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Journal of Molecular Biology Jun 2024Nucleosomes are the basic compaction unit of chromatin and nucleosome structure, and their higher-order assemblies regulate genome accessibility. Many post-translational...
Nucleosomes are the basic compaction unit of chromatin and nucleosome structure, and their higher-order assemblies regulate genome accessibility. Many post-translational modifications alter nucleosome dynamics, nucleosome-nucleosome interactions, and ultimately chromatin structure and gene expression. Here, we investigate the role of two post-translational modifications associated with actively transcribed regions, H3K36me3 and H4K5/8/12/16ac, in the contexts of tri-nucleosome arrays that provide a tractable model system for quantitative single-molecule analysis, while enabling us to probe nucleosome-nucleosome interactions. Direct visualization by AFM imaging reveals that H3K36me3 and H4K5/8/12/16ac nucleosomes adopt significantly more open and loose conformations than unmodified nucleosomes. Similarly, magnetic tweezers force spectroscopy shows a reduction in DNA outer turn wrapping and nucleosome-nucleosome interactions for the modified nucleosomes. The results suggest that for H3K36me3 the increased breathing and outer DNA turn unwrapping seen in mononucleosomes propagates to more open conformations in nucleosome arrays. In contrast, the even more open structures of H4K5/8/12/16ac nucleosome arrays do not appear to derive from the dynamics of the constituent mononucleosomes, but are driven by reduced nucleosome-nucleosome interactions, suggesting that stacking interactions can overrule DNA breathing of individual nucleosomes. We anticipate that our methodology will be broadly applicable to reveal the influence of other post-translational modifications and to observe the activity of nucleosome remodelers.
PubMed: 38908785
DOI: 10.1016/j.jmb.2024.168671 -
Journal of Dairy Science Jun 2024Due to its beneficial effects on human health, Bifidobacterium is commonly added to milk powder. Accurate quantification of viable Bifidobacterium is essential for...
Rapid and accurate quantification of viable Bifidobacterium cells in milk powder with a propidium monoazide - antibiotic fluorescence in situ hybridization - flow cytometry method.
Due to its beneficial effects on human health, Bifidobacterium is commonly added to milk powder. Accurate quantification of viable Bifidobacterium is essential for assessing the therapeutic efficacy of milk powder. In this study, we introduced a novel propidium monoazide (PMA) - antibiotic fluorescence in situ hybridization (AFISH) - flow cytometry (FCM) method to rapidly and accurately quantify viable Bifidobacterium cells in milk powder. Briefly, Bifidobacterium cells were treated with chloramphenicol (CM) to increase their rRNA content, followed by staining with RNA-binding oligonucleotide probes, based on the AFISH technique. Then, the DNA-binding dye PMA was used to differentiate between viable and non-viable cells. The PMA-AFISH-FCM method, including sample pretreatment, CM treatment, dual staining, and FCM analysis, required around 2 h and was found to be better than the current methods. This is the first study to implement FCM combined with PMA and oligonucleotide probe for detecting Bifidobacterium.
PubMed: 38908696
DOI: 10.3168/jds.2024-24876 -
Experimental Cell Research Jun 2024Pelvic organ prolapse (POP) is a group of diseases caused by extracellular matrix (ECM) degradation in pelvic supportive tissues. Cysteine and serine rich nuclear...
Silencing of Cysteine and serine rich nuclear protein 1 inhibits apoptosis, senescence and collagen degradation in human-derived vaginal fibroblasts in response to oxidative stress or DNA damage.
