Biology subjects

Fleming, P.

Publications and source records attributed to Fleming, P..

5 recordsLinked to original sources

bioRxiv2025

Pseudomonas superinfection drives Pf phage transmission within airway infections in patients with cystic fibrosis

Pf bacteriophages, lysogenic viruses that infect Pseudomonas aeruginosa (Pa), are implicated in the pathogenesis of chronic Pa infections; phage-infected (Pf+) strains are known to predominate in people with cystic fibrosis (pwCF) who are older and have more severe disease. However, the transmission patterns of Pf underlying the progressive dominance of Pf+ strains are unclear. In particular, it is unknown whether phage transmission commonly occurs horizontally between bacteria within the airway via viral particles or if Pf+ bacteria are mostly acquired via new Pseudomonas infections. Here, we have studied Pa genomic sequences from 3 patient cohorts totaling 663 clinical isolates from 105 pwCF. We identify Pf+ isolates and analyze transmission patterns of Pf within patients between genetically similar groups of bacteria called "clone types". We find that Pf is predominantly passed down vertically within Pa lineages and rarely via horizontal transfer between clone types within the airway. Conversely, we find extensive evidence of Pa superinfection by a new, genetically distinct Pa that is Pf+. Finally, we find that clinical isolates show reduced activity of the type IV pilus and reduced susceptibility to Pf in vitro. These results cast new light on the transmission of virulence-associated phages in the clinical setting.

microbiology↗

bioRxiv2024

The Molecular Basis for Hydrodynamic Properties of PEGylated Human Serum Albumin

Polyethylene glycol conjugation provides a protective modification that enhances the pharmacokinetics and solubility of proteins for therapeutic use. A knowledge of the structural ensemble of these PEGylated proteins is necessary to understand the molecular details that contribute to their hydrodynamic and colligative properties. Because of the large size and dynamic flexibility of pharmaceutically important PEGylated proteins, the determination of structure is challenging. Here we demonstrate that structural ensembles, generated by coarse-grained simulations, can be analyzed with HullRad and used to predict sedimentation coefficients and concentration dependent hydrodynamic and diffusion nonideality coefficients of PEGylated proteins. A knowledge of these properties enhances the ability to design and analyze new modified protein therapeutics. STATEMENT OF SIGNIFICANCEProteins constitute a growing class of biotherapeutics. Chemical modification(s) with inert polymers are known to enhance the serum half-life and formulation of these biological therapeutics but the effects of modification on protein-protein interactions in solution have been difficult to predict. Here we describe methods for predicting the molecular basis for the hydrodynamic properties of polymer conjugated proteins that determine their solution behavior.

biophysics↗

bioRxiv2023

An adapted stem cell-derived microglia protocol for the study of microgliopathies and other neurological disorders

BackgroundAdult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a primary microgliopathy caused by pathogenic variants in the colony-stimulating factor 1 receptor (CSF1R) gene. Since CSF1R signaling is crucial for microglia development, survival and function, induced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial defects caused by ALSP patient-specific CSF1R variants. MethodsSerial modifications to an existing iMGL protocol were made, including but not limited to changes in growth factor combination to drive microglial differentiation, until successful derivation of microglia-like cells from an ALSP patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines, the quality of the new iMGL protocol was validated through cell yield assessment, measurement of microglia marker expression, transcriptomic comparison to primary microglia, and evaluation of inflammatory and phagocytic activities. Similarly, molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL. ResultsThe newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to primary human microglia and with higher phagocytic and inflammatory competence at [~]3-fold greater yield compared to the original protocol. Using this protocol, decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally, ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 (P2RY12) expression. Finally, ALSP patient-derived cells showed surprisingly high phagocytic capacity, which was associated with higher lysosomal content. ConclusionsWe optimized a pre-existing iMGL protocol, generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol, we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant, with preliminary characterization pointing toward functional alterations in migratory and phagocytic activities.

neuroscience↗

bioRxiv2019

Perinatal Inflammation Influences but Does Not Arrest Rapid Immune Development in Preterm Babies

Infection and infection-related complications are important causes of death and morbidity following preterm birth. Despite this, there is limited understanding of the development of the immune system in those born prematurely and how it is influenced by perinatal factors. To investigate this, we prospectively and longitudinally followed a cohort of babies born before 32 weeks of gestation. We demonstrated that preterm babies, including those born extremely prematurely, were capable of rapidly acquiring adult levels of immune functionality; that immune maturation appeared to occur independently of the developing microbiome, which was highly heterogeneous across different infants; and that the biggest drivers of change in the trajectory of perinatal immune development was exposure to infection in utero or post-natally. Conspicuously, a unifying factor among infants who developed infection despite their growing immune potentials was an inability to mount adequate T cell CXCL8 responses. Because this defect was present at birth, it may predict those babies likely to have poor clinical outcomes.

immunology↗

bioRxiv2018

Environmental DNA reveals the structure of phytoplankton assemblages along a 2900-km transect in the Mississippi River

The environmental health of aquatic ecosystems is critical to society, yet traditional assessments of water quality have limited utility for some bodies of water such as large rivers. Sequencing of environmental DNA (eDNA) has the potential to complement if not replace traditional sampling of biotic assemblages for the purposes of reconstructing aquatic assemblages and, by proxy, assessing water quality. Despite this potential, there has been little testing of the ability of eDNA to reconstruct assemblages and their absolute and relative utility to infer water quality metrics. Here, we reconstruct phytoplankton communities by amplifying and sequencing DNA from a portion of the 23S rRNA region from filtered water samples along a 2900-km transect in the Mississippi River. Across the entire length, diatoms dominated the assemblage (72.6%) followed by cryptophytes (8.7%) and cyanobacteria (7.0%). There were no general trends in the abundances of these major taxa along the length of the river, but individual taxon abundance peaked in different regions. For example, the abundance of taxa genetically similar to Melosira tropica peaked at approximately 60% of all reads 2750 km upstream from the Gulf of Mexico, while taxa similar to Skeletonema marinoi began to increase below the confluence with the Missouri River until it reached approximately 30% of the reads at the Gulf of Mexico. There were four main clusters of samples based on phytoplankton abundance, two above the confluence with the Missouri and two below. Phytoplankton abundance was a poor predictor of NH4 + concentrations in the water, but predicted 61% and 80% of the variation in observed NO3 -and PO4 3-concentrations, respectively. Phytoplankton richness increased with increasing distance along the river, but was best explained by phosphate concentrations and water clarity. Along the Mississippi transect, there was similar structure to phytoplankton and bacterial assemblages, indicating that the two sets of organisms are responding to similar environmental factors. In all, the research here demonstrates the potential utility of metabarcoding for reconstructing aquatic assemblages, which might aid in conducting water quality assessments.

ecology↗