Biology subjects

Drake, K.

Publications and source records attributed to Drake, K..

12 recordsLinked to original sources

bioRxiv2026

Network Formation Dynamics in Thiol-ene Crosslinked Hyaluronic Acid Hydrogels: Design Principles for In Vitro Tissue Models

Hydrogels are widely used as three-dimensional cell culture systems to understand the impact of cellular mechanotransduction for tissue engineering applications. Photoinitiated thiol-ene click chemistry is a commonly utilized hydrogel crosslinking mechanism that provides spatial and temporal control over hydrogel network formation and resulting mesh size and compressive properties. Despite historically documented efficiency as step-growth reactions, these reactions do not always proceed as predicted. To understand the impact of cell confinement and microenvironmental mechanics on cellular function, thiol-ene network formation must be thoroughly characterized. To this end, the objective of this work was to investigate the crosslinking dynamics to determine hydrogel network formation as assessed via mesh size and mechanical properties using a pentenoate-functionalized hyaluronic acid thiol-ene reaction. Hydrogel parameters including polymer concentration and thiol:-ene crosslinker molar ratio were modulated (4, 6, or 8 polymer weight percent and 0.15:1, 0.5:1, or 1:1 molar ratio of thiol groups to reactive -ene groups) to tune network properties including shear storage modulus and relative mesh size. Molecular Dynamics (MD) simulations were used to simulate the thiol-ene crosslinking reaction and establish a method for predicting thiol-ene reaction efficiency. Lastly, the feasibility of this hydrogel system for in vitro modeling was confirmed via assessment of metabolic activity of encapsulated primary human meniscal cells.

bioengineering↗

bioRxiv2025

Untangling the effects of within-host and between-host natural selection in viruses with applications to evolution of HIV-1 co-receptor usage

The fitness of a pathogen can be characterised at multiple levels corresponding to different stages of its life cycle, including replication within target cells (replicative capacity), immune evasion, and the capacity to infect new hosts (transmissibility). Pathogens evolve towards greater fitness within hosts, which can potentially compromise transmissibility. Typically, epidemiological studies for measuring pathogen fitness compute a single selection coefficient for pathogen variants that encompasses all stages of the pathogen life cycle. To address this problem, we develop a population-genetic model and software for the inference of multi-level selection coefficients which represent relative within-host fitness and transmissibility. Our approach builds on population genetic models previously applied to macro-evolution, whereby speciation and extinction rates are state-dependent. We relate the state-dependence in speciation and extinction rates to competing within- and between-host selection. Evolution of beneficial phenotypes within hosts may occur rapidly (higher speciation), but reduce rates of dispersal between hosts (higher extinction). This new coalescent-based method also accounts for highly nonlinear-population dynamics and variable sampling through time making it suitable for epidemiological studies. We derive the equilibrium proportion of a variant with multi-level selection under mutation-selection balance and show how this can be used in combination with the coalescent to greatly increase accuracy and precision of estimated selection coefficients. We characterise the accuracy of this method using a variety of birth-death and coalescent simulations before analysing a real dataset of HIV-1 genetic sequences from a large population-based sample of 4056 people living with HIV in Zambia. We quantified the multi-level relative fitness underlying HIV-1 co-receptor switching. In accordance with previous observations, we estimate that the relative transmissibility of X4-tropic virus is 9%, while within-host switching to X4-tropism is approximately seven-fold higher than switching to R5-tropism, indicating a strong within-host and between-host tradeoff of co-receptor usage. Our methods are available in an open-source R package musseco, providing a general-purpose tool for inferring multi-level selection coefficients for epidemiological sequence data. Author SummaryThe fitness of a pathogen can be characterised at each stage of its life cycle. However, epidemiological studies measuring pathogen fitness typically estimate a single value encompassing the entire life cycle. Pathogens evolve towards greater fitness within hosts, but this can compromise the capacity to infect new hosts. Our new method quantifies relative fitness at both levels. This method accounts for rapidly changing pathogen populations and variable sampling through time making it suitable for epidemiological studies. We tested our method using simulations and demonstrate its application by analysing real HIV-1 genetic sequences from people living with HIV in Zambia. This method sheds new light on long-standing questions about the relative fitness of HIV-1 variants which utilise different host-cell co-receptors. Using our new method, we estimate that the capacity of X4 variants to infect new people is just 9% of that of R5 variants, while within-host switching from R5 to X4 usage is seven times greater than the reverse switch. This indicates a strong within-host and between-host trade-off in co-receptor usage.