Pelvic organ prolapse (POP) is a group of diseases caused by extracellular matrix (ECM) degradation in pelvic supportive tissues. Cysteine and serine rich nuclear protein 1 (CSRNP1) is involved in cell proliferation and survival regulation, and reportedly facilitates collagen breakdown in human chondrocytes. The present study aimed to probe the effect of CSRNP1 on collagen metabolism in human-derived vaginal fibroblasts. High expression of CSRNP1 was found in POP patient-derived vaginal fibroblasts in comparison to normal-derived vaginal fibroblasts. Following functional experiments revealed that CSRNP1 overexpression led to proliferation inhibition, apoptosis and collagen degradation in normal vaginal fibroblasts. In line with this, silencing of CSRNP1 inhibited hydrogen peroxide (HO)-triggered apoptosis, ROS generation and collagen loss in normal vaginal fibroblasts. Silencing of CSRNP1 also reduced the expression of cell senescence markers p21 and γ-H2Ax (the histone H2Ax phosphorylated at Ser139), as well as curbed collagen breakdown in normal vaginal fibroblasts caused by a DNA damage agent etoposide. Transcriptomic analysis of vaginal fibroblasts showed that differentially expressed genes affected by CSRNP1 overexpression were mainly enriched in the Wnt signaling pathway. Treatment with a Wnt pathway inhibitor DKK1 blocked CSRNP1 knockdown-caused collagen deposition. Mechanistically, CSRNP1 was identified to be a target of Snail family transcriptional repressor 2 (SNAI2). Forced expression of CSRNP1 reversed the anti-apoptotic, anti-senescent and anti-collagen loss effects of SNAI2 in normal vaginal fibroblasts exposed to HO or etoposide. Our study indicates that the SNAI2/CSRNP1 axis may be a key driver in POP progression, which provides a potential therapeutic strategy for POP.
PubMed: 38908423
DOI: 10.1016/j.yexcr.2024.114139 -
Methods in Molecular Biology (Clifton,... 2024Single-cell RNA sequencing supports the isolation of individual cells and barcoding of cDNA, specific to each cell of origin. Subsequent sequencing of the generated...
Single-cell RNA sequencing supports the isolation of individual cells and barcoding of cDNA, specific to each cell of origin. Subsequent sequencing of the generated library yields both the gene expression sequences and the cellular barcode, allowing distinction of gene expression patterns across individual cells. The 10X Genomics 3' HT assay uses a droplet-based method to isolate individual cells within oil emulsions, combined with a gel bead coated in uniquely barcoded primers, specific to each bead. The high-throughput, HT, assay is similar to its predecessor (3' v3.1) in reaction chemistry but utilizes (a) higher numbers of cellular barcodes, (b) a new, proprietary chip designed to target up to 60,000 cells per lane, and (c) captures up to 16 samples per run. The 3' HT assay supports whole cells and nuclei as input, with an approximate 60% capture rate. Here we describe the methods for sample quality control (QC) assays, loading and operation of the Chromium X instrument for cell capture, and cDNA synthesis and library preparation for downstream Illumina sequencing.
Topics: Humans; High-Throughput Nucleotide Sequencing; Genomics; Gene Library; Single-Cell Analysis; Sequence Analysis, RNA; Gene Expression Profiling; DNA, Complementary
PubMed: 38907921
DOI: 10.1007/978-1-0716-3918-4_15 -
Methods in Molecular Biology (Clifton,... 2024Oligonucleotide probe tagging and reverse transcriptase polymerase-chain reaction (RT-PCR) are the most widely used techniques currently used for detecting and analyzing...
Oligonucleotide probe tagging and reverse transcriptase polymerase-chain reaction (RT-PCR) are the most widely used techniques currently used for detecting and analyzing RNA. RNA detection using labeled oligonucleotide probe-based approaches is suitable for point-of-care (POC) applications but lacks assay sensitivity, whereas RT-PCR requires complex instrumentation. As an alternative, immunoassay detection formats coupled with isothermal RNA amplification techniques have been proposed for handheld assay development. In this chapter, we describe a robust technique comprising of: (a) target RNA tagging with a complementary oligonucleotide probe labeled with a hapten moiety to form a DNA/RNA duplex hybrid; (b) complexing the DNA/RNA duplex with a pre-coated antibody (Ab) directed at the hapten moiety; (c) sandwich complex formation with an Ab that selectively recognizes the DNA/RNA structural motif; and (d) detection of the sandwich complex using a secondary Ab enzyme conjugate targeting the anti-DNA/RNA Ab followed by standard enzyme-linked immunosorbent assay (ELISA) visualization.
