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1.  Compaction of rolling circle amplification products increases signal integrity and signal-to-noise ratio 
Scientific Reports  2015;5:12317.
Rolling circle amplification (RCA) for generation of distinct fluorescent signals in situ relies upon the self-collapsing properties of single-stranded DNA in commonly used RCA-based methods. By introducing a cross-hybridizing DNA oligonucleotide during rolling circle amplification, we demonstrate that the fluorophore-labeled RCA products (RCPs) become smaller. The reduced size of RCPs increases the local concentration of fluorophores and as a result, the signal intensity increases together with the signal-to-noise ratio. Furthermore, we have found that RCPs sometimes tend to disintegrate and may be recorded as several RCPs, a trait that is prevented with our cross-hybridizing DNA oligonucleotide. These effects generated by compaction of RCPs improve accuracy of visual as well as automated in situ analysis for RCA based methods, such as proximity ligation assays (PLA) and padlock probes.
PMCID: PMC4511876  PMID: 26202090
2.  The possibility of lifestyle and biological risk markers to predict morbidity and mortality in a cohort of young men after 26 years follow-up 
BMJ Open  2015;5(5):e006798.
To study the association between lifestyle and biological risk markers measured at one occasion, morbidity and mortality from cardiovascular disease (CVD) and cancer, and morbidity from diabetes approximately 26 years later.
A follow-up study of a cohort of men, 33–42 years old at baseline.
Primary healthcare centre in Sweden.
All 757 men, living in the community of Habo in Sweden in 1985, and all 652 of these participating in a health examination in 1985–1987.
Health profile and a health dialogue with a nurse. A doctor invited the high-risk group to further dialogue and examination. Intervention programmes were carried out in the primary healthcare centre and in cooperation with local associations.
Main outcome measures
CVD and cancer diagnoses from the Swedish National Board of Health and Welfare. Data from pharmacy registers of sold drugs concerning diabetes mellitus.
The participants were divided in three groups based on summarised risk points from lifestyle (smoking, physical activity, alcohol consumption) and biological risk markers (body mass index (BMI), blood pressure, serum cholesterol) selected from the health profile. Comparisons were done between these groups. The group with the lowest summarised total risk points had a significantly lower risk for CVD and cancer compared with the group with the highest summarised risk points. The group with the lowest risk points concerning lifestyle had a significantly lower risk for CVD, and the group with lowest risk points for biological risk markers had a significantly lower risk for both CVD and cancer compared with the groups with the highest risk points. Smoking and serum cholesterol were the most important risk factors. In association to diabetes, BMI and smoking were the most important risk factors.
Risk factors measured on one occasion seemed to be able to predict CVD, cancer and diabetes 26 years later.
PMCID: PMC4431125  PMID: 25948404
3.  Fourth-generation sequencing in the cell and the clinic 
Genome Medicine  2014;6(4):31.
Nearly 40 years ago, DNA was sequenced for the first time. Since then, DNA sequencing has undergone continuous development, passing through three generations of sequencing technology. We are now entering the beginning of a new phase of genomic analysis in which massively parallel sequencing is performed directly in the cell. Two methods have recently been described for in situ RNA sequencing, one targeted and one untargeted, that rely on ligation chemistry. This fourth generation of sequencing technology opens up prospects for transcriptomic analysis, biomarker validation, diagnosis and patient stratification for cancer treatment.
PMCID: PMC4062057  PMID: 25031621
4.  Diagnostics of Primary Immunodeficiency Diseases: A Sequencing Capture Approach 
PLoS ONE  2014;9(12):e114901.
Primary Immunodeficiencies (PID) are genetically inherited disorders characterized by defects of the immune system, leading to increased susceptibility to infection. Due to the variety of clinical symptoms and the complexity of current diagnostic procedures, accurate diagnosis of PID is often difficult in daily clinical practice. Thanks to the advent of “next generation” sequencing technologies and target enrichment methods, the development of multiplex diagnostic assays is now possible. In this study, we applied a selector-based target enrichment assay to detect disease-causing mutations in 179 known PID genes. The usefulness of this assay for molecular diagnosis of PID was investigated by sequencing DNA from 33 patients, 18 of which had at least one known causal mutation at the onset of the experiment. We were able to identify the disease causing mutations in 60% of the investigated patients, indicating that the majority of PID cases could be resolved using a targeted sequencing approach. Causal mutations identified in the unknown patient samples were located in STAT3, IGLL1, RNF168 and PGM3. Based on our results, we propose a stepwise approach for PID diagnostics, involving targeted resequencing, followed by whole transcriptome and/or whole genome sequencing if causative variants are not found in the targeted exons.
