Integrative Multi-Omics Research in Cerebral Palsy: Current Progress and Future Prospects.

Cerebral palsy (CP) describes a heterogeneous group of non-progressive neurodevelopmental disorders affecting movement and posture. The etiology and diagnostic biomarkers of CP are a hot topic in clinical research. Recent advances in omics techniques, including genomics, epigenomics, transcriptomics, metabolomics and proteomics, have offered new insights to further understand the pathophysiology of CP and have allowed for identification of diagnostic biomarkers of CP. In present study, we reviewed the latest multi-omics investigations of CP and provided an in-depth summary of current research progress in CP. This review will offer the basis and recommendations for future fundamental research on the pathogenesis of CP, identification of diagnostic biomarkers, and prevention strategies for CP.

Long Non-coding RNAs in Parkinson's Disease.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder and is associated with a range of motor and non-motor clinical symptoms. The underlying molecular pathogenesis of PD involves a variety of pathways and mechanisms, including α-synuclein proteostasis, mitochondrial dysfunction, oxidative stress, autophagy and apoptosis, neuroinflammation, and epigenetic regulation. Long non-coding RNAs (lncRNAs) are involved in the regulation of multiple pathological processes of PD. In this review, we provide an overview of large-scale studies on lncRNA expression profiling in PD patients and models, as well as highlight the impacts of lncRNAs on the pathogenesis of PD, which could provide basic information regarding the putative lncRNA-based biomarkers and therapeutic targets for the early diagnosis and treatment strategies for PD.

Cutibacterium acnes Type II strains are associated with acne in Chinese patients.

Acne is a common inflammatory skin disease, especially in adolescents. Certain Cutibacterium acnes subtypes are associated with acne, although more than one subtype of C. acnes strains may simultaneously reside on the surface of the skin of an individual. To better understand the relationship between the genomic characteristics of C. acnes subtypes and acnes, we collected 50 C. acnes strains from the facial skin of 10 people (5 healthy individuals, 5 patients with acne) in Liaoning, China and performed whole genome sequencing of all strains. We demonstrated that the six potential pathogenic C. acnes strains were all Type II subtype, and discovered 90 unique genes of the six strains related to acne using pan-genome analysis. The distribution of 2 of the 90 genes was identified by PCR in bacterial cultures collected from the facial skin of 171 individuals (55 healthy individuals, 52 patients with mild acne and 64 patients with moderate to severe acne). Both the genes were significantly associated with acne (Chi square test, P < 0.01). We conclude that Type II strains are associated with acne in Chinese patients.

Identification of novel KMT2D mutations in two Chinese children with Kabuki syndrome: a case report and systematic literature review.

BACKGROUND: Kabuki syndrome (KS) is a rare pediatric congenital disorder with multiple congenital anomalies and intellectual disabilities, which is inherited in an autosomal dominant manner. Mutations in KMT2D and KDM6A have been proven to be the primary cause in most cases of KS. CASE PRESENTATION: Here we report two Chinese boys with clinical features of KS referred to our hospital for clinical diagnosis. Next-generation sequencing was performed on MiSeq to analyze the genetic mutations in both patients. In both, two novel de novo mutations in KMT2D gene (c.5235delA, p.(A1746Lfs*39) and c.7048G > A, p.(Q2350*)) were detected, both of which were subsequently confirmed by the two-generation pedigree analysis based on Sanger sequencing. A systematic literature review of previously reported mutational spectrum of KMT2D was also conducted. CONCLUSIONS: Two novel de novo mutations in KMT2D gene were identified and considered to be pathogenic in both of KS patients. Our data adds information to the growing knowledge on the mutational spectrum of KS.

Profiling microRNA expression during multi-staged date palm (Phoenix dactylifera L.) fruit development.

MicroRNAs (miRNAs) play crucial roles in multiple stages of plant development and regulate gene expression at posttranscriptional and translational levels. In this study, we first identified 238 conserved miRNAs in date palm (Phoenix dactylifera) based on a high-quality genome assembly and defined 78 fruit-development-associated (FDA) miRNAs, whose expression profiles are variable at different fruit development stages. Using experimental data, we subsequently detected 276 novel P. dactylifera-specific FDA miRNAs and predicted their targets. We also revealed that FDA miRNAs function mainly in regulating genes involved in starch/sucrose metabolisms and other carbon metabolic pathways; among them, 221 FDA miRNAs exhibit negative correlation with their corresponding targets, which suggests their direct regulatory roles on mRNA targets. Our data define a comprehensive set of conserved and novel FDA miRNAs along with their expression profiles, which provide a basis for further experimentation in assigning discrete functions of these miRNAs in P. dactylifera fruit development.

