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Circular RNAs (circRNAs) constitute an abundant class of covalently closed noncoding RNA molecules that are formed by backsplicing from eukaryotic protein-coding genes. Recent studies have shown that circRNAs can act as microRNA or protein decoys, as well as transcriptional regulators. However, the functions of most circRNAs are still poorly understood. Because circRNA sequences overlap with their linear parent transcripts, depleting specific circRNAs without affecting host gene expression remains a challenge. In this study, we assessed the utility of LNA-modified antisense oligonucleotides (ASOs) to knock down circRNAs for loss-of-function studies. We found that, while most RNase H-dependent gapmer ASOs mediate effective knockdown of their target circRNAs, some gapmers reduce the levels of the linear parent transcript. The circRNA targeting specificity can be enhanced using design-optimized gapmer ASOs, which display potent and specific circRNA knockdown with a minimal effect on the host genes. In summary, our results demonstrate that LNA-modified ASOs complementary to backsplice-junction sequences mediate robust knockdown of circRNAs in vitro and, thus, represent a useful tool to explore the biological roles of circRNAs in loss-of-function studies in cultured cells and animal models.

Hypocretin (Hcrt), also known as orexin, neuropeptide signaling stabilizes sleep and wakefulness in all vertebrates. A lack of Hcrt causes the sleep disorder narcolepsy, and increased Hcrt signaling has been speculated to cause insomnia, but while the signaling pathways of Hcrt are relatively well-described, the intracellular mechanisms that regulate its expression remain unclear. Here, we tested the role of microRNAs (miRNAs) in regulating Hcrt expression. We found that miR-137, miR-637, and miR-654-5p target the human HCRT gene. miR-137 is evolutionarily conserved and also targets mouse Hcrt as does miR-665. Inhibition of miR-137 specifically in Hcrt neurons resulted in Hcrt upregulation, longer episodes of wakefulness, and significantly longer wake bouts in the first 4 h of the active phase. IL-13 stimulation upregulated endogenous miR-137, while Hcrt mRNA decreased both in vitro and in vivo. Furthermore, knockdown of miR-137 in zebrafish substantially increased wakefulness. Finally, we show that in humans, the MIR137 locus is genetically associated with sleep duration. In conclusion, these results show that an evolutionarily conserved miR-137:Hcrt interaction is involved in sleep–wake regulation.

Small interfering RNA (siRNA)-based therapeutics holds the promise to treat a wide range of human diseases that are currently incurable using conventional therapies. Most siRNA therapeutic efforts to date have focused on the treatment of liver diseases due to major breakthroughs in the development of efficient strategies for delivering siRNA drugs to the liver. Indeed, the development of lipid nanoparticle-formulated and GalNAc-conjugated siRNA therapeutics has resulted in recent FDA approvals of the first siRNA-based drugs, patisiran for the treatment of hereditary transthyretin amyloidosis and givosiran for the treatment of acute hepatic porphyria, respectively. Here, we describe the current strategies for delivering siRNA drugs to the liver and summarize recent advances in clinical development of siRNA therapeutics for the treatment of liver diseases.

Small interfering RNA (siRNA)-based therapeutics holds the promise to treat a wide range of human diseases that are currently incurable using conventional therapies. Most siRNA therapeutic efforts to date have focused on the treatment of liver diseases due to major breakthroughs in the development of efficient strategies for delivering siRNA drugs to the liver. Indeed, the development of lipid nanoparticle-formulated and GalNAc-conjugated siRNA therapeutics has resulted in recent FDA approvals of the first siRNA-based drugs, patisiran for the treatment of hereditary transthyretin amyloidosis and givosiran for the treatment of acute hepatic porphyria, respectively. Here, we describe the current strategies for delivering siRNA drugs to the liver and summarize recent advances in clinical development of siRNA therapeutics for the treatment of liver diseases.

