| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
- Kraus, Robert H. S., et al.
(författare)
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Avian influenza surveillance with FTA cards : field methods, biosafety, and transportation issues solved
- 2011
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Ingår i: Journal of Visualized Experiments. - : MyJove Corporation. - 1940-087X. ; :54
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Tidskriftsartikel (refereegranskat)abstract
- Avian Influenza Viruses (AIVs) infect many mammals, including humans(1). These AIVs are diverse in their natural hosts, harboring almost all possible viral subtypes(2). Human pandemics of flu originally stem from AIVs(3). Many fatal human cases during the H5N1 outbreaks in recent years were reported. Lately, a new AIV related strain swept through the human population, causing the 'swine flu epidemic'(4). Although human trading and transportation activity seems to be responsible for the spread of highly pathogenic strains(5), dispersal can also partly be attributed to wild birds(6, 7). However, the actual reservoir of all AIV strains is wild birds. In reaction to this and in face of severe commercial losses in the poultry industry, large surveillance programs have been implemented globally to collect information on the ecology of AIVs, and to install early warning systems to detect certain highly pathogenic strains(8-12). Traditional virological methods require viruses to be intact and cultivated before analysis. This necessitates strict cold chains with deep freezers and heavy biosafety procedures to be in place during transport. Long-term surveillance is therefore usually restricted to a few field stations close to well equipped laboratories. Remote areas cannot be sampled unless logistically cumbersome procedures are implemented. These problems have been recognised(13, 14) and the use of alternative storage and transport strategies investigated (alcohols or guanidine)(15-17). Recently, Kraus et al.(18) introduced a method to collect, store and transport AIV samples, based on a special filter paper. FTA cards(19) preserve RNA on a dry storage basis(20) and render pathogens inactive upon contact(21). This study showed that FTA cards can be used to detect AIV RNA in reverse-transcription PCR and that the resulting cDNA could be sequenced and virus genes and determined. In the study of Kraus et al.(18) a laboratory isolate of AIV was used, and samples were handled individually. In the extension presented here, faecal samples from wild birds from the duck trap at the Ottenby Bird Observatory (SE Sweden) were tested directly to illustrate the usefulness of the methods under field conditions. Catching of ducks and sample collection by cloacal swabs is demonstrated. The current protocol includes up-scaling of the work flow from single tube handling to a 96-well design. Although less sensitive than the traditional methods, the method of FTA cards provides an excellent supplement to large surveillance schemes. It allows collection and analysis of samples from anywhere in the world, without the need to maintaining a cool chain or safety regulations with respect to shipping of hazardous reagents, such as alcohol or guanidine.
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| 3. |
- Kraus, Robert H. S., et al.
(författare)
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Genome wide SNP discovery, analysis and evaluation in mallard (Anas platyrhynchos)
- 2011
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Ingår i: BMC Genomics. - 1471-2164 .- 1471-2164. ; 12, s. 150-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Next generation sequencing technologies allow to obtain at low cost the genomic sequence information that currently lacks for most economically and ecologically important organisms. For the mallard duck genomic data is limited. The mallard is, besides a species of large agricultural and societal importance, also the focal species when it comes to long distance dispersal of Avian Influenza. For large scale identification of SNPs we performed Illumina sequencing of wild mallard DNA and compared our data with ongoing genome and EST sequencing of domesticated conspecifics. This is the first study of its kind for waterfowl. Results: More than one billion base pairs of sequence information were generated resulting in a 16x coverage of a reduced representation library of the mallard genome. Sequence reads were aligned to a draft domesticated duck reference genome and allowed for the detection of over 122,000 SNPs within our mallard sequence dataset. In addition, almost 62,000 nucleotide positions on the domesticated duck reference showed a different nucleotide compared to wild mallard. Approximately 20,000 SNPs identified within our data were shared with SNPs identified in the sequenced domestic duck or in EST sequencing projects. The shared SNPs were considered to be highly reliable and were used to benchmark non-shared SNPs for quality. Genotyping of a representative sample of 364 SNPs resulted in a SNP conversion rate of 99.7%. The correlation of the minor allele count and observed minor allele frequency in the SNP discovery pool was 0.72. Conclusion: We identified almost 150,000 SNPs in wild mallards that will likely yield good results in genotyping. Of these, similar to 101,000 SNPs were detected within our wild mallard sequences and similar to 49,000 were detected between wild and domesticated duck data. In the similar to 101,000 SNPs we found a subset of similar to 20,000 SNPs shared between wild mallards and the sequenced domesticated duck suggesting a low genetic divergence. Comparison of quality metrics between the total SNP set (122,000 + 62,000 = 184,000 SNPs) and the validated subset shows similar characteristics for both sets. This indicates that we have detected a large amount (similar to 150,000) of accurately inferred mallard SNPs, which will benefit bird evolutionary studies, ecological studies (e. g. disentangling migratory connectivity) and industrial breeding programs.