Severe infection by Vibrio anguillarum following a bite by a marine fish: a case report.

Vibrio anguillarum is a cause of vibriosis in marine fisheries worldwide, but only one previous study reported human pathogenicity of this species. Here, we report a 70-year-old man from Dalian, a coastal city in northeast China, who experienced a severe infection with V. anguillarum due to a bite on his left hand when handling hairtail, a marine fish. This patient had low immunity because of the long-term use of glucocorticoids due to nephrotic syndrome. Despite treatments consisting of a strong antibiotic, continuous veno-venous hemofiltration, debridement, and fasciotomy, his condition deteriorated and he died of septic shock and multiple organ dysfunction syndrome. His death might be partly due to the delayed amputation of the left forearm, because he seemed to get better for the first several days. This case report emphasizes the possibility of human infection by V. anguillarum, which is likely to be more lethal in immunocompromised individuals.

AAV vectors applied to the treatment of CNS disorders: Clinical status and challenges.

In recent years, adeno-associated virus (AAV) has become the most important vector for central nervous system (CNS) gene therapy. AAV has already shown promising results in the clinic, for several CNS diseases that cannot be treated with drugs, including neurodegenerative diseases, neuromuscular diseases, and lysosomal storage disorders. Currently, three of the four commercially available AAV-based drugs focus on neurological disorders, including Upstaza for aromatic l-amino acid decarboxylase deficiency, Luxturna for hereditary retinal dystrophy, and Zolgensma for spinal muscular atrophy. All these studies have provided paradigms for AAV-based therapeutic intervention platforms. AAV gene therapy, with its dual promise of targeting disease etiology and enabling 'long-term correction' of disease processes, has the advantages of immune privilege, high delivery efficiency, tissue specificity, and cell tropism in the CNS. Although AAV-based gene therapy has been shown to be effective in most CNS clinical trials, limitations have been observed in its clinical applications, which are often associated with side effects. In this review, we summarized the therapeutic progress, challenges, limitations, and solutions for AAV-based gene therapy in 14 types of CNS diseases. We focused on viral vector technologies, delivery routes, immunosuppression, and other relevant clinical factors. We also attempted to integrate several hurdles faced in clinical and preclinical studies with their solutions, to seek the best path forward for the application of AAV-based gene therapy in the context of CNS diseases. We hope that these thoughtful recommendations will contribute to the efficient translation of preclinical studies and wide application of clinical trials.

High-throughput sequencing-based gene profiling on multi-staged fruit development of date palm (Phoenix dactylifera, L.).

Date palm provides both staple food and gardening for the Middle East and North African countries for thousands of years. Its fruits have diversified significantly, such as nutritional content, size, length, weight color, and ripping process. Dates palm represent an excellent model system for the study of fruit development and diversity of fruit-bearing palm species that produce the most versatile fruit types as compared to other plant families. Using Roche/454 GS FLX instrument, we acquired 7.6 million sequence tags from seven fruiting stages (F1-F7). Over 99% of the raw reads are assembled, and the numbers of isotigs (equivalent to transcription units or unigenes) range from 30,684 to 40,378 during different fruiting stages. We annotated isotigs using BLASTX and BLASTN, and mapped 74% of the isotigs to known functional sequences or genes. Based on gene ontology categorization and pathway analysis, we have identified 10 core cell division genes, 18 ripening related genes, and 7 starch metabolic enzymes, which are involved as nutrition storage and sugar/starch metabolisms. We noticed that many metabolic pathways vary significantly during fruit development, and carbohydrate metabolism (especially sugar synthesis) is particularly prominent during fruit ripening. Transcriptomics study on various fruiting stages of date palm shows complicated metabolic activities during fruit development, ripening, synthesis and accumulation of starch enzymes and other related sugars. Most Genes are highly expressed in early stages of development, while late developmental stages are critical for fruit ripening including most of the metabolism associated ones.

Large-scale collection and annotation of gene models for date palm (Phoenix dactylifera, L.).

