Biology subjects

O'Donnell, A.

Publications and source records attributed to O'Donnell, A..

7 recordsLinked to original sources

bioRxiv2026

Design to Data for Mutant of β-Glucosidase B from Paenibacillus polymyxa: G23S

{beta}-glucosidase (BglB) from Paenibacillus polymyxa was mutated (G23S, Rosetta/Foldit numbering; G26S, conventional numbering) to assess structural and functional changes. Foldit modeling and prior Design 2 Data (D2D) database results led us to hypothesize that this mutation would increase substrate binding affinity and catalytic efficiency, with a moderate reduction in thermal stability. The mutant protein was expressed, purified, and analyzed using kinetics and thermal stability assays. Relative to the wild-type (WT), G23S exhibited a similar binding affinity (similar Km), an approximately 2-fold increase in turnover number (kcat) and catalytic efficiency (kcat/Km), an almost 14-fold increase in maximum reaction velocity (Vmax) and a slight decrease in thermostability (T50). The results largely support the hypothesis, indicating that changes in residue 23 can enhance catalytic power while minimally compromising stability.

biochemistry↗

bioRxiv2026

DNA Adenine Methylation Clock in Brain Aging and Alzheimer's Disease Progression

N6-methyldeoxyadenosine (N6medA) is a recently identified endogenous DNA modification widely found in bacteria, plants, and eukaryotes. In mammals, N6medA has been implicated in brain function, immunity, and response to environmental stress, but its relevance to gene regulation and mammalian aging remains controversial due to its extremely low abundance (< 1 per 10 million adenines) and an uncertainty regarding its genomic origin. We have developed and validated an ultrasensitive isotope dilution nano liquid chromatography-nanospray ionization Orbitrap mass spectrometry methodology to quantify N6medA in genomic DNA. Applying this approach to human prefrontal cortex tissues, we found that genomic N6medA levels increase linearly with chronological age (Pearson correlation coefficient, 0.95). Individuals with mild cognitive impairment (MCI) and Alzheimers disease (AD) exhibited a trend toward elevated cortical N6medA levels relative to age-matched controls. Genome-wide profiling of N6medA in human prefrontal cortex was conducted using two independent methods: NAME-Seq and MeDIP-Seq, which revealed age associated adenine methylation changes reminiscent of established epigenetic aging signatures such as the 5-methylcytosine clocks. N6medA mapping experiments identified a subset of genomic loci that were altered in MCI and AD. Pathway analysis of cross-validated adenine methylation sites revealed an enrichment of genes involved in neuronal function and age-related neurological processes, including glutamatergic synapse, axon guidance, and long-term depression. Finally, mass-spectrometry-based photoaffinity proteomics with synthetic DNA representing a region of the APP gene identified N6medA reader proteins with known roles in DNA repair, replication and transcription. Together, these findings identify N6medA as an age-associated DNA modification in the human brain and suggest that its accumulation and recognition by specific protein readers may contribute to molecular processes underlying brain aging and age-related neurodegeneration.

biochemistry↗

bioRxiv2023

Adapting antibacterial display to identify serum active macrocyclic peptide antibiotics

The lack of available treatments for many antimicrobial resistant infections highlights the critical need for antibiotic discovery innovation. Peptides are an underappreciated antibiotic scaffold because they often suffer from proteolytic instability and toxicity towards human cells, making in vivo use challenging. To investigate sequence factors related to serum activity, we adapt an antibacterial display technology to screen a library of peptide macrocycles for antibacterial potential directly in human serum. We identify dozens of new macrocyclic peptide antibiotic sequences and find that serum activity within our library is influenced by peptide length, cationic charge, and the number of disulfide bonds present. Interestingly, an optimized version of our most active lead peptide permeates the outer membrane of gram-negative bacteria without strong inner membrane disruption and kills bacteria slowly while causing cell elongation. This contrasts with traditional cationic antimicrobial peptides, which kill rapidly via lysis of both bacterial membranes. Notably, this optimized variant is not toxic to mammalian cells and retains its function in vivo, suggesting therapeutic promise. Our results support the use of more physiologically relevant conditions when screening peptides for antimicrobial activity which retain in vivo functionality. SignificanceTraditional methods of natural antibiotic discovery are low throughput and cannot keep pace with the development of antimicrobial resistance. Synthetic peptide display technologies offer a high-throughput means of screening drug candidates, but rarely consider functionality beyond simple target binding and do not consider retention of function in vivo. Here, we adapt a function-based, antibacterial display technology to screen a large library of peptide macrocycles directly for bacterial growth inhibition in human serum. This screen identifies an optimized non-toxic macrocyclic peptide antibiotic retaining in vivo function, suggesting this advancement could increase clinical antibiotic discovery efficiency.

