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Introduction: Among gynecological cancers, ovarian cancer has a high mortality rate. Cisplatin-based chemotherapy is commonly used for the treatment of ovarian cancer. However, the clinical efficacy of cisplatin in ovarian cancer is limited due to the development of chemo-resistance during treatment. Objective: In the study, we aimed to investigate the synergistic anti-cancer activity and targets of the FDA-approved drug disulfiram combined with cisplatin in ovarian cancer. Methods: The cell viability was determined by Celltier-Glo luminescent assay. The synergistic anti-cancer activity was assessed by combination index. Cell cycle and apoptosis were detected by flow cytometry. The in vivo anti-tumor activity and side effects were evaluated using a xenografted mice model. The synergistic anti-cancer targets were identified by a mass spectrometry-based proteomics analysis. Results: In this study, we first found that disulfiram synergistically enhanced the anti-tumor activity of cisplatin in chemo-resistant ovarian cancer cells, which was accompanied by the enhanced induction of cellular apoptosis. Secondly, the in vivo study demonstrated that the combination treatment of disulfiram and cisplatin dramatically inhibited tumor growth and had no apparent side effects in ovarian cancer xenografted mice. Finally, proteomics analysis identified SMAD3 as a potential target of disulfiram-cisplatin combined treatment, and the down-regulation of SMAD3 could increase cisplatin-induced cell death in ovarian cancer. Conclusion: Combination treatment of disulfiram and cisplatin synergistically inhibited the growth of ovarian cancer through down-regulating SMAD3. As a repurposed drug, disulfiram could be quickly transformed into a clinic to overcome cisplatin resistance for the treatment of ovarian cancer.

Cell‐free methylated DNA immunoprecipitation and high‐throughput sequencing (cfMeDIP‐seq) is a new bisulfite‐free technique, which can detect the whole‐genome methylation of blood cell‐free DNA (cfDNA). Using this technique, we identified differentially methylated regions (DMR) of cfDNA between lung tumors and normal controls. Based on the top 300 DMR, we built a random forest prediction model, which was able to distinguish malignant lung tumors from normal controls with high sensitivity and specificity of 91.0% and 93.3% (AUROC curve of 0.963). In summary, we reported a non–invasive prediction model that had good ability to distinguish malignant pulmonary nodules., This study reported the whole‐genome methylation profiling of plasma cfDNA of patients with pulmonary nodules by cfMeDIP‐seq. A random forest model was used to distinguish pulmonary malignant tumors from normal controls, based on the whole‐genome methylation profiling.

Over the past decades, low‐dose computed tomography (LD‐CT) screening has been widely used for the early detection of lung cancer. Increasing numbers of indeterminate pulmonary nodules are now being discovered. However, it remains challenging to distinguish malignant from benign pulmonary nodules, especially those considered to be small or ground‐glass (GGN) nodules. Liquid biopsies have been successfully applied in the diagnosis of advanced lung cancer, and the potential value for early detection of lung cancer has made great progress. Recent studies have demonstrated the value of various blood‐based tumor biomarkers in determining the nature of pulmonary nodules, including cell‐free DNA (cfDNA), microRNAs (miRNAs), circulating tumor cells (CTCs) and tumor‐associated autoantibodies (AAbs). In this review, we summarize the latest progress of liquid biopsies, and their potential applications and challenges in the diagnosis of malignant pulmonary nodules., Schematic representation of liquid biopsies for aiding in the diagnosis of pulmonary nodules detected by LD‐CT. The biomarkers for liquid biopsies include cell‐free DNA (cfDNA), microRNAs (miRNAs), circulating tumor cells (CTCs) and autoantibodies (AAbs).

