Systematic identification and characterization of chicken (Gallus gallus) ncRNAs

Recent studies have demonstrated that non-coding RNAs (ncRNAs) play important roles during development and evolution. Chicken, the first genome-sequenced non-mammalian amniote, possesses unique features for developmental and evolutionary studies. However, apart from microRNAs, information on chicken ncRNAs has mainly been obtained from computational predictions without experimental validation. In the present study, we performed a systematic identification of intermediate size ncRNAs (50–500 nt) by ncRNA library construction and identified 125 chicken ncRNAs. Importantly, through the bioinformatics and expression analysis, we found the chicken ncRNAs has several novel features: (i) comparative genomic analysis against 18 sequenced vertebrate genomes revealed that the majority of the newly identified ncRNA candidates is not conserved and most are potentially bird/chicken specific, suggesting that ncRNAs play roles in lineage/species specification during evolution. (ii) The expression pattern analysis of intronic snoRNAs and their host genes suggested the coordinated expression between snoRNAs and their host genes. (iii) Several spatio-temporal specific expression patterns suggest involvement of ncRNAs in tissue development. Together, these findings provide new clues for future functional study of ncRNAs during development and evolution.     

Identification of ncRNAs as potential therapeutic targets in multiple sclerosis through differential ncRNA – mRNA network analysis

Background

Several studies have revealed a potential role for both small nucleolar RNAs (snoRNAs) and microRNAs (miRNAs) in the physiopathology of relapsing-remitting multiple sclerosis (RRMS). This potential implication has been mainly described through differential expression studies. However, it has been suggested that, in order to extract additional information from large-scale expression experiments, differential expression studies must be complemented with differential network studies. Thus, the present work is aimed at the identification of potential therapeutic ncRNA targets for RRMS through differential network analysis of ncRNA – mRNA coexpression networks. ncRNA – mRNA coexpression networks have been constructed from both selected ncRNA (specifically miRNAs, snoRNAs and sdRNAs) and mRNA large-scale expression data obtained from 22 patients in relapse, the same 22 patients in remission and 22 healthy controls. Condition-specific (relapse, remission and healthy) networks have been built and compared to identify the parts of the system most affected by perturbation and aid the identification of potential therapeutic targets among the ncRNAs.

Results

All the coexpression networks we built present a scale-free topology and many snoRNAs are shown to have a prominent role in their architecture. The differential network analysis (relapse vs. remission vs. controls’ networks) has revealed that, although both network topology and the majority of the genes are maintained, few ncRNA – mRNA links appear in more than one network. We have selected as potential therapeutic targets the ncRNAs that appear in the disease-specific network and were found to be differentially expressed in a previous study.

Conclusions

Our results suggest that the diseased state of RRMS has a strong impact on the ncRNA – mRNA network of peripheral blood leukocytes, as a massive rewiring of the network happens between conditions. Our findings also indicate that the role snoRNAs have in targeted gene silencing is a widespread phenomenon. Finally, among the potential therapeutic target ncRNAs, SNORA40 seems to be the most promising candidate.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1396-5) contains supplementary material, which is available to authorized users.     

Protein interaction network for Alzheimer's disease using computational approach

Alzheimer''s disease (AD) is the most common form of dementia. It is the sixth leading cause of death in old age people. Despite recent advances in the field of drug design, the medical treatment for the disease is purely symptomatic and hardly effective. Thus there is a need to understand the molecular mechanism behind the disease in order to improve the drug aspects of the disease. We provided two contributions in the field of proteomics in drug design. First, we have constructed a protein-protein interaction network for Alzheimer''s disease reviewed proteins with 1412 interactions predicted among 969 proteins. Second, the disease proteins were given confidence scores to prioritize and then analyzed for their homology nature with respect to paralogs and homologs. The homology persisted with the mouse giving a basis for drug design phase. The method will create a new drug design technique in the field of bioinformatics by linking drug design process with protein-protein interactions via signal pathways. This method can be improvised for other diseases in future.     

