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The Journal of Thoracic and... Dec 2023Thoracic aortic aneurysm and dissection has a genetic predisposition and a variety of clinical manifestations. This study aimed to investigate the clinical and molecular...
OBJECTIVES
Thoracic aortic aneurysm and dissection has a genetic predisposition and a variety of clinical manifestations. This study aimed to investigate the clinical and molecular characterizations of patients with thoracic aortic aneurysm and dissection and further explore the relationship between the genotype and phenotype, as well as their postoperative outcomes.
METHODS
A total of 1095 individuals with thoracic aortic aneurysm and dissection admitted to our hospital between 2013 and 2022 were included. Next-generation sequencing and multiplex ligation-dependent probe amplification were performed, and mosaicism analysis was additionally implemented to identify the genetic causes.
RESULTS
A total of 376 causative variants were identified in 83.5% of patients with syndromic thoracic aortic aneurysm and dissection and 18.7% of patients with nonsyndromic thoracic aortic aneurysm and dissection, including 8 copy number variations and 2 mosaic variants. Patients in the "pathogenic" and "variant of uncertain significance" groups had younger ages of aortic events and higher aortic reintervention risks compared with genetically negative cases. In addition, patients with FBN1 haploinsufficiency variants had shorter reintervention-free survival than those with FBN1 dominant negative variants.
CONCLUSIONS
Our data expanded the genetic spectrum of heritable thoracic aortic aneurysm and dissection and indicated that copy number variations and mosaic variants contributed to a small proportion of the disease-causing alterations. Moreover, positive genetic results might have a possible predictive value for aortic event severity and postoperative risk stratification.
Topics: Humans; Aortic Dissection; DNA Copy Number Variations; Aortic Aneurysm, Thoracic; Genetic Predisposition to Disease; Aorta
PubMed: 36517271
DOI: 10.1016/j.jtcvs.2022.11.004 -
Sheng Wu Gong Cheng Xue Bao = Chinese... May 2024To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly...
To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly methods and validated the established method by using fructose-1,6-diphosphatase 1 (FBP1) with 4 mutant sites. The fragments containing mutations were assembled by introducing mutant sites and I recognition sequences. After digestion/ligation, the ligated fragment was amplified with the primers containing overlap region to the linearized vector. The amplified fragment was ligated to the linearized vector and the ligation product was transformed into . After screening and sequencing, the recombinant plasmid with 4 mutant sites was obtained. This protocol overcame the major defects of Gibson assembly and Golden Gate assembly, serving as an efficient solution for multi-fragment assembly and multi-site mutagenesis.
Topics: Escherichia coli; Fructose-Bisphosphatase; Homologous Recombination; Plasmids; Genetic Vectors; DNA; Mutation; Mutagenesis, Site-Directed; Cloning, Molecular
PubMed: 38783816
DOI: 10.13345/j.cjb.230793 -
Gastroenterology Feb 2024The highly heterogeneous cellular and molecular makeup of pancreatic ductal adenocarcinoma (PDAC) not only fosters exceptionally aggressive tumor biology, but...
BACKGROUND & AIMS
The highly heterogeneous cellular and molecular makeup of pancreatic ductal adenocarcinoma (PDAC) not only fosters exceptionally aggressive tumor biology, but contradicts the current concept of one-size-fits-all therapeutic strategies to combat PDAC. Therefore, we aimed to exploit the tumor biological implication and therapeutic vulnerabilities of a clinically relevant molecular PDAC subgroup characterized by SMAD4 deficiency and high expression of the nuclear factor of activated T cells (SMAD4/NFATc1).
METHODS
Transcriptomic and clinical data were analyzed to determine the prognostic relevance of SMAD4/NFATc1 cancers. In vitro and in vivo oncogenic transcription factor complex formation was studied by immunoprecipitation, proximity ligation assays, and validated cross model and species. The impact of SMAD4 status on therapeutically targeting canonical KRAS signaling was mechanistically deciphered and corroborated by genome-wide gene expression analysis and genetic perturbation experiments, respectively. Validation of a novel tailored therapeutic option was conducted in patient-derived organoids and cells and transgenic as well as orthotopic PDAC models.
RESULTS
Our findings determined the tumor biology of an aggressive and chemotherapy-resistant SMAD4/NFATc1 subgroup. Mechanistically, we identify SMAD4 deficiency as a molecular prerequisite for the formation of an oncogenic NFATc1/SMAD3/cJUN transcription factor complex, which drives the expression of RRM1/2. RRM1/2 replenishes nucleoside pools that directly compete with metabolized gemcitabine for DNA strand incorporation. Disassembly of the NFATc1/SMAD3/cJUN complex by mitogen-activated protein kinase signaling inhibition normalizes RRM1/2 expression and synergizes with gemcitabine treatment in vivo to reduce the proliferative index.
