The active form of vitamin D, 1α,25-(OH)2D3, has been associated with metabolism control, cell growth, differentiation, antiproliferation, apoptosis, and adaptive/innate immune responses, besides its ...functions in the integrity of bone and calcium homeostasis. The circadian rhythm regulates a variety of biological processes, many of them related to the functions associated with 1α,25-(OH)2D3. In the present study, we determine whether 1α,25-(OH)2D3 alters the expression of circadian genes in adipose-derived stem cells (ADSCs). The effect of 1α,25-(OH)2D3 on the expression of circadian genes BMAL1 and PER2 was measured by qPCR, over a 60-h period every 4 h, in serum shocked ADSCs, serum shocked ADSCs supplemented with 1α,25-(OH)2D3, and ADSCs under the presence of only 1α,25-(OH)2D3. The results showed that 1α,25-(OH)2D3 was able to synchronize circadian clock gene expression in ADSCs. The expression of circadian genes BMAL1 and PER2 in ADSCs that contained only 1α,25-(OH)2D3 has a profile similar to that found in the ADSCs synchronized by a serum shock. The results suggest an important role of 1α,25-(OH)2D3 in the regulation of the molecular clock.
In mammalian cells, gene transcription is regulated in a cell type specific manner by the interactions of transcriptional factors with genomic DNA. Lineage-specific transcription factors are ...considered to play essential roles in cell specification and differentiation during development. ChIP coupled with high-throughput DNA sequencing (ChIP-seq) is widely used to analyze genome-wide binding sites of transcription factors (or its associated complex) to genomic DNA. However, a large number of cells are required for one standard ChIP reaction, which makes it difficult to study the limited number of isolated primary cells or rare cell populations. In order to understand the regulatory mechanism of oligodendrocyte lineage-specific transcription factor Olig2 in acutely purified mouse OPCs, a detailed method using ChIP-seq to identify the genome-wide binding sites of Olig2 (or Olig2 complex) is shown. First, the protocol explains how to purify the platelet-derived growth factor receptor alpha (PDGFRα) positive OPCs from mouse brains. Next, Olig2 antibody mediated ChIP and library construction are performed. The last part describes the bioinformatic software and procedures used for Olig2 ChIP-seq analysis. In summary, this paper reports a method to analyze the genome-wide bindings of transcriptional factor Olig2 in acutely purified brain OPCs.
Systematic study of the regulatory mechanisms of Hematopoietic Stem Cell and Progenitor Cell (HSPC) self-renewal is fundamentally important for understanding hematopoiesis and for manipulating HSPCs ...for therapeutic purposes. Previously, we have characterized gene expression and identified important transcription factors (TFs) regulating the switch between self-renewal and differentiation in a multipotent Hematopoietic Progenitor Cell (HPC) line, EML (Erythroid, Myeloid, and Lymphoid) cells. Herein, we report binding maps for additional TFs (SOX4 and STAT3) by using chromatin immunoprecipitation (ChIP)-Sequencing, to address the underlying mechanisms regulating self-renewal properties of lineage-CD34+ subpopulation (Lin-CD34+ EML cells). Furthermore, we applied the Assay for Transposase Accessible Chromatin (ATAC)-Sequencing to globally identify the open chromatin regions associated with TF binding in the self-renewing Lin-CD34+ EML cells. Mass spectrometry (MS) was also used to quantify protein relative expression levels. Finally, by integrating the protein-protein interaction database, we built an expanded transcriptional regulatory and interaction network. We found that MAPK (Mitogen-activated protein kinase) pathway and TGF-β/SMAD signaling pathway components were highly enriched among the binding targets of these TFs in Lin-CD34+ EML cells. The present study integrates regulatory information at multiple levels to paint a more comprehensive picture of the HSPC self-renewal mechanisms.
We integrated datasets from transcriptomic, proteomic, epigenomic, and protein-protein interaction levels to build a global regulatory network to better characterize the mechanisms underlying HSPCs self-renewal.
The active form of vitamin D, 1α,25-(OH)^sub 2^D^sub 3^, has been associated with metabolism control, cell growth, differentiation, antiproliferation, apoptosis, and adaptive/innate immune responses, ...besides its functions in the integrity of bone and calcium homeostasis. The circadian rhythm regulates a variety of biological processes, many of them related to the functions associated with 1α,25-(OH)^sub 2^D^sub 3^. In the present study, we determine whether 1α,25-(OH)^sub 2^D^sub 3^ alters the expression of circadian genes in adipose-derived stem cells (ADSCs). The effect of 1α,25-(OH)^sub 2^D^sub 3^ on the expression of circadian genes BMAL1 and PER2 was measured by qPCR, over a 60-h period every 4 h, in serum shocked ADSCs, serum shocked ADSCs supplemented with 1α,25-(OH)^sub 2^D^sub 3^, and ADSCs under the presence of only 1α,25-(OH)^sub 2^D^sub 3^. The results showed that 1α,25-(OH)^sub 2^D^sub 3^ was able to synchronize circadian clock gene expression in ADSCs. The expression of circadian genes BMAL1 and PER2 in ADSCs that contained only 1α,25-(OH)^sub 2^D^sub 3^ has a profile similar to that found in the ADSCs synchronized by a serum shock. The results suggest an important role of 1α,25-(OH)^sub 2^D^sub 3^ in the regulation of the molecular clock.