PMCID: PMC4263707  PMID: 25502423
5.  Overall Accessibility to Traveling by Rail for the Elderly with and without Functional Limitations: The Whole-Trip Perspective 
Elderly persons’ perceived accessibility to railway traveling depends on their functional limitations/diseases, their functional abilities and their travel behaviors in interaction with the barriers encountered during whole trips. A survey was conducted on a random sample of 1000 city residents (65–85 years old; 57% response rate). The travels were perceived least accessible by respondents with severely reduced functional ability and by those with more than one functional limitation/disease (e.g., restricted mobility and chronic pain). Those who traveled “often”, perceived the accessibility to be better than those who traveled less frequently. For travelers with high functional ability, the main barriers to more frequent traveling were travel costs and low punctuality. For those with low functional ability, one’s own health was reported to be the main barrier. Our results clarify the links among existing functional limitations/functional abilities, the barriers encountered, the travel behavior, and the overall accessibility to traveling. By operationalizing the whole-trip concept as a chain of events, we deliver practical knowledge on vulnerable groups for decision-making to improve the transport environment for all.
PMCID: PMC4276655  PMID: 25514149
accessibility; travel behavior; functional limitation; barrier; railway travel; older persons
6.  A General Method for Rapid Determination of Antibiotic Susceptibility and Species in Bacterial Infections 
Journal of Clinical Microbiology  2014;53(2):425-432.
To ensure correct antibiotic treatment and reduce the unnecessary use of antibiotics, there is an urgent need for new rapid methods for species identification and determination of antibiotic susceptibility in infectious pathogenic bacteria. We have developed a general method for the rapid identification of the bacterial species causing an infection and the determination of their antibiotic susceptibility profiles. An initial short cultivation step in the absence and presence of different antibiotics was combined with sensitive species-specific padlock probe detection of the bacterial target DNA to allow a determination of growth (i.e., resistance) and no growth (i.e., susceptibility). A proof-of-concept was established for urinary tract infections in which we applied the method to determine the antibiotic susceptibility profiles of Escherichia coli for two drugs with 100% accuracy in 3.5 h. The short assay time from sample to readout enables fast appropriate treatment with effective drugs and minimizes the need to prescribe broad-spectrum antibiotics due to unknown resistance profiles of the treated infection.
PMCID: PMC4298551  PMID: 25411178
7.  Detection of Rotavirus Using Padlock Probes and Rolling Circle Amplification 
PLoS ONE  2014;9(11):e111874.
Rotavirus infections are one of the most common reasons for hospitalizations due to gastrointestinal diseases. Rotavirus is often diagnosed by latex agglutination assay, chromatography immunoassay, or by electron microscopy, which are all quite insensitive. Reverse transcription polymerase chain reaction, on the other hand, is very sensitive to variations at the genomic level. We developed a novel assay based on a set of 58 different padlock probes with a detection limit of 1,000 copies. Twenty-two patient samples were analyzed and the assay showed high concordance with a PCR-based assay. In summary, we present a new assay for sensitive and variation tolerant detection of rotavirus.
PMCID: PMC4219791  PMID: 25369034
8.  Redox State and Mitochondrial Respiratory Chain Function in Skeletal Muscle of LGMD2A Patients 
PLoS ONE  2014;9(7):e102549.
Calpain-3 deficiency causes oxidative and nitrosative stress-induced damage in skeletal muscle of LGMD2A patients, but mitochondrial respiratory chain function and anti-oxidant levels have not been systematically assessed in this clinical population previously.
We identified 14 patients with phenotypes consistent with LGMD2A and performed CAPN3 gene sequencing, CAPN3 expression/autolysis measurements, and in silico predictions of pathogenicity. Oxidative damage, anti-oxidant capacity, and mitochondrial enzyme activities were determined in a subset of muscle biopsies.
