Customizable BAC-based DNA markers for pulsed-field gel electrophoresis.

DNA markers are used as a size reference and sample loading control during gel electrophoresis. Most markers are designed for conventional gel electrophoresis to separate DNA smaller than 20 kb. For larger molecules, pulsed-field gel electrophoresis (PFGE) marker is required. Limited PFGE markers are available because large DNA are prone to nicking and degradation, causing smeary bands. Here, we developed a robust marker based on bacterial artificial chromosomes (BACs) with bands up to 184 kb. This marker could consistently confer intense and distinct bands for accurate gel analysis in molecular biology studies, laboratory validations or clinical diagnosis.

An antibody binding-based fluorescent assay for the rapid quantification of globotriaosylceramide levels in human Fabry cells.

Fabry disease is caused by reduced α-GAL A activity and accumulation of globotriaosylceramide (Gb3). Here, we describe a microplate Gb3 assay using fluorophore-tagged antibody and crude cellular lipid extracts. The assay is able to detect higher Gb3 concentrations in human Fabry cells compared to non-diseased cells. This result was verified by immunofluorescence staining that revealed large amounts of Gb3 deposits in Fabry cell lines, demonstrating the accuracy of this method. This assay may provide the basis for detecting Fabry disease by quantifying Gb3 deposits from human biological samples, for example, from urine and blood.

Phage N15 protelomerase resolves its tos recognition site into hairpin telomeres within mammalian cells.

Phage N15 protelomerase (TelN) cleaves double-stranded circular DNA containing a telomerase-occupancy-site (tos) and rejoins the resulting linear-ends to form closed-hairpin-telomeres in Escherichia coli (E. coli). Continued TelN expression is essential to support resolution of the linear structure. In mammalian cells, no enzyme with TelN-like activities has been found. In this work, we show that phage TelN, expressed transiently and stably in human and mouse cells, recapitulates its native activities in these exogenous environments. We found TelN to accurately resolve tos-DNA in vitro and in vivo within human and mouse cells into linear DNA-containing terminal telomeres that are resistant to RecBCD degradation, a hallmark of protelomerase processing. In stable cells, TelN activity was detectable for at least 60 days, which suggests the possibility of limited silencing of its expression. Correspondingly, linear plasmid containing a 100 kb human ß-globin gene expressed for at least 120 h in non-ß-globin-expressing mouse cells with TelN presence. Our results demonstrate TelN is able to cut and heal DNA as hairpin-telomeres within mammalian cells, providing a tool for creating novel structures by DNA resolution in these hosts. The TelN protelomerase may be useful for exploring novel technologies for genome interrogation and chromosome engineering.

A closer look at ligand specificity for cellular activation of NOD2 with synthetic muramyl dipeptide analogues.

To further understand the specificity of muramyl dipeptide (MDP) sensing by NOD2, we evaluated the compatibility of synthetic MDP analogues for cellular uptake and NAGK phosphorylation, the pre-requisite steps of intracellular NOD2 activation. Our results revealed that these two prior steps do not confer ligand stereoselectivity; yet NAGK strictly discriminates against the disaccharide NOD2 agonists for phosphorylation in vitro, despite it being indispensable for the cellular NOD2-stimulating effects of these analogues, implying potential glycosidase cleavage as a novel intermediate step for cellular activation of NOD2.

Correction: A closer look at ligand specificity for cellular activation of NOD2 with synthetic muramyl dipeptide analogues.

Correction for 'A closer look at ligand specificity for cellular activation of NOD2 with synthetic muramyl dipeptide analogues' by Christopher Adamson et al., Chem. Commun., 2024, 60, 2212-2215, https://doi.org/10.1039/D3CC05807G.

Molecular Docking Reveals Critical Residues in Candida albicans Cyr1 for Peptidoglycan Recognition and Hyphal Growth.

