Pan-cancer analysis of transcripts encoding novel open-reading frames (nORFs) and their potential biological functions.

Uncharacterized and unannotated open-reading frames, which we refer to as novel open reading frames (nORFs), may sometimes encode peptides that remain unexplored for novel therapeutic opportunities. To our knowledge, no systematic identification and characterization of transcripts encoding nORFs or their translation products in cancer, or in any other physiological process has been performed. We use our curated nORFs database (nORFs.org), together with RNA-Seq data from The Cancer Genome Atlas (TCGA) and Genotype-Expression (GTEx) consortiums, to identify transcripts containing nORFs that are expressed frequently in cancer or matched normal tissue across 22 cancer types. We show nORFs are subject to extensive dysregulation at the transcript level in cancer tissue and that a small subset of nORFs are associated with overall patient survival, suggesting that nORFs may have prognostic value. We also show that nORF products can form protein-like structures with post-translational modifications. Finally, we perform in silico screening for inhibitors against nORF-encoded proteins that are disrupted in stomach and esophageal cancer, showing that they can potentially be targeted by inhibitors. We hope this work will guide and motivate future studies that perform in-depth characterization of nORF functions in cancer and other diseases.

Evolutionary divergence of novel open reading frames in cichlids speciation.

Novel open reading frames (nORFs) with coding potential may arise from noncoding DNA. Not much is known about their emergence, functional role, fixation in a population or contribution to adaptive radiation. Cichlids fishes exhibit extensive phenotypic diversification and speciation. Encounters with new environments alone are not sufficient to explain this striking diversity of cichlid radiation because other taxa coexistent with the Cichlidae demonstrate lower species richness. Wagner et al. analyzed cichlid diversification in 46 African lakes and reported that both extrinsic environmental factors and intrinsic lineage-specific traits related to sexual selection have strongly influenced the cichlid radiation, which indicates the existence of unknown molecular mechanisms responsible for rapid phenotypic diversification, such as emergence of novel open reading frames (nORFs). In this study, we integrated transcriptomic and proteomic signatures from two tissues of two cichlids species, identified nORFs and performed evolutionary analysis on these nORF regions. Our results suggest that the time scale of speciation of the two species and evolutionary divergence of these nORF genomic regions are similar and indicate a potential role for these nORFs in speciation of the cichlid fishes.

Behavioural analysis of single-cell aneural ciliate, Stentor roeseli, using machine learning approaches.

There is still a significant gap between our understanding of neural circuits and the behaviours they compute-i.e. the computations performed by these neural networks (Carandini 2012 Nat. Neurosci.15, 507-509. (doi:10.1038/nn.3043)). Cellular decision-making processes, learning, behaviour and memory formation-all that have been only associated with animals with neural systems-have also been observed in many unicellular aneural organisms, namely Physarum, Paramecium and Stentor (Tang & Marshall2018 Curr. Biol.28, R1180-R1184. (doi:10.1016/j.cub.2018.09.015)). As these are fully functioning organisms, yet being unicellular, there is a much better chance to elucidate the detailed mechanisms underlying these learning processes in these organisms without the complications of highly interconnected neural circuits. An intriguing learning behaviour observed in Stentor roeseli (Jennings 1902 Am. J. Physiol. Legacy Content8, 23-60. (doi:10.1152/ajplegacy.1902.8.1.23)) when stimulated with carmine has left scientists puzzled for more than a century. So far, none of the existing learning paradigm can fully encapsulate this particular series of five characteristic avoidance reactions. Although we were able to observe all responses described in the literature and in a previous study (Dexter et al. 2019), they do not conform to any particular learning model. We then investigated whether models inferred from machine learning approaches, including decision tree, random forest and feed-forward artificial neural networks could infer and predict the behaviour of S. roeseli. Our results showed that an artificial neural network with multiple 'computational' neurons is inefficient at modelling the single-celled ciliate's avoidance reactions. This has highlighted the complexity of behaviours in aneural organisms. Additionally, this report will also discuss the significance of elucidating molecular details underlying learning and decision-making processes in these unicellular organisms, which could offer valuable insights that are applicable to higher animals.

A new R package and web application for detecting bilateral asymmetry in parasitic infections.

