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Cell Death & Disease Apr 2024Acute liver failure (ALF) is a deadly illness due to insufficient detoxification in liver induced by drugs, toxins, and other etiologies, and the effective treatment for...
Acute liver failure (ALF) is a deadly illness due to insufficient detoxification in liver induced by drugs, toxins, and other etiologies, and the effective treatment for ALF is very limited. Among the drug-induced ALF, acetaminophen (APAP) overdose is the most common cause. However, the molecular mechanisms underlying APAP hepatoxicity remain incompletely understood. Sirtuin 6 (Sirt6) is a stress responsive protein deacetylase and plays an important role in regulation of DNA repair, genomic stability, oxidative stress, and inflammation. Here, we report that genetic and pharmacological activation of Sirt6 protects against ALF in mice. We first observed that Sirt6 expression was significantly reduced in the liver tissues of human patients with ALF and mice treated with an overdose of APAP. Then we developed an inducible Sirt6 transgenic mice for Cre-mediated overexpression of the human Sirt6 gene in systemic (Sirt6-Tg) and hepatic-specific (Sirt6-HepTg) manners. Both Sirt6-Tg mice and Sirt6-HepTg mice exhibited the significant protection against APAP hepatoxicity. In contrast, hepatic-specific Sirt6 knockout mice exaggerated APAP-induced liver damages. Mechanistically, Sirt6 attenuated APAP-induced hepatocyte necrosis and apoptosis through downregulation of oxidative stress, inflammation, the stress-activated kinase JNK activation, and apoptotic caspase activation. Moreover, Sirt6 negatively modulated the level and activity of poly (ADP-ribose) polymerase 1 (PARP1) in APAP-treated mouse liver tissues. Importantly, the specific Sirt6 activator MDL-800 exhibited better therapeutic potential for APAP hepatoxicity than the current drug acetylcysteine. Furthermore, in the model of bile duct ligation induced ALF, hepatic Sirt6-KO exacerbated, but Sirt6-HepTg mitigated liver damage. Collectively, our results demonstrate that Sirt6 protects against ALF and suggest that targeting Sirt6 activation could be a new therapeutic strategy to alleviate ALF.
Topics: Animals; Humans; Male; Mice; Acetaminophen; Apoptosis; Hepatocytes; Liver; Liver Failure, Acute; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Oxidative Stress; Sirtuins
PubMed: 38649362
DOI: 10.1038/s41419-024-06537-5 -
Nature Communications Apr 2024Recurrent DNA break clusters (RDCs) are replication-transcription collision hotspots; many are unique to neural progenitor cells. Through high-resolution replication...
Recurrent DNA break clusters (RDCs) are replication-transcription collision hotspots; many are unique to neural progenitor cells. Through high-resolution replication sequencing and a capture-ligation assay in mouse neural progenitor cells experiencing replication stress, we unravel the replication features dictating RDC location and orientation. Most RDCs occur at the replication forks traversing timing transition regions (TTRs), where sparse replication origins connect unidirectional forks. Leftward-moving forks generate telomere-connected DNA double-strand breaks (DSBs), while rightward-moving forks lead to centromere-connected DSBs. Strand-specific mapping for DNA-bound RNA reveals co-transcriptional dual-strand DNA:RNA hybrids present at a higher density in RDC than in other actively transcribed long genes. In addition, mapping RNA polymerase activity uncovers that head-to-head interactions between replication and transcription machinery result in 60% DSB contribution to the head-on compared to 40% for co-directional. Taken together we reveal TTR as a fragile class and show how the linear interaction between transcription and replication impacts genome stability.
