 2619 patients, of whom 59.9% suffered from anterior, 14.8% from intermediate, 18.3% from posterior and 7.0% from panuveitis. 37.2% of all cases showed an association between uveitis and extra-organ diseases; diseases with primarily arthritic manifestations were seen in 10.1% of all cases, non-infectious systemic diseases (i.e., Behçet´s disease, sarcoidosis or multiple sclerosis) in 8.4% and infectious uveitis in 18.7%. 49.4% of subjects suffering from anterior uveitis tested positively for the HLA-B27 antigen. In posterior uveitis cases 29% were caused by ocular toxoplasmosis and 17.7% by multifocal choroiditis.
Ophthalmologists, rheumatologists, infectiologists, neurologists and general practitioners should be familiar with the differential diagnosis of uveitis. A better interdisciplinary approach could help in tailoring of the work-up, earlier diagnosis of co-existing diseases and management of uveitis patients.
Uveitis; Etiology; Systemic associations; Arthritis; Infections
H3K4me3 is a histone modification that accumulates at the transcription-start site (TSS) of active genes and is known to be important for transcription activation. The way in which H3K4me3 is regulated at TSS and the actual molecular basis of its contribution to transcription remain largely unanswered. To address these questions, we have analyzed the contribution of dKDM5/LID, the main H3K4me3 demethylase in Drosophila, to the regulation of the pattern of H3K4me3. ChIP-seq results show that, at developmental genes, dKDM5/LID localizes at TSS and regulates H3K4me3. dKDM5/LID target genes are highly transcribed and enriched in active RNApol II and H3K36me3, suggesting a positive contribution to transcription. Expression-profiling show that, though weakly, dKDM5/LID target genes are significantly downregulated upon dKDM5/LID depletion. Furthermore, dKDM5/LID depletion results in decreased RNApol II occupancy, particularly by the promoter-proximal Pol lloser5 form. Our results also show that ASH2, an evolutionarily conserved factor that locates at TSS and is required for H3K4me3, binds and positively regulates dKDM5/LID target genes. However, dKDM5/LID and ASH2 do not bind simultaneously and recognize different chromatin states, enriched in H3K4me3 and not, respectively. These results indicate that, at developmental genes, dKDM5/LID and ASH2 coordinately regulate H3K4me3 at TSS and that this dynamic regulation contributes to transcription.
A patient presenting with an atypical manifestation of cutaneous leishmaniasis after travel to Cyprus was successfully treated with miltefosine. The K26 typing revealed a hitherto undescribed strain of the Leishmania donovani/infantum complex as the causing agent.
Neurocysticercosis (NCC) is a major cause of epilepsy in regions where pigs are free-ranging and hygiene is poor. Pork production is expected to increase in the next decade in sub-Saharan Africa, hence NCC will likely become more prevalent. In this study, people with epilepsy (PWE, n = 212) were followed up 28.6 months after diagnosis of epilepsy. CT scans were performed, and serum and cerebrospinal fluid (CSF) of selected PWE were analysed. We compared the demographic data, clinical characteristics, and associated risk factors of PWE with and without NCC. PWE with NCC (n = 35) were more likely to be older at first seizure (24.3 vs. 16.3 years, p = 0.097), consumed more pork (97.1% vs. 73.6%, p = 0.001), and were more often a member of the Iraqw tribe (94.3% vs. 67.8%, p = 0.005) than PWE without NCC (n = 177). PWE and NCC who were compliant with anti-epileptic medications had a significantly higher reduction of seizures (98.6% vs. 89.2%, p = 0.046). Other characteristics such as gender, seizure frequency, compliance, past medical history, close contact with pigs, use of latrines and family history of seizures did not differ significantly between the two groups. The number of NCC lesions and active NCC lesions were significantly associated with a positive antibody result. The electroimmunotransfer blot, developed by the Centers for Disease Control and Prevention, was more sensitive than a commercial western blot, especially in PWE and cerebral calcifications. This is the first study to systematically compare the clinical characteristics of PWE due to NCC or other causes and to explore the utility of two different antibody tests for diagnosis of NCC in sub-Saharan Africa.
