ALBERT

Description: Background: Fish oil is a popular nutritional product consumed in Hong Kong. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are the two main bioactive components responsible for the health benefits of fish oil. Market survey in Hong Kong demonstrated that various fish oil capsules with different origins and prices are sold simultaneously. However, these capsules are labelled with same ingredient levels, namely EPA 180 mg/g and DHA 120 mg/g. This situation makes the consumers very confused. To evaluate the quality of various fish oil capsules, a comparative analysis of the contents of EPA and DHA in fish oil is crucial. Methods: A gas chromatography-mass spectrometry (GC-MS) method was developed for identification and determination of EPA and DHA in fish oil capsules. A comprehensive validation of the developed method was conducted. Ten batches of fish oil capsules samples purchased from drugstores of Hong Kong were analyzed by using the developed method. Results: The present method presented good sensitivity, precision and accuracy. The limits of detection (LOD) for EPA and DHA were 0.08 ng and 0.21 ng, respectively. The relative standard deviation (RSD) values of EPA and DHA for precision tests were both less than 1.05%; and the recovery for accuracy test of EPA and DHA were 100.50% and 103.83%, respectively. In ten fish oil samples, the contents of EPA ranged from 39.52 mg/g to 509.16 mg/g, and the contents of DHA ranged from 35.14 mg/g to 645.70 mg/g. Conclusion: The present method is suitable for the quantitative analysis of EPA and DHA in fish oil capsules. There is a significant variation in the contents of the quantified components in fish oil samples, and there is not a linear relationship between price and contents of EPA and DHA. Strict supervision of the labelling of the fish oil capsules is urgently needed.

Description: Background: In eukaryotic genomes, about 10% of genes are arranged in a head-to-head (H2H) orientation, and the distance between the transcription start sites of each gene pair is closer than 1 kb. Two genes in an H2H pair are prone to co-express and co-function. There have been many studies on bidirectional promoters. However, the mechanism by which H2H genes are regulated at the transcriptional level still needs further clarification, especially with regard to the co-regulation of H2H pairs. In this study, we first used the Hi-C data of chromatin linkages to identify spatially interacting H2H pairs, and then integrated ChIP-seq data to compare H2H gene pairs with and without evidence of spatial interactions in terms of their binding transcription factors (TFs). Using ChIP-seq and DNase-seq data, histones and DNase associated with H2H pairs were identified. Furthermore, we looked into the connections between H2H genes in a human co-expression network. Results: We found that i) Similar to the behaviour of two genes within an H2H pair (intra-H2H pair), a gene pair involving two distinct H2H pairs (inter-H2H pair) which interact with each other spatially, share common transcription factors (TFs); ii) TFs of intra- and inter-H2H pairs are distributed differently. Factors such as HEY1, GABP, Sin3Ak-20, POL2, E2F6, and c-MYC are essential for the bidirectional transcription of intra-H2H pairs; while factors like CTCF, BDP1, GATA2, RAD21, and POL3 play important roles in coherently regulating inter-H2H pairs; iii) H2H gene blocks are enriched with hypersensitive DNase and modified histones, which participate in active transcriptions; and iv) H2H genes tend to be highly connected compared with non-H2H genes in the human co-expression network. Conclusions: Our findings shed new light on the mechanism of the transcriptional regulation of H2H genes through their linear and spatial interactions. For intra-H2H gene pairs, transcription factors regulate their transcriptions through bidirectional promoters, whereas for inter-H2H gene pairs, transcription factors are likely to regulate their activities depending on the spatial interaction of H2H gene pairs. In this way, two distinctive groups of transcription factors mediate intra- and inter-H2H gene transcriptions respectively, resulting in a highly compact gene regulatory network.

