1,164 results on '"Micrococcal Nuclease metabolism"'
Search Results
2. A fluorogenic micrococcal nuclease-based probe for fast detection and optical imaging of Staphylococcus aureus in prosthetic joint and fracture-related infections.
- Author
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Schoenmakers JWA, López-Álvarez M, IJpma FFA, Wouthuyzen-Bakker M, McNamara JO 2nd, van Oosten M, Jutte PC, and van Dijl JM
- Subjects
- Humans, Optical Imaging methods, Staphylococcal Infections diagnostic imaging, Fractures, Bone diagnostic imaging, Time Factors, Female, Staphylococcus aureus isolation & purification, Fluorescent Dyes chemistry, Biofilms, Prosthesis-Related Infections diagnostic imaging, Prosthesis-Related Infections microbiology, Synovial Fluid microbiology, Micrococcal Nuclease metabolism
- Abstract
Purpose: Staphylococcus aureus is the most common and impactful multi-drug resistant pathogen implicated in (periprosthetic) joint infections (PJI) and fracture-related infections (FRI). Therefore, the present proof-of-principle study was aimed at the rapid detection of S. aureus in synovial fluids and biofilms on extracted osteosynthesis materials through bacteria-targeted fluorescence imaging with the 'smart-activatable' DNA-based AttoPolyT probe. This fluorogenic oligonucleotide probe yields large fluorescence increases upon cleavage by micrococcal nuclease, an enzyme secreted by S. aureus., Methods: Synovial fluids from patients with suspected PJI and extracted osteosynthesis materials from trauma patients with suspected FRI were inspected for S. aureus nuclease activity with the AttoPolyT probe. Biofilms on osteosynthesis materials were imaged with the AttoPolyT probe and a vancomycin-IRDye800CW conjugate (vanco-800CW) specific for Gram-positive bacteria., Results: 38 synovial fluid samples were collected and analyzed. Significantly higher fluorescence levels were measured for S. aureus-positive samples compared to, respectively, other Gram-positive bacterial pathogens (p < 0.0001), Gram-negative bacterial pathogens (p = 0.0038) and non-infected samples (p = 0.0030), allowing a diagnosis of S. aureus-associated PJI within 2 h. Importantly, S. aureus-associated biofilms on extracted osteosynthesis materials from patients with FRI were accurately imaged with the AttoPolyT probe, allowing their correct distinction from biofilms formed by other Gram-positive bacteria detected with vanco-800CW within 15 min., Conclusion: The present study highlights the potential clinical value of the AttoPolyT probe for fast and accurate detection of S. aureus infection in synovial fluids and biofilms on extracted osteosynthesis materials., (© 2023. The Author(s).)
- Published
- 2024
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3. nucMACC: An MNase-seq pipeline to identify structurally altered nucleosomes in the genome.
- Author
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Wernig-Zorc S, Kugler F, Schmutterer L, Räß P, Hausmann C, Holzinger S, Längst G, and Schwartz U
- Subjects
- Animals, Chromatin Assembly and Disassembly, Genome, Promoter Regions, Genetic, RNA Polymerase II metabolism, RNA Polymerase II genetics, Chromatin genetics, Chromatin metabolism, Sequence Analysis, DNA methods, Nucleosomes metabolism, Nucleosomes genetics, Micrococcal Nuclease metabolism, Drosophila melanogaster genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism
- Abstract
Micrococcal nuclease sequencing is the state-of-the-art method for determining chromatin structure and nucleosome positioning. Data analysis is complex due to the AT-dependent sequence bias of the endonuclease and the requirement for high sequencing depth. Here, we present the nucleosome-based MNase accessibility (nucMACC) pipeline unveiling the regulatory chromatin landscape by measuring nucleosome accessibility and stability. The nucMACC pipeline represents a systematic and genome-wide approach for detecting unstable ("fragile") nucleosomes. We have characterized the regulatory nucleosome landscape in Drosophila melanogaster , Saccharomyces cerevisiae , and mammals. Two functionally distinct sets of promoters were characterized, one associated with an unstable nucleosome and the other being nucleosome depleted. We show that unstable nucleosomes present intermediate states of nucleosome remodeling, preparing inducible genes for transcriptional activation in response to stimuli or stress. The presence of unstable nucleosomes correlates with RNA polymerase II proximal pausing. The nucMACC pipeline offers unparalleled precision and depth in nucleosome research and is a valuable tool for future nucleosome studies.
- Published
- 2024
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4. Engineering the bacteriophage 80 alpha endolysin as a fast and ultrasensitive detection toolbox against Staphylococcus aureus.
- Author
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Zhao F, Yang Y, Zhan W, Li Z, Yin H, Deng J, Li W, Li R, Zhao Q, and Li J
- Subjects
- Bacteriophages chemistry, Bacteriophages genetics, Bacteriophages isolation & purification, Humans, Staphylococcus Phages genetics, Staphylococcus Phages chemistry, Staphylococcus Phages isolation & purification, Animals, Nucleic Acid Amplification Techniques methods, Staphylococcal Infections microbiology, DNA, Bacterial genetics, DNA, Bacterial isolation & purification, Micrococcal Nuclease chemistry, Micrococcal Nuclease metabolism, Micrococcal Nuclease genetics, Viral Proteins chemistry, Viral Proteins metabolism, Staphylococcus aureus isolation & purification, Staphylococcus aureus virology, Biosensing Techniques methods, Endopeptidases chemistry, Endopeptidases isolation & purification, Endopeptidases genetics
- Abstract
The isolation and identification of pathogenic bacteria from a variety of samples are critical for controlling bacterial infection-related health problems. The conventional methods, such as plate counting and polymerase chain reaction-based approaches, tend to be time-consuming and reliant on specific instruments, severely limiting the effective identification of these pathogens. In this study, we employed the specificity of the cell wall-binding (CBD) domain of the Staphylococcus aureus bacteriophage 80 alpha (80α) endolysin towards the host bacteria for isolation. Amidase 3-CBD conjugated magnetic beads successfully isolated as few as 1 × 10
2 CFU/mL of S. aureus cells from milk, blood, and saliva. The cell wall hydrolyzing activity of 80α endolysin promoted the genomic DNA extraction efficiency by 12.7 folds on average, compared to the commercial bacterial genomic DNA extraction kit. Then, recombinase polymerase amplification (RPA) was exploited to amplify the nuc gene of S. aureus from the extracted DNA at 37 °C for 30 min. The RPA product activated Cas12a endonuclease activity to cleave fluorescently labeled ssDNA probes. We then converted the generated signal into a fluorescent readout, detectable by either the naked eye or a portable, self-assembled instrument with ultrasensitivity. The entire procedure, from isolation to identification, can be completed within 2 h. The simplicity and sensitivity of the method developed in this study make it of great application value in S. aureus detection, especially in areas with limited resource supply., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)- Published
- 2024
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5. Napthalimide-based nuclease inhibitor: A multifunctional therapeutic material to bolster MRSA uptake by macrophage-like cells and mitigate pathogen adhesion on orthopaedic implant.
- Author
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Konwar B, De S, Das G, and Ramesh A
- Subjects
- Humans, Macrophages drug effects, Macrophages metabolism, Macrophages microbiology, Bacterial Adhesion drug effects, Biofilms drug effects, Micrococcal Nuclease metabolism, Anti-Bacterial Agents pharmacology, Anti-Bacterial Agents chemistry, Prostheses and Implants microbiology, Enzyme Inhibitors pharmacology, Enzyme Inhibitors chemistry, THP-1 Cells, Staphylococcal Infections drug therapy, Poloxamer chemistry, Poloxamer pharmacology, Methicillin-Resistant Staphylococcus aureus drug effects
- Abstract
The healthcare burden rendered by methicillin-resistant Staphylococcus aureus (MRSA) warrants the development of therapeutics that offer a distinct benefit in the clinics as compared to conventional antibiotics. The present study describes the potential of napthalimide-based synthetic ligands (C1-C3) as inhibitors of the staphylococcal nuclease known as micrococcal nuclease (MNase), a key virulence factor of the pathogen. Amongst the ligands, the most potent MNase inhibitor C1 rendered non-competitive inhibition, reduced MNase turnover number (K
cat ) and catalytic efficiency (Kcat /Km ) with an IC50 value of ~950 nM. CD spectroscopy suggested distortion of MNase conformation in presence of C1. Flow cytometry and confocal microscopy indicated that C1 restored the ability of activated THP-1 cells to engulf DNA-entrapped MRSA cells. Interestingly, C1 could inhibit MRSA adhesion onto collagen. For potential application, C1-loaded pluronic F-127 micellar nanocarrier (C1-PMC) was generated, wherein the anti-adhesion activity of the pluronic carrier (PMC) and C1 was harnessed in tandem to deter MRSA cell adhesion onto collagen. MRSA biofilm formation was hindered on C1-PMC-coated titanium (Ti) wire, while eluates from C1-PMC-coated Ti wires were non-toxic to HEK 293, MG-63 and THP-1 cells. The multifunctional C1 provides a blueprint for designing therapeutic materials that hold translational potential for mitigation of MRSA infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)- Published
- 2024
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6. Simultaneous Dual-Gene Test of Methicillin-Resistant Staphylococcus Aureus using an Argonaute-Centered Portable and Visual Biosensor.
- Author
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Kou J, Li Y, Zhao Z, Qiao J, Zhang Q, Han X, Cheng X, Man S, and Ma L
- Subjects
- Argonaute Proteins metabolism, Argonaute Proteins genetics, Bacterial Proteins genetics, Bacterial Proteins metabolism, Penicillin-Binding Proteins genetics, Animals, Micrococcal Nuclease metabolism, Micrococcal Nuclease genetics, Methicillin-Resistant Staphylococcus aureus genetics, Methicillin-Resistant Staphylococcus aureus isolation & purification, Biosensing Techniques methods, Nucleic Acid Amplification Techniques methods
- Abstract
The development of novel method for drug-resistant bacteria detection is imperative. A simultaneous dual-gene Test of methicillin-resistant Staphylococcus aureus (MRSA) is developed using an Argonaute-centered portable biosensor (STAR). This is the first report concerning Argonaute-based pathogenic bacteria detection. Simply, the species-specific mecA and nuc gene are isothermally amplified using loop-mediated isothermal amplification (LAMP) technique, followed by Argonaute-based detection enabled by its programmable, guided, sequence-specific recognition and cleavage. With the strategy, the targeted nucleic acid signals gene are dexterously converted into fluorescent signals. STAR is capable of detecting the nuc gene and mecA gene simultaneously in a single reaction. The limit of detection is 10 CFU/mL with a dynamic range from 10 to 10
7 CFU/mL. The sample-to-result time is <65 min. This method is successfully adapted to detect clinical samples, contaminated foods, and MRSA-infected animals. This work broadens the reach of Argonaute-based biosensing and presents a novel bacterial point-of-need (PON) detection platform., (© 2024 Wiley‐VCH GmbH.)- Published
- 2024
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7. Micrococcal nuclease regulates biofilm formation and dispersal in methicillin-resistant Staphylococcus aureus USA300.
