Biology subjects

Samuel, M. S.

Publications and source records attributed to Samuel, M. S..

5 recordsLinked to original sources

bioRxiv2026

In Utero CFTR Modulation Alleviates Disease in G551D Cystic Fibrosis Pigs

Previous studies indicate that pigs with CFTR-null and CFTR-{Delta}F508 mutations develop multiorgan disease similar to that in people with cystic fibrosis (CF). At birth, their airways exhibit host defense defects that predispose to airway infection, inflammation, and mucus accumulation. The CFTR-G551D mutation causes CF by producing CFTR channels that localize correctly but have reduced channel activity. Ivacaftor (VX-770) is a small molecule drug developed to potentiate CFTR activity. To test the phenotype of the CFTR-G551D mutation in pigs and determine whether ivacaftor can rescue CF abnormalities, we developed CFTRG551D/G551D (CF-G551D) pigs through homologous recombination in fetal fibroblasts and somatic cell nuclear transfer. Newborn CF-G551D piglets exhibited phenotypes similar to CF-null piglets, including meconium ileus, exocrine pancreatic destruction, micro-gallbladder, vas deferens destruction, and airway structural abnormalities. Compared to wild-type pigs, CF-G551D pigs had reduced forskolin-stimulated short-circuit current in airway and intestinal tissues. Ivacaftor increased the single-channel open state probability of CFTR-G551D and increased short-circuit current to near wild-type levels. Similar to our other CF pig models, we found that 100% of CF-G551D pigs were born with meconium ileus. To test whether in utero ivacaftor treatment could prevent or alleviate meconium ileus, pregnant sows were treated with ivacaftor beginning at day 35 of gestation and continuing until delivery. This treatment rescued the pancreas, gallbladder, and vas deferens phenotype in the majority of CF-G551D pigs. Animals that were spared from meconium ileus were able to survive without ivacaftor treatment. Airway disease developed similar to other CF pig models. These findings indicate that this model may be useful for studies in which CFTR function can be reversed, for investigating in utero CFTR correction strategies, and for longitudinal studies in CF pigs.

physiology↗

bioRxiv2025

ROCK2 inhibition has a dual role in reducing ECM remodelling and cell growth, while impairing migration and invasion

The Rho-associated coiled-coil containing protein kinases 1/2 (ROCK1/2) are key signalling proteins involved in the regulation of the actin cytoskeleton and control a variety of cellular processes. This includes cell proliferation, stemness, cell migration and invasion as well as actomyosin contraction and extracellular matrix (ECM) remodelling. Due to a lack of ROCK2 specific inhibitors, previous pharmacological studies of ROCK function have relied on pan-ROCK1/2 inhibition. Here, we use the ROCK2 specific inhibitor, GV101, in the context of breast cancer (BC) models to uncouple the effects of ROCK2 pharmacological inhibition on both epithelial and stromal fibroblast cell populations. We demonstrate that ROCK2 inhibition with GV101 reduces fibroblast-mediated remodelling of pre-existing and de novo synthesised ECM resulting in a reduction in biomechanical stiffness, while also limiting epithelial cell growth in 2D and 3D settings. We also demonstrate that ROCK2 inhibition exposes epithelial cell vulnerability to fluid flow-induced shear stress. Furthermore, using 3D co-cultures we reveal that GV101 reduces single-cell migratory capacity, while the disruption of stromal ECM architecture is sufficient to impede collective cell migration and invasion. Finally, we assessed ROCK2 expression in human BC patient tumours revealing that ROCK2 expression is up-regulated during BC progression and that high ROCK2 expression correlates with poor patient outcome in the triple-negative subtype of BC. Together, these results demonstrate that ROCK2 specific inhibition disrupts key functions in both epithelial cell and stromal fibroblast compartments warranting further assessment of ROCK2 in highly proliferative, fibrotic or metastatic diseases, such as BC. HighlightsO_LIFibroblast-mediated remodelling of pre-existing and de novo synthesised extracellular matrix is reduced upon ROCK2 specific targeting. C_LIO_LIROCK2 inhibition impairs epithelial cancer cell growth and colony formation in 2D and 3D contexts, while also limiting single-cell migration and cell survival upon fluid flow-induced shear stress. C_LIO_LIROCK2 targeting during fibroblast-mediated ECM remodelling is sufficient to disrupt collective cell migration as well as 3D invasion into organotypic fibroblast-contracted collagen matrices. C_LIO_LIROCK2 expression is up-regulated during breast cancer progression and high expression correlates with poor patient outcome in triple-negative breast cancer. C_LI

cell biology↗

bioRxiv2024

Development and Initial Characterization of Pigs with DNAI1 Mutations and Primary Ciliary Dyskinesia