On September 12, 2019 at 12:49:48 (UT) a bolide was observed by hundreds of eye-witnesses from the Netherlands, Germany, Belgium, Denmark and the UK. One day later a small meteorite stone was found by accident in Flensburg. The presence of short-lived cosmogenic radionuclides with half-lives as short as 16 days proves the recent exposure of the found object to cosmic rays in space linking it clearly to the bolide event. An exceptionally short exposure time of ~5000 years was determined. The 24.5 g stone has a fresh black fusion crust, a low density of -9 m³/kg). The rock consists of relict chondrules and clusters of sulfide and magnetite grains set in a fine-grained matrix. The most abundant phases are phyllosilicates. Carbonates (~3.9 vol.%) occur as calcites, dolomites, and a Na-rich phase. The relict chondrules (often surrounded by sulfide laths) are free of anhydrous silicates and contain abundant serpentine. Lithic clasts are also surrounded by similar sulfide laths partly intergrown with carbonates. ⁵³Mn-⁵³Cr ages of carbonates in Flensburg indicate that brecciation and contemporaneous formation of the pyrrhotite-carbonate intergrowths by hydrothermal activities happened no later than 4564.6±1.0 Ma (using D'Orbigny as Mn-Cr anchor). This corresponds to 2.6±1.0 or 3.4±1.0 Ma after CAIs, depending on the exact age CAI age. This is the oldest dated evidence for brecciation and carbonate formation, which likely occurred during parent body growth and incipient heating due to ²⁶Al. The O isotope composition of Flensburg reveals that it plots at the ¹⁶O-rich end of the CM chondrite field and in the transition field to the CV-CK-CR chondrites. This is unexpected since the O-isotope compositions of strongly aqueously-altered chondrites are generally found to be at the ¹⁶O-poor end of the CM range. The mass-dependent Te isotopic compositionof Flensburg is slightly different from mean CM chondrites and is most similar to those of the ungrouped C2 chondrite Tagish Lake. On the other hand, the 50Ti and 54Cr isotope nomalies indicate that Flensburg is similar to CM chondrites, as do the ~10 wt.% H₂O. Yet, the bulk Zn, Cu, and Pb concentrations are about 30% lower than those of mean CM chondrites. The He, Ne and, Ar isotopes of Flensburg show no solar wind contribution; its trapped noble gas signature is similar to that of CMs with a slightly lower concentration of ²⁰Netr. Based on the bulk H, C and N elemental abundances and isotopic compositions, Flensburg is clearly unique among the analyzed chondrites as it has the lightest bulk H and N isotopic compositions of any type 1 or 2 chondrite. The number of soluble organic compounds in Flensburg is even lower than that of the brecciated CI chondrite Orgueil. The extraordinary significance of Flensburg is given by the observation that it represents the oldest chondrite sample, in which the contemporaneous episodes of aqueous alteration and brecciation are 81 perfectly visible. The characterization of a large variety of carbonaceous chondrites with different alteration histories is important for interpreting returned samples from the OSIRIS-REx and Hayabusa 2 missions.

With the dramatic background of a newly emerged virus (SARS-CoV-2) spreading around the world, Coronavirus and other infectious health threats for the human and animal populations were illustrated and debated in excellent presentations at the IABS meeting 26-28 of February 2020. Historical evidence of pandemics and lessons learned from recent epidemics or epizootics caused by many pathogens (e.g., Ebola, Zika, and African Swine Fever viruses) illustrated the overarching need for close international cooperation. New and old technologies in vaccine development and their use were presented, resulting in a call for greater interaction between the human and the veterinary fields in order to leverage the expertise and knowledge in both human and animal medicine. The One Health concept was also emphasized for eliminating the 59,000 fatal human rabies cases annually attributed to unvaccinated dogs. For preventable, infectious diseases commonly spreading in the poorer regions of the world, a new regulatory approach and governance structure was called for to give access to affordable vaccines. Vaccines were touted as one of the most successful health invention ever introduced; on a similar level to health improvements due to clean water.

Narcolepsy type 1, a neurological sleep disorder strongly associated with Human Leukocyte Antigen (HLA-)DQB1*06:02, is caused by the loss of hypothalamic neurons producing the wake-promoting neuropeptide hypocretin (hcrt, also known as orexin). This loss is believed to be caused by an autoimmune reaction. To test whether hcrt itself could be a possible target in the autoimmune attack, CD4+ T-cell reactivity towards six different 15-mer peptides from prepro-hypocretin with high predicted affinity to the DQA1*01:02/DQB1*06:02 MHC class II dimer was tested using EliSpot in a cohort of 22 narcolepsy patients with low CSF hcrt levels, and 23 DQB1*06:02 positive healthy controls. Our ELISpot assay had a detection limit of 1:10,000 cells. We present data showing that autoreactive CD4+ T-cells targeting epitopes from the hcrt precursor in the context of MHC-DQA1*01:02/DQB1*06:02 are either not present or present in a frequency is

On the 17th of October 2019, a workshop was held at Wageningen Bioveterinary Research in Lelystad, the Netherlands, to discuss the obstacles to vaccination in the veterinary field. Participants from academia, OIE, FAO, EC, EMA, USDA, national regulatory and veterinary health authorities, and the animal health industry discussed how availability and access to animal vaccines can be improved not just in the EU and US but also in Low to Middle Income Countries (LMIC) across the world and agreed that this requires innovations in both the scientific and the regulatory field. The workshop called for engaging all stakeholders to improve regulatory acceptance of novel vaccine technologies and encourage their registration. There is a need for better mutual understanding between academia, industry and regulators, and more openness to discuss framework, requirements, and product authorisations, and to converge the regulatory rules between regions. The next leap forward could be a broader application of novel technologies using RNA- or DNA-based vaccine platforms, where the "backbone" is maintained, while the gene of interest coding for an immunogenic protein can be exchanged in a standardised manner. This approach enables rapid response in outbreak situations and should lower the risk and cost of vaccine development.