- Author
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Kaplan JB and Horswill AR
- Subjects
- Anti-Bacterial Agents pharmacology, Bacterial Proteins genetics, Bacterial Proteins metabolism, DNA, Bacterial genetics, Virulence Factors genetics, Microbial Sensitivity Tests, Amoxicillin pharmacology, Biofilms growth & development, Biofilms drug effects, Methicillin-Resistant Staphylococcus aureus genetics, Methicillin-Resistant Staphylococcus aureus drug effects, Methicillin-Resistant Staphylococcus aureus physiology, Micrococcal Nuclease genetics, Micrococcal Nuclease metabolism
- Abstract
Biofilm formation is an important virulence factor for methicillin-resistant Staphylococcus aureus (MRSA). The extracellular matrix of MRSA biofilms contains significant amounts of double-stranded DNA that hold the biofilm together. MRSA cells secrete micrococcal nuclease (Nuc1), which degrades double-stranded DNA. In this study, we used standard methodologies to investigate the role of Nuc1 in MRSA biofilm formation and dispersal. We quantified biofilm formation and extracellular DNA (eDNA) levels in broth and agar cultures. In some experiments, cultures were supplemented with sub-MIC amoxicillin to induce biofilm formation. Biofilm erosion was quantitated by culturing biofilms on rods and enumerating detached colony-forming units (CFUs), and biofilm sloughing was investigated by perfusing biofilms cultured in glass tubes with fresh broth and measuring the sizes of the detached cell aggregates. We found that an MRSA nuc1
- mutant strain produced significantly more biofilm and more eDNA than a wild-type strain, both in the absence and presence of sub-MIC amoxicillin. nuc1- mutant biofilms grown on rods detached significantly less than wild-type biofilms. Detachment was restored by exogenous DNase or complementing the nuc1- mutant. In the sloughing assay, nuc1- mutant biofilms released cell aggregates that were significantly larger than those released by wild-type biofilms. Our results suggest that Nuc1 modulates biofilm formation, biofilm detachment, and the sizes of detached cell aggregates. These processes may play a role in the spread and subsequent survival of MRSA biofilms during biofilm-related infections.IMPORTANCEInfections caused by antibiotic-resistant bacteria known as methicillin-resistant Staphylococcus aureus (MRSA) are a significant problem in hospitals. MRSA forms adherent biofilms on implanted medical devices such as catheters and breathing tubes. Bacteria can detach from biofilms on these devices and spread to other parts of the body such as the blood or lungs, where they can cause life-threatening infections. In this article, researchers show that MRSA secretes an enzyme known as thermonuclease that causes bacteria to detach from the biofilm. This is important because understanding the mechanism by which MRSA detaches from biofilms could lead to the development of procedures to mitigate the problem., Competing Interests: The authors declare no conflict of interest.- Published
- 2024
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8. C2c: Predicting Micro-C from Hi-C.
- Author
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Zhu H, Liu T, and Wang Z
- Subjects
- Humans, Computational Biology methods, Neural Networks, Computer, Micrococcal Nuclease metabolism, Micrococcal Nuclease genetics, Nucleosomes genetics, Software, Chromatin genetics
- Abstract
Motivation: High-resolution Hi-C data, capable of detecting chromatin features below the level of Topologically Associating Domains (TADs), significantly enhance our understanding of gene regulation. Micro-C, a variant of Hi-C incorporating a micrococcal nuclease (MNase) digestion step to examine interactions between nucleosome pairs, has been developed to overcome the resolution limitations of Hi-C. However, Micro-C experiments pose greater technical challenges compared to Hi-C, owing to the need for precise MNase digestion control and higher-resolution sequencing. Therefore, developing computational methods to derive Micro-C data from existing Hi-C datasets could lead to better usage of a large amount of existing Hi-C data in the scientific community and cost savings., Results: We developed C2c ("high" or upper case C to "micro" or lower case c), a computational tool based on a residual neural network to learn the mapping between Hi-C and Micro-C contact matrices and then predict Micro-C contact matrices based on Hi-C contact matrices. Our evaluation results show that the predicted Micro-C contact matrices reveal more chromatin loops than the input Hi-C contact matrices, and more of the loops detected from predicted Micro-C match the promoter-enhancer interactions. Furthermore, we found that the mutual loops from real and predicted Micro-C better match the ChIA-PET data compared to Hi-C and real Micro-C loops, and the predicted Micro-C leads to more TAD-boundaries detected compared to the Hi-C data. The website URL of C2c can be found in the Data Availability Statement.
- Published
- 2024
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9. Inhibition of staphylococcal nuclease by benzimidazole-based Ligand: Implications in DNA-Mediated entrapment and uptake of MRSA by Macrophage-like cells.
- Author
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Konwar B, De S, Das G, and Ramesh A
- Subjects
- Micrococcal Nuclease analysis, Micrococcal Nuclease chemistry, Micrococcal Nuclease metabolism, Ligands, DNA chemistry, Macrophages metabolism, Benzimidazoles pharmacology, Methicillin-Resistant Staphylococcus aureus
- Abstract
The staphylococcal nuclease also referred as micrococcal nuclease (MNase) is a key drug target as the enzyme degrades the neutrophil extracellular trap (NET) and empowers the pathogen to subvert the host innate immune system. To this end, the current study presents a critical evaluation of MNase inhibition rendered by benzimidazole-based ligands (C1 and C2) and probes its therapeutic implications. A nuclease assay indicated that MNase inhibition rendered by C1 and C2 was ∼ 55 % and ∼ 72 %, respectively, at the highest tested concentration of 10 µM. Studies on enzyme kinetics revealed that C2 rendered non-competitive inhibition and significantly reduced MNase turnover number (K
cat ) and catalytic efficiency (Kcat /Km ) with an IC50 value of ∼ 1122 nM. In CD spectroscopy, a notable perturbation in the β-sheet content of MNase was observed in presence of C2. Fluorescence-microscope analysis indicated that MNase inhibition by C2 could restore entrapment of methicillin-resistant Staphylococcus aureus (MRSA) in calf-thymus DNA (CT-DNA). Flow cytometry and confocal microscope analysis revealed that uptake of DNA-entrapped MRSA by activated THP-1 cells was reinstated by MNase inhibition rendered by C2. Inhibition of nuclease by the non-toxic ligand C2 holds therapeutic prospect as it has the potential to bolster the DNA-mediated entrapment machinery and mitigate MRSA infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)- Published
- 2024
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10. MNase-Seq Analysis for Identifying Stress-Altered Nucleosome Occupancy in Plants.
- Author
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Watkins C, Willyerd KL, Liao CP, Ruhl DR, and Chen C
- Subjects
- Droughts, Gene Expression Regulation, Plant, Chromatin metabolism, Chromatin genetics, Plants genetics, Plants metabolism, Nucleosomes metabolism, Nucleosomes genetics, Micrococcal Nuclease metabolism, High-Throughput Nucleotide Sequencing methods, Stress, Physiological genetics, Triticum genetics, Triticum metabolism
- Abstract
Nucleosome occupancy plays an important role in chromatin compaction, affecting biological processes by hampering the binding of cis-acting elements such as transcription factors, RNA polymerase machinery, and coregulatory. Accessible regions allow for cis-acting elements to bind DNA and regulate transcription. Here, we detail our protocol to profile nucleosome occupancy and chromatin structure dynamics under drought stress at the genome-wide scale using micrococcal nuclease (MNase) digestion. Combining variable MNase concentration treatments and high-throughput sequencing, we investigate the changes in the overall chromatin state using bread wheat samples from an exemplary drought experiment., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2024
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11. ChEC-Seq: A Comprehensive Guide for Scalable and Cost-Efficient Genome-Wide Profiling in Saccharomyces cerevisiae.
- Author
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Gera T, Kumar DK, Yaakov G, Barkai N, and Jonas F
- Subjects
- Chromatin genetics, Chromatin metabolism, Binding Sites, Sequence Analysis, DNA methods, Micrococcal Nuclease metabolism, Micrococcal Nuclease genetics, Computational Biology methods, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, High-Throughput Nucleotide Sequencing methods, Genome, Fungal
- Abstract
Chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq) is a profiling method for protein-DNA interactions that can detect binding locations in vivo, does not require antibodies or fixation, and provides genome-wide coverage at near nucleotide resolution.The core of this method is an MNase fusion of the target protein, which allows it, when triggered by calcium exposure, to cut DNA at its binding sites and to generate small DNA fragments that can be readily separated from the rest of the genome and sequenced.Improvements since the original protocol have increased the ease, lowered the costs, and multiplied the throughput of this method to enable a scale and resolution of experiments not available with traditional methods such as ChIP-seq. This method describes each step from the initial creation and verification of the MNase-tagged yeast strains, over the ChEC MNase activation and small fragment purification procedure to the sequencing library preparation. It also briefly touches on the bioinformatic steps necessary to create meaningful genome-wide binding profiles., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2024
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12. Analysis of HBV cccDNA Minichromosome Accessibility by MNase-qPCR and High-Throughput Sequencing.
- Author
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Hu K, Zai W, Wang Y, Ye J, Yuan Z, and Chen J
- Subjects
- Humans, Real-Time Polymerase Chain Reaction methods, Micrococcal Nuclease metabolism, Micrococcal Nuclease genetics, Hepatitis B virus genetics, High-Throughput Nucleotide Sequencing methods, DNA, Circular genetics, DNA, Viral genetics
- Abstract
The covalently closed circular DNA (cccDNA) of the hepatitis B virus (HBV) is organized as a minichromosome structure in the nucleus of infected hepatocytes and considered the major obstacle to the discovery of a cure for HBV. Until now, no strategies directly targeting cccDNA have been advanced to clinical stages as much is unknown about the accessibility and activity regulation of the cccDNA minichromosome. We have described the method for evaluation of the cccDNA minichromosome accessibility using micrococcal nuclease-quantitative polymerase chain reaction and high-throughput sequencing, which could be useful tools for cccDNA research and HBV cure studies., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2024
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13. Hsa_circ_0001583 fuels bladder cancer metastasis by promoting staphylococcal nuclease and tudor domain containing 1-mediated MicroRNA decay.