Description: Background: Berberine is a natural alkaloid derived from a traditional Chinese herbal medicine. It is known to modulate microRNA (miRNA) levels, although the mechanism for this action is unknown. Here, we previously demonstrate that the expression of 87 miRNAs is differentially affected by berberine in multiple myeloma cells. Among 49 miRNAs that are down-regulated, nine act as oncomirs, including miR-21. Integrative analysis showed that 28 of the down-regulated miRNAs participate in tumor protein p53 (TP53) signaling and other cancer pathways. miR-21 is involved in all these pathways, and is one of the most important oncomirs to be affected by berberine in multiple myeloma cells. Results: We confirmed that berberine down-regulated miRNA-21 expression and significantly up-regulated the expression of programmed cell death 4 (PDCD4), a predicted miR-21 target. Luciferase reporter assays confirmed that PDCD4 was directly regulated by miR-21. Bioinformatic analysis revealed that the miR-21 promoter can be targeted by signal transducer and activator of transcription 3 (STAT3). Down-regulation of interleukin 6 (IL6) by berberine might lead to inhibition of miR-21 transcription through STAT3 down-regulation in multiple myeloma. Furthermore, both berberine and seed-targeting anti-miR-21 oligonucleotide induced apoptosis, G2-phase cell cycle arrest and colony inhibition in multiple myeloma cell lines. Depletion of PDCD4 by short interfering RNA could rescue berberine-induced cytotoxicity in multiple myeloma cells. Conclusions: Our results suggest that berberine suppresses multiple myeloma cell growth, at least in part, by down-regulating miR-21 levels possibly through IL6/STAT3. This led to increased PDCD4 expression, which is likely to result in suppression of the p53 signaling pathway. These findings may also provide new mechanistic insight into the anti-cancer effects of certain compounds in traditional Chinese herbal medicines.

Description: The majority of human embryonic stem cell lines depend on a feeder cell layer for continuous growth in vitro, so that they can remain in an undifferentiated state. Limited knowledge is available concerning the molecular mechanisms that underlie the capacity of feeder cells to support both the proliferation and pluripotency of these cells. Importantly, feeder cells generally lose their capacity to support human embryonic stem cell proliferation in vitro following long-term culture. In this study, we performed large-scale gene expression profiles of human foreskin fibroblasts during early, intermediate and late passages using a custom DNA microarray platform (NeuroStem 2.0 Chip). The microarray data was validated using RT-PCR and virtual SAGE analysis. Our comparative gene expression study identified a limited number of molecular targets potentially involved in the ability of human neonatal foreskin fibroblasts to serve as feeder cells for human embryonic stem cell cultures. Among these, the C-KIT, leptin and pigment epithelium-derived factor (PEDF) genes were the most interesting candidates.

Description: Background Analyses of mitochondrial (mt) genome sequences in recent years challenge the current working hypothesis of Nematoda phylogeny proposed from morphology, ecology and nuclear small subunit rRNA gene sequences, and raise the need to sequence additional mt genomes for a broad range of nematode lineages. Results We sequenced the complete mt genomes of three Ascaridia species (family Ascaridiidae) that infest chickens, pigeons and parrots, respectively. These three Ascaridia species have an identical arrangement of mt genes to each other but differ substantially from other nematodes. Phylogenetic analyses of the mt genome sequences of the Ascaridia species, together with 62 other nematode species, support the monophylies of seven high-level taxa of the phylum Nematoda: 1) the subclass Dorylaimia; 2) the orders Rhabditida, Trichinellida and Mermithida; 3) the suborder Rhabditina; and 4) the infraorders Spiruromorpha and Oxyuridomorpha. Analyses of mt genome sequences, however, reject the monophylies of the suborders Spirurina and Tylenchina, and the infraorders Rhabditomorpha, Panagrolaimomorpha and Tylenchomorpha. Monophyly of the infraorder Ascaridomorpha varies depending on the methods of phylogenetic analysis. The Ascaridomorpha was more closely related to the infraorders Rhabditomorpha and Diplogasteromorpha (suborder Rhabditina) than they were to the other two infraorders of the Spirurina: Oxyuridorpha and Spiruromorpha. The closer relationship among Ascaridomorpha, Rhabditomorpha and Diplogasteromorpha was also supported by a shared common pattern of mitochondrial gene arrangement. Conclusions Analyses of mitochondrial genome sequences and gene arrangement has provided novel insights into the phylogenetic relationships among several major lineages of nematodes. Many lineages of nematodes, however, are underrepresented or not represented in these analyses. Expanding taxon sampling is necessary for future phylogenetic studies of nematodes with mt genome sequences.

