The discovery of the CRISPR/Cas genome-editing system has revolutionized our understanding of the plant genome. CRISPR/Cas has been used for over a decade to modify plant genomes for the study of specific genes and biosynthetic pathways as well as to speed up breeding in many plant species, including both model and non-model crops. Although the CRISPR/Cas system is very efficient for genome editing, many bottlenecks and challenges slow down further improvement and applications. In this review we discuss the challenges that can occur during tissue culture, transformation, regeneration, and mutant detection. We also review the opportunities provided by new CRISPR platforms and specific applications related to gene regulation, abiotic and biotic stress response improvement, and de novo domestication of plants.
- Klíčová slova
- CA18111, CRISPR applications, CRISPR platforms, PlantEd, gene regulation, mutant detection, plant regeneration,
- MeSH
- CRISPR-Cas systémy * genetika MeSH
- editace genu * MeSH
- geneticky modifikované rostliny genetika MeSH
- genom rostlinný genetika MeSH
- šlechtění rostlin MeSH
- zemědělské plodiny genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Recent developments in high-throughput sequencing (HTS) technologies and bioinformatics have drastically changed research in virology, especially for virus discovery. Indeed, proper monitoring of the viral population requires information on the different isolates circulating in the studied area. For this purpose, HTS has greatly facilitated the sequencing of new genomes of detected viruses and their comparison. However, bioinformatics analyses allowing reconstruction of genome sequences and detection of single nucleotide polymorphisms (SNPs) can potentially create bias and has not been widely addressed so far. Therefore, more knowledge is required on the limitations of predicting SNPs based on HTS-generated sequence samples. To address this issue, we compared the ability of 14 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 21 variants of pepino mosaic virus (PepMV) in three samples through large-scale performance testing (PT) using three artificially designed datasets. To evaluate the impact of bioinformatics analyses, they were divided into three key steps: reads pre-processing, virus-isolate identification, and variant calling. Each step was evaluated independently through an original, PT design including discussion and validation between participants at each step. Overall, this work underlines key parameters influencing SNPs detection and proposes recommendations for reliable variant calling for plant viruses. The identification of the closest reference, mapping parameters and manual validation of the detection were recognized as the most impactful analysis steps for the success of the SNPs detections. Strategies to improve the prediction of SNPs are also discussed.
- Klíčová slova
- Bioinformatic, Genomic, Plant, Variant, Virus,
- MeSH
- genom virový genetika MeSH
- jednonukleotidový polymorfismus * genetika MeSH
- lidé MeSH
- výpočetní biologie MeSH
- vysoce účinné nukleotidové sekvenování * MeSH
- znalosti MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Non-alcoholic fatty liver disease (NAFLD), encompassing fatty liver and its progression into nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC), is one of the rapidly rising health concerns worldwide. SIRT6 is an essential nuclear sirtuin that regulates numerous pathological processes including insulin resistance and inflammation, and recently it has been implicated in the amelioration of NAFLD progression. SIRT6 overexpression protects from formation of fibrotic lesions. However, the underlying molecular mechanisms are not fully delineated. Moreover, new allelic variants of SIRT6 (N308K/A313S) were recently associated with the longevity in Ashkenazi Jews by improving genome maintenance and DNA repair, suppressing transposons and killing cancer cells. Whether these new SIRT6 variants play different or enhanced roles in liver diseases is currently unknown. In this study, we aimed to clarify how these new centenarian-associated SIRT6 genetic variants affect liver metabolism and associated diseases. We present evidence that overexpression of centenarian-associated SIRT6 variants dramatically altered the metabolomic and secretomic profiles of unchallenged immortalized human hepatocytes (IHH). Most amino acids were increased in the SIRT6 N308K/A313S overexpressing IHH when compared to IHH transfected with the SIRT6 wild-type sequence. Several unsaturated fatty acids and glycerophospholipids were increased, and ceramide tended to be decreased upon SIRT6 N308K/A313S overexpression. Furthermore, we found that overexpression of SIRT6 N308K/A313S in a 3D hepatic spheroid model formed by the co-culture of human immortalized hepatocytes (IHH) and hepatic stellate cells (LX2) inhibited collagen deposition and fibrotic gene expression in absence of metabolic or dietary challenges. Hence, our findings suggest that novel longevity associated SIRT6 N308K/A313S variants could favor the prevention of NASH by altering hepatocyte proteome and lipidome.