Mitochondrial quality control plays an important role in maintaining mitochondrial homeostasis and function. Disruption of mitochondrial quality control degrades brain function. We found that flunarizine (FNZ), a drug whose chronic use causes parkinsonism, led to a parkinsonism-like motor dysfunction in mice. FNZ induced mitochondrial dysfunction and decreased mitochondrial mass specifically in the brain. FNZ decreased mitochondrial content in both neurons and astrocytes, without affecting the number of nigral dopaminergic neurons. In human neural progenitor cells, FNZ also induced mitochondrial depletion. Mechanistically, independent of ATG5- or RAB9-mediated mitophagy, mitochondria were engulfed by lysosomes, followed by a vesicle-associated membrane protein 2– and syntaxin-4–dependent extracellular secretion. A genome-wide CRISPR knockout screen identified genes required for FNZ-induced mitochondrial elimination. These results reveal not only a previously unidentified lysosome-associated exocytosis process of mitochondrial quality control that may participate in the FNZ-induced parkinsonism but also a drug-based method for generating mitochondria-depleted mammal cells.

Background Tumor cells undergoing epithelial-mesenchymal transition (EMT) display enhanced ability to enter the circulation, thereby being major source of circulating tumor cells (CTCs). In this study, we aimed to better understand the roles of CTC undergoing EMT in monitoring cancer progression. Methods We analyzed gene expression profiling of epithelial and mesenchymal markers in lung or colon tumor samples by mining TCGA database. We detected CTCs and classify their EMT phenotypes of 31 patients with lung or colon cancer by using a CanPatrol CTC-enrichment technique. Results The bioinformatic analysis indicated that mesenchymal markers were expressed in a subset of lung tumor samples, and its high expression was associated with poor survival of lung cancer patients. However, in colon cancer, majority of tumor samples expressed hybrid epithelial/mesenchymal markers. CTC analysis with EMT classification showed that the number of CTCs with mesenchymal phenotype was high in lung cancer patients with the advanced stage. Dynamic CTC analysis in a lung cancer patient indicated that CTC with mesenchymal phenotype was effective to monitor tumor progression. In a colon cancer patient, dynamic CTC analysis indicated that CTC with hybrid epithelial/mesenchymal phenotypes was an effective biomarker to guide therapy. Conclusions Encouraging results from this proof-of-concept study show that CTC with mesenchymal phenotype or hybrid epithelial/mesenchymal phenotypes could be a potential biomarker for monitoring tumor progression in lung or colon cancer respectively.

Neurogenesis, a highly orchestrated process, entails the transition from a pluripotent to neural state and involves neural progenitor cells (NPCs) and neuronal/glial subtypes. However, the precise epigenetic mechanisms underlying fate decision remain poorly understood. Here, we delete KDM6s (JMJD3 and/or UTX), the H3K27me3 demethylases, in human embryonic stem cells (hESCs) and show that their deletion does not impede NPC generation from hESCs. However, KDM6-deficient NPCs exhibit poor proliferation and a failure to differentiate into neurons and glia. Mechanistically, both JMJD3 and UTX are found to be enriched in gene loci essential for neural development in hNPCs, and KDM6 impairment leads to H3K27me3 accumulation and blockade of DNA accessibility at these genes. Interestingly, forced expression of neuron-specific chromatin remodelling BAF (nBAF) rescues the neuron/glia defect in KDM6-deficient NPCs despite H3K27me3 accumulation. Our findings uncover the differential requirement of KDM6s in specifying NPCs and neurons/glia and highlight the contribution of individual epigenetic regulators in fate decisions in a human development model., Neurogenesis is an ordered transition from pluriptotent cells to neural precursor cells (NPCs) to neurons. Here the authors show that loss of the lysine demethylases JMJD3 and UTX leads reduced DNA accessibility at neurogenesis loci in human NPCs, and that the chromatin remodeller BAF can rescue differentiation defects.