An international workshop, held in Wiesbaden, Germany on 15-17 May 2019 provided an overview of existing and new methods and approaches to diagnostics in animal health and their benefits and challenges. The variability in quality and authority review of test kits across the world is a concern for the reliability of test results and the decisions that are based on the diagnostic data. In countries or regions without regulatory oversight, there is an urgent need for international harmonisation of quality requirements and licensing procedures. This would increase the validity of the diagnostic methods and allow mutual recognition of test results within the network of official control laboratories and amongst animal health officials. Regional cooperation, as well as the OIE Laboratory Network, should be used to support licensing procedures, pool resources for serum and sample banks, survey outbreak responses, and coordinate research and development of new veterinary diagnostics. The end-users must have clear information on a test's performance, limitations, and interpretation of results.

Narcolepsy Type 1 (NT1) is a neurological sleep disorder, characterized by the loss of hypocretin/orexin signaling in the brain. Genetic, epidemiological and experimental data support the hypothesis that NT1 is a T-cell-mediated autoimmune disease targeting the hypocretin producing neurons. While autoreactive CD4+ T cells have been detected in patients, CD8+ T cells have only been examined to a minor extent. Here we detect CD8+ T cells specific toward narcolepsy-relevant peptides presented primarily by NT1-associated HLA types in the blood of 20 patients with NT1 as well as in 52 healthy controls, using peptide-MHC-I multimers labeled with DNA barcodes. In healthy controls carrying the disease-predisposing HLA-DQB1*06:02 allele, the frequency of autoreactive CD8+ T cells was lower as compared with both NT1 patients and HLA-DQB1*06:02-negative healthy individuals. These findings suggest that a certain level of CD8+ T-cell reactivity combined with HLA-DQB1*06:02 expression is important for NT1 development., Autoreactive T cells are suspected to destroy hypocretin-producing neurons in narcolepsy. Here the authors detect CD8 T cells recognizing narcolepsy-related proteins in healthy individuals and in patients with narcolepsy, and show that the frequency of self-reactive CD8 T cells differs between patients and controls sharing the same HLA-II risk allele.

The hypocretin/orexin system regulates, among other things, sleep and energy homeostasis. The system is likely regulated by both homeostatic and circadian mechanisms. Little is known about local differences in the regulation of hypocretin activity. The aim of this study was to establish an optimized peptide quantification method for hypocretin-1 extracted from different mouse brain areas and use this method for investigating circadian fluctuations of hypocretin-1 levels in these areas. The results show that hypocretin-1 peptide can be extracted from small pieces of intact tissue, with sufficient yield for measurements in a standard radioimmunoassay. Utilizing the optimized method, it was found that prepro-hypocretin mRNA and peptide show circadian fluctuations in the mouse brain. This study further demonstrates that the hypocretin-1 peptide level in the frontal brain peaks during dark as does prepro-hypocretin mRNA in the hypothalamus. However, in midbrain and brainstem tissue caudal to the hypothalamus, there was less circadian fluctuation and a tendency for higher levels during the light phase. These data suggest that regulation of the hypocretin system differs between brain areas.

International audience; Since its introduction in the 1950s, colistin has been used mainly as a topical treatment in human medicine owing to its toxicity when given systemically. Sixty years later, colistin is being used as a last-resort drug to treat infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacteriaceae (e.g., Escherichia coli, Klebsiella pneumoniae), for which mortality can be high. In veterinary medicine, colistin has been used for decades for the treatment and prevention of infectious diseases. Colistin has been administered frequently as a group treatment for animal gastrointestinal infections caused by Gram-negative bacteria within intensive husbandry systems. Given the ever-growing need to retain the efficacy of antimicrobials used to treat MDR infections in humans, the use of colistin in veterinary medicine is being re-evaluated. Despite extensive use in veterinary medicine, there is limited evidence for the development of resistance to colistin and no evidence has been found for the transmission of resistance in bacteria that have been spread from animals to humans. Since surveillance for colistin resistance in animals is limited and the potential for such transmission exists, there is a clear need to reinforce systematic monitoring of bacteria from food-producing animals for resistance to colistin (polymyxins). Furthermore, colistin should only be used for treatment of clinically affected animals and no longer for prophylaxis of diseases, in line with current principles of responsible use of antibiotics.