- Klíčová slova
- Aging, Hepatic stellate cells, Hepatocytes, Liver, Metabolomics, SIRT6, Spheroids,
- MeSH
- hepatocelulární karcinom * metabolismus patologie MeSH
- hepatocyty metabolismus patologie MeSH
- kolagen metabolismus MeSH
- lidé MeSH
- nádory jater * metabolismus patologie MeSH
- nealkoholová steatóza jater * genetika metabolismus patologie MeSH
- senioři nad 80 let MeSH
- sirtuiny * genetika metabolismus MeSH
- století lidé MeSH
- Check Tag
- lidé MeSH
- senioři nad 80 let MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- kolagen MeSH
- SIRT6 protein, human MeSH Prohlížeč
- sirtuiny * MeSH
DNA damage repair (DDR) is a safeguard for genome integrity maintenance. Increasing DDR efficiency could increase the yield of induced pluripotent stem cells (iPSC) upon reprogramming from somatic cells. The epigenetic mechanisms governing DDR during iPSC reprogramming are not completely understood. Our goal was to evaluate the splicing isoforms of histone variant macroH2A1, macroH2A1.1, and macroH2A1.2, as potential regulators of DDR during iPSC reprogramming. GFP-Trap one-step isolation of mtagGFP-macroH2A1.1 or mtagGFP-macroH2A1.2 fusion proteins from overexpressing human cell lines, followed by liquid chromatography-tandem mass spectrometry analysis, uncovered macroH2A1.1 exclusive interaction with Poly-ADP Ribose Polymerase 1 (PARP1) and X-ray cross-complementing protein 1 (XRCC1). MacroH2A1.1 overexpression in U2OS-GFP reporter cells enhanced specifically nonhomologous end joining (NHEJ) repair pathway, while macroH2A1.1 knock-out (KO) mice showed an impaired DDR capacity. The exclusive interaction of macroH2A1.1, but not macroH2A1.2, with PARP1/XRCC1, was confirmed in human umbilical vein endothelial cells (HUVEC) undergoing reprogramming into iPSC through episomal vectors. In HUVEC, macroH2A1.1 overexpression activated transcriptional programs that enhanced DDR and reprogramming. Consistently, macroH2A1.1 but not macroH2A1.2 overexpression improved iPSC reprogramming. We propose the macroH2A1 splicing isoform macroH2A1.1 as a promising epigenetic target to improve iPSC genome stability and therapeutic potential.
- Klíčová slova
- DNA damage, cell reprogramming, induced pluripotent stem cells, macroH2A1.1,
- MeSH
- DNA MeSH
- endoteliální buňky metabolismus MeSH
- histony * metabolismus MeSH
- indukované pluripotentní kmenové buňky * metabolismus MeSH
- lidé MeSH
- myši MeSH
- oprava DNA MeSH
- protein XRCC1 genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA MeSH
- histony * MeSH
- MACROH2A1 protein, human MeSH Prohlížeč
- protein XRCC1 MeSH
- XRCC1 protein, human MeSH Prohlížeč
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death, which develops in the context of fibrosis and cirrhosis caused by chronic inflammation, in turn due to non-alcoholic fatty liver disease (NAFLD), alcohol consumption and/or hepatitis viral infection. An increased number of senescent cells are associated with age-related tissue degeneration during NAFLD-induced HCC, or during chemotherapeutic treatment. Senolytic agents target selectively senescent cells. A combination of the senolytic drugs dasatinib and quercetin (D+Q) reduced hepatic lipid accumulation and alleviated age-associated physical dysfunction in mice. However, whether D+Q can impact the treatment of HCC, at the end-stage of the NAFLD inflammatory spectrum, is unknown. Here, using two well-established HCC cell lines (HepG2, Huh-7), we demonstrate that the maximal cytostatic doses for D and/or Q (1 + 1 μM) lacked efficacy in removing doxorubicin-induced β-gal-positive senescent cells. Moreover, D+Q did not affect doxorubicin-dependent induction of flattened morphology, activation of p16, expression of SASP-associated genes or formation of γH2AX foci. We then investigated the antitumor efficacy of doxorubicin, D+Q, or the combination, in xenograft studies conducted with HCC cells inoculated in athymic nude mice. Doxorubicin reduced tumor growth by 30% compared to control mice, while D+Q was ineffective in synergizing with doxorubicin and in clearing doxorubicin-induced HCC senescent cells. Unexpectedly, D+Q alone appeared to have acute pro-tumorigenic effects in control mice. While our data need to be confirmed in animal models that fully recapitulate NAFLD, we demonstrate that these compounds are ineffective, alone or in synergy with senescence-inducing chemotherapy, against experimental HCC.
- Klíčová slova
- HCC (hepatocellular carcinoma), NAFLD (non-alcoholic fatty liver disease), dasatinib, quercertin, senolytic agents,
- Publikační typ
- časopisecké články MeSH