The date palm (Phoenix dactylifera L.), famed for its sugar-rich fruits (dates) and cultivated by humans since 4,000 B.C., is an economically important crop in the Middle East, Northern Africa, and increasingly other places where climates are suitable. Despite a long history of human cultivation, the understanding of P. dactylifera genetics and molecular biology are rather limited, hindered by lack of basic data in high quality from genomics and transcriptomics. Here we report a large-scale effort in generating gene models (assembled expressed sequence tags or ESTs and mapped to a genome assembly) for P. dactylifera, using the long-read pyrosequencing platform (Roche/454 GS FLX Titanium) in high coverage. We built fourteen cDNA libraries from different P. dactylifera tissues (cultivar Khalas) and acquired 15,778,993 raw sequencing reads-about one million sequencing reads per library-and the pooled sequences were assembled into 67,651 non-redundant contigs and 301,978 singletons. We annotated 52,725 contigs based on the plant databases and 45 contigs based on functional domains referencing to the Pfam database. From the annotated contigs, we assigned GO (Gene Ontology) terms to 36,086 contigs and KEGG pathways to 7,032 contigs. Our comparative analysis showed that 70.6 % (47,930), 69.4 % (47,089), 68.4 % (46,441), and 69.3 % (47,048) of the P. dactylifera gene models are shared with rice, sorghum, Arabidopsis, and grapevine, respectively. We also assigned our gene models into house-keeping and tissue-specific genes based on their tissue specificity.

A comparison between ribo-minus RNA-sequencing and polyA-selected RNA-sequencing.

To compare the two RNA-sequencing protocols, ribo-minus RNA-sequencing (rmRNA-seq) and polyA-selected RNA-sequencing (mRNA-seq), we acquired transcriptomic data-52 and 32 million alignable reads of 35 bases in length-from the mouse cerebrum, respectively. We found that a higher proportion, 44% and 25%, of the uniquely alignable rmRNA-seq reads, is in intergenic and intronic regions, respectively, as compared to 23% and 15% from the mRNA-seq dataset. Further analysis made an additional discovery of transcripts of protein-coding genes (such as Histone, Heg1, and Dux), ncRNAs, snoRNAs, snRNAs, and novel ncRNAs as well as repeat elements in rmRNA-seq dataset. This result suggests that rmRNA-seq method should detect more polyA- or bimorphic transcripts. Finally, through comparative analyses of gene expression profiles among multiple datasets, we demonstrated that different RNA sample preparations may result in significant variations in gene expression profiles.

Hydroxyurea-induced global transcriptional suppression in mouse ES cells.

Hydroxyurea (HU), as a therapeutic medicine, has been extensively used clinically. To further survey molecular mechanisms of HU treatment, we analyzed global transcriptomic alteration of mouse ES cells in response to the treatment using high-throughput sequencing. We show that the global transcriptional activity is significantly suppressed as cells are exposed to HU treatment and alters multiple key cellular pathways, including cell cycle, apoptosis and DNAs. HU treatment also alters alternative splicing mechanisms and suppresses non-coding RNA expression. Our result provides novel clues for the understanding of how cells respond to HU and further suggests that high-throughput sequencing technology provides a powerful tool to study mechanisms of clinical drugs at the cellular level.

A novel mechanism of epigenetic regulation: nucleosome-space occupancy.

Nucleosome positioning around the gene space (or transcriptional unit) plays a crucial role for gene regulation but we do not know if the spatial organization--nucleosome-space occupancy or nucleosome density in a defined sequence unit length--contributes to the regulation complexity of mammalian gene expression. Using our own rmRNA-Seq (ribosomal RNA-minus RNA sequencing) and publically available ChIP-Seq (H3) data from mouse stem cells, we discovered a non-random distribution of nucleosomes along chromosomes, and further genome-wide studies on histone modifications, DNA methylation, transcriptional activity, gene density, and base compositional dynamics, demonstrated that nucleosome-space occupancy of genomic regions--clustered genes and their intergenic spaces--show distinctive features, where a high occupancy coincides with active transcription, intensive histone modifications, poor DNA methylation, and higher GC contents as compared to the nucleosome-poor regions. We therefore proposed that nucleosome-space occupancy as a novel mechanism of epigenetic gene regulation, creating a vital environment for transcriptional activation.

MicroRNA Expression in Multistage Date Fruit Development.

MicroRNAs (miRNAs) are small endogenous noncoding RNAs. Plant miRNAs are known to play important regulatory roles in homeostasis, stress response, and diverse developmental processes. Here, we describe the identification of conserved miRNAs in date palm (Phoenix dactylifera L.) based on transcriptomic data acquired across multistage fruit development and genome sequences, which include 238 plant conserved miRNAs and 276 novel P. dactylifera-specific miRNAs.

