Biology subjects

Smith, N. M.

Publications and source records attributed to Smith, N. M..

6 recordsLinked to original sources

bioRxiv2026

Translational Quantitative Proteomic Assay for Bacteriophages: A New Frontier in Phage Pharmaceutical Development

Accurate quantitation of therapeutic bacteriophages (phages) remains a challenge for clinical development. Plaque-based enumeration is the current standard but is laborious, host-dependent, and variable, particularly when distinguishing individual phages in cocktails. Targeted mass spectrometry of virion structural proteins offers an orthogonal, structure-based approach amenable to reproducible and scalable phage quantitation. Here, we describe a targeted proteomic liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for host-independent quantitation of the Pseudomonas aeruginosa podovirus LUZ19. Proteomic characterization was performed on an LTQ Orbitrap XL to assess sequence coverage and select surrogate peptide candidates based on specificity and sensitivity. High-resolution peptide mapping identified multiple structural proteins of LUZ19 and provided 55% sequence coverage for the major head protein (YP_001671977.1). Fifteen peptides were detected and evaluated, from which the tryptic peptide EVAELDGQELAR was selected based on abundance, stability, and chromatographic performance. Quantitative analysis was conducted on a QTRAP 7500+ using optimized multiple reaction monitoring transitions for targeted peptide detection. Back-calculated concentrations met accuracy criteria across a validated range of 0.008 to 80 pg/mL, with bias spanning -8.2 to 8.2%, intra-day precision ranging from 0.5 to 9.8%, and inter-day precision ranging from 6.3 to 9.7%. Peptide concentrations from digested lysate samples were related to phage concentrations determined by double layer agar assay, yielding an estimated three copies of the major head protein per virion. ImportanceBacteriophages are the most abundant biological entities on the planet and represent a promising therapeutic class for combating drug-resistant bacterial infections. Realizing the clinical potential of bacteriophage therapy requires analytical methods capable of meeting the standards of modern drug development. Targeted mass spectrometry offers unmatched specificity and resolution for precise quantitation of individual bacteriophages within complex biological samples, a capability that conventional enumeration methods cannot match. Only one prior study has applied mass spectrometry to bacteriophage quantitation, using a well-characterized model bacteriophage at a single concentration without calibration or a validated analytical range. Using Pseudomonas aeruginosa podovirus LUZ19, we present the first targeted mass spectrometry-based bacteriophage quantitation assay developed and validated following FDA bioanalytical guidance. This work establishes a rigorous analytical foundation that moves bacteriophage therapy closer to the standards required for informed dose selection, candidate evaluation, and clinical development.

pharmacology and toxicology↗

bioRxiv2023

Polymer-mediated oligonucleotide delivery enables construction of barcoded 3D cultures for spatial single-cell analysis

Spatial transcriptomics has been widely used to capture gene expression profiles, realised as a two-dimensional (2D) projection of RNA captured from tissue sections. Wree-dimensional (3D) cultures such as spheroids and organoids are highly promising alternatives to oversimplified and homogeneous 2D cell culture models, but existing spatial transcriptomic platforms do not currently have sufficient resolution for robust analysis of 3D cultures. We present a transfection-based method for fluorescent DNA barcoding of cell populations, and the subsequent construction of spheroidal cellular architectures using barcoded cells in a layer-by-layer approach. For the first time, changes in gene expression throughout this 3D culture architecture are interrogated using multiplex single-cell RNA sequencing in which DNA barcodes are used to encode the spatial positioning of cells. We show that transfection with fluorophore-conjugated barcode oligonucleotides enables both imaging and sequencing at single-cell resolution, providing spatial maps of gene expression and drug response. Additionally, we show that fluorophore-conjugated DNA barcodes support correlative imaging studies such as mechano-microscopy to capture information about spatially-varying mechanical heterogeneity in 3D cultures. We ability to create customised, encoded cellular assemblies is a general approach that can resolve spatial differences in gene expression in 3D cell culture models.

bioengineering↗

bioRxiv2023

Metastable Intermediates Identified in Epithelial to Mesenchymal Transition are Regulated by G-Quadruplex DNA Structures