CONCLUSIONS
Our results suggest that PDAC characterized by SMAD4 deficiency and oncogenic NFATc1/SMAD3/cJUN complex formation exposes sensitivity to a mitogen-activated protein kinase signaling inhibition and gemcitabine combination therapy.
Topics: Humans; Gemcitabine; Cell Line, Tumor; Pancreatic Neoplasms; Carcinoma, Pancreatic Ductal; Smad4 Protein; Mitogen-Activated Protein Kinases; Smad3 Protein
PubMed: 37913894
DOI: 10.1053/j.gastro.2023.10.026 -
Cell Reports Nov 2023Fibrosis, characterized by sustained activation of myofibroblasts and excessive extracellular matrix (ECM) deposition, is known to be associated with chronic...
Fibrosis, characterized by sustained activation of myofibroblasts and excessive extracellular matrix (ECM) deposition, is known to be associated with chronic inflammation. Receptor-interacting protein kinase 3 (RIPK3), the central kinase of necroptosis signaling, is upregulated in fibrosis and contributes to tumor necrosis factor (TNF)-mediated inflammation. In bile-duct-ligation-induced liver fibrosis, we found that myofibroblasts are the major cell type expressing RIPK3. Genetic ablation of β1 integrin, the major profibrotic ECM receptor in fibroblasts, not only abolished ECM fibrillogenesis but also blunted RIPK3 expression via a mechanism mediated by the chromatin-remodeling factor chromodomain helicase DNA-binding protein 4 (CHD4). While the function of CHD4 has been conventionally linked to the nucleosome-remodeling deacetylase (NuRD) and CHD4-ADNP-HP1(ChAHP) complexes, we found that CHD4 potently repressed a set of genes, including Ripk3, with high locus specificity but independent of either the NuRD or the ChAHP complex. Thus, our data uncover that β1 integrin intrinsically links fibrotic signaling to RIPK3-driven inflammation via a novel mode of action of CHD4.
Topics: Humans; Integrin beta1; Necroptosis; Mi-2 Nucleosome Remodeling and Deacetylase Complex; Transcription Factors; Nucleosomes; Fibrosis; Inflammation
PubMed: 37883227
DOI: 10.1016/j.celrep.2023.113322 -
Nature Communications Feb 2024Nonhomologous end joining (NHEJ), the primary pathway of vertebrate DNA double-strand-break (DSB) repair, directly re-ligates broken DNA ends. Damaged DSB ends that...
Nonhomologous end joining (NHEJ), the primary pathway of vertebrate DNA double-strand-break (DSB) repair, directly re-ligates broken DNA ends. Damaged DSB ends that cannot be immediately re-ligated are modified by NHEJ processing enzymes, including error-prone polymerases and nucleases, to enable ligation. However, DSB ends that are initially compatible for re-ligation are typically joined without end processing. As both ligation and end processing occur in the short-range (SR) synaptic complex that closely aligns DNA ends, it remains unclear how ligation of compatible ends is prioritized over end processing. In this study, we identify structural interactions of the NHEJ-specific DNA Ligase IV (Lig4) within the SR complex that prioritize ligation and promote NHEJ fidelity. Mutational analysis demonstrates that Lig4 must bind DNA ends to form the SR complex. Furthermore, single-molecule experiments show that a single Lig4 binds both DNA ends at the instant of SR synapsis. Thus, Lig4 is poised to ligate compatible ends upon initial formation of the SR complex before error-prone processing. Our results provide a molecular basis for the fidelity of NHEJ.