Twenty-one disease-causing variants were detected along the entire CAPN3 gene, five of which were novel (c.338 T>C, c.500 T>C, c.1525-1 G>T, c.2115+4 T>G, c.2366 T>A). Protein- and mRNA-based tests confirmed in silico predictions and the clinical diagnosis in 75% of patients. Reductions in antioxidant defense mechanisms (SOD-1 and NRF-2, but not SOD-2), coupled with increased lipid peroxidation and protein ubiquitination, were observed in calpain-3 deficient muscle, indicating a redox imbalance primarily affecting non-mitochondrial compartments. Although ATP synthase levels were significantly lower in LGMD2A patients, citrate synthase, cytochrome c oxidase, and complex I+III activities were not different from controls.
Despite significant oxidative damage and redox imbalance in cytosolic/myofibrillar compartments, mitochondrial respiratory chain function is largely maintained in skeletal muscle of LGMD2A patients.
PMCID: PMC4117472  PMID: 25079074
9.  Gold Nanowire Based Electrical DNA Detection Using Rolling Circle Amplification 
ACS Nano  2014;8(2):1147-1153.
We present an electrical sensor that uses rolling circle amplification (RCA) of DNA to stretch across the gap between two electrodes, interact with metal nanoparticle seeds to generate an electrically conductive nanowire, and produce electrical signals upon detection of specific target DNA sequences. RCA is a highly specific molecular detection mechanism based on DNA probe circularization. With this technique, long single-stranded DNA with simple repetitive sequences are produced. Here we show that stretched RCA products can be metalized using silver or gold solutions to form metal wires. Upon metallization, the resistance drops from TΩ to kΩ for silver and to Ω for gold. Metallization is seeded by gold nanoparticles aligned along the single-stranded DNA product through hybridization of functionalized oligonucleotides. We show that combining RCA with electrical DNA detection produces results in readout with very high signal-to-noise ratio, an essential feature for sensitive and specific detection assays. Finally, we demonstrate detection of 10 ng of Escherichia coli genomic DNA using the sensor concept.
PMCID: PMC3936482  PMID: 24433087
gold nanoparticles; rolling circle amplification; nanoelectronics; gold conjugation; metal enhancement
10.  DNA repair genes are selectively mutated in diffuse large B cell lymphomas 
The Journal of Experimental Medicine  2013;210(9):1729-1742.
Mutations in DNA damage response and repair genes correlate with genomic instability in diffuse large B cell lymphomas.
DNA repair mechanisms are fundamental for B cell development, which relies on the somatic diversification of the immunoglobulin genes by V(D)J recombination, somatic hypermutation, and class switch recombination. Their failure is postulated to promote genomic instability and malignant transformation in B cells. By performing targeted sequencing of 73 key DNA repair genes in 29 B cell lymphoma samples, somatic and germline mutations were identified in various DNA repair pathways, mainly in diffuse large B cell lymphomas (DLBCLs). Mutations in mismatch repair genes (EXO1, MSH2, and MSH6) were associated with microsatellite instability, increased number of somatic insertions/deletions, and altered mutation signatures in tumors. Somatic mutations in nonhomologous end-joining (NHEJ) genes (DCLRE1C/ARTEMIS, PRKDC/DNA-PKcs, XRCC5/KU80, and XRCC6/KU70) were identified in four DLBCL tumors and cytogenetic analyses revealed that translocations involving the immunoglobulin-heavy chain locus occurred exclusively in NHEJ-mutated samples. The novel mutation targets, CHEK2 and PARP1, were further screened in expanded DLBCL cohorts, and somatic as well as novel and rare germline mutations were identified in 8 and 5% of analyzed tumors, respectively. By correlating defects in a subset of DNA damage response and repair genes with genomic instability events in tumors, we propose that these genes play a role in DLBCL lymphomagenesis.
PMCID: PMC3754869  PMID: 23960188
11.  In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics 
Oncotarget  2013;4(12):2407-2418.
Current assays for somatic mutation analysis are based on extracts from tissue sections that often contain morphologically heterogeneous neoplastic regions with variable contents of genetically normal stromal and inflammatory cells, obscuring the results of the assays. We have developed an RNA-based in situ mutation assay that targets oncogenic mutations in a multiplex fashion that resolves the heterogeneity of the tissue sample. Activating oncogenic mutations are targets for a new generation of cancer drugs. For anti-EGFR therapy prediction, we demonstrate reliable in situ detection of KRAS mutations in codon 12 and 13 in colon and lung cancers in three different types of routinely processed tissue materials. High-throughput screening of KRAS mutation status was successfully performed on a tissue microarray. Moreover, we show how the patterns of expressed mutated and wild-type alleles can be studied in situ in tumors with complex combinations of mutated EGFR, KRAS and TP53. This in situ method holds great promise as a tool to investigate the role of somatic mutations during tumor progression and for prediction of response to targeted therapy.