Metabolic switch is critical for cell fate determination through metabolic functions, epigenetic modifications, and gene expression. However, the mechanisms underlying these alterations and their functional roles remain unclear. Here, we show that Plin2-mediated moderate lipid hydrolysis is critical for pluripotency of embryonic stem cells (ESCs). Upon exit from pluripotency, lipid droplet (LD)-associated protein Plin2 is recognized by Hsc70 and degraded via chaperone-mediated autophagy to facilitate LD mobilization. Enhancing lipid hydrolysis by Plin2 knockout promotes pluripotency exit, which is recovered by ATGL inhibition. Mechanistically, excessive lipid hydrolysis induces a dramatic lipidomic remodeling characterized by decreased cardiolipin and phosphatidylethanolamine, which triggers defects in mitochondrial cristae and fatty acid oxidation, resulting in reduced acetyl-CoA and histone acetylation. Our results reveal how LD mobilization is regulated and its critical role in ESC pluripotency, and indicate the mechanism linking LD homeostasis to mitochondrial remodeling and epigenetic regulation, which might shed light on development and diseases.

Hematopoietic stem cells (HSCs)/progenitor cells (HPCs) are generated from hemogenic endothelial cells (HECs) during the endothelial-to-hematopoietic transition (EHT); however, the underlying mechanism remains poorly understood. Here, using an array of approaches, including CRSPR/Cas9 gene knockouts, RNA-Seq, ChIP-Seq, ATAC-Seq etc., we report that vitamin C (Vc) is essential in HPC generation during human pluripotent stem cell (hPSC) differentiation in defined culture conditions. Mechanistically, we found that the endothelial cells generated in the absence of Vc fail to undergo the EHT because of an apparent failure in opening up genomic loci essential for hematopoiesis. Under Vc deficiency, these loci exhibited abnormal accumulation of histone H3 trimethylation at Lys-27 (H3K27me3), a repressive histone modification that arose because of lower activities of demethylases that target H3K27me3. Consistently, deletion of the two H3K27me3 demethylases, Jumonji domain-containing 3 (JMJD3 or KDM6B) and histone demethylase UTX (UTX or KDM6A), impaired HPC generation even in the presence of Vc. Furthermore, we noted that Vc and jmjd3 are also important for HSC generation during zebrafish development. Together, our findings reveal an essential role for Vc in the EHT for hematopoiesis, and identify KDM6-mediated chromatin demethylation as an important regulatory mechanism in hematopoietic cell differentiation.

Multiple primary tumors are defined by the presence of two or more independent primary tumors in the same or different organs of an individual patient. However, the underlying genetic cause for the development of multiple primary tumors is largely unknown. In the study, we report a rare case with four synchronous distinct histological cancer types in a 26 years old Chinese female. In the patient, whole-exome sequencing identified a homozygous germline insertion mutation in WWOX which encodes the DNA repair-related enzyme, WW domain containing oxidoreductase. The mutation was found in a heterozygous state in her parents and brother without any cancer phenotype thus far. Surprisingly, we found multiple novel aberrant WWOX transcripts in the patient's normal colon tissue. The patient's colon metastasis from clear cell adenocarcinoma of the ovary showed a nonhypermutated profile enriched for C-T transition, and harbored somatic pathogenic mutations of HRAS, BRCA2, SMAD4, CHEK2, and AKT1 genes. To our knowledge, this is the first study reporting WWOX gene aberrations in a young patient with the early occurrence of multiple primary tumors. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly due in large part to age-dependent atrophy of retinal pigment epithelium (RPE) cells. RPE cells form a monolayer located between the choroid and the outer segments of photoreceptors, playing multifarious roles in maintenance of visual function. Allogeneically induced pluripotent stem cell-derived RPE (iPSC-RPE or iRPE) has become a potential approach for providing an abundant source of donors for clinical cell products. Transplantation of iRPE has been proven effective in rescuing impaired retinas in Royal College of Surgeons (RCS) rats after approximately 5 to 6 weeks. Here, we explore the long-term (19 weeks) safety and efficacy of human iRPE cell transplantation in pre-clinical animal models. METHODS: The expression of human RPE-specific markers in iRPE cells was determined using immunofluorescence staining. For the proliferative test, Ki-67 expression was also verified by immunofluorescence and flow cytometric analysis. Then, iRPE cells were transplanted into the subretinal space of immune-deficient NOD/SCID/IL-2Rgc(null) (NSG) mice to assess their safety. To evaluate whether the transplanted cells could survive and rescue visual function, we performed color fundus photography, focal electroretinogram and immunostaining after delivering iRPE cells into the subretinal space of RCS rats. RESULTS: Human iRPE cells expressed native RPE-specific markers, such as microphthalmia-associated transcription factor (MiTF), retinal pigment epithelium-specific 65-kDa protein (RPE65) and tight-junction associated structural protein (ZO-1), and their proliferative capacity (Ki-67 expression) was poor after 25 days of induction. A tumorigenicity test revealed no tumor formation or abnormal proliferation in the immunodeficient mice after subretinal injection of 5×10(5) iRPE cells. The transplanted iRPE cells survived for at least 19 weeks and maintained visual function for 15 weeks. CONCLUSIONS: In the present study, we provided further evidence for the use of human iRPE transplantation to treat retinal degenerative disease in pre-clinical animal models. Therefore, we consider human iRPE cells a promising source of cell replacement therapy for AMD.

