ExpressAnalyst: A unified platform for RNA-sequencing analysis in non-model species.

The increasing application of RNA sequencing to study non-model species demands easy-to-use and efficient bioinformatics tools to help researchers quickly uncover biological and functional insights. We developed ExpressAnalyst ( www.expressanalyst.ca ), a web-based platform for processing, analyzing, and interpreting RNA-sequencing data from any eukaryotic species. ExpressAnalyst contains a series of modules that cover from processing and annotation of FASTQ files to statistical and functional analysis of count tables or gene lists. All modules are integrated with EcoOmicsDB, an ortholog database that enables comprehensive analysis for species without a reference transcriptome. By coupling ultra-fast read mapping algorithms with high-resolution ortholog databases through a user-friendly web interface, ExpressAnalyst allows researchers to obtain global expression profiles and gene-level insights from raw RNA-sequencing reads within 24 h. Here, we present ExpressAnalyst and demonstrate its utility with a case study of RNA-sequencing data from multiple non-model salamander species, including two that do not have a reference transcriptome.

Using MetaboAnalyst 5.0 for LC-HRMS spectra processing, multi-omics integration and covariate adjustment of global metabolomics data.

Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) has become a workhorse in global metabolomics studies with growing applications across biomedical and environmental sciences. However, outstanding bioinformatics challenges in terms of data processing, statistical analysis and functional interpretation remain critical barriers to the wider adoption of this technology. To help the user community overcome these barriers, we have made major updates to the well-established MetaboAnalyst platform ( www.metaboanalyst.ca ). This protocol extends the previous 2011 Nature Protocol by providing stepwise instructions on how to use MetaboAnalyst 5.0 to: optimize parameters for LC-HRMS spectra processing; obtain functional insights from peak list data; integrate metabolomics data with transcriptomics data or combine multiple metabolomics datasets; conduct exploratory statistical analysis with complex metadata. Parameter optimization may take ~2 h to complete depending on the server load, and the remaining three stages may be executed in ~60 min.

Native Microbiome Members of C. elegans Act Synergistically in Biosynthesis of Pyridoxal 5'-Phosphate.

The roles of the healthy microbiome on the host and the relationships between members of the microbiome remain to be fully characterized. Due to the complexity of the interactions between the mammalian microbiome and its host, the use of model organisms such as the nematode worm Caenorhabditis elegans is a promising strategy to study host-microbiome interactions in vivo, as well as bacterial crosstalk within the host. Previously it was found that native bacterial isolates of the worm, Chryseobacterium sp. CHNTR56 MYb120 and Comamonas sp. 12022 MYb131, possess genomic diversity in the biosynthesis of the active form of vitamin B6, pyridoxal 5'-phosphate (PLP), and contribute to host fitness and lifespan extension. However, the relative contribution of PLP from each isolate, as well as the existence of interbacterial relationships within the worm gut remain to be characterized. In the present work, we investigated the presence and measured the abundance of PLP in the isolates and in the worms grown with the isolates using ultraperformance liquid chromatography tandem-mass spectrometry (UPLC-MS/MS). Our analyses confirmed the presence of PLP in vitro and in vivo. The elevated abundance of PLP in the isolates (which reached statistically significant levels when the two isolates were combined), and within worms grown with the combination of bacterial isolates, compared to control, indicated synergism between the isolates in the production of PLP. Isotope labeling revealed that Comamonas sp. 12022 MYb131 was the main provider of PLP in worms grown with the combination of bacterial isolates. The dominance of this isolate inside the worm was further confirmed by a colonization assay. An untargeted metabolomics analysis of the bacteria showed that the pathways related to cell growth, protein synthesis and lipid synthesis/energy production were regulated in the combination group in comparison with Comamonas sp. 12022 MYb131 alone. Furthermore, glutamine, involved in the de novo synthesis of purine and pyrimidines, was specifically abundant in this group, indicating the potential role of this metabolite in initiating and sustaining bacterial growth. This bacterial crosstalk is suggested to promote the growth of Comamonas sp. 12022 MYb131 in vivo, and synthesis of bacterial metabolites such as PLP in the worm gut.

Oxidative stress suppression in C. elegans by peptides from dogfish skin via regulation of transcription factors DAF-16 and HSF-1.