Other than hypocretin-1 (HCRT-1) deficiency in narcolepsy type 1 (NT1), the neurochemical imbalance of NT1 and narcolepsy type 2 (NT2) with normal HCRT-1 levels is largely unknown. The neuropeptide melanin-concentrating hormone (MCH) is mainly secreted during sleep and is involved in rapid eye movement (REM) and non-rapid eye movement (NREM) sleep regulation. Hypocretin neurons reciprocally interact with MCH neurons. We hypothesized that altered MCH secretion contributes to the symptoms and sleep abnormalities of narcolepsy and that this is reflected in morning cerebrospinal fluid (CSF) MCH levels, in contrast to previously reported normal evening/afternoon levels.Lumbar CSF and plasma were collected from 07:00 to 10:00 from 57 patients with narcolepsy (subtypes: 47 NT1; 10 NT2) diagnosed according to International Classification of Sleep Disorders, Third Edition (ICSD-3) and 20 healthy controls. HCRT-1 and MCH levels were quantified by radioimmunoassay and correlated with clinical symptoms, polysomnography (PSG), and Multiple Sleep Latency Test (MSLT) parameters.CSF and plasma MCH levels were not significantly different between narcolepsy patients regardless of ICSD-3 subtype, HCRT-1 levels, or compared to controls. CSF MCH and HCRT-1 levels were not significantly correlated. Multivariate regression models of CSF MCH levels, age, sex, and body mass index predicting clinical, PSG, and MSLT parameters did not reveal any significant associations to CSF MCH levels.Our study shows that MCH levels in CSF collected in the morning are normal in narcolepsy and not associated with the clinical symptoms, REM sleep abnormalities, nor number of muscle movements during REM or NREM sleep of the patients. We conclude that morning lumbar CSF MCH measurement is not an informative diagnostic marker for narcolepsy.

MicroRNAs (miRNAs) are involved in the pathogenesis of many human diseases, including some neurological disorders. Recently, we have reported dysregulated miRNAs in plasma from patients with central hypersomnias including type 1 and type 2 narcolepsy, and idiopathic hypersomnia. This study addressed whether miRNA levels are altered in the cerebrospinal fluid (CSF) of patients with central hypersomnias. We conducted high-throughput analyses of miRNAs in CSF from patients using quantitative real-time polymerase chain reaction panels. We identified 13, 9, and 11 miRNAs with a more than two-fold change in concentration in CSF from patients with type 1 and type 2 narcolepsy and idiopathic hypersomnia, respectively, compared with matched healthy controls. Most miRNAs differed in more than one of the sleep disorders. However, all miRNAs were detected at low levels in CSF and varied between individuals. None of them showed significant differences in concentrations between groups after correcting for multiple testing, and none could be validated in an independent cohort. Nevertheless, approximately 60% of the most abundant miRNAs in the profile reported here have previously been identified in the CSF of healthy individuals, showing consistency with previous miRNA profiles found in CSF. In conclusion, we were not able to demonstrate distinct levels or patterns of miRNAs in CSF from central hypersomnia patients.

Colorectal cancer (CRC) is one of the leading causes of cancer deaths in Western countries. A significant number of CRC patients undergoing curatively intended surgery subsequently develop recurrence and die from the disease. MicroRNAs (miRNAs) are aberrantly expressed in cancers and appear to have both diagnostic and prognostic significance. In this study, we identified novel miRNAs associated with recurrence of CRC, and their possible mechanism of action. TaqMan(®) Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosas and 46 microsatellite stable CRC tumors. Four miRNAs (miR-362-3p, miR-570, miR-148 a* and miR-944) were expressed at a higher level in tumors from patients with no recurrence (p

Narcolepsy type 1, a neurological sleep disorder strongly associated with Human Leukocyte Antigen (HLA-)DQB1*06:02, is caused by the loss of hypothalamic neurons producing the wake-promoting neuropeptide hypocretin (hcrt, also known as orexin). This loss is believed to be caused by an autoimmune reaction. To test whether hcrt itself could be a possible target in the autoimmune attack, CD4

Objectives To investigate whether cerebrospinal fluid (CSF) biomarkers of neurodegeneration are altered in narcolepsy in order to evaluate whether the hypocretin deficiency and abnormal sleep-wake pattern in narcolepsy leads to neurodegeneration. Methods Twenty-one patients with central hypersomnia (10 type 1 narcolepsy, 5 type 2 narcolepsy, and 6 idiopathic hypersomnia cases), aged 33 years on average and with a disease duration of 2-29 years, and 12 healthy controls underwent CSF analyses of the levels of β-amyloid, total tau protein (T-tau), phosphorylated tau protein (P-tau181), α-synuclein, neurofilament light chain (NF-L), and chitinase 3-like protein-1 (CHI3L1). Results Levels of β-amyloid were lower in patients with type 1 narcolepsy (375.4 ± 143.5 pg/mL) and type 2 narcolepsy (455.9 ± 65.0 pg/mL) compared to controls (697.9 ± 167.3 pg/mL, p < .05). Furthermore, in patients with type 1 narcolepsy, levels of T-tau (79.0 ± 27.5 pg/mL) and P-tau181 (19.1 ± 4.3 pg/mL) were lower than in controls (162.2 ± 49.9 pg/mL and 33.8 ± 9.2 pg/mL, p < .05). Levels of α-synuclein, NF-L, and CHI3L1 in CSF from narcolepsy patients were similar to those of healthy individuals. Conclusion Six CSF biomarkers of neurodegeneration were decreased or normal in narcolepsy indicating that taupathy, synucleinopathy, and immunopathy are not prevalent in narcolepsy patients with a disease duration of 2-29 years. Lower CSF levels of β-amyloid, T-tau protein, and P-tau181 in narcolepsy may indicate that hypocretin deficiency and an abnormal sleep-wake pattern alter the turnover of these proteins in the central nervous system.

