An RNA-Seq analysis of coronavirus in the skin of the Pangolin.

Protection of the Critically Endangered East Asian Pangolin species is hampered by the vulnerability of captive individuals to infection. Studies have previously shown the pangolin to have a unique pseudogenisation of many immunity genes (including IFNE, IFIH1, cGAS, STING, TLR5, and TLR11), and we suspected that these losses could account for this vulnerability. Here we used RNA-Seq data to show the effect of these gene losses on the transcriptional response to a viral skin infection in a deceased pangolin. This virus is very closely related to the one causing the current COVID-19 pandemic in the human population (SARS-CoV2), and we found the most upregulated pathway was the same one previously identified in the lungs of SARS-CoV2-infected humans. As predicted, we found that the pathways downstream of the lost genes were not upregulated. For example, the pseudogenised interferon epsilon (IFNE) is known to be particularly important in epithelial immunity, and we show that interferon-related responses were not upregulated in the infected pangolin skin. We suggest that the pangolin's innate gene pseudogenisation is indeed likely to be responsible for the animal's vulnerability to infection.

BreakAlign: a Perl program to align chimaeric (split) genomic NGS reads and allow visual confirmation of novel retroviral integrations.

BACKGROUND: Retroviruses replicate by integrating a DNA copy into a host chromosome. Detecting novel retroviral integrations (ones not in the reference genome sequence of the host) from genomic NGS data is bioinformatically challenging and frequently produces many false positives. One common method of confirmation is visual inspection of an alignment of the chimaeric (split) reads that span a putative novel retroviral integration site. We perceived the need for a program that would facilitate this by producing a multiple alignment containing both the viral and host regions that flank an integration. RESULTS: BreakAlign is a Perl program that uses blastn to produce such a multiple alignment. In addition to the NGS dataset and a reference viral sequence, the program requires either (a) the ~ 500nt host genome sequence that spans the putative integration or (b) coordinates of this putative integration in an installed copy of the reference human genome (multiple integrations can be processed automatically). BreakAlign is freely available from https://github.com/marchiem/breakalign and is accompanied by example files allowing a test run. CONCLUSION: BreakAlign will confirm and facilitate characterisation of both (a) germline integrations of endogenous retroviruses and (b) somatic integrations of exogenous retroviruses such as HIV and HTLV. Although developed for use with genomic short-read NGS (second generation) data and retroviruses, it should also be useful for long-read (third generation) data and any mobile element with at least one conserved flanking region.

Human Endogenous Retrovirus Type K Promotes Proliferation and Confers Sensitivity to Antiretroviral Drugs in Merlin-Negative Schwannoma and Meningioma.

Deficiency of the tumor suppressor Merlin causes development of schwannoma, meningioma, and ependymoma tumors, which can occur spontaneously or in the hereditary disease neurofibromatosis type 2 (NF2). Merlin mutations are also relevant in a variety of other tumors. Surgery and radiotherapy are current first-line treatments; however, tumors frequently recur with limited treatment options. Here, we use human Merlin-negative schwannoma and meningioma primary cells to investigate the involvement of the endogenous retrovirus HERV-K in tumor development. HERV-K proteins previously implicated in tumorigenesis were overexpressed in schwannoma and all meningioma grades, and disease-associated CRL4DCAF1 and YAP/TEAD pathways were implicated in this overexpression. In normal Schwann cells, ectopic overexpression of HERV-K Env increased proliferation and upregulated expression of c-Jun and pERK1/2, which are key components of known tumorigenic pathways in schwannoma, JNK/c-Jun, and RAS/RAF/MEK/ERK. Furthermore, FDA-approved retroviral protease inhibitors ritonavir, atazanavir, and lopinavir reduced proliferation of schwannoma and grade I meningioma cells. These results identify HERV-K as a critical regulator of progression in Merlin-deficient tumors and offer potential strategies for therapeutic intervention. SIGNIFICANCE: The endogenous retrovirus HERV-K activates oncogenic signaling pathways and promotes proliferation of Merlin-deficient schwannomas and meningiomas, which can be targeted with antiretroviral drugs and TEAD inhibitors.

Correction to: Characterising a human endogenous retrovirus(HERV)-derived tumour-associated antigen: enriched RNA-Seq analysis of HERV-K(HML-2) in mantle cell lymphoma cell lines.

