Targeted insertion and reporter transgene activity at a gene safe harbor of the human blood fluke, Schistosoma mansoni.

The identification and characterization of genomic safe harbor sites (GSHs) can facilitate consistent transgene activity with minimal disruption to the host cell genome. We combined computational genome annotation and chromatin structure analysis to predict the location of four GSHs in the human blood fluke, Schistosoma mansoni, a major infectious pathogen of the tropics. A transgene was introduced via CRISPR-Cas-assisted homology-directed repair into one of the GSHs in the egg of the parasite. Gene editing efficiencies of 24% and transgene-encoded fluorescence of 75% of gene-edited schistosome eggs were observed. The approach advances functional genomics for schistosomes by providing a tractable path for generating transgenics using homology-directed, repair-catalyzed transgene insertion. We also suggest that this work will serve as a roadmap for the development of similar approaches in helminths more broadly.

[Putting freelancing in the medical profession to the test]. / Der Freie Beruf auf dem Prüfstand.

Hallmarks of freelancing in the medical profession include special professional qualifications or expertise. Identification with the activity is also reflected in a physician's responsibility for patients that goes beyond a mere business relationship. At the same time, this responsibility requires that a physician can act independently of economic aspects. Privileges of self-employed are, in addition to a fee schedule, the possibility to establish one's own pension fund and to exercise self-governance in medical associations. An important aspect of being self-employed is self-governance. The goal of the independence of those who are self-employed is to avoid the social and irresolvable conflict of values in state- or market-based systems. Physicians work in a field of tension between an empathetic, time-taking medical activity and a fast "economically sensible, expedient and necessary" medicine. Enduring this dilemma is the original task of the liberal profession.

Identification and characterisation of the tegument-expressed aldehyde dehydrogenase SmALDH_312 of Schistosoma mansoni, a target of disulfiram.

Schistosomiasis is an infectious disease caused by blood flukes of the genus Schistosoma and affects approximately 200 million people worldwide. Since Praziquantel (PZQ) is the only drug for schistosomiasis, alternatives are needed. By a biochemical approach, we identified a tegumentally expressed aldehyde dehydrogenase (ALDH) of S. mansoni, SmALDH_312. Molecular analyses of adult parasites showed Smaldh_312 transcripts in both genders and different tissues. Physiological and cell-biological experiments exhibited detrimental effects of the drug disulfiram (DSF), a known ALDH inhibitor, on larval and adult schistosomes in vitro. DSF also reduced stem-cell proliferation and caused severe tegument damage in treated worms. In silico-modelling of SmALDH_312 and docking analyses predicted DSF binding, which we finally confirmed by enzyme assays with recombinant SmALDH_312. Furthermore, we identified compounds of the Medicine for Malaria Venture (MMV) pathogen box inhibiting SmALDH_312 activity. Our findings represent a promising starting point for further development towards new drugs for schistosomiasis.

Metabolic reprogramming of hepatocytes by Schistosoma mansoni eggs.

Background & Aims: Schistosomiasis is a parasitic infection which affects more than 200 million people globally. Schistosome eggs, but not the adult worms, are mainly responsible for schistosomiasis-specific morbidity in the liver. It is unclear if S. mansoni eggs consume host metabolites, and how this compromises the host parenchyma. Methods: Metabolic reprogramming was analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging, liquid chromatography with high-resolution mass spectrometry, metabolite quantification, confocal laser scanning microscopy, live cell imaging, quantitative real-time PCR, western blotting, assessment of DNA damage, and immunohistology in hamster models and functional experiments in human cell lines. Major results were validated in human biopsies. Results: The infection with S. mansoni provokes hepatic exhaustion of neutral lipids and glycogen. Furthermore, the distribution of distinct lipid species and the regulation of rate-limiting metabolic enzymes is disrupted in the liver of S. mansoni infected animals. Notably, eggs mobilize, incorporate, and store host lipids, while the associated metabolic reprogramming causes oxidative stress-induced DNA damage in hepatocytes. Administration of reactive oxygen species scavengers ameliorates these deleterious effects. Conclusions: Our findings indicate that S. mansoni eggs completely reprogram lipid and carbohydrate metabolism via soluble factors, which results in oxidative stress-induced cell damage in the host parenchyma. Impact and implications: The authors demonstrate that soluble egg products of the parasite S. mansoni induce hepatocellular reprogramming, causing metabolic exhaustion and a strong redox imbalance. Notably, eggs mobilize, incorporate, and store host lipids, while the metabolic reprogramming causes oxidative stress-induced DNA damage in hepatocytes, independent of the host's immune response. S. mansoni eggs take advantage of the host environment through metabolic reprogramming of hepatocytes and enterocytes. By inducing DNA damage, this neglected tropical disease might promote hepatocellular damage and thus influence international health efforts.