Neurocysticercosis, a preventable and treatable disease, is one of the main causes of epilepsy in low income countries. In these countries, the diagnosis of epilepsy is often based on clinical presentation and interviews as neuroimaging is rarely available. It is crucial to distinguish people with epilepsy due to neurocysticercosis from other people with epilepsy by clinical symptoms and/or serology, because the former warrants a specific approach both in terms of diagnosis and treatment. The authors compared the demographic and clinical data of the two groups and found that people with epilepsy due to neurocysticercosis are older, more likely to consume pork, and respond better to anti-epileptic treatment. Additionally, the authors compared two antibody tests for cysticercosis with computed tomography images, which showed a higher sensitivity of the CDC electroimmunotransfer blot compared to a commercial western blot. The number of neurocysticercosis lesions was significantly associated with a positive antibody result in both tests. In summary, this research describes clinical characteristics of people with epilepsy and neurocysticercosis and assesses the usefulness of two immunoblots in those patients. This has implications not only for the diagnosis of neurocysticercosis in low income countries, but also for future epidemiological research.
Anamnesis data of 104 patients with Cystic Echinococcosis were correlated retrospectively with the detected species/strain of Echinococcus. Ninety-two percent (N = 23) of autochthonous Austrian and 33% (N = 9) of patients with former Yugoslavian (YU) origin were infected with E. canadensis G7, the pig strain. All patients originating from Turkey harbored E. granulosus G1, the sheep strain. All E. canadensis G7-infected patients showed small liver cysts (ø 5.9 cm), only one of them an additional lung cyst. The median age at the time of operation of the Austrian patients was 55 years, of the Turkish patients 30 years, and of the former YU patients 23 years in the E. canadensis and 42 years in the E. granulosus-infected patients, respectively. The unexpected high number of E. canadensis G7-infected patients and the immigrants' young age show the importance of E. canadensis as a cause of human Cystic Echinococcosis in Central Europe and accordingly this new species has to be included into future echinococcosis control programs.
Linguatula serrata, the so-called tongue worm, is a worm-like,
bloodsucking parasite belonging to the Pentastomida group. Infections with
L. serrata tongue worms are rare in Europe. We describe a
case of ocular linguatulosis in central Europe and provide molecular data on
L. serrata tongue worms.
Linguatula; Pentastomida; anterior chamber; uveitis; 18S ribosomal DNA; tongue worm; parasites; molecular data; Austria; dispatch
A 49-year-old immunocompetent white man had a painful ulcer (1.5 cm in diameter) on the left ventrolateral surface of a grossly enlarged tongue. The ulcer was present for two months. Impaired swallowing resulted in substantial weight loss and fatigue. Histopathologic analysis of a punch biopsy specimen indicated numerous Leishman Donovan bodies within macrophages. A polymerase chain reaction confirmed the presence of L. donovani. Therapy with two cycles of liposomal amphotericin B over a three-month period was administered. Four months after discharge, the ulcer had healed completely and the tongue returned to its normal size and function.
Expression profiling, the measurement of all transcripts of a cell or tissue type, is currently the most comprehensive method to describe their physiological states. Given that accurate profiling methods currently available require RNA amounts found in thousands to millions of cells, many fields of biology working with specialized cell types cannot use these techniques because available cell numbers are limited. Currently available alternative methods for expression profiling from nanograms of RNA or from very small cell populations lack a broad validation of results to provide accurate information about the measured transcripts.
Methods and Findings
We provide evidence that currently available methods for expression profiling of very small cell populations are prone to technical noise and therefore cannot be used efficiently as discovery tools. Furthermore, we present Pico Profiling, a new expression profiling method from as few as ten cells, and we show that this approach is as informative as standard techniques from thousands to millions of cells. The central component of Pico Profiling is Whole Transcriptome Amplification (WTA), which generates expression profiles that are highly comparable to those produced by others, at different times, by standard protocols or by Real-time PCR. We provide a complete workflow from RNA isolation to analysis of expression profiles.
Pico Profiling, as presented here, allows generating an accurate expression profile from cell populations as small as ten cells.
We identified Onchocerca jakutensis as the causative agent of an unusual human filariasis in a patient with lupus erythematosus. To our knowledge, this is the first case of human infection with O. jakutensis and the first human case of zoonotic onchocercosis involving >1 worm.