Mutations in more than 50 different genes cause primary ciliary dyskinesia (PCD) by disrupting the activity of motile cilia that facilitate mucociliary transport (MCT). Knowledge of PCD has come from studies identifying disease-causing mutations, characterizing structural cilia abnormalities, finding genotype-phenotype relationships, and studying the cell biology of cilia. Despite these important findings, we still lack effective treatments and people with PCD have significant pulmonary impairment. As with many other diseases, a better understanding of pathogenic mechanisms may lead to effective treatments. To pursue disease mechanisms, we used CRISPR-Cas9 to develop a PCD pig with a disrupted DNAI1 gene. PCD pig airway cilia lacked the outer dynein arm and had impaired beating. MCT was impaired under both baseline conditions and after cholinergic stimulation in PCD pigs. Neonatal PCD pigs developed neonatal respiratory distress with evidence of atelectasis, air trapping, and airway mucus obstruction. Despite airway mucus accumulation, lung bacterial counts were similar between neonatal wild-type and PCD pigs. Sinonasal disease was present in all neonatal PCD pigs. Older PCD pigs developed worsening airway mucus obstruction, inflammation, and bacterial infection. This pig model closely mimics the disease phenotype seen in people with PCD and can be used to better understand the pathophysiology of PCD airway disease.

physiology↗

bioRxiv2022

Actomyosin-mediated cellular tension promotes Yap nuclear translocation and myocardial proliferation through alpha5 integrin signaling

The cardiomyocyte phenotypic switch from a proliferative to terminally differentiated state results in the loss of regenerative potential of the mammalian heart shortly after birth. Yet, the molecular mechanisms that regulate this critical developmental process are incompletely understood. Nonmuscle myosin IIB (NM IIB)-mediated actomyosin contractility regulates cardiomyocyte cytokinesis in the embryonic heart, and NM IIB levels decline after birth suggesting a role for cellular tension in the regulation of cardiomyocyte cell cycle activity in the postnatal heart. The Rho kinase (ROCK) serine/threonine protein kinases that act downstream of the RhoA small GTP-binding protein regulate nonmuscle myosin contractile force generation. To investigate the role of actomyosin contractility in cardiomyocyte maturation and cell cycle arrest, we conditionally-activated ROCK2 kinase domain (ROCK2:ER) in the murine postnatal heart. Here we show that cardiac-specific activation of actomyosin contractility shifts the balance from cell-cell to cell-matrix adhesions. Specifically, 5/{beta}1 integrin and fibronectin matrix increase in response to actomyosin-mediated tension. Moreover, activation of ROCK2:ER promotes nuclear translocation of Yap, a mechanosensitive transcriptional co-activator, and enhances cardiomyocyte proliferation. Finally, we show that reduction of myocardial 5 integrin rescues the myocardial proliferation phenotype in ROCK2:ER hearts. These data demonstrate that cardiomyocytes respond to increase intracellular tension by altering their intercellular contacts in favor of cell-matrix interactions leading to Yap nuclear translocation, thus uncovering a novel function for nonmuscle myosin contractility in promoting cardiomyocyte cell cycle activity in the postnatal heart.

developmental biology↗

bioRxiv2021

14-3-3ζ-depletion impairs mammary gland development in the mouse

The 14-3-3 family of proteins have roles in regulating several key cellular processes. While their significant structural and functional homology had informed the idea that these proteins acted redundantly, it is now becoming clear that individual family members may have tissue and context specific functions, highlighting the need for a more nuanced understanding of these important proteins. Here, we demonstrate that mice deficient in 14-3-3{zeta} exhibit developmental defects of the mammary epithelium, associated with dysregulation of key transcription factors involved in the maintenance of mammary stem cell populations. We believe that this model will be prove useful for investigating the role of 14-3-3{zeta} in the maintenance of mammary stem cell populations and elucidating the transcriptional networks driving specification of the mammary epithelium.

cell biology↗