Changes in the lipid profile of hamster liver after Schistosoma mansoni infection, characterized by mass spectrometry imaging and LC-MS/MS analysis.

Schistosomiasis, caused by the human parasite Schistosoma mansoni, is one of the WHO-listed neglected tropical diseases (NTDs), and it has severe impact on morbidity and mortality, especially in Africa. Not only the adult worms but also their eggs are responsible for health problems. Up to 50% of the eggs produced by the female worms are not excreted with the feces but are trapped in the host tissue, such as the liver, where they provoke immune responses and a change in the lipid profile. We built up a database with 372 infection markers found in livers of S. mansoni-infected hamsters, using LC-MS/MS for identification, followed by statistical analysis. Most of them belong to the lipid classes of phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), and triglycerides (TGs). We assigned some of these markers to specific anatomical structures by applying high-resolution MALDI MSI to cryosections of hamster liver and generating ion images based on the marker list from the LC-MS/MS experiments. Furthermore, enrichment and depletion of several markers were visualized.

[What do urologist do in daily practice? : A first "unfiltered" look at patient care]. / Was machen Urologen in der Praxis? : Ein erster "ungefilterter" Blick auf die Versorgung.

The topic of routine medical care data and healthcare science has gained in relevance and provides an important basis for both healthcare policymakers and those providing care. Access to relevant data and the ability to analyze these is highly competitive as it yields the most compelling arguments and strong facts in any discourse on the ultimately limited resources of the entire healthcare sector. All randomized clinical trials and prospective data collections harbor the inherent similarity that they contain data within a predefined frame of data elements in order to control for any confounding factors. In addition, analyses using retrospective data collections use a predefined evaluation matrix and filter the existing data according to these established data elements. However, an unfiltered and un(pre)specified view to all data would be ideal. An approximation to this goal as part of this project could be the unfiltered collection of as much data as possible and their collection in a data pool, which then could be processed, in a constantly improving analyses algorithm. The automated self-extraction of data from the private-practice information technology (IT) system to UROscience will create a data pool which could be used to answer many different questions related to the reality of healthcare. The preliminary analyses presented here demonstrate that, on basis of the existing data, this versatile sample is available to provide insight into the treatment reality of urologic outpatient care.

Schistosoma mansoni eggs induce Wnt/ß-catenin signaling and activate the protooncogene c-Jun in human and hamster colon.

Schistosomiasis (bilharzia) is a neglected tropical disease caused by parasitic flatworms of the genus Schistosoma, with considerable morbidity in parts of the Middle East, South America, Southeast Asia, in sub-Saharan Africa, and particularly also in Europe. The WHO describes an increasing global health burden with more than 290 million people threatened by the disease and a potential to spread into regions with temperate climates like Corsica, France. The aim of our study was to investigate the influence of S. mansoni infection on colorectal carcinogenic signaling pathways in vivo and in vitro. S. mansoni infection, soluble egg antigens (SEA) and the Interleukin-4-inducing principle from S. mansoni eggs induce Wnt/ß-catenin signaling and the protooncogene c-Jun as well as downstream factor Cyclin D1 and markers for DNA-damage, such as Parp1 and γH2a.x in enterocytes. The presence of these characteristic hallmarks of colorectal carcinogenesis was confirmed in colon biopsies from S. mansoni-infected patients demonstrating the clinical relevance of our findings. For the first time it was shown that S. mansoni SEA may be involved in the induction of colorectal carcinoma-associated signaling pathways.

Tissue- and sex-specific lipidomic analysis of Schistosoma mansoni using high-resolution atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging.

Schistosomes are human pathogens causing the neglected tropical disease schistosomiasis, which occurs worldwide in (sub-)tropical regions. This infectious disease is often associated with poverty, and more than 700 million people are at risk of infection. Exploitation of novel habitats and limited therapeutic options brought schistosomes into research focus. Schistosomes are the only trematodes that have evolved separate sexes. They are covered by their metabolically active tegument, a surface area representing the interface between male and female in their permanent mating contact but also between parasite and host. The tegument comprises, besides others, numerous specific lipid compounds. Limited information is available on the exact lipid composition and its spatial distribution. We used atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) to characterize the Schistosoma mansoni tegument surface in comparison to tissue sections of whole worms or couples. We found that phosphatidylcholines (PC) and specific phosphatidylethanolamines (PE) are significantly more abundant inside the worm body compared to the tegument. On the other hand, the latter was found to be enriched in sphingomyelins (SM), phosphatidylserines (PS), lysophosphatidylcholines (LPC), and specific PE species. We further investigated lipid classes concerning number of carbon atoms in fatty acyl chains as well as the degree of unsaturation and found pronounced differences between the tegument and whole-worm body. Furthermore, differences between male and female teguments were found. The lipid composition of S. mansoni tissues has been investigated in an untargeted, spatially resolved manner for the first time.