- Author
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Liu C, Cong Y, Chen L, Lv F, Cheng L, Song Y, and Xing Y
- Subjects
- Humans, RNA, Circular genetics, RNA, Circular metabolism, Micrococcal Nuclease genetics, Micrococcal Nuclease metabolism, Tudor Domain, Biomarkers, Tumor genetics, Cell Proliferation, Cell Movement genetics, Cell Line, Tumor, Gene Expression Regulation, Neoplastic, Membrane Proteins genetics, ADAM Proteins genetics, ADAM Proteins metabolism, Endonucleases genetics, Endonucleases metabolism, MicroRNAs genetics, MicroRNAs metabolism, Urinary Bladder Neoplasms genetics
- Abstract
Muscle-invasive and metastatic bladder cancer indicates extra worse prognosis. Accumulating evidence roots for the prominent role of circular RNAs(circRNAs) in bladder cancer, while the mechanisms linking circRNAs and bladder cancer metastasis remain limitedly investigated. Here, we identified a significantly upregulated circRNA candidate, hsa_circ_0001583, from online datasets. Validated by qRT-PCR, PCR, sanger sequencing, actinomycin D and RNase R digestion experiments, hsa_circ_0001583 was proved to be a genuine circular RNA with higher expression levels in bladder cancer tissue. Through gain and loss of function experiments, hsa_circ_0001583 exhibited potent migration and invasion powers both in vitro and in vivo. The staphylococcal nuclease and Tudor domain containing 1 (SND1) was identified as an authentic binding partner for hsa_circ_0001583 through RNA pulldown and RIP experiments. Elevated levels of hsa_circ_0001583 could bind more to SND1 and protect the latter from degradation. Rescue experiments demonstrated that such interaction-induced increased in SND1 levels in bladder cancer cells enabled the protein to pump its endonuclease activity, leading to the degradation of tumor-suppressing MicroRNAs (miRNAs) including miR-126-3p, the suppressor of Disintegrin And Metalloproteinase Domain-Containing Protein 9 (ADAM9), ultimately driving cells into a highly migrative and invasive state. In summary, our study is the first to highlight the upregulation of hsa_circ_0001583 in bladder cancer and its role in downregulating miR-126-3p by binding to and stabilizing the SND1 protein, thereby promoting bladder cancer cell migration and invasion. This study adds hsa_circ_0001583 to the pool of bladder cancer metastasis biomarkers and therapeutic targets., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)
- Published
- 2024
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14. Improving genome-wide mapping of nucleosomes in Trypanosome cruzi.
- Author
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Beati P, Massimino Stepñicka M, Vilchez Larrea SC, Smircich P, Alonso GD, and Ocampo J
- Subjects
- Chromatin genetics, Histones genetics, DNA, Micrococcal Nuclease metabolism, Nucleosomes genetics, Trypanosoma genetics
- Abstract
In Trypanosoma cruzi DNA is packaged into chromatin by octamers of histone proteins that form nucleosomes. Transcription of protein coding genes in trypanosomes is constitutive producing polycistronic units and gene expression is primarily regulated post-transcriptionally. However, chromatin organization influences DNA dependent processes. Hence, determining nucleosome position is of uppermost importance to understand the peculiarities found in trypanosomes. To map nucleosomes genome-wide in several organisms, digestion of chromatin with micrococcal nuclease followed by deep sequencing has been applied. Nonetheless, the special requirements for cell manipulation and the uniqueness of the chromatin organization in trypanosomes entails a customized analytical approach. In this work, we adjusted this broadly used method to the hybrid reference strain, CL Brener. Particularly, we implemented an exhaustive and thorough computational workflow to overcome the difficulties imposed by this complex genome. We tested the performance of two aligners, Bowtie2 and HISAT2, and discuss their advantages and caveats. Specifically, we highlight the relevance of using the whole genome as a reference instead of the commonly used Esmeraldo-like haplotype to avoid spurious alignments. Additionally, we show that using the whole genome refines the average nucleosome representation, but also the quality of mapping for every region represented. Moreover, we show that the average nucleosome organization around trans-splicing acceptor site described before, is not just an average since the same chromatin pattern is detected for most of the represented regions. In addition, we extended the study to a non-hybrid strain applying the experimental and analytical approach to Sylvio-X10 strain. Furthermore, we provide a source code for the construction of 2D plots and heatmaps which are easy to adapt to any T. cruzi strain., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Beati et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
- Published
- 2023
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15. FXR1 impedes the development of osteoarthritis by targeting SND1.
- Author
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Wang H, Li Z, Chen L, Lv H, Sun Y, Yan F, and Lu Y
- Subjects
- Mice, Animals, Micrococcal Nuclease metabolism, Chondrocytes metabolism, Apoptosis, Cartilage, Articular metabolism, Osteoarthritis metabolism, Osteoarthritis, Knee
- Abstract
Objectives: To investigate the role of fragile X mental retardation syndrome-related protein 1 (FXR1), an RNA binding protein, in the development of osteoarthritis (OA), to define its mechanism of action in cartilage, and to determine whether targeting FXR1 can prevent OA in mice., Methods: Western blot analysis and quantitative polymerase chain reaction were performed using cartilage tissue from control and osteoarthritic mice. FXR1 expression was detected by immunofluorescence staining using cartilage tissue from mice. OA was induced by destabilising the medial meniscus in the mice. Infection of mouse chondrocytes with FXR1 lentivirus, as well as viral injection into the mouse knee joint cavity, resulted in high FXR1 protein expression. Chondrocyte apoptosis was detected by TUNEL assay and cell senescence was detected by SA-β-gal staining assay., Results: FXR1 expression was significantly reduced in cartilage and soft tissue from mice with OA compared with the controls. FXR1 overexpression reduced staphylococcal nuclease domain protein 1 (SND1) levels. Furthermore, FXR1 is able to inhibit apoptosis and senescence of chondrocytes via SND1 and hinder the development of OA in mice., Conclusions: FXR1 down-regulates SND1 expression, thereby alleviating osteoarthritic symptoms in mice. In summary, FXR1 may have a therapeutic approach to the treatment of OA.
- Published
- 2023
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16. Folding of Staphylococcal Nuclease Induced by Binding of Chemically Modified Substrate Analogues Sheds Light on Mechanisms of Coupled Folding/Binding Reactions.
- Author
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Mori Y, Mizukami T, Segawa S, Roder H, and Maki K
- Subjects
- Ligands, Kinetics, Protein Conformation, Protein Folding, Micrococcal Nuclease metabolism
- Abstract
Several proteins have been shown to undergo a shift in the mechanism of ligand binding-induced folding from conformational selection (CS; folding precedes binding) to induced fit (IF; binding precedes folding) with increasing ligand concentration. In previous studies of the coupled folding/binding reaction of staphylococcal nuclease (SNase) in the presence of a substrate analogue, adenosine-3',5'-diphosphate (prAp), we found that the two phosphate groups make important energetic contributions toward stabilizing its complex with the native protein as well as transient conformational states encountered at high ligand concentrations favoring IF. However, the structural contributions of each phosphate group during the reaction remain unclear. To address this question, we relied on fluorescence, nuclear magnetic resonance (NMR), absorption, and isothermal titration calorimetry to study the effects of deletion of the phosphate groups of prAp on the kinetics of ligand-induced folding, using a strategy analogous to mutational ϕ-value analysis to interpret the results. Kinetic measurements over a wide range of ligand concentrations, together with structural characterization of a transient protein-ligand encounter complex using 2D NMR, indicated that, at high ligand concentrations favoring IF, (i) the 5'-phosphate group interacts weakly with denatured SNase during early stages of the reaction, resulting in loose docking of the two domains of SNase, and (ii) the 3'-phosphate group engages in some specific contacts with the polypeptide in the transition state prior to formation of the native SNase-prAp complex.
- Published
- 2023
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17. Profiling Accessible Chromatin and Nucleosomes in the Mammalian Genome.
- Author
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Lim HW and Iwafuchi M
- Subjects
- Animals, Micrococcal Nuclease metabolism, Genome, Histones genetics, Histones metabolism, Mammals genetics, Mammals metabolism, Nucleosomes genetics, Chromatin genetics
- Abstract
Genomic DNA wraps around core histones to form nucleosomes, which provides steric constraints on how transcription factors (TFs) can interact with gene regulatory sequences. It is increasingly apparent that well-positioned, accessible nucleosomes are an inherent feature of active enhancers and can facilitate cooperative TF binding, referred to as nucleosome-mediated cooperativity. Thus, profiling chromatin and nucleosome properties (accessibility, positioning, and occupancy) on the genome is crucial to understand cell-type-specific gene regulation. Here we describe a simplified protocol to profile accessible nucleosomes in the mammalian genome using low-level and high-level micrococcal nuclease (MNase) digestion followed by genome-wide sequencing., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2023
- Full Text
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18. Genome-Wide Identification of Open Chromatin in Plants Using MH-Seq.
- Author
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Zhang A, Li X, Zhao H, Jiang J, and Zhang W
- Subjects
- Animals, Nucleosomes, Micrococcal Nuclease metabolism, High-Throughput Nucleotide Sequencing methods, DNA genetics, Plants genetics, Transcription Factors metabolism, Mammals genetics, Chromatin genetics, Arabidopsis genetics, Arabidopsis metabolism
- Abstract
Functional cis-regulatory elements (CREs) act as precise transcriptional switches for fine-tuning gene transcription. Identification of CREs is critical for understanding regulatory mechanisms of gene expression associated with various biological processes in eukaryotes. It is well known that CREs reside in open chromatin that exhibits hypersensitivity to enzyme cleavage and physical shearing. Currently, high-throughput methodologies, such as DNase-seq, ATAC-seq, and FAIRE-seq, have been widely applied in mapping open chromatin in various eukaryotic genomes. More recently, differential MNase (micrococcal nuclease) treatment has been successfully employed to map open chromatin in addition to profiling nucleosome landscape in both mammalian and plant species. We have developed a MNase hypersensitivity sequencing (MH-seq) technique in plants. The MH-seq procedure includes plant nuclei fixation and purification, differential treatments of purified nuclei with MNase, specific recovery of MNase-trimmed small DNA fragments within 20~100 bp in length, and MH-seq library construction followed by Illumina sequencing and data analysis. MH-seq has been successfully applied for global identification of open chromatin in both Arabidopsis thaliana and maize. It has been proven to be an attractive alternative for profiling open chromatin. Thus, MH-seq is expected to be valuable in probing chromatin accessibility on a genome-wide scale for other plants with sequenced genomes. Moreover, MHS data allow to implement footprinting assays to unveil binding sites of transcription factors., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2023
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19. Identification of Small Inhibitors for Human Metadherin, an Oncoprotein, through in silico Approach.