When parasites invade paired structures of their host non-randomly, the resulting asymmetry may have both pathological and ecological significance. To facilitate the detection and visualisation of asymmetric infections we have developed a free software tool, Analysis of Symmetry of Parasitic Infections (ASPI). This tool has been implemented as an R package (https://cran.r-project.org/package=aspi) and a web application (https://wayland.shinyapps.io/aspi). ASPI can detect both consistent bias towards one side, and inconsistent bias in which the left side is favoured in some hosts and the right in others. Application of ASPI is demonstrated using previously unpublished data on the distribution of metacercariae of species of Diplostomum von Nordmann, 1832 in the eyes of ruffe Gymnocephalus cernua (Linnaeus). Invasion of the lenses appeared to be random, with the proportion of metacercariae in the left and right lenses showing the pattern expected by chance. However, analysis of counts of metacercariae from the humors, choroid and retina revealed asymmetry between eyes in 38% of host fish.

Fast imaging of live organisms with sculpted light sheets.

Light-sheet microscopy is an increasingly popular technique in the life sciences due to its fast 3D imaging capability of fluorescent samples with low photo toxicity compared to confocal methods. In this work we present a new, fast, flexible and simple to implement method to optimize the illumination light-sheet to the requirement at hand. A telescope composed of two electrically tuneable lenses enables us to define thickness and position of the light-sheet independently but accurately within milliseconds, and therefore optimize image quality of the features of interest interactively. We demonstrated the practical benefit of this technique by 1) assembling large field of views from tiled single exposure each with individually optimized illumination settings; 2) sculpting the light-sheet to trace complex sample shapes within single exposures. This technique proved compatible with confocal line scanning detection, further improving image contrast and resolution. Finally, we determined the effect of light-sheet optimization in the context of scattering tissue, devising procedures for balancing image quality, field of view and acquisition speed.

The systematics of Echinorhynchus Zoega in Müller, 1776 (Acanthocephala, Echinorhynchidae) elucidated by nuclear and mitochondrial sequence data from eight European taxa.

The acanthocephalan genus Echinorhynchus Zoega in Müller, 1776 (sensuYamaguti 1963) is a large and widespread group of parasites of teleost fish and malacostracan crustaceans, distributed from the Arctic to the Antarctic in habitats ranging from freshwaters to the deep-sea. A total of 52 species are currently recognised based on the conventional morphological species concept; however, the true diversity in the genus is masked by cryptic speciation. The considerable diversity within Echinorhynchus is an argument for subdividing the genus if monophyletic groups with supporting morphological characters can be identified. With this objective in mind, partial sequences of two genes with different rates of evolution and patterns of inheritance (nuclear 28S rRNA and mitochondrial cytochrome c oxidase subunit I) were used to infer the phylogenetic relationships among eight taxa of Echinorhynchus. These included representatives of each of three genus group taxa proposed in a controversial revision of the genus based on cement gland pattern, namely Echinorhynchus (sensu stricto), Metechinorhynchus Petrochenko, 1956 and Pseudoechinorhynchus Petrochenko, 1956. These groupings have previously been rejected by some authorities, because the diagnostic character is poorly defined; this study shows that Echinorhynchus (sensu stricto) and Metechinorhynchus are not natural, monophyletic groups. A revision of Echinorhynchus will require tandem molecular phylogenetic and morphological analyses of a larger sample of taxa, but this study has identified two morhological characters that might potentially be used to define new genera. The estimated phylogeny also provides insight into the zoogeographical history of Echinorhynchus spp. We postulate that the ancestral Echinorhynchus had a freshwater origin and the genus subsequently invaded the sea, probably several times. The freshwater taxa of the Echinorhynchusbothniensis Zdzitowiecki & Valtonen, 1987 clade may represent a reinvasion of freshwater by one or more ancestral marine species.

Spotting the differences: probing host/microbiota interactions with a dedicated software tool for the analysis of faecal outputs in Drosophila.

The intestinal physiology of Drosophila melanogaster can be monitored in an integrative, non-invasive manner by analysing graphical features of the excreta produced by flies fed on a dye-supplemented diet. This assay has been used by various labs to explore gut function and its regulation. To facilitate its use, we present here a free, stand-alone dedicated software tool for the analysis of fly excreta. The Ultimate Reader of Dung (T.U.R.D.) is designed to offer a flexible environment for a wide range of experimental designs, with special attention to automation and high-throughput processing. This software detects the distinctive changes in acid-base and water balance previously reported to occur in response to dietary challenges and mating. We have used T.U.R.D. to test the contribution of the bacterial environment of the flies to various intestinal parameters including the established diet- and mating-triggered responses. To this end, we have analysed the faecal patterns of flies reared in germ-free conditions, upon re-association with controlled microbiota and subjected to food-borne or systemic, non-lethal bacterial infections. We find that the tested faecal outputs are unchanged in all these conditions, suggesting that the impact of the bacterial environment on the intestinal features highlighted by faecal deposit analysis is minimal.

