Simultaneous Dual-Gene Test of Methicillin-Resistant Staphylococcus Aureus using an Argonaute-Centered Portable and Visual Biosensor.

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 107 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.

Inhibition of staphylococcal nuclease by benzimidazole-based Ligand: Implications in DNA-Mediated entrapment and uptake of MRSA by Macrophage-like cells.

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 âˆ¼ 55 % and âˆ¼ 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 (Kcat) and catalytic efficiency (Kcat/Km) with an IC50 value of âˆ¼ 1122 nM. In CD spectroscopy, a notable perturbation in the ß-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.

C2c: Predicting Micro-C from Hi-C.

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.

Analysis of HBV cccDNA Minichromosome Accessibility by MNase-qPCR and High-Throughput Sequencing.

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.

nucMACC: An MNase-seq pipeline to identify structurally altered nucleosomes in the genome.

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.

ChEC-Seq: A Comprehensive Guide for Scalable and Cost-Efficient Genome-Wide Profiling in Saccharomyces cerevisiae.

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.

Micrococcal nuclease regulates biofilm formation and dispersal in methicillin-resistant Staphylococcus aureus USA300.

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.

Hsa_circ_0001583 fuels bladder cancer metastasis by promoting staphylococcal nuclease and tudor domain containing 1-mediated MicroRNA decay.

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.

MNase-Seq Analysis for Identifying Stress-Altered Nucleosome Occupancy in Plants.

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.

Improvement of bovine ß-lactoglobulin production and secretion by Lactococcus lactis

The stabilizing effects of staphylococcal nuclease (Nuc) and of a synthetic propeptide (LEISSTCDA, hereafter called LEISS) on the production of a model food allergen, bovine ß-lactoglobulin (BLG), in Lactococcus lactis were investigated. The fusion of Nuc to BLG (Nuc-BLG) results in higher production and secretion of the hybrid protein. When LEISS was fused to BLG, the production of the resulting protein LEISS-BLG was only slightly improved compared to the one obtained with Nuc-BLG. However, the secretion of LEISS-BLG was dramatically enhanced (~10- and 4-fold higher than BLG and Nuc-BLG, respectively). Finally, the fusion of LEISS to Nuc-BLG resulting in the protein LEISS-Nuc-BLG led to the highest production of the hybrid protein, estimated at ~8 æg/ml (~2-fold higher than Nuc-BLG). In conclusion, the fusions described here led to the improvement of the production and secretion of BLG. These tools will be used to modulate the immune response against BLG via delivery of recombinant lactococci at the mucosal level, in a mouse model of cow's milk allergy.