Topics: RNA; Enzyme-Linked Immunosorbent Assay; Humans; Immunoassay; Oligonucleotide Probes; Antibodies; Nucleic Acid Hybridization; DNA
PubMed: 38907919
DOI: 10.1007/978-1-0716-3918-4_13 -
Methods in Molecular Biology (Clifton,... 2024RNA in situ hybridization reveals the abundance and location of gene expression in cells or tissues, providing a technical basis for the clinical diagnosis of diseases....
RNA in situ hybridization reveals the abundance and location of gene expression in cells or tissues, providing a technical basis for the clinical diagnosis of diseases. In this chapter, we show a "V" shape probe-mediated single-molecule chromogenic in situ hybridization (vsmCISH) technique for bright-field visualization of individual RNA molecules. In our method, several pairs of target hybridization probes are hybridized to RNA molecules and each probe pair forms a "V" shape overhang. The overhang oligonucleotides then mediated the proximity ligation to form DNA circles, followed by rolling circle amplification for signal enhancement and enzyme-catalyzed chromogenic reaction-based readout. The colorimetric assay avoids problems such as photobleaching and autofluorescence of current fluorescent in situ hybridization-based single-molecule RNA detection techniques. Furthermore, the relatively straightforward protocol makes the method useful for biological research and clinical diagnosis applications.
Topics: In Situ Hybridization; RNA; Humans; Chromogenic Compounds; Colorimetry; Single Molecule Imaging
PubMed: 38907917
DOI: 10.1007/978-1-0716-3918-4_11 -
Analytical Chemistry Jun 2024The quantitative detection of antibodies is crucial for the diagnosis of infectious and autoimmune diseases, while the traditional methods experience high background...
The quantitative detection of antibodies is crucial for the diagnosis of infectious and autoimmune diseases, while the traditional methods experience high background signal noise and restricted signal gain. In this work, we have developed a highly efficient electrochemical biosensor by constructing a programmable DNA nanomachine integrated with electrochemically controlled atom transfer radical polymerization (eATRP). The sensor works by binding the target antidigoxin antibody (anti-Dig) to the epitope of the recognization probe, which then initiates the cascaded strand displacement reaction on a magnetic bead, leading to the capture of cupric oxide (CuO) nanoparticles through magnetic separation. After CuO was dissolved, the eATRP initiators were attached to the electrode based on the Cu-catalyzed azide-alkyne cycloaddition. The subsequent eATRP reaction results in the formation of long electroactive polymers (poly-FcMMA), producing an amplified current response for sensitive detection of anti-Dig. This method achieved a detection limit at clinically relevant picomolar concentration in human serum, offering a sensitive, convenient, and cost-effective tool for detecting various biomarkers in a wide range of applications.
PubMed: 38904276
DOI: 10.1021/acs.analchem.4c01176 -
Inorganic Chemistry Jun 2024As a typical RNA virus, the genetic information on HIV-1 is entirely stored in RNA. The reverse transcription activity of HIV-1 reverse transcriptase (RT) plays a...
As a typical RNA virus, the genetic information on HIV-1 is entirely stored in RNA. The reverse transcription activity of HIV-1 reverse transcriptase (RT) plays a crucial role in the replication and transmission of the virus. Non-nucleoside RT inhibitors (NNRTIs) block the function of RT by binding to the RNA binding site on RT, with very few targeting viral RNA. In this study, by transforming planar conjugated ligands into a spiro structure, we convert classical Ru(II) DNA intercalators into a nonintercalator. This enables selective binding to HIV-1 transactivation response (TAR) RNA on the outer side of nucleic acids through dual interactions involving hydrogen bonds and electrostatic attraction, effectively inhibiting HIV-1 RT and serving as a selective fluorescence probe for TAR RNA.