Epstein-Barr Virus-Induced Expression of a Novel Human Vault RNA

Non-protein-coding RNAs (ncRNAs) have recently emerged on the scene of genomic research as prominent players in the regulation of gene expression. Many functionally characterized ncRNAs have been shown to be differentially expressed in various organisms during specific environmental or developmental conditions, thus establishing regulatory networks crucial for shaping cellular life. Here, we show that the expression of vault RNAs (vtRNAs) is specifically up-regulated in human lymphocytes upon infection by γ-herpesviruses, such as the Epstein-Barr virus and Kaposi's sarcoma virus. vtRNAs are ncRNAs that are integral to the vault complex, a gigantic (13 MDa) hollow ribonucleoprotein particle with a thus far elusive biological role. Stimulation of vtRNA expression by the Epstein-Barr virus was evident for all three canonical vtRNAs (hvg1-hvg3) and also for a novel ncRNA candidate, initially termed CBL-3. This ncRNA shares clear primary- and secondary-structure similarities with the three known vtRNAs. Importantly, CBL-3 co-sediments with intact vault particles in density gradients of various human cell lines, thus strongly indicating this ncRNA as a novel, fourth vault-complex-associated RNA.     

miR-200c suppression increases tau hyperphosphorylation by targeting 14-3-3γ in early stage of 5xFAD mouse model of Alzheimer's disease

Background and Purpose: Recently, several abnormally regulated microRNAs (miRNAs) have been identified in patients with Alzheimer''s disease (AD). The purpose of this study was to identify abnormally expressed miRNAs and to investigate whether they affect pathological changes in AD in the 5xFAD AD mouse model.Experimental Approach: Using microarray analysis and RT-qPCR, miRNA expression in the hippocampus of a 4-month-old 5xFAD mouse model of AD was investigated. A dual-luciferase assay was performed to determine whether the altered miR-200c regulates the translation of the target mRNA, Ywhag. Whether miR-200c modulates AD pathology was determined in primary hippocampal neurons and C57BL/6J mice transfected with miR-200c inhibitor. In addition, total miRNAs were extracted from the serums of 28 healthy age-matched controls and 22 individual participants with cognitive impairment, and RT-qPCR was performed.Key results: miR-200c expression was reduced in the hippocampus of 5xFAD mice. In primary hippocampal neurons, miR-200c regulated the translation of 14-3-3γ and increased tau phosphorylation (p-tau) by increasing p-GSK-3β (GSK-3β phosphorylation). It was also confirmed that miR-200c inhibition in the hippocampus of C57BL/6J mice induces cognitive impairment and increases tau phosphorylation through 14-3-3γ activation. Finally, aberrant expression of miR-200c was confirmed in the blood serum of human AD patients.Conclusion and Implications: Our results strongly suggest that dysregulation of miR-200c expression contributes to the pathogenesis of AD, including cognitive impairment through hyperphosphorylated tau.     

Experimental RNomics and genomic comparative analysis reveal a large group of species-specific small non-message RNAs in the silkworm Bombyx mori

Accumulating evidences show that small non-protein coding RNAs (ncRNAs) play important roles in development, stress response and other cellular processes. The silkworm is an important model for studies on insect genetics and control of lepidopterous pests. Here, we have performed the first systematic identification and analysis of intermediate size ncRNAs (50-500 nt) in the silkworm. We identified 189 novel ncRNAs, including 141 snoRNAs, six snRNAs, three tRNAs, one SRP and 38 unclassified ncRNAs. Forty ncRNAs showed significantly altered expression during silkworm development or across specific stage transitions. Genomic comparisons revealed that 123 of these ncRNAs are potentially silkworm-specific. Analysis of the genomic organization of the ncRNA loci showed that 32.62% of the novel snoRNA loci are intergenic, and that all the intronic snoRNAs follow the pattern of one-snoRNA-per-intron. Target site analysis predicted a total of 95 2'-O-methylation and pseudouridylation modification sites of rRNAs, snRNAs and tRNAs. Together, these findings provide new clues for future functional study of ncRNA during insect development and evolution.     