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| 4. |
- Kraus, Robert H. S., et al.
(författare)
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Genome wide SNP discovery, analysis and evaluation in mallard (Anas platyrhynchos)
- 2011
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Ingår i: BMC Genomics. - : BioMed Central Ltd.. - 1471-2164. ; 12
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Next generation sequencing technologies allow to obtain at low cost the genomic sequence information that currently lacks for most economically and ecologically important organisms. For the mallard duck genomic data is limited. The mallard is, besides a species of large agricultural and societal importance, also the focal species when it comes to long distance dispersal of Avian Influenza. For large scale identification of SNPs we performed Illumina sequencing of wild mallard DNA and compared our data with ongoing genome and EST sequencing of domesticated conspecifics. This is the first study of its kind for waterfowl. Results: More than one billion base pairs of sequence information were generated resulting in a 16x coverage of a reduced representation library of the mallard genome. Sequence reads were aligned to a draft domesticated duck reference genome and allowed for the detection of over 122,000 SNPs within our mallard sequence dataset. In addition, almost 62,000 nucleotide positions on the domesticated duck reference showed a different nucleotide compared to wild mallard. Approximately 20,000 SNPs identified within our data were shared with SNPs identified in the sequenced domestic duck or in EST sequencing projects. The shared SNPs were considered to be highly reliable and were used to benchmark non-shared SNPs for quality. Genotyping of a representative sample of 364 SNPs resulted in a SNP conversion rate of 99.7%. The correlation of the minor allele count and observed minor allele frequency in the SNP discovery pool was 0.72. Conclusion: We identified almost 150,000 SNPs in wild mallards that will likely yield good results in genotyping. Of these, similar to 101,000 SNPs were detected within our wild mallard sequences and similar to 49,000 were detected between wild and domesticated duck data. In the similar to 101,000 SNPs we found a subset of similar to 20,000 SNPs shared between wild mallards and the sequenced domesticated duck suggesting a low genetic divergence. Comparison of quality metrics between the total SNP set (122,000 + 62,000 = 184,000 SNPs) and the validated subset shows similar characteristics for both sets. This indicates that we have detected a large amount (similar to 150,000) of accurately inferred mallard SNPs, which will benefit bird evolutionary studies, ecological studies (e. g. disentangling migratory connectivity) and industrial breeding programs.
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| 5. |
- Kraus, Robert, et al.
(författare)
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Widespread horizontal genomic exchange does not erode species barriers among sympatric ducks
- 2012
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Ingår i: BMC Evolutionary Biology. - 1471-2148 .- 1471-2148. ; 12, s. 45-
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND:The study of speciation and maintenance of species barriers is at the core of evolutionary biology. During speciation the genome of one population becomes separated from other populations of the same species, which may lead to genomic incompatibility with time. This separation is complete when no fertile offspring is produced from inter-population matings, which is the basis of the biological species concept. Birds, in particular ducks, are recognised as a challenging and illustrative group of higher vertebrates for speciation studies. There are many sympatric and ecologically similar duck species, among which fertile hybrids occur relatively frequently in nature, yet these species remain distinct.RESULTS:We show that the degree of shared single nucleotide polymorphisms (SNPs) between five species of dabbling ducks (genus Anas) is an order of magnitude higher than that previously reported between any pair of eukaryotic species with comparable evolutionary distances. We demonstrate that hybridisation has led to sustained exchange of genetic material between duck species on an evolutionary time scale without disintegrating species boundaries. Even though behavioural, genetic and ecological factors uphold species boundaries in ducks, we detect opposing forces allowing for viable interspecific hybrids, with long-term evolutionary implications. Based on the superspecies concept we here introduce the novel term "supra-population" to explain the persistence of SNPs identical by descent within the studied ducks despite their history as distinct species dating back millions of years.CONCLUSIONS:By reviewing evidence from speciation theory, palaeogeography and palaeontology we propose a fundamentally new model of speciation to accommodate our genetic findings in dabbling ducks. This model, we argue, may also shed light on longstanding unresolved general speciation and hybridisation patterns in higher organisms, e.g. in other bird groups with unusually high hybridisation rates. Observed parallels to horizontal gene transfer in bacteria facilitate the understanding of why ducks have been such an evolutionarily successful group of animals. There is large evolutionary potential in the ability to exchange genes among species and the resulting dramatic increase of effective population size to counter selective constraints.
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| 6. |
- Kraus, Robert, et al.