Schizophrenia: metabolic aspects of aetiology, diagnosis and future treatment strategies.

Despite decades of research, the pathophysiology and aetiology of schizophrenia remains incompletely understood. The disorder is frequently accompanied by metabolic symptoms including dyslipidaemia, hyperinsulinaemia, type 2 diabetes and obesity. These symptoms are a common side effect of currently available antipsychotic medications. However, reports of metabolic dysfunction in schizophrenia predate the antipsychotic era and have also been observed in first onset patients prior to antipsychotic treatment. Here, we review the evidence for abnormalities in metabolism in schizophrenia patients, both in the central nervous system and periphery. Molecular analysis of post mortem brain tissue has pointed towards alterations in glucose metabolism and insulin signalling pathways, and blood-based molecular profiling analyses have demonstrated hyperinsulinaemia and abnormalities in secretion of insulin and co-released factors at first presentation of symptoms. Nonetheless, such features are not observed for all subjects with the disorder and not all individuals with such abnormalities suffer the symptoms of schizophrenia. One interpretation of these data is the presence of an underlying metabolic vulnerability in a subset of individuals which interacts with environmental or genetic factors to produce the overt symptoms of the disorder. Further investigation of metabolic aspects of schizophrenia may prove critical for diagnosis, improvement of existing treatment based on patient stratification/personalised medicine strategies and development of novel antipsychotic agents.

Morphological variation in Echinorhynchustruttae Schrank, 1788 and the Echinorhynchusbothniensis Zdzitowiecki & Valtonen, 1987 species complex from freshwater fishes of northern Europe.

Echinorhynchustruttae and the Echinorhynchusbothniensis species complex are common parasites of salmoniform and other fishes in northern Europe. Echinorhynchusbothniensis and its sibling species Echinorhynchus 'bothniensis' are thought to be closely related to the Nearctic Echinorhynchusleidyi Van Cleave, 1924 based on morphological similarity and common usage of a mysid intermediate host. This study provides the first analysis of morphological and meristic variation in Echinorhynchustruttae and expands our knowledge of anatomical variability in the Echinorhynchusbothniensis group. Morphological variability in Echinorhynchustruttae was found to be far greater than previously reported, with part of the variance attributable to sexual dimorphism. Echinorhynchustruttae, the two species of the Echinorhynchusbothniensis group and Echinorhynchusleidyi displayed considerable interspecific overlap in the ranges of all conventional morphological characters. However, Proboscis profiler, a tool for detecting acanthocephalan morphotypes using multivariate analysis of hook morphometrics, successfully separated Echinorhynchustruttae from the other taxa. The Echinorhynchusbothniensis species group could not be reliably distinguished from Echinorhynchusleidyi (or each other), providing further evidence of the affinity of these taxa. Observations on the distribution of Echinorhynchustruttae in its definitive host population are also reported.

Evolution of neuronal and endothelial transcriptomes in primates.

The study of gene expression evolution in vertebrates has hitherto focused on the analysis of transcriptomes in tissues of different species. However, because a tissue is made up of different cell types, and cell types differ with respect to their transcriptomes, the analysis of tissues offers a composite picture of transcriptome evolution. The isolation of individual cells from tissue sections opens up the opportunity to study gene expression evolution at the cell type level. We have stained neurons and endothelial cells in human brains by antibodies against cell type-specific marker proteins, isolated the cells using laser capture microdissection, and identified genes preferentially expressed in the two cell types. We analyze these two classes of genes with respect to their expression in 62 different human tissues, with respect to their expression in 44 human "postmortem" brains from different developmental stages and with respect to between-species brain expression differences. We find that genes preferentially expressed in neurons differ less across tissues and developmental stages than genes preferentially expressed in endothelial cells. We also observe less expression differences within primate species for neuronal transcriptomes. In stark contrast, we see more gene expression differences between humans, chimpanzees, and rhesus macaques relative to within-species differences in genes expressed preferentially in neurons than in genes expressed in endothelial cells. This suggests that neuronal and endothelial transcriptomes evolve at different rates within brain tissue.

Proboscis profiler: a tool for detecting acanthocephalan morphotypes.

