Multiplexed CRISPR-Cas9 protocol for large transgene integration into the Schistosoma mansoni genome.

Precise, on-target CRISPR-Cas9 genome editing has been shown in Schistosoma mansoni, involving both non-homology end joining and homology-directed repair pathways. Here, we present a multiplexed CRISPR-Cas9 protocol for large transgene integration into the S. mansoni genome. We describe steps for deploying multiplexed ribonucleoprotein complexes (RNPs) and donor DNA preparation. We then detail procedures for introducing RNPs into schistosome eggs by square-wave electroporation in the presence of a 5' phosphorothioate-modified double-stranded donor transgene. For complete details on the use and execution of this protocol, please refer to Ittiprasert et al. (2023).1.

Time trends and geographical variation in major lower limb amputation related to peripheral arterial disease in England.

BACKGROUND: Above and below knee amputation (AKA, BKA) are treatments of last resort for peripheral arterial disease (PAD). The aim was to examine amputation rates, AKA:BKA ratios, previous revascularization and minor amputation, lengths of stay in hospital, mortality following amputation, and regional variation in people with and without diabetes in England. METHODS: The study used population-based ecological and cohort study designs, 31 672 census areas, hospital admissions from 2006 to 2018 and Poisson, logistic and Cox regression. RESULTS: There were 47 249 major lower limb amputations (50.7% AKA; 48% had diabetes), giving an annual PAD-related amputation rate of 11 per 100 000 in the population aged 25+ years. Amputation rates were higher in men and substantially higher in people with diabetes. The AKA:BKA ratio was 0.63 in patients with diabetes (n = 22 702) and 1.62 in patients without diabetes (n = 24 547). Of patients having AKA, 25.3% died within 90 days of amputation compared with 11.9% for BKA. Median survival following amputation ranged from only 1.68 years following AKA in patients with diabetes to 5.72 years following BKA in patients without diabetes. Amputation rates decreased over time mainly in the population with diabetes. Short-term mortality and lengths of stay in hospital also decreased over time, while the percentage with previous revascularization generally increased. Amputation rates and AKA:BKA ratios were highest in the North. Adjustment for age, sex and deprivation did not substantially alter geographical patterns. Adjusted 90-day mortality was generally higher in the North and the Midlands but also high in London. There were also regional variations in adjusted duration from admission to amputation, duration from amputation to discharge or death in hospital, previous revascularization and previous minor amputation. CONCLUSIONS: There were large variations in amputation rates and survival following amputation in relation to diabetes status and amputation level, and regional variations which remained after adjustment for deprivation.

Tetraspanins from the liver fluke Opisthorchis viverrini stimulate cholangiocyte migration and inflammatory cytokine production.

The liver fluke Opisthorchis viverrini (Poirier, 1886) (Digenea) secretes extracellular vesicles (EVs) bearing CD63-like tetraspanins on their surface. Fluke EVs are actively internalised by host cholangiocytes in the bile ducts, where they drive pathology and promote neoplasia through induction of cellular proliferation and secretion of inflammatory cytokines. We investigated the effects of tetraspanins of the CD63 superfamily by co-culturing recombinant forms of the large extracellular loop (LEL) of O. viverrini tetraspanin-2 (rLEL-Ov-TSP-2) and tetraspanin-3 (rLEL-Ov-TSP-3) with non-cancerous human bile duct (H69) and cholangiocarcinoma (CCA, M213) cell lines. The results showed that cell lines co-cultured with excretory/secretory products from adult O. viverrini (Ov-ES) underwent significantly increased cell proliferation at 48 hours but not 24 hours compared to untreated control cells (P < 0.05), whereas rLEL-Ov-TSP-3 co-culture resulted in significantly increased cell proliferation at both 24 hours (P < 0.05) and 48 hours (P < 0.01) time points. In like fashion, H69 cholangiocytes co-cultured with both Ov-ES and rLEL-Ov-TSP-3 underwent significantly elevated Il-6 and Il-8 gene expression for at least one of the time points assessed. Finally, both rLEL-Ov-TSP-2 and rLEL-Ov-TSP-3 significantly enhanced migration of both M213 and H69 cell lines. These findings indicated that O. viverrini CD63 family tetraspanins can promote a cancerous microenvironment by enhancing innate immune responses and migration of biliary epithelial cells.

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.

Tetraspanins from Opisthorchis viverrini stimulate cholangiocyte migration and inflammatory cytokine production.