Functional peptides were obtained via enzymatic hydrolysis of smooth dogfish (Mustelus canis) skin. The enzyme-assisted process was optimized to achieve high yield of smooth dogfish skin peptides (SDSP). Fractions of SDSP (MW < 2 kDa, 2-5 kDa, 5-10 kDa and >10 kDa) showed in vitro antioxidant activities. The peptides <2 kDa (SDSP<2 kDa) significantly improved motility, reduced ROS and H2O2 levels of Caenorhabditis elegans, and increased its resistance to oxidative stress compared to the other peptide fractions. In vivo function of SDSP<2 kDa could be explained by their capacity to increase the expression of stress-response genes. The enhanced resistance to oxidative stress mediated by SDSP<2 kDa was dependent on DAF-16 and HSF-1. The amino acid residues and sequences of SDSP<2 kDa were characterized and revealed a higher content of hydrophobic versus polar amino acid contents. This study (especially the in vivo investigation) explored new potent antioxidant peptides derived from dogfish skin.

The symbiotic relationship between Caenorhabditis elegans and members of its microbiome contributes to worm fitness and lifespan extension.

BACKGROUND: A healthy microbiome influences host physiology through a mutualistic relationship, which can be important for the host to cope with cellular stress by promoting fitness and survival. The mammalian microbiome is highly complex and attributing host phenotypes to a specific member of the microbiome can be difficult. The model organism Caenorhabditis elegans and its native microbiome, discovered recently, can serve as a more tractable, experimental model system to study host-microbiome interactions. In this study, we investigated whether certain members of C. elegans native microbiome would offer a benefit to their host and putative molecular mechanisms using a combination of phenotype screening, omics profiling and functional validation. RESULTS: A total of 16 members of C. elegans microbiome were screened under chemically-induced toxicity. Worms grown with Chryseobacterium sp. CHNTR56 MYb120 or Comamonas sp. 12022 MYb131, were most resistant to oxidative chemical stress (SiO2 nanoparticles and juglone), as measured by progeny output. Further investigation showed that Chryseobacterium sp. CHNTR56 positively influenced the worm's lifespan, whereas the combination of both isolates had a synergistic effect. RNAseq analysis of young adult worms, grown with either isolate, revealed the enrichment of cellular detoxification mechanisms (glutathione metabolism, drug metabolism and metabolism of xenobiotics) and signaling pathways (TGF-beta and Wnt signaling pathways). Upregulation of cysteine synthases (cysl genes) in the worms, associated with glutathione metabolism, was also observed. Nanopore sequencing uncovered that the genomes of the two isolates have evolved to favor the specific route of the de novo synthesis pathway of vitamin B6 (cofactor of cysl enzymes) through serC or pdxA2 homologs. Finally, co-culture with vitamin B6 extended worm lifespan. CONCLUSIONS: In summary, our study indicates that certain colonizing members of C. elegans have genomic diversity in vitamin B6 synthesis and promote host fitness and lifespan extension. The regulation of host cellular detoxification genes (i.e. gst) along with cysl genes at the transcriptome level and the bacterium-specific vitamin B6 synthesis mechanism at the genome level are in an agreement with enhanced host glutathione-based cellular detoxification due to this interspecies relationship. C. elegans is therefore a promising alternative model to study host-microbiome interactions in host fitness and lifespan.

Comprehensive phenotyping and transcriptome profiling to study nanotoxicity in C. elegans.

Engineered nanoparticles are used at an increasing rate in both industry and medicine without fully understanding their impact on health and environment. The nematode Caenorhabditis elegans is a suitable model to study the toxic effects of nanoparticles as it is amenable to comprehensive phenotyping, such as locomotion, growth, neurotoxicity and reproduction. In this study, we systematically evaluated the effects of silver (Ag) and five metal oxide nanoparticles: SiO2, CeO2, CuO, Al2O3 and TiO2. The results showed that Ag and SiO2 exposures had the most toxic effects on locomotion velocity, growth and reproduction, whereas CeO2, Al2O3 and CuO exposures were mostly neurotoxic. We further performed RNAseq to compare the gene expression profiles underlying Ag and SiO2toxicities. Gene set enrichment analyses revealed that exposures to Ag and SiO2consistently downregulated several biological processes (regulations in locomotion, reproductive process and cell growth) and pathways (neuroactive ligand-receptor interaction, wnt and MAPK signaling, etc.), with opposite effects on genes involved in innate immunity. Our results contribute to mechanistic insights into toxicity of Ag and SiO2 nanoparticles and demonstrated that C. elegans as a valuable model for nanotoxicity assessment.