microbiology↗

bioRxiv2023

PROX1 inhibits PDGF-B expression to prevent myxomatous degeneration of heart valves

BackgroundCardiac valve disease (CVD) is observed in 2.5% of the general population and 10% of the elderly people. Effective pharmacological treatments are currently not available, and patients with severe CVD require surgery. PROX1 and FOXC2 are transcription factors that are required for the development of lymphatic and venous valves. We found that PROX1 and FOXC2 are expressed in a subset of valvular endothelial cells (VECs) that are located on the downstream (fibrosa) side of cardiac valves. Whether PROX1 and FOXC2 regulate cardiac valve development and disease is not known. MethodsWe used histology, electron microscopy and echocardiography to investigate the structure and functioning of heart valves from Prox1{Delta}VEC mice in which Prox1 was conditionally deleted from VECs. Isolated valve endothelial cells and valve interstitial cells were used to identify the molecular mechanisms in vitro, which were tested in vivo by RNAScope, additional mouse models and pharmacological approaches. The significance of our findings was tested by evaluation of human samples of mitral valve prolapse (MVP) and aortic valve insufficiency. ResultsHistological analysis revealed that the aortic and mitral valves of Prox1{Delta}VEC mice become progressively thick and myxomatous. Echocardiography revealed that the aortic valves of Prox1{Delta}VEC mice are stenotic. FOXC2 was downregulated and platelet-derived growth factor-B (PDGF-B) was upregulated in the VECs of Prox1{Delta}VEC mice. Conditional knockdown of FOXC2 and conditional overexpression of PDGF-B in VECs recapitulated the phenotype of Prox1{Delta}VEC mice. PDGF-B was also increased in mice lacking FOXC2 and in human MVP and insufficient aortic valve samples. Pharmacological inhibition of PDGF-B signaling with imatinib partially ameliorated the valve defects of Prox1{Delta}VEC mice. ConclusionPROX1 antagonizes PDGF-B signaling partially via FOXC2 to maintain the extracellular matrix composition and prevent myxomatous degeneration of cardiac valves. Novelty and SignificanceWhat Is Known? O_LIThe transcription factors PROX1 and FOXC2 are critical regulators of lymphatic and venous valve development. C_LIO_LIPROX1 and FOXC2 are expressed in the downstream valvular endothelial cells of heart valves. C_LI What Is New? O_LIDeletion of Prox1 from the valvular endothelial cells of mice results in enlarged and myxomatous aortic and mitral valves. Aortic valves of the mutant (Prox1{Delta}VEC) mice were stenotic. C_LIO_LIFOXC2 is partially responsible for the phenotype of Prox1{Delta}VEC mice. C_LIO_LIPROX1 and FOXC2 inhibit the expression of the cytokine PDGF-B in heart valves. C_LIO_LIHyperactivation of PDGF-B signaling results in aortic and mitral valve thickening. C_LIO_LIInhibition of PDGF-B signaling ameliorates aortic valve stenosis in Prox1{Delta}VEC mice. C_LIO_LIPDGFB is overexpressed and PROX1 is downregulated in human mitral valve prolapse (MVP) samples. C_LI Our findings suggest that PROX1 is an inhibitor of myxomatous valve disease that afflicts ~10% of the elderly population. We have also identified PDGF-B as a potential target for treating myxomatous valve disease.