The key virulent characteristic of Candida albicans, the major fungal pathogen in humans, lies in its ability to switch between the benign yeast state and the invasive hyphal form upon exposure to specific stimuli. Among the numerous hyphal-inducing signals, bacterial peptidoglycan fragments (PGNs) represent the most potent inducers of C. albicans hyphal growth. The sole adenylyl cyclase Cyr1 in C. albicans is a known sensor for PGNs and activates downstream signaling of hyphal growth, yet the molecular details of PGN-Cyr1 interactions have remained unclear. In this study, we performed in silico docking of a PGN motif to the modeled structure of the Cyr1 leucine-rich repeat (LRR) domain and uncovered four putative PGN-interacting residues in Cyr1_LRR. The critical roles of these residues in PGN binding and supporting C. albicans hyphal growth were demonstrated by in-gel fluorescence binding assay and hyphal induction assay, respectively. Remarkably, the C. albicans mutant harboring the cyr1 variant allele that is defective for PGN recognition exhibits significantly reduced cytotoxicity in macrophage infection assay. Overall, our work offered important insights into the molecular recognition of PGNs by C. albicans Cyr1 sensor protein, establishing that disruption of PGN recognition by Cyr1 results in defective hyphal growth and reduced virulence of C. albicans. Our findings provide an exciting starting point for the future development of Cyr1 antagonists as novel anti-virulence therapeutics to combat C. albicans invasive growth and infection.

Phage N15-Based Vectors for Gene Cloning and Expression in Bacteria and Mammalian Cells.

Bacteriophage N15 is the first virus known to deliver linear prophage into Escherichia coli. During its lysogenic cycle, N15 protelomerase (TelN) resolves its telomerase occupancy site (tos) into hairpin telomeres. This protects the N15 prophage from bacterial exonuclease degradation, enabling it to stably replicate as a linear plasmid in E. coli. Interestingly, purely proteinaceous TelN can retain phage DNA linearization and hairpin formation without involving host- or phage-derived intermediates or cofactors in the heterologous environment. This unique feature has led to the advent of synthetic linear DNA vector systems derived from the TelN-tos module for the genetic engineering of bacterial and mammalian cells. This review will focus on the development and advantages of N15-based novel cloning and expression vectors in the bacterial and mammalian environments. To date, N15 is the most widely exploited molecular tool for the development of linear vector systems, especially the production of therapeutically useful miniDNA vectors without a bacterial backbone. Compared to typical circular plasmids, linear N15-based plasmids display remarkable cloning fidelity in propagating unstable repetitive DNA sequences and large genomic fragments. Additionally, TelN-linearized vectors with the relevant origin of replication can replicate extrachromosomally and retain transgenes functionality in bacterial and mammalian cells without compromising host cell viability. Currently, this DNA linearization system has shown robust results in the development of gene delivery vehicles, DNA vaccines and engineering mammalian cells against infectious diseases or cancers, highlighting its multifaceted importance in genetic studies and gene medicine.

Chemoenzymatic Probes Reveal Peptidoglycan Recognition and Uptake Mechanisms in Candida albicans.

Candida albicans, the major fungal pathogen in humans, is under the strong influence of bacterial peptidoglycan fragments to undergo the yeast-to-hyphae transition, a key virulent step in C. albicans pathogenesis and infections. However, due to the synthetic difficulties of obtaining peptidoglycan fragments for biological studies, mechanistic details of how C. albicans recognizes and uptakes these peptidoglycan fragments have not been well elucidated. Notably, previous works have solely focused on the synthetic peptidoglycan ligand, muramyl dipeptide (MDP), despite its poor hyphal-inducing activity in C. albicans. In this work, we isolated and purified natural peptidoglycan fragments via enzymatic degradation of bacteria cell wall sacculi and chemoenzymatically installed a series of functional d-amino acids into the natural muropeptide, creating peptidoglycan probes that bear photoaffinity, bio-orthogonal, or fluorescent functionality. Using these chemoenzymatic peptidoglycan probes, we established that natural peptidoglycan fragments, which are potent hyphal-inducers, interact with the C. albicans Cyr1 sensor protein in the in-gel fluorescence assay as well as in in vitro pulldown studies. Moreover, we established that bacterial peptidoglycan probes enter C. albicans cells via an energy-dependent endocytic process.

Visualization of Bacteria-Mediated Gene Delivery Using High-Resolution Electron and Confocal Microscopy.