Topics: Animals; Transcription, Genetic; DNA Breaks, Double-Stranded; Mice; DNA Replication; Genomic Instability; Neural Stem Cells; DNA; Replication Origin; Telomere; Centromere
PubMed: 38678011
DOI: 10.1038/s41467-024-47934-w -
Proceedings of the National Academy of... Aug 2023Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, uses an RNA-dependent RNA polymerase along with several accessory factors...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, uses an RNA-dependent RNA polymerase along with several accessory factors to replicate its genome and transcribe its genes. Nonstructural protein (nsp) 13 is a helicase required for viral replication. Here, we found that nsp13 ligates iron, in addition to zinc, when purified anoxically. Using inductively coupled plasma mass spectrometry, UV-visible absorption, EPR, and Mössbauer spectroscopies, we characterized nsp13 as an iron-sulfur (Fe-S) protein that ligates an FeS cluster in the treble-clef metal-binding site of its zinc-binding domain. The Fe-S cluster in nsp13 modulates both its binding to the template RNA and its unwinding activity. Exposure of the protein to the stable nitroxide TEMPOL oxidizes and degrades the cluster and drastically diminishes unwinding activity. Thus, optimal function of nsp13 depends on a labile Fe-S cluster that is potentially targetable for COVID-19 treatment.
Topics: Humans; SARS-CoV-2; COVID-19 Drug Treatment; COVID-19; DNA Helicases; RNA; Sulfur; Viral Nonstructural Proteins; RNA Helicases
PubMed: 37552760
DOI: 10.1073/pnas.2303860120 -
Frontiers in Bioengineering and... 2023Molecular cloning is used in a wide variety of biological and medical research. Here, we developed a rapid and efficient DNA-assembling method for routine laboratory...
Molecular cloning is used in a wide variety of biological and medical research. Here, we developed a rapid and efficient DNA-assembling method for routine laboratory work. We discovered that the cleavage speed of T5 exonuclease is approximately 3 nt/min at 0°C and hence developed a T5 exonuclease-mediated low-temperature sequence- and ligation-independent cloning method (TLTC). Two homologous regions of 15 bp-25 bp compatible with the ends of the vector backbones were introduced into the inserts through PCR. Approximately 120 fmol of inserts and linear vectors was mixed at a molar ratio of approximately 3:1 and treated with 0.5 U of T5 exonuclease at 0°C for 5 min. Then, the mixture was transformed into to generate recombinant plasmids. Single segment and multi-segments can be assembled efficiently using TLTC. For single segment, the overall cloning efficiency is above 95%. Moreover, extra nucleotides in the vectors can be removed during TLTC. In conclusion, an extremely simple and fast DNA cloning/assembling method was established in the present study. This method facilitates routine DNA cloning and synthesis of DNA fragments.
PubMed: 37635997
DOI: 10.3389/fbioe.2023.1167534 -
Journal of Molecular Histology Feb 2024Sepsis has a systemic inflammatory response syndrome caused by infection. While neutrophils play contradictory roles in different stages of sepsis. Neutrophils have been...
Sepsis has a systemic inflammatory response syndrome caused by infection. While neutrophils play contradictory roles in different stages of sepsis. Neutrophils have been proven to play an antibacterial role by producing neutrophil extracellular traps (NETs). Although the NET is beneficial to bacteria resistance, abnormal NET increases tissue damage. The complement C5a receptor 1 (C5ar1) is a gene related to strong inflammatory reactions and is found to be associated with inflammatory factors. This study found that there were 45 down-regulated genes and 704 up-regulated genes in sepsis rats by transcriptome sequencing. And those genes were significantly related to inflammation and immunity by GO and KEGG enrichment analysis involving the chemokine signaling pathway, the Toll-like receptor (TLR) signaling pathway, and the Fc gamma R-mediated phagocytosis. Additionally, the C5ar1 gene was significantly upregulated with interesting potential in sepsis and used for further study. This study used cecum ligation and puncture (CLP) rats that were respectively injected intravenously with PBS or the lentivirus vector to explore the effect of C5ar1 on CLP rats. It demonstrated that silenced- C5ar1 inhibited the ALT, AST, BUN, and CREA levels, improved the lung and spleen injury, and reduced the TNF-α, IL-6, IL-1β, IL-10, cf-DNA, and cfDNA/MPO levels. Additionally, silenced C5ar1 inhibited the TLR2, TLR4, and peptidylarginine deiminase 4 expression levels, which suggested the improvement of silenced C5ar1 on sepsis via inhibiting NETs and the TLR signaling pathway. This study provides a basis and new direction for the study of treatment on sepsis.