(författare)
-
Widespread horizontal genomic exchange does not erode species barriers among sympatric ducks
- 2012
-
Ingår i: BMC Evolutionary Biology. - : BioMed Central Ltd.. - 1471-2148. ; 12
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND:The study of speciation and maintenance of species barriers is at the core of evolutionary biology. During speciation the genome of one population becomes separated from other populations of the same species, which may lead to genomic incompatibility with time. This separation is complete when no fertile offspring is produced from inter-population matings, which is the basis of the biological species concept. Birds, in particular ducks, are recognised as a challenging and illustrative group of higher vertebrates for speciation studies. There are many sympatric and ecologically similar duck species, among which fertile hybrids occur relatively frequently in nature, yet these species remain distinct.RESULTS:We show that the degree of shared single nucleotide polymorphisms (SNPs) between five species of dabbling ducks (genus Anas) is an order of magnitude higher than that previously reported between any pair of eukaryotic species with comparable evolutionary distances. We demonstrate that hybridisation has led to sustained exchange of genetic material between duck species on an evolutionary time scale without disintegrating species boundaries. Even though behavioural, genetic and ecological factors uphold species boundaries in ducks, we detect opposing forces allowing for viable interspecific hybrids, with long-term evolutionary implications. Based on the superspecies concept we here introduce the novel term "supra-population" to explain the persistence of SNPs identical by descent within the studied ducks despite their history as distinct species dating back millions of years.CONCLUSIONS:By reviewing evidence from speciation theory, palaeogeography and palaeontology we propose a fundamentally new model of speciation to accommodate our genetic findings in dabbling ducks. This model, we argue, may also shed light on longstanding unresolved general speciation and hybridisation patterns in higher organisms, e.g. in other bird groups with unusually high hybridisation rates. Observed parallels to horizontal gene transfer in bacteria facilitate the understanding of why ducks have been such an evolutionarily successful group of animals. There is large evolutionary potential in the ability to exchange genes among species and the resulting dramatic increase of effective population size to counter selective constraints.
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| 7. |
- Quandt, Dagmar, et al.
(författare)
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Implementing liquid biopsies into clinical decision making for cancer immunotherapy
- 2017
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Ingår i: Oncotarget. - : Impact Journals, LLC. - 1949-2553. ; 8:29, s. 48507-48520
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Forskningsöversikt (refereegranskat)abstract
- During the last decade, novel immunotherapeutic strategies, in particular antibodies directed against immune checkpoint inhibitors, have revolutionized the treatment of different malignancies leading to an improved survival of patients. Identification of immune-related biomarkers for diagnosis, prognosis, monitoring of immune responses and selection of patients for specific cancer immunotherapies is urgently required and therefore areas of intensive research. Easily accessible samples in particular liquid biopsies (body fluids), such as blood, saliva or urine, are preferred for serial tumor biopsies. Although monitoring of immune and tumor responses prior, during and post immunotherapy has led to significant advances of patients' outcome, valid and stable prognostic biomarkers are still missing. This might be due to the limited capacity of the technologies employed, reproducibility of results as well as assay stability and validation of results. Therefore solid approaches to assess immune regulation and modulation as well as to follow up the nature of the tumor in liquid biopsies are urgently required to discover valuable and relevant biomarkers including sample preparation, timing of the collection and the type of liquid samples. This article summarizes our knowledge of the well-known liquid material in a new context as liquid biopsy and focuses on collection and assay requirements for the analysis and the technical developments that allow the implementation of different high-throughput assays to detect alterations at the genetic and immunologic level, which could be used for monitoring treatment efficiency, acquired therapy resistance mechanisms and the prognostic value of the liquid biopsies.
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| 8. |
- Söderquist, Pär, et al.
(författare)
-
Released game birds cause introgression in European mallard
- 2015
-
Konferensbidrag (refereegranskat)abstract
- The practice of restocking already viable populations to improve harvest has since long been common in forestry, fisheries and wildlife management. The potential risks with restocking of native species has for a long time been overshadowed by the related issue of invasive species. However, during the last decade releases of native species with a potential non-native genome have attained more attention. A suitable model species for studying genetic effects of large-scale releases of a native species is the Mallard, Anas platyrhynchos; it is the most widespread duck in the world, it is a migrating species, and an important game bird. In several European countries it is also farmed and released to increase the harvestable population, and more than 3 million unfledged hatchlings are released each year around Europe. The aims of this study were to determine if wild and released farmed Mallards differed genetically among subpopulations in Europe, if there are signs of previous or ongoing introgression between wild and farmed Mallards, and if the genetic structure of the wild Mallard population has changed since large-scale releases started in the 1970s. We used 360 Single Nucleotide Polymorphisms (SNPs) to analyze the genetic structure of historical wild, present-day wild, and farmed Mallards. We found a clear genetic difference between wild and farmed Mallards in Europe. We also found signs of introgression of farmed genes in the wild Mallard population, however, the rate of hybridization is probably minor due to low survival of released farmed Mallards and a change of the wild population since the start of large-scale releases is therefore limited. A low level of hybridization between farmed and wild Mallard is desired as introgressed genes may be detrimental for wild Mallards, and efforts to increase survival of farmed Mallards should therefore not be encouraged.