evolutionary biology↗

bioRxiv2024

Longitudinal prevalence and co-carriage of pathogens associated with nursing home acquired pneumonia in three long-term care facilities

Nursing home acquired pneumonia (NHAP), and its subset - aspiration-associated pneumonia, is a leading cause of morbidity and mortality among residents in long-term care facilities (LTCFs). Understanding colonization dynamics of respiratory pathogens in LTCF residents is essential for effective infection control. This study examines the longitudinal trends in prevalence, persistence, bacterial load, and co-colonization patterns of five respiratory pathogens in three LTCFs in Phoenix, Arizona. Anterior nares and oral swabs were collected every other week and tested using qPCR for Haemophilus influenzae, Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus, and Chlamydia pneumoniae. Weekly average positivity rates were 17.75% for H. influenzae (0% - 39.39%), 9.95% for P. aeruginosa (0% - 37.74%), 31.89% for S. pneumoniae (1.79% - 41.67%), and for 28.00% for S. aureus (0% - 55.36%). C. pneumoniae was not detected. H. influenzae and S. pneumoniae predominantly colonized the oral cavity, while P. aeruginosa and S. aureus predominantly colonized the nasal cavity. S. pneumoniae and S. aureus colonizations were significantly more persistent than H. influenzae and P. aeruginosa, with persistence correlating with significantly higher bacterial loads. Co-colonization did occur in [~]20% of positive samples, but appeared to be due to random chance. This study reveals distinct colonization patterns among respiratory pathogens in LTCF residents, highlighting differences in site-specific prevalence, persistence, and bacterial load. These findings underscore the importance of longitudinal monitoring to inform targeted infection control strategies in LTCFs.

microbiology↗

bioRxiv2024

RNA methylation controls stress granule formation and function during erythropoiesis

Stress granules (SGs) are crucial in RNA regulation, affecting cell fate and function. SGs contain RNAs, some of which can be methylated. We studied m6A RNA modifications during the human CD34+ HSPCs (hCD34+) differentiating into erythroid cells and found that mRNAs encoding many erythroid-specific proteins had decreased methylation during differentiation. Increased levels ALKBH5 demethylase during erythropoiesis controls the levels of the 3UTR methylation of these mRNAs. hCD34+ carrying ALKBH5 mutations demonstrated a block in erythropoiesis, and mass-spectrometry studies of the mutant cells showed decreased levels of SG proteins, including the core granule protein ATXN2. ALKBH5 directly regulates the methylation of the mRNA of ATXN2. ATXN2 overexpression accelerated the erythroid differentiation of HSPCs and rescued the erythroid differentiation of ALKBH5 mutant cells. Very few SGs are found in normal human erythroid progenitors. SGs accumulated substantially in ALKBH5 mutant cells, and surprisingly overexpression of ATNX2 reduced SG numbers to normal. Polysome analysis demonstrated m6A-modified RNAs to be enriched in the pre-polysome fractions that were less translated. This work establishes a mechanism by which during stress, ATXN2 facilitates the release of SG-stored m6A-modified RNAs including erythroid-specific and SG-enriched RNAs that are loaded onto functional ribosomes, allowing better translation and accelerated erythroid differentiation during stress.

cell biology↗

bioRxiv2024

High-throughput targeted amplicon screening tool for characterizing intrahost diversity in Staphylococcus aureus directly from sample