Topics: Rats; Animals; Extracellular Traps; Neutrophils; Inflammation; Lung; Sepsis
PubMed: 38165570
DOI: 10.1007/s10735-023-10172-3 -
Nature Communications Nov 2023The blood proteome holds great promise for precision medicine but poses substantial challenges due to the low abundance of most plasma proteins and the vast dynamic...
The blood proteome holds great promise for precision medicine but poses substantial challenges due to the low abundance of most plasma proteins and the vast dynamic range of the plasma proteome. Here we address these challenges with NUcleic acid Linked Immuno-Sandwich Assay (NULISA™), which improves the sensitivity of traditional proximity ligation assays by ~10,000-fold to attomolar level, by suppressing assay background via a dual capture and release mechanism built into oligonucleotide-conjugated antibodies. Highly multiplexed quantification of both low- and high-abundance proteins spanning a wide dynamic range is achieved by attenuating signals from abundant targets with unconjugated antibodies and next-generation sequencing of barcoded reporter DNA. A 200-plex NULISA containing 124 cytokines and chemokines and other proteins demonstrates superior sensitivity to a proximity extension assay in detecting biologically important low-abundance biomarkers in patients with autoimmune diseases and COVID-19. Fully automated NULISA makes broad and in-depth proteomic analysis easily accessible for research and diagnostic applications.
Topics: Humans; Proteomics; Proteome; Blood Proteins; Antibodies; Cytokines
PubMed: 37945559
DOI: 10.1038/s41467-023-42834-x -
International Journal of Molecular... Sep 2023Approximately 20-30% of endometrial carcinomas (EC) are characterized by mismatch repair (MMR) deficiency (dMMR) or microsatellite instability (MSI), and their testing...
Approximately 20-30% of endometrial carcinomas (EC) are characterized by mismatch repair (MMR) deficiency (dMMR) or microsatellite instability (MSI), and their testing has become part of the routine diagnosis. The aim of this study was to establish and compare the MMR status using various approaches. Immunohistochemistry (IHC), PCR-based MSI, and the detection of defects in the four key MMR genes (MLH1, PMS2, MSH2, and MSH6) via methylation-specific multiplex ligation-dependent probe amplification (MLPA) and targeted next-generation sequencing (NGS) were performed. MSH3 expression was also evaluated. A set of 126 early-stage EC samples were analyzed, 53.2% of which were dMMR and 46.8% of which were proficient MMR (pMMR) as determined using IHC, whereas 69.3% were classified as microsatellite stable, while 8.8% and 21.9% were classified MSI-low (MSI-L) and MSI-high (MSI-H), respectively. In total, 44.3% of the samples showed genetic or epigenetic alterations in one or more genes; MLH1 promoter methylation was the most common event. Although acceptable concordance was observed, there were overall discrepancies between the three testing approaches, mainly associated with the dMMR group. IHC had a better correlation with MMR genomic status than the MSI status determined using PCR. Further studies are needed to establish solid conclusions regarding the best MMR assessment technique for EC.
Topics: Female; Humans; DNA Mismatch Repair; Endometrial Neoplasms; Colorectal Neoplasms; Neoplastic Syndromes, Hereditary; Microsatellite Instability
PubMed: 37833916
DOI: 10.3390/ijms241914468 -
Genes To Cells : Devoted To Molecular &... Aug 2023The cloning of DNA fragments to plasmid vectors is at the heart of molecular biology. Recent developments have led to various methods utilizing homologous recombination...
The cloning of DNA fragments to plasmid vectors is at the heart of molecular biology. Recent developments have led to various methods utilizing homologous recombination of homology arms. Among them, Seamless Ligation Cloning Extract (SLiCE) is an affordable alternative solution that uses simple Escherichia coli lysates. However, the underlying molecular mechanisms remain unclear and the reconstitution of the extract by defined factors has not yet been reported. We herein show that the key factor in SLiCE is Exonuclease III (ExoIII), a double-strand (ds) DNA-dependent 3'-5' exonuclease, encoded by XthA. SLiCE prepared from the xthAΔ strain is devoid of recombination activity, whereas purified ExoIII alone is sufficient to assemble two blunt-ended dsDNA fragments with homology arms. In contrast to SLiCE, ExoIII is unable to digest (or assemble) fragments with 3' protruding ends; however, the addition of single-strand DNA-targeting Exonuclease T overcomes this issue. Through the combination of commercially available enzymes under optimized conditions, we achieved the efficient, reproducible, and affordable cocktail, "XE cocktail," for seamless DNA cloning. By reducing the cost and time required for DNA cloning, researchers will devote more resources to advanced studies and the careful validation of their own findings.