Genererating a core cluster of Fasciola hepatica virulence and immunomodulation-related genes using a comparative in silico approach.

A total of 71 virulence and immunomodulation-related transcripts (VIRs) of Fasciola hepatica have been previously proposed (Haçariz et al., 2015). In an attempt to further refine this cohort, an in silico meta analysis approach was carried out using publicly available sequence data of related liver flukes, Clonorchis sinensis and Opisthorchis viverrini. Data of both liver flukes were investigated in terms of sequential homology with data of non-parasitic organisms, pathogens and VIRs of F. hepatica, directional selection (Ka/Ks), and cytokine signaling relation (protein motif based). Some VIRs of F. hepatica [showing homology with immune receptors (for toll/interleukin-1, TGF-ß or TNF-α), TGF-ß, TNF-α, CD147, or relation with suppressors of cytokine signaling/IKBKE 1 or stimulation of TGF-ß (through thrombospondin similarity)] were found to be orthologous with those of both C. sinensis and O. viverrini. The in silico analysis indicates that on the basis of genetic commonality, a total of 30 VIRs of F. hepatica are highlighted as of foremost importance in the parasite evasion strategy, through controlling of host immune system. Findings in this study could be important to further enhance our understanding of the parasitic mechanisms and develop effective control strategies against F. hepatica and other related parasites.

The omic approach to parasitic trematode research-a review of techniques and developments within the past 5 years.

The evolution of technologies to explore parasite biology at a detailed level has made significant advances in recent years, particularly with the development of omic-based strategies. Whilst extensive efforts have been made in the past to develop therapeutic and prophylactic control strategies for trematode parasites, only the therapeutic anthelmintic approach can be regarded as usable in clinical practice. Currently, there is no commercialised prophylactic strategy (such as vaccination) for protection of the definitive host against any trematode parasite. Since 2010 in particular, the integration of omic technologies, including liquid chromatography-mass spectrometry (LC-MS) and next-generation sequencing (NGS), has been increasingly reported in trematode-related studies. Both LC-MS and NGS facilitate a better understanding of the biology of trematodes and provide a promising route to identifying clinically important biological characteristics of parasitic trematodes. In this review, we focus on the application, advantages, and disadvantages of omic technologies (LC-MS and NGS) in trematode research within the past 5 years and explore the use and translation of the omic-based research results into practical tools to deal with infection.

Comparative transcriptome profiling approach to glean virulence and immunomodulation-related genes of Fasciola hepatica.

BACKGROUND: Fasciola hepatica causes chronic liver disease, fasciolosis, leading to significant losses in the livestock economy and concerns for human health in many countries. The identification of F. hepatica genes involved in the parasite's virulence through modulation of host immune system is utmost important to comprehend evasion mechanisms of the parasite and develop more effective strategies against fasciolosis. In this study, to identify the parasite's putative virulence genes which are associated with host immunomodulation, we explored whole transcriptome of an adult F. hepatica using current transcriptome profiling approaches integrated with detailed in silico analyses. In brief, the comparison of the parasite transcripts with the specialised public databases containing sequence data of non-parasitic organisms (Dugesiidae species and Caenorhabditis elegans) or of numerous pathogens and investigation of the sequences in terms of nucleotide evolution (directional selection) and cytokine signaling relation were conducted. RESULTS: NGS of the whole transcriptome resulted in 19,534,766 sequence reads, yielding a total of 40,260 transcripts (N50 = 522 bp). A number of the parasite transcripts (n = 1,671) were predicted to be virulence-related on the basis of the exclusive homology with the pathogen-associated data, positive selection or relationship with cytokine signaling. Of these, a group of the virulence-related genes (n = 62), not previously described, were found likely to be associated with immunomodulation based on in silico functional categorisation, showing significant sequence similarities with various immune receptors (i.e. MHC I class, TGF-ß receptor, toll/interleukin-1 receptor, T-cell receptor, TNF receptor, and IL-18 receptor accessory protein), cytokines (i.e. TGF-ß, interleukin-4/interleukin-13 and TNF-α), cluster of differentiations (e.g. CD48 and CD147) or molecules associated with other immunomodulatory mechanisms (such as regulation of macrophage activation). Some of the genes (n = 5) appeared to be under positive selection (Ka/Ks > 1), imitating proteins associated with cytokine signaling (through sequence homologies with thrombospondin type 1, toll/interleukin-1 receptor, TGF-ß receptor and CD147). CONCLUSIONS: With a comparative transcriptome profiling approach, we have identified a number of potential immunomodulator genes of F. hepatica (n = 62), which are firstly described here, could be employed for the development of better strategies (including RNAi) in the battle against both zoonotically and economically important disease, fasciolosis.