- Author
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Khattak AA, Ahmad A, Khattak HA, and Irshad Khan MZ
- Subjects
- Male, Humans, Molecular Docking Simulation, Oncogene Proteins metabolism, Endonucleases metabolism, Membrane Proteins metabolism, RNA-Binding Proteins, Micrococcal Nuclease metabolism, Neoplasms
- Abstract
Aims: Cancer is a disease that takes lives of thousands of people each year. There are more than 100 different types of cancers known to man. This fatal disease is one of the leading causes of death today., Background: Astrocyte elevated gene-1(AEG-1)/Metadherin (MTDH) activates multiple oncogenic signaling pathways and leads to different types of cancers. MTDH interacting with staphylococcal nuclease domain containing 1(SND1) supports the survival and growth of mammary epithelial cells under oncogenic conditions., Objective: Silencing MTDH or SND1 individually or disrupting their interaction compromises the tumorigenic potential of tumor-initiating cells. The aim of our present study was to investigate novel interactions of staphylococcal nuclease domain containing 1 (SND1) binding domain of AEG-1/MTDH with different lead compounds through molecular docking approach using MOE software., Methods: Molecular docking was done by docking the ChemBridge database against important residues of MTDH involved in interaction with SND1. After docking the whole ChemBridge database, the top 200 interactive compounds were selected based on docking scores. After applying Lipinski's rule, all the remaining chosen compounds were studied on the basis of binding affinity, binding energy, docking score and protein-ligand interactions. Finally, 10 compounds showing multiple interactions with different amino acid residues were selected as the top interacting compounds., Results: Three compounds were selected for simulation studies after testing these compounds using topkat toxicity and ADMET studies. The simulation study indicated that compound 32538601 is a lead compound for inhibiting MTDH-SND1 complex formation., Conclusion: These novels, potent inhibitors of MTDH-SND1 complex can ultimately help us in controlling cancer up to some extent., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)
- Published
- 2023
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20. The Drosophila BEAF insulator protein interacts with the polybromo subunit of the PBAP chromatin remodeling complex.
- Author
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McKowen JK, Avva SVSP, Maharjan M, Duarte FM, Tome JM, Judd J, Wood JL, Negedu S, Dong Y, Lis JT, and Hart CM
- Subjects
- Animals, Chromatin Assembly and Disassembly, Nucleosomes genetics, Nucleosomes metabolism, Micrococcal Nuclease genetics, Micrococcal Nuclease metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Transcription Factors genetics, Transcription Factors metabolism, Drosophila genetics, Drosophila metabolism, Chromatin genetics, Chromatin metabolism, Insulator Elements, Drosophila Proteins metabolism
- Abstract
The Drosophila Boundary Element-Associated Factor of 32 kDa (BEAF) binds in promoter regions of a few thousand mostly housekeeping genes. BEAF is implicated in both chromatin domain boundary activity and promoter function, although molecular mechanisms remain elusive. Here, we show that BEAF physically interacts with the polybromo subunit (Pbro) of PBAP, a SWI/SNF-class chromatin remodeling complex. BEAF also shows genetic interactions with Pbro and other PBAP subunits. We examine the effect of this interaction on gene expression and chromatin structure using precision run-on sequencing and micrococcal nuclease sequencing after RNAi-mediated knockdown in cultured S2 cells. Our results are consistent with the interaction playing a subtle role in gene activation. Fewer than 5% of BEAF-associated genes were significantly affected after BEAF knockdown. Most were downregulated, accompanied by fill-in of the promoter nucleosome-depleted region and a slight upstream shift of the +1 nucleosome. Pbro knockdown caused downregulation of several hundred genes and showed a correlation with BEAF knockdown but a better correlation with promoter-proximal GAGA factor binding. Micrococcal nuclease sequencing supports that BEAF binds near housekeeping gene promoters while Pbro is more important at regulated genes. Yet there is a similar general but slight reduction of promoter-proximal pausing by RNA polymerase II and increase in nucleosome-depleted region nucleosome occupancy after knockdown of either protein. We discuss the possibility of redundant factors keeping BEAF-associated promoters active and masking the role of interactions between BEAF and the Pbro subunit of PBAP in S2 cells. We identify Facilitates Chromatin Transcription (FACT) and Nucleosome Remodeling Factor (NURF) as candidate redundant factors., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)
- Published
- 2022
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21. Phospho-Tudor-SN coordinates with STAT5 to regulate prolactin-stimulated milk protein synthesis and proliferation of bovine mammary epithelial cells.
- Author
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Ao J, Ma Z, Li R, Zhang S, Gao X, and Zhang M
- Subjects
- Cattle, Animals, Prolactin pharmacology, Prolactin metabolism, Micrococcal Nuclease metabolism, Mammary Glands, Animal metabolism, Signal Transduction physiology, Epithelial Cells metabolism, TOR Serine-Threonine Kinases genetics, TOR Serine-Threonine Kinases metabolism, Cell Proliferation, Milk Proteins, STAT5 Transcription Factor genetics, STAT5 Transcription Factor metabolism
- Abstract
Tudor staphylococcal nuclease (Tudor-SN) participates in milk synthesis and cell proliferation in response to prolactin (PRL) and plays a regulatory role on mTOR phosphorylation. However, the complicated molecular mechanism of Tudor-SN regulating milk protein synthesis and cell proliferation still remains to be illustrated. In present study, we observed that the proteins level of phosphorylated Tudor-SN and phosphorylated STAT5 were simultaneously enhanced upon PRL treatment in bovine mammary epithelial cells (BMECs). Tudor-SN overexpression and knockdown experiment showed that Tudor-SN positively regulated the synthesis of milk protein, cell proliferation and the phosphorylation of STAT5, which was dependent on Tudor-SN phosphorylation. STAT5 knockdown experiment showed that Tudor-SN stimulated mTOR pathway through regulating STAT5 activation, which was required for PRL to activate the mTOR pathway. Thus, these results demonstrate the primary mechanism of Tudor-SN coordinating with STAT5 to regulate milk protein synthesis and cell proliferation under stimulation of PRL in BMECs, which may provide some new perspectives for increasing milk production.
- Published
- 2022
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22. The palmitoylation of AEG-1 dynamically modulates the progression of hepatocellular carcinoma.
- Author
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Zhou B, Wang Y, Zhang L, Shi X, Kong H, Zhang M, Liu Y, Shao X, Liu Z, Song H, Li W, Gao X, Chang Y, Dou C, Guo W, Zhang S, Kang X, Gao J, Liang Y, Zheng J, and Kong E
- Subjects
- Humans, Mice, Animals, Lipoylation, Cysteine metabolism, Micrococcal Nuclease metabolism, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Cell Adhesion Molecules genetics, Cell Adhesion Molecules metabolism, Membrane Proteins genetics, Membrane Proteins metabolism, Cell Line, Tumor, Endonucleases metabolism, Carcinoma, Hepatocellular pathology, Liver Neoplasms pathology
- Abstract
Rationale: Protein palmitoylation is tightly related to tumorigenesis or tumor progression as many oncogenes or tumor suppressors are palmitoylated. AEG-1, an oncogene, is commonly elevated in a variety of human malignancies, including hepatocellular carcinoma (HCC). Although AEG-1 was suggested to be potentially modified by protein palmitoylation, the regulatory roles of AEG-1 palmitoylation in tumor progression of HCC has not been explored. Methods: Techniques as Acyl-RAC assay and point mutation were used to confirm that AEG-1 is indeed palmitoylated. Moreover, biochemical experiments and immunofluorescent microscopy were applied to examine the cellular functions of AEG-1 palmitoylation in several cell lines. Remarkably, genetically modified knock-in (AEG-1-C75A) and knockout (Zdhhc6-KO) mice were established and subjected to the treatment of DEN to induce the HCC mice model, through which the roles of AEG-1 palmitoylation in HCC is directly addressed. Last, HCQ, a chemical compound, was introduced to prove in principal that elevating the level of AEG-1 palmitoylation might benefit the treatment of HCC in xenograft mouse model. Results: We showed that AEG-1 undergoes palmitoylation on a conserved cysteine residue, Cys-75. Blocking AEG-1 palmitoylation exacerbates the progression of DEN-induced HCC in vivo . Moreover, it was demonstrated that AEG-1 palmitoylation is dynamically regulated by zDHHC6 and PPT1/2. Accordingly, suppressing the level of AEG-1 palmitoylation by the deletion of Zdhhc6 reproduces the enhanced tumor-progression phenotype in DEN-induced HCC mouse model. Mechanistically, we showed that AEG-1 palmitoylation adversely regulates its protein stability and weakens AEG-1 and staphylococcal nuclease and tudor domain containing 1 (SND1) interaction, which might contribute to the alterations of the RISC activity and the expression of tumor suppressors. For intervention, HCQ, an inhibitor of PPT1, was applied to augment the level of AEG-1 palmitoylation, which retards the tumor growth of HCC in xenograft model. Conclusion: Our study suggests an unknown mechanism that AEG-1 palmitoylation dynamically manipulates HCC progression and pinpoints that raising AEG-1 palmitoylation might confer beneficial effect on the treatment of HCC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)
- Published
- 2022
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23. MiOS, an integrated imaging and computational strategy to model gene folding with nucleosome resolution.
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Neguembor MV, Arcon JP, Buitrago D, Lema R, Walther J, Garate X, Martin L, Romero P, AlHaj Abed J, Gut M, Blanc J, Lakadamyali M, Wu CT, Brun Heath I, Orozco M, Dans PD, and Cosma MP
- Subjects
- Chromatin genetics, DNA genetics, Histones genetics, Humans, RNA Polymerase II genetics, Micrococcal Nuclease metabolism, Nucleosomes genetics
- Abstract
The linear sequence of DNA provides invaluable information about genes and their regulatory elements along chromosomes. However, to fully understand gene function and regulation, we need to dissect how genes physically fold in the three-dimensional nuclear space. Here we describe immuno-OligoSTORM, an imaging strategy that reveals the distribution of nucleosomes within specific genes in super-resolution, through the simultaneous visualization of DNA and histones. We combine immuno-OligoSTORM with restraint-based and coarse-grained modeling approaches to integrate super-resolution imaging data with Hi-C contact frequencies and deconvoluted micrococcal nuclease-sequencing information. The resulting method, called Modeling immuno-OligoSTORM, allows quantitative modeling of genes with nucleosome resolution and provides information about chromatin accessibility for regulatory factors, such as RNA polymerase II. With Modeling immuno-OligoSTORM, we explore intercellular variability, transcriptional-dependent gene conformation, and folding of housekeeping and pluripotency-related genes in human pluripotent and differentiated cells, thereby obtaining the highest degree of data integration achieved so far to our knowledge., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)
- Published
- 2022
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24. N-Glycosylation on Asn50 of SND1 Is Required for Glioma U87 Cell Proliferation and Metastasis.
- Author
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Zhou Y, Li Q, Zheng J, and Lin N
- Subjects
- Biomarkers metabolism, Cell Proliferation, Endonucleases genetics, Endonucleases metabolism, Glycosylation, Humans, Micrococcal Nuclease metabolism, Nuclear Proteins genetics, Nuclear Proteins metabolism, Endoplasmic Reticulum-Associated Degradation, Glioma metabolism
- Abstract
Staphylococcal nuclease domain-containing protein 1 (SND1) is an evolutionarily conserved multidomain protein, which has gained attention recently due to its positive regulation in several cancer progression and metastatic spread. However, the specific contribution of SND1 glycosylation in glioma remains uncertain. In the current study, we confirmed that SND1 was highly expressed in human glioma. Using site-directed mutagenesis, we created four predicted N-glycosylation site mutants for SND1 and provided the first evidence that SND1 undergoes N-glycosylation on its Asn50, Asn168, Asn283, and Asn416 residues in human glioma U87 cells. In addition, we found that removing the N-glycans on the Asn50 site destabilized SND1 and led to its endoplasmic reticulum-associated degradation. Furthermore, destabilized SND1 inhibits the glioma cell proliferation and metastasis. Collectively, our results reveal that N-glycosylation at Asn50 is essential for SND1 folding and trafficking, thus essential for the glioma process, providing new insights for SND1 as a potential disease biomarker for glioma., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 Ying Zhou et al.)