The liver fluke Opsithorchis viverrini secretes extracellular vesicles (EVs) bearing CD63-like tetraspanins on their surface. Fluke EVs are actively internalized by host cholangiocytes in the bile ducts, where they drive pathology and promote neoplasia through induction of cellular proliferation and secretion of inflammatory cytokines. We investigated the effects of tetraspanins of the CD63 superfamily by co-culturing recombinant forms of the large extracellular loop (LEL) of O. viverrini tetraspanin-2 (rLEL- Ov -TSP-2) and tetraspanin-3 (rLEL- Ov -TSP-3) with non-cancerous human bile duct (H69) and cholangiocarcinoma (CCA, M213) cell lines. The results showed that cell lines co-cultured with excretory/secretory products from adult O. viverrini ( Ov- ES) underwent significantly increased cell proliferation at 48 hours but not 24 hours compared to untreated control cells ( P <0.05), whereas rLEL- Ov -TSP-3 co-culture resulted in significantly increased cell proliferation at both 24 hr ( P <0.05) and 48 hr ( P <0.01) time points. In like fashion, H69 cholangiocytes co-cultured with both Ov -ES and rLEL- Ov -TSP-3 underwent significantly elevated Il-6 and Il-8 gene expression for at least one of the time points assessed. Finally, both rLEL- Ov -TSP-and rLEL- Ov -TSP-3 significantly enhanced migration of both M213 and H69 cell lines. These findings indicated that O. viverrini CD63 family tetraspanins can promote a cancerous microenvironment by enhancing innate immune responses and migration of biliary epithelial cells.

Socioeconomic disparities in surgery for carotid artery disease in England.

BACKGROUND: Carotid artery disease and stroke are more prevalent in socioeconomically deprived areas. The aim was to investigate socioeconomic disparities in carotid artery disease surgery rates and in outcomes following surgery. METHODS: The study used population-based ecological and cohort study designs, 31 672 census areas in England, hospital admissions from April 2006 to March 2018, the Index of Multiple Deprivation 2010 as the area-level deprivation indicator, and Poisson, logistic, and Cox regression. RESULTS: A total of 54 377 patients (67 per cent men) from a population aged 55 years and older of 14.7 million had carotid artery disease procedures (95 per cent carotid endarterectomy). Carotid endarterectomy rates were 116 per cent (95% c.i. 101 to 132) higher in men and 180 per cent (95% c.i. 155 to 207) higher in women aged 55-64 years in the most compared with the least socioeconomically deprived areas by quintile. However, this difference diminished and appeared to reverse with increasing age, with 24 per cent (95% c.i. 14 to 33) and 12 per cent (95% c.i. -3 to 24) lower carotid endarterectomy rates respectively in men and women aged 85 years and older in the most deprived areas. Patients in deprived areas having carotid endarterectomy were more likely to have been admitted as symptomatic emergency carotid artery disease admissions. Mortality, and a combined outcome of mortality or stroke-related re-admission, were both worse in patients living in more deprived areas and were only partially accounted for by the higher prevalence of co-morbidities. There was, however, no clear pattern of association between deprivation and elective waiting time for carotid endarterectomy. CONCLUSIONS: These results provide evidence of socioeconomic disparities in surgery for carotid artery disease. Clear policies are needed to address these disparities.

Small extracellular vesicles but not microvesicles from Opisthorchis viverrini promote cell proliferation in human cholangiocytes.

Chronic infection with O. viverrini has been linked to the development of cholangiocarcinoma (CCA), which is a major public health burden in the Lower Mekong River Basin countries, including Thailand, Lao PDR, Vietnam and Cambodia. Despite its importance, the exact mechanisms by which O. viverrini promotes CCA are largely unknown. In this study, we characterized different extracellular vesicle populations released by O. viverrini (OvEVs) using proteomic and transcriptomic analyses and investigated their potential role in host-parasite interactions. While 120k OvEVs promoted cell proliferation in H69 cells at different concentrations, 15k OvEVs did not produce any effect compared to controls. The proteomic analysis of both populations showed differences in their composition that could contribute to this differential effect. Furthermore, the miRNAs present in 120k EVs were analysed and their potential interactions with human host genes was explored by computational target prediction. Different pathways involved in inflammation, immune response and apoptosis were identified as potentially targeted by the miRNAs present in this population of EVs. This is the first study showing specific roles for different EV populations in the pathogenesis of a parasitic helminth, and more importantly, an important advance towards deciphering the mechanisms used in establishment of opisthorchiasis and liver fluke infection-associated malignancy.