An amendment to this paper has been published and can be accessed via the original article.

Characterising a human endogenous retrovirus(HERV)-derived tumour-associated antigen: enriched RNA-Seq analysis of HERV-K(HML-2) in mantle cell lymphoma cell lines.

BACKGROUND: The cell-surface attachment protein (Env) of the HERV-K(HML-2) lineage of endogenous retroviruses is a potentially attractive tumour-associated antigen for anti-cancer immunotherapy. The human genome contains around 100 integrated copies (called proviruses or loci) of the HERV-K(HML-2) virus and we argue that it is important for therapy development to know which and how many of these contribute to protein expression, and how this varies across tissues. We measured relative provirus expression in HERV-K(HML-2), using enriched RNA-Seq analysis with both short- and long-read sequencing, in three Mantle Cell Lymphoma cell lines (JVM2, Granta519 and REC1). We also confirmed expression of the Env protein in two of our cell lines using Western blotting, and analysed provirus expression data from all other relevant published studies. RESULTS: Firstly, in both our and other reanalysed studies, approximately 10% of the transcripts mapping to HERV-K(HML-2) came from Env-encoding proviruses. Secondly, in one cell line the majority of the protein expression appears to come from one provirus (12q14.1). Thirdly, we find a strong tissue-specific pattern of provirus expression. CONCLUSIONS: A possible dependency of Env expression on a single provirus, combined with the earlier observation that this provirus is not present in all individuals and a general pattern of tissue-specific expression among proviruses, has serious implications for future HERV-K(HML-2)-targeted immunotherapy. Further research into HERV-K(HML-2) as a possible tumour-associated antigen in blood cancers requires a more targeted, proteome-based, screening protocol that will consider these polymorphisms within HERV-K(HML-2). We include a plan (and necessary alignments) for such work.

OncoSim and OncoWiki: an authentic learning approach to teaching cancer genomics.

BACKGROUND: Personalised medicine is rapidly changing the clinical environment, especially in regard to the management of cancer. However, for the large part, methods used to educate undergraduate students as future biomedical scientists and medical doctors have not reflected these changes. In order to make effective use of advances in cancer genomic knowledge, there is a need to expose students to the challenges of genomic medicine and to do so in a manner that makes this complex information accessible. METHODS: The teaching method developed, OncoSim, is a scaffolded 'Personal Research' module option for final year biomedical undergraduate students. It uses an authentic learning approach to teach cancer genomics via simulated cancer patient case studies that have identifiable potential therapeutic targets with associated drug therapies (so-called targeted therapy/precision oncology). In addition, these simulated case studies can be uploaded to a dedicated learning website (OncoWiki) where they can be freely downloaded and used to teach medical students the principles of targeted therapy. A preliminary evaluation of OncoSim was carried out using 3 research tools: (1) online questionnaires; (2) semi-structured interviews; and (3) analysis of whole cohort mark ranges. Thematic analysis was used to code and categorise interview data. RESULTS: The teaching materials for OncoSim and the OncoWiki site are freely accessible at https://www.oncowiki.co.uk. Questionnaire data and comparison of whole cohort marks showed OncoSim was at least as effective as alternative choices, and suggested OncoSim provided a valued alternative to traditional laboratory-based projects. No barriers to receptiveness were found. Interview analysis provided 5 broad themes (authentic learning experience; individual challenges; interest in cancer; positive learning experience; supportive structure) supporting the authentic learning aspect of the project, the strong scaffolding provided and the overall effectiveness of the approach. CONCLUSIONS: Our preliminary, proof-of-concept, evaluation suggests that OncoSim will be effective in supporting the teaching of genomic medicine to undergraduate students. We plan and hope our study will encourage further formal evaluation in a larger cohort of students, including a control group. The OncoWiki site has the capacity to grow independently as future students create and upload simulated case studies for other students to then download and analyse.

Variable Baseline Papio cynocephalus Endogenous Retrovirus (PcEV) Expression Is Upregulated in Acutely SIV-Infected Macaques and Correlated to STAT1 Expression in the Spleen.