Transcriptomic study of the red palm weevil Rhynchophorus ferrugineus embryogenesis.

The red palm weevil (RPW), Rhynchophorus ferrugineus (Coleoptera: Curculionidae), is an invasive, concealed and destructive tissue borer, and it becomes a lethal pest of the palm family of plants and has been reported to attack 20 palm species around the globe. Here we report a systematic transcriptomic study on embryogenesis of RPW, where we analyze the transcriptomes across five developmental stages of RPW embryogenesis, involving four embryonic stages (E1, E2, E3 and E4) and one larval stage (L1). Using the RNA-seq and next-generation platforms, we generated 80 to 91 million reads for each library and assemble 22 532 genes that are expressed at different embryonic stages. Among the total transcripts from the five embryonic development stages, we found that 30.45 % are differentially expressed, 10.10 % show stage-specificity and even a larger fraction, 62.88 %, exhibit constitutive expression in all the stages. We also analyzes the expression dynamics of several conserved signaling pathways (such as Hedgehog, JAK-STAT, Notch, TGF-ß, Ras/MAPK and Wnt), as well as key developmental genes, including those related to apoptosis, axis formation, Hox complex, neurogenesis and segmentation. The datasets provide an essential resource for gene annotation and RPW functional genomics, including studies by using tools and concepts from multiple disciplines, such as development, physiology, biochemistry, molecular biology and genetics.

Adolescent mouse takes on an active transcriptomic expression during postnatal cerebral development.

Postnatal cerebral development is a complicated biological process precisely controlled by multiple genes. To understand the molecular mechanism of cerebral development, we compared dynamics of mouse cerebrum transcriptome through three developmental stages using high-throughput RNA-seq technique. Three libraries were generated from the mouse cerebrum at infancy, adolescence and adulthood, respectively. Consequently, 44,557,729 (infancy), 59,257,530 (adolescence) and 72,729,636 (adulthood) reads were produced, which were assembled into 15,344, 16,048 and 15,775 genes, respectively. We found that the overall gene expression level increased from infancy to adolescence and decreased later on upon reaching adulthood. The adolescence cerebrum has the most active gene expression, with expression of a large number of regulatory genes up-regulated and some crucial pathways activated. Transcription factor (TF) analysis suggested the similar dynamics as expression profiling, especially those TFs functioning in neurogenesis differentiation, oligodendrocyte lineage determination and circadian rhythm regulation. Moreover, our data revealed a drastic increase in myelin basic protein (MBP)-coding gene expression in adolescence and adulthood, suggesting that the brain myelin may be generated since mouse adolescence. In addition, differential gene expression analysis indicated the activation of rhythmic pathway, suggesting the function of rhythmic movement since adolescence; Furthermore, during infancy and adolescence periods, gene expression related to axonrepulsion and attraction showed the opposite trends, indicating that axon repulsion was activated after birth, while axon attraction might be activated at the embryonic stage and declined during the postnatal development. Our results from the present study may shed light on the molecular mechanism underlying the postnatal development of the mammalian cerebrum.

Transcriptome profiling of the developing postnatal mouse testis using next-generation sequencing.

Mammalian testis development is a complex and highly sophisticated process. To study the dynamic change of normal testis development at the transcriptional level, we investigated mouse testes at three postnatal ages: 6 days postnatal, 4 weeks old, and 10 weeks old, representing infant (PN1), juvenile (PN2), and adult (PN3) stages, respectively. Using ultra high-throughput RNA sequencing (RNA-seq) technology, we obtained 211 million reads with a length of 35 bp. We identified 18837 genes that were expressed in mouse testes, and found that genes expressed at the highest level were involved in spermatogenesis. The gene expression pattern in PN1 was distinct from that in PN2 and PN3, which indicates that spermatogenesis has commenced in PN2. We analyzed a large number of genes related to spermatogenesis and somatic development of the testis, which play important roles at different developmental stages. We also found that the MAPK, Hedgehog, and Wnt signaling pathways were significantly involved at different developmental stages. These findings further our understanding of the molecular mechanisms that regulate testis development. Our study also demonstrates significant advantages of RNA-seq technology for studying transcriptome during development.

Genome sequence of the date palm Phoenix dactylifera L.