A significant proportion of people are asymptomatic carriers of Staphylococcus aureus (SA), an important risk factor for development of opportunistic infections. SA colonization is dynamic, appearing and disappearing, with strains evolving and potentially shifting in composition over time and between body sites. These changes make detection challenging and the numerous potential sources of reintroduction from other people and even other body site reservoirs preclude efficient efforts to prevent transmission and spread. Identifying typical sources is therefore critical for mitigation. Whole-genome sequencing (WGS), ideally of multiple colonies from multiple body sites, is the gold standard for characterizing SA strains and confirming transmission. However, this is often too resource-intensive for initial assessments of transmission and not feasible for large-scale studies involving various body sites from multiple individuals over time. To address these challenges, we developed a low-cost, custom, species-specific amplicon sequencing (AmpSeq) assay, optimized to provide high resolution discrimination of SA genotypes directly from samples. We tested this approach on a subset of samples that were a part of a large-scale longitudinal study of SA carriage. Oral and nasal samples were collected from 9 participants every two weeks for up to 18 weeks and qPCR positive samples were analyzed using our AmpSeq assay directly from the sample without culturing. The longitudinal sampling strategy enabled us to characterize changes in SA colonization patterns over time, detect potential strain mixtures, and identify rare variants that may serve as signatures of transmission between different body sites or among individuals. Without using WGS, we were able to rapidly eliminate the possibility of transmission between sampled residents. Participants that had positive oral and nasal samples had no fixed SNP differences between the two body sites, suggesting likely within-person spread. In these cases, we were able to infer the most likely direction of spread (nasal to oral sites) by analyzing segregating rare variants. While WGS can be used to provide higher resolution to colonization patterns and validate these findings, our amplicon sequencing approach offers a rapid, cost-effective, direct-from-sample method for species-specific screening intended for population-level characterization that allows researchers to characterize strain types, identify or eliminate likely transmission cases, and identify potential reservoirs before resorting to more expensive WGS methods. Authors summaryColonizing opportunistic pathogens like Staphylococcus aureus present a unique challenge for disease study because rather than causing acute infections upon transmission, they persist asymptomatically for long periods of time allowing the bacterial population to evolve and differentiate. Characterizing the diversity within these populations is important for choosing correct treatments, quantifying the risk of horizontal gene transfer, and understanding paths of transmission between people and spread to different body sites. The gold-standard approach for characterizing population diversity is through culturing and whole-genome sequencing of multiple colonies per sample which is labor-intensive and expensive for any large-scale study. Using a custom-designed species-specific amplicon sequencing assay, we offer a cost-effective method for characterizing the diversity in Staphylococcus aureus populations directly from samples without the need for labor-intensive culturing or whole-genome sequencing. Our small-scale study highlights how this method provides a scalable tool for large epidemiological studies ideal for systematically exploring broader patterns of carriage and transmission.

genomics↗

bioRxiv2022

Minor intron containing genes: Achilles' heel of viruses?

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed the worlds unpreparedness to deal with the emergence of novel pathogenic viruses, pointing to the urgent need to identify targets for broad-spectrum antiviral strategies. Here, we report that proteins encoded by Minor Intron-containing Genes (MIGs) are significantly enriched in datasets of cellular proteins that are leveraged by SARS-CoV-2 and other viruses. Pointing to a general gateway for viruses to tap cellular machinery, MIG-encoded proteins (MIG-Ps) that react to the disruption of the minor spliceosome are most important points of viral attack, suggesting that MIG-Ps may pan-viral drug targets. While contemporary anti-viral drugs shun MIG-Ps, we surprisingly found that anti-cancer drugs that have been repurposed to combat SARS-CoV-2, indeed target MIG-Ps, suggesting that such genes can potentially be tapped to efficiently fight viruses.

systems biology↗

bioRxiv2021

Minor intron splicing efficiency increases with the development of lethal prostate cancer

Here we explored the role of minor spliceosome (MiS) function and minor intron-containing gene (MIG) expression in prostate cancer (PCa). We show MIGs are enriched as direct interactors of cancer-causing genes and their expression discriminates PCa progression. Increased expression of MiS U6atac snRNA, including others, and 6x more efficient minor intron splicing was observed in castration-resistant PCa (CRPC) versus primary PCa. Notably, androgen receptor signalling influenced MiS activity. Inhibition of MiS through siU6atac in PCa caused minor intron mis-splicing and aberrant expression of MIG transcripts and encoded proteins, which enriched for MAPK activity, DNA repair and cell cycle. Single cell-RNAseq confirmed cell cycle defects and lineage dependency on the MiS from primary to CRPC and neuroendocrine PCa. siU6atac was [~]50% more efficient in lowering tumor burden of CRPC cells and organoids versus current state-of-the-art combination therapy. In all, MiS is a strong therapeutic target for lethal PCa and potentially other cancers. Graphical AbstractU6atac expression, MiS activity, and minor intron splicing correlate with PCa therapy resistance and PCa progression to CRPC-adeno and transdifferentiation to CRPC-NE. One major MiS regulator during that process is the AR-axis, which is re-activated during CRPC-adeno and blocked in CRPC-NE. Molecularly, an increase in MiS dependent splicing promotes changes of transcriptome and proteome. This results in cell cycle activation, increased MAPK signalling and increased DNA repair. U6atac mediated MiS inhibition renders MiS splicing error-prone through increased intron retention and alternative splicing events, which results in cell cycle block and decreased MAPK signalling and DNA repair. MiS inhibition blocks all stages of PCa. Figure created with BioRender.com. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/471104v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@1c1c296org.highwire.dtl.DTLVardef@1ce1416org.highwire.dtl.DTLVardef@88d842org.highwire.dtl.DTLVardef@2ddf96_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2020