Definitive hematopoiesis generates hematopoietic stem/progenitor cells (HSPCs) that give rise to all mature blood and immune cells, but remains poorly defined in human. Here, we resolve human hematopoietic populations at the earliest hematopoiesis stage by single-cell RNA-seq. We characterize the distinct molecular profiling between early primitive and definitive hematopoiesis in both human embryonic stem cell (hESC) differentiation and early embryonic development. We identify CD44 to specifically discriminate definitive hematopoiesis and generate definitive HSPCs from hESCs. The multipotency of hESCs-derived HSPCs for various blood and immune cells is validated by single-cell clonal assay. Strikingly, these hESCs-derived HSPCs give rise to blood and lymphoid lineages in vivo. Lastly, we characterize gene-expression dynamics in definitive and primitive hematopoiesis and reveal an unreported role of ROCK-inhibition in enhancing human definitive hematopoiesis. Our study provides a prospect for understanding human early hematopoiesis and a firm basis for generating blood and immune cells for clinical purposes.

The purpose of the article is to establish a quick enrichment and detection method using immunomagnetic beads and flow cytometry to analyze circulating tumor cells (CTCs) in the peripheral blood. After incubation with CD326-PE and CD45-APC antibodies, more than 60% MCF7 cells in M-Buffer could be detected while less than 10% of the same cells could be detected by flow cytometry (FCM) if spiked into blood. However, in combination with CD326 and CD45 immunomagnetic beads, detection rate of MCF7 cells in blood reached 57%. For circulating tumor cells, enrichment by CD326 and CD45 immunomagnetic beads improve the detection rate from nearly undetectable to more than 24.14%. Live CTCs in peripheral blood can be effectively and sensitively detected by using a combination of immunomagnetic beads (CD45 and CD326) and flow cytometry.

Adventitious root (AR) formation is critically important in vegetative propagation through cuttings in some plants, especially woody species. However, the underlying molecular mechanisms remain elusive. Here, we report the identification of a poplar homeobox gene, PuHox52, which was induced rapidly (within 15 min) at the basal ends of stems upon cutting and played a key regulatory role in adventitious rooting. We demonstrated that overexpression of PuHox52 significantly increased the number of ARs while suppression of PuHox52 had the opposite effect. A multilayered hierarchical gene regulatory network (ML-hGRN) mediated by PuHox52 was reverse-engineered and demonstrated to govern AR formation. PuHox52 regulated AR formation through upregulation of nine hub regulators, including a jasmonate signaling pathway gene, PuMYC2, and an auxin signaling pathway gene, PuAGL12. We also identified coherent type 4 feed-forward loops within this ML-hGRN; PuHox52 repressed PuHDA9, which encodes a histone deacetylase, and led to an increase in acetylation and presumably expression of three hub regulators, PuWRKY51, PuLBD21 and PuIAA7. Our results indicate that the ML-hGRN mediated by PuHox52 governs AR formation at the basal ends of stem cuttings from poplar trees.