Retroviral replication leaves a DNA copy in the host cell chromosome, which over millions of years of infection of germline cells has led to 5% of the human genome sequence being comprised of endogenous retroviruses (ERVs), distributed throughout an estimated 100,000 loci. Over time these loci have accrued mutations such as premature stop codons that prevent continued replication. However, many loci remain both transcriptionally and translationally active and ERVs have been implicated in interacting with the host immune system. Using archived plasma and tissue samples from past macaque studies, experimentally infected with simian immunodeficiency virus (SIV), the expression of one macaque ERV in response to acute viral infection was explored together with a measure of the innate immune response. Specifically, RNA levels were determined for (a) Papio cynocephalus Endogenous Retrovirus (PcEV), an ERV (b) STAT1, a key gene in the interferon signaling pathway, and (c) SIV, an exogenous pathogen. Bioinformatic analysis of DNA sequences of the PcEV loci within the macaque reference genome revealed the presence of open reading frames (ORFs) consistent with potential protein expression but not ERV replication. Quantitative RT-PCR analysis of DNase-treated RNA extracts from plasma derived from acute SIV-infection detected PcEV RNA at low levels in 7 of 22 macaques. PcEV RNA levels were significantly elevated in PBMC and spleen samples recovered during acute SIV infection, but not in the thymus and lymph nodes. A strong positive correlation was identified between PcEV and STAT1 RNA levels in spleen samples recovered from SIV-positive macaques. One possibility is that SIV infection induces PcEV expression in infected lymphoid tissue that contributes to induction of an antiviral response.

Systematic Analysis and Biomarker Study for Alzheimer's Disease.

Revealing the relationship between dysfunctional genes in blood and brain tissues from patients with Alzheimer's Disease (AD) will help us to understand the pathology of this disease. In this study, we conducted the first such large systematic analysis to identify differentially expressed genes (DEGs) in blood samples from 245 AD cases, 143 mild cognitive impairment (MCI) cases, and 182 healthy control subjects, and then compare these with DEGs in brain samples. We evaluated our findings using two independent AD blood datasets and performed a gene-based genome-wide association study to identify potential novel risk genes. We identified 789 and 998 DEGs common to both blood and brain of AD and MCI subjects respectively, over 77% of which had the same regulation directions across tissues and disease status, including the known ABCA7, and the novel TYK2 and TCIRG1. A machine learning classification model containing NDUFA1, MRPL51, and RPL36AL, implicating mitochondrial and ribosomal function, was discovered which discriminated between AD patients and controls with 85.9% of area under the curve and 78.1% accuracy (sensitivity = 77.6%, specificity = 78.9%). Moreover, our findings strongly suggest that mitochondrial dysfunction, NF-κB signalling and iNOS signalling are important dysregulated pathways in AD pathogenesis.

Overlapping genes and the proteins they encode differ significantly in their sequence composition from non-overlapping genes.

Overlapping genes represent a fascinating evolutionary puzzle, since they encode two functionally unrelated proteins from the same DNA sequence. They originate by a mechanism of overprinting, in which point mutations in an existing frame allow the expression (the "birth") of a completely new protein from a second frame. In viruses, in which overlapping genes are abundant, these new proteins often play a critical role in infection, yet they are frequently overlooked during genome annotation. This results in erroneous interpretation of mutational studies and in a significant waste of resources. Therefore, overlapping genes need to be correctly detected, especially since they are now thought to be abundant also in eukaryotes. Developing better detection methods and conducting systematic evolutionary studies require a large, reliable benchmark dataset of known cases. We thus assembled a high-quality dataset of 80 viral overlapping genes whose expression is experimentally proven. Many of them were not present in databases. We found that overall, overlapping genes differ significantly from non-overlapping genes in their nucleotide and amino acid composition. In particular, the proteins they encode are enriched in high-degeneracy amino acids and depleted in low-degeneracy ones, which may alleviate the evolutionary constraints acting on overlapping genes. Principal component analysis revealed that the vast majority of overlapping genes follow a similar composition bias, despite their heterogeneity in length and function. Six proven mammalian overlapping genes also followed this bias. We propose that this apparently near-universal composition bias may either favour the birth of overlapping genes, or/and result from selection pressure acting on them.

Discovery of Novel Biomarkers for Alzheimer's Disease from Blood.

