| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
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| 3. |
- Frank, Stefan, et al.
(författare)
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yylncT Defines a Class of Divergently Transcribed lncRNAs and Safeguards the T-mediated Mesodermal Commitment of Human PSCs.
- 2019
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Ingår i: Cell stem cell. - : Elsevier BV. - 1875-9777 .- 1934-5909. ; 24:2
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Tidskriftsartikel (refereegranskat)abstract
- Human protein-coding genes are often accompanied by divergently transcribed non-coding RNAs whose functions, especially in cell fate decisions, are poorly understood. Using an hESC-based cardiac differentiation model, we define a class of divergent lncRNAs, termed yin yang lncRNAs (yylncRNAs), that mirror the cell-type-specific expression pattern of their protein-coding counterparts. yylncRNAs arepreferentially encoded from the genomic loci ofkey developmental cell fate regulators. Most yylncRNAs are spliced polyadenylated transcripts showing comparable expression patterns invivo inmouse and in human embryos. Signifying theirdevelopmental function, the key mesoderm specifier BRACHYURY (T) is accompanied by yylncT, whichlocalizes to the active T locus during mesoderm commitment. yylncT binds the de novo DNA methyltransferase DNMT3B, and its transcript is required for activation of the T locus, with yylncTdepletion specifically abolishing mesodermal commitment. Collectively, we report a lncRNA-mediated regulatory layer safeguarding embryonic cell fate transitions.
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| 6. |
- Kuo, Chao-Chung, et al.
(författare)
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Detection of RNA-DNA binding sites in long noncoding RNAs.
- 2019
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Ingår i: Nucleic acids research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 47:6
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Tidskriftsartikel (refereegranskat)abstract
- Long non-coding RNAs (lncRNAs) can act as scaffolds that promote the interaction of proteins, RNA, and DNA. There is increasing evidence of sequence-specific interactions of lncRNAs with DNA via triple-helix (triplex) formation. This process allows lncRNAs to recruit protein complexes to specific genomic regions and regulate gene expression. Here we propose a computational method called Triplex Domain Finder (TDF) to detect triplexes and characterize DNA-binding domains and DNA targets statistically. Case studies showed that this approach can detect the known domains of lncRNAs Fendrr, HOTAIR and MEG3. Moreover, we validated a novel DNA-binding domain in MEG3 by a genome-wide sequencing method. We used TDF to perform a systematic analysis of the triplex-forming potential of lncRNAs relevant to human cardiac differentiation. We demonstrated that the lncRNA with the highest triplex-forming potential, GATA6-AS, forms triple helices in the promoter of genes relevant to cardiac development. Moreover, down-regulation of GATA6-AS impairs GATA6 expression and cardiac development. These data indicate the unique ability of our computational tool to identify novel triplex-forming lncRNAs and their target genes.
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| 7. |
- Mitra, Sanhita, et al.
(författare)
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Subcellular distribution of p53 by the p53-responsive lncRNA NBAT1 determines chemotherapeutic response in neuroblastoma.
- 2021
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Ingår i: Cancer research. - 1538-7445. ; 81:6, s. 1457-1471
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Tidskriftsartikel (refereegranskat)abstract
- Neuroblastoma has a low mutation rate for the p53 gene. Alternative ways of p53 inactivation have been proposed in neuroblastoma, such as abnormal cytoplasmic accumulation of wild-type p53. However, mechanisms leading to p53 inactivation via cytoplasmic accumulation are not well investigated. Here we show that the neuroblastoma risk-associated locus 6p22.3-derived tumor suppressor NBAT1 is a p53-responsive lncRNA that regulates p53 subcellular levels. Low expression of NBAT1 provided resistance to genotoxic drugs by promoting p53 accumulation in cytoplasm and loss from mitochondrial and nuclear compartments. Depletion of NBAT1 altered CRM1 function and contributed to the loss of p53-dependent nuclear gene expression during genotoxic drug treatment. CRM1 inhibition rescued p53-dependent nuclear functions and sensitized NBAT1-depleted cells to genotoxic drugs. Combined inhibition of CRM1 and MDM2 was even more effective in sensitizing aggressive neuroblastoma cells with p53 cytoplasmic accumulation. Thus, our mechanistic studies uncover an NBAT1-dependent CRM1/MDM2-based potential combination therapy for high-risk neuroblastoma patients.
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| 8. |
- Mondal, Tanmoy, 1981, et al.
(författare)
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Sense-antisense lncRNA pair encoded by locus 6p22.3 determines neuroblastoma susceptibility via the USP36-CHD7-SOX9 regulatory axis
- 2018
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Ingår i: Cancer Cell. - : Elsevier BV. - 1535-6108 .- 1878-3686. ; 33:3
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Tidskriftsartikel (refereegranskat)abstract
- Trait-associated loci often map to genomic regions encoding long noncoding RNAs (lncRNAs), but the role of these lncRNAs in disease etiology is largely unexplored. We show that a pair of sense/antisense lncRNA (6p22lncRNAs) encoded by CASC15 and NBAT1 located at the neuroblastoma (NB) risk-associated 6p22.3 locus are tumor suppressors and show reduced expression in high-risk NBs. Loss of functional synergy between 6p22lncRNAs results in an undifferentiated state that is maintained by a gene-regulatory network, including SOX9 located on 17q, a region frequently gained in NB. 6p22lncRNAs regulate SOX9 expression by controlling CHD7 stability via modulating the cellular localization of USP36, encoded by another 17q gene. This regulatory nexus between 6p22.3 and 17q regions may lead to potential NB treatment strategies.
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