- Published
- 2022
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25. A modified CUT&RUN-seq technique for qPCR analysis of chromatin-protein interactions.
- Author
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Panday A, Elango R, Willis NA, and Scully R
- Subjects
- Chromatin Immunoprecipitation methods, Endonucleases, Micrococcal Nuclease metabolism, Chromatin genetics, Chromosomes metabolism
- Abstract
Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) even with optimization may give low signal-to-background ratio and spatial resolution. Here, we adapted Cleavage Under Targets and Release Using Nuclease (CUT&RUN) (originally developed by the Henikoff group) to develop CUT&RUN-qPCR. By studying the recruitment of selected proteins (but amenable to other proteins), we find that CUT&RUN-qPCR is more sensitive and gives better spatial resolution than ChIP-qPCR. For complete details on the use and execution of this protocol, please refer to Skene et al. (2018) and Skene and Henikoff (2017)., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)
- Published
- 2022
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26. Mechanism for Bioactive Nanomaterial circ0024831 Regulation of Staphylococcal Nuclease Domain Containing 1 via RNA Methylation Recognition in Osteosarcoma.
- Author
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Zheng H, Wang J, Zhang W, He B, Wang Y, Zhang X, Mao H, and Fan L
- Subjects
- Endonucleases genetics, Endonucleases metabolism, Humans, Methylation, Micrococcal Nuclease metabolism, Nuclear Proteins genetics, Nuclear Proteins metabolism, RNA, Circular genetics, RNA, Messenger genetics, RNA, Messenger metabolism, Bone Neoplasms genetics, Nanostructures, Osteosarcoma genetics
- Abstract
Bioactive nanomaterial circular RNA (circRNA) is an important non-coding RNA with a strong specificity, stable structure and high expression abundance. It can affect many diseases and physiological processes and may become a new way of disease diagnosis and targeted therapy. Recent studies have shown that Staphylococcal Nuclease Domain-Containing Protein 1 (SND1) can recognize N6-methyladenine (M6A) modified mRNA and regulate target mRNA stability. It can then control the expression of a series of downstream genes. However, whether SND1 can directly combine with circRNA and regulate its stability and function are new issues to be discussed. Results showed bioactive nanomaterial circ0024831 could directly bind to the Tudor domain of SND1 in the cytoplasm to block the recognition of SND1 to M6A modified RNA thus reducing the stability of downstream target gene mRNA and inhibiting the expression of downstream regulatory proteins. The down-regulation of circ0024831 expression in osteosarcoma cells relieved inhibition of SND1 which lead to change of tumor-related gene expression profile, promoting the occurrence and development of osteosarcoma.
- Published
- 2022
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27. Single-Cell Factor Localization on Chromatin using Ultra-Low Input Cleavage Under Targets and Release using Nuclease.
- Author
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Lardo SM and Hainer SJ
- Subjects
- Chromatin Immunoprecipitation, Micrococcal Nuclease metabolism, Nucleosomes, Transcription Factors metabolism, Chromatin genetics, DNA metabolism
- Abstract
Determining the binding locations of a protein on chromatin is essential for understanding its function and potential regulatory targets. Chromatin Immunoprecipitation (ChIP) has been the gold standard for determining protein localization for over 30 years and is defined by the use of an antibody to pull out the protein of interest from sonicated or enzymatically digested chromatin. More recently, antibody tethering techniques have become popular for assessing protein localization on chromatin due to their increased sensitivity. Cleavage Under Targets & Release Under Nuclease (CUT&RUN) is the genome-wide derivative of Chromatin Immunocleavage (ChIC) and utilizes recombinant Protein A tethered to micrococcal nuclease (pA-MNase) to identify the IgG constant region of the antibody targeting a protein of interest, therefore enabling site-specific cleavage of the DNA flanking the protein of interest. CUT&RUN can be used to profile histone modifications, transcription factors, and other chromatin-binding proteins such as nucleosome remodeling factors. Importantly, CUT&RUN can be used to assess the localization of either euchromatic- or heterochromatic-associated proteins and histone modifications. For these reasons, CUT&RUN is a powerful method for determining the binding profiles of a wide range of proteins. Recently, CUT&RUN has been optimized for transcription factor profiling in low populations of cells and single cells and the optimized protocol has been termed ultra-low input CUT&RUN (uliCUT&RUN). Here, a detailed protocol is presented for single-cell factor profiling using uliCUT&RUN in a manual 96-well format.
- Published
- 2022
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28. Tethered MNase Structure Probing as Versatile Technique for Analyzing RNPs Using Tagging Cassettes for Homologous Recombination in Saccharomyces cerevisiae.
- Author
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Teubl F, Schwank K, Ohmayer U, Griesenbeck J, Tschochner H, and Milkereit P
- Subjects
- Chromatin metabolism, DNA genetics, Homologous Recombination, Micrococcal Nuclease metabolism, Nucleosomes metabolism, RNA metabolism, Ribonucleoproteins genetics, Ribonucleoproteins metabolism, Calcium metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism
- Abstract
Micrococcal nuclease (MNase) originating from Staphylococcus aureus is a calcium dependent ribo- and desoxyribonuclease which has endo- and exonucleolytic activity of low sequence preference. MNase is widely used to analyze nucleosome positions in chromatin by probing the enzyme's DNA accessibility in limited digestion reactions. Probing reactions can be performed in a global way by addition of exogenous MNase , or locally by "chromatin endogenous cleavage " (ChEC ) reactions using MNase fusion proteins . The latter approach has recently been adopted for the analysis of local RNA environments of MNase fusion proteins which are incorporated in vivo at specific sites of ribonucleoprotein (RNP ) complexes. In this case, ex vivo activation of MNase by addition of calcium leads to RNA cleavages in proximity to the tethered anchor protein thus providing information about the folding state of its RNA environment.Here, we describe a set of plasmids that can be used as template for PCR-based MNase tagging of genes by homologous recombination in S. cerevisiae . The templates enable both N- and C-terminal tagging with MNase in combination with linker regions of different lengths and properties. In addition, an affinity tag is included in the recombination cassettes which can be used for purification of the particle of interest before or after induction of MNase cleavages in the surrounding RNA or DNA. A step-by-step protocol is provided for tagging of a gene of interest, followed by affinity purification of the resulting fusion protein together with associated RNA and subsequent induction of local MNase cleavages., (© 2022. The Author(s).)
- Published
- 2022
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29. Micrococcal Nuclease Digestion Assays for the Analysis of Chromosome Structure in Archaea.
- Author
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Maruyama H
- Subjects
- Chromatin genetics, DNA genetics, Digestion, Nucleosomes, Archaea genetics, Archaea metabolism, Micrococcal Nuclease metabolism
- Abstract
The digestion of chromosomes using micrococcal nuclease (MNase) enables the analysis of their fundamental structural units. For example, the digestion of eukaryotic chromatin using MNase results in laddered DNA fragments (~150 bp increment), which reflects the length of the DNA wrapped around regularly spaced nucleosomes. Here, we describe the application of MNase to examine the chromosome structure in Archaea. We used Thermococcus kodakarensis, a hyperthermophilic euryarchaeon that encodes proteins homologous to eukaryotic histones. Methods for chromosome extraction and agarose gel electrophoresis of MNase-digested DNA including small fragments (~30 bp) are also described., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2022
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30. Pharmacological disruption of the MTDH-SND1 complex enhances tumor antigen presentation and synergizes with anti-PD-1 therapy in metastatic breast cancer.
- Author
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Shen M, Smith HA, Wei Y, Jiang YZ, Zhao S, Wang N, Rowicki M, Tang Y, Hang X, Wu S, Wan L, Shao ZM, and Kang Y
- Subjects
- Antigen Presentation, Endonucleases metabolism, Female, Humans, Membrane Proteins metabolism, Micrococcal Nuclease metabolism, Nuclear Proteins genetics, RNA-Binding Proteins metabolism, Transcription Factors metabolism, Breast Neoplasms drug therapy
- Abstract
Despite increased overall survival rates, curative options for metastatic breast cancer remain limited. We have previously shown that metadherin (MTDH) is frequently overexpressed in poor prognosis breast cancer, where it promotes metastasis and therapy resistance through its interaction with staphylococcal nuclease domain-containing 1 (SND1). Through genetic and pharmacological targeting of the MTDH-SND1 interaction, we reveal a key role for this complex in suppressing antitumor T cell responses in breast cancer. The MTDH-SND1 complex reduces tumor antigen presentation and inhibits T cell infiltration and activation by binding to and destabilizing Tap1/2 messenger RNAs, which encode key components of the antigen-presentation machinery. Following small-molecule compound C26-A6 treatment to disrupt the MTDH-SND1 complex, we showed enhanced immune surveillance and sensitivity to anti-programmed cell death protein 1 therapy in preclinical models of metastatic breast cancer, in support of this combination therapy as a viable approach to increase immune-checkpoint blockade therapy responses in metastatic breast cancer., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)
- Published
- 2022
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31. Early Strides in NMR Dynamics Measurements.