High-dose of vitamin C supplementation reduces amyloid plaque burden and ameliorates pathological changes in the brain of 5XFAD mice

Blood–brain barrier (BBB) breakdown and mitochondrial dysfunction have been implicated in the pathogenesis of Alzheimer''s disease (AD), a neurodegenerative disease characterized by cognitive deficits and neuronal loss. Besides vitamin C being as one of the important antioxidants, recently, it has also been reported as a modulator of BBB integrity and mitochondria morphology. Plasma levels of vitamin C are decreased in AD patients, which can affect disease progression. However, investigation using animal models on the role of vitamin C in the AD pathogenesis has been hampered because rodents produce with no dependence on external supply. Therefore, to identify the pathogenic importance of vitamin C in an AD mouse model, we cross-bred 5 familial Alzheimer''s disease mutation (5XFAD) mice (AD mouse model) with ι-gulono-γ-lactone oxidase (Gulo) knockout (KO) mice, which are unable to synthesize their own vitamin C, and produced Gulo KO mice with 5XFAD mice background (KO-Tg). These mice were maintained on either low (0.66 g/l) or high (3.3 g/l) supplementation of vitamin C. We found that the higher supplementation of vitamin C had reduced amyloid plaque burden in the cortex and hippocampus in KO-Tg mice, resulting in amelioration of BBB disruption and mitochondrial alteration. These results suggest that intake of a larger amount of vitamin C could be protective against AD-like pathologies.     

Alterations of brain and cerebellar proteomes linked to A�� and tau pathology in a female triple-transgenic murine model of Alzheimer's disease

The triple-transgenic Alzheimer (3 × Tg-AD) mouse expresses mutant PS1_M146V, APP_swe, and tau_P301L transgenes and progressively develops plaques and neurofibrillary tangles with a temporal- and region-specific profile that resembles the neuropathological progression of Alzheimer''s disease (AD). In this study, we used proteomic approaches such as two-dimensional gel electrophoresis and mass spectrometry to investigate the alterations in protein expression occurring in the brain and cerebellum of 3 × Tg-AD and presenilin-1 (PS1) knock-in mice (animals that do not develop Aβ- or tau-dependent pathology nor cognitive decline and were used as control). Finally, using the Ingenuity Pathway Analysis we evaluated novel networks and molecular pathways involved in this AD model. We identified several differentially expressed spots and analysis of 3 × Tg-AD brains showed a significant downregulation of synaptic proteins that are involved in neurotransmitter synthesis, storage and release, as well as a set of proteins that are associated with cytoskeleton assembly and energy metabolism. Interestingly, in the cerebellum, a structure not affected by AD, we found an upregulation of proteins involved in carbohydrate metabolism and protein catabolism. Our findings help to unravel the pathogenic brain mechanisms set in motion by mutant amyloid precursor protein (APP) and hyperphosphorylated tau. These data also reveal cerebellar pathways that may be important to counteract the pathogenic actions of Aβ and tau, and ultimately offer novel targets for therapeutic intervention.     

基于BP神经网络和RBF神经网络预测老年痴呆症疾病进展的对比研究

目的:比较反向传播算法(BP)神经网络和径向基函数(RBF)神经网络预测老年痴呆症疾病进展的效果。方法:以老年痴呆症随访数据为研究对象,以性别、年龄、受教育程度、有无高血压、有无高胆固醇、有无心脏病、有无中风史、有无家族史8个指标作为输入变量,以五年随访的MMSE差值为输出变量,构建基于BP神经网络和RBF神经网络的老年痴呆症疾病进展预测模型。结果:与BP神经网络模型相比,RBF神经网络预测的结果更好,能够有效地预测老年痴呆症疾病进展。结论:神经网络模型将老年痴呆症疾病进展预测问题转化为随访数据中相关测量指标与MMSE差值的非线性问题,为复杂的老年痴呆症疾病进展预测提供了新思路。     

Epitope mapping of novel monoclonal antibodies to human angiotensin I‐converting enzyme