Blood-based biomarkers for Alzheimer's disease would be very valuable because blood is a more accessible biofluid and is suitable for repeated sampling. However, currently there are no robust and reliable blood-based biomarkers for practical diagnosis. In this study we used a knowledge-based protein feature pool and two novel support vector machine embedded feature selection methods to find panels consisting of two and three biomarkers. We validated these biomarker sets using another serum cohort and an RNA profile cohort from the brain. Our panels included the proteins ECH1, NHLRC2, HOXB7, FN1, ERBB2, and SLC6A13 and demonstrated promising sensitivity (>87%), specificity (>91%), and accuracy (>89%).

Integrated genomic approaches identify major pathways and upstream regulators in late onset Alzheimer's disease.

Previous studies have evaluated gene expression in Alzheimer's disease (AD) brains to identify mechanistic processes, but have been limited by the size of the datasets studied. Here we have implemented a novel meta-analysis approach to identify differentially expressed genes (DEGs) in published datasets comprising 450 late onset AD (LOAD) brains and 212 controls. We found 3124 DEGs, many of which were highly correlated with Braak stage and cerebral atrophy. Pathway Analysis revealed the most perturbed pathways to be (a) nitric oxide and reactive oxygen species in macrophages (NOROS), (b) NFkB and (c) mitochondrial dysfunction. NOROS was also up-regulated, and mitochondrial dysfunction down-regulated, in healthy ageing subjects. Upstream regulator analysis predicted the TLR4 ligands, STAT3 and NFKBIA, for activated pathways and RICTOR for mitochondrial genes. Protein-protein interaction network analysis emphasised the role of NFKB; identified a key interaction of CLU with complement; and linked TYROBP, TREM2 and DOK3 to modulation of LPS signalling through TLR4 and to phosphatidylinositol metabolism. We suggest that NEUROD6, ZCCHC17, PPEF1 and MANBAL are potentially implicated in LOAD, with predicted links to calcium signalling and protein mannosylation. Our study demonstrates a highly injurious combination of TLR4-mediated NFKB signalling, NOROS inflammatory pathway activation, and mitochondrial dysfunction in LOAD.

The decline of human endogenous retroviruses: extinction and survival.

BACKGROUND: Endogenous Retroviruses (ERVs) are retroviruses that over the course of evolution have integrated into germline cells and eventually become part of the host genome. They proliferate within the germline of their host, making up ~5% of the human and mouse genome sequences. Several lines of evidence have suggested a decline in the rate of ERV integration into the human genome in recent evolutionary history but this has not been investigated quantitatively or possible causes explored. RESULTS: By dating the integration of ERV loci in 40 mammal species, we show that the human genome and that of other hominoids (great apes and gibbons) have experienced an approximately four-fold decline in the ERV integration rate over the last 10 million years. A major cause is the recent extinction of one very large ERV lineage (HERV-H), which is responsible for most of the integrations over the last 30 million years. The decline however affects most other ERV lineages. Only about 10% of the decline might be attributed to an accompanying increase in body mass (a trait we have shown recently to be negatively correlated with ERV integration rate). Humans are unusual compared to related species - Old World monkeys, great apes and gibbons - in (a) having not acquired any new ERV lineages during the last 30 million years and (b) the possession of an old ERV lineage that has continued to replicate up until at least the last few hundred thousand years - the potentially medically significant HERVK(HML2). CONCLUSIONS: The human genome shares with the genome of other great apes and gibbons a recent decline in ERV integration that is not typical of other primates and mammals. The human genome differs from that of related species both in maintaining up until at least recently a replicating old ERV lineage and in not having acquired any new lineages. We speculate that the decline in ERV integration in the human genome has been exacerbated by a relatively low burden of horizontally-transmitted retroviruses and subsequent reduced risk of endogenization.

Larger mammalian body size leads to lower retroviral activity.

Retroviruses have been infecting mammals for at least 100 million years, leaving descendants in host genomes known as endogenous retroviruses (ERVs). The abundance of ERVs is partly determined by their mode of replication, but it has also been suggested that host life history traits could enhance or suppress their activity. We show that larger bodied species have lower levels of ERV activity by reconstructing the rate of ERV integration across 38 mammalian species. Body size explains 37% of the variance in ERV integration rate over the last 10 million years, controlling for the effect of confounding due to other life history traits. Furthermore, 68% of the variance in the mean age of ERVs per genome can also be explained by body size. These results indicate that body size limits the number of recently replicating ERVs due to their detrimental effects on their host. To comprehend the possible mechanistic links between body size and ERV integration we built a mathematical model, which shows that ERV abundance is favored by lower body size and higher horizontal transmission rates. We argue that because retroviral integration is tumorigenic, the negative correlation between body size and ERV numbers results from the necessity to reduce the risk of cancer, under the assumption that this risk scales positively with body size. Our model also fits the empirical observation that the lifetime risk of cancer is relatively invariant among mammals regardless of their body size, known as Peto's paradox, and indicates that larger bodied mammals may have evolved mechanisms to limit ERV activity.