Topics: DNA Ligase ATP; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Repair; DNA Ligases; DNA
PubMed: 38341432
DOI: 10.1038/s41467-024-45553-z -
International Journal of Biochemistry... 2023Metagenomics is defined as the study of the genome of the total microbiota found in nature and is often referred to as microbial environmental genomics because it... (Review)
Review
Metagenomics is defined as the study of the genome of the total microbiota found in nature and is often referred to as microbial environmental genomics because it entails the examination of a group of genetic components (genomes) from a diverse community of organisms in a particular setting. It is a sub-branch of omics technology that encompasses Deoxyribonucleic Acid (DNA), Ribonucleic acid (DNA), proteins, and various components associated with comprehensive analysis of all aspects of biological molecules in a system-wide manner. Clustered regularly interspaced palindromic repeats and its endonuclease, CRISPR-associated protein which forms a complex called CRISPR-cas9 technology, though it is a different technique used to make precise changes to the genome of an organism, it can be used in conjunction with metagenomic approaches to give a better, rapid, and more accurate description of genomes and sequence reads. There have been ongoing improvements in sequencing that have deepened our understanding of microbial genomes forever. From the time when only a small amount of gene could be sequenced using traditional methods (e.g., "the plus and minus" method developed by Allan and Sanger and the "chemical cleavage" method that is known for its use in the sequencing the phiX174 bacteriophage genome via radio-labeled DNA polymerase-primer in a polymerization reaction aided by polyacrylamide gel) to the era of total genomes sequencing which includes "sequencing-by-ligation" and the "sequencing-by-synthesis" that detects hydrogen ions when new DNA is synthesized (Second Generation) and then Next Generation Sequencing technologies (NGS). With these technologies, the Human Genome Project (HGP) was made possible. The study looks at recent advancements in metagenomics in plants and animals by examining findings from randomly selected research papers. All selected case studies examined the functional and taxonomical analysis of different microbial communities using high-throughput sequencing to generate different sequence reads. In animals, five studies indicated how Zebrafish, Livestock, Poultry, cattle, niches, and the human microbiome were exploited using environmental samples, such as soil and water, to identify microbial communities and their functions. It has also been used to study the microbiome of humans and other organisms, including gut microbiomes. Recent studies demonstrated how these technologies have allowed for faster and more accurate identification of pathogens, leading to improved disease diagnostics. They have also enabled the development of personalized medicine by allowing for the identification of genetic variations that can impact drug efficacy and toxicity. Continued advancements in sequencing techniques and the refinement of CRISPR-Cas9 tools offer even greater potential for transformative breakthroughs in scientific research and applications. On the other hand, metagenomic data are always large and uneasy to handle. The complexity of taxonomical profiling, functional annotation, and mechanisms of complex interaction still needs better bioinformatics tools. Current review focuses on better (e.g., AI-driven algorithms) tools that can predict metabolic pathways and interactions, and manipulate complex data to address potential bias for accurate interpretation.
PubMed: 37736390
DOI: No ID Found -
Epigenetics Dec 2023Sepsis is the primary cause of acute kidney injury (AKI) and is associated with high mortality rates. Growing evidence suggests that noncoding RNAs are vitally involved...
Sepsis is the primary cause of acute kidney injury (AKI) and is associated with high mortality rates. Growing evidence suggests that noncoding RNAs are vitally involved in kidney illnesses, whereas the role of circular RNAs (circRNAs) in sepsis-induced AKI (SAKI) remains largely unknown. In this present study, caecal ligation and puncture (CLP) in mice was performed to establish an SAKI model. The expression of circRNAs and mRNAs was analysed using circRNA microarray or next-generation sequencing. The results revealed that the expressions of 197 circRNAs and 2509 mRNAs were dysregulated. Validation of the selected circRNAs was performed by qRT-PCR. Bioinformatics analyses and chromatin immunoprecipitation demonstrated that NF-κB/p65 signalling induced the upregulation of circC3, circZbtb16, and circFkbp5 and their linear counterparts by p65 transcription in mouse tubular epithelial cells (mTECs). Furthermore, competitive endogenous RNA (ceRNA) networks demonstrated that some components of NF-κB signalling were potential targets of these dysregulated circRNAs. Among them, Tnf-α was increased by circFkbp5 through the downregulation of miR-760-3p in lipopolysaccharide (LPS)-stimulated mTECs. Knocking down circFkbp5 inhibited the p65 phosphorylation and apoptosis in injured mTECs. These findings suggest that the selected circRNAs and the related ceRNA networks provide new knowledge into the fundamental mechanism of SAKI and circFkbp5/miR-760-3p/Tnf-α axis might be therapeutic targets.
Topics: Mice; Animals; RNA, Circular; MicroRNAs; NF-kappa B; Tumor Necrosis Factor-alpha; DNA Methylation; RNA, Messenger; Acute Kidney Injury; Sepsis
PubMed: 37979155
DOI: 10.1080/15592294.2023.2278960 -
Molecular Cancer Jan 2024The ATM kinase constitutes a master regulatory hub of DNA damage and activates the p53 response pathway by phosphorylating the MDM2 protein, which develops an affinity...
BACKGROUND
The ATM kinase constitutes a master regulatory hub of DNA damage and activates the p53 response pathway by phosphorylating the MDM2 protein, which develops an affinity for the p53 mRNA secondary structure. Disruption of this interaction prevents the activation of the nascent p53. The link of the MDM2 protein-p53 mRNA interaction with the upstream DNA damage sensor ATM kinase and the role of the p53 mRNA in the DNA damage sensing mechanism, are still highly anticipated.
