A large-scale CRISPR screen reveals context-specific genetic regulation of retinal ganglion cell regeneration.

Many genes are known to regulate retinal regeneration after widespread tissue damage. Conversely, genes controlling regeneration after limited cell loss, as per degenerative diseases, are undefined. As stem/progenitor cell responses scale to injury levels, understanding how the extent and specificity of cell loss impact regenerative processes is important.

Structural Evaluation of a Nitroreductase Engineered for Improved Activation of the 5-Nitroimidazole PET Probe SN33623.

Bacterial nitroreductase enzymes capable of activating imaging probes and prodrugs are valuable tools for gene-directed enzyme prodrug therapies and targeted cell ablation models. We recently engineered a nitroreductase ( NfsB F70A/F108Y) for the substantially enhanced reduction of the 5-nitroimidazole PET-capable probe, SN33623, which permits the theranostic imaging of vectors labeled with oxygen-insensitive bacterial nitroreductases. This mutant enzyme also shows improved activation of the DNA-alkylation prodrugs CB1954 and metronidazole.

The Catalysis Mechanism of Nitroreductase A, a Candidate for Gene-Directed Prodrug Therapy: Potentiometric and Substrate Specificity Studies.

nitroreductase A (NfsA) is a candidate for gene-directed prodrug cancer therapy using bioreductively activated nitroaromatic compounds (ArNO). In this work, we determined the standard redox potential of FMN of NfsA to be -215 ± 5 mV at pH 7.0.

Molecular regulation of retinal regeneration is context specific.

Unlabelled: Many genes are known to regulate retinal regeneration following widespread tissue damage. Conversely, genes controlling regeneration following limited retinal cell loss, akin to disease conditions, are undefined. Combining a novel retinal ganglion cell (RGC) ablation-based glaucoma model, single cell omics, and rapid CRISPR/Cas9-based knockout methods to screen 100 genes, we identified 18 effectors of RGC regeneration kinetics.

Preparation, analysis and toxicity characterisation of the redox metabolites of the azo food dye tartrazine.

Tartrazine (E102, FD&C Yellow 5) is a vibrant yellow azo dye added to many processed foods. The safety of this ubiquitous chemical has not been fully elucidated, and it has been linked to allergic reactions and ADHD in some individuals. In our study, bacterial species isolated from human stool decolourised tartrazine and, upon exposure to air, a purple compound formed.

A metagenomic library cloning strategy that promotes high-level expression of captured genes to enable efficient functional screening.

Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole.

An inducible model of chronic hyperglycemia.

Transgene driven expression of Escherichia coli nitroreductase (NTR1.0) renders animal cells susceptible to the antibiotic metronidazole (MTZ). Many NTR1.

The Crystal Structure of Engineered Nitroreductase NTR 2.0 and Impact of F70A and F108Y Substitutions on Substrate Specificity.

Bacterial nitroreductase enzymes that convert prodrugs to cytotoxins are valuable tools for creating transgenic targeted ablation models to study cellular function and cell-specific regeneration paradigms. We recently engineered a nitroreductase ("NTR 2.0") for substantially enhanced reduction of the prodrug metronidazole, which permits faster cell ablation kinetics, cleaner interrogations of cell function, ablation of previously recalcitrant cell types, and extended ablation paradigms useful for modelling chronic diseases.

A metagenomic library cloning strategy that promotes high-level expression of captured genes to enable efficient functional screening.

Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole.

Development of a compartmentalised self-replication protocol for selection of superior blunt-end DNA ligases.

DNA ligases are widely used in molecular biology to generate recombinant DNA. However, having evolved for nick-sealing, they are inefficient at catalysing the blunt-ended ligations that are critical to many biotechnological applications, including next-generation sequencing. To facilitate engineering of superior blunt-ended DNA ligases, we have developed and validated a compartmentalised self-replication protocol that can select for the most effective ligases from a library of variants.

Metathramycin, a new bioactive aureolic acid discovered by heterologous expression of a metagenome derived biosynthetic pathway.

Bacterial natural products have been a rich source of bioactive compounds for drug development, and advances in DNA sequencing, informatics and molecular biology have opened new avenues for their discovery. Here, we describe the isolation of an aureolic acid biosynthetic gene cluster from a metagenome library derived from a New Zealand soil sample. Heterologous expression of this pathway in resulted in the production and isolation of two new aureolic acid compounds, one of which (metathramycin, ) possesses potent bioactivity against a human colon carcinoma cell line (HCT-116, IC = 14.

Large-scale phenotypic drug screen identifies neuroprotectants in zebrafish and mouse models of retinitis pigmentosa.

Retinitis pigmentosa (RP) and associated inherited retinal diseases (IRDs) are caused by rod photoreceptor degeneration, necessitating therapeutics promoting rod photoreceptor survival. To address this, we tested compounds for neuroprotective effects in multiple zebrafish and mouse RP models, reasoning drugs effective across species and/or independent of disease mutation may translate better clinically. We first performed a large-scale phenotypic drug screen for compounds promoting rod cell survival in a larval zebrafish model of inducible RP.

Engineering the Nitroreductase NfsA to Create a Flexible Enzyme-Prodrug Activation System.