Peptide derived from progranulin of the carcinogenic liver fluke, Opisthorchis viverrini stimulates cell hyperproliferation and proinflammatory cytokine production.

Purpose: Progranulin (PGRN) is a secreted glycoprotein growth factor with roles in wound healing, inflammation, angiogenesis and malignancy. An orthologue of the gene encoding human PGRN was identified in the carcinogenic liver fluke Opisthorchis viverrini. Methods: Sequence structure, general characteristics and possible function of O. viverrini PGRN was analyzed using bioinformatics. Expression profiles were investigated with quantitative RT-PCR, western blot and immunolocalization. A specific peptide of Ov-PGRN was used to investigate a role for this molecule in pathogenesis. Results: The structure of the gene coding for O. viverrini PGRN was 36,463 bp in length, and comprised of 13 exons, 12 introns, and a promoter sequence. The Ov-pgrn mRNA is 2,768 bp in length and encodes an 846 amino acids with a predicted molecular mass of 91.61 kDa. Ov-PGRN exhibited one half and seven complete granulin domains. Phylogenetic analysis revealed that Ov-PGRN formed its closest relationship with PGRN of liver flukes in the Opisthorchiidae. Transcripts of Ov-pgrn were detected in several developmental stages, with highest expression in the metacercaria, indicating that Ov-PGRN may participate as a growth factor in the early development of O. viverrini. Western blot analysis revealed the presence of detected Ov-PGRN in both soluble somatic or excretory/secretory products, and immunolocalization indicated high levels of expression in the tegument and parenchyma of the adult fluke. Co-culture of a human cholangiocyte cell line and a peptide fragment of Ov-PGRN stimulated proliferation of cholangiocytes and upregulation of expression of the cytokines IL6 and IL8. Conclusion: Ov-PGRN is expressed throughout the life cycle of liver fluke, and likely plays a key role in development and growth.

Excretory/Secretory Proteome of Females and Males of the Hookworm Ancylostoma ceylanicum.

The dynamic host-parasite mechanisms underlying hookworm infection establishment and maintenance in mammalian hosts remain poorly understood but are primarily mediated by hookworm's excretory/secretory products (ESPs), which have a wide spectrum of biological functions. We used ultra-high performance mass spectrometry to comprehensively profile and compare female and male ESPs from the zoonotic human hookworm Ancylostoma ceylanicum, which is a natural parasite of dogs, cats, and humans. We improved the genome annotation, decreasing the number of protein-coding genes by 49% while improving completeness from 92 to 96%. Compared to the previous genome annotation, we detected 11% and 10% more spectra in female and male ESPs, respectively, using this improved version, identifying a total of 795 ESPs (70% in both sexes, with the remaining sex-specific). Using functional databases (KEGG, GO and Interpro), common and sex-specific enriched functions were identified. Comparisons with the exclusively human-infective hookworm Necator americanus identified species-specific and conserved ESPs. This is the first study identifying ESPs from female and male A. ceylanicum. The findings provide a deeper understanding of hookworm protein functions that assure long-term host survival and facilitate future engineering of transgenic hookworms and analysis of regulatory elements mediating the high-level expression of ESPs. Furthermore, the findings expand the list of potential vaccine and diagnostic targets and identify biologics that can be explored for anti-inflammatory potential.

Special considerations for studies of extracellular vesicles from parasitic helminths: A community-led roadmap to increase rigour and reproducibility.

Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross-species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well-recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth-derived EVs in these processes and highlights EVs as an important participant in cross-phylum communication. While the mammalian EV field is guided by a community-agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan-helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth-derived EVs to complement the MISEV guidelines. We summarise community-agreed standards for studying EVs derived from this broad set of non-model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved.

Urban centre green metrics in Great Britain: A geospatial and socioecological study.