Onchocerca jakutensis; zoonosis; filariasis; PCR; paraffin-embedded tissue; lupus erythematosus; dispatch
Microarrays have revolutionized many areas of biology due to our technical ability to quantify tens of thousands of transcripts within a single experiment. However, there are still many areas that cannot benefit from this technology due to the amount of biological material needed for microarray analysis. In response to this demand, chemistries have been developed that boast the capability of generating targets from nanogram amounts of total RnA, reflecting minimal amounts of biological material, on the order of several hundred or thousand cells. Herein, we describe the evaluation of four chemistries for RnA amplification in terms of reproducibility, sensitivity, accuracy, and comparability to results from a single round of T7 amplification. No evidence for false-positive measurements of differential expression was observed. In contrast, clear differences between chemistries in sensitivity and accuracy were detected. PCR validation showed an interaction of probe sequence on the array and target labeling chemistry, resulting in a chemistry-dependent probe set sensitivity varying over an order of magnitude.
microarray; gene expression; RNA amplification; small sample; GeneChip; quantitative PCR
Array-based comparative genomic hybridization (aCGH) is a high-throughput method for measuring genome-wide DNA copy number changes. Current aCGH methods have limited resolution, sensitivity and reproducibility. Microarrays for aCGH are available only for a few organisms and combination of aCGH data with expression data is cumbersome.
We present a novel method of using commercial oligonucleotide expression microarrays for aCGH, enabling DNA copy number measurements and expression profiles to be combined using the same platform. This method yields aCGH data from genomic DNA without complexity reduction at a median resolution of approximately 17,500 base pairs. Due to the well-defined nature of oligonucleotide probes, DNA amplification and deletion can be defined at the level of individual genes and can easily be combined with gene expression data.
A novel method of gene resolution analysis of copy number variation (graCNV) yields high-resolution maps of DNA copy number changes and is applicable to a broad range of organisms for which commercial oligonucleotide expression microarrays are available. Due to the standardization of oligonucleotide microarrays, graCNV results can reliably be compared between laboratories and can easily be combined with gene expression data using the same platform.
Over the past several years, microarray technology has evolved into a critical component of any discovery-based program. Since 1999, the Association of Biomolecular Resource Facilities (ABRF) Microarray Research Group (MARG) has conducted biennial surveys designed to generate a profile of microarray service laboratories and, more importantly, an overview of technology development and implementation. Survey questions addressed instrumentation, protocols, staffing, funding, and work flow in a microarray facility. Presented herein are the results of the MARG 2005 survey; where possible, trends in the field are discussed and compared to data collected from previous surveys.
Microarray; survey; Affymetrix; protein array; bioinformatics
DNA methylation is the best-studied epigenetic modification and describes the conversion of cytosine to 5-methylcytosine. The importance of this phenomenon is that aberrant promoter hypermethylation is a common occurrence in cancer and is frequently associated with gene silencing. Various techniques are currently available for the analysis of DNA methylation. However, accurate and reproducible quantification of DNA methylation remains challenging. In this report, we describe Bio-COBRA (combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform), as a novel approach to quantitative DNA methylation analysis. The combination of a well-established method, COBRA, which interrogates DNA methylation via the restriction enzyme analysis of PCR-amplified bisulfite treated DNAs, with the Bioanalyzer platform allows for the rapid and quantitative assessment of DNA methylation patterns in large sample sets. The sensitivity and reproducibility of Bio-COBRA make it a valuable tool for the analysis of DNA methylation in clinical samples, which could aid in the development of diagnostic and prognostic parameters with respect to disease detection and management.
Surveillance for alveolar echinococcosis in central Europe was initiated in 1998. On a voluntary basis, 559 patients were reported to the registry. Most cases originated from rural communities in regions from eastern France to western Austria; single cases were reported far away from the disease-“endemic” zone throughout central Europe. Of 210 patients, 61.4% were involved in vocational or part-time farming, gardening, forestry, or hunting. Patients were diagnosed at a mean age of 52.5 years; 78% had symptoms. Alveolar echinococcosis primarily manifested as a liver disease. Of the 559 patients, 190 (34%) were already affected by spread of the parasitic larval tissue. Of 408 (73%) patients alive in 2000, 4.9% were cured. The increasing prevalence of Echinococcus multilocularis in foxes in rural and urban areas of central Europe and the occurrence of cases outside the alveolar echinococcosis–endemic regions suggest that this disease deserves increased attention.
alveolar echinococcosis; alveolar hydatid disease; Echinococcus multilocularis; epidemiology; geographic distribution; risk factors; clinical characteristics; case registry; research
In this paper, a serological assay for the detection of antibodies to Capillaria hepatica, a zoonotic parasite, is described. In the past, the only way of detecting Capillaria hepatica was to perform a liver biopsy. The indirect immunofluorescence (IIF) assay, based on liver sections of naturally infected mice and human serum samples, is suitable for detecting early stages of human infections and for screening purposes. No cross-reactivity with other parasitic infections was detected. We have applied the IIF assay to serum samples of 60 employees of the Zoological Garden of Vienna, Schönbrunn, Austria, and found one positive and one questionable sample.