Single cell transcriptome analysis of MCF-7 reveals consistently and inconsistently expressed gene groups each associated with distinct cellular localization and functions.

Single cell transcriptome (SCT) analysis provides superior resolution to illustrate tumor cell heterogeneity for clinical implications. We characterized four SCTs of MCF-7 using 143 housekeeping genes (HKGs) as control, of which lactate dehydrogenase B (LDHB) expression is silenced. These SCT libraries mapped to 11,423, 11,486, 10,380, and 11,306 RefSeq genes (UCSC), respectively. High consistency in HKG expression levels across all four SCTs, along with transcriptional silencing of LDHB, was observed, suggesting a high sensitivity and reproducibility of the SCT analysis. Cross-library comparison on expression levels by scatter plotting revealed a linear correlation and an 83-94% overlap in transcript isoforms and expressed genes were also observed. To gain insight of transcriptional diversity among the SCTs, expressed genes were split into consistently expressed (CE) (expressed in all SCTs) and inconsistently expressed (IE) (expressed in some but not all SCTs) genes for further characterization, along with the 142 expressed HKGs as a reference. Distinct transcriptional strengths were found among these groups, with averages of 1,612.0, 88.0 and 1.2 FPKM for HKGs, CE and IE, respectively. Comparison between CE and IE groups further indicated that expressions of CE genes vary more significantly than that of IE genes. Gene Ontology analysis indicated that proteins encoded by CE genes are mainly involved in fundamental intracellular activities, while proteins encoded by IE genes are mainly for extracellular activities, especially acting as receptors or ion channels. The diversified gene expressions, especially for those encoded by IE genes, may contribute to cancer drug resistance.

Stage-Specific Demethylation in Primordial Germ Cells Safeguards against Precocious Differentiation.

Remodeling DNA methylation in mammalian genomes can be global, as seen in preimplantation embryos and primordial germ cells (PGCs), or locus specific, which can regulate neighboring gene expression. In PGCs, global and locus-specific DNA demethylation occur in sequential stages, with an initial global decrease in methylated cytosines (stage I) followed by a Tet methylcytosine dioxygenase (Tet)-dependent decrease in methylated cytosines that act at imprinting control regions (ICRs) and meiotic genes (stage II). The purpose of the two-stage mechanism is unclear. Here we show that Dnmt1 preserves DNA methylation through stage I at ICRs and meiotic gene promoters and is required for the pericentromeric enrichment of 5hmC. We discovered that the functional consequence of abrogating two-stage DNA demethylation in PGCs was precocious germline differentiation leading to hypogonadism and infertility. Therefore, bypassing stage-specific DNA demethylation has significant consequences for progenitor germ cell differentiation and the ability to transmit DNA from parent to offspring.

Exosomal miR-10a derived from amniotic fluid stem cells preserves ovarian follicles after chemotherapy.

Chemotherapy (CTx)-induced premature ovarian failure (POF) in woman remains clinically irreversible. Amniotic fluid stem cells (AFSCs) have shown the potential to treat CTx-induced POF; however, the underlying mechanism is unclear. Here we demonstrate that AFSC-derived exosomes recapitulate the anti-apoptotic effect of AFSCs on CTx-damaged granulosa cells (GCs), which are vital for the growth of ovarian follicles. AFSC-derived exosomes prevent ovarian follicular atresia in CTx-treated mice via the delivery of microRNAs in which both miR-146a and miR-10a are highly enriched and their potential target genes are critical to apoptosis. The down-regulation of these two miRNAs in AFSC-derived exosomes attenuates the anti-apoptotic effect on CTx-damaged GCs in vitro. Further, the administration of these miRNAs recapitulates the effects both in vitro and in vivo, in which miR-10a contributes a dominant influence. Our findings illustrate that miR-10a has potential as a novel therapeutic agent for the treatment of POF.

TEA: the epigenome platform for Arabidopsis methylome study.

BACKGROUND: Bisulfite sequencing (BS-seq) has become a standard technology to profile genome-wide DNA methylation at single-base resolution. It allows researchers to conduct genome-wise cytosine methylation analyses on issues about genomic imprinting, transcriptional regulation, cellular development and differentiation. One single data from a BS-Seq experiment is resolved into many features according to the sequence contexts, making methylome data analysis and data visualization a complex task. RESULTS: We developed a streamlined platform, TEA, for analyzing and visualizing data from whole-genome BS-Seq (WGBS) experiments conducted in the model plant Arabidopsis thaliana. To capture the essence of the genome methylation level and to meet the efficiency for running online, we introduce a straightforward method for measuring genome methylation in each sequence context by gene. The method is scripted in Java to process BS-Seq mapping results. Through a simple data uploading process, the TEA server deploys a web-based platform for deep analysis by linking data to an updated Arabidopsis annotation database and toolkits. CONCLUSIONS: TEA is an intuitive and efficient online platform for analyzing the Arabidopsis genomic DNA methylation landscape. It provides several ways to help users exploit WGBS data. TEA is freely accessible for academic users at: http://tea.iis.sinica.edu.tw .

