Matthias Wittwer

Prolonged unloading of rat soleus muscle causes distinct adaptations of the gene profile

Using commercially available microarray technology, we investigated a series of transcriptional adaptations caused by atrophy of rat m. soleus due to 35 days of hindlimb suspension. We detected 395 out of 1,200 tested transcripts, which reflected 1%–5% of totally expressed genes. From various cellular functional pathways, we detected multiple genes that spanned a 200‐fold range of gene expression levels. Statistical analysis combining L1 regression with the sign test based on the conservative Bonferroni correction identified 105 genes that underwent transcriptional adaptations with atrophy. Generally, expressional changes were discrete (<50%) and pointed in the same direction for genes belonging to the same cellular functional units. In particular, a distinct expressional adaptation of genes involved in fiber transformation; that is, metabolism, protein turnover, and cell regulation were noted and matched to corresponding transcriptional changes in nutrient trafficking. Expressional changes of extracellular proteases, and of genes involved in nerve‐muscle interaction and excitation‐contraction coupling identify previously not recognized adaptations that occur in atrophic m. soleus. Considerations related to technical and statistical aspects of the array approach for profiling the skeletal muscle genome and the impact of observed novel adaptations of the m. soleus transcriptome are put into perspective of the physiological adaptations occurring with muscular atrophy.

A hypoxia complement differentiates the muscle response to endurance exercise.

Metabolic stress is believed to constitute an important signal for training‐induced adjustments of gene expression and oxidative capacity in skeletal muscle. We hypothesized that the effects of endurance training on expression of muscle‐relevant transcripts and ultrastructure would be specifically modified by a hypoxia complement during exercise due to enhanced glycolytic strain. Endurance training of untrained male subjects in conditions of hypoxia increased subsarcolemmal mitochondrial density in the recruited vastus lateralis muscle and power output in hypoxia more than training in normoxia, i.e. 169 versus 91% and 10 versus 6%, respectively, and tended to differentially elevate sarcoplasmic volume density (42 versus 20%, P= 0.07). The hypoxia‐specific ultrastructural adjustments with training corresponded to differential regulation of the muscle transcriptome by single and repeated exercise between both oxygenation conditions. Fine‐tuning by exercise in hypoxia comprised gene ontologies connected to energy provision by glycolysis and fat metabolism in mitochondria, remodelling of capillaries and the extracellular matrix, and cell cycle regulation, but not fibre structure. In the untrained state, the transcriptome response during the first 24 h of recovery from a single exercise bout correlated positively with changes in arterial oxygen saturation during exercise and negatively with blood lactate. This correspondence was inverted in the trained state. The observations highlight that the expression response of myocellular energy pathways to endurance work is graded with regard to metabolic stress and the training state. The exposed mechanistic relationship implies that the altitude specificity of improvements in aerobic performance with a ‘living low–training high’ regime has a myocellular basis.

Identification of Cryptic Anopheles Mosquito Species by Molecular Protein Profiling

Vector control is the mainstay of malaria control programmes. Successful vector control profoundly relies on accurate information on the target mosquito populations in order to choose the most appropriate intervention for a given mosquito species and to monitor its impact. An impediment to identify mosquito species is the existence of morphologically identical sibling species that play different roles in the transmission of pathogens and parasites. Currently PCR diagnostics are used to distinguish between sibling species. PCR based methods are, however, expensive, time-consuming and their development requires a priori DNA sequence information. Here, we evaluated an inexpensive molecular proteomics approach for Anopheles species: matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS is a well developed protein profiling tool for the identification of microorganisms but so far has received little attention as a diagnostic tool in entomology. We measured MS spectra from specimens of 32 laboratory colonies and 2 field populations representing 12 Anopheles species including the A. gambiae species complex. An important step in the study was the advancement and implementation of a bioinformatics approach improving the resolution over previously applied cluster analysis. Borrowing tools for linear discriminant analysis from genomics, MALDI-TOF MS accurately identified taxonomically closely related mosquito species, including the separation between the M and S molecular forms of A. gambiae sensu stricto. The approach also classifies specimens from different laboratory colonies; hence proving also very promising for its use in colony authentication as part of quality assurance in laboratory studies. While being exceptionally accurate and robust, MALDI-TOF MS has several advantages over other typing methods, including simple sample preparation and short processing time. As the method does not require DNA sequence information, data can also be reviewed at any later stage for diagnostic or functional patterns without the need for re-designing and re-processing biological material.