[Investigation of the abundance of proteins secreted by Fasciola hepatica, which is exposed to environmental change in experimental studies, with an advanced proteomic approach]. / Deneysel Çalismalarda Çevresel Degisiklige Maruz Kalan Fasciola hepatica'nin Salgiladigi Protein Miktarlarinin Bir Ileri Proteomik Yaklasimla Incelenmesi.

OBJECTIVE: Investigation of the abundance of proteins secreted by Fasciola hepatica, which is exposed to environmental change after it is removed from the main host, with an advanced proteomic approach. METHODS: Adult Fasciola hepatica parasites, obtained from the main host, were directly placed in phosphate-buffered saline (PBS, at room temperature) and incubated at 37°C for 2 hours (after arrival at the Institute within 1 hour). After this, without applying extra procedures, such as washing the parasites, secreted parasite proteins in PBS were investigated using an advanced proteomic method [a mass spectrometry system with electrospray ionization and quadrupole time-of-flight source coupled to ultra performance liquid chromatography, nano UPLC-ESI-QTOF-MS] with a reviewed F. hepatica protein database (Universal Protein Resource; UniProt) and data-independent acquisition method. RESULTS: With the proteomic analysis of the PBS, after incubation with the parasites, cathepsin L protease 1, fatty acid-binding protein 1 and 2, thioredoxin peroxidase (TPx), and kunitz-type proteinase inhibitor were identified. The abundance of Fasciola hepatica TPx was approximately 2-6 times higher than that of the other proteins identified in this study (p<0.01). CONCLUSION: The stress on the parasite stem from environmental change could be associated with the stimulation of the secretion of TPx. The application of advanced proteomic approaches could provide useful data in the development of effective protective methods against the parasite.

Generating a detailed protein profile of Fasciola hepatica during the chronic stage of infection in cattle.

Fasciola hepatica is a trematode helminth causing a damaging disease, fasciolosis, in ruminants and humans. Comprehensive proteomic studies broaden our knowledge of the parasite's protein profile, and provide new insights into the development of more effective strategies to deal with fasciolosis. The objective of this study was to generate a comprehensive profile of F. hepatica proteins expressed during the chronic stage of infection in cattle by building on previous efforts in this area. The approach included an improved sample preparation procedure for surface and internal layers of the parasite, the application of nano-UPLC-ESI-qTOF-MS (nano-ultra-performance LC and ESI quadrupole TOF MS) integrated with different acquisition methods and in silico database search against various protein databases and a transcript database including a new assembly of publically available EST. Of a total of 776 identified proteins, 206 and 332 were specific to the surface and internal layers of the parasite, respectively. Furthermore, 238 proteins were common to both layers, with comparative differences of 172 proteins detected. Specific proteins not previously identified in F. hepatica, but shown to be immunomodulatory or potential drug targets for other parasites, are discussed.

TMCO1 deficiency causes autosomal recessive cerebrofaciothoracic dysplasia.