- Author
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Dyson HJ
- Subjects
- Crystallography, X-Ray, History, 20th Century, Micrococcal Nuclease metabolism, Micrococcal Nuclease ultrastructure, Nuclear Magnetic Resonance, Biomolecular methods, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Nuclear Magnetic Resonance, Biomolecular history
- Abstract
The study of protein dynamics using the measurement of relaxation times by NMR was based on a set of studies in the mid-20th century that outlined theories and methods. However, the complexity of protein NMR was such that these simple experiments were not practical for application to proteins. The advent of techniques in the 1980s for isotopic labeling of proteins meant that pulse sequences could now be applied in multidimensional NMR experiments to enable per-residue information about the local relaxation times. One of the earliest advances was published in Biochemistry in 1989. The paper "Backbone dynamics of proteins as studied by
15 N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease" by Lewis Kay, Dennis Torchia, and Ad Bax delineated a set of pulse sequences that are used with minor modifications even today. This paper, with others from a limited number of other laboratories, forms the basis for the experimental determination of the backbone dynamics of proteins. The biological insights obtained from such measurements have only increased in the past 30 years. Sometimes, the best and perhaps only way to advance a field is an advancement in the technical capabilities that allows new perspectives to be reached.- Published
- 2021
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- View/download PDF
32. Accurate and Transferable Reactive Molecular Dynamics Models from Constrained Density Functional Theory.
- Author
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Li C and Voth GA
- Subjects
- Amino Acids, Density Functional Theory, Proteins, Micrococcal Nuclease metabolism, Molecular Dynamics Simulation
- Abstract
Chemical reactions constitute the central feature of many liquid, material, and biomolecular processes. Conventional molecular dynamics (MD) is inadequate for simulating chemical reactions given the fixed bonding topology of most force fields, while modeling chemical reactions using ab initio molecular dynamics is limited to shorter time and length scales given its high computational cost. As such, the multiscale reactive molecular dynamics method provides one promising alternative for simulating complex chemical systems at atomistic detail on a reactive potential energy surface. However, the parametrization of such models is a key barrier to their applicability and success. In this work, we present reactive MD models derived from constrained density functional theory that are both accurate and transferable. We illustrate the features of these models for proton dissociation reactions of amino acids in both aqueous and protein environments. Specifically, we present models for ionizable glutamate and lysine that predict accurate absolute p K
a values in water as well as their significantly shifted p Ka in staphylococcal nuclease (SNase) without any modification of the models. As one outcome of the new methodology, the simulations show that the deprotonation of ionizable residues in SNase can be closely coupled with side chain rotations, which is a concept likely generalizable to many other proteins. Furthermore, the present approach is not limited to only p Ka prediction but can enable the fully atomistic simulation of many other reactive systems along with a determination of the key aspects of the reaction mechanisms.- Published
- 2021
- Full Text
- View/download PDF
33. MNase Digestion Protection Patterns of the Linker DNA in Chromatosomes.
- Author
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Shen CH and Allan J
- Subjects
- Animals, Binding Sites, Chickens, DNA genetics, Histones genetics, Hydrolysis, Nucleic Acid Conformation, Nucleosomes genetics, beta-Globins genetics, Chromatin Assembly and Disassembly, DNA metabolism, Histones metabolism, Micrococcal Nuclease metabolism, Nucleosomes metabolism, beta-Globins metabolism
- Abstract
The compact nucleosomal structure limits DNA accessibility and regulates DNA-dependent cellular activities. Linker histones bind to nucleosomes and compact nucleosomal arrays into a higher-order chromatin structure. Recent developments in high throughput technologies and structural computational studies provide nucleosome positioning at a high resolution and contribute to the information of linker histone location within a chromatosome. However, the precise linker histone location within the chromatin fibre remains unclear. Using monomer extension, we mapped core particle and chromatosomal positions over a core histone-reconstituted, 1.5 kb stretch of DNA from the chicken adult β-globin gene, after titration with linker histones and linker histone globular domains. Our results show that, although linker histone globular domains and linker histones display a wide variation in their binding affinity for different positioned nucleosomes, they do not alter nucleosome positions or generate new nucleosome positions. Furthermore, the extra ~20 bp of DNA protected in a chromatosome is usually symmetrically distributed at each end of the core particle, suggesting linker histones or linker histone globular domains are located close to the nucleosomal dyad axis.
- Published
- 2021
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- View/download PDF
34. Pressure Unfolding of Proteins: New Insights into the Role of Bound Water.
- Author
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Arsiccio A and Shea JE
- Subjects
- Micrococcal Nuclease metabolism, Pressure, Protein Denaturation, Thermodynamics, Protein Folding, Water
- Abstract
High pressures can be detrimental for protein stability, resulting in unfolding and loss of function. This phenomenon occurs because the unfolding transition is accompanied by a decrease in volume, which is typically attributed to the elimination of cavities that are present within the native state as a result of packing defects. We present a novel computational approach that enables the study of pressure unfolding in atomistically detailed protein models in implicit solvent. We include the effect of pressure using a transfer free energy term that allows us to decouple the effect of protein residues and bound water molecules on the volume change upon unfolding. We discuss molecular dynamics simulations results using this protocol for two model proteins, Trp-cage and staphylococcal nuclease (SNase). We find that the volume reduction of bound water is the key energetic term that drives protein denaturation under the effect of pressure, for both Trp-cage and SNase. However, we note differences in unfolding mechanisms between the smaller Trp-cage and the larger SNase protein. Indeed, the unfolding of SNase, but not Trp-cage, is seen to be further accompanied by a reduction in the volume of internal cavities. Our results indicate that, for small peptides, like Trp-cage, pressure denaturation is driven by the increase in solvent accessibility upon unfolding, and the subsequent increase in the number of bound water molecules. For larger proteins, like SNase, the cavities within the native fold act as weak spots, determining the overall resistance to pressure denaturation. Our simulations display a striking agreement with the pressure-unfolding profile experimentally obtained for SNase and represent a promising approach for a computationally efficient and accurate exploration of pressure-induced denaturation of proteins.
- Published
- 2021
- Full Text
- View/download PDF
35. Influence of sub-inhibitory concentrations of antimicrobials on micrococcal nuclease and biofilm formation in Staphylococcus aureus.
- Author
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Rosman CWK, van der Mei HC, and Sjollema J
- Subjects
- Biofilms growth & development, Ciprofloxacin pharmacology, Doxycycline pharmacology, Erythromycin pharmacology, Gentamicins pharmacology, Micrococcal Nuclease metabolism, Polysaccharides, Bacterial metabolism, Resveratrol pharmacology, Staphylococcus aureus enzymology, Staphylococcus aureus growth & development, Stem Cells drug effects, Vancomycin pharmacology, Anti-Infective Agents pharmacology, Biofilms drug effects, Micrococcal Nuclease drug effects, Staphylococcus aureus drug effects
- Abstract
A major contributor to biomaterial associated infection (BAI) is Staphylococcus aureus. This pathogen produces a protective biofilm, making eradication difficult. Biofilms are composed of bacteria encapsulated in a matrix of extracellular polymeric substances (EPS) comprising polysaccharides, proteins and extracellular DNA (eDNA). S. aureus also produces micrococcal nuclease (MN), an endonuclease which contributes to biofilm composition and dispersion, mainly expressed by nuc1. MN expression can be modulated by sub-minimum inhibitory concentrations of antimicrobials. We investigated the relation between the biofilm and MN expression and the impact of the application of antimicrobial pressure on this relation. Planktonic and biofilm cultures of three S. aureus strains, including a nuc1 deficient strain, were cultured under antimicrobial pressure. Results do not confirm earlier findings that MN directly influences total biomass of the biofilm but indicated that nuc1 deletion stimulates the polysaccharide production per CFU in the biofilm in in vitro biofilms. Though antimicrobial pressure of certain antibiotics resulted in significantly increased quantities of polysaccharides per CFU, this did not coincide with significantly reduced MN activity. Erythromycin and resveratrol significantly reduced MN production per CFU but did not affect total biomass or biomass/CFU. Reduction of MN production may assist in the eradication of biofilms by the host immune system in clinical situations.
- Published
- 2021
- Full Text
- View/download PDF
36. Oral delivery of staphylococcal nuclease ameliorates DSS induced ulcerative colitis in mice via degrading intestinal neutrophil extracellular traps.
- Author
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Dong W, Liu D, Zhang T, You Q, Huang F, and Wu J
- Subjects
- Administration, Oral, Animals, Colitis, Ulcerative chemically induced, Colitis, Ulcerative drug therapy, Colitis, Ulcerative metabolism, Cytokines metabolism, Disease Models, Animal, Inflammation chemically induced, Intestines, Male, Mice, Micrococcal Nuclease metabolism, Tight Junction Proteins metabolism, Extracellular Traps metabolism, Micrococcal Nuclease administration & dosage
- Abstract
Recent studies have revealed that neutrophil extracellular traps (NETs) may contribute directly to the initiation of ulcerative colitis (UC), a typical inflammatory bowel disease (IBD) characterized by mucosal damage. Staphylococcal nuclease (SNase), a nonspecific phosphodiesterase, has a strong ability to degrade DNA. Here we investigate whether intestinal NET degradation with an oral preparation of SNase can ameliorate dextran sulfate sodium (DSS)-induced UC in mice. SNase encapsulated with calcium alginate (ALG-SNase) was formulated using crosslinking technology with sodium alginate and calcium chloride. ALG-SNase were orally administered to DSS-induced UC mice, and their therapeutic efficacy was evaluated. The expression of inflammatory cytokines and biomarkers of NETs was also assessed, as well as the intestinal permeability in mice. The results showed that ALG-SNase nanoparticles were successfully prepared and delivered to the colon of UC mice. In addition, oral administration of ALG-SNase nanoparticles decreased NET levels in the colon and effectively alleviated the clinical colitis index and tissue inflammation in UC mice. Moreover, the SNase nanoparticles reduced intestinal permeability and regulated the expression of proinflammatory cytokines. Furthermore, the markers of NETs were strongly correlated with the expression levels of tight junction proteins in colon tissue. In conclusion, our data showed that oral administration of ALG-SNase can effectively ameliorate colitis in UC mice via NET degradation and suggested SNase as a candidate therapy for the treatment of UC., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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- View/download PDF
37. Rapid and inexpensive preparation of genome-wide nucleosome footprints from model and non-model organisms.
- Author
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McKnight LE, Crandall JG, Bailey TB, Banks OGB, Orlandi KN, Truong VN, Donovan DA, Waddell GL, Wiles ET, Hansen SD, Selker EU, and McKnight JN
- Subjects
- Animals, Cell Culture Techniques methods, Cells, Cultured, DNA chemistry, DNA genetics, DNA metabolism, Genomics, Micrococcal Nuclease metabolism, Saccharomyces cerevisiae genetics, DNA Footprinting methods, Nucleosomes chemistry, Nucleosomes genetics, Nucleosomes metabolism, Sequence Analysis, DNA methods
- Abstract
MNase-seq (micrococcal nuclease sequencing) is used to map nucleosome positions in eukaryotic genomes to study the relationship between chromatin structure and DNA-dependent processes. Current protocols require at least two days to isolate nucleosome-protected DNA fragments. We have developed a streamlined protocol for S. cerevisiae and other fungi which takes only three hours. Modified protocols were developed for wild fungi and mammalian cells. This method for rapidly producing sequencing-ready nucleosome footprints from several organisms makes MNase-seq faster and easier, with less chemical waste., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)
- Published
- 2021
- Full Text
- View/download PDF
38. Multiple roles of H2A.Z in regulating promoter chromatin architecture in human cells.
- Author
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Cole L, Kurscheid S, Nekrasov M, Domaschenz R, Vera DL, Dennis JH, and Tremethick DJ
- Subjects
- Binding Sites, Cell Line, Tumor, Chromatin genetics, Epigenomics, Gene Expression, Humans, Micrococcal Nuclease metabolism, Nucleosomes metabolism, RNA Polymerase II metabolism, Transcription Factors, Chromatin metabolism, Histones genetics, Histones metabolism, Promoter Regions, Genetic
- Abstract
Chromatin accessibility of a promoter is fundamental in regulating transcriptional activity. The histone variant H2A.Z has been shown to contribute to this regulation, but its role has remained poorly understood. Here, we prepare high-depth maps of the position and accessibility of H2A.Z-containing nucleosomes for all human Pol II promoters in epithelial, mesenchymal and isogenic cancer cell lines. We find that, in contrast to the prevailing model, many different types of active and inactive promoter structures are observed that differ in their nucleosome organization and sensitivity to MNase digestion. Key aspects of an active chromatin structure include positioned H2A.Z MNase resistant nucleosomes upstream or downstream of the TSS, and a MNase sensitive nucleosome at the TSS. Furthermore, the loss of H2A.Z leads to a dramatic increase in the accessibility of transcription factor binding sites. Collectively, these results suggest that H2A.Z has multiple and distinct roles in regulating gene expression dependent upon its location in a promoter.