METHODS
The proximity ligation assay (PLA) has been extensively used to reveal the sub-cellular localisation of the protein-mRNA and protein-protein interactions. ELISA and co-immunoprecipitation confirmed the interactions in vitro and in cells.
RESULTS
This study provides a novel mechanism whereby the p53 mRNA interacts with the ATM kinase enzyme and shows that the L22L synonymous mutant, known to alter the secondary structure of the p53 mRNA, prevents the interaction. The relevant mechanistic roles in the DNA Damage Sensing pathway, which is linked to downstream DNA damage response, are explored. Following DNA damage (double-stranded DNA breaks activating ATM), activated MDMX protein competes the ATM-p53 mRNA interaction and prevents the association of the p53 mRNA with NBS1 (MRN complex). These data also reveal the binding domains and the phosphorylation events on ATM that regulate the interaction and the trafficking of the complex to the cytoplasm.
CONCLUSION
The presented model shows a novel interaction of ATM with the p53 mRNA and describes the link between DNA Damage Sensing with the downstream p53 activation pathways; supporting the rising functional implications of synonymous mutations altering secondary mRNA structures.
Topics: Humans; Polynucleotide 5'-Hydroxyl-Kinase; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53; DNA Damage; DNA Repair; Ataxia Telangiectasia Mutated Proteins
PubMed: 38263180
DOI: 10.1186/s12943-024-01933-z -
Small Methods Jan 2024There have been limited efforts to ligate the staple nicks in DNA origami which is crucial for their stability against thermal and mechanical treatments, and chemical...
There have been limited efforts to ligate the staple nicks in DNA origami which is crucial for their stability against thermal and mechanical treatments, and chemical and biological environments. Here, two near quantitative ligation methods are demonstrated for the native backbone linkage at the nicks in origami: i) a cosolvent dimethyl sulfoxide (DMSO)-assisted enzymatic ligation and ii) enzyme-free chemical ligation by CNBr. Both methods achieved over 90% ligation in 2D origami, only CNBr-method resulted in ≈80% ligation in 3D origami, while the enzyme-alone yielded 31-55% (2D) or 22-36% (3D) ligation. Only CNBr-method worked efficiently for 3D origami. The CNBr-mediated reaction is completed within 5 min, while DMSO-method took overnight. Ligation by these methods improved the structural stability up to 30 °C, stability during the electrophoresis and subsequent extraction, and against nuclease and cell lysate. These methods are straightforward, non-tedious, and superior in terms of cost, reaction time, and efficiency.
Topics: Nanostructures; Dimethyl Sulfoxide; Nucleic Acid Conformation; DNA; Endonucleases
PubMed: 37736703
DOI: 10.1002/smtd.202300999 -
BioRxiv : the Preprint Server For... Aug 2023Errors in mitosis can generate micronuclei that entrap mis-segregated chromosomes, which are susceptible to catastrophic fragmentation through a process termed...
Errors in mitosis can generate micronuclei that entrap mis-segregated chromosomes, which are susceptible to catastrophic fragmentation through a process termed chromothripsis. The reassembly of fragmented chromosomes by error-prone DNA double-strand break (DSB) repair generates a spectrum of simple and complex genomic rearrangements that are associated with human cancers and disorders. How specific DSB repair pathways recognize and process these lesions remains poorly understood. Here we used CRISPR/Cas9 to systematically inactivate distinct DSB processing or repair pathways and interrogated the rearrangement landscape of fragmented chromosomes from micronuclei. Deletion of canonical non-homologous end joining (NHEJ) components, including DNA-PKcs, LIG4, and XLF, substantially reduced the formation of complex rearrangements and shifted the rearrangement landscape toward simple alterations without the characteristic patterns of cancer-associated chromothripsis. Following reincorporation into the nucleus, fragmented chromosomes localize within micronuclei bodies (MN bodies) and undergo successful ligation by NHEJ within a single cell cycle. In the absence of NHEJ, chromosome fragments were rarely engaged by polymerase theta-mediated alternative end-joining or recombination-based mechanisms, resulting in delayed repair kinetics and persistent 53BP1-labeled MN bodies in the interphase nucleus. Prolonged DNA damage signaling from unrepaired fragments ultimately triggered cell cycle arrest. Thus, we provide evidence supporting NHEJ as the exclusive DSB repair pathway generating complex rearrangements following chromothripsis from mitotic errors.
PubMed: 37609143
DOI: 10.1101/2023.08.10.552800