miRNAs are small 22-nucleotide RNAs that can post-transcriptionally regulate gene expression. It has been proposed that dietary plant miRNAs can enter the human bloodstream and regulate host transcripts; however, these findings have been widely disputed. We here conduct the first comprehensive meta-study in the field, surveying the presence and abundances of cross-species miRNAs (xenomiRs) in 824 sequencing data sets from various human tissues and body fluids. We find that xenomiRs are commonly present in tissues (17%) and body fluids (69%); however, the abundances are low, comprising 0.001% of host human miRNA counts. Further, we do not detect a significant enrichment of xenomiRs in sequencing data originating from tissues and body fluids that are exposed to dietary intake (such as liver). Likewise, there is no significant depletion of xenomiRs in tissues and body fluids that are relatively separated from the main bloodstream (such as brain and cerebro-spinal fluids). Interestingly, the majority (81%) of body fluid xenomiRs stem from rodents, which are a rare human dietary contribution but common laboratory animals. Body fluid samples from the same studies tend to group together when clustered by xenomiR compositions, suggesting technical batch effects. Last, we performed carefully designed and controlled animal feeding studies, in which we detected no transfer of plant miRNAs into rat blood, or bovine milk sequences into piglet blood. In summary, our comprehensive computational and experimental results indicate that xenomiRs originate from technical artifacts rather than dietary intake.

Aberrant activation of the Wnt signaling pathway is causally involved in the formation of most colorectal cancers (CRCs). Although detailed knowledge exists regarding Wnt-regulated protein-coding genes, much less is known about the possible involvement of non-coding RNAs. Here we used TaqMan Array MicroRNA Cards, capable of detecting 664 unique human microRNAs (miRNAs), to describe changes of the miRNA transcriptome following disruption of beta-catenin/TCF4 activity in DLD1 CRC cells. Most miRNAs appeared to respond independent of host gene regulation and proximal TCF4 chromatin occupancy as inferred from expression microarray and ChIP–chip data. A module of miRNAs induced by abrogated Wnt signaling in vitro was downregulated in two independent series of human primary CRCs (n=76) relative to normal adjacent mucosa (n=34). Several of these miRNAs (miR-145, miR-126, miR-30e-3p and miR-139-5p) markedly inhibited CRC cell growth in vitro when ectopically expressed. By using an integrative approach of proteomics and expression microarrays, we found numerous mRNAs and proteins to be affected by ectopic miR-30e-3p levels. This included HELZ and PIK3C2A that were directly repressed by several miRNA binding sites as confirmed by luciferase reporter assays in combination with mutational analyses. Finally, small interfering RNA-mediated downregulation of PIK3C2A, but not HELZ, was sufficient on its own to restrict CRC cell growth. Collectively, our study demonstrates that multiple miRNAs are upregulated as a consequence of forced attenuation of Wnt signaling in CRC cells, and some of these miRNAs inhibit cell growth with concomitant suppression of several growth-stimulatory cancer-related genes. Aberrant activation of the Wnt signaling pathway is causally involved in the formation of most colorectal cancers (CRCs). Although detailed knowledge exists regarding Wnt-regulated protein-coding genes, much less is known about the possible involvement of non-coding RNAs. Here we used TaqMan Array MicroRNA Cards, capable of detecting 664 unique human microRNAs (miRNAs), to describe changes of the miRNA transcriptome following disruption of beta-catenin/TCF4 activity in DLD1 CRC cells. Most miRNAs appeared to respond independent of host gene regulation and proximal TCF4 chromatin occupancy as inferred from expression microarray and ChIP-chip data. A module of miRNAs induced by abrogated Wnt signaling in vitro was downregulated in two independent series of human primary CRCs (n=76) relative to normal adjacent mucosa (n=34). Several of these miRNAs (miR-145, miR-126, miR-30e-3p and miR-139-5p) markedly inhibited CRC cell growth in vitro when ectopically expressed. By using an integrative approach of proteomics and expression microarrays, we found numerous mRNAs and proteins to be affected by ectopic miR-30e-3p levels. This included HELZ and PIK3C2A that were directly repressed by several miRNA binding sites as confirmed by luciferase reporter assays in combination with mutational analyses. Finally, small interfering RNA-mediated downregulation of PIK3C2A, but not HELZ, was sufficient on its own to restrict CRC cell growth. Collectively, our study demonstrates that multiple miRNAs are upregulated as a consequence of forced attenuation of Wnt signaling in CRC cells, and some of these miRNAs inhibit cell growth with concomitant suppression of several growth-stimulatory cancer-related genes.Oncogene advance online publication, 3 October 2011; doi:10.1038/onc.2011.453.

We have generated Artificial Neural Networks (ANN) capable of performing sensitive, quantitative predictions of peptide binding to the MHC class I molecule, HLA-A*0204. We have shown that such quantitative ANN are superior to conventional classification ANN, that have been trained to predict binding vs non-binding peptides. Furthermore, quantitative ANN allowed a straightforward application of a 'Query by Committee' (QBC) principle whereby particularly information-rich peptides could be identified and subsequently tested experimentally. Iterative training based on QBC-selected peptides considerably increased the sensitivity without compromising the efficiency of the prediction. This suggests a general, rational and unbiased approach to the development of high quality predictions of epitopes restricted to this and other HLA molecules. Due to their quantitative nature, such predictions will cover a wide range of MHC-binding affinities of immunological interest, and they can be readily integrated with predictions of other events involved in generating immunogenic epitopes. These predictions have the capacity to perform rapid proteome-wide searches for epitopes. Finally, it is an example of an iterative feedback loop whereby advanced, computational bioinformatics optimize experimental strategy, and vice versa.

Study Objectives: MicroRNAs (miRNAs) have been implicated in the pathogenesis of human diseases including neurological disorders. The aim is to address the involvement of miRNAs in the pathophysiology of central hypersomnias including autoimmune narcolepsy with cataplexy and hypocretin deficiency (type 1 narcolepsy), narcolepsy without cataplexy (type 2 narcolepsy), and idiopathic hypersomnia. Design: We conducted high-throughput analysis of miRNA in plasma from three groups of patients—with type 1 narcolepsy, type 2 narcolepsy, and idiopathic hypersomnia, respectively—in comparison with healthy controls using quantitative real-time polymerase chain reaction (qPCR) panels. Setting: University hospital based sleep clinic and research laboratories. Patients: Twelve patients with type 1 narcolepsy, 12 patients with type 2 narcolepsy, 12 patients with idiopathic hypersomnia, and 12 healthy controls. Measurements and Results: By analyzing miRNA in plasma with qPCR we identified 50, 24, and 6 miRNAs that were different in patients with type 1 narcolepsy, type 2 narcolepsy, and idiopathic hypersomnia, respectively, compared with healthy controls. Twenty miRNA candidates who fulfilled the criteria of at least two-fold difference and p-value < 0.05 were selected to validate the miRNA changes in an independent cohort of patients. Four miRNAs differed significantly between type 1 narcolepsy patients and healthy controls. Levels of miR-30c, let-7f, and miR-26a were higher, whereas the level of miR-130a was lower in type 1 narcolepsy than healthy controls. The miRNA differences were not specific for type 1 narcolepsy, since the levels of the four miRNAs were also altered in patients with type 2 narcolepsy and idiopathic hypersomnia compared with healthy controls. Conclusion: The levels of four miRNAs differed in plasma from patients with type 1 narcolepsy, type 2 narcolepsy and idiopathic hypersomnia suggesting that alterations of miRNAs may be involved in the pathophysiology of central hypersomnias.

Type 1 narcolepsy, an autoimmune disease affecting hypocretin (orexin) neurons, is strongly associated with HLA-DQB1*06:02. Among polymorphisms associated with the disease is single-nucleotide polymorphism rs2305795 (c.*638G>A) located within the P2RY11 gene. P2RY11 is in a region of synteny conserved in mammals and zebrafish containing PPAN, EIF3G and DNMT1 (DNA methyltransferase 1). As mutations in DNMT1 cause a rare dominant form of narcolepsy in association with deafness, cerebellar ataxia and dementia, we questioned whether the association with P2RY11 in sporadic narcolepsy could be secondary to linkage disequilibrium with DNMT1. Based on genome-wide association data from two cohorts of European and Chinese ancestry, we found that the narcolepsy association signal drops sharply between P2RY11/EIF3G and DNMT1, suggesting that the association with narcolepsy does not extend into the DNMT1 gene region. Interestingly, using transethnic mapping, we identified a novel single-nucleotide polymorphism rs3826784 (c.596–260A>G) in the EIF3G gene also associated with narcolepsy. The disease-associated allele increases EIF3G mRNA expression. EIF3G is located in the narcolepsy risk locus and EIF3G expression correlates with PPAN and P2RY11 expression. This suggests shared regulatory mechanisms that might be affected by the polymorphism and are of relevance to narcolepsy.

Peptides are key immune targets. They are generated by fragmentation of antigenic proteins, selected by major histocompatibility complex (MHC) molecules and subsequently presented to T cells. One of the most selective requirements is that of peptide binding to MHC. Accurate descriptions and predictions of peptide-MHC interactions are therefore important. Quantitative matrices representing MHC class I specificity can be used to search any query protein for the presence of MHC binding peptides. Assuming that each peptide residue contributes to binding in an additive and sequence independent manner, such "crude" matrix-driven predictions can be expressed as a quantitative estimates of binding strength. Crude matrix-driven predictions are reasonably uniform (i.e. precise), however, there is a general tendency towards overestimating binding (i.e. being inaccurate). To evaluate and possibly improve predictions, we have measured the MHC class I binding of a large number of peptides. In an attempt to further improve predictions and to include sequence dependency, we subdivided the panel of peptides according to whether the peptides had zero, one or two primary anchor residues. This allowed us to define unique anchor-stratified calibrations, which led to predictions of improved precision and accuracy.

Introduction A previous GWA study found an association between narcolepsy and SNPs in P2RY11. Later a missense mutation in DNMT1 was discovered to cause a rare familial form of narcolepsy in association with ataxia, deafness and dementia. Since P2RY11 and DNMT1 are located in close proximity we speculated if rs2305795 located in P2RY11 could be in linkage disequilibrium (LD) with a SNP located in DNMT1. Here we investigated SNPs in the P2RY11- DNMT1 region in several narcolepsy cohorts and examined if different genotypes were correlated with a changed gene expression in the same locus. Materials and methods GWAS: Caucasian cohort: 807 cases and 1074 controls, Chinese cohort: 1078 cases and 1903 controls, African American cohort: 249 cases and 1048 controls. Fine mapping was performed using Taqman SNP genotyping assays for rs1551750, rs12460842, rs2305795, and rs3826784. RNA sequencing on blood cells combined with a GWA dataset from patients with depression and healthy controls (n = 1000) were used to analyze gene expression. As disease status did not influence expression levels, we analyzed the combined cohort. Results Based on the LOD plots of GWA datasets we found that the narcolepsy association signal drops sharply between P2RY11 and DNMT1. Interestingly, however, we identified a novel SNP rs3826784 in EIF3G which was associated with narcolepsy and in high LD with rs2305795 in Caucasian and Chinese, but lower LD in African Americans. Genotyping showed that surprisingly rs3826784 but not rs2305795 was associated with narcolepsy in African Americans. We further found that EIF3G gene expression correlated with PPAN and P2RY11, but not DNMT1. Conclusion We found a novel association between SNP rs3826784 in EIF3G and narcolepsy. rs3826784 and the previously reported rs2305795 are in high LD in Caucasian and Chinese, but lower in African Americans. Since rs3826784 but not rs2305795 is associated with narcolepsy in African Americans, rs3826784 is a better marker for the genetic association and could be the functional variant involved. The strong correlation between the different genes in the locus suggests a shared regulatory mechanism that might be affected by the polymorphism. At the genetic level, however, the P2RY11/EIF3G association to narcolepsy does not extend into the DNMT1 gene region. Acknowledgements The Stanford Center for Sleep Sciences and Medicine, especially Ling Lin and Juliette Faraco. The Computer Science Department, espcially Alexis Battle and Xiaowei Zhu. The Lundbeck Foundation for financial support.

Introduction miRNAs have been implicated in the pathogenesis of human diseases including neurological disorders. The aim is to address the involvement of miRNAs in the pathophysiology of central hypersomnias including narcolepsy with cataplexy and hypocretin deficiency (NC), narcolepsy without cataplexy (NwC) and idiopathic hypersomnia (IH) in comparison with healthy controls (HC). Materials and methods We conducted high-throughput analysis of miRNA in plasma from patients with NC, NwC and IH in comparison with HC using quantitative real-time polymerase chain reaction (qRT-PCR) panels. Data were analyzed with the following softwares: GenEx qRT-PCR data analysis, miRNA expression atlas in normal tissues and DIANA-mirPath pathway analysis. Results Using analysis of miRNA in plasma with qRT-PCR we identified 50, 24 and 6 miRNAs that were changed in patients with NC, NwC, IH, respectively, compared to HC. Twenty miRNA candidates which fulfilled the criteria of twofold change and p-value 0.05 were selected for validation of miRNA changes in an independent cohort of patients. Four miRNAs were significantly changed between NC patients and HC. Levels of miR-30c, let-7f and miR- 26a were increased, whereas the level of miR-130a was decreased in NC compared to HC. The miRNAs changes were not specific for NC, since the levels of the four miRNAs were also altered in patients with NwC and IH compared with HC. Conclusion The levels of four miRNAs are changed in plasma from patients with NC, NwC and IH suggesting that alterations of miRNAs can be involved in the pathophysiology of central hypersomnias. Acknowledgements Lundbeck Foundation is acknowledged for financial support. Birte Kofoed is thanked for technical assistance.

It has been speculated that humpback whales, Megaptera novaeangliae, from the northeastern North Atlantic breed in tropical waters off the coast of West Africa and therefore that they represent a separate breeding population from that which winters in the West Indies. We determined the genotype at six microsatellite loci as well as the sequence of the first 288 nucleotides in the mitochondrial control region of 133 skin biopsies collected from humpback whales in the central North Atlantic (Iceland and Jan Mayen) and the northeastern North Atlantic (Bear Island and the northern coast of Norway). We detected no significant deviations from Hardy-Weinberg proportions nor any differences in genotype frequencies between localities at the nuclear loci. However, the mitochondrial analyses revealed two distinct matrilineal aggregations: the central and the northeastern North Atlantic. Our findings were not compatible with the idea of a separate eastern North Atlantic breeding ground unless one has been established recently. The proposed alternative hypothesis of a common North Atlantic panmictic population (wintering primarily in the West Indies) in which individual whales display maternally directed site-fidelity to specific feeding grounds was supported by re-sightings of two northeastern North Atlantic humpback whales in the West Indies.

Colorectal cancer (CRC) is one of the leading causes of cancer death in Western countries. A significant number of CRC patients undergoing curative-intented surgery subsequently develop recurrence and die from the disease. MicroRNAs (miRNAs) are aberrantly expressed in cancers and appear to have both diagnostic and prognostic significance. In this study we identified novel miRNAs associated with recurrence of CRC. TaqMan® Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosas and 46 microsatellite stable CRC tumors. Four miRNAs (miR-362-3p, miR-570, miR-148a* and miR-944) were expressed at a lower level in the tumors from patients with recurrence (p < 0.015) compared to tumors from patients with no recurrence. A significant association of low expression with reduced disease free survival was confirmed for miR-362-3p in a second independent cohort of 43 CRC patients using single TaqMan® microRNA assays. In vitro functional analysis showed that over-expression of miR-362-3p in colon cancer cell lines (HCT116, LS174T, and DLD1) reduced cell viability and proliferation mainly due to cell cycle arrest. E2F1, USF2 and PTPN1 were identified as potential miR-362-3p targets by mRNA profiling of HCT116 cells over-expressing miR-362-3p. Subsequently, these genes were confirmed as direct targets by Luciferase reporter assays. In vitro knock-down confirmed that these targets are important players in the miR-362-3p target network. We conclude that miR-362-3p may be a novel prognostic marker in CRC and hypothesize that augmented expression of miR-362-3p may prevent the development of distant metastasis by targeting E2F1, USF2 and PTPN1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4138. doi:1538-7445.AM2012-4138

Aberrant activation of the Wnt signaling pathway is causally involved in the formation of most colorectal cancers (CRCs). Whereas detailed knowledge exists regarding Wnt regulated protein coding genes, much less is known about the possible involvement of non-coding RNAs. Here we used TaqMan Array MicroRNA Cards, capable of detecting 664 unique human microRNAs (miRNAs), to describe changes of the miRNA transcriptome following disruption of beta-catenin/TCF4 activity in DLD1 CRC cells. Most miRNAs appeared to respond independently of host gene regulation and proximal TCF4 chromatin occupancy. A module of miRNAs suppressed by Wnt signaling in vitro was downregulated in a series of human primary CRCs (n = 46), and CRC cell lines (n = 7), relative to normal adjacent mucosa (n = 14). Several of these miRNAs (miR-145, miR-126, miR-30e-3p, and miR-139-5p) markedly inhibited CRC cell growth in vitro when ectopically expressed. By using an integrative approach of proteomics and microarrays, together interrogating thousands of proteins and genome-wide mRNA levels, we also provide detailed insight into the target gene network controlled by miR-30e-3p. Specifically HELZ and PIK3C2A were directly repressed via several functional seed matching binding motifs located within the transcripts. siRNA-mediated silencing of PIK3C2A partly phenocopied the growth suppressive phenotype of miR-30e-3p suggesting that miR-30e-3p restricts CRC cell growth in part by targeting PIK3C2A. Collectively, our study demonstrates that multiple miRNAs are upregulated as a consequence of forced attenuation of Wnt signaling in CRC cells, and some of these miRNAs inhibit cell growth, possibly through repression of several growth stimulatory cancer-related genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3964. doi:10.1158/1538-7445.AM2011-3964