Unfixed endogenous retroviral insertions in the human population.

UNLABELLED: One lineage of human endogenous retroviruses (HERVs), HERV-K(HML2), is upregulated in many cancers, some autoimmune/inflammatory diseases, and HIV-infected cells. Despite 3 decades of research, it is not known if these viruses play a causal role in disease, and there has been recent interest in whether they can be used as immunotherapy targets. Resolution of both these questions will be helped by an ability to distinguish between the effects of different integrated copies of the virus (loci). Research so far has concentrated on the 20 or so recently integrated loci that, with one exception, are in the human reference genome sequence. However, this viral lineage has been copying in the human population within the last million years, so some loci will inevitably be present in the human population but absent from the reference sequence. We therefore performed the first detailed search for such loci by mining whole-genome sequences generated by next-generation sequencing. We found a total of 17 loci, and the frequency of their presence ranged from only 2 of the 358 individuals examined to over 95% of them. On average, each individual had six loci that are not in the human reference genome sequence. Comparing the number of loci that we found to an expectation derived from a neutral population genetic model suggests that the lineage was copying until at least ∼250,000 years ago. IMPORTANCE: About 5% of the human genome sequence is composed of the remains of retroviruses that over millions of years have integrated into the chromosomes of egg and/or sperm precursor cells. There are indications that protein expression of these viruses is higher in some diseases, and we need to know (i) whether these viruses have a role in causing disease and (ii) whether they can be used as immunotherapy targets in some of them. Answering both questions requires a better understanding of how individuals differ in the viruses that they carry. We carried out the first careful search for new viruses in some of the many human genome sequences that are now available thanks to advances in sequencing technology. We also compared the number that we found to a theoretical expectation to see if it is likely that these viruses are still replicating in the human population today.

'There and back again': revisiting the pathophysiological roles of human endogenous retroviruses in the post-genomic era.

Almost 8% of the human genome comprises endogenous retroviruses (ERVs). While they have been shown to cause specific pathologies in animals, such as cancer, their association with disease in humans remains controversial. The limited evidence is partly due to the physical and bioethical restrictions surrounding the study of transposons in humans, coupled with the major experimental and bioinformatics challenges surrounding the association of ERVs with disease in general. Two biotechnological landmarks of the past decade provide us with unprecedented research artillery: (i) the ultra-fine sequencing of the human genome and (ii) the emergence of high-throughput sequencing technologies. Here, we critically assemble research about potential pathologies of ERVs in humans. We argue that the time is right to revisit the long-standing questions of human ERV pathogenesis within a robust and carefully structured framework that makes full use of genomic sequence data. We also pose two thought-provoking research questions on potential pathophysiological roles of ERVs with respect to immune escape and regulation.

Integrating phylodynamics and epidemiology to estimate transmission diversity in viral epidemics.

The epidemiology of chronic viral infections, such as those caused by Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV), is affected by the risk group structure of the infected population. Risk groups are defined by each of their members having acquired infection through a specific behavior. However, risk group definitions say little about the transmission potential of each infected individual. Variation in the number of secondary infections is extremely difficult to estimate for HCV and HIV but crucial in the design of efficient control interventions. Here we describe a novel method that combines epidemiological and population genetic approaches to estimate the variation in transmissibility of rapidly-evolving viral epidemics. We evaluate this method using a nationwide HCV epidemic and for the first time co-estimate viral generation times and superspreading events from a combination of molecular and epidemiological data. We anticipate that this integrated approach will form the basis of powerful tools for describing the transmission dynamics of chronic viral diseases, and for evaluating control strategies directed against them.

Dating the origin and dispersal of hepatitis B virus infection in humans and primates.