Long non-coding RNAs (lncRNAs) (> 200 bp) play crucial roles in transcriptional regulation during numerous biological processes. However, it is challenging to comprehensively identify lncRNAs, ...because they are often expressed at low levels and with more cell-type specificity than are protein-coding genes. In the present study, we performed ab initio transcriptome reconstruction using eight purified cell populations from mouse cortex and detected more than 5000 lncRNAs. Predicting the functions of lncRNAs using cell-type specific data revealed their potential functional roles in Central Nervous System (CNS) development. We performed motif searches in ENCODE DNase I digital footprint data and Mouse ENCODE promoters to infer transcription factor (TF) occupancy. By integrating TF binding and cell-type specific transcriptomic data, we constructed a novel framework that is useful for systematically identifying lncRNAs that are potentially essential for brain cell fate determination. Based on this integrative analysis, we identified lncRNAs that are regulated during Oligodendrocyte Precursor Cell (OPC) differentiation from Neural Stem Cells (NSCs) and that are likely to be involved in oligodendrogenesis. The top candidate, lnc-OPC, shows highly specific expression in OPCs and remarkable sequence conservation among placental mammals. Interestingly, lnc-OPC is significantly up-regulated in glial progenitors from experimental autoimmune encephalomyelitis (EAE) mouse models compared to wild-type mice. OLIG2-binding sites in the upstream regulatory region of lnc-OPC were identified by ChIP (chromatin immunoprecipitation)-Sequencing and validated by luciferase assays. Loss-of-function experiments confirmed that lnc-OPC plays a functional role in OPC genesis. Overall, our results substantiated the role of lncRNA in OPC fate determination and provided an unprecedented data source for future functional investigations in CNS cell types. We present our datasets and analysis results via the interactive genome browser at our laboratory website that is freely accessible to the research community. This is the first lncRNA expression database of collective populations of glia, vascular cells, and neurons. We anticipate that these studies will advance the knowledge of this major class of non-coding genes and their potential roles in neurological development and diseases.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The analysis of biological networks has become essential to study functional genomic data. Compadre is a tool to estimate pathway/gene sets activity indexes using sub-matrix decompositions for ...biological networks analyses. The Compadre pipeline also includes one of the direct uses of activity indexes to detect altered gene sets. For this, the gene expression sub-matrix of a gene set is decomposed into components, which are used to test differences between groups of samples. This procedure is performed with and without differentially expressed genes to decrease false calls. During this process, Compadre also performs an over-representation test. Compadre already implements four decomposition methods principal component analysis (PCA), Isomaps, independent component analysis (ICA) and non-negative matrix factorization (NMF), six statistical tests (t- and f-test, SAM, Kruskal-Wallis, Welch and Brown-Forsythe), several gene sets (KEGG, BioCarta, Reactome, GO and MsigDB) and can be easily expanded. Our simulation results shown in Supplementary Information suggest that Compadre detects more pathways than over-representation tools like David, Babelomics and Webgestalt and less false positives than PLAGE. The output is composed of results from decomposition and over-representation analyses providing a more complete biological picture. Examples provided in Supplementary Information show the utility, versatility and simplicity of Compadre for analyses of biological networks.
Compadre is freely available at http://bioinformatica.mty.itesm.mx:8080/compadre. The R package is also available at https://sourceforge.net/p/compadre.
Background
Tissue engineering research aims to address the global shortage of donated corneal tissue, yet challenges persist in clinical translation. This study assesses the pathway from basic ...research to clinical adoption in corneal tissue engineering.
Methods
Bibliometric and patent analyses were conducted using the Web of Science-Core Collection and Lens databases to identify top authors, countries, journals, publication trends, inventors, patent statuses, and affiliated companies. A quality-adjusted life year (QALY) analysis compared engineered corneal endothelium to full keratoplasty. A pilot study surveyed thirty ophthalmologist surgeons from eight Latin American countries.
Results
A strong upward publication trend (R2 = 0.89, p = 1.53x10^-9) in corneal endothelium engineering was observed over the past decade, led by the USA, China, and Japan. Among 614 research papers, 26 patents and 10 companies were identified. Engineered corneal endothelium showed a QALY gain of 0.74 versus 0.07 of corneal transplants. Most survey respondents (97%) expressed interest in adopting engineered corneal endothelium for transplantation if affordability, biocompatibility, and functionality were assured.
Conclusions
While tissue engineering offers promise in alleviating corneal scarcity, a significant gap remains between scientific advancements and clinical adoption, presenting "death valleys." Addressing this requires more efficient navigation of the interplay between scientific progress, technology adoption, and clinical practice.
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