PubMed

Recent Publications

Characterization of a cancer-associated Epstein-Barr virus EBNA1 variant reveals a novel interaction with PLOD1 and PLOD3

Virology. 2021 Jul 21;562:103-109. doi: 10.1016/j.virol.2021.07.009. Online ahead of print.

ABSTRACT

Whole genome sequence analysis of Epstein-Barr virus genomes from tumours and healthy individuals identified three amino acid changes in EBNA1 that are strongly associated with gastric carcinoma and nasopharyngeal carcinoma. Here we show that, while these mutations do not impact EBNA1 plasmid maintenance function, one of them (Thr85Ala) decreases transcriptional activation and results in a gain of function interaction with PLOD1 and PLOD3. PLOD family proteins are strongly linked to multiple cancers, and PLOD1 is recognized as a prognostic marker of gastric carcinoma. We identified the PLOD1 binding site in EBNA1as the N-terminal transactivation domain and show that lysine 83 is critical for this interaction. The results provide a novel link between EBV infection and the cancer-associated PLOD proteins.

PMID:34304093 | DOI:10.1016/j.virol.2021.07.009



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Topoisomerase II deficiency leads to a postreplicative structural shift in all Saccharomyces cerevisiae chromosomes

Sci Rep. 2021 Jul 22;11(1):14940. doi: 10.1038/s41598-021-93875-5.

ABSTRACT

The key role of Topoisomerase II (Top2) is the removal of topological intertwines between sister chromatids. In yeast, inactivation of Top2 brings about distinct cell cycle responses. In the case of the conditional top2-5 allele, interphase and mitosis progress on schedule but cells suffer from a chromosome segregation catastrophe. We here show that top2-5 chromosomes fail to enter a Pulsed-Field Gel Electrophoresis (PFGE) in the first cell cycle, a behavior traditionally linked to the presence of replication and recombination intermediates. We distinguished two classes of affected chromosomes: the rDNA-bearing chromosome XII, which fails to enter a PFGE at the beginning of S-phase, and all the other chromosomes, which fail at a postreplicative stage. In synchronously cycling cells, this late PFGE retention is observed in anaphase; however, we demonstrate that this behavior is independent of cytokinesis, stabilization of anaphase bridges, spindle pulling forces and, probably, anaphase onset. Strikingly, once the PFGE retention has occurred it becomes refractory to Top2 re-activation. DNA combing, two-dimensional electrophoresis, genetic analyses, and GFP-tagged DNA damage markers suggest that neither recombination intermediates nor unfinished replication account for the postreplicative PFGE shift, which is further supported by the fact that the shift does not trigger the G2/M checkpoint. We propose that the absence of Top2 activity leads to a general chromosome structural/topological change in mitosis.

PMID:34294749 | DOI:10.1038/s41598-021-93875-5



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Retention of duplicated genes in evolution

Trends Genet. 2021 Jul 20:S0168-9525(21)00186-4. doi: 10.1016/j.tig.2021.06.016. Online ahead of print.

ABSTRACT

Gene duplication is a prevalent phenomenon across the tree of life. The processes that lead to the retention of duplicated genes are not well understood. Functional genomics approaches in model organisms, such as yeast, provide useful tools to test the mechanisms underlying retention with functional redundancy and divergence of duplicated genes, including fates associated with neofunctionalization, subfunctionalization, back-up compensation, and dosage amplification. Duplicated genes may also be retained as a consequence of structural and functional entanglement. Advances in human gene editing have enabled the interrogation of duplicated genes in the human genome, providing new tools to evaluate the relative contributions of each of these factors to duplicate gene retention and the evolution of genome structure.

PMID:34294428 | DOI:10.1016/j.tig.2021.06.016



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Nicotinamide phosphoribosyltransferase inhibitors selectively induce apoptosis of AML stem cells by disrupting lipid homeostasis

Cell Stem Cell. 2021 Jul 12:S1934-5909(21)00274-5. doi: 10.1016/j.stem.2021.06.004. Online ahead of print.