UNLABELLED: The origin of hepatitis B virus (HBV) infection in humans and other primates remains largely unresolved. Understanding the origin of HBV is crucial because it provides a framework for studying the burden, and subsequently the evolution, of HBV pathogenicity with respect to changes in human population size and life expectancy. To investigate this controversy we examined the relationship between HBV phylogeny and genetic diversity of modern humans, investigated the timescale of global HBV dispersal, and tested the hypothesis of HBV-human co-divergence. We find that the global distribution of HBV genotypes and subgenotypes are consistent with the major prehistoric modern human migrations. We calibrate the HBV molecular clock using the divergence times of different indigenous human populations based on archaeological and genetic evidence and show that HBV jumped into humans around 33,600 years ago; 95% higher posterior density (HPD): 22,000-47,100 years ago (estimated substitution rate: 2.2 × 10(-6) ; 95% HPD: 1.5-3.0 × 10(-6) substitutions/site/year). This coincides with the origin of modern non-African humans. Crucially, the most pronounced increase in the HBV pandemic correlates with the global population increase over the last 5,000 years. We also show that the non-human HBV clades in orangutans and gibbons resulted from cross-species transmission events from humans that occurred no earlier than 6,100 years ago. CONCLUSION: Our study provides, for the first time, an estimated timescale for the HBV epidemic that closely coincides with dates of human dispersals, supporting the hypothesis that HBV has been co-expanding and co-migrating with human populations for the last 40,000 years. (HEPATOLOGY 2013).

Env-less endogenous retroviruses are genomic superspreaders.

Endogenous retroviruses (ERVs) differ from typical retroviruses in being inherited through the host germline and therefore are a unique combination of pathogen and selfish genetic element. Some ERV lineages proliferate by infecting germline cells, as do typical retroviruses, whereas others lack the env gene required for virions to enter cells and thus behave like retrotransposons. We wished to know what factors determined the relative abundance of different ERV lineages, so we analyzed ERV loci recovered from 38 mammal genomes by in silico screening. By modeling the relationship between proliferation and replication mechanism in detail within one group, the intracisternal A-type particles (IAPs), and performing simple correlations across all ERV lineages, we show that when ERVs lose the env gene their proliferation within that genome is boosted by a factor of ∼30. We also show that ERV abundance follows the Pareto principle or 20/80 rule, with ∼20% of lineages containing 80% of the loci. This rule is observed in many biological systems, including infectious disease epidemics, where commonly ∼20% of the infected individuals are responsible for 80% of onward infection. We thus borrow simple epidemiological and ecological models and show that retrotransposition and loss of env is the trait that leads endogenous retroviruses to becoming genomic superspreaders that take over a significant proportion of their host's genome.

Detecting remote sequence homology in disordered proteins: discovery of conserved motifs in the N-termini of Mononegavirales phosphoproteins.

Paramyxovirinae are a large group of viruses that includes measles virus and parainfluenza viruses. The viral Phosphoprotein (P) plays a central role in viral replication. It is composed of a highly variable, disordered N-terminus and a conserved C-terminus. A second viral protein alternatively expressed, the V protein, also contains the N-terminus of P, fused to a zinc finger. We suspected that, despite their high variability, the N-termini of P/V might all be homologous; however, using standard approaches, we could previously identify sequence conservation only in some Paramyxovirinae. We now compared the N-termini using sensitive sequence similarity search programs, able to detect residual similarities unnoticeable by conventional approaches. We discovered that all Paramyxovirinae share a short sequence motif in their first 40 amino acids, which we called soyuz1. Despite its short length (11-16aa), several arguments allow us to conclude that soyuz1 probably evolved by homologous descent, unlike linear motifs. Conservation across such evolutionary distances suggests that soyuz1 plays a crucial role and experimental data suggest that it binds the viral nucleoprotein to prevent its illegitimate self-assembly. In some Paramyxovirinae, the N-terminus of P/V contains a second motif, soyuz2, which might play a role in blocking interferon signaling. Finally, we discovered that the P of related Mononegavirales contain similarly overlooked motifs in their N-termini, and that their C-termini share a previously unnoticed structural similarity suggesting a common origin. Our results suggest several testable hypotheses regarding the replication of Mononegavirales and suggest that disordered regions with little overall sequence similarity, common in viral and eukaryotic proteins, might contain currently overlooked motifs (intermediate in length between linear motifs and disordered domains) that could be detected simply by comparing orthologous proteins.