Green infrastructure plays a vital role in urban ecosystems. This includes sustaining biodiversity and human health. Despite a large number of studies investigating greenspace disparities in suburban areas, no known studies have compared the green attributes (e.g., trees, greenness, and greenspaces) of urban centres. Consequently, there may be uncharacterised socioecological disparities between the cores of urban areas (e.g., city centres). This is important because people spend considerable time in urban centres due to employment, retail and leisure opportunities. Therefore, the availability of--and disparities in--green infrastructure in urban centres can affect many lives and potentially underscore a socio-ecological justice issue. To facilitate comparisons between urban centres in Great Britain, we analysed open data of urban centre boundaries with a central business district and population of ≥100,000 (n = 68). Given the various elements that contribute to 'greenness', we combine a range of different measurements (trees, greenness, and accessible greenspaces) into a single indicator. We applied the normalised difference vegetation index (NDVI) to estimate the mean greenness of urban centres and the wider urban area (using a 1 km buffer) and determined the proportion of publicly accessible greenspace within each urban centre with Ordnance Survey Open Greenspace data. Finally, we applied a land cover classification algorithm using i-Tree Canopy to estimate tree coverage. This is the first study to define and rank urban centres based on multiple green attributes. The results suggest important differences in the proportion of green attributes between urban centres. For instance, Exeter scored the highest with a mean NDVI of 0.15, a tree coverage of 11.67%, and an OS Greenspace coverage of 0.05%, and Glasgow the lowest with a mean NDVI of 0.02, a tree cover of 1.95% and an OS Greenspace coverage of 0.00%. We also demonstrated that population size negatively associated with greenness and tree coverage, but not greenspaces, and that green attributes negatively associated with deprivation. This is important because it suggests that health-promoting and biodiversity-supporting resources diminish as population and deprivation increase. Disparities in green infrastructure across the country, along with the population and deprivation-associated trends, are important in terms of socioecological and equity justice. This study provides a baseline and stimulus to help local authorities and urban planners create and monitor equitable greening interventions in urban/city centres.

An Innovative Test for the Rapid Detection of Specific IgG Antibodies in Human Whole-Blood for the Diagnosis of Opisthorchis viverrini Infection.

Chronic human liver fluke infections caused by Opisthorchis viverrini and Clonorchis sinensis can last for decades and cause liver and biliary diseases, including life-threatening pathology prior to cholangiocarcinoma (CCA). CCA generally has a poor prognosis. Serological diagnosis can support parasitological examination in diagnosing disease and screening for the risk of CCA. Here, we present an improved and innovative lateral flow immunochromatographic test (ICT) kit that uses whole-blood samples (WBS) rather than serum to diagnose human opisthorchiasis, which also successfully diagnosed human clonorchiasis. This ICT includes a soluble worm extract of O. viverrini adults and colloidal-gold-labeled conjugates of the IgG antibody to evaluate the diagnostic values with simulated WBS (n = 347). Simulated WBS were obtained by the spiking infection sera with red blood cells. The diagnostic sensitivity, specificity, positive and negative predictive values, and accuracy for detecting opisthorchiasis were 95.5%, 87.0%, 80.5%, 97.2%, and 90.1%, respectively. For clonorchiasis, these findings were 85.7%, 87.0%, 53.6%, 97.2%, and 86.8%, respectively. Combined for both diseases, they were 93.2%, 87.0%, 84.0%, 94.6%, and 89.6%, respectively. The ICT kit can possibly replace the ICT platforms for antibody detection in serum samples in field surveys in remote areas where sophisticated equipment is not available.

Knockout of liver fluke granulin, Ov-grn-1, impedes malignant transformation during chronic infection with Opisthorchis viverrini.

Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor for cholangiocarcinoma (CCA) in the Mekong Basin countries of Thailand, Lao PDR, Vietnam, Myanmar and Cambodia. Using a novel model of CCA, involving infection with gene-edited liver flukes in the hamster during concurrent exposure to dietary nitrosamine, we explored the role of the fluke granulin-like growth factor Ov-GRN-1 in malignancy. We derived RNA-guided gene knockout flukes (ΔOv-grn-1) using CRISPR/Cas9/gRNA materials delivered by electroporation. Genome sequencing confirmed programmed Cas9-catalyzed mutations of the targeted genes, which was accompanied by rapid depletion of transcripts and the proteins they encode. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes. However, less hepatobiliary tract disease manifested during chronic infection with ΔOv-grn-1 worms in comparison to hamsters infected with control gene-edited and mock-edited parasites. Specifically, immuno- and colorimetric-histochemical analysis of livers revealed markedly less periductal fibrosis surrounding the flukes and less fibrosis globally within the hepatobiliary tract during infection with ΔOv-grn-1 genotype worms, minimal biliary epithelial cell proliferation, and significantly fewer mutations of TP53 in biliary epithelial cells. Moreover, fewer hamsters developed high-grade CCA compared to controls. The clinically relevant, pathophysiological phenotype of the hepatobiliary tract confirmed a role for this secreted growth factor in malignancy and morbidity during opisthorchiasis.