Angiotensin I‐converting enzyme (ACE, CD143) plays a crucial role in blood pressure regulation, vascular remodeling, and immunity. A wide spectrum of mAbs to different epitopes on the N and C domains of human ACE have been generated and used to study different aspects of ACE biology, including establishing a novel approach–conformational fingerprinting. Here we characterized a novel set of 14 mAbs, developed against human seminal fluid ACE. The epitopes for these novel mAbs were defined using recombinant ACE constructs with truncated N and C domains, species cross‐reactivity, ACE mutagenesis, and competition with the previously mapped anti‐ACE mAbs. Nine mAbs recognized regions on the N domain, and 5 mAbs–on the C domain of ACE. The epitopes for most of these novel mAbs partially overlap with epitopes mapped onto ACE by the previously generated mAbs, whereas mAb 8H1 recognized yet unmapped region on the C domain where three ACE mutations associated with Alzheimer''s disease are localized and is a marker for ACE mutation T877M. mAb 2H4 could be considered as a specific marker for ACE in dendritic cells. This novel set of mAbs can identify even subtle changes in human ACE conformation caused by tissue‐specific glycosylation of ACE or mutations, and can detect human somatic and testicular ACE in biological fluids and tissues. Furthermore, the high reactivity of these novel mAbs provides an opportunity to study changes in the pattern of ACE expression or glycosylation in different tissues, cells, and diseases, such as sarcoidosis and Alzheimer''s disease.     

New insights into the role of microRNAs and long noncoding RNAs in most common neurodegenerative diseases

Neurodegenerative diseases (NDs) are a diversity of neurological disorders characterized by the progressive degeneration of the structure and function of the central nervous system (CNS). The most common NDs are Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). Recently, many studies have investigated associations between common NDs with noncoding RNAs (ncRNAs) molecules. ncRNAs are regulatory molecules in the normal functioning of the CNS. Two of the most important ncRNAs are microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). These types of ncRNAs are involved in different biological processes including brain development, maturation, differentiation, neuronal cell specification, neurogenesis, and neurotransmission. Increasing data has demonstrated that miRNAs and lncRNAs have strong correlations with the development of NDs, particularly gene expression. Besides, ncRNAs can be introduced as new biomarkers for diagnosis and prognosis of NDs. Hence, in this review, we summarized the involvement of various miRNAs and lncRNAs in most common NDs followed by a correlation of ncRNAs dysregulation with the AD, PD, and HD.     

Methodological obstacles in knocking down small noncoding RNAs

In the recent past, several thousand noncoding RNA (ncRNA) genes have been predicted within eukaryal genomes. However, for their functional analysis only a few high-throughput methods are currently available to knock down selected ncRNA species, such as microRNAs, which are targeted by antisense probes, termed antagomirs. We thus compared the efficiencies of four knockdown strategies, previously mainly employed for the analysis of protein-coding genes, to study the function of ncRNAs, in particular, small nucleolar RNAs (snoRNAs). Thereby, the class of snoRNAs represents one of the most abundant ncRNA species. The majority of snoRNAs has been shown to mediate nucleotide modifications by targeting ribosomal RNAs (rRNAs) through complementary antisense elements. However, some snoRNAs, termed “orphan snoRNAs,” lack telltale complementarities to rRNAs and thus their function remains elusive. We therefore applied RNA interference (RNAi), locked nucleic acid (LNA), or peptide nucleic acid antisense approaches, as well as a ribozyme-based strategy to knock down a snoRNA. As a proof of principle, we targeted the canonical U81 snoRNA, which has been shown to mediate modification of nucleotide A₃₉₁ within eukaryal 28S rRNA. Our results demonstrate that while RNAi is an unsuitable tool for snoRNA knockdown, a ribozyme-based strategy, as well as an LNA-antisense oligonucleotide approach, resulted in a decrease of U81 snoRNA expression levels up to 60%. However, no concomitant decrease in enzymatic activity of U81 snoRNA was observed, indicating that improvement of more efficient knockdown techniques for ncRNAs will be required in the future.