Bacterial nitroreductase enzymes that can efficiently convert nitroaromatic prodrugs to a cytotoxic form have numerous applications in targeted cellular ablation. For example, the generation of cytotoxic metabolites that have low bystander potential (i.e.

Hydrated Rubrolides from the New Zealand Tunicate .

LCMS analysis of an extract of the New Zealand tunicate showed evidence for numerous new rubrolides. Following a mass spectrometry-guided isolation procedure, new hydrated rubrolides V and W ( and ), along with previously reported rubrolide G (), were isolated and characterized using MS and NMR. The anti-bacterial and cell cytotoxic activity of the compounds were compared to the potent anti-MRSA compound rubrolide A; hydration across the C-5/C-6 bond was shown to abrogate antibacterial activity.

Directed evolution of the B. subtilis nitroreductase YfkO improves activation of the PET-capable probe SN33623 and CB1954 prodrug.

Objectives: To use directed evolution to improve YfkO-mediated reduction of the 5-nitroimidazole PET-capable probe SN33623 without impairing conversion of the anti-cancer prodrug CB1954.

Results: Two iterations of error-prone PCR, purifying selection, and FACS sorting in a DNA damage quantifying GFP reporter strain were used to identify three YfkO variants able to sensitize E. coli host cells to at least 2.

A cofactor consumption screen identifies promising NfsB family nitroreductases for dinitrotoluene remediation.

Objectives: To survey a library of over-expressed nitroreductases to identify those most active with 2,4- and 2,6-dinitrotoluene substrates, as promising candidates for phytoremediation of soils and groundwater contaminated with poly-nitro toluene pollutants.

Results: To indirectly monitor dinitrotoluene reduction we implemented a nitroblue tetrazolium dye screen to compare relative rates of NADPH consumption for 58 nitroreductase candidates, over-expressed in a nitroreductase-deleted strain of Escherichia coli. Although the screen only provides activity data at a single substrate concentration, by altering the substrate concentration and duration of incubation we showed we could first distinguish between more-active and less-active enzymes and then discriminate between the relative rates of reduction exhibited by the most active nitroreductases in the collection.

Lamellarin Sulfates from the Pacific Tunicate .

Six new lamellarin sulfates (-) were isolated from the methanolic extract of the Pacific tunicate , collected from the Kingdom of Tonga. Mass spectrometric molecular networking through the GNPS platform was used to target the isolation of -. Planar structures were elucidated through a combination of NMR and MS experiments.

VapC proteins from Mycobacterium tuberculosis share ribonuclease sequence specificity but differ in regulation and toxicity.

The chromosome of Mycobacterium tuberculosis (Mtb) contains a large number of Type II toxin-antitoxin (TA) systems. The majority of these belong to the VapBC TA family, characterised by the VapC protein consisting of a PIN domain with four conserved acidic residues, and proposed ribonuclease activity. Characterisation of five VapC (VapC1, 19, 27, 29 and 39) proteins from various regions of the Mtb chromosome using a combination of pentaprobe RNA sequences and mass spectrometry revealed a shared ribonuclease sequence-specificity with a preference for UAGG sequences.

Evaluation of NfsA-like nitroreductases from Neisseria meningitidis and Bartonella henselae for enzyme-prodrug therapy, targeted cellular ablation, and dinitrotoluene bioremediation.

Objectives: To characterize the activities of two candidate nitroreductases, Neisseria meningitidis NfsA (NfsA_Nm) and Bartonella henselae (PnbA_Bh), with the nitro-prodrugs, CB1954 and metronidazole, and the environmental pollutants 2,4- and 2,6-dinitrotoluene.

Results: NfsA_Nm and PnbA_Bh were evaluated in Escherichia coli over-expression assays and as His-tagged proteins in vitro. With the anti-cancer prodrug CB1954, both enzymes were more effective than the canonical O-insensitive nitroreductase E.

Engineering a Multifunctional Nitroreductase for Improved Activation of Prodrugs and PET Probes for Cancer Gene Therapy.

Gene-directed enzyme-prodrug therapy (GDEPT) is a promising anti-cancer strategy. However, inadequate prodrugs, inefficient prodrug activation, and a lack of non-invasive imaging capabilities have hindered clinical progression. To address these issues, we used a high-throughput Escherichia coli platform to evolve the multifunctional nitroreductase E.

Structure and Function of AmtR in Mycobacterium smegmatis: Implications for Post-Transcriptional Regulation of Urea Metabolism through a Small Antisense RNA.

Soil-dwelling bacteria of the phylum actinomycetes generally harbor either GlnR or AmtR as a global regulator of nitrogen metabolism. Mycobacterium smegmatis harbors both of these canonical regulators; GlnR regulates the expression of key genes involved in nitrogen metabolism, while the function and signal transduction pathway of AmtR in M. smegmatis remains largely unknown.

U-2012: An improved Lowry protein assay, insensitive to sample color, offering reagent stability and enhanced sensitivity.

Traditional colorimetric protein assays such as Biuret, Lowry, and modified Lowry (U-1988) are unsuitable for colored biological samples. Here we describe an improved Lowry protein assay (U-2012), which utilizes stable reagents and offers enhanced sensitivity over the U-1988 assay. U-2012 circumvents interference from colored pigments and other substances (for example sugars) bound to perchloric acid (PCA) precipitated proteins by hydrogen peroxide (H2O2) induced oxidation at 50°C.