Socioeconomic disparities in abdominal aortic aneurysm repair rates and survival.

BACKGROUND: Abdominal aortic aneurysm (AAA) is more prevalent in socioeconomically disadvantaged areas. This study investigated socioeconomic disparities in AAA repair rates and survival. METHODS: The study used ecological and cohort study designs, from 31 672 census areas in England (April 2006 to March 2018), the Index of Multiple Deprivation 2010 as the area-level deprivation indicator, and Poisson, logistic and Cox regression. RESULTS: Some 77 606 patients (83.4 per cent men) in four age categories (55-64, 65-74, 75-84, 85 or more years) were admitted with AAA from a population aged at least 55 years of 14.7 million. Elective open and endovascular repair rates were 41 (95 per cent c.i. 23 to 61) and 60 (36 to 89) per cent higher respectively among men aged 55-64 years in the most versus least deprived areas by quintile. This differences diminished and appeared to reverse with increasing age, with 26 (-1 to 45) and 25 (13 to 35) per cent lower rates respectively in men aged 85 years or more in the most deprived areas. Men admitted from more deprived areas were more likely to die in hospital without aneurysm repair. Among those who had aneurysm repair, this was more likely to be for a ruptured aneurysm than among men from less deprived areas. For intact aneurysm repair, they were relatively more likely to have this during an emergency admission. The mortality rate after repair was higher for men from more deprived areas, although the hazard diminished with age. Patterns were unclear for women. CONCLUSION: There were clear socioeconomic disparities in operation rates, mode of presentation, and outcome for AAA surgery. Policies are needed to address these disparities.

Varicose veins treatment in England: population-based study of time trends and disparities related to demographic, ethnic, socioeconomic, and geographical factors.

BACKGROUND: Varicose vein (VV) treatments have changed significantly in recent years leading to potential disparities in service provision. The aim of this study was to examine the trends in VV treatment in England and to identify disparities in the provision of day-case and inpatient treatments related to deprivation, ethnicity, and other demographic, and geographical factors. METHOD: A population-based study using linked hospital episode statistics for England categorized VV procedures and compared population rates and procedure characteristics by ethnicity, deprivation quintile, and geographical area. RESULTS: A total of 311 936 people had 389 592 VV procedures between 2006/07 and 2017/18, with a further 63 276 procedures between 2018/19 and 2020/21. Procedure rates have reduced in all but the oldest age groups, whereas endovenous procedures have risen to more than 60 per cent of the total in recent years. In younger age groups there was a 20-30 per cent reduction in procedure rates for the least-deprived compared with the most-deprived quintiles. Non-white ethnicity was associated with lower procedure rates. Large regional and local differences were identified in standardized rates of VV procedures. In the most recent 5-year interval, the North-East region had a three-fold higher rate than the South-East region with evidence of greater variation between commissioners in overall rates, the proportion of endovenous procedures, and policies regarding bilateral treatments. CONCLUSIONS: There are substantial geographical variations in the provision of treatment for VVs, which are not explained by demographic differences. These have persisted, despite the publication of guidelines from the National Institute for Health and Care Excellence, and many commissioners, and providers would seem to implement policies that are contrary to this guidance. Lower rates of procedures in less-deprived areas may reflect treatments carried out in private practice, which are not included in these data.

Adherence of Helicobacter pylori to Opisthorchis viverrini gut epithelium and the tegument mediated via L-fucose binding adhesin.

Recent reports implicate both the liver fluke Opisthorchis viverrini as a reservoir of Helicobacter pylori within the human gastrointestinal tract and H. pylori in the pathogenesis of opisthorchiasis-associated cholangiocarcinoma. We postulated that adherence of bacterial ligands to host receptors initiates colonization of the live fluke by H. pylori and here we aimed to assess the molecular interaction between O. viverrini and H. pylori by investigating host receptors for H. pylori in the fluke. Several known receptors of H. pylori including Lewis B, sialyl-Lewis X, Toll-like receptor 4 and L-fucose were detected immunohistochemically and histochemically by focusing analysis on the gut epithelium and tegument of the adult stage of the fluke. The frequency of detection of Lewis B, sialyl-Lewis X, TLR4 and L-fucose in 100 individual worms was 3, 3, 19 and 70%, respectively. Detection of H. pylori by a diagnostic ureA gene-based PCR assay revealed the presence of H. pylori in individual O. viverrini worms in 41 of 49 (79%) worms examined. In addition, numbers of bacteria decreased in a dose- and time-dependent fashion following exposure to fucosidase. These findings suggested that L-fucose represents a tractable receptor for H. pylori that can mediate bacterial colonization of the gut of O. viverrini.