Topics: Cloning, Molecular; DNA; Escherichia coli; Homologous Recombination; DNA, Single-Stranded; Plasmids
PubMed: 37132531
DOI: 10.1111/gtc.13034 -
Nature Protocols Jun 2024Microbial split-pool ligation transcriptomics (microSPLiT) is a high-throughput single-cell RNA sequencing method for bacteria. With four combinatorial barcoding rounds,... (Review)
Review
Microbial split-pool ligation transcriptomics (microSPLiT) is a high-throughput single-cell RNA sequencing method for bacteria. With four combinatorial barcoding rounds, microSPLiT can profile transcriptional states in hundreds of thousands of Gram-negative and Gram-positive bacteria in a single experiment without specialized equipment. As bacterial samples are fixed and permeabilized before barcoding, they can be collected and stored ahead of time. During the first barcoding round, the fixed and permeabilized bacteria are distributed into a 96-well plate, where their transcripts are reverse transcribed into cDNA and labeled with the first well-specific barcode inside the cells. The cells are mixed and redistributed two more times into new 96-well plates, where the second and third barcodes are appended to the cDNA via in-cell ligation reactions. Finally, the cells are mixed and divided into aliquot sub-libraries, which can be stored until future use or prepared for sequencing with the addition of a fourth barcode. It takes 4 days to generate sequencing-ready libraries, including 1 day for collection and overnight fixation of samples. The standard plate setup enables single-cell transcriptional profiling of up to 1 million bacterial cells and up to 96 samples in a single barcoding experiment, with the possibility of expansion by adding barcoding rounds. The protocol requires experience in basic molecular biology techniques, handling of bacterial samples and preparation of DNA libraries for next-generation sequencing. It can be performed by experienced undergraduate or graduate students. Data analysis requires access to computing resources, familiarity with Unix command line and basic experience with Python or R.
PubMed: 38886529
DOI: 10.1038/s41596-024-01007-w -
ELife Apr 2024Poly(ADP-ribose)ylation or PARylation by PAR polymerase 1 (PARP1) and dePARylation by poly(ADP-ribose) glycohydrolase (PARG) are equally important for the dynamic...
Poly(ADP-ribose)ylation or PARylation by PAR polymerase 1 (PARP1) and dePARylation by poly(ADP-ribose) glycohydrolase (PARG) are equally important for the dynamic regulation of DNA damage response. PARG, the most active dePARylation enzyme, is recruited to sites of DNA damage via pADPr-dependent and PCNA-dependent mechanisms. Targeting dePARylation is considered an alternative strategy to overcome PARP inhibitor resistance. However, precisely how dePARylation functions in normal unperturbed cells remains elusive. To address this challenge, we conducted multiple CRISPR screens and revealed that dePARylation of S phase pADPr by PARG is essential for cell viability. Loss of dePARylation activity initially induced S-phase-specific pADPr signaling, which resulted from unligated Okazaki fragments and eventually led to uncontrolled pADPr accumulation and PARP1/2-dependent cytotoxicity. Moreover, we demonstrated that proteins involved in Okazaki fragment ligation and/or base excision repair regulate pADPr signaling and cell death induced by PARG inhibition. In addition, we determined that PARG expression is critical for cellular sensitivity to PARG inhibition. Additionally, we revealed that PARG is essential for cell survival by suppressing pADPr. Collectively, our data not only identify an essential role for PARG in normal proliferating cells but also provide a potential biomarker for the further development of PARG inhibitors in cancer therapy.
Topics: Cell Survival; S Phase; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerase Inhibitors; Antineoplastic Agents
PubMed: 38578205
DOI: 10.7554/eLife.89303