PMCID: PMC3926836  PMID: 24280411
Padlock probes; RCA; in situ; KRAS; cancer diagnostics
12.  The Effects on Absorbed Dose Distribution in Intraoral X-ray Imaging When Using Tube Voltages of 60 and 70 kV for Bitewing Imaging 
Efforts are made in radiographic examinations to obtain the best image quality with the lowest possible absorbed dose to the patient. In dental radiography, the absorbed dose to patients is very low, but exposures are relatively frequent. It has been suggested that frequent low-dose exposures can pose a risk for development of future cancer. It has previously been reported that there was no significant difference in the diagnostic accuracy of approximal carious lesions in radiographs obtained using tube voltages of 60 and 70 kV. The aim of this study was, therefore, to evaluate the patient dose resulting from exposures at these tube voltages to obtain intraoral bitewing radiographs.
Material and Methods
The absorbed dose distributions resulting from two bitewing exposures were measured at tube voltages of 60 and 70 kV using Gafchromic® film and an anatomical head phantom. The dose was measured in the occlusal plane, and ± 50 mm cranially and caudally to evaluate the amount of scattered radiation. The same entrance dose to the phantom was used. The absorbed dose was expressed as the ratio of the maximal doses, the mean doses and the integral doses at tube voltages of 70 and 60 kV.
The patient receives approximately 40 - 50% higher (mean and integral) absorbed dose when a tube voltage of 70 kV is used.
The results of this study clearly indicate that 60 kV should be used for dental intraoral radiographic examinations for approximal caries detection.
PMCID: PMC3887573  PMID: 24422035
dental radiography; dental digital radiography; bitewing radiography; radiation dosage; radiographic image enhancement.
13.  Improving Precision of Proximity Ligation Assay by Amplified Single Molecule Detection 
PLoS ONE  2013;8(7):e69813.
Proximity ligation assay (PLA) has been proven to be a robust protein detection method. The technique is characterized by high sensitivity and specificity, but the assay precision is probably limited by the PCR readout. To investigate this potential limitation and to improve precision, we developed a digital proximity ligation assay for protein measurement in fluids based on amplified single molecule detection. The assay showed significant improvements in precision, and thereby also detection sensitivity, over the conventional real-time PCR readout.
PMCID: PMC3713053  PMID: 23874999
14.  National surveys: a way to manage treatment strategies in Parkinson’s disease? Pharmaceutical prescribing patterns and patient experiences of symptom control and their impact on disease 
The purpose of this study was to draw conclusions from patient-reported experiences in two national surveys from Scandinavia with the intention of comparing treatment strategies and increasing our knowledge of factors that affect the experiences of patients with Parkinson’s disease (PD).
A total of 2000 individuals in Sweden and 1300 in Norway were invited to complete postal surveys covering PD-related issues. Patient experiences of diagnostic procedures, symptom control, and follow-up in PD and the effects on symptom-related quality of life were collected. Pharmaceutical prescription data on anti-PD drugs and administrative data were collected from national registries.
The surveys were completed by 1553 (78%) of the Swedish cohort and 1244 (96%) of the Norwegian cohort. Only small differences were seen in disease duration and age distribution. Statistically as well as clinically significant differences in symptom control, diagnostic, and follow-up procedures, as well as in pharmacological treatment and impact on quality of life, were found between the national cohorts independent of disease duration.
Information from separate national surveys has the potential to increase our knowledge of patient experiences in PD and can be used to compare, evaluate, educate, and guide health care staff and administrators in optimizing health care for patients with the disease.