ABSTRACT

Current treatments for acute myeloid leukemia (AML) are often ineffective in eliminating leukemic stem cells (LSCs), which perpetuate the disease. Here, we performed a metabolic drug screen to identify LSC-specific vulnerabilities and found that nicotinamide phosphoribosyltransferase (NAMPT) inhibitors selectively killed LSCs, while sparing normal hematopoietic stem and progenitor cells. Treatment with KPT-9274, a NAMPT inhibitor, suppressed the conversion of saturated fatty acids to monounsaturated fatty acids, a reaction catalyzed by the stearoyl-CoA desaturase (SCD) enzyme, resulting in apoptosis of AML cells. Transcriptomic analysis of LSCs treated with KPT-9274 revealed an upregulation of sterol regulatory-element binding protein (SREBP)-regulated genes, including SCD, which conferred partial protection against NAMPT inhibitors. Inhibition of SREBP signaling with dipyridamole enhanced the cytotoxicity of KPT-9274 on LSCs in vivo. Our work demonstrates that altered lipid homeostasis plays a key role in NAMPT inhibitor-induced apoptosis and identifies NAMPT inhibition as a therapeutic strategy for targeting LSCs in AML.

PMID:34293334 | DOI:10.1016/j.stem.2021.06.004



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Seeds of their own destruction: Dominant-negative peptide screening yields functional insight and therapeutic leads

Cell Syst. 2021 Jul 21;12(7):691-693. doi: 10.1016/j.cels.2021.06.003.

ABSTRACT

Systematic, high-throughput screening for "dominant-negative" protein fragments is an emerging method for mapping functional regions of the parental protein in vivo. In this issue of Cell Systems, Ford et al. apply this approach to 65 cancer drivers, providing functional insights and demonstrating therapeutic potential for several dominant-negative peptides.

PMID:34293323 | DOI:10.1016/j.cels.2021.06.003



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A Roadmap to the Structure-Related Metabolism Pathways of Per- and Polyfluoroalkyl Substances in the Early Life Stages of Zebrafish (<em>Danio rerio</em>)

Environ Health Perspect. 2021 Jul;129(7):77004. doi: 10.1289/EHP7169. Epub 2021 Jul 21.

ABSTRACT

BACKGROUND: Thousands of per- and polyfluoroalkyl substances (PFAS) with diverse structures have been detected in the ambient environment. Apart from a few well-studied PFAS, the structure-related toxicokinetics of a broader set of PFAS remain unclear.

OBJECTIVES: To understand the toxicokinetics of PFAS, we attempted to characterize the metabolism pathways of 74 structurally diverse PFAS samples from the U.S. Environmental Protection Agency's PFAS screening library.

METHODS: Using the early life stages of zebrafish (Danio rerio) as a model, we determined the bioconcentration factors and phenotypic toxicities of 74 PFAS. Then, we applied high-resolution mass spectrometry-based nontargeted analysis to identify metabolites of PFAS in zebrafish larvae after 5 d of exposure by incorporating retention time and mass spectra. In vitro enzymatic activity experiments with human recombinant liver carboxylesterase (hCES1) were employed to validate the structure-related hydrolysis of 11 selected PFAS.

RESULTS: Our findings identified five structural categories of PFAS prone to metabolism. The metabolism pathways of PFAS were highly related to their structures as exemplified by fluorotelomer alcohols that the predominance of β-oxidation or taurine conjugation pathways were primarily determined by the number of hydrocarbons. Hydrolysis was identified as a major metabolism pathway for diverse PFAS, and perfluoroalkyl carboxamides showed the highest in vivo hydrolysis rates, followed by carboxyesters and sulfonamides. The hydrolysis of PFAS was verified with recombinant hCES1, with strong substrate preferences toward perfluoroalkyl carboxamides.

CONCLUSIONS: We suggest that the roadmap of the structure-related metabolism pathways of PFAS established in this study would provide a starting point to inform the potential health risks of other PFAS. https://doi.org/10.1289/EHP7169.

PMID:34288731 | PMC:PMC8294803 | DOI:10.1289/EHP7169



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High-content imaging and analysis to quantify the nuclear to cytoplasmic ratio of TGFβ and hippo effectors in mammalian cells

STAR Protoc. 2021 Jun 28;2(3):100632. doi: 10.1016/j.xpro.2021.100632. eCollection 2021 Sep 17.

ABSTRACT

Automated high-content immunofluorescence (IF) microscopy is used to monitor and quantify localization of the TGFβ/Smads and Taz/Yap Hippo effectors in mouse epithelial EpH4 cells transfected with Taz/Yap siRNAs. The nuclear-to-cytoplasmic protein ratios obtained by IF are converted into normalized masses by estimating the ratio of the compartment volumes. This method has the advantage that endogenous rather than tagged proteins are tracked and that knockdown of Taz/Yap can be simultaneously monitored at the single-cell level. For complete details on the use and execution of this protocol, please refer to Labibi et al. (2020).