Molecular studies conducted over the past 25 years have revealed previously unrecognised diversity in the phylum Acanthocephala. Several nominal species have been shown to represent complexes of morphologically cryptic biological species, a situation potentially confounding the analysis of ecological data. A software tool, 'Proboscis profiler', was developed to detect morphological heterogeneity in collections of superficially similar acanthocephalan worms based on the multivariate statistical analysis of proboscis hook dimensions. Proboscis profiler identifies objective, natural groups in a collection of acanthocephalans which may correspond to distinct biological species or populations. Initial trials demonstrate that Proboscis profiler can discriminate biological acanthocephalan species of the Echinorhynchus gadi Zoega in Müller, 1776 complex and differentiate between dorsal and ventral hook rows from the proboscis of E. salmonis Müller, 1784. Proboscis profiler is free software and can be downloaded from http://acanthocephala.sourceforge.net.

Metabolic changes in schizophrenia and human brain evolution.

BACKGROUND: Despite decades of research, the molecular changes responsible for the evolution of human cognitive abilities remain unknown. Comparative evolutionary studies provide detailed information about DNA sequence and mRNA expression differences between humans and other primates but, in the absence of other information, it has proved very difficult to identify molecular pathways relevant to human cognition. RESULTS: Here, we compare changes in gene expression and metabolite concentrations in the human brain and compare them to the changes seen in a disorder known to affect human cognitive abilities, schizophrenia. We find that both genes and metabolites relating to energy metabolism and energy-expensive brain functions are altered in schizophrenia and, at the same time, appear to have changed rapidly during recent human evolution, probably as a result of positive selection. CONCLUSION: Our findings, along with several previous studies, suggest that the evolution of human cognitive abilities was accompanied by adaptive changes in brain metabolism, potentially pushing the human brain to the limit of its metabolic capabilities.

Improved grading and survival prediction of human astrocytic brain tumors by artificial neural network analysis of gene expression microarray data.

Histopathologic grading of astrocytic tumors based on current WHO criteria offers a valuable but simplified representation of oncologic reality and is often insufficient to predict clinical outcome. In this study, we report a new astrocytic tumor microarray gene expression data set (n = 65). We have used a simple artificial neural network algorithm to address grading of human astrocytic tumors, derive specific transcriptional signatures from histopathologic subtypes of astrocytic tumors, and asses whether these molecular signatures define survival prognostic subclasses. Fifty-nine classifier genes were identified and found to fall within three distinct functional classes, that is, angiogenesis, cell differentiation, and lower-grade astrocytic tumor discrimination. These gene classes were found to characterize three molecular tumor subtypes denoted ANGIO, INTER, and LOWER. Grading of samples using these subtypes agreed with prior histopathologic grading for both our data set (96.15%) and an independent data set. Six tumors were particularly challenging to diagnose histopathologically. We present an artificial neural network grading for these samples and offer an evidence-based interpretation of grading results using clinical metadata to substantiate findings. The prognostic value of the three identified tumor subtypes was found to outperform histopathologic grading as well as tumor subtypes reported in other studies, indicating a high survival prognostic potential for the 59 gene classifiers. Finally, 11 gene classifiers that differentiate between primary and secondary glioblastomas were also identified.

Altered T-cell function in schizophrenia: a cellular model to investigate molecular disease mechanisms.

Despite decades of research into the aetiology and pathophysiology of schizophrenia, our understanding of this devastating disorder remains incomplete, with adverse consequences for both diagnosis and treatment. Here we investigate whether differences between patients and controls can be observed in peripheral patient tissue, with a view of establishing a means for dynamic investigations into cell function. In vitro stimulation of peripheral blood CD3+ pan T cells with anti-CD3 (clone OKT3) was used to investigate disease-associated cell responses. T cells from both medicated (n = 39), unmedicated (n = 6) and minimally medicated (n = 5) schizophrenia patients were found to have significantly lower proliferative responses to stimulation, compared to well-matched controls (n = 32). Expression of CD3 and TCR (T cell receptor) alphabeta chains was equivalent between patients and controls, ensuring equal stimulation with anti-CD3, and there was no significant difference in the proportions of CD4+ and CD8+ T cells between samples (n = 12). Lower T cell proliferation in schizophrenia patients was not found to result from deficient early tyrosine phosphorylation signalling or lower IL-2 (interleukin-2) production, as these parameters were similar between patients and controls, as was the expression of CD25, the IL-2 receptor alpha chain. Analysis of CD45 isoforms, however, revealed that patients had a significantly greater percentage of CD8+ and CD4+ CD45RA+ cells before stimulation and significantly higher fluorescence intensity of CD45RA on CD4+ and CD8+ cells before and after stimulation. There was significantly higher expression of CD45 RB on both CD4+ and CD8+ unstimulated cells, with a trend towards lower numbers of CD45RO+ T cells in patient blood. Gene expression analysis in freshly isolated T cells from six minimally treated or first onset patients and six controls was carried out using human whole-genome CodeLink microarrays to identify functional pathways that may affect the ability of patient cells to respond to stimulation. Functional profiling showed prominent transcript changes in categories pertaining to cell cycle machinery, intracellular signalling, oxidative stress and metabolism. Intriguingly, chromosomal location analysis of genes significantly altered between schizophrenia and controls revealed clusters at 1p36, 1q42 and 6p22, which have previously been identified as strong susceptibility loci for schizophrenia.