Cerebrofaciothoracic dysplasia (CFT) (OMIM #213980) is a multiple congenital anomaly and intellectual disability syndrome involving the cranium, face, and thorax. The characteristic features are cranial involvement with macrocrania at birth, brachycephaly, various CT/MRI findings including hypoplasia of corpus callosum, enlargement of septum pellicidum, and diffuse hypodensity of the grey matter, flat face, hypertelorism, cleft lip and cleft palate, low-set, posteriorly rotated ears, short neck, and multiple costal and vertebral anomalies. The underlying genetic defect remains unknown. Using combination of homozygosity mapping and whole-exome sequencing, we identified a homozygous nonsense founder mutation, p.Arg87Ter (c.259 C>T), in the human transmembrane and coiled-coil domains protein 1 (TMCO1) in four out of five families of Turkish origin. The entire critical region on chromosome 1q24 containing TMCO1 was excluded in the fifth family with characteristic findings of CFT providing evidence for genetic heterogeneity of CFT spectrum. Another founder TMCO1 mutation has recently been reported to cause a unique genetic condition, TMCO1-defect syndrome (OMIM #614132). TMCO1-defect syndrome shares many features with CFT. This study supports the fact that "TMCO1-defect syndrome," initially thought to represent a distinct disorder, indeed belongs to the genetically heterogeneous CFT dysplasia spectrum.

Fasciola hepatica - where is 28S ribosomal RNA?

Advanced molecular biology techniques are currently used to develop new effective strategies against fasciolosis. Assessment of the quality of extracted total RNA is an important step prior to commencing many molecular biology methods such as transcriptomics. However, RNA quality assessment is complicated for some organisms, including Fasciola hepatica, by the absence of a 28S rRNA peak/band, when assessed with modern protocols. In this study, electrophoretic profiles of F. hepatica ribosomal RNAs were evaluated using microfluidics capillary based and conventional non-denaturing gel electrophoresis methods. An important modification to recommended protocols, the exclusion of heat-denaturation step, in the microfluidics capillary based electrophoresis is critical to visualise the expected 28S rRNA and obtain an RNA integrity number (RIN). The intensity of the 28S rRNA band is reduced by the effect of non-denaturing gel electrophoresis.

A proteomic approach to investigate the distribution and abundance of surface and internal Fasciola hepatica proteins during the chronic stage of natural liver fluke infection in cattle.

Fasciola hepatica, a trematode helminth, causes an economically important disease (fasciolosis) in ruminants worldwide. Proteomic analysis of the parasite provides valuable information to understand the relationship between the parasite and its host. Previous studies have identified various parasite proteins, some of which are considered as vaccine candidates or important drug targets. However, the approximate distribution and abundance of the proteins on the surface and within internal parts of the liver fluke are unknown. In this study, two fractions including surface protein fraction (representing surface part of the parasite, near subplasma membrane of the tegument and above the basal membrane of the tegument) and internal protein fraction (representing internal part of the parasite, mainly deeper sides of the tegument including subbasal membrane and other further internal elements of the parasite) were obtained. Components of these two fractions were investigated by an advanced proteomics approach using a high-definition mass spectrometer with nano electrospray ionization source coupled to a high-performance liquid chromatography system (nanoUPLC-ESI-qTOF-MS). FABP1 was found highly abundant in the SPF fraction. Potentially novel F. hepatica proteins showing homology with AKT interacting protein (Xenopus tropicalis), sterol O-acyltransferase 2 (Homo sapiens), and integrin beta 7 (Mus musculus) were identified with high quantities in only the surface fraction of the parasite and may be possible candidates for future control strategies.

Experimental Fasciola hepatica infection alters responses to tests used for diagnosis of bovine tuberculosis.

Fasciola hepatica is a prevalent helminth parasite of livestock. Infection results in polarization of the host's immune response and generation of type 2 helper (Th2) immune responses, which are known to be inhibitory to Th1 responses. Bovine tuberculosis (BTB) is a bacterial disease of economic and zoonotic importance. Control polices for this disease rely on extensive annual testing and a test-and-slaughter policy. The correct diagnosis of BTB relies on cell-mediated immune responses. We established a model of coinfection of F. hepatica and Mycobacterium bovis BCG to examine the impact of helminth infection on correct diagnosis. We found the predictive capacity of tests to be compromised in coinfected animals and that F. hepatica infection altered macrophage function. Interleukin-4 and gamma interferon expression in whole-blood lymphocytes restimulated in vitro with M. bovis antigen was also altered in coinfected animals. These results raise the question of whether F. hepatica infection can affect the predictive capacity of tests for the diagnosis of BTB and possibly also influence susceptibility to BTB and other bacterial diseases. Further studies on the interplay between helminth infection and BTB are warranted.