Schistosoma mansoni Egg-Secreted Antigens Activate Hepatocellular Carcinoma-Associated Transcription Factors c-Jun and STAT3 in Hamster and Human Hepatocytes.

Clinical data have provided evidence that schistosomiasis can promote hepatocellular carcinogenesis. c-Jun and STAT3 are critical regulators of liver cancer development and progression. The aim of the present study was to investigate the hepatocellular activation of c-Jun and STAT3 by Schistosoma mansoni infection. Expression and function of c-Jun and STAT3 as well as proliferation and DNA repair were analyzed by western blotting, electrophoretic mobility-shift assay, and immunohistochemistry in liver of S. mansoni-infected hamsters, Huh7 cells, primary hepatocytes, and human liver biopsies. Hepatocellular activation of c-Jun was demonstrated by nuclear translocation of c-Jun, enhanced phosphorylation (Ser73), and AP-1/DNA-binding in response to S. mansoni infection. Nuclear c-Jun staining pattern around lodged eggs without ambient immune reaction, and directionally from granuloma to the central veins, suggested that substances released from schistosome eggs were responsible for the observed effects. In addition, hepatocytes with c-Jun activation show cell activation and DNA double-strand breaks. These findings from the hamster model were confirmed by analyses of human biopsies from patients with schistosomiasis. Cell culture experiments finally demonstrated that activation of c-Jun and STAT3 as well as DNA repair were induced by an extract from schistosome eggs (soluble egg antigens) and culture supernatants of live schistosome egg (egg-conditioned medium), and in particular by IPSE/alpha-1, the major component secreted by live schistosome eggs. The permanent activation of hepatocellular carcinoma-associated proto-oncogenes such as c-Jun and associated transcription factors including STAT3 by substances released from tissue-trapped schistosome eggs may be important factors contributing to the development of liver cancer in S. mansoni-infected patients. Therefore, identification and therapeutic targeting of the underlying pathways is a useful strategy to prevent schistosomiasis-associated carcinogenesis.

Author Correction: Arylmethylamino steroids as antiparasitic agents.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Identification of a new panel of reference genes to study pairing-dependent gene expression in Schistosoma mansoni.

Facilitated by the Schistosoma mansoni genome project, multiple transcriptomic studies were performed over the last decade to elucidate gene expression patterns among different developmental stages of the complex schistosome life cycle. While these analyses enable the identification of candidate genes with key functions in schistosome biology, a diverse molecular tool set is needed that allows comprehensive functional characterization at the single gene level. This includes the availability of reliable reference genes to confirm changes in the transcription of genes of interest over different biological samples and experimental conditions. In particular, the investigation of one key aspect of schistosome biology, the pairing-dependent gene expression in females and males, requires knowledge on reference genes that are expressed independently of both pairing and of in vitro culture effects. Therefore, the present study focused on the identification of quantitative reverse transcription (qRT)-PCR reference genes suitable for the investigation of pairing-dependent gene expression in the S. mansoni male. The "pipeline" we present here is based on qRT-PCR analyses of high biological replication combined with three different statistical analysis tools, BestKeeper, geNorm, and NormFinder. Our approach resulted in a statistically robust ranking of 15 selected reference genes with respect to their transcription stability between pairing-unexperienced and -experienced males. We further tested the top seven candidate genes for their transcription stability during invitro culture of adult S. mansoni. Of these, the two most suitable reference genes were used to investigate the influence of the pairing contact on the transcription of genes of interest, comprising a tyrosine decarboxylase gene Smtdc1, an ebony ortholog Smebony, and the follistatin ortholog Smfst in S. mansoni males. Performing pairing, separation and re-pairing experiments with adult S. mansoni in vitro, our results indicate for the first time that pairing can act as a molecular on/off-switch of specific genes to strictly control their expression in schistosome males.

Lipid Topography in Schistosoma mansoni Cryosections, Revealed by Microembedding and High-Resolution Atmospheric-Pressure Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry Imaging.

