Biology subjects

Meyerholz, D. K.

Publications and source records attributed to Meyerholz, D. K..

At least 19 recordsLinked to original sources

bioRxiv2026

Efficient colonic colonization by Campylobacter jejuni requires the heme receptor ChuA

Previous research demonstrated that Campylobacter jejuni encodes a heme utilization system that facilitates heme-dependent growth under iron-limiting conditions and that transcription of this system is induced during human infection. Despite these observations, it remained unknown whether the heme transport system is required for colonization and disease in a susceptible host. To address this, we created individual non-polar deletion mutants of each component of the heme transport system and examined their ability to promote heme-dependent growth and iron uptake. From this work, we found that only the heme receptor, ChuA, was required for heme-dependent growth and iron acquisition, which supports the earlier work of another group. Further, we examined whether intestinal colonization, immune activation, and pathology were altered during infection with these mutants. After establishing that elevated heme and chuABCD expression occur during C. jejuni infection of IL-10-/- mice, we found that a mutant of the heme receptor, ChuA, exhibited significantly reduced colonization of the colon. In addition, we found that neutrophil and circulating monocyte recruitment were significantly reduced in the colon during infection with the ChuA mutant, but that populations of self-maintaining tissue macrophages remained high. Loss of ChuA reduced colonic colonization and was accompanied by diminished innate immune cell recruitment and intestinal pathology. Together, these findings identify ChuA-dependent heme acquisition as a key determinant of efficient colonic colonization and the associated inflammatory disease.

microbiology↗

bioRxiv2026

Intratumoral plasma cells are required for activation of CD8+ T cells and success of immune checkpoint blockade therapy in de novo MPNSTs

BackgroundThe role of intratumoral plasma cells in immune checkpoint blockade (ICB) therapy has never been tested although their presence is linked with improved patient response and survival. Malignant peripheral nerve sheath tumors (MPNSTs) are deadly sarcomas with minimal responsiveness to ICB therapies. Strikingly, drugs inhibiting cyclin-dependent kinases 4/6 (CDK4/6) and MEK sensitize de novo MPNSTs to immunotherapy targeting programmed death-ligand 1 (PD-L1), which correlates with increased intratumoral plasma cells. Here, we tested if plasma cells mediate the MPNST response to anti-PD-L1 therapy. MethodsAnti-tumor activity of PD-L1 inhibition, with or without CDK4/6-MEK inhibition, was measured in de novo MPNSTs within wild-type versus plasma cell-deficient mice. Plasma cell-dependent effects of CDK4/6-MEK inhibition on priming the MPNST immune environment were determined by single cell transcriptomics and immunostaining. FindingsMPNSTs lacking plasma cells failed to respond to anti-PD-L1 monotherapy and were no longer sensitized to immunotherapy by CDK4/6-MEK inhibition. Plasma cell-deficient MPNSTs exposed to CDK4/6-MEK inhibitors had impaired antigen presentation on major histocompatibility class I (MHC-I) and decreased CD8+ T cell infiltration and activation. Complementary analyses of human sarcomas showed increased intratumoral plasma cell signatures prognose better patient survival. InterpretationPlasma cells favorably remodel the tumor immune environment by increasing CD8+ T cell infiltration and are critical for successful ICB therapy in MPNSTs. This work may help inform ICB treatment strategies and cancer patient stratification for many different tumor types. FundingThis research was supported by University of Iowa Sarcoma Research Program awards and NIH grants T34-GM141143, T32-GM067795, F31-CA281312, P30-CA086862, and R01-NS119322. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSFor many types of cancer, intratumoral plasma cells have been correlated with better patient survival and improved response to immune checkpoint blockade (ICB) therapies. However, the biology underlying those associations is not understood and no study has examined the requirement of plasma cells in immunotherapy response. Compelling data in malignant peripheral nerve sheath tumors (MPNSTs) showed that dual kinase inhibition of oncogenic CDK4/6 and MEK induced intratumoral plasma cell accumulation and sensitized tumors to ICB therapy. While CDK4/6-MEK inhibition is known to enhance antitumor immunity in other tumor types by CD8+ T cells or natural killer (NK) cells, a role for plasma cells has never been explored. Added value of this studyStudies were performed in MPNSTs, an under-researched cancer that normally responds poorly to ICB monotherapies. This is the first investigation to show that intratumoral plasma cells are essential for successful ICB therapy and they support anti-tumor immunity by promoting a pro-inflammatory, CD8+ T cell state involving MHC-I antigen presentation. Findings provide new insight into immunomodulatory effects of CDK4/6-MEK inhibitor therapies, revealing plasma cells are needed for those drugs to activate CD8+ T cell mediated antitumor immunity. Implications of all the available evidenceThe fundamental advance in understanding how plasma cells promote successful ICB immunotherapy is likely applicable to other solid tumors and may guide novel therapeutic strategies in which plasma cell-inducing agents are combined with ICB antibodies. Moreover, an increased presence of intratumoral plasma cells in tumor specimens may streamline clinical decisions regarding which patients are most likely to benefit from ICB therapy.

cancer biology↗

bioRxiv2026

Defining Functional Correction Thresholds in Primary Ciliary Dyskinesia for Effective Gene Therapies

RationalePrimary ciliary dyskinesia (PCD) is an inherited disorder characterized by defective motile cilia and impaired mucociliary clearance. Mutations in CCDC40 disrupt axonemal organization, resulting in dyskinetic or immotile cilia. While emerging therapies may restore function in only a subset of cells, the functional consequences of mixed populations of mutant and healthy cilia are not well understood. ObjectivesTo determine how defined mixtures of CCDC40-deficient and wild-type ciliated cells influence mucociliary transport. MethodsHuman bronchial epithelial cells were combined at varying ratios of CCDC40-deficient and wild-type cells and differentiated to model heterogeneous epithelia. High-speed video microscopy and particle-tracking algorithms were used to assess ciliary motion and mucus transport and combined with electron microscopy to evaluate cilia ultrastructure. Measurements and Main ResultsAirway differentiation was largely preserved with marked ultrastructural defects observed in CCDC40 cells, including multiple central centrioles, absent inner dynein arms, and basal body misorientation. Ciliated surface coverage decreased, and goblet coverage increased with higher mutant representation. Mucociliary transport declined nonlinearly, with speeds dropping from [~]56 {micro}m/s (100% WT) to [~]9 {micro}m/s in 100% mutant cultures. Clearance-per-beat and flow coordination decreased sharply with rising mutant burden. Modeling revealed that transport efficiency was equivalent to that recorded in ex vivo human tissues and plateaued when [~]75% of the ciliated population was WT. ConclusionsTogether, these findings define a PCD-specific functional correction threshold and show that effective therapy must overcome the disruptive biomechanical and cellular influences of mutant epithelial cells, providing a quantitative benchmark to guide gene-therapy design and clinical translation.

cell biology↗

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

A SARS-CoV-2 entry inhibitor trimerizes to lock the spike protein in a closed conformation

The SARS-CoV-2 spike protein binds its receptor ACE2 to initiate target-cell infection. To engage ACE2, at least one of the three receptor-binding domains (RBDs) of the spike must adopt the up orientation. Here we describe S22, a potent, bioavailable, and non-toxic inhibitor of BA.2 and all subsequent Omicron variants. Cryo-EM analyses showed that S22 assembled as a trimer in a previously uncharacterized pocket of the spike apex, stabilizing all three RBDs in the down orientation, thereby preventing ACE2 association. Binding studies, especially those using mixed S22-sensitive and -resistant spikes, imply a cooperative assembly of three S22 molecules with three RBDs, resulting in an unusually slow S22 off-rate. Consistent with its slow dissociation and favorable pharmacokinetics, S22 suppressed viral replication 100-fold in the lungs of XBB.1.5-infected mice. Thus, S22 potently inhibits Omicron entry through a distinct mechanism whereby a small compound assembles cooperatively as a trimer to stabilize spike in an inactive conformation.

microbiology↗