- Published
- 2021
- Full Text
- View/download PDF
39. High-Resolution ChIP-MNase Mapping of Nucleosome Positions at Selected Genomic Loci and Alleles.
- Author
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van Essen D, Oruba A, and Saccani S
- Subjects
- Binding Sites, Cells, Cultured, Chromatin genetics, Chromatin metabolism, Chromatin Assembly and Disassembly, Protein Binding, Quality Control, Alleles, Chromatin Immunoprecipitation methods, Chromatin Immunoprecipitation Sequencing methods, Chromosome Mapping methods, Genetic Loci, Micrococcal Nuclease metabolism, Nucleosomes metabolism
- Abstract
The positioning of nucleosomes regulates the accessibility of genomic DNA and can impact the activities of functional elements. Nucleosome positioning is highly consistent at each genomic location in any particular cell-type, but can vary in an orchestrated fashion between different cell-types and between genomic loci according to their activities. Here, we describe a technique-"ChIP-MNase" (chromatin immunoprecipitation linked to micrococcal nuclease mapping)-to determine nucleosome positions at chosen sets of genomic features that can be defined by their molecular composition and recovered by chromatin immunoprecipitation. ChIP-MNase enables high-resolution analysis of nucleosome positioning at genomic regions-of-interest and can allow differential analysis of alleles undergoing distinct molecular processes., (© 2021. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2021
- Full Text
- View/download PDF
40. Detection and Quantification of Secreted Nuclease Activity in Staphylococcus aureus Culture Supernatants.
- Author
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Wiemels RE, Keogh RA, and Carroll RK
- Subjects
- Animals, Bacterial Proteins metabolism, Bacteriological Techniques, Extracellular Traps metabolism, Immune Evasion, Male, Micrococcal Nuclease metabolism, Spermatozoa chemistry, Staphylococcus aureus pathogenicity, Virulence Factors analysis, Virulence Factors metabolism, Bacterial Proteins analysis, Micrococcal Nuclease analysis, Salmon genetics, Staphylococcus aureus enzymology
- Abstract
Staphylococcal secreted nuclease contributes to S. aureus virulence by degrading neutrophil extracellular traps (NETs), which allows the bacterium to evade the host immune system and has also been shown to promote biofilm dispersal. In this chapter, two methods for detecting nuclease activity are described, both of which have increased sensitivity compared to the traditional nuclease agar method., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2021
- Full Text
- View/download PDF
41. pH-Dependent Conformational Changes Lead to a Highly Shifted p K a for a Buried Glutamic Acid Mutant of SNase.
- Author
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Sarkar A and Roitberg AE
- Subjects
- Hydrogen-Ion Concentration, Hydrophobic and Hydrophilic Interactions, Protein Conformation, Thermodynamics, Glutamic Acid genetics, Micrococcal Nuclease genetics, Micrococcal Nuclease metabolism
- Abstract
Ionizable residues are rarely present in the hydrophobic interior of proteins, but when they are, they play important roles in biological processes such as energy transduction and enzyme catalysis. Internal ionizable residues have anomalous experimental p K
a values with respect to their p Ka in bulk water. This work investigates the atomistic cause of the highly shifted p Ka of the internal Glu23 in the artificially mutated variant V23E of Staphylococcal Nuclease (SNase) using pH replica exchange molecular dynamics (pH-REMD) simulations. The p Ka of Glu23 obtained from our calculations is 6.55, which is elevated with respect to the glutamate p Ka of 4.40 in bulk water. The calculated value is close to the experimental p Ka of 7.10. Our simulations show that the highly shifted p Ka of Glu23 is the product of a pH-dependent conformational change, which has been observed experimentally and also seen in our simulations. We carry out an analysis of this pH-dependent conformational change in response to the protonation state change of Glu23. Using a four-state thermodynamic model, we estimate the two conformation-specific p Ka values of Glu23 and describe the coupling between the conformational and ionization equilibria.- Published
- 2020
- Full Text
- View/download PDF
42. Genome-wide off-rates reveal how DNA binding dynamics shape transcription factor function.
- Author
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de Jonge WJ, Brok M, Lijnzaad P, Kemmeren P, and Holstege FC
- Subjects
- Binding Sites, Chromatin genetics, Chromatin Assembly and Disassembly genetics, DNA-Binding Proteins genetics, Gene Expression Regulation, Fungal genetics, Genome, Fungal, Micrococcal Nuclease metabolism, Nucleosomes genetics, Promoter Regions, Genetic, Protein Binding, RNA Polymerase II genetics, RNA Polymerase II metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Transcription Factors genetics, Chromatin metabolism, Chromatin Immunoprecipitation Sequencing methods, DNA-Binding Proteins metabolism, Nucleosomes metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Transcription Factors metabolism, Transcription, Genetic genetics
- Abstract
Protein-DNA interactions are dynamic, and these dynamics are an important aspect of chromatin-associated processes such as transcription or replication. Due to a lack of methods to study on- and off-rates across entire genomes, protein-DNA interaction dynamics have not been studied extensively. Here, we determine in vivo off-rates for the Saccharomyces cerevisiae chromatin organizing factor Abf1, at 191 sites simultaneously across the yeast genome. Average Abf1 residence times span a wide range, varying between 4.2 and 33 min. Sites with different off-rates are associated with different functional characteristics. This includes their transcriptional dependency on Abf1, nucleosome positioning and the size of the nucleosome-free region, as well as the ability to roadblock RNA polymerase II for termination. The results show how off-rates contribute to transcription factor function and that DIVORSEQ (Determining In Vivo Off-Rates by SEQuencing) is a meaningful way of investigating protein-DNA binding dynamics genome-wide., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)
- Published
- 2020
- Full Text
- View/download PDF
43. MNase Profiling of Promoter Chromatin in Salmonella typhimurium -Stimulated GM12878 Cells Reveals Dynamic and Response-Specific Nucleosome Architecture.
- Author
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Cole L and Dennis J
- Subjects
- Humans, Micrococcal Nuclease metabolism, Promoter Regions, Genetic, Salmonella typhimurium genetics, Salmonella typhimurium metabolism, Chromatin, Nucleosomes
- Abstract
The nucleosome is the primary unit of chromatin structure and commonly imputed as a regulator of nuclear events, although the exact mechanisms remain unclear. Recent studies have shown that certain nucleosomes can have different sensitivities to micrococcal nuclease (MNase) digestion, resulting in the release of populations of nucleosomes dependent on the concentration of MNase. Mapping MNase sensitivity of nucleosomes at transcription start sites genome-wide reveals an important functional nucleosome organization that correlates with gene expression levels and transcription factor binding. In order to understand nucleosome distribution and sensitivity dynamics during a robust genome response, we mapped nucleosome position and sensitivity using multiple concentrations of MNase. We used the innate immune response as a model system to understand chromatin-mediated regulation. Herein we demonstrate that stimulation of a human lymphoblastoid cell line (GM12878) with heat-killed Salmonella typhimurium (HKST) results in changes in nucleosome sensitivity to MNase. We show that the HKST response alters the sensitivity of -1 nucleosomes at highly expressed promoters. Finally, we correlate the increased sensitivity with response-specific transcription factor binding. These results indicate that nucleosome sensitivity dynamics reflect the cellular response to HKST and pave the way for further studies that will deepen our understanding of the specificity of genome response., (Copyright © 2020 Lauren and Jonathan.)
- Published
- 2020
- Full Text
- View/download PDF
44. Thermonuclease test accuracy is preserved in methicillin-resistant Staphylococcus aureus isolates.
- Author
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Canning B, Mohamed I, Wickramasinghe N, Swindells J, and O'Shea MK
- Subjects
- Bacterial Proteins genetics, Enzyme Assays, Humans, Methicillin-Resistant Staphylococcus aureus genetics, Methicillin-Resistant Staphylococcus aureus isolation & purification, Micrococcal Nuclease genetics, Micrococcal Nuclease metabolism, Retrospective Studies, Bacterial Proteins analysis, Bacterial Proteins metabolism, Methicillin-Resistant Staphylococcus aureus enzymology, Micrococcal Nuclease analysis, Staphylococcal Infections microbiology
- Abstract
Introduction. The nuc gene encodes a thermonuclease which is present in Staphylococcus aureus but not in coagulase-negative staphylococci (CoNS) and is the target of the rapid phenotypic thermonuclease test. The effect of nuc gene variation in methicillin-resistant S. aureus (MRSA) on the performance of PCR testing has been noted, although there are no reports about the effect of MRSA on the activity of the thermonuclease enzyme. Aim. Our goals were to examine the sensitivity and specificity of the thermonuclease test used to distinguish S. aureus from CoNS cultured from blood. In addition, we aimed to assess differences in the sensitivity, specificity and accuracy of the thermonuclease test between methicillin-sensitive S. aureus (MSSA) and MRSA isolates. Methodology. We performed a retrospective analysis of 1404 isolates. Each isolate from a positive blood culture was identified as a Gram-positive coccus by microscopy then analysed with the thermonuclease test (Southern Group Laboratory) prior to confirmatory identification using VITEK microbial identification platforms (bioMérieux) and cefoxitin disc diffusion testing. Results. Of 1331 samples included in the final analysis, 189 were thermonuclease-positive, of which 176 were identified as S. aureus . Of the 1142 thermonuclease-negative samples, 13 were finally identified as S. aureus , giving a sensitivity of 93.1 % (95 % confidence interval [CI] 88.5-96.3) and specificity of 98.9 % (95 % CI 98.1-99.4). Of the nine proven MRSA samples, eight were thermonuclease-positive, giving a sensitivity of 88.9 % (95 % CI 51.8-99.7). Thermonuclease test accuracy for MSSA and MRSA isolates was 98.1 % (95 % CI 97.2-98.8) versus 98.8 % (95 % CI 98.0-99.3), respectively. Conclusions. In the era of increasing use of molecular-based microbiology assays, the thermonuclease test remains a simple, inexpensive and robust test for the presumptive identification of S. aureus cultured from blood, irrespective of methicillin sensitivity.
- Published
- 2020
- Full Text
- View/download PDF
45. Staphylococcus aureus Specific FRET Probe-Based Antibacterial Susceptibility Testing (SF-AST) by Detection of Micrococcal Nuclease Activity.