Description: Background Transmembrane receptor kinases play critical roles in both animal and plant signaling pathways regulating growth, development, differentiation, cell death, and pathogenic defense responses. In Arabidopsis thaliana, there are at least 223 Leucine-rich repeat receptor-like kinases (LRR-RLKs), representing one of the largest protein families. Although functional roles for a handful of LRR-RLKs have been revealed, the functions of the majority of members in this protein family have not been elucidated. Results As a resource for the in-depth analysis of this important protein family, the complementary DNA sequences (cDNAs) of 194 LRR-RLKs were cloned into the GatewayR donor vector pDONR/ZeoR and analyzed by DNA sequencing. Among them, 157 clones showed sequences identical to the predictions in the Arabidopsis sequence resource, TAIR8. The other 37 cDNAs showed gene structures distinct from the predictions of TAIR8, which was mainly caused by alternative splicing of pre-mRNA. Most of the genes have been further cloned into GatewayR destination vectors with GFP or FLAG epitope tags and have been transformed into Arabidopsis for in planta functional analysis. All clones from this study have been submitted to the Arabidopsis Biological Resource Center (ABRC) at Ohio State University for full accessibility by the Arabidopsis research community. Conclusions Most of the Arabidopsis LRR-RLK genes have been isolated and the sequence analysis showed a number of alternatively spliced variants. The generated resources, including cDNA entry clones, expression constructs and transgenic plants, will facilitate further functional analysis of the members of this important gene family.

Description: Background First identified in fruit flies with temperature-sensitive paralysis phenotypes, the Drosophila melanogaster TipE locus encodes four voltage-gated sodium (NaV) channel auxiliary subunits. This cluster of TipE-like genes on chromosome 3L, and a fifth family member on chromosome 3R, are important for the optional expression and functionality of the Para NaV channel but appear quite distinct from auxiliary subunits in vertebrates. Here, we exploited available arthropod genomic resources to trace the origin of TipE-like genes by mapping their evolutionary histories and examining their genomic architectures. Results We identified a remarkably conserved synteny block of TipE-like orthologues with well-maintained local gene arrangements from 21 insect species. Homologues in the water flea, Daphnia pulex, suggest an ancestral pancrustacean repertoire of four TipE-like genes; a subsequent gene duplication may have generated functional redundancy allowing gene losses in the silk moth and mosquitoes. Intronic nesting of the insect TipE gene cluster probably occurred following the divergence from crustaceans, but in the flour beetle and silk moth genomes the clusters apparently escaped from nesting. Across Pancrustacea, TipE gene family members have experienced intronic nesting, escape from nesting, retrotransposition, translocation, and gene loss events while generally maintaining their local gene neighbourhoods. D. melanogaster TipE-like genes exhibit coordinated spatial and temporal regulation of expression distinct from their host gene but well-correlated with their regulatory target, the Para NaV channel, suggesting that functional constraints may preserve the TipE gene cluster. We identified homology between TipE-like NaV channel regulators and vertebrate Slo-beta auxiliary subunits of big-conductance calcium-activated potassium (BKCa) channels, which suggests that ion channel regulatory partners have evolved distinct lineage-specific characteristics. Conclusions TipE-like genes form a remarkably conserved genomic cluster across all examined insect genomes. This study reveals likely structural and functional constraints on the genomic evolution of insect TipE gene family members maintained in synteny over hundreds of millions of years of evolution. The likely common origin of these NaV channel regulators with BKCa auxiliary subunits highlights the evolutionary plasticity of ion channel regulatory mechanisms.

Description: Background In the studies incorporating worldwide sampling of A. thaliana populations, the samples from East Asia, especially from China, were very scattered; and the studies focused on global patterns of cpDNA genetic variation among accessions of A. thaliana are very few. In this study, chloroplast DNA sequence variability was used to infer phylogenetic relationships among Arabidopsis thaliana accessions from around the world, with the emphasis on samples from China. Results A data set comprising 77 accessions of A. thaliana, including 19 field-collected Chinese accessions together with three related species (A. arenosa, A. suecica, and Olimarabidopsis cabulica) as the out-group, was compiled. The analysis of the nucleotide sequences showed that the 77 accessions of A. thaliana were partitioned into two major differentiated haplotype classes (MDHCs). The estimated divergence time of the two MDHCs was about 0.39 mya. Forty-nine haplotypes were detected among the 77 accessions, which exhibited nucleotide diversity (π) of 0.00169. The Chinese populations along the Yangtze River were characterized by five haplotypes, and the two accessions collected from the middle range of the Altai Mountains in China shared six specific variable sites. Conclusions The dimorphism in the chloroplast DNA could be due to founder effects during late Pleistocene glaciations and interglacial periods, although introgression cannot be ruled out. The Chinese populations along the Yangtze River may have dispersed eastwards to their present-day locations from the Himalayas. These populations originated from a common ancestor, and a rapid demographic expansion began approximately 90,000 years ago. Two accessions collected from the middle range of the Altai Mountains in China may have survived in a local refugium during late Pleistocene glaciations. The natural populations from China with specific genetic characteristics enriched the gene pools of global A. thaliana collections.