Silencing of Opisthorchis viverrini Tetraspanin Gene Expression Results in Reduced Secretion of Extracellular Vesicles.

Inter-phylum transfer of molecular information is exquisitely exemplified in the uptake of parasite extracellular vesicles (EVs) by their target mammalian host tissues. The oriental liver fluke, Opisthorchis viverrini is the major cause of bile duct cancer in people in Southeast Asia. A major mechanism by which O. viverrini promotes cancer is through the secretion of excretory/secretory products which contain extracellular vesicles (OvEVs). OvEVs contain microRNAs that are predicted to impact various mammalian cell proliferation pathways, and are internalized by cholangiocytes that line the bile ducts. Upon uptake, OvEVs drive relentless proliferation of cholangiocytes and promote a tumorigenic environment, but the underlying mechanisms of this process are unknown. Moreover, purification and characterization methods for helminth EVs in general are ill defined. We therefore compared different purification methods for OvEVs and characterized the sub-vesicular compartment proteomes. Two CD63-like tetraspanins (Ov-TSP-2 and TSP-3) are abundant on the surface of OvEVs, and could serve as biomarkers for these parasite vesicles. Anti-TSP-2 and -TSP-3 IgG, as well as different endocytosis pathway inhibitors significantly reduced OvEV uptake and subsequent proliferation of cholangiocytes in vitro. Silencing of Ov-tsp-2 and tsp-3 gene expression in adult flukes using RNA interference resulted in substantial reductions in OvEV secretion, and those vesicles that were secreted were deficient in their respective TSP proteins. Our findings shed light on the importance of tetraspanins in fluke EV biogenesis and/or stability, and provide a conceivable mechanism for the efficacy of anti-tetraspanin subunit vaccines against a range of parasitic helminth infections.

Chromosome-level genome of Schistosoma haematobium underpins genome-wide explorations of molecular variation.

Urogenital schistosomiasis is caused by the blood fluke Schistosoma haematobium and is one of the most neglected tropical diseases worldwide, afflicting > 100 million people. It is characterised by granulomata, fibrosis and calcification in urogenital tissues, and can lead to increased susceptibility to HIV/AIDS and squamous cell carcinoma of the bladder. To complement available treatment programs and break the transmission of disease, sound knowledge and understanding of the biology and ecology of S. haematobium is required. Hybridisation/introgression events and molecular variation among members of the S. haematobium-group might effect important biological and/or disease traits as well as the morbidity of disease and the effectiveness of control programs including mass drug administration. Here we report the first chromosome-contiguous genome for a well-defined laboratory line of this blood fluke. An exploration of this genome using transcriptomic data for all key developmental stages allowed us to refine gene models (including non-coding elements) and annotations, discover 'new' genes and transcription profiles for these stages, likely linked to development and/or pathogenesis. Molecular variation within S. haematobium among some geographical locations in Africa revealed unique genomic 'signatures' that matched species other than S. haematobium, indicating the occurrence of introgression events. The present reference genome (designated Shae.V3) and the findings from this study solidly underpin future functional genomic and molecular investigations of S. haematobium and accelerate systematic, large-scale population genomics investigations, with a focus on improved and sustained control of urogenital schistosomiasis.

RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni.

The efficiency of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. was compared with SpCas9 from Streptococcus pyogenes, for functional genomics in Schistosoma mansoni. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a and Cas9 resulted in staggered- and blunt-ended strand breaks, respectively. AsCas12a was more efficient than SpCas9 for gene knockout, as determined by TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout efficiency of both nucleases markedly increased in the presence of single-stranded oligodeoxynucleotide (ssODN) template. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands were tested, resulting in KO efficiencies of 15.67, 28.71, and 21.43% in the SpCas9 plus ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans-cleavage against the ssODNs by activated AsCas12a was not apparent in vitro. SpCas9 catalyzed more precise transgene insertion, with knock-in efficiencies of 17.07% for the KI_Cas9 group, 14.58% for KI_Cas12a-NT-ssODN, and 12.37% for KI_Cas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases.