Analysis of the transcriptome of adult Dictyocaulus filaria and comparison with Dictyocaulus viviparus, with a focus on molecules involved in host–parasite interactions

Parasitic nematodes cause diseases of major economic importance in animals. Key representatives are species of Dictyocaulus (=lungworms), which cause bronchitis (=dictyocaulosis, commonly known as “husk”) and have a major adverse impact on the health of livestock. In spite of their economic importance, very little is known about the immunomolecular biology of these parasites. Here, we conducted a comprehensive investigation of the adult transcriptome of Dictyocaulus filaria of small ruminants and compared it with that of Dictyocaulus viviparus of bovids. We then identified a subset of highly transcribed molecules inferred to be linked to host–parasite interactions, including cathepsin B peptidases, fatty-acid and/or retinol-binding proteins, β-galactoside-binding galectins, secreted protein 6 precursors, macrophage migration inhibitory factors, glutathione peroxidases, a transthyretin-like protein and a type 2-like cystatin. We then studied homologues of D. filaria type 2-like cystatin encoded in D. viviparus and 24 other nematodes representing seven distinct taxonomic orders, with a particular focus on their proposed role in immunomodulation and/or metabolism. Taken together, the present study provides new insights into nematode–host interactions. The findings lay the foundation for future experimental studies and could have implications for designing new interventions against lungworms and other parasitic nematodes. The future characterisation of the genomes of Dictyocaulus spp. should underpin these endeavours

The Interference of Notch1 target Hes1 affects cell growth, differentiation and invasiveness of glioblastoma stem cells through modulation of multiple oncogenic targets

The invasive and lethal nature of Glioblastoma multiforme (GBM) necessitates the continuous identification of molecular targets and search of efficacious therapies to inhibit GBM growth. The GBM resistance to chemotherapy and radiation it is attributed to the existence of a rare fraction of cancer stem cells (CSC) that we have identified within the tumor core and in peritumor tissue of GBM. Since Notch1 pathway is a potential therapeutic target in brain cancer, earlier we highlighted that pharmacological inhibition of Notch1 signalling by γ-secretase inhibitor-X (GSI-X), reduced cell growth of some c-CSC than to their respective p-CSC, but produced negligible effects on cell cycle distribution, apoptosis and cell invasion. In the current study, we assessed the effects of Hes1-targeted shRNA, a Notch1 gene target, specifically on GBM CSC refractory to GSI-X. Depletion of Hes1 protein induces major changes in cell morphology, cell growth rate and in the invasive ability of shHes1-CSC in response to growth factor EGF. shHes1-CSC show a decrease of the stemness marker Nestin concurrently to a marked increase of neuronal marker MAP2 compared to pLKO.1-CSC. Those effects correlated with repression of EGFR protein and modulation of Stat3 phosphorylation at Y705 and S727 residues. In the last decade Stat3 has gained attention as therapeutic target in cancer but there is not yet any approved Stat3-based glioma therapy. Herein, we report that exposure to a Stat3/5 inhibitor, induced apoptosis either in shHes1-CSC or control cells. Taken together, Hes1 seems to be a favorable target but not sufficient itself to target GBM efficaciously, therefore a possible pharmacological intervention should provide for the use of anti-Stat3/5 drugs either alone or in combination regimen

Androgen deprivation therapy promotes an obesity-like microenvironment in periprostatic fat

Prostate cancer is a leading cause of morbidity and cancer-related death worldwide. Androgen deprivation therapy (ADT) is the cornerstone of management for advanced disease. The use of these therapies is associated with multiple side effects, including metabolic syndrome and truncal obesity. At the same time, obesity has been associated with both prostate cancer development and disease progression, linked to its effects on chronic inflammation at a tissue level. The connection between ADT, obesity, inflammation and prostate cancer progression is well established in clinical settings; however, an understanding of the changes in adipose tissue at the molecular level induced by castration therapies is missing. Here, we investigated the transcriptional changes in periprostatic fat tissue induced by profound ADT in a group of patients with high-risk tumours compared to a matching untreated cohort. We find that the deprivation of androgen is associated with a pro-inflammatory and obesity-like adipose tissue microenvironment. This study suggests that the beneficial effect of therapies based on androgen deprivation may be partially counteracted by metabolic and inflammatory side effects in the adipose tissue surrounding the prostate

Detection of ctDNA in plasma of patients with clinically localised prostate cancer is associated with rapid disease progression.

BACKGROUND DNA originating from degenerate tumour cells can be detected in the circulation in many tumour types, where it can be used as a marker of disease burden as well as to monitor treatment response. Although circulating tumour DNA (ctDNA) measurement has prognostic/predictive value in metastatic prostate cancer, its utility in localised disease is unknown. METHODS We performed whole-genome sequencing of tumour-normal pairs in eight patients with clinically localised disease undergoing prostatectomy, identifying high confidence genomic aberrations. A bespoke DNA capture and amplification panel against the highest prevalence, highest confidence aberrations for each individual was designed and used to interrogate ctDNA isolated from plasma prospectively obtained pre- and post- (24 h and 6 weeks) surgery. In a separate cohort (n = 189), we identified the presence of ctDNA TP53 mutations in preoperative plasma in a retrospective cohort and determined its association with biochemical- and metastasis-free survival. RESULTS Tumour variants in ctDNA were positively identified pre-treatment in two of eight patients, which in both cases remained detectable postoperatively. Patients with tumour variants in ctDNA had extremely rapid disease recurrence and progression compared to those where variants could not be detected. In terms of aberrations targeted, single nucleotide and structural variants outperformed indels and copy number aberrations. Detection of ctDNA TP53 mutations was associated with a significantly shorter metastasis-free survival (6.2 vs. 9.5 years (HR 2.4; 95% CIs 1.2-4.8, p = 0.014). CONCLUSIONS CtDNA is uncommonly detected in localised prostate cancer, but its presence portends more rapidly progressive disease

Investigation of the tumour microenvironment of prostate cancer

© 2018 Dr. Stefano MangiolaOne in seven men in Australia is at risk of developing prostate cancer before the age of 75. This disease is a leading cause of male death worldwide, with a mortality rate of 62 men per 100,000. Treatments for prostate cancer exist, including surgery, radiotherapy and androgen deprivation therapy. However, the results achieved by the combination of these therapies can lead to variable outcomes, mainly due to the genetic heterogeneity of tumour cells and/or emergence of resistance. In contrast to the cancer cells, the non-cancerous portion of the tumour microenvironment, such as immune cells, fibroblasts and endothelial cells, is a genetically stable target that has a key role in cancer development. Improving our knowledge of the genetic and molecular interactions existing between cancer cells and other non-cancerous cell populations, both in primary or metastatic prostate cancer will provide new key insights in the biology of the disease and give new treatment opportunity

Developing a CITE-sequencing analysis pipeline by investigating a COVID-19 dataset

Curs 2021-2022Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-sequencing) is a multimodal highthroughput single-cell technology that measures contemporaneously gene and surface protein expression levels for each single cell sequenced. By using a CITE-sequencing dataset of patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we aimed at developing a robust analysis pipeline that will later serve for more ambitious purposes. Our study included the analysis of peripheral blood mononuclear cells (PBMCs) derived from 6 COVID-19 donors (3 moderate, 3 severe) and 6 healthy donors. Our study design included a panel of 277 Antibody-derived tags (ADTs) including 9 isotype control antibodies. To decrease confounders and sequencing costs, samples were pooled before sequencing after being labeled using the cell hashing technique. Cells were also designed to be demultiplexed and assigned to their donor using vireo. After a deep quality control and extensive assessment of demultiplexing, RNA and Protein matrices were further pre-processed by maintaining only high-quality sequenced cells and doublets removal. These underwent separate normalization and finally integration using the Weighted Nearest Neighbored analysis. A final annotation completed this initial analysis.Associate Editor: Mireia Olivell

Probabilistic outlier identification for RNA sequencing generalized linear models

Relative transcript abundance has proven to be a valuable tool for understanding the function of genes in biological systems. For the differential analysis of transcript abundance using RNA sequencing data, the negative binomial model is by far the most frequently adopted. However, common methods that are based on a negative binomial model are not robust to extreme outliers, which we found to be abundant in public datasets. So far, no rigorous and probabilistic methods for detection of outliers have been developed for RNA sequencing data, leaving the identification mostly to visual inspection. Recent advances in Bayesian computation allow large-scale comparison of observed data against its theoretical distribution given in a statistical model. Here we propose ppcseq, a key quality-control tool for identifying transcripts that include outlier data points in differential expression analysis, which do not follow a negative binomial distribution. Applying ppcseq to analyse several publicly available datasets using popular tools, we show that from 3 to 10 percent of differentially abundant transcripts across algorithms and datasets had statistics inflated by the presence of outliers.Peer reviewe

<i>IL17RB</i> and <i>IL17REL</i> Expression Are Associated with Improved Prognosis in HPV-Infected Head and Neck Squamous Cell Carcinomas

Changes in the cellular secretome are implicated in virus infection, malignancy, and anti-tumor immunity. We analyzed the association between transcriptional signatures (TS) from 24 different immune and stromal cell types on the prognosis of HPV-infected and HPV-free head and neck squamous carcinoma (HNSCC) patients from The Cancer Genome Atlas (TCGA) cohort. We found that HPV-positive HNSCC patients have tumors with elevated immune cell TS and improved prognosis, which was specifically associated with an increased tumor abundance of memory B and activated natural killer (NK) cell TS, compared to HPV-free HNSCC patients. HPV-infected patients upregulated many transcripts encoding secreted factors, such as growth factors, hormones, chemokines and cytokines, and their cognate receptors. Analysis of secretome transcripts and cognate receptors revealed that tumor expression of IL17RB and IL17REL are associated with a higher viral load and memory B and activated NK cell TS, as well as improved prognosis in HPV-infected HNSCC patients. The transcriptional parameters that we describe may be optimized to improve prognosis and risk stratification in the clinic and provide insights into gene and cellular targets that may potentially enhance anti-tumor immunity mediated by NK cells and memory B cells in HPV-infected HNSCC patients

Identification of G protein-coupled receptors in Schistosoma haematobium and S. mansoni by comparative genomics

BACKGROUND: Schistosomiasis is a parasitic disease affecting ~200 million people worldwide. Schistosoma haematobium and S. mansoni are two relatively closely related schistosomes (blood flukes), and the causative agents of urogenital and hepatointestinal schistosomiasis, respectively. The availability of genomic, transcriptomic and proteomic data sets for these two schistosomes now provides unprecedented opportunities to explore their biology, host interactions and schistosomiasis at the molecular level. A particularly important group of molecules involved in a range of biological and developmental processes in schistosomes and other parasites are the G protein-coupled receptors (GPCRs). Although GPCRs have been studied in schistosomes, there has been no detailed comparison of these receptors between closely related species. Here, using a genomic-bioinformatic approach, we identified and characterised key GPCRs in S. haematobium and S. mansoni (two closely related species of schistosome). METHODS: Using a Hidden Markov Model (HMM) and Support Vector Machine (SVM)-based pipeline, we classified and sub-classified GPCRs of S. haematobium and S. mansoni, combined with phylogenetic and transcription analyses. RESULTS: We identified and classified classes A, B, C and F as well as an unclassified group of GPCRs encoded in the genomes of S. haematobium and S. mansoni. In addition, we characterised ligand-specific subclasses (i.e. amine, peptide, opsin and orphan) within class A (rhodopsin-like). CONCLUSIONS: Most GPCRs shared a high degree of similarity and conservation, except for members of a particular clade (designated SmGPR), which appear to have diverged between S. haematobium and S. mansoni and might explain, to some extent, some of the underlying biological differences between these two schistosomes. The present set of annotated GPCRs provides a basis for future functional genomic studies of cellular GPCR-mediated signal transduction and a resource for future drug discovery efforts in schistosomes

Bioinformatic exploration of RIO protein kinases of parasitic and free-living nematodes

Despite right open reading frame kinases being essential for life, their functions, substrates and cellular pathways remain enigmatic. In the present study, gene structures were characterised for 26 right open reading frame kinase from draft genomes of parasitic and free-living nematodes. RNA-seq transcription profiles of right open reading frame kinase genes were investigated for selected parasitic nematodes and showed that these kinases are transcribed in developmental stages that infect their mammalian host. Three-dimensional structural models of Caenorhabditis elegans right open reading frame kinases were predicted, and elucidated functional domains and conserved regions in nematode homologs. These findings provide prospects for functional studies of RIO kinase genes in C. elegans and an opportunity for the design and validation of nematode-specific inhibitors of these enzymes in socioeconomic parasitic worms