Palindromic sequence impedes sequencing-by-ligation mechanism.

BACKGROUND: Current next-generation sequencing (NGS) platforms adopt two types of sequencing mechanisms: by synthesis or by ligation. The former is employed by 454 and Solexa systems, while the latter by SOLiD system. Although the pros and cons for each sequencing mechanism have more or less been discussed in a number of occasions, the potential obstacle imposed by palindromic sequences has not yet been addressed. METHODS: To test the effect of the palindromic region on sequencing efficacy, we clonally amplified a paired-end ditag sequence composed of a 24-bp palindromic sequence flanked by a pair of tags from the E. coli genome. We used the near homogeneous fragments produced from MmeI digestion of the amplified clone to generate a sequencing library for SOLiD 5500xl sequencer. RESULTS: Results showed that, traditional ABI sequencers, which adopt sequencing-by-synthesis mechanism, were able to read through the palindromic region. However, SOLiD 5500xl was unable to do so. Instead, the palindromic region was read as miscellaneous random sequences. Moreover, readable tag sequence turned obscure ~2 bp prior to the palindromic region. CONCLUSIONS: Taken together, we demonstrate that SOLiD machines, which employ sequencing-by-ligation mechanism, are unable to read through the palindromic region. On the other hand, sequencing-by-synthesis sequencers had no difficulty in doing so.

New classification of supersecondary structures of sandwich-like proteins uncovers strict patterns of strand assemblage.

To describe the supersecondary structure (SSS) of beta sandwich-like proteins (SPs), we introduce a structural unit called the "strandon." A strandon is defined as a set of sequentially consecutive strands connected by hydrogen bonds in 3D structures. Representing beta-proteins as the assembly of strandons exposes the underlying similarities in their SSS and enables us to construct a novel classification scheme of SPs. Classification of all known SPs is based on shared supersecondary structural features and is presented in the SSS database (http://binfs.umdnj.edu/sssdb/). Analysis of the SSS reveals two common specific patterns. The first pattern defines the arrangement of strandons and was found in 95% of all examined SPs. The second pattern establishes the ordering of strands in the protein domain and was observed in 82% of the analyzed SPs. Knowledge of these two patterns that uncover the spatial arrangement of strands will likely prove useful in protein structure prediction.

Early predict the outcomes of refractive accommodative esotropia by initial presentations.

BACKGROUND: The differential diagnosis between fully and partially refractive accommodative esotropia (Ac-ET) depends on outcomes after intervention with refraction correction. Whether the differences exist in terms of initial clinical features between these two variants has not been fully explored. METHODS: Children between the ages of 6 months and 8 years with esotropia and spherical equivalent greater than +3.00 (D) were included in this study. After wearing diopters glasses for at least 2 years, children were classified according to the indexed criteria into the fully Ac-ET group (group A, N=28) partially Ac-ET (group or the B, N=17). Six clinical parameters, including age at onset, age at first visit, visual acuity, refractive error, angle of esodeviation, and presence or absence of inferior oblique overaction at initial presentation were compared between these two groups. RESULTS: The angle of esodeviation (31.4+/-11.6 PD vs. 42.6+/-12.6 PD, p = 0.004) was significantly different between the fully and partially Ac-ET groups, while refraction (+5.79+/-1.84 D vs. +4.79+/-1.40 D, p = 0.062) had borderline significance. On the contrary, the age at onset (2.35+/-1.74 yrs vs. 2.01+/-1.96 yrs, p=0.539), age at first visit (3.51+/-1.36 yrs vs. 3.01+/-1.70 yrs, p =0.285), inferior oblique overaction (32% vs. 47%, p =0.317), and visual acuity before (LogMAR: 0.40+/-0.25 vs. LogMAR: 0.34+/-0.25, p =0.544) and after treatment (LogMAR: 0.057+/-0.079 vs. LogMAR: 0.051+/-0.19, p = 0.088) were similar in the two groups. CONCLUSIONS: Children with a smaller angle of esodeviation and higher hyperopia were more likely belonging to fully Ac-ET, which can be treated with glasses without the need of surgical intervention. Early detection and early treatment of accommodative esotropia are needed to prevent strabismus and amblyopia.