Background Autophagy is a programmed cell degradation mechanism that has been associated with several physiological and pathophysiological processes, including malignancy. Improper induction of autophagy has been proposed to play a pivotal role in the progression of hepatocellular carcinoma (HCC). Methods Univariate Cox regression analysis of overall survival (OS) was performed to identify risk-associated autophagy-related genes (ARGs) in HCC data set from The Cancer Genome Atlas (TCGA). Multivariate cox regression was then performed to develop a risk prediction model for the prognosis of 370 HCC patients. The multi-target receiver operating characteristic (ROC) curve was used to determine the model’s accuracy. Besides, the relationship between drug sensitivity and ARGs expression was also examined. Results A total of 62 differentially expressed ARGs were identified in HCC patients. Univariate and multivariate regression identified five risk-associated ARGs (HDAC1, RHEB, ATIC, SPNS1 and SQSTM1) that were correlated with OS in HCC patients. Of importance, the risk-associated ARGs were independent risk factors in the multivariate risk model including clinical parameters such as malignant stage (HR = 1.433, 95% CI = 1.293–1.589, P Conclusion We developed a novel prognostic risk model by integrating the molecular signature and clinical parameters of HCC, which can effectively predict the outcomes of HCC patients.

The success of Yamanaka factor reprogramming of somatic cells into induced pluripotent stem cells suggests that some factor(s) must remodel the nuclei from a condensed state to a relaxed state. How factor‐dependent chromatin opening occurs remains unclear. Using FRAP and ATAC‐seq, we found that Oct4 acts as a pioneer factor that loosens heterochromatin and facilitates the binding of Klf4 and the expression of epithelial genes in early reprogramming, leading to enhanced mesenchymal‐to‐epithelial transition. A mutation in the Oct4 linker, L80A, which shows impaired interaction with the BAF complex component Brg1, is inactive in heterochromatin loosening. Oct4‐L80A also blocks the binding of Klf4 and retards MET. Finally, vitamin C or Gadd45a could rescue the reprogramming deficiency of Oct4‐L80A by enhancing chromatin opening and Klf4 binding. These studies reveal a cooperation between Oct4 and Klf4 at the chromatin level that facilitates MET at the cellular level and shed light into the research of multiple factors in cell fate determination.

Somatic cell reprogramming provides insight into basic principles of cell fate determination, which remain poorly understood. Here we show that the transcription factor Glis1 induces multi-level epigenetic and metabolic remodelling in stem cells that facilitates the induction of pluripotency. We find that Glis1 enables reprogramming of senescent cells into pluripotent cells and improves genome stability. During early phases of reprogramming, Glis1 directly binds to and opens chromatin at glycolytic genes, whereas it closes chromatin at somatic genes to upregulate glycolysis. Subsequently, higher glycolytic flux enhances cellular acetyl-CoA and lactate levels, thereby enhancing acetylation (H3K27Ac) and lactylation (H3K18la) at so-called 'second-wave' and pluripotency gene loci, opening them up to facilitate cellular reprogramming. Our work highlights Glis1 as a powerful reprogramming factor, and reveals an epigenome-metabolome-epigenome signalling cascade that involves the glycolysis-driven coordination of histone acetylation and lactylation in the context of cell fate determination.

