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Long non-coding (lnc)RNAs are a class of eukaryotic RNA that do not code for protein and are linked with transcriptional regulation, amongst a myriad of other functions. Using a customin silicopipeline we have identified 6,436 putative lncRNA transcripts in the liver fluke parasite,Fasciola hepatica; none of which are conserved with those previously described fromSchistosoma mansoni. F. hepaticalncRNAs were distinct fromF. hepaticamRNAs in transcript length, coding probability, exon/intron composition, expression patterns, and genome distribution. RNA-Seq and digital droplet PCR measurements demonstrated developmentally regulated expression of lncRNAs between intra-mammalian life stages; a similar proportion of lncRNAs (14.2 %) and mRNAs (12.8 %) were differentially expressed (pF. hepaticalife stages. While most lncRNAs (81%) were intergenic, we identified some that overlapped protein coding loci in antisense (13%) or intronic (6%) configurations. We found no unequivocal evidence for correlated developmental expression within positionally correlated lncRNA:mRNA pairs, but global co-expression analysis identified five lncRNA that were inversely co-regulated with 89 mRNAs, including a large number of functionally essential proteases. The presence of micro (mi)RNA binding sites in 3135 lncRNAs indicates the potential for miRNA-based post-transcriptional regulation of lncRNA, and/or their function as competing endogenous (ce)RNAs. This first description of lncRNAs inF. hepaticaprovides an avenue to future functional and comparative genomics studies that will provide a new perspective on a poorly understood aspect of parasite biology.

For over a decade RNA interference (RNAi) has been an important molecular tool for functional genomics studies in parasitic flatworms. Despite this, our understanding of RNAi dynamics in many flatworm parasites, such as the temperate liver fluke (Fasciola hepatica), remains rudimentary. The ability to maintain developing juvenile fluke in vitro provides the opportunity to perform functional studies during development of the key pathogenic life stage. Here, we investigate the RNAi competence of developing juvenile liver fluke. Firstly, all life stages examined possess, and express, core candidate RNAi effectors encouraging the hypothesis that all life stages of F. hepatica are RNAi competent. RNAi effector analyses supported growing evidence that parasitic flatworms have evolved a separate clade of RNAi effectors with unknown function. Secondly, we assessed the impact of growth/development during in vitro culture on RNAi in F. hepatica juveniles and found that during the first week post-excystment liver fluke juveniles exhibit quantitatively lower RNAi mediated transcript knockdown when maintained in growth inducing media. This did not appear to occur in older in vitro juveniles, suggesting that rapidly shifting transcript dynamics over the first week following excystment alters RNAi efficacy after a single 24 h exposure to double stranded (ds)RNA. Finally, RNAi efficiency was found to be improved through use of a repeated dsRNA exposure methodology that has facilitated silencing of genes in a range of tissues, thereby increasing the utility of RNAi as a functional genomics tool in F. hepatica., Graphical abstract Image 1, Highlights • All F. hepatica life stages express the RNAi machinery for a functional pathway. • RNAi-mediated knockdown in juvenile F. hepatica is affected by in vitro growth. • Repeated dsRNA exposures ensure longevity of transcript and protein knockdown. • This provides a basis for a robust RNAi platform in F. hepatica juveniles.

Fasciola spp. liver fluke have significant impacts in veterinary and human medicine. The absence of a vaccine and increasing anthelmintic resistance threaten sustainable control and underscore the need for novel flukicides. Functional genomic approaches underpinned by in vitro culture of juvenile Fasciola hepatica facilitate control target validation in the most pathogenic life stage. Comparative transcriptomics of in vitro and in vivo maintained 21 day old F. hepatica finds that 86% of genes are expressed at similar levels across maintenance treatments suggesting commonality in core biological functioning within these juveniles. Phenotypic comparisons revealed higher cell proliferation and growth rates in the in vivo juveniles compared to their in vitro counterparts. These phenotypic differences were consistent with the upregulation of neoblast-like stem cell and cell-cycle associated genes in in vivo maintained worms. The more rapid growth/development of in vivo juveniles was further evidenced by a switch in cathepsin protease expression profiles, dominated by cathepsin B in in vitro juveniles and then by cathepsin L in in vivo juveniles. Coincident with more rapid growth/development was the marked downregulation of both classical and peptidergic neuronal signalling components in in vivo maintained juveniles, supporting a role for the nervous system in regulating liver fluke growth and development. Differences in the miRNA complements of in vivo and in vitro juveniles identified 31 differentially expressed miRNAs, notably fhe-let-7a-5p, fhe-mir-124-3p and, miRNAs predicted to target Wnt-signalling, supporting a key role for miRNAs in driving the growth/developmental differences in the in vitro and in vivo maintained juvenile liver fluke. Widespread differences in the expression of neuronal genes in juvenile fluke grown in vitro and in vivo expose significant interplay between neuronal signalling and the rate of growth/development, encouraging consideration of neuronal targets in efforts to dysregulate growth/development for parasite control.Author SummaryParasitic worms are notoriously difficult to study outside of a host organism. However, recent developments in culture methods for Fasciola hepatica liver fluke juveniles support growth and development of these parasites in the laboratory (in vitro) towards adult parasites. Having the ability to grow pathogenic juvenile stages in vitro enables functional studies to validate potential drug and vaccine targets. However, comparison of in vitro grown juveniles to juveniles retrieved from infected hosts (in vivo) shows considerable size differences suggesting at least some differences in biology that could undermine the relevance of data generated from in vitro maintained parasites. This study examines gene expression differences between in vitro and in vivo maintained F. hepatica juveniles via transcriptomic analysis to identify similarities and differences in their biology which may help explain differences in the rate of growth and development. 86% of genes were shown to be expressed at similar levels across treatment groups suggesting a high level of biological similarity between in vitro and in vivo juveniles. However, the genes that are expressed differently between these juveniles will help improve current culture methods and provide a new group of potential drug targets that impact on juvenile growth and development.