PMCID: PMC3704404  PMID: 23847426
parkinson’s disease; diagnosis; follow-up; pharmaceutical prescription; quality of life; survey
15.  Human Enterovirus Species B in Ileocecal Crohn's Disease 
Advanced ileocecal Crohn's disease (ICD) is characterized by strictures, inflammation in the enteric nervous system (myenteric plexitis), and a high frequency of NOD2 mutations. Recent findings implicate a role of NOD2 and another CD susceptibility gene, ATG16L1, in the host response against single-stranded RNA (ssRNA) viruses. However, the role of viruses in CD is unknown. We hypothesized that human enterovirus species B (HEV-B), which are ssRNA viruses with dual tropism both for the intestinal epithelium and the nervous system, could play a role in ICD.
We used immunohistochemistry and in situ hybridization to study the general presence of HEV-B and the presence of the two HEV-B subspecies, Coxsackie B virus (CBV) and Echovirus, in ileocecal resections from 9 children with advanced, stricturing ICD and 6 patients with volvulus, and in intestinal biopsies from 15 CD patients at the time of diagnosis.
All patients with ICD had disease-associated polymorphisms in NOD2 or ATG16L1. Positive staining for HEV-B was detected both in the mucosa and in myenteric nerve ganglia in all ICD patients, but in none of the volvulus patients. Expression of the cellular receptor for CBV, CAR, was detected in nerve cell ganglia.
The common presence of HEV-B in the mucosa and enteric nervous system of ICD patients in this small cohort is a novel finding that warrants further investigation to analyze whether HEV-B has a role in disease onset or progress. The presence of CAR in myenteric nerve cell ganglia provides a possible route of entry for CBV into the enteric nervous system.
PMCID: PMC3696939  PMID: 23804031
16.  Correction: The Influence of Sulphate Deposition on the Seasonal Variation of Peat Pore Water Methyl Hg in a Boreal Mire 
PLoS ONE  2013;8(5):10.1371/annotation/c81e29f5-0042-4155-bcba-19839ed573b3.
PMCID: PMC3654722
17.  Detection of Rifampicin Resistance in Mycobacterium tuberculosis by Padlock Probes and Magnetic Nanobead-Based Readout 
PLoS ONE  2013;8(4):e62015.
Control of the global epidemic tuberculosis is severely hampered by the emergence of drug-resistant Mycobacterium tuberculosis strains. Molecular methods offer a more rapid means of characterizing resistant strains than phenotypic drug susceptibility testing. We have developed a molecular method for detection of rifampicin-resistant M. tuberculosis based on padlock probes and magnetic nanobeads. Padlock probes were designed to target the most common mutations associated with rifampicin resistance in M. tuberculosis, i.e. at codons 516, 526 and 531 in the gene rpoB. For detection of the wild type sequence at all three codons simultaneously, a padlock probe and two gap-fill oligonucleotides were used in a novel assay configuration, requiring three ligation events for circularization. The assay also includes a probe for identification of the M. tuberculosis complex. Circularized probes were amplified by rolling circle amplification. Amplification products were coupled to oligonucleotide-conjugated magnetic nanobeads and detected by measuring the frequency-dependent magnetic response of the beads using a portable AC susceptometer.
PMCID: PMC3632517  PMID: 23630621
18.  Automated Genotyping of Biobank Samples by Multiplex Amplification of Insertion/Deletion Polymorphisms 
PLoS ONE  2012;7(12):e52750.
The genomic revolution in oncology will entail mutational analyses of vast numbers of patient-matched tumor and normal tissue samples. This has meant an increased risk of patient sample mix up due to manual handling. Therefore, scalable genotyping and sample identification procedures are essential to pathology biobanks. We have developed an efficient alternative to traditional genotyping methods suited for automated analysis. By targeting 53 prevalent deletions and insertions found in human populations with fluorescent multiplex ligation dependent genome amplification, followed by separation in a capillary sequencer, a peak spectrum is obtained that can be automatically analyzed. 24 tumor-normal patient samples were successfully matched using this method. The potential use of the developed assay for forensic applications is discussed.
PMCID: PMC3531329  PMID: 23300761
19.  Non-Random mtDNA Segregation Patterns Indicate a Metastable Heteroplasmic Segregation Unit in m.3243A>G Cybrid Cells 
PLoS ONE  2012;7(12):e52080.