SUMMARYPhysiological interconversion between specialized cell types has only been described in a few mammalian tissues and the mechanisms remain obscure. Using genetic lineage tracing during postnatal development and in-vitro models we demonstrate conversion of gastric interstitial cells of Cajal (ICC), regulatory cells that electrically pace phasic contractions and mediate nitrergic and cholinergic neural control of smooth muscle cells, into phenotypically distinct “fibroblast-like” interstitial cells (FLC), which only mediate purinergic signaling. Mechanistically, we find this transition to be epigenetically governed by H3K27 trimethylation of cell identity-related promoters whose susceptibility to repression is predicted by H3K27 acetylation patterns in ICC. The phenotypic switch was reversible by inhibition, knockdown or in-vivo genomic inactivation of the polycomb H3K27 methyl-transferase Ezh2. These results demonstrate a role for Ezh2-mediated epigenetic repression in physiological mammalian transdifferentiation and identify FLC as a reserve from which ICC can potentially be restored in common gastrointestinal disorders where ICC are depleted.GRAPHICAL ABSTRACTHIGHLIGHTSGastric pacemaker cells (ICC) transdifferentiate into quiescent cells (FLC) in vivoICC-to-FLC shift switches neural control from nitrergic/cholinergic to purinergicEzh2-mediated H3K27me3 represses cell-identity genes during ICC-to-FLC transitionEzh2 inhibition restores ICC numbers, phenotype and functioneTOC BLURBSyed et al. find aging to cause transdifferentiation of gastric pacemaker cells (interstitial cells of Cajal, ICC), which also communicate cholinergic and nitrergic neurotransmission to smooth muscle cells, into quiescent “fibroblast-like cells” (FLC), which only mediate purinergic signals. This switch is governed by Ezh2, whose inhibition can reverse ICC depletion.

Ovarian cancer has a high death rate and is often not detected until late stages. While some studies found high expressions of MMPs correlated with cancer invasion, metastasis, and poor prognosis, however, several other studies indicated MMPs might inhibit cancer rather than promote cancer in certain situations. Thus, the role of different MMPs in different cancer types needs a systematic re-evaluation. In this study, we used RNA-Seq and corresponding clinical data downloaded from TCGA and analyzed the correlations between MMP expressions and the clinicopathologic characteristics and outcome in ovarian serous cystadenocarcinoma (OSC) patients. Among the MMPs investigated, MMP-3 was significantly increased in high-grade and high-stage tumors compared with low-grade and low-stage ones. Using univariate analysis and multivariate Cox model, high expressions of MMP-19 and -20 were found to associate with poor overall survival independent of clinicopathologic characteristics. Moreover, using in vitro studies, we found ovarian cancer cell lines with higher MMP-19 and -20 protein expressing levels were associated with anti-cancer drugs resistance, while knockdown of MMP-19 or -20 increased ovarian cancer cell sensitivities to several clinical using chemotherapy agents. Finally, knockdown of MMP-19 or -20 also decreased the invasion abilities of several ovarian cancer cell lines. These in vitro studies provided potential mechanisms of high MMP-19 and -20 expressions in the poor prognosis of ovarian cancer.

microRNAs (miRNAs) are endogenous small non-coding RNAs that bind to mRNAs and target them for cleavage and/or translational repression, leading to gene silencing. We previously developed short tandem target mimic (STTM) technology to deactivate endogenous miRNAs in Arabidopsis. Here, we created hundreds of STTMs that target both conserved and species-specific miRNAs in Arabidopsis, tomato, rice, and maize, providing a resource for the functional interrogation of miRNAs. We not only revealed the functions of several miRNAs in plant development, but also demonstrated that tissue-specific inactivation of a few miRNAs in rice leads to an increase in grain size without adversely affecting overall plant growth and development. RNA-seq and small RNA-seq analyses of STTM156/157 and STTM165/166 transgenic plants revealed the roles of these miRNAs in plant hormone biosynthesis and activation, secondary metabolism, and ion-channel activity-associated electrophysiology, demonstrating that STTM technology is an effective approach for studying miRNA functions. To facilitate the study and application of STTM transgenic plants and to provide a useful platform for storing and sharing of information about miRNA-regulated gene networks, we have established an online Genome Browser (https://blossom.ffr.mtu.edu/designindex2.php) to display the transcriptomic and miRNAomic changes in STTM-induced miRNA knockdown plants.