Schistosomes are parasitic platyhelminthes that cause schistosomiasis, which is a life-threatening infectious disease for humans in the tropics and subtropics worldwide. Within the human host, female and male schistosomes develop and pair as a prerequisite for egg production. Part of the eggs get lodged in organs such as the gut, spleen, and liver, where they cause severe inflammatory processes, including liver fibrosis, which is one of the most serious pathological symptoms. High-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) has been used as a powerful tool to investigate adult schistosomes at the topographic molecular level. An MSI-compatible protocol was developed, covering critical sample preparation steps and focusing on obtaining artifact-free, longitudinal cryosections. Planar, consecutive sections were prepared from ∼400 µm thick S. mansoni worm couples, comparing several microembedding approaches. High-resolution MSI at both, 10 and 5 µm lateral resolution unraveled anatomical structures and differential abundances of glycerophospholipids and saccharides in females and males. In addition, glycerophospholipids occurred differentially abundant in worm tissues of the female, such as the gut, which is essential for nutrient uptake and subsequent metabolism. Fragment ions of isobaric phospholipids were investigated by on-tissue MS2 imaging experiments, unambiguously showing isomer-specific ion signals. This study provides a solid basis for investigating schistosome parasites in chemical detail at the whole-worm level by MSI.

Evolution of gene dosage on the Z-chromosome of schistosome parasites.

XY systems usually show chromosome-wide compensation of X-linked genes, while in many ZW systems, compensation is restricted to a minority of dosage-sensitive genes. Why such differences arose is still unclear. Here, we combine comparative genomics, transcriptomics and proteomics to obtain a complete overview of the evolution of gene dosage on the Z-chromosome of Schistosoma parasites. We compare the Z-chromosome gene content of African (Schistosoma mansoni and S. haematobium) and Asian (S. japonicum) schistosomes and describe lineage-specific evolutionary strata. We use these to assess gene expression evolution following sex-linkage. The resulting patterns suggest a reduction in expression of Z-linked genes in females, combined with upregulation of the Z in both sexes, in line with the first step of Ohno's classic model of dosage compensation evolution. Quantitative proteomics suggest that post-transcriptional mechanisms do not play a major role in balancing the expression of Z-linked genes.

Transplantation of schistosome sporocysts between host snails: A video guide.

Schistosomiasis is an important parasitic disease, touching roughly 200 million people worldwide. The causative agents are different Schistosoma species. Schistosomes have a complex life cycle, with a freshwater snail as intermediate host. After infection, sporocysts develop inside the snail host and give rise to human dwelling larvae. We present here a detailed step-by-step video instruction in English, French, Spanish and Portuguese that shows how these sporocysts can be manipulated and transferred from one snail to another. This procedure provides a technical basis for different types of ex vivo modifications, such as those used in functional genomics studies.

Tackling Hypotheticals in Helminth Genomes.

Advancements in genome sequencing have led to the rapid accumulation of uncharacterized 'hypothetical proteins' in the public databases. Here we provide a community perspective and some best-practice approaches for the accurate functional annotation of uncharacterized genomic sequences.

A gene expression atlas of adult Schistosoma mansoni and their gonads.

RNA-Seq has proven excellence in providing information about the regulation and transcript levels of genes. We used this method for profiling genes in the flatworm Schistosoma mansoni. This parasite causes schistosomiasis, an infectious disease of global importance for human and animals. The pathology of schistosomiasis is associated with the eggs, which are synthesized as a final consequence of male and female adults pairing. The male induces processes in the female that lead to the full development of its gonads as a prerequisite for egg production. Unpaired females remain sexually immature. Based on an organ-isolation method we obtained gonad tissue for RNA extraction from paired and unpaired schistosomes, with whole adults included as controls. From a total of 23 samples, we used high-throughput cDNA sequencing (RNA-Seq) on the Illumina platform to profile gene expression between genders and tissues, with and without pairing influence. The data obtained provide a wealth of information on the reproduction biology of schistosomes and a rich resource for exploitation through basic and applied research activities.

Arylmethylamino steroids as antiparasitic agents.