PMID:34258593 | PMC:PMC8254078 | DOI:10.1016/j.xpro.2021.100632



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Opinion: Independent third-party entities as a model for validation of commercial antibodies

N Biotechnol. 2021 Jul 8;65:1-8. doi: 10.1016/j.nbt.2021.07.001. Online ahead of print.

ABSTRACT

A vast array of commercial antibodies covers a large percentage of human gene products, but determining which among them is most appropriate for any given application is challenging. This leads to use of non-specific antibodies that contributes to issues with reproducibility. It is our opinion that the community of scientists who use commercial antibodies in their biomedical research would benefit from third-party antibody characterization entities that use standardized operating procedures to assess and compare antibody performance. Ideally, such entities would follow the principles of open science, such that all antibodies against any given protein target would be tested in parallel, and all data generated released to the public domain without bias. Furthermore, there should be no financial incentive for the entity beyond cost-recovery. Such non-profit organizations, combined with other scientific efforts, could catalyse new discoveries by providing scientists with better validated antibody tools.

PMID:34246180 | DOI:10.1016/j.nbt.2021.07.001



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Sphingosine-1-Phosphate Receptor 3 Potentiates Inflammatory Programs in Normal and Leukemia Stem Cells to Promote Differentiation

Cancer Discov. 2021 Jan;2(1):32-53. doi: 10.1158/2643-3230.BCD-20-0155. Epub 2020 Dec 1.

ABSTRACT

Acute myeloid leukemia (AML) is a caricature of normal hematopoiesis driven from leukemia stem cells (LSC) that share some hematopoietic stem cell (HSC) programs including responsiveness to inflammatory signaling. Although inflammation dysregulates mature myeloid cells and influences stemness programs and lineage determination in HSCs by activating stress myelopoiesis, such roles in LSCs are poorly understood. Here, we show that S1PR3, a receptor for the bioactive lipid sphingosine-1-phosphate, is a central regulator that drives myeloid differentiation and activates inflammatory programs in both HSCs and LSCs. S1PR3-mediated inflammatory signatures varied in a continuum from primitive to mature myeloid states across cohorts of patients with AML, each with distinct phenotypic and clinical properties. S1PR3 was high in LSCs and blasts of mature myeloid samples with linkages to chemosensitivity, whereas S1PR3 activation in primitive samples promoted LSC differentiation leading to eradication. Our studies open new avenues for therapeutic target identification specific for each AML subset. SIGNIFICANCE: S1PR3 is a novel regulator of myeloid fate in normal hematopoiesis that is heterogeneously expressed in AML. S1PR3 marks a subset of less primitive AML cases with a distinct inflammatory signature and therefore has clinical implications as both a therapeutic target and a biomarker to distinguish primitive from mature AML.See related commentary by Yang et al., p. 3.This article is highlighted in the In This Issue feature, p. 1.

PMID:34238756 | DOI:10.1158/2643-3230.BCD-20-0155



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A Colorimetric Test to Differentiate Patients Infected with Influenza from COVID-19

Small Struct. 2021 Jun 6:2100034. doi: 10.1002/sstr.202100034. Online ahead of print.

ABSTRACT

Patients infected with SARS-CoV-2 and influenza display similar symptoms, but treatment requirements are different. Clinicians need to accurately distinguish SARS-CoV-2 from influenza to provide appropriate treatment. Here, the authors develope a color-based technique to differentiate between patients infected with SARS-CoV-2 and influenza A using a nucleic acid enzyme-gold nanoparticle (GNP) molecular test requiring minimal equipment. The MNAzyme and GNP probes are designed to be robust to viral mutations. Conserved regions of the viral genomes are targeted, and two MNAzymes are created for each virus. The ability of the system to distinguish between SARS-CoV-2 and influenza A using 79 patient samples is tested. When detecting SARS-CoV-2 positive patients, the clinical sensitivity is 90%, and the specificity is 100%. When detecting influenza A, the clinical sensitivity and specificity are 93% and 100%, respectively. The high clinical performance of the MNAzyme-GNP assay shows that it can be used to help clinicians choose effective treatments.

PMID:34230923 | PMC:PMC8250260 | DOI:10.1002/sstr.202100034



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