Morphometric discrimination of two allozymically diagnosed sibling species of the Echinorhynchus gadi Zoega in Muller complex (Acanthocephala) in the North Sea.

Allozyme electrophoresis was used to detect biological species of the E. gadi complex from gadids from the northern North Sea. A fixed difference at one of nine enzyme loci surveyed confirmed the existence of two reproductively isolated, sympatric species. Mixed infections of two E. gadi spp. (termed A and B) were observed in Gadus morhua and Pollachius virens. E. gadi sp. B was also found in Melanogrammus aeglefinus and Merlangius merlangus. The presence of gravid females of E. gadi spp. A and B in the same host species, P. virens, and sometimes in the same host individual, indicates that neither differential host-specificity nor seasonal differences in mating time are responsible for their reproductive isolation. Morphological study of probosces from electrophoretically identified specimens demonstrated that the two species can be discriminated in graphical and cluster analyses of hook morphometrics. E. gadi sp. I (of Vainola etal., 1994) and E. gadi sp. A are probably conspecific.

Echinorhynchus salmonis Müller, 1784 (Acanthocephala: Echinorhynchidae) from the Bothnian Bay, Baltic Sea: morphological variability and radial asymmetry of proboscis hooks.

Echinorhynchus salmonis is a common parasite of salmoniform and other fishes, occurring in fresh and brackish waters throughout the Holarctic. Presented here is the first analysis of the morphometric and meristic variation in a Palaearctic population of E. salmonis, collected from whitefish Coregonus lavaretus L. and smelt Osmerus eperlaus (L.) from the Bothnian Bay, northern Baltic Sea. Morphological data were compared with published descriptions of congeneric taxa. Nearctic populations of salmonid echinorhynchids considered by some to represent a distinct species, E. coregoni Linkins in Van Cleave, 1919, did not show any morphological divergence from Palaearctic populations, indicating that the name E. coregoni should be suppressed. Similarly, E. alpinus Linstow, 1901 has been considered to be a junior synonym of E. salmonis. However, E. alpinus should be regarded as a valid species, because it has a longer and more elongate body. The armature of the acanthocephalan proboscis typically displays radial symmetry. However, in E. salmonis the hooks on the dorsal surface of the proboscis are smaller than those on the ventral surface. Size differences between dorsal and ventral hooks are most pronounced at the base of the proboscis. The systematic and functional significance of radial asymmetry of proboscis hooks is discussed.

Gene expression profiling of mammary gland development reveals putative roles for death receptors and immune mediators in post-lactational regression.

INTRODUCTION: In order to gain a better understanding of the molecular processes that underlie apoptosis and tissue regression in mammary gland, we undertook a large-scale analysis of transcriptional changes during the mouse mammary pregnancy cycle, with emphasis on the transition from lactation to involution. METHOD: Affymetrix microarrays, representing 8618 genes, were used to compare mammary tissue from 12 time points (one virgin, three gestation, three lactation and five involution stages). Six animals were used for each time point. Common patterns of gene expression across all time points were identified and related to biological function. RESULTS: The majority of significantly induced genes in involution were also differentially regulated at earlier stages in the pregnancy cycle. This included a marked increase in inflammatory mediators during involution and at parturition, which correlated with leukaemia inhibitory factor-Stat3 (signal transducer and activator of signalling-3) signalling. Before involution, expected increases in cell proliferation, biosynthesis and metabolism-related genes were observed. During involution, the first 24 hours after weaning was characterized by a transient increase in expression of components of the death receptor pathways of apoptosis, inflammatory cytokines and acute phase response genes. After 24 hours, regulators of intrinsic apoptosis were induced in conjunction with markers of phagocyte activity, matrix proteases, suppressors of neutrophils and soluble components of specific and innate immunity. CONCLUSION: We provide a resource of mouse mammary gene expression data for download or online analysis. Here we highlight the sequential induction of distinct apoptosis pathways in involution and the stimulation of immunomodulatory signals, which probably suppress the potentially damaging effects of a cellular inflammatory response while maintaining an appropriate antimicrobial and phagocytic environment.