Visual analysis of the gene delivery process when using invasive bacteria as a vector has been conventionally performed using standard light and fluorescence microscopy. These microscopes can provide basic information on the invasiveness of the bacterial vector including the ability of the vector to successfully adhere to the cell membrane. Standard microscopy techniques however fall short when finer details including membrane attachment as well as internalization into the cytoplasm are desired. High-resolution visual analysis of bacteria-mediated gene delivery can allow accurate measurement of the adherence and internalization capabilities of engineered vectors. Here, we describe the use of scanning electron microscopy (SEM) to directly quantify vectors when they are external to the cell wall, and confocal microscopy to evaluate the vectors when they have internalized into the cytoplasm. By performing the invasion procedure on microscope coverslips, cells can be easily prepared for analysis using electron or confocal microscopes. Imaging the invasion complexes in high resolution can provide important insights into the behavior of bacterial vectors including E. coli, Listeria, and Salmonella when invading their target cells to deliver DNA and other molecules.

In silico-guided sequence modifications of K-ras epitopes improve immunological outcome against G12V and G13D mutant KRAS antigens.

BACKGROUND: Somatic point substitution mutations in the KRAS proto-oncogene primarily affect codons 12/13 where glycine is converted into other amino acids, and are highly prevalent in pancreatic, colorectal, and non-small cell lung cancers. These cohorts are non-responsive to anti-EGFR treatments, and are left with non-specific chemotherapy regimens as their sole treatment options. In the past, the development of peptide vaccines for cancer treatment was reported to have poor AT properties when inducing immune responses. Utilization of bioinformatics tools have since become an interesting approach in improving the design of peptide vaccines based on T- and B-cell epitope predictions. METHODS: In this study, the region spanning exon 2 from the 4th to 18th codon within the peptide sequence of wtKRAS was chosen for sequence manipulation. Mutated G12V and G13D K-ras controls were generated in silico, along with additional single amino acid substitutions flanking the original codon 12/13 mutations. IEDB was used for assessing human and mouse MHC class I/II epitope predictions, as well as linear B-cell epitopes predictions, while RNA secondary structure prediction was performed via CENTROIDFOLD. A scoring and ranking system was established in order to shortlist top mimotopes whereby normalized and reducing weighted scores were assigned to peptide sequences based on seven immunological parameters. Among the top 20 ranked peptide sequences, peptides of three mimotopes were synthesized and subjected to in vitro and in vivo immunoassays. Mice PBMCs were treated in vitro and subjected to cytokine assessment using CBA assay. Thereafter, mice were immunized and sera were subjected to IgG-based ELISA. RESULTS: In silico immunogenicity prediction using IEDB tools shortlisted one G12V mimotope (68-V) and two G13D mimotopes (164-D, 224-D) from a total of 1,680 candidates. Shortlisted mimotopes were predicted to promote high MHC-II and -I affinities with optimized B-cell epitopes. CBA assay indicated that: 224-D induced secretions of IL-4, IL-5, IL-10, IL-12p70, and IL-21; 164-D triggered IL-10 and TNF-α; while 68-V showed no immunological responses. Specific-IgG sera titers against mutated K-ras antigens from 164-D immunized Balb/c mice were also elevated post first and second boosters compared to wild-type and G12/G13 controls. DISCUSSION: In silico-guided predictions of mutated K-ras T- and B-cell epitopes were successful in identifying two immunogens with high predictive scores, Th-bias cytokine induction and IgG-specific stimulation. Developments of such immunogens are potentially useful for future immunotherapeutic and diagnostic applications against KRAS(+) malignancies, monoclonal antibody production, and various other research and development initiatives.

Partial purification and characterization of polyphenol oxidase from Chinese parsley (Coriandrum sativum).

Purification and characterization of polyphenol oxidase (PPO) from Chinese parsley (Coriandrum sativum) were achieved. Crude PPO exhibited an enzyme activity of 1,952.24 EU/mL. PPO was partially purified up to 6.52x with a 10.89% yield using gel filtration chromatography. Maximal PPO activity was found at 35°C, pH 8.0 for 4-methylcatechol and at 40°C, pH 7.0 for catechol. PPO showed a higher affinity towards 4-methylcatechol, but a higher thermal stability when reacting with catechol. LCysteine was a better inhibitor than citric acid for reducing PPO activity at concentrations of 1 and 3mM in the presence of either substrate. Two 46 kDa isoenzymes were identified using SDS-PAGE. Isolation and characterization of Chinese parsley serves as a guideline for prediction of enzyme behavior leading to effective prevention of enzymatic browning during processing and storage, including inhibition and inactivation of PPO.