Background Stored biological samples with pathology information and medical records are invaluable resources for translational medical research. However, RNAs extracted from the archived clinical tissues are often substantially degraded. RNA degradation distorts the RNA-seq read coverage in a gene-specific manner, and has profound influences on whole-genome gene expression profiling. Result We developed the transcript integrity number (TIN) to measure RNA degradation. When applied to 3 independent RNA-seq datasets, we demonstrated TIN is a reliable and sensitive measure of the RNA degradation at both transcript and sample level. Through comparing 10 prostate cancer clinical samples with lower RNA integrity to 10 samples with higher RNA quality, we demonstrated that calibrating gene expression counts with TIN scores could effectively neutralize RNA degradation effects by reducing false positives and recovering biologically meaningful pathways. When further evaluating the performance of TIN correction using spike-in transcripts in RNA-seq data generated from the Sequencing Quality Control consortium, we found TIN adjustment had better control of false positives and false negatives (sensitivity = 0.89, specificity = 0.91, accuracy = 0.90), as compared to gene expression analysis results without TIN correction (sensitivity = 0.98, specificity = 0.50, accuracy = 0.86). Conclusion TIN is a reliable measurement of RNA integrity and a valuable approach used to neutralize in vitro RNA degradation effect and improve differential gene expression analysis. Electronic supplementary material The online version of this article (doi:10.1186/s12859-016-0922-z) contains supplementary material, which is available to authorized users.

Concordance between RIN and median TIN score for 20 peripheral blood mononuclear cell (PBMC) samples [4]. The PBMC samples were stored at room temperature for 0 h (blue), 12 h (green), 24 h (orange), 48 h (purple) and 84 h (red). Each time point contains 4 individuals (replicates). r, Pearson correlation coefficient. (PDF 143 kb)

Relationship between 5â ˛UTR (untranslated regoin) size and TIN score for 12 Glioblastoma (GBM) samples. (a)-(c) three samples with RIN value of 10; (d)-(e), two samples with RIN value of 8; (f)-(h) three samples with RIN value of 6; (i)-(k) three samples with RIN value of 4; (l) one sample with RIN value of 2. r, Pearson correlation coefficient. r, Pearson correlation coefficient. Linear regression lines fitted to data are indicated as black dashed lines. (PDF 7140 kb)

Smoothed scatter plots showing correlation between TIN score and transcript size. (a)-(c) three samples with RIN value of 10; (d)-(e), two samples with RIN value of 8; (f)-(h) three samples with RIN value of 6; (i)-(k) three samples with RIN value of 4; (l) one sample with RIN value of 2. Blue, orange and red represents low, median and high density of data points, respectively. Transcripts with no read coverage or smaller then 100 nucleotide were removed. r, Pearson correlation coefficient. Linear regression lines fitted to data are indicated as black dashed lines. (PDF 6875 kb)

Venn diagram showing the overlapping between 665 DEGs (before TIN correction) and 289 DEGs (after TIN correction). DEG, differentially expressed gene. (PDF 101 kb)

Evaluating TIN (x-axis) metric using transcript level RNA fragment size (y-axis) for 12 Glioblastoma (GBM) samples [5]. (a)-(c) Three samples with RIN value of 10 (red); (d)-(e), two samples with RIN value of 8 (purple); (f)-(h) three samples with RIN value of 6 (orange); (i)-(k) three samples with RIN value of 4 (blue); (l) one sample with RIN value of 2 (cyan). Each dot represents 50 transcripts. Black curves indicate locally weighted polynomial regression curves. r, Pearson correlation coefficient. (PDF 1490 kb)

Evaluating RIN and median TIN score using sample level RNA fragment size as benchmark. Only 28 mCRPC samples with RINâ

Barplot showing Pearson correlation coefficients between TIN and RNA fragment size. 12 Glioblastoma (GBM) samples were stratified by RIN score; RIN = 10 (red), RIN = 8 (purple), RIN = 6 (orange), RIN = 4 (blue) and RIN = 2 (cyan). (PDF 120 kb)