- Author
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Yi SY, Jeong J, Kim KE, Park K, and Shin YB
- Subjects
- DNA Probes, Fluorescence Resonance Energy Transfer, Methicillin Resistance, Microbial Sensitivity Tests, Predictive Value of Tests, Reproducibility of Results, Sensitivity and Specificity, Anti-Bacterial Agents pharmacology, Micrococcal Nuclease metabolism, Staphylococcus aureus drug effects, Staphylococcus aureus enzymology
- Abstract
In this study, we describe a simple and rapid antibacterial susceptibility testing (AST) method for Staphylococcus aureus called S. aureus specific fluorescence resonance energy transfer (FRET) probe-based AST (SF-AST), which is based on an S. aureus specific FRET probe (SF probe) that detects micrococcal nuclease (MNase) activity secreted from S. aureus . The SF-AST was tested with an S. aureus quality control (QC) strain against six relevant antibiotics, and the minimum inhibitory concentration (MIC) values obtained with the broth microdilution (BMD) method were compared, as a gold standard AST. Results were obtained with high accuracy in 4-6 h. The MIC for the methicillin resistance using 20 clinical S. aureus isolates of SF-AST showed 100% sensitivity, specificity, positive predictive value, and negative predictive value, as compared to BMD. Thus, the SF-AST method is a simple, rapid, and useful antibiotic resistance test for S. aureus , and it provides a basis for clinical treatment in a short time.
- Published
- 2020
- Full Text
- View/download PDF
46. Genome-wide profiling of nucleosome position and chromatin accessibility in single cells using scMNase-seq.
- Author
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Gao W, Lai B, Ni B, and Zhao K
- Subjects
- Animals, Mice, NIH 3T3 Cells, Chromatin genetics, Genomics, Micrococcal Nuclease metabolism, Nucleosomes genetics, Sequence Analysis, DNA methods, Single-Cell Analysis methods
- Abstract
Nucleosome organization is important for chromatin compaction and accessibility. Profiling nucleosome positioning genome-wide in single cells provides critical information to understand the cell-to-cell heterogeneity of chromatin states within a cell population. This protocol describes single-cell micrococcal nuclease sequencing (scMNase-seq), a method for detecting genome-wide nucleosome positioning and chromatin accessibility simultaneously from a small number of cells or single cells. To generate scMNase-seq libraries, single cells are isolated by FACS sorting, lysed and digested by MNase. DNA is purified, end-repaired and ligated to Y-shaped adaptors. Following PCR amplification with indexing primers, the subnucleosome-sized (fragments with a length of ≤80 bp) and mononucleosome-sized (fragments with a length between 140 and 180 bp) DNA fragments are recovered and sequenced on Illumina HiSeq platforms. On average, 0.5-1 million unique mapped reads are obtained for each single cell. The mononucleosome-sized DNA fragments precisely define genome-wide nucleosome positions in single cells, while the subnucleosome-sized DNA fragments provide information on chromatin accessibility. Library preparation of scMNase-seq takes only 2 d, requires only standard molecular biology techniques and does not require sophisticated laboratory equipment. Processing of high-throughput sequencing data requires basic bioinformatics skills and uses publicly available bioinformatics software.
- Published
- 2020
- Full Text
- View/download PDF
47. Cell-free transcription in Xenopus egg extract.
- Author
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Barrows JK and Long DT
- Subjects
- Animals, Cell Nucleus metabolism, Cytoplasm metabolism, Fertilization, Genome, Histones chemistry, Micrococcal Nuclease metabolism, Plasmids metabolism, Polyadenylation, RNA Polymerase II metabolism, Sequence Analysis, RNA, Transcription Factors metabolism, Transcription, Genetic, Cell-Free System, Gene Expression Regulation, Oocytes chemistry, Xenopus laevis
- Abstract
Soluble extracts prepared from Xenopus eggs have been used extensively to study various aspects of cellular and developmental biology. During early egg development, transcription of the zygotic genome is suppressed. As a result, traditional extracts derived from unfertilized and early stage eggs possess little or no intrinsic transcriptional activity. In this study, we show that Xenopus nucleoplasmic extract (NPE) supports robust transcription of a chromatinized plasmid substrate. Although prepared from eggs in a transcriptionally inactive state, the process of making NPE resembles some aspects of egg fertilization and early embryo development that lead to transcriptional activation. With this system, we observed that promoter-dependent recruitment of transcription factors and RNA polymerase II leads to conventional patterns of divergent transcription and pre-mRNA processing, including intron splicing and 3' cleavage and polyadenylation. We also show that histone density controls transcription factor binding and RNA polymerase II activity, validating a mechanism proposed to regulate genome activation during development. Together, these results establish a new cell-free system to study the regulation, initiation, and processing of mRNA transcripts., (© 2019 Barrows and Long.)
- Published
- 2019
- Full Text
- View/download PDF
48. During the Early Stages of Staphylococcus aureus Biofilm Formation, Induced Neutrophil Extracellular Traps Are Degraded by Autologous Thermonuclease.
- Author
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Sultan AR, Hoppenbrouwers T, Lemmens-den Toom NA, Snijders SV, van Neck JW, Verbon A, de Maat MPM, and van Wamel WJB
- Subjects
- Fluorescence Resonance Energy Transfer, Humans, Microbial Viability, Polysaccharides, Bacterial metabolism, Reactive Oxygen Species metabolism, Staphylococcal Infections immunology, Staphylococcal Infections pathology, Staphylococcus aureus metabolism, Biofilms growth & development, Extracellular Traps metabolism, Micrococcal Nuclease metabolism, Neutrophils immunology, Staphylococcus aureus growth & development, Staphylococcus aureus pathogenicity
- Abstract
Staphylococcus aureus extracellular DNA (eDNA) plays a crucial role in the structural stability of biofilms during bacterial colonization; on the contrary, host immune responses can be induced by bacterial eDNA. Previously, we observed production of S. aureus thermonuclease during the early stages of biofilm formation in a mammalian cell culture medium. Using a fluorescence resonance energy transfer (FRET)-based assay, we detected thermonuclease activity of S. aureus biofilms grown in Iscove's modified Dulbecco's medium (IMDM) earlier than that of widely studied biofilms grown in tryptic soy broth (TSB). The thermonuclease found was Nuc1, confirmed by mass spectrometry and competitive Luminex assay. These results indicate that biofilm development in IMDM may not rely on eDNA for structural stability. A bacterial viability assay in combination with wheat germ agglutinin (WGA) staining confirmed the accumulation of dead cells and eDNA in biofilms grown in TSB. However, in biofilms grown in IMDM, minimal amounts of eDNA were found; instead, polysaccharide intercellular adhesin (PIA) was detected. To investigate if this early production of thermonuclease plays a role in immune modulation by biofilm, we studied the effect of thermonuclease on human neutrophil extracellular trap (NET) formation using a nuc knockout and complemented strain. We confirmed that thermonuclease produced by early-stage biofilms grown in IMDM degraded biofilm-induced NETs. Additionally, neither the presence of biofilms nor thermonuclease stimulated an increase in reactive oxygen species (ROS) production by neutrophils. Our findings indicated that S. aureus , during the early stages of biofilm formation, actively evades the host immune responses by producing thermonuclease., (Copyright © 2019 American Society for Microbiology.)
- Published
- 2019
- Full Text
- View/download PDF
49. Accurate Calculation of Barnase and SNase Folding Energetics Using Short Molecular Dynamics Simulations and an Atomistic Model of the Unfolded Ensemble: Evaluation of Force Fields and Water Models.
- Author
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Galano-Frutos JJ and Sancho J
- Subjects
- Computer Simulation, Models, Molecular, Molecular Dynamics Simulation, Protein Conformation, Protein Folding, Protein Stability, Thermodynamics, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Micrococcal Nuclease chemistry, Micrococcal Nuclease metabolism, Ribonucleases chemistry, Ribonucleases metabolism
- Abstract
As proteins perform most cellular functions, quantitative understanding of protein energetics is required to gain control of biological phenomena. Accurate models of native proteins can be obtained experimentally, but the lack of equally fine models of unfolded ensembles impedes the calculation of protein folding energetics from first principles. Here, we show that an atomistic unfolded ensemble model, consisting of a few dozen conformations built from a protein sequence, can be used in conjunction with an X-ray structure of its native state to calculate accurately by difference the changes in enthalpy and heat capacity of the polypeptide upon folding. The calculation is done using molecular dynamics simulations, popular force fields, and water models, and for the two model proteins studied (barnase and SNase), the results agree within error or are very close to their experimentally determined properties. The enthalpy sampling of the unfolded ensemble is done through short 2 ns simulations that do not significantly modify the representative distribution of R
g of the starting conformations. The impressive accuracy obtained opens the possibility to investigate quantitatively systems or phenomena not amenable to experiment and paves the way for addressing the calculation of protein conformational stability (i.e., the change in Gibbs energy upon folding), a central goal of structural biology. So far, these calculated enthalpy and heat capacity changes, combined with the experimentally determined melting temperatures of the corresponding protein, allow us to reproduce the stability curves of both barnase and SNase.- Published
- 2019
- Full Text
- View/download PDF
50. Dissection of acute stimulus-inducible nucleosome remodeling in mammalian cells.
- Author
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Comoglio F, Simonatto M, Polletti S, Liu X, Smale ST, Barozzi I, and Natoli G
- Subjects
- Animals, Cells, Cultured, Chromatin Immunoprecipitation, High-Throughput Nucleotide Sequencing, Macrophages metabolism, Mice, Mice, Inbred C57BL, Micrococcal Nuclease metabolism, Nucleosomes metabolism, Regulatory Elements, Transcriptional physiology, Transcription Factors metabolism
- Abstract
Accessibility of the genomic regulatory information is largely controlled by the nucleosome-organizing activity of transcription factors (TFs). While stimulus-induced TFs bind to genomic regions that are maintained accessible by lineage-determining TFs, they also increase accessibility of thousands of cis -regulatory elements. Nucleosome remodeling events underlying such changes and their interplay with basal positioning are unknown. Here, we devised a novel quantitative framework discriminating different types of nucleosome remodeling events in micrococcal nuclease ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) data sets and used it to analyze nucleosome dynamics at stimulus-regulated cis -regulatory elements. At enhancers, remodeling preferentially affected poorly positioned nucleosomes while sparing well-positioned nucleosomes flanking the enhancer core, indicating that inducible TFs do not suffice to overrule basal nucleosomal organization maintained by lineage-determining TFs. Remodeling events appeared to be combinatorially driven by multiple TFs, with distinct TFs showing, however, different remodeling efficiencies. Overall, these data provide a systematic view of the impact of stimulation on nucleosome organization and genome accessibility in mammalian cells., (© 2019 Comoglio et al.; Published by Cold Spring Harbor Laboratory Press.)
- Published
- 2019
- Full Text
- View/download PDF
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