In search of antiparasitic agents, we here identify arylmethylamino steroids as potent compounds and characterize more than 60 derivatives. The lead compound 1o is fast acting and highly active against intraerythrocytic stages of chloroquine-sensitive and resistant Plasmodium falciparum parasites (IC50 1-5 nM) as well as against gametocytes. In P. berghei-infected mice, oral administration of 1o drastically reduces parasitaemia and cures the animals. Furthermore, 1o efficiently blocks parasite transmission from mice to mosquitoes. The steroid compounds show low cytotoxicity in mammalian cells and do not induce acute toxicity symptoms in mice. Moreover, 1o has a remarkable activity against the blood-feeding trematode parasite Schistosoma mansoni. The steroid and the hydroxyarylmethylamino moieties are essential for antimalarial activity supporting a chelate-based quinone methide mechanism involving metal or haem bioactivation. This study identifies chemical scaffolds that are rapidly internalized into blood-feeding parasites.

Schistosome sex matters: a deep view into gonad-specific and pairing-dependent transcriptomes reveals a complex gender interplay.

As a key event for maintaining life cycles, reproduction is a central part of platyhelminth biology. In case of parasitic platyhelminths, reproductive processes can also contribute to pathology. One representative example is the trematode Schistosoma, which causes schistosomiasis, an infectious disease, whose pathology is associated with egg production. Among the outstanding features of schistosomes is their dioecious lifestyle and the pairing-dependent differentiation of the female gonads which finally leads to egg synthesis. To analyze the reproductive biology of Schistosoma mansoni in-depth we isolated complete ovaries and testes from paired and unpaired schistosomes for comparative RNA-seq analyses. Of >7,000 transcripts found in the gonads, 243 (testes) and 3,600 (ovaries) occurred pairing-dependently. Besides the detection of genes transcribed preferentially or specifically in the gonads of both genders, we uncovered pairing-induced processes within the gonads including stem cell-associated and neural functions. Comparisons to work on neuropeptidergic signaling in planarian showed interesting parallels but also remarkable differences and highlights the importance of the nervous system for flatworm gonad differentiation. Finally, we postulated first functional hints for 235 hypothetical genes. Together, these results elucidate key aspects of flatworm reproductive biology and will be relevant for basic as well as applied, exploitable research aspects.

Biarylalkyl Carboxylic Acid Derivatives as Novel Antischistosomal Agents.

Parasitic platyhelminths are responsible for serious infectious diseases, such as schistosomiasis, which affect humans as well as animals across vast regions of the world. The drug arsenal available for the treatment of these diseases is limited; for example, praziquantel is the only drug currently used to treat ≥240 million people each year infected with Schistosoma spp., and there is justified concern about the emergence of drug resistance. In this study, we screened biarylalkyl carboxylic acid derivatives for their antischistosomal activity against S. mansoni. These compounds showed significant influence on egg production, pairing stability, and vitality. Tegumental lesions or gut dilatation was also observed. Substitution of the terminal phenyl residue in the biaryl scaffold with a 3-hydroxy moiety and derivatization of the terminal carboxylic acid scaffold with carboxamides yielded compounds that displayed significant antischistosomal activity at concentrations as low as 10 µm with satisfying cytotoxicity values. The present study provides detailed insight into the structure-activity relationships of biarylalkyl carboxylic acid derivatives and thereby paves the way for a new drug-hit moiety for fighting schistosomiasis.

Derivatives of biarylalkyl carboxylic acid induce pleiotropic phenotypes in adult Schistosoma mansoni in vitro.

Schistosomes and other parasitic platyhelminths cause infectious diseases of worldwide significance for humans and animals. Despite their medical and economic importance, vaccines are not available and the number of drugs is alarmingly limited. For most platyhelminths including schistosomes, Praziquantel (PZQ) is the commonly used drug. With respect to its regular application in mass treatment programs, however, there is increasing concern about resistance development.Previous studies demonstrated that inhibitors used to treat non-parasitic human diseases may be useful to be tested for their effects on parasites. To this end, we focused on biarylalkyl carboxylic acids (BACAs) as basis, which had been shown before to be interesting candidates in the context of finding alternative approaches to treat diabetes mellitus. We tested 32 chemically modified derivatives of these substances (biarylalkyl carboxylic acid derivatives (BACADs)) for their effects on adult Schistosoma mansoni in vitro. Treatment with 18 BACADs resulted in egg production-associated phenotypes and reduced pairing stability. In addition, 12 of these derivatives affected vitality and/or caused severe tegument damage, gut dilatation, or other forms of tissue disintegration which led to the death of worms. In most cases (10/12), one derivative caused more than one phenotype at a time. In vitro experiments in the presence of serum albumin (SA) and alpha-acidic glycoprotein (AGP) indicated a varying influence of these blood components on the effects of two selected derivatives. The variety of observed phenotypes suggested that different targets were hit. The results demonstrated that BACADs are interesting substances with respect to their anti-schistosomal effects.