PubMed: 38904258
DOI: 10.1021/acs.inorgchem.4c01815 -
Analytical Chemistry Jun 2024Tumor-derived extracellular vesicles (TEVs) are rich in cellular information and hold great promise as a biomarker for noninvasive cancer diagnosis. However, accurate...
Ultrasensitive and Wash-Free Detection of Tumor Extracellular Vesicles by Aptamer-Proximity-Ligation-Activated Rolling Circle Amplification Coupled to Single Particle ICP-MS.
Tumor-derived extracellular vesicles (TEVs) are rich in cellular information and hold great promise as a biomarker for noninvasive cancer diagnosis. However, accurate measurement of TEVs presents challenges due to their low abundance and potential interference from a high number of EVs derived from normal cells. Herein, an aptamer-proximity-ligation-activated rolling circle amplification (RCA) method for EV membrane recognition, coupled with single particle inductively coupled plasma mass spectrometry (sp-ICP-MS) for the quantification of TEVs, is developed. When DNA-labeled ultrasmall gold nanoparticle (AuNP) probes bind to the long chains formed by RCA, they aggregate to form large particles. Notably, small AuNPs scarcely produce pulse signals in sp-ICP-MS, thereby detecting TEVs in a wash-free manner. By leveraging the strong binding affinity of aptamers, dual aptamers for EpCAM and PD-L1 recognition, and the sp-ICP-MS technique, this method offers remarkable sensitivity and selectivity in tracing TEVs. Under optimized conditions, the present method shows a favorable linear relationship between the pulse signal frequency of sp-ICP-MS and TEV concentration within the range of 10-10 particles/mL, along with a detection limit of 1.1 × 10 particles/mL. The pulse signals from sp-ICP-MS combined with machine learning algorithms are used to discriminate cancer patients from healthy donors with 100% accuracy. Due to its simple and fast operation and excellent sensitivity and accuracy, this approach holds significant potential for diverse applications in life sciences and personalized medicine.
PubMed: 38904228
DOI: 10.1021/acs.analchem.4c02066 -
Spectrochimica Acta. Part A, Molecular... Jun 2024Herein, two simple fluorescent signal-on sensing strategies for detecting lead ions (Pb) were established based on structure-switching aptamer probes and...
Herein, two simple fluorescent signal-on sensing strategies for detecting lead ions (Pb) were established based on structure-switching aptamer probes and exonuclease-assisted signal amplification strategies. Two hairpin-structure fluorescent probes with blunt-ended stem arms were designed by extending the base sequence of Pb aptamer (PS2.M) and labelling the probes with FAM (in probe 1) and 2-aminopurine (2-AP) (in probe 2), respectively. In method 1, graphene oxide (GO) was added to adsorb probe 1 and quench the fluorescence emission of FAM to achieve low fluorescent background. In method 2, fluorescent 2-AP molecule inserted into the double-stranded DNA of probe 2 was quenched as a result of base stacking interactions, leading to a simplified, quencher-free approach. The addition of Pb can induce the probes to transform into G-quadruplex structures, exposing single DNA strands at the 3' end (the extended sequences). This exposure enables the activation of exonuclease I (Exo I) on the probes, leading to the cleavage effect and subsequent release of free bases and fluorophores, thereby resulting in amplified fluorescence signals. The two proposed methods exhibit good specificity and sensitivity, with detection limits of 0.327 nM and 0.049 nM Pb for method 1 and method 2, respectively, and have been successfully applied to detect Pb in river water and fish samples. Both detection methods employ the structure-switching aptamer probes and can be completed in two or three steps without the need for complex analytical instruments. Therefore, they have a broad prospect in the sensitive and simple detection of lead ion contamination in food and environmental samples.
PubMed: 38901233
DOI: 10.1016/j.saa.2024.124643