Date palm (Phoenix dactylifera L.) is a cultivated woody plant species with agricultural and economic importance. Here we report a genome assembly for an elite variety (Khalas), which is 605.4 Mb in size and covers >90% of the genome (~671 Mb) and >96% of its genes (~41,660 genes). Genomic sequence analysis demonstrates that P. dactylifera experienced a clear genome-wide duplication after either ancient whole genome duplications or massive segmental duplications. Genetic diversity analysis indicates that its stress resistance and sugar metabolism-related genes tend to be enriched in the chromosomal regions where the density of single-nucleotide polymorphisms is relatively low. Using transcriptomic data, we also illustrate the date palm's unique sugar metabolism that underlies fruit development and ripening. Our large-scale genomic and transcriptomic data pave the way for further genomic studies not only on P. dactylifera but also other Arecaceae plants.

The association between H3K4me3 and antisense transcription.

Histone H3 lysine 4 trimethylation (H3K4me3) is well known to occur in the promoter region of genes for transcription activation. However, when investigating the H3K4me3 profiles in the mouse cerebrum and testis, we discovered that H3K4me3 also has a significant enrichment at the 3' end of actively transcribed (sense) genes, named as 3'-H3K4me3. 3'-H3K4me3 is associated with ~15% of protein-coding genes in both tissues. In addition, we examined the transcriptional initiation signals including RNA polymerase II (RNAPII) binding sites and 5'-CAGE-tag that marks transcriptional start sites. Interestingly, we found that 3'-H3K4me3 is associated with the initiation of antisense transcription. Furthermore, 3'-H3K4me3 modification levels correlate positively with the antisense expression levels of the associated sense genes, implying that 3'-H3K4me3 is involved in the activation of antisense transcription. Taken together, our findings suggest that H3K4me3 may be involved in the regulation of antisense transcription that initiates from the 3' end of sense genes. In addition, a positive correlation was also observed between the expression of antisense and the associated sense genes with 3'-H3K4me3 modification. More importantly, we observed the 3'-H3K4me3 enrichment among genes in human, fruitfly and Arabidopsis, and found that the sequences of 3'-H3K4me3-marked regions are highly conserved and essentially indistinguishable from known promoters in vertebrate. Therefore, we speculate that these 3'-H3K4me3-marked regions may serve as potential promoters for antisense transcription and 3'-H3K4me3 appear to be a universal epigenetic feature in eukaryotes. Our results provide a novel insight into the epigenetic roles of H3K4me3 and the regulatory mechanism of antisense transcription.

Comparative analyses of H3K4 and H3K27 trimethylations between the mouse cerebrum and testis.

The global features of H3K4 and H3K27 trimethylations (H3K4me3 and H3K27me3) have been well studied in recent years, but most of these studies were performed in mammalian cell lines. In this work, we generated the genome-wide maps of H3K4me3 and H3K27me3 of mouse cerebrum and testis using ChIP-seq and their high-coverage transcriptomes using ribominus RNA-seq with SOLiD technology. We examined the global patterns of H3K4me3 and H3K27me3 in both tissues and found that modifications are closely-associated with tissue-specific expression, function and development. Moreover, we revealed that H3K4me3 and H3K27me3 rarely occur in silent genes, which contradicts the findings in previous studies. Finally, we observed that bivalent domains, with both H3K4me3 and H3K27me3, existed ubiquitously in both tissues and demonstrated an invariable preference for the regulation of developmentally-related genes. However, the bivalent domains tend towards a "winner-takes-all" approach to regulate the expression of associated genes. We also verified the above results in mouse ES cells. As expected, the results in ES cells are consistent with those in cerebrum and testis. In conclusion, we present two very important findings. One is that H3K4me3 and H3K27me3 rarely occur in silent genes. The other is that bivalent domains may adopt a "winner-takes-all" principle to regulate gene expression.

The disequilibrium of nucleosomes distribution along chromosomes plays a functional and evolutionarily role in regulating gene expression.

To further understand the relationship between nucleosome-space occupancy (NO) and global transcriptional activity in mammals, we acquired a set of genome-wide nucleosome distribution and transcriptome data from the mouse cerebrum and testis based on ChIP (H3)-seq and RNA-seq, respectively. We identified a nearly consistent NO patterns among three mouse tissues--cerebrum, testis, and ESCs--and found, through clustering analysis for transcriptional activation, that the NO variations among chromosomes are closely associated with distinct expression levels between house-keeping (HK) genes and tissue-specific (TS) genes. Both TS and HK genes form clusters albeit the obvious majority. This feature implies that NO patterns, i.e. nucleosome binding and clustering, are coupled with gene clustering that may be functionally and evolutionarily conserved in regulating gene expression among different cell types.