Background: Expansion of intronic (CTG·CAG)n repeats in TCF4 is found in most Fuchs endothelial corneal dystrophy (FECD) patients. Results: RNA foci co-localizing with the splicing factor MBNL1 are found in FECD cells, and changes in mRNA splicing occur. Conclusion: Trinucleotide repeat expansion in FECD is associated with RNA focus formation and missplicing. Significance: RNA toxicity occurs in a disease affecting millions of patients., Fuchs endothelial corneal dystrophy (FECD) is an inherited degenerative disease that affects the internal endothelial cell monolayer of the cornea and can result in corneal edema and vision loss in severe cases. FECD affects ∼5% of middle-aged Caucasians in the United States and accounts for >14,000 corneal transplantations annually. Among the several genes and loci associated with FECD, the strongest association is with an intronic (CTG·CAG)n trinucleotide repeat expansion in the TCF4 gene, which is found in the majority of affected patients. Corneal endothelial cells from FECD patients harbor a poly(CUG)n RNA that can be visualized as RNA foci containing this condensed RNA and associated proteins. Similar to myotonic dystrophy type 1, the poly(CUG)n RNA co-localizes with and sequesters the mRNA-splicing factor MBNL1, leading to missplicing of essential MBNL1-regulated mRNAs. Such foci and missplicing are not observed in similar cells from FECD patients who lack the repeat expansion. RNA-Seq splicing data from the corneal endothelia of FECD patients and controls reveal hundreds of differential alternative splicing events. These include events previously characterized in the context of myotonic dystrophy type 1 and epithelial-to-mesenchymal transition, as well as splicing changes in genes related to proposed mechanisms of FECD pathogenesis. We report the first instance of RNA toxicity and missplicing in a common non-neurological/neuromuscular disease associated with a repeat expansion. The FECD patient population with this (CTG·CAG)n trinucleotide repeat expansion exceeds that of the combined number of patients in all other microsatellite expansion disorders.

DNA methyltransferase 1 (DNMT1) is essential for DNA methylation, gene regulation and chromatin stability. We previously discovered DNMT1 mutations cause hereditary sensory and autonomic neuropathy type 1 with dementia and hearing loss (HSAN1E; OMIM 614116). HSAN1E is the first adult-onset neurodegenerative disorder caused by a defect in a methyltransferase gene. HSAN1E patients appear clinically normal until young adulthood, then begin developing the characteristic symptoms involving central and peripheral nervous systems. Some HSAN1E patients also develop narcolepsy and it has recently been suggested that HSAN1E is allelic to autosomal dominant cerebellar ataxia, deafness, with narcolepsy (ADCA-DN; OMIM 604121), which is also caused by mutations in DNMT1. A hotspot mutation Y495C within the targeting sequence domain of DNMT1 has been identified among HSAN1E patients. The mutant DNMT1 protein shows premature degradation and reduced DNA methyltransferase activity. Herein, we investigate genome-wide DNA methylation at single-base resolution through whole-genome bisulfite sequencing of germline DNA in 3 pairs of HSAN1E patients and their gender- and age-matched siblings. Over 1 billion 75-bp single-end reads were generated for each sample. In the 3 affected siblings, overall methylation loss was consistently found in all chromosomes with X and 18 being most affected. Paired sample analysis identified 564,218 differentially methylated CpG sites (DMCs; P < 0.05), of which 300 134 were intergenic and 264 084 genic CpGs. Hypomethylation was predominant in both genic and intergenic regions, including promoters, exons, most CpG islands, L1, L2, Alu, and satellite repeats and simple repeat sequences. In some CpG islands, hypermethylated CpGs outnumbered hypomethylated CpGs. In 201 imprinted genes, there were more DMCs than in non-imprinted genes and most were hypomethylated. Differentially methylated region (DMR) analysis identified 5649 hypomethylated and 1872 hypermethylated regions. Importantly, pathway analysis revealed 1693 genes associated with the identified DMRs were highly associated in diverse neurological disorders and NAD+/NADH metabolism pathways is implicated in the pathogenesis. Our results provide novel insights into the epigenetic mechanism of neurodegeneration arising from a hotspot DNMT1 mutation and reveal pathways potentially important in a broad category of neurological and psychological disorders.

RIN (RNA integrity number) score distribution for 120 metastatic castration resistant prostate cancer (mCRPC) samples. (PDF 9 kb)

RIN (RNA integrity number) score distribution for 120 metastatic castration resistant prostate cancer (mCRPC) samples. (PDF 9 kb)