Characterization of interstitial heterogeneity in the developing kidney

Kidney formation requires the coordinated growth of multiple cell types including the collecting ducts, nephrons, vasculature and interstitium. There has been a long-held belief that interactions between the progenitors of the collecting ducts and nephrons are primarily responsible for development of this organ. However, over the last several years, it has become increasingly clear that multiple aspects of kidney development require signaling from the interstitium. How the interstitium orchestrates these various roles is still poorly understood. We show that during development, the interstitium is a highly heterogeneous, patterned population of cells that occupies distinct positions correlated to the adjacent parenchyma. Our analysis indicates that the heterogeneity is not a mere reflection of different stages in a linear developmental trajectory but instead represents several novel differentiated cell states. Further, we find that beta-catenin has a cell autonomous role in the development of a medullary subset of the interstitium and that this non-autonomously affects the development of the adjacent epithelia. These findings suggest the intriguing possibility that the different interstitial subtypes may create microenvironments that play unique roles in development of the adjacent epithelia and endothelia. Graphical AbstractThe developing interstitium is a highly heterogeneous, patterned population of cells that occupies distinct positions correlated to the adjacent parenchyma. O_FIG O_LINKSMALLFIG WIDTH=161 HEIGHT=200 SRC="FIGDIR/small/973966v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1c79dc3org.highwire.dtl.DTLVardef@469934org.highwire.dtl.DTLVardef@f43787org.highwire.dtl.DTLVardef@1698bd6_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2019

Single Cell Profiling of Acute Kidney Injury Reveals Novel Transcriptional Signatures, Mixed Identities and Epithelial-to-Stromal Crosstalk

Acute kidney injury (AKI) is a rapid decline of renal function, with an incidence of up to 67% of intensive care unit patients. Current treatments are merely supportive, emphasizing the need for deeper understanding that could lead to improved therapies. We used single cell RNA sequencing, in situ hybridization and protein expression analyses to create comprehensive renal cell specific transcriptional profiles of multiple AKI stages. We revealed that AKI induces marked dedifferentiation, renal developmental gene activation and mixed identities in injured renal tubules. Moreover, we identified potential pathologic crosstalk between epithelial and stromal cells, and several novel genes involved in AKI. We also demonstrated the definitive effects of age on AKI outcome, and showed that renal developmental genes hold a potential as novel AKI markers. Moreover, our study provides the resource power which will aid in unraveling the molecular genetics of AKI.

developmental biology↗

bioRxiv2019

A Bigenic mouse model of FSGS reveals perturbed pathways in podocytes, mesangial cells and endothelial cells

Focal segmental glomerulosclerosis is a major cause of end stage renal disease. Many patients prove unresponsive to available therapies. An improved understanding of the molecular basis of the disease process could provide insights leading to novel therapeutic approaches. In this study we carried out an RNA-seq analysis of the altered gene expression patterns of podocytes, mesangial cells and glomerular endothelial cells of the bigenic Cd2ap+/-, Fyn-/- mutant mouse model of FSGS. In the podocytes we observed upregulation of many genes related to the Tgf{beta} family/pathway, including Gdnf, Tgf{beta}1, Tgf{beta}2, Snai2, Vegfb, Bmp4, and Tnc. The mutant podocytes also showed upregulation of Acta2, a marker of smooth muscle and associated with myofibroblasts, which are implicated in driving fibrosis. GO analysis of the podocyte upregulated genes identified elevated protein kinase activity, increased expression of growth factors, and negative regulation of cell adhesion, perhaps related to the observed podocyte loss. Both podocytes and mesangial cells showed strong upregulation of aldehyde dehydrogenase genes involved in the synthesis of retinoic acid. Similarly, the Cd2ap+/-, Fyn-/- mesangial cells, as well as podocytes in other genetic models, and the glomeruli of human FSGS patients, all show upregulation of the serine protease Prss23, with the common thread suggesting important functionality. Another gene with strong upregulation in the Cd2ap+/-, Fyn-/- mutant mesangial cells as well as multiple other mutant mouse models of FSGS was thrombospondin, which activates the secreted inactive form of Tgf{beta}. The Cd2ap+/-, Fyn-/- mutant endothelial cells showed elevated expression of genes involved in cell proliferation, angioblast migration, angiogenesis, and neovasculature, all consistent with the formation of new blood vessels in the diseased glomerulus. The resulting global definition of the perturbed molecular pathways in the three major cell types of the mutant glomerulus provide deeper understanding of the molecular pathogenic pathways.

pathology↗

bioRxiv2018

Multivariate Computational Analysis of Gamma Delta T cell Inhibitory Receptor Signatures Reveals the Divergence of Healthy and ART-Suppressed HIV+ Aging

Even with effective viral control, HIV-infected individuals are at a higher risk for morbidities associated with older age than the general population, and these serious non-AIDS events (SNAEs) track with plasma inflammatory and coagulation markers. The cell subsets driving inflammation in aviremic HIV infection are not yet elucidated. Also, whether ART-suppressed HIV infection causes premature induction of the inflammatory events found in uninfected elderly or if a novel inflammatory network ensues when HIV and older age co-exist is unclear. In this study we measured combinational expression of five inhibitory receptors (IRs) on seven immune cell subsets and 16 plasma markers from peripheral blood mononuclear cells (PBMC) and plasma samples, respectively, from a HIV and Aging cohort comprised of ART-suppressed HIV-infected and uninfected controls stratified by age ([≤]35 or [≥]50 years old). For data analysis, multiple multivariate computational algorithms (cluster identification, characterization, and regression (CITRUS), partial least squares regression (PLSR), and partial least squares-discriminant analysis (PLS-DA)) were used to determine if immune parameter disparities can distinguish the subject groups and to investigate if there is a cross-impact of aviremic HIV and age on immune signatures. IR expression on gamma delta ({gamma}{delta}) T cells exclusively separated HIV+ subjects from controls in CITRUS analyses and secretion of inflammatory cytokines and cytotoxic mediators from {gamma}{delta} T cells tracked with TIGIT expression among HIV+ subjects. Also, plasma markers predicted the percentages of TIGIT+ {gamma}{delta} T cells in subjects with and without HIV in PSLR models, and a PLS-DA model of {gamma}{delta} T cell IR signatures and plasma markers significantly stratified all four of the subject groups (uninfected younger, uninfected older, HIV+ younger, and HIV+ older). These data implicate {gamma}{delta} T cells as an inflammatory driver in ART-suppressed HIV infection and provide evidence of distinct inflamm-aging processes with and without ART-suppressed HIV infection.

immunology↗

bioRxiv2017

Minor spliceosome inactivation in the developing mouse cortex causes self-amplifying radial glial cell death and microcephaly.

Inactivation of the minor spliceosome has been linked to microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1). To interrogate how minor intron splicing regulates cortical development, we employed Emx1-Cre to ablate Rnu11, which encodes the minor spliceosome-specific U11 small nuclear RNA (snRNA), in the developing cortex (pallium). Rnu11 cKO mice were born with microcephaly, caused by death of self-amplifying radial glial cells (RGCs). However, both intermediate progenitor cells (IPCs) and neurons were produced in the U11-null pallium. RNAseq of the pallium revealed elevated minor intron retention in the mutant, particularly in genes regulating cell cycle. Moreover, the only downregulated minor intron-containing gene (MIG) was Spc24, which regulates kinetochore assembly. These findings were consistent with the observation of fewer RGCs entering cytokinesis prior to RGC loss, underscoring the requirement of minor splicing for cell cycle progression in RGCs. Overall, we provide a potential explanation of how disruption of minor splicing might cause microcephaly in MOPD1.\n\nSummary StatementHere we report the first mammalian model to investigate the role of the minor spliceosome in cortical development and microcephaly.\n\nList of abbreviations usedMOPD1=microcephalic osteodysplastic primordial dwarfism type 1; snRNA=small nuclear RNA; cKO=conditional knockout; NPC=neural progenitor cell; RGC=radial glial cell; IPC=intermediate progenitor cell; MIG=minor intron-containing gene

developmental biology↗