Biology subjects

Clark, F.

Publications and source records attributed to Clark, F..

12 recordsLinked to original sources

bioRxiv2026

Lymphangiogenesis is Critical for Healing and Survival in a Murine Model of Laryngotracheal Injury

Objective: Lymphatic dysfunction has been implicated in exacerbating fibrosis in numerous diseases, yet the role of the lymphatic system in laryngotracheal injury has not previously been explored. This study aims to evaluate lymphatic vascular remodeling in a murine model of laryngotracheal stenosis (LTS) and determine how pharmacologic blockade of lymphangiogenesis impacts airway healing after mucosal injury. Methods: LTS was induced in C57BL6 mice using an established chemomechanical injury model. Lymphatic density was quantified using LYVE-1 immunohistochemistry. Mice were treated with the VEGFR-3-selective tyrosine kinase inhibitor SAR131675 to block lymphangiogenesis after injury. Outcomes assessed included survival, histopathology, immunohistochemistry, and Evans blue dye vascular leakage. Results: Laryngotracheal injury induced a substantial increase in subepithelial lymphatic vessel density concomitant with fibrotic remodeling. Pharmacologic inhibition of VEGFR-3 signaling with SAR131675 abrogated this lymphangiogenic response and resulted in markedly increased mortality, impaired epithelial repair with obstructive sloughing, increased edema, and persistent histopathologic evidence of tissue injury. A qualitative increase in pathologic fibrocellular remodeling was also observed, though with no measurable difference in lamina propria thickness. Conclusion: These findings establish lymphatic remodeling as an essential component of successful airway repair following mucosal injury. Lymphatic dysfunction is a common feature of known risk factors for LTS including diabetes, obesity, and prematurity, and can be exacerbated by positive pressure ventilation. Disruption of the lymphangiogenic response to airway injury may lead to stasis of pro-inflammatory factors that result in chronic inflammation, maladaptive remodeling, and pathologic tissue changes. The lymphatic vasculature is a viable target for future mechanistic study and potential therapeutic intervention following airway injury.

physiology↗

bioRxiv2025

Predicting Human Pharmacokinetic Parameters of Drugs using a Multi-Tissue Chip Platform Integrating Liver, Kidney, and Skeletal Muscle Microphysiological Systems

Accurate prediction of human pharmacokinetic (PK) parameters remains a critical challenge in drug discovery. While simple in vitro assays provide insight into discrete metabolic processes, they cannot recapitulate the complex multi-organ interactions that involve absorption, distribution, metabolism, and excretion (ADME) processes. This limitation has perpetuated a heavy reliance on animal experimentation to predict integrated outcomes like hepatic and renal clearance in humans. In this study, we developed a multi-tissue chip (MTC) to evaluate the PK parameters of intravenously (IV) administered drugs. The MTC platform provides continuous oxygenation through media recirculation and interconnects three MPSs (liver, kidney, skeletal muscle) to study drug metabolism, excretion and distribution within a single in vitro system. The functionality of each MPS was characterized for more than 15 days with single- and multi-tissue chips. A diverse set of drugs from all extended clearance classification system (ECCS) classes with various clearance mechanisms was evaluated on the MTCs and on-chip PK parameters were evaluated, such as hepatic metabolism, uptake, and disposition, tubular secretion and reabsorption, and muscle distribution. These PK parameters were successfully correlated to clinical parameters for IV drugs: hepatic clearance (CLH), renal clearance (CLR), volume of distribution (VD) and then these parameters were extrapolated to human drug exposures using physiologically-based pharmacokinetic (PBPK) modeling. This study demonstrated that the MTC platform could deliver accurate prediction for clinical pharmacokinetics of intravenously administered small molecules without the need for animal studies.

bioengineering↗

bioRxiv2025

APETALA2 controls seed growth by regulating outer integument mechanical identity during seed coat differentiation

Organ morphogenesis requires differentiating cells to acquire mechanical properties that precisely regulate growth. In Arabidopsis thaliana, seed size and shape are largely controlled by the outer integument of the seed coat, whose two layers develop distinct mechanical behaviors critical for growth regulation. Here, we show that the cell identity factor APETALA2 (AP2) both promotes and restricts seed growth by coordinating the post-fertilization differentiation of these layers into distinct seed coat tissues. AP2 drives layer-specific remodeling of cell wall composition, notably affecting pectin and xyloglucan, thereby establishing contrasting mechanical properties between the two layers. Unexpectedly, these changes in wall composition and mechanics do not alter mechanosensitive responses, revealing a surprising uncoupling between cell wall remodeling and mechanotransduction. Our findings identify AP2 as a key coordinator of tissue-specific mechanical differentiation during organ morphogenesis.

plant biology↗

bioRxiv2025

A developmentally regulated long-range enhancer-promoter contact mediates human neural development

SOX2 is a core pluripotency factor in human embryonic stem cells (hESCs), but upon differentiation to the three germ layers, its expression is preserved selectively in neuroectoderm. The mechanisms regulating SOX2 transcription in distinct developmental stages remain incompletely understood. Here, we demonstrate that a distant enhancer 550 kb from the human SOX2 locus is selectively activated in neural stem cells (NSCs) and establishes long-range contact with the SOX2 gene. CRISPR-Cas9 excision of the enhancer has no effect in hESCs but reduces SOX2 transcription in NSCs and impairs neuroectodermal differentiation and forebrain specification in teratomas and cerebral organoids. CRISPR excision of a CTCF recognition motif adjacent to the enhancer does not affect enhancer activation in neuroectoderm but reduces chromatin looping and SOX2 transcription to partially reproduce phenotypes seen with enhancer deletion. Our findings indicate that the development of the human nervous system depends on a developmentally regulated long-range contact between a distant enhancer and the SOX2 locus.

developmental biology↗

bioRxiv2025

A Cell Type-Specific Role for Tubb6 in Ciliogenesis of Xenopus Epidermal Multiciliated Cells

Cilia are microtubule-based organelles found on the surface of most eukaryotic cells. These microtubules are composed of - and {beta}-tubulin heterodimers, and different tubulin isotypes can confer distinct properties to microtubules. Despite their importance, the contribution of individual tubulin isotype to cilia formation and function remains largely unexplored in vertebrates. Here, we identify a critical role for the {beta}-tubulin isotype Tubb6 in the formation of motile cilia in Xenopus epidermal multiciliated cells (MCCs). Tubb6 mRNA is selectively expressed in MCCs, and its protein product localizes to ciliary axonemes. Loss of Tubb6 leads to a marked reduction in cilia number and length, resulting in defective MCC function. In contrast, mono-motile cilia in the gastrocoel roof plate are unaffected by Tubb6 depletion, suggesting a selective requirement for ciliogenesis in MCCs. Together, our findings uncover a cell type-specific role for Tubb6 in motile cilia formation and highlight the functional specialization of tubulin isotypes in vertebrate cilia assembly.

developmental biology↗

bioRxiv2025

Beyond the Axoneme: Whole-Cell Proteomics Identifies Novel Regulators of Ciliogenesis in Xenopus Epidermal Multiciliated Cells

Motile cilia generate directional fluid flow critical for development and tissue homeostasis, and their dysfunction underlies a range of human diseases. Prior proteomic studies have focused on isolated axonemes, leaving the regulatory components of ciliogenesis located in other cellular compartments of multiciliated cells (MCCs) largely unidentified. To bridge this gap, we performed whole-cell proteomic profiling of MCCs in Xenopus using an inducible system that enables selective MCC-enrichment. Leveraging a single-vessel in-cell proteomics approach with ultra-high sample preservation, we identified over 9,000 proteins, including 832 upregulated in MCCs. These encompassed both established ciliary components and novel candidates localized to distinct ciliary structures. Functional validation confirmed the involvement of previously uncharacterized proteins in ciliogenesis. This study presents a high-resolution, whole-cell proteome of vertebrate MCCs, providing critical insights into the molecular networks that govern ciliogenesis and offering a valuable resource for understanding the mechanisms of cilia-related disorders.

developmental biology↗

bioRxiv2025

The Role of SIAMESE in G2 Checkpoint Regulation in Arabidopsis thaliana

In Arabidopsis, SIAMESE (SIM) is a cyclin-dependent kinase inhibitor that restricts progression through mitosis. SIM is well known as a regulator of endoreplication in trichomes and roots. Mathematical modeling of the cell cycle has indicated that SIM may also modulate the length of G2 during mitotic cycles, potentially replacing the WEE1/CDC25 circuit that regulates G2 timing in animals and fungi, which is absent in plants. The predictions of this model were tested in several ways. First, the root meristem is longer in sim mutant roots than in wild-type (WT) roots. Second, two independent methods of measuring cell cycle phases, long-term live-cell imaging using fluorescent protein cell cycle markers and 5-ethynyl-2-deoxyuridine (EdU) pulse-chase imaging, showed that the length of G2 in root meristem cortex cells is shorter in sim mutant plants compared to WT, consistent with the view that these changes in G2 length are due to greater G2 CDK activity in sim mutants. Additionally, the fluorescence of a CYCB:GFP fusion both rose and declined more sharply in the mutant than in wild-type. Because both transcription and degradation of this fusion are directly affected by G2 CDK activity, this result is consistent with sim mutants having greater G2 CDK activity. Taken together, these results suggest that, in addition to its known role in inducing endoreplication, SIM may play a role in regulating the length of G2 during mitotic cycles, at least partially replacing the absence of WEE1/CDC25 regulation of the mitotic cell cycle.

plant biology↗

bioRxiv2023

Puzzle cell shape emerges from the interaction of growth with mechanical constraints

Puzzle-shaped epidermal cells not only reduce mechanical stress during organ growth but also record the expansion history of the tissue in their outlines. By combining mechanical simulations with time-lapse imaging, we show that transitions from directional to isotropic expansion induce new lobes to form along the previous growth axis, and that switching the order of anisotropic and isotropic phases yields hybrid shapes that reliably preserve those transitions. In maize, model predictions and live imaging coincide precisely, and in Ara-bidopsis, final lobe patterns correlate more with growth history than with cell size alone. Ge-netic or pharmacological disruption of lobe formation constrains leaf expansion or drives compensatory elongation, which underscores a mechanical function. A broad survey of living and fossil vascular plants reveals that the mechanism to make puzzle-shaped cells is both widespread and developmentally plastic, suggesting that single snapshots of leaves can give insight into their growth history. Together, these findings demonstrate that puzzle cells trans-form cell geometry into a living record of how tissues grow.

plant biology↗

bioRxiv2023

A Novel Milli-fluidic Liver Tissue Chip with continuous Recirculation for Predictive pharmacokinetics Applications

A crucial step in lead selection during drug development is accurate estimation and optimization of hepatic clearance using in vitro methods. However, current methods are limited by factors such as lack of physiological relevance, short culture/incubation times that are not consistent with drug exposure patterns in patients, use of drug absorbing materials, and evaporation during long-term incubation. To address these technological needs, we developed a novel milli-fluidic human liver tissue chip (LTC) that was designed with continuous media recirculation and optimized for hepatic cultures using human primary hepatocytes. Here, we characterized the LTC using a series of physiologically relevant metrics and test compounds to demonstrate that we could accurately predict the PK of both low and high clearance compounds. The non-biological characterization indicated that the cyclic olefin copolymer (COC)-based LTC exhibited negligible evaporation and minimal non-specific binding of drugs of varying ionic states and lipophilicity. Biologically, the LTC exhibited functional and polarized hepatic culture with sustained metabolic CYP activity for at least 15 days. This long-term culture was then used for drug clearance studies for low and high-clearance compounds for at least 12 days and clearance was estimated for a range of compounds with high in vitro in vivo correlation (IVIVC). We also demonstrated that LTC can be induced by rifampicin, and the culture age had insignificant effect on depletion kinetic and predicted clearance value. Thus, we used advances in bioengineering to develop a novel purpose-built platform with high reproducibility and minimal variability to address unmet needs for PK applications.

pharmacology and toxicology↗

bioRxiv2022

Co-evolution of human influenza A and Epstein Barr virus-specific CD8 ex vivo memory T cell receptor BV repertoires with increasing age

CD8 memory T cells are generated during primary infection with intracellular pathogens, such as viruses. These cells play an important role in the protection of the host upon re-infection with the same pathogen. In this study, we compare CD8 memory T cell receptor (TCR) BV repertoires directly ex vivo for two common human viruses, influenza A virus (IAV), an RNA virus that frequently re-infects due to a high rate of genetic mutation, and Epstein-Barr virus (EBV), a DNA virus, which persists in B cells for life, in the 95% of people that become infected. In cross- sectional and longitudinal studies of EBV seropositive, HLA-A2+, young (18-22 years), middle age (25-59 years), and older (>60 years) donors, we demonstrate that CD8 memory TCR repertoires to three immunodominant epitopes, known to have cross-reactive responses, IAV- M158-66 (M1), EBV-BMLF1280-288 (BM), and EBV-BRLF109-117 (BR) co-evolve as individuals age. Cross-sectional studies showed that IAV-M1- and both EBV-specific repertoires narrowed their TRBV usage with increasing age manifesting to different degrees for each epitope. In fact, narrowing of EBV-BM and EBV-BR-specific TRBV usage correlated with increasing age. IAV- M1-specific TRBV usage was significantly narrowed by middle-age. There was evidence that TRBV usage was changing with increasing age. For instance, IAV-M1-specific dominant BV19 usage appeared to become bimodal showing either high or low frequency of usage in the older age group, while BV30 usage frequency directly correlated with age. For the EBV epitope-specific responses there was preferential usage of particular TRBV and changes in the hierarchy of BV family usage in the different age groups. There appeared to be focusing of the TRBV repertoire by all 3 epitopes to three common BV in the older donors, which would be consistent with retention of crossreactive TCR suggesting co-evolution. Longitudinal studies tracking two donors over 14- 15 years (middle age to older) showed that there were continuous modulations in the TCR repertoire of IAV-M1, EBV-BM and EBV-BR-specific responses over time. There was evidence that acute IAV infection could contribute to these changes in TCR repertoire. This could be occurring by the TCR cross-reactivity that is known to exist between these 3 epitopes, and which appeared to be enhanced during acute IAV infection based on increased usage of common shared TRBV. These studies suggest that virus-specific TCR repertoires change over time as individuals age leading to narrowing of the repertoire favoring retention of potentially crossreactive TCR.

immunology↗

bioRxiv2022

A modular intramolecular triplex photo-switching motif that enables rapid and reversible control of aptamer binding activity

DNA switches that can change conformation in response to certain wavelengths of light could enable rapid and non-invasive control of chemical processes for a wide range of applications. However, most current photo-responsive DNA switches are limited either by irreversible switching or reversible switching with impractically slow kinetics. Here, we report the design of an intramolecular triplex photoswitch (TPS) design based on single-stranded DNA that undergoes rapid and reversible photoswitching between folded and unfolded states through isomerization of internal azobenzene modifications. After optimizing the performance of our photoswitch design, we used molecular dynamics (MD) simulations to reveal how individual azobenzenes contribute to the stabilization or destabilization of the triplex depending on their photoisomerization state. By coupling our TPS to an existing aptamer, we can reversibly modulate its binding affinity with less than 15 seconds of UV light exposure. We further demonstrate reproducible shifting in affinity over multiple cycles of UV and blue light irradiation without substantial photobleaching. Given that our TPS can introduce switching functionality to aptamers without manipulating the aptamer sequence itself, we believe our design methodology should offer a versatile means for integrating photo-responsive properties into DNA nanostructures.

bioengineering↗

bioRxiv2021

Increasing Animal Cognition Research in Zoos

Animal cognition covers various mental processes including perception, learning, decision-making and memory, and animal behavior is often used as a proxy for measuring cognition. Animal cognition/behavior research has multiple benefits; it provides fundamental knowledge on animal biology and evolution but can also have applied conservation and welfare applications. Zoos provide an excellent yet relatively untapped resource for animal cognition research, because they house a wide variety of species - many of which are under threat - and allow close observation and relatively high experimental control compared to the wild. Multi-zoo collaboration leads to increased sample size and species representation, which in turn leads to more robust science. However, there are salient challenges associated with zoo-based cognitive research, which are subject-based (e.g., small sample sizes at single zoos, untrained/unhabituated subjects, site effects) and human-based (e.g., time restrictions, safety concerns, and perceptions of animals interacting with unnatural technology or apparatus). We aim to increase the understanding and subsequent uptake of animal cognition research in zoos, by transparently outlining the main benefits and challenges. Importantly, we use our own research (1) a study on novelty responses in hornbills, and (2) a multi-site collaboration called the ManyBirds project to demonstrate how challenges may be overcome. These potential options include using drop and go apparatuses that require no training, close human contact or animal separation. This article is aimed at zoo animal care and research staff, as well as external researchers interested in zoo-based studies. RESEARCH HIGHLIGHTSO_LIZoos are an excellent yet relatively untapped resource for animal cognition research. C_LIO_LIZoo cognition research has historically been challenging, and traditional laboratory paradigms often do not translate well to the majority of zoos. C_LIO_LISalient challenges of zoo-based cognitive research can be overcome by using less restrictive test apparatuses, limiting animal training and isolation, and subscribing to multi-zoo collaborative programs. C_LI

scientific communication and education↗