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| 9. |
- Söderquist, Pär, et al.
(författare)
-
Released game birds cause introgression in European mallard
- 2015
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The practice of restocking already viable populations to improve harvest has since long been common in forestry, fisheries and wildlife management. The potential risks with restocking of native species has for a long time been overshadowed by the related issue of invasive species. However, during the last decade releases of native species with a potential non-native genome have attained more attention. A suitable model species for studying genetic effects of large-scale releases of a native species is the Mallard, Anas platyrhynchos; it is the most widespread duck in the world, it is a migrating species, and an important game bird. In several European countries it is also farmed and released to increase the harvestable population, and more than 3 million unfledged hatchlings are released each year around Europe. The aims of this study were to determine if wild and released farmed Mallards differed genetically among subpopulations in Europe, if there are signs of previous or ongoing introgression between wild and farmed Mallards, and if the genetic structure of the wild Mallard population has changed since large-scale releases started in the 1970s. We used 360 Single Nucleotide Polymorphisms (SNPs) to analyze the genetic structure of historical wild, present-day wild, and farmed Mallards. We found a clear genetic difference between wild and farmed Mallards in Europe. We also found signs of introgression of farmed genes in the wild Mallard population, however, the rate of hybridization is probably minor due to low survival of released farmed Mallards and a change of the wild population since the start of large-scale releases is therefore limited. A low level of hybridization between farmed and wild Mallard is desired as introgressed genes may be detrimental for wild Mallards, and efforts to increase survival of farmed Mallards should therefore not be encouraged.
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| 10. |
- Söderquist, Pär, et al.
(författare)
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Released gamebrds cause introgression in European mallard
- 2015
-
Ingår i: 4th Pan-European Duck Symposium, Hangö, Finland. ; , s. 75-
-
Konferensbidrag (refereegranskat)abstract
- The practice of restocking already viable populations to improve harvest has since long been common in forestry, fisheries and wildlife management. The potential risks with restocking of native species has for a long time been overshadowed by the related issue of invasive species. However, during the last decade releases of native species with a potential non-native genome have attained more attention. A suitable model species for studying genetic effects of large-scale releases of a native species is the Mallard, Anas platyrhynchos; it is the most widespread duck in the world, it is a migrating species, and an important game bird. In several European countries it is also farmed and released to increase the harvestable population, and more than 3 million unfledged hatchlings are released each year around Europe. The aims of this study were to determine if wild and released farmed Mallards differed genetically among subpopulations in Europe, if there are signs of previous or ongoing introgression between wild and farmed Mallards, and if the genetic structure of the wild Mallard population has changed since large-scale releases started in the 1970s. We used 360 Single Nucleotide Polymorphisms (SNPs) to analyze the genetic structure of historical wild, present-day wild, and farmed Mallards. We found a clear genetic difference between wild and farmed Mallards in Europe. We also found signs of introgression of farmed genes in the wild Mallard population, however, the rate of hybridization is probably minor due to low survival of released farmed Mallards and a change of the wild population since the start of large-scale releases is therefore limited. A low level of hybridization between farmed and wild Mallard is desired as introgressed genes may be detrimental for wild Mallards, and efforts to increase survival of farmed Mallards should therefore not be encouraged.
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| 11. |
- Yin, Shenglai, et al.
(författare)
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No evidence that migratory geese disperse avian influenza viruses from breeding to wintering ground
- 2017
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Ingår i: PLOS ONE. - : PLOS. - 1932-6203. ; 12:5
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Tidskriftsartikel (refereegranskat)abstract
- Low pathogenic avian influenza virus can mutate to a highly pathogenic strain that causes severe clinical signs in birds and humans. Migratory waterfowl, especially ducks, are considered the main hosts of low pathogenic avian influenza virus, but the role of geese in dispersing the virus over long-distances is still unclear. We collected throat and cloaca samples from three goose species, Bean goose (Anser fabalis), Barnacle goose (Branta leucopsis) and Greater white-fronted goose (Anser albifrons), from their breeding grounds, spring stopover sites, and wintering grounds. We tested if the geese were infected with low pathogenic avian influenza virus outside of their wintering grounds, and analysed the spatial and temporal patterns of infection prevalence on their wintering grounds. Our results show that geese were not infected before their arrival on wintering grounds. Barnacle geese and Greater white-fronted geese had low prevalence of infection just after their arrival on wintering grounds in the Netherlands, but the prevalence increased in successive months, and peaked after December. This suggests that migratory geese are exposed to the virus after their arrival on wintering grounds, indicating that migratory geese might not disperse low pathogenic avian influenza virus during autumn migration.
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