Cancer is a heterogenous disease, with multiple cellular subpopulations present within a single tumour mass that differ genetically and morphologically, and thus respond differently to chemotherapeutics. Epithelial-to-Mesenchymal transition (EMT) has been shown to play a role in tumour heterogeneity. Single-cell sequencing is critical to identify cell-type-specific transcriptomic differences with multiplexing methods increasing experimental scope with reduced cost. Cell hashing with barcoded antibodies is commonly used to multiplex samples but is limited to samples expressing target antigens. Antigen-independent methods of barcoding cells, such as barcoded lipid-anchors, have gained traction but present substantial populations that cannot be unambiguously demultiplexed. Herein we report a multiplexed single-cell transfection-enabled cell hashing sequencing (scTECH-seq) platform, which uses antigen-independent endocytic uptake to barcode cells, resulting in efficient, uniform barcoding with high cell recovery. We apply this methodology to identify distinct metastable cell states in human mammary cells undergoing EMT and show that stabilisation of G-quadruplex DNA has the potential to inhibit EMT.

genomics↗

bioRxiv2022

The imprinted Mir483 is a growth suppressor and metabolic regulator functioning through IGF1

Mir483 is a conserved and highly expressed microRNA in placental mammals, embedded within the Igf2 gene. Here, we uncover the control mechanisms and physiological functions of Mir483 in vivo, by generating constitutive loss-of-function and over-expressing mice. Mir483 expression is imprinted and dependent on the Igf2 promoters and Igf2/H19 imprinting control region. Over-expression of Mir483 causes severe mid-gestation fetal, but not placental, growth restriction, and late lethality. Fetal death is prevented by restoring Mir483 to endogenous levels using an inducible transgenic system. Continuous postnatal Mir483 over-expression induces growth stunting, elevated hepatic lipid content, increased adiposity, reduced local and systemic IGF1 levels and increased GH. The growth phenotypes are rescued by IGF1 infusion. Our findings provide evidence for a novel functional antagonism between a growth-suppressor microRNA and its growth-promoter host gene, and suggest that Mir483 evolved to limit excessive tissue growth through repression of IGF ligand signalling.

developmental biology↗

bioRxiv2022

Human genomic DNA is widely interspersed with i-motif structures

DNA i-motif structures are formed in the nucleus of human cells and are believed to provide critical genomic regulation. While the existence of i-motif structures in human cells has been demonstrated by immunofluorescent staining and by characterisation of select model genes, the abundance and distribution of such structures in the human genome has remained unclear. Here we utilize high affinity i-motif immunoprecipitation followed by sequencing to map i-motifs in human genomic DNA. Validated by biolayer interferometry and circular dichroism spectroscopy, our approach identified over 650,000 i-motif structures in human genomic DNA. The i-motif structures are widely distributed throughout the human genome and are common among highly expressed genes and in genes upregulated in G0/G1 cell cycle phase. Our findings provide experimental evidence for the widespread formation of i-motif structures in human genomic DNA.

genetics↗

bioRxiv2019

Mesenchymal Igf2 is a major paracrine regulator of pancreatic growth and function

The genetic mechanisms that determine the size of the adult pancreas are poorly understood. Here we demonstrate that many imprinted genes are highly expressed in the pancreatic mesenchyme, and explore the role of Igf2 in-vivo. Mesenchyme-specific Igf2 deletion results in acinar and beta-cell hypoplasia, postnatal whole-body growth restriction and maternal glucose intolerance during pregnancy. Surprisingly, mesenchymal mass is unaffected, suggesting that the mesenchyme is a developmental reservoir of IGF2 used for paracrine signalling. The unique actions of mesenchymal IGF2 are demonstrated by the absence of phenotypes upon Igf2 deletion in the developing pancreatic epithelium. Furthermore, increased IGF2 activity specifically in the mesenchyme, through Igf2 loss-of-imprinting or Igf2r deletion, leads to pancreatic acinar overgrowth. Ex-vivo exposure of primary acinar cells to exogenous IGF2 increases cell proliferation and amylase production through AKT signalling. We propose that mesenchymal Igf2, and perhaps other imprinted genes, are key developmental regulators of adult pancreas size and function.

developmental biology↗