The liver fluke

1ABSTRACTThe liver fluke, Fasciola hepatica, is a global burden on the wellbeing and productivity of farmed ruminants, and a zoonotic threat to human health. Despite the clear need for accelerated discovery of new drug and vaccine treatments for this pathogen, we still have a relatively limited understanding of liver fluke biology and host interactions. Noncoding RNAs, including micro (mi)RNAs, are key to transcriptional regulation in all eukaryotes, such that an understanding of miRNA biology can shed light on organismal function at a systems level. Four previous publications have reported up to 89 mature miRNA sequences from F. hepatica, but our data show that this does not represent a full account of this species miRNome. We have expanded on previous studies by sequencing, for the first time, miRNAs from multiple life stages (adult, newly excysted juvenile (NEJ), metacercariae and adult-derived extracellular vesicles (EVs)). These experiments detected an additional 61 high-confidence miRNAs, most of which have not been described in any other species, expanding the F. hepatica miRNome to 150 mature sequences. We used quantitative (q)PCR assays to provide the first developmental profile of miRNA expression across metacercariae, NEJ, adult and adult-derived Evs. The majority of miRNAs were expressed most highly in metacercariae, with at least six distinct expression clusters apparent across life stages. Intracellular miRNAs were functionally analysed to identify target mRNAs with inversely correlated expression in F. hepatica tissue transcriptomes, highlighting regulatory interactions with key virulence transcripts including cathepsin proteases, and neuromuscular genes that control parasite growth, development and motility. We also linked 28 adult-derived EV miRNAs with downregulation of 397 host genes in F. hepatica-infected transcriptomes from ruminant lymph node, peripheral blood mononuclear cell (PBMC) and liver tissue transcriptomes. These included genes involved in signal transduction, immune and metabolic pathways, adding to the evidence for miRNA-based immunosuppression during fasciolosis. These data expand our understanding of the F. hepatica miRNome, provide the first data on developmental miRNA regulation in this species, and provide a set of testable hypotheses for functional genomics interrogations of liver fluke miRNA biology.CONTRIBUTION TO THE FIELDPrevious studies identified 89 micro (mi)RNAs (non-coding RNAs responsible for regulating gene expression) in the trematode parasite, Fasciola hepatica, and attached functional annotations to the miRNAs that are secreted by liver fluke in vitro. This study expands the known miRNA complement of F. hepatica by 40%, adding an additional 61 miRNAs, many of which appear Fasciola-specific. We used this expanded dataset to perform the first analysis of developmental miRNA expression across intra-mammalian parasites, showing clear developmentally regulated expression profiles across infectious metacercariae, juvenile and adult parasites, and extracellular vesicles secreted by adult parasites. We performed rigorous functional annotation of cellular miRNAs, correlating their expression with mRNA transcriptomes to identify potential roles for specific miRNAs in regulating expression of important proteases and nerve/muscle transcripts, all of which are important for parasite virulence. We also functionally analysed miRNAs secreted by adult parasites in terms of interactions with host transcripts; for the first time this analysis was supported by host transcriptome data, linking secreted miRNAs with mRNAs that are downregulated by fluke infection in sheep and cattle. This work contributes new data, insights and analyses to the field, providing a rich source of hypotheses for future experimental validation.

For over a decade RNA interference (RNAi) has been an important molecular tool for functional genomics studies in parasitic flatworms. Despite this, our understanding of RNAi dynamics in many flatworm parasites, such as the temperate liver fluke (Fasciola hepatica), remains rudimentary. The ability to maintain developing juvenile fluke in vitro provides the opportunity to perform functional studies during development of the key pathogenic life stage. Here, we investigate the RNAi competence of developing juvenile liver fluke. Firstly, all life stages examined possess, and express, core candidate RNAi effectors encouraging the hypothesis that all life stages of F. hepatica are RNAi competent. RNAi effector analyses supported growing evidence that parasitic flatworms have evolved a separate clade of RNAi effectors with unknown function. Secondly, we assessed the impact of growth / development during in vitro culture on RNAi in F. hepatica juveniles and found that during the first week post-excystment liver fluke juveniles exhibit quantitatively lower RNAi mediated transcript knockdown when maintained in growth inducing media. This did not appear to occur in older in vitro juveniles, suggesting that rapidly shifting transcript dynamics over the first week following excystment alters RNAi efficacy after a single 24 hour exposure to double stranded (ds)RNA. Finally, RNAi efficiency was found to be improved through use of a repeated dsRNA exposure methodology that has facilitated silencing of genes in a range of tissues, thereby increasing the utility of RNAi as a functional genomics tool in F. hepatica.

Background: \ud Borderline Personality Disorder (BPD) is a psychiatric disorder associated with significant morbidity and mortality. However, the neurobiological alterations underlying the condition remain poorly understood. As a result, existing treatments remain inadequate. One of the main risk factors for the development of BPD is a history of childhood maltreatment. However, it is considered neither causative nor specific to the condition. Current theory is therefore increasingly moving toward a ‘Gene x Environment’ (GxE) model of the condition. The purpose of the current work was to conduct a systematic literature review, which comprehensively identifies all published molecular level GxE studies that have explored the role of specific genetic loci, in influencing the risk of BPD following exposure to childhood abuse or neglect.\ud \ud Methods: \ud Four electronic databases were used to systematically search for molecular level GxE studies of any design, which focused on the development of BPD following exposure to childhood abuse or neglect, without language or date restrictions. Articles were screened independently by two reviewers and results were synthesised narratively.\ud \ud Results: \ud A total of 473 articles were screened of which sixteen were selected for inclusion in our review. Implicated genes were categorised according to their influence on; Neurotransmitter Systems, Neurodevelopment and Neuroendocrine Systems.\ud \ud Conclusions: \ud The identified studies have produced several relevant and statistically significant results. Of particular note, is the repeated finding that genes involved in HPA axis regulation, may be altered by exposure to childhood maltreatment, influencing subsequent susceptibility to BPD. This is both biologically plausible and of potential clinical significance.

Pollinators use multiple cues whilst foraging including direct cues from flowers and indirect cues from other pollinators. The use of indirect social cues is common in social insects, such as honeybees and bumblebees, where a social environment facilitates the ability to use such cues. Bumblebees use cues to forage on flowers according to previous foraging experiences. Flowers are an essential food source for pollinators but also pose a high risk of parasite infection through the shared use of flowers leading to parasite spillover. Nevertheless, bumblebees have evolved behavioral defense mechanisms to limit parasite infection by avoiding contaminated flowers. Mechanisms underlying the avoidance of contaminated flowers by bumblebees are poorly understood. Bumblebees were recorded having the choice to forage on non-contaminated flowers and flowers contaminated by a trypan osome gut parasite, Crithidia bombi. The use of different treatments with presence or absence of conspecifics on both contaminated and non-contaminated flowers allowed to investigate the role of social visual cues on their pathogen avoidance behavior. Bumblebees are expected to use social visual cues to avoid contaminated flowers. Our study reveals that the presence of a conspecific on flowers either contaminated or not does not help bumblebee foragers avoiding contaminated flowers. Nevertheless, bumblebees whereas gaining experience tend to avoid their conspecific when placed on contaminated flower and copy it when on the non-contaminated flower. Our experiment suggests a detrimental impact of floral scent on disease avoidance behavior.

The majority of anthelmintics dysregulate neuromuscular function, a fact most prominent for drugs against nematode parasites. In contrast to the strong knowledge base for nematode neurobiology, resource and tool deficits have prevented similar advances in flatworm parasites since those driven by bioimaging, immunocytochemistry, and neuropeptide biochemistry 20-30 years ago. However, recent developments are encouraging a renaissance in liver fluke neurobiology that can now support flukicide discovery. Emerging data promote neuromuscular signalling components, and especially G protein-coupled receptors (GPCRs), as next-generation targets. Here, we summarise these data and expose some of the new opportunities to accelerate progress towards GPCR-targeted flukicides for Fasciola hepatica.