Rapid diagnosis of experimental meningitis by bacterial heat production in cerebrospinal fluid

BackgroundCalorimetry is a nonspecific technique which allows direct measurement of heat generated by biological processes in the living cell. We evaluated the potential of calorimetry for rapid detection of bacterial growth in cerebrospinal fluid (CSF) in a rat model of bacterial meningitis.MethodsInfant rats were infected on postnatal day 11 by direct intracisternal injection with either Streptococcus pneumoniae, Neisseria meningitidis or Listeria monocytogenes. Control animals were injected with sterile saline or heat-inactivated S. pneumoniae. CSF was obtained at 18 hours after infection for quantitative cultures and heat flow measurement. For calorimetry, 10 μl and 1 μl CSF were inoculated in calorimetry ampoules containing 3 ml trypticase soy broth (TSB).ResultsThe mean bacterial titer (± SD) in CSF was 1.5 ± 0.6 × 108 for S. pneumoniae, 1.3 ± 0.3 × 106 for N. meningitidis and 3.5 ± 2.2 × 104 for L. monocytogenes. Calorimetric detection time was defined as the time until heat flow signal exceeded 10 μW. Heat signal was detected in 10-μl CSF samples from all infected animals with a mean (± SD) detection time of 1.5 ± 0.2 hours for S. pneumoniae, 3.9 ± 0.7 hours for N. meningitidis and 9.1 ± 0.5 hours for L. monocytogenes. CSF samples from non-infected animals generated no increasing heat flow (<10 μW). The total heat was the highest in S. pneumoniae ranging from 6.7 to 7.5 Joules, followed by L. monocytogenes (5.6 to 6.1 Joules) and N. meningitidis (3.5 to 4.4 Joules). The lowest detectable bacterial titer by calorimetry was 2 cfu for S. pneumoniae, 4 cfu for N. meningitidis and 7 cfu for L. monocytogenes.ConclusionBy means of calorimetry, detection times of <4 hours for S. pneumoniae and N. meningitidis and <10 hours for Listeria monocytogenes using as little as 10 μl CSF were achieved. Calorimetry is a new diagnostic method allowing rapid and accurate diagnosis of bacterial meningitis from a small volume of CSF.

Genotyping of human and porcine Yersinia enterocolitica, Yersinia intermedia, and Yersinia bercovieri strains from Switzerland by amplified fragment length polymorphism analysis

ABSTRACT In this study, 231 strains of Yersinia enterocolitica, 25 strains of Y. intermedia, and 10 strains of Y. bercovieri from human and porcine sources (including reference strains) were analyzed using amplified fragment length polymorphism (AFLP), a whole-genome fingerprinting method for subtyping bacterial isolates. AFLP typing distinguished the different Yersinia species examined. Representatives of Y. enterocolitica biotypes 1A, 1B, 2, 3, and 4 belonged to biotype-related AFLP clusters and were clearly distinguished from each other. Y. enterocolitica biotypes 2, 3, and 4 appeared to be more closely related to each other (83% similarity) than to biotypes 1A (11%) and 1B (47%). Biotype 1A strains exhibited the greatest genetic heterogeneity of the biotypes studied. The biotype 1A genotypes were distributed among four major clusters, each containing strains from both human and porcine sources, confirming the zoonotic potential of this organism. The AFLP technique is a valuable genotypic method for identification and typing of Y. enterocolitica and other Yersinia spp.

Gene expression in cortex and hippocampus during acute pneumococcal meningitis

BackgroundPneumococcal meningitis is associated with high mortality (~30%) and morbidity. Up to 50% of survivors are affected by neurological sequelae due to a wide spectrum of brain injury mainly affecting the cortex and hippocampus. Despite this significant disease burden, the genetic program that regulates the host response leading to brain damage as a consequence of bacterial meningitis is largely unknown.We used an infant rat model of pneumococcal meningitis to assess gene expression profiles in cortex and hippocampus at 22 and 44 hours after infection and in controls at 22 h after mock-infection with saline. To analyze the biological significance of the data generated by Affymetrix DNA microarrays, a bioinformatics pipeline was used combining (i) a literature-profiling algorithm to cluster genes based on the vocabulary of abstracts indexed in MEDLINE (NCBI) and (ii) the self-organizing map (SOM), a clustering technique based on covariance in gene expression kinetics.ResultsAmong 598 genes differentially regulated (change factor ≥ 1.5; p ≤ 0.05), 77% were automatically assigned to one of 11 functional groups with 94% accuracy. SOM disclosed six patterns of expression kinetics. Genes associated with growth control/neuroplasticity, signal transduction, cell death/survival, cytoskeleton, and immunity were generally upregulated. In contrast, genes related to neurotransmission and lipid metabolism were transiently downregulated on the whole. The majority of the genes associated with ionic homeostasis, neurotransmission, signal transduction and lipid metabolism were differentially regulated specifically in the hippocampus. Of the cell death/survival genes found to be continuously upregulated only in hippocampus, the majority are pro-apoptotic, while those continuously upregulated only in cortex are anti-apoptotic.ConclusionTemporal and spatial analysis of gene expression in experimental pneumococcal meningitis identified potential targets for therapy.

Mycobacterium ulcerans Persistence at a Village Water Source of Buruli Ulcer Patients

Buruli ulcer (BU), a neglected tropical disease of the skin and subcutaneous tissue, is caused by Mycobacterium ulcerans and is the third most common mycobacterial disease after tuberculosis and leprosy. While there is a strong association of the occurrence of the disease with stagnant or slow flowing water bodies, the exact mode of transmission of BU is not clear. M. ulcerans has emerged from the environmental fish pathogen M. marinum by acquisition of a virulence plasmid encoding the enzymes required for the production of the cytotoxic macrolide toxin mycolactone, which is a key factor in the pathogenesis of BU. Comparative genomic studies have further shown extensive pseudogene formation and downsizing of the M. ulcerans genome, indicative for an adaptation to a more stable ecological niche. This has raised the question whether this pathogen is still present in water-associated environmental reservoirs. Here we show persistence of M. ulcerans specific DNA sequences over a period of more than two years at a water contact location of BU patients in an endemic village of Cameroon. At defined positions in a shallow water hole used by the villagers for washing and bathing, detritus remained consistently positive for M. ulcerans DNA. The observed mean real-time PCR Ct difference of 1.45 between the insertion sequences IS2606 and IS2404 indicated that lineage 3 M. ulcerans, which cause human disease, persisted in this environment after successful treatment of all local patients. Underwater decaying organic matter may therefore represent a reservoir of M. ulcerans for direct infection of skin lesions or vector-associated transmission.

Hepatic Gene Expression Profile in Mice Perorally Infected with Echinococcus multilocularis Eggs

Background Alveolar echinococcosis (AE) is a severe chronic hepatic parasitic disease currently emerging in central and eastern Europe. Untreated AE presents a high mortality (>90%) due to a severe hepatic destruction as a result of parasitic metacestode proliferation which behaves like a malignant tumor. Despite this severe course and outcome of disease, the genetic program that regulates the host response leading to organ damage as a consequence of hepatic alveolar echinococcosis is largely unknown. Methodology/Principal Findings We used a mouse model of AE to assess gene expression profiles in the liver after establishment of a chronic disease status as a result of a primary peroral infection with eggs of the fox tapeworm Echinococcus multilocularis. Among 38 genes differentially regulated (false discovery rate adjusted p≤0.05), 35 genes were assigned to the functional gene ontology group <immune response>, while 3 associated with the functional group <intermediary metabolism>. Upregulated genes associated with <immune response> could be clustered into functional subgroups including <macrophages>, <APCs>, <lymphocytes, chemokines and regulation>, <B-cells> and <eosinophils>. Two downregulated genes related to <lymphocytes, chemokines and regulation> and <intermediary metabolism>, respectively. The <immune response> genes either associated with an <immunosupression> or an <immunostimulation> pathway. From the overexpressed genes, 18 genes were subsequently processed with a Custom Array microfluidic card system in order to assess respective expression status at the mRNA level relative to 5 reference genes (Gapdh, Est1, Rlp3, Mdh-1, Rpl37) selected upon a constitutive and stable expression level. The results generated by the two independent tools used for the assessment of gene expression, i.e., microarray and microfluidic card system, exhibited a high level of congruency (Spearman correlation rho = 0.81, p = 7.87e-5) and thus validated the applied methods. Conclusions/Significance Based on this set of biomarkers, new diagnostic targets have been made available to predict disease status and progression. These biomarkers may also offer new targets for immuno-therapeutic intervention.

Identification of Highly Pathogenic Microorganisms by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: Results of an Interlaboratory Ring Trial

ABSTRACT In the case of a release of highly pathogenic bacteria (HPB), there is an urgent need for rapid, accurate, and reliable diagnostics. MALDI-TOF mass spectrometry is a rapid, accurate, and relatively inexpensive technique that is becoming increasingly important in microbiological diagnostics to complement classical microbiology, PCR, and genotyping of HPB. In the present study, the results of a joint exercise with 11 partner institutions from nine European countries are presented. In this exercise, 10 distinct microbial samples, among them five HPB, Bacillus anthracis, Brucella canis, Burkholderia mallei, Burkholderia pseudomallei, and Yersinia pestis, were characterized under blinded conditions. Microbial strains were inactivated by high-dose gamma irradiation before shipment. Preparatory investigations ensured that this type of inactivation induced only subtle spectral changes with negligible influence on the quality of the diagnosis. Furthermore, pilot tests on nonpathogenic strains were systematically conducted to ensure the suitability of sample preparation and to optimize and standardize the workflow for microbial identification. The analysis of the microbial mass spectra was carried out by the individual laboratories on the basis of spectral libraries available on site. All mass spectra were also tested against an in-house HPB library at the Robert Koch Institute (RKI). The averaged identification accuracy was 77% in the first case and improved to >93% when the spectral diagnoses were obtained on the basis of the RKI library. The compilation of complete and comprehensive databases with spectra from a broad strain collection is therefore considered of paramount importance for accurate microbial identification.

Comparison of rapid methods for detection of Giardia spp. and Cryptosporidium spp. (oo)cysts using transportable instrumentation in a field deployment.

Reliable, sensitive, quantitative, and mobile rapid screening methods for pathogenic organisms are not yet readily available, but would provide a great benefit to humanitarian intervention units in disaster situations. We compared three different methods (immunofluorescent microscopy, IFM; flow cytometry, FCM; polymerase chain reaction, PCR) for the rapid and quantitative detection of Giardia lamblia and Cryptosporidium parvum (oo)cysts in a field campaign. For this we deployed our mobile instrumentation and sampled canal water and vegetables during a 2 week field study in Thailand. For purification and concentrations of (oo)cysts, we used filtration and immunomagnetic separation. We were able to detect considerably high oo(cysts) concentrations (ranges: 15-855 and 0-240 oo(cysts)/liter for Giardia and Cryptosporidium, respectively) in 85 to 300 min, with FCM being fastest, followed by PCR, and IFM being slowest due to the long analysis time per sample. FCM and IFM performed consistently well, whereas PCR reactions often failed. The recovery, established by FCM, was around 30% for Giardia and 13% for Cryptosporidium (oo)cysts. It was possible to track (oo)cysts from the wastewater further downstream to irrigation waters and confirm contamination of salads and water vegetables. We believe that rapid detection, in particular FCM-based methods, can substantially help in disaster management and outbreak prevention.