Search

The neglected tropical disease trichuriasis is caused by the whipworm Trichuris trichiura, a soil-transmitted helminth that has infected humans for millennia. Today, T. trichiura infects as many as 500 million people, predominantly in communities with poor sanitary infrastructure enabling sustained faecal-oral transmission. Using whole-genome sequencing of geographically distributed worms collected from human and other primate hosts, together with ancient samples preserved in archaeologically-defined latrines and deposits dated up to one thousand years old, we present the first population genomics study of T. trichiura. We describe the continent-scale genetic structure between whipworms infecting humans and baboons relative to those infecting other primates. Admixture and population demographic analyses support a stepwise distribution of genetic variation that is highest in Uganda, consistent with an African origin and subsequent translocation with human migration. Finally, genome-wide analyses between human samples and between human and non-human primate samples reveal local regions of genetic differentiation between geographically distinct populations. These data provide insight into zoonotic reservoirs of human-infective T. trichiura and will support future efforts toward the implementation of genomic epidemiology of this globally important helminth.

The combined analyses of grains, seeds, fruits, pollen, animal bones and parasite eggs from a latrine dating from the late 1680s provides us with a detailed view of the diet in a Renaissance neighbourhood in Copenhagen. Analyses show that the residents had access to a varied diet of primarily fish, bread/porridge, and a range of fruits, nuts and herbs, including exotic products deriving from a global trade network. This study also shows that combining strands of evidence in a multi-proxy analysis of latrine deposits leads to much more nuanced results than with single-evidence analysis. Botanical evidence from seeds and pollen is combined here with results from previously published DNA analysis of plastids (Søe et al., 2018). Pollen of myrtle family, possibly cloves, as well as that of citrus family, have been observed, neither of which have been recorded archaeologically in Denmark before. Also, human tapeworm is recorded here for the first time in a Danish archaeological context.

High-resolution insight into parasitic infections and diet of past populations in Northern Europe and the Middle East (500 BC- 1700 AD) was obtained by pre-concentration of parasite eggs from ancient latrines and deposits followed by shotgun sequencing of DNA. Complementary profiling of parasite, vertebrate and plant DNA proved highly informative in the study of ancient health, human-animal interactions as well as animal and plant dietary components. Most prominent were finding of soil-borne parasites transmitted directly between humans, but also meat-borne parasites that require consumption of raw or undercooked fish and pork. The detection of parasites for which sheep, horse, dog, pig, and rodents serves as definitive hosts are clear markers of domestic and synanthropic animals living in closer proximity of the respective sites. Finally, the reconstruction of full mitochondrial parasite genomes from whipworm (Ascaris lumbricoides) and roundworm species (Trichuris trichiura and Trichuris muris) and estimates of haplotype frequencies elucidates the genetic diversity and provides insights into epidemiology and parasite biology.

The demographic history of Greenland is characterized by recurrent migrations and extinctions since the first humans arrived 4,500 years ago. Our current understanding of these extinct cultures relies primarily on preserved fossils found in their archaeological deposits, which hold valuable information on past subsistence practices. However, some exploited taxa, though economically important, comprise only a small fraction of these sub-fossil assemblages. Here we reconstruct a comprehensive record of past subsistence economies in Greenland by sequencing ancient DNA from four well-described midden deposits. Our results confirm that the species found in the fossil record, like harp seal and ringed seal, were a vital part of Inuit subsistence, but also add a new dimension with evidence that caribou, walrus and whale species played a more prominent role for the survival of Paleo-Inuit cultures than previously reported. Most notably, we report evidence of bowhead whale exploitation by the Saqqaq culture 4,000 years ago.

Human trichuriasis is a neglected tropical disease which affects hundreds of millions of people worldwide and is particularly prevalent among children living in areas where sanitation is poor. This review examines the current knowledge on the taxonomy, genetics and phylogeography of human Trichuris and its relationship to whipworm parasites in other host species. The evidence for zoonotic transmission of Trichuris and the emergence of anthelmintic resistance is assessed. In addition, the implications of the recent publication of the genomes and transcriptomes of multiple Trichuris species are discussed. Finally, priorities for future research in Trichuris genetics are proposed.

Human worm infections have, to a large extent, been eradicated in countries with high sanitary standards by preventing the fecal-oral transmission of infective eggs. It is possible to study parasite infections among past populations by retrieving and analyzing parasite eggs using paleoparasitological techniques such as morphological examination and molecular identification. Hard-shelled parasite eggs can be recovered from the environment even after extended periods of time and they have shown to be excellent reservoirs of ancient DNA (aDNA). aDNA analysis has enabled identifying which species of parasite an egg originates from. This is impossible solely using morphological examination. One example is the whipworm, Trichuris spp. that is known to have narrow host ranges, which makes it particularly suited to determine from which host an egg originates. A case study will be presented, in which parasite eggs from environmental samples collected at a Viking-age settlement (1018-1030 A.D.) are DNA typed to the species level. The human whipworm (Trichuris trichiura) and the human roundworm (Ascaris lumbricoides) are identified indicating that these parasites were endemic in Denmark in the Viking-age. Further, eggs of the Liver Fluke (Fasciola hepatica), whose primary hosts are cows and sheep, are identified indicating that grazing animals were kept in close proximity of the settlement.

Helminth infections used to be common in the humans in Denmark. The whipworm (Trichuris sp.) and roundworm (Ascaris sp.) are of particular interest as they produce very hard-shelled eggs, which allow them to be recovered in the environment after extended periods of time. In this project we will investigate how the diversity of food-borne parasitic infections has changed with cultural and dietary habits, hunting practice and intensity of animal husbandry. This is done by isolating and typing ancient DNA remains from parasite eggs found in archeological samples from across Denmark.

Background: The development of microarray-based genetic tests for diseases that are caused by known mutations is becoming increasingly important. The key obstacle to developing functional genotyping assays is that such mutations need to be genotyped regardless of their location in genomic regions. These regions include large variations in G+C content, and structural features like hairpins. Methods/Findings: We describe a rational, stable method for screening and combining assay conditions for the genetic analysis of 42 Phenylketonuria-associated mutations in the phenylalanine hydroxylase gene. The mutations are located in regions with large variations in G+C content (20-75%). Custom-made microarrays with different lengths of complementary probe sequences and spacers were hybridized with pooled PCR products of 12 exons from each of 38 individual patient DNA samples. The arrays were washed with eight buffers with different stringencies in a custom-made microfluidic system. The data were used to assess which parameters play significant roles in assay development. Conclusions: Several assay development methods found suitable probes and assay conditions for a functional test for all investigated mutation sites. Probe length, probe spacer length, and assay stringency sufficed as variable parameters in the search for a functional multiplex assay. We discuss the optimal assay development methods for several different scenarios.

A microfluidic device (the HistoFlex) designed to perform and monitor molecular biological assays under dynamic flow conditions on microscope slide-substrates, with special emphasis on analyzing histological tissue sections, is presented. Microscope slides were reversibly sealed onto a cast polydimethylsiloxane (PDMS) insert, patterned with distribution channels and reaction chambers. Topology optimization was used to design reaction chambers with uniform flow conditions. The HistoFlex provided uniform hybridization conditions, across the reaction chamber, as determined by hybridization to microscope slides of spotted DNA microarrays when applying probe concentrations generally used in in situ hybridization (ISH) assays. The HistoFlex's novel ability in online monitoring of an in situ hybridization assay was demonstrated using direct fluorescent detection of hybridization to 18S rRNA. Tissue sections were not visually damaged during assaying, which enabled adapting a complete ISH assay for detection of microRNAs (miRNA). The effects of flow based incubations on hybridization, antibody incubation and Tyramide Signal Amplification (TSA) steps were investigated upon adapting the ISH assay for performing in the HistoFlex. The hybridization step was significantly enhanced using flow based incubations due to improved hybridization efficiency. The HistoFlex device enabled a fast miRNA ISH assay (3 hours) which provided higher hybridization signal intensity compared to using conventional techniques (5 h 40 min). We further demonstrate that the improved hybridization efficiency using the HistoFlex permits more complex assays e. g. those comprising sequential hybridization and detection of two miRNAs to be performed with significantly increased sensitivity. The HistoFlex provides a new histological analysis platform that will allow multiple and sequential assays to be performed under their individual optimum assay conditions. Images can subsequently be recorded either in comb

The use of short, high-affinity probes consisting of a combination of DNA and locked nucleic acid (LNA) has enabled the specific detection of microRNAs (miRNAs) by in situ hybridization (ISH). However, detection of low–copy number miRNAs is still not always possible. Here the authors show that probes consisting of 2'-O-methyl RNAs (2OMe) and LNA at every third base (2:1 ratio), under optimized hybridization conditions, excluding yeast RNA from the hybridization buffer, can provide superior performance in detection of miRNA targets in terms of sensitivity and signal-to-noise ratio compared to DNA + LNA probes. Furthermore, they show that hybridizations can be performed in buffers of 4M urea instead of 50% formamide, thereby yielding an equally specific but nontoxic assay. The use of 2OMe + LNA–based probes and the optimized ISH assay enable simple and fast detection of low–copy number miRNA targets, such as miR-130a in mouse brain.

MikroRNA (miRNA) er små ikke kodende RNA stykker, der er involveret i post-transkriptionel regulering af gen-ekspression. Ekspressionen af specifikke miRNAer er reguleret i forskellige kræftformer og kan derfor betragtes som nye biomarkører, der kan bruges til klassifikation og mulig diagnostik. Nye histologiske teknikker som er mere følsomme og i stand til at detekterer multiple miRNAer samtidigt eller i kombination med protein biomarkører, anses for at være nødvendige i udviklingen af nye diagnostiske analyser. På trods af disse lovende udsigter har implementering af miRNA detektion i eksisterende histologiske teknikker vist sig at være problematiske. Udviklingen af locked nukleinsyre (LNA) prober med høj affinitet for miRNA har muliggjort in situ detektion i forskellige typer af væv. Begrænset følsomhed i sådanne teknikker kan dog forhindre detektion af meget lavt udtrykte miRNAer. Dette kan være forårsaget af utilstrækkelig masse transport af reagenser ved inkubering med konventionel teknikker, da disse er hæmmet af diffusion. Til løsning af disse problemstillinger er der i dette studie fremsat to modeller. Den første model involverede udvikling af teknikker med øget følsomhed for in situ detektion af miRNAer. Disse teknikker blev udført med prober bestående af 2’O-Methyl-RNA og LNA i en optimeret, giftfri buffer, hvor uspecifik RNA var udeladt. Den optimerede teknik gav højere følsomhed i detektion af fem ud af otte miRNAer, samt muliggjorde detektion af den lavt udtrykte miRNA-130a sammenlignet med konventionelle teknikker, hvor der benyttes prober af DNA og LNA. Desuden kunne multiple miRNAer detekteres ved brug af sekventielle inkuberinger af TSA reagens. Dette tillod detektion af to miRNA og et protein på samme vævssnit. Den anden model involverede design og konstruktion af en mikrofluid enhed, som blev betegnet HistoFlex. HistoFlex tillader automatiseret og sekventiel indsprøjtning af reagenser i reaktionskamre, der er reversibelt forseglet til objektglas