Ras protein plays a critical role in cell growth and proliferation by transmitting signal from cell surface receptor tyrosine kinase to downstream cellular proteins such as RAF and MEK. KRas is one of the most frequently mutated oncogenes, with mutations at residues G12, G13, and Q61, found in lung, colon, pancreatic and other solid malignancies. We have developed an orally bioavailable, covalent inhibitor of KRas G12C, D-1553, with potent in vitro biochemical, cellular activity and in vivo efficacy. Here we report the efficacy of D-1553 in a large panel of patient-derived xenograft (PDX) tumor models with KRas G12C mutation. In the lung cancer PDX models, D-1553 exhibited tumor growth inhibition (TGI) from 43.6% to 124.3%, with 4 out of 8 models showing tumor regression. In the colorectal cancer PDX models, the range of TGI was from 60.9% to 105.7%, with 3 out of 9 models showing regression. Combination treatment of D-1553 with chemotherapy and targeted agents demonstrated enhanced anti-tumor activity resulting in more tumor regression compared to single agent treatment. These observations from PDX models support the combination treatment as a key to increased overall response rate to KRas G12C inhibitor in clinical trial. D-1553 is currently in a Phase 1/2 clinical trial for patients with advanced solid tumors harboring KRas G12C mutation (NCT04585035). Citation Format: Zhe Shi, Jifang Weng, Xiaochong Fan, Qingqing Zhu, Emily Robb, Alyssa Moriarty, Michael Wick, Yueheng Jiang, Ling Zhang, Xing Dai, Yaolin Wang. Potent in vivo anti-tumor activity of D-1553 as a single agent and in combination with targeted therapeutics in a broad spectrum of patient-derived xenograft tumor models with KRas G12C mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1056.

Background: Patient-derived xenografts (PDX) as xenotransplantation of human tumors into athymic nude mice have become the hallmark of preclinical modelling in cancer research. They are particularly useful in the characterization of targeted therapies in drug development. We sough to build a collection of PDX connected to our early phase trials facility for preclinical testing of new drugs. Methods: Between March 2017 and October 2019, a total of 483 samples from patients with cancer were obtained for implant. A full review of the characteristics including histological particularities, molecular findings and treatment experience was reviewed for a full description of the collection. Results: 483 tumor samples were implanted. A total of 157 achieved tumor growth (defined as a target volume of 150 mm3 in at least three consecutive measurements). Here, we present the description of 121 successfully engrafted models passed at least 2 times from initial implant. Most common type of cancer is colorectal adenocarcinoma (CRC=63; 52%) (MSI-H=8, 13%). 20 are molecularly characterized: KRAS: 9 (45%); NRAS=1 (5%) and BRAF=4 (20%). Prior therapy: chemo=16, VEGFi=9, EGFRi=1, ICi=1. Fifteen ovarian carcinomas (12,39%). Three BRCA2m (2) and EPCAMm (1). Eight breast carcinomas (6,6%): 6 Invasive Ductal Carcinomas (IDC) (three are triple negative with BRCA2m; 3 resistant to aromatase inhibitors, and 1 to a CDK inhibitor), 1 papillary carcinoma and 1 medullary carcinoma harboring a TP53 mutation. Eleven non-small cell lung cancers grew successfully, all EGFRwt (KRASm=2). One squamous cell carcinoma with a BRD4/NUTM1 fusion treated with cisplatin/vinorelbine and atezolizumab prior to implant. Eight glioblastomas (prior temozolomide=3 (37%), MGMTmt=5 (62%) and IDHm=1 (12%). One uveal melanoma with a MET fusion. The collection also includes endometrial MSI-H cancers (2), bladder (2), head and neck (1), kidney (4), pancreas (2), biliary tract (1) and cervix carcinomas (1) and one osteosarcoma. Conclusions: The importance of PDX development programs relies on the reproducibility of tumors with specific oncogenic drivers potentially targetable. A full comprehensive characterization of PDX shareable collections are paramount for tracking of molecular, diagnostic, prognostic and predictive markers of drug response. Citation Format: Natalia Baños, Tatiana Hernandez-Guerrero, Victoria Bonilla, Bernard Doger, Laura Del Puerto, Emily Robb, Cecilio Cadena, Jesus Garcia-Foncillas, Victor Moreno, Michael Wick. Establishment of a comprehensive patient derived xenograft (PDX) collection for use in precision medicine; A project from START Madrid- FJD [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5061.

Expanding 'omics' datasets for parasitic nematodes have accelerated the identification of putative drug targets derived from the nematode nervous system. However, novel drug target validation is hampered by the absence of adequate localisation, functional characterisation, and receptor deorphanisation tools in key nematode pathogens. Reverse genetics techniques have advanced to encompass transgenic, targeted mutagenesis, gene silencing (RNA interference), and genome editing (CRISPR/Cas9) approaches in Caenorhabditis elegans. Unfortunately the translation to nematode pathogens has been slow, such that parasite-focused toolbox development and optimisation is critical. Here we review the discovery, localisation, and functional characterisation toolkit available for parasitic nematode neuropeptide research, and assess the scope and limitations of the tools and techniques for novel nematicide discovery.