developmental biology↗

bioRxiv2021

Complexities in the role of acetylation dynamics in modifying inducible gene activation parameters

High levels of histone acetylation are associated with the regulatory elements of active genes, suggesting a link between acetylation and gene activation. However, several studies have shown that histone acetylation dynamics rather than hyperacetylation per se are important determinants in gene activation, particularly at inducible genes. We revisited this model, in the context of EGF-inducible gene expression and found that rather than a simple unifying model, there are two broad classes of genes; one in which high lysine acetylation activity is required for efficient gene activation, and a second group where the opposite occurs and high acetylation activity is inhibitory. We examined the latter class in more detail using EGR2 as a model gene and found that lysine acetylation levels are critical for several activation parameters, including the timing of expression onset, and overall amplitudes of the transcriptional response. In contrast, DUSP1 responds in the canonical manner and its transcriptional activity is promoted by acetylation. Single cell approaches demonstrate heterogenous DUSP1 activation kinetics and that acetylation levels influence allele activation frequencies. Our data therefore point to a complex interplay between acetylation dynamics and target gene induction, which cannot simply be explained by a unified response to acetylation activity. Instead, acetylation level thresholds are an important determinant of transcriptional induction dynamics that are sensed in a gene-specific manner.

molecular biology↗

bioRxiv2020

Vascular dimorphism ensured by regulated proteoglycan dynamics favors rapid umbilical artery closure at birth

The umbilical artery lumen occludes rapidly at birth, preventing blood loss, whereas the umbilical vein remains patent, providing the newborn with a placental infusion. Here, we identify differential arterial-venous proteoglycan dynamics as a determinant of these contrasting vascular responses. We show that the umbilical artery, unlike the vein, has an inner layer enriched in the hydrated proteoglycan aggrecan, external to which lie contraction-primed smooth muscle cells (SMC). At birth, SMC contraction drives inner layer buckling and centripetal displacement to occlude the arterial lumen, a mechanism elicited by biomechanical and computational analysis. Vascular dimorphism arises from spatially regulated proteoglycan expression and breakdown in umbilical vessels. Mice lacking aggrecan or the metalloprotease ADAMTS1, which degrades proteoglycans, demonstrated their opposing roles in umbilical cord arterial-venous dimorphism and contrasting effects on SMC differentiation. Umbilical vessel dimorphism is conserved in mammals, suggesting that their differential proteoglycan dynamics were a positive selection step in mammalian evolution.Competing Interest StatementThe authors have declared no competing interest.View Full Text

developmental biology↗

bioRxiv2019

The versican-hyaluronan complex provides an essential extracellular matrix niche for Flk1+ precursors in vasculogenesis and primitive hematopoiesis

Little is known about extracellular matrix (ECM) contributions to formation of the earliest cell lineages in the embryo. Here, we show that the proteoglycan versican and glycosaminoglycan hyaluronan are associated with emerging Flk1+ hematoendothelial progenitors at gastrulation. The mouse versican mutant Vcanhdf lacks yolk sac vasculature, with attenuated yolk sac hematopoiesis. CRISPR/Cas9-mediated Vcan inactivation in mouse embryonic stem cells reduced vascular endothelial and hematopoietic differentiation in embryoid bodies, which generated fewer blood colonies, and had an impaired angiogenic response to VEGF165. HA was severely depleted in Vcanhdf embryos, with corresponding increase in the HA-depolymerase TMEM2. Conversely, HA-deficient mouse embryos also had vasculogenic suppression but with increased versican proteolysis. VEGF165 and Indian hedgehog, crucial vasculogenic factors, utilized the versican-HA matrix, specifically versican chondroitin sulfate chains, for binding. Versican-HA ECM is an obligate requirement for vasculogenesis and primitive hematopoiesis, acts as an vasculogenic factor-enriching microniche for Flk1+ progenitors from their origin at gastrulation.

developmental biology↗