Many pathogenic mitochondrial DNA mutations are heteroplasmic, with a mixture of mutated and wild-type mtDNA present within individual cells. The severity and extent of the clinical phenotype is largely due to the distribution of mutated molecules between cells in different tissues, but mechanisms underpinning segregation are not fully understood. To facilitate mtDNA segregation studies we developed assays that measure m.3243A>G point mutation loads directly in hundreds of individual cells to determine the mechanisms of segregation over time. In the first study of this size, we observed a number of discrete shifts in cellular heteroplasmy between periods of stable heteroplasmy. The observed patterns could not be parsimoniously explained by random mitotic drift of individual mtDNAs. Instead, a genetically metastable, heteroplasmic mtDNA segregation unit provides the likely explanation, where stable heteroplasmy is maintained through the faithful replication of segregating units with a fixed wild-type/m.3243A>G mutant ratio, and shifts occur through the temporary disruption and re-organization of the segregation units. While the nature of the physical equivalent of the segregation unit remains uncertain, the factors regulating its organization are of major importance for the pathogenesis of mtDNA diseases.
PMCID: PMC3525564  PMID: 23272214
20.  Regeneration-associated WNT Signaling Is Activated in Long-term Reconstituting AC133bright Acute Myeloid Leukemia Cells12 
Neoplasia (New York, N.Y.)  2012;14(12):1236-2124.
Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disorder characterized by two molecularly distinct self-renewing leukemic stem cell (LSC) populations most closely related to normal progenitors and organized as a hierarchy. A requirement for WNT/β-catenin signaling in the pathogenesis of AML has recently been suggested by a mouse model. However, its relationship to a specific molecular function promoting retention of self-renewing leukemia-initiating cells (LICs) in human remains elusive. To identify transcriptional programs involved in the maintenance of a self-renewing state in LICs, we performed the expression profiling in normal (n = 10) and leukemic (n = 33) human long-term reconstituting AC133+ cells, which represent an expanded cell population in most AML patients. This study reveals the ligand-dependent WNT pathway activation in AC133bright AML cells and shows a diffuse expression and release of WNT10B, a hematopoietic stem cell regenerative-associated molecule. The establishment of a primary AC133+ AML cell culture (A46) demonstrated that leukemia cells synthesize and secrete WNT ligands, increasing the levels of dephosphorylated β-catenin in vivo. We tested the LSC functional activity in AC133+ cells and found significant levels of engraftment upon transplantation of A46 cells into irradiated Rag2-/-γc-/- mice. Owing to the link between hematopoietic regeneration and developmental signaling, we transplanted A46 cells into developing zebrafish. This system revealed the formation of ectopic structures by activating dorsal organizer markers that act downstream of the WNT pathway. In conclusion, our findings suggest that AC133bright LSCs are promoted by misappropriating homeostatic WNT programs that control hematopoietic regeneration.
PMCID: PMC3540948  PMID: 23308055
21.  Targeted enrichment of genomic DNA regions for next-generation sequencing 
Briefings in Functional Genomics  2011;10(6):374-386.
In this review, we discuss the latest targeted enrichment methods and aspects of their utilization along with second-generation sequencing for complex genome analysis. In doing so, we provide an overview of issues involved in detecting genetic variation, for which targeted enrichment has become a powerful tool. We explain how targeted enrichment for next-generation sequencing has made great progress in terms of methodology, ease of use and applicability, but emphasize the remaining challenges such as the lack of even coverage across targeted regions. Costs are also considered versus the alternative of whole-genome sequencing which is becoming ever more affordable. We conclude that targeted enrichment is likely to be the most economical option for many years to come in a range of settings.
PMCID: PMC3245553  PMID: 22121152
targeted enrichment; next-generation sequencing; genome partitioning; exome; genetic variation
22.  The Influence of Sulphate Deposition on the Seasonal Variation of Peat Pore Water Methyl Hg in a Boreal Mire 
PLoS ONE  2012;7(9):e45547.
In this paper we investigate the hypothesis that long-term sulphate (SO42−) deposition has made peatlands a larger source of methyl mercury (MeHg) to remote boreal lakes. This was done on experimental plots at a boreal, low sedge mire where the effect of long-term addition of SO42− on peat pore water MeHg concentrations was observed weekly throughout the snow-free portion of 1999. The additions of SO42− started in 1995. The seasonal mean of the pore water MeHg concentrations on the plots with 17 kg ha−1 yr−1 of sulphur (S) addition (1.3±0.08 ng L−1, SE; n = 44) was significantly (p<0.0001) higher than the mean MeHg concentration on the plots with 3 kg ha−1 yr−1 of ambient S deposition (0.6±0.02 ng L−1, SE; n = 44). The temporal variation in pore water MeHg concentrations during the snow free season was larger in the S-addition plots, with an amplitude of >2 ng L−1 compared to +/−0.5 ng L−1 in the ambient S deposition plots. The concentrations of pore water MeHg in the S-addition plots were positively correlated (r2 = 0.21; p = 0.001) to the groundwater level, with the lowest concentrations of MeHg during the period with the lowest groundwater levels. The pore water MeHg concentrations were not correlated to total Hg, DOC concentration or pH. The results from this study indicate that the persistently higher pore water concentrations of MeHg in the S-addition plots are caused by the long-term additions of SO42− to the mire surface. Since these waters are an important source of runoff, the results support the hypothesis that SO42− deposition has increased the contribution of peatlands to MeHg in downstream aquatic systems. This would mean that the increased deposition of SO42− in acid rain has contributed to the modern increase in the MeHg burdens of remote lakes hydrologically connected to peatlands.
PMCID: PMC3448652  PMID: 23029086
23.  Genome-Wide Detection of Spontaneous Chromosomal Rearrangements in Bacteria 
PLoS ONE  2012;7(8):e42639.
Genome rearrangements have important effects on bacterial phenotypes and influence the evolution of bacterial genomes. Conventional strategies for characterizing rearrangements in bacterial genomes rely on comparisons of sequenced genomes from related species. However, the spectra of spontaneous rearrangements in supposedly homogenous and clonal bacterial populations are still poorly characterized. Here we used 454 pyrosequencing technology and a ‘split mapping’ computational method to identify unique junction sequences caused by spontaneous genome rearrangements in chemostat cultures of Salmonella enterica Var. Typhimurium LT2. We confirmed 22 unique junction sequences with a junction microhomology more than 10 bp and this led to an estimation of 51 true junction sequences, of which 28, 12 and 11 were likely to be formed by deletion, duplication and inversion events, respectively. All experimentally confirmed rearrangements had short inverted (inversions) or direct (deletions and duplications) homologous repeat sequences at the endpoints. This study demonstrates the feasibility of genome wide characterization of spontaneous genome rearrangements in bacteria and the very high steady-state frequency (20–40%) of rearrangements in bacterial populations.
PMCID: PMC3411829  PMID: 22880062
24.  Colorimetric Nucleic Acid Testing Assay for RNA Virus Detection Based on Circle-to-Circle Amplification of Padlock Probes▿ 
Journal of Clinical Microbiology  2011;49(12):4279-4285.
We developed a molecular diagnostic method for detection of RNA virus based on padlock probes and colorimetric readout. The feasibility of our approach was demonstrated by using detection of Crimean-Congo hemorrhagic fever (CCHF) virus as a model. Compared with conventional PCR-based methods, our approach does not require advanced equipment, involves easier assay design, and has a sensitivity of 103 viral copies/ml. By using a cocktail of padlock probes, synthetic templates representing different viral strain variants could be detected. We analyzed 34 CCHF patient samples, and all patients were correctly diagnosed when the results were compared to those of the current real-time PCR method. This is the first time that highly specific padlock probes have been applied to detection of a highly variable target sequence typical of RNA viruses.
PMCID: PMC3232973  PMID: 21956984
25.  Methods for analysis of the cancer microenvironment and their potential for disease prediction, monitoring and personalized treatments 
The EPMA Journal  2012;3(1):7.
A tumor does not consist of a homogenous population of cancer cells. Therefore, to understand cancer, the tumor microenvironment and the interplay between the different cell types present in the tumor has to be taken into account, and how this regulates the growth and survival of the cancer cells. To achieve a full picture of this complex interplay, analysis of tumor tissue should ideally be performed with cellular resolution, providing activity status of individual cells in this heterogeneous population of different cell-types. In addition, in situ analysis provides information on the architecture of the tissue wherein the cancer cells thrive, providing information of the identity of neighboring cells that can be used to understand cell-cell communication. Herein we describe how padlock probes and in situ PLA can be used for visualization of nucleic acids and protein activity, respectively, directly in tissue sections, and their potential future role in personalized medicine.
PMCID: PMC3384241  PMID: 22738217
PLA; Padlock probes; Tumor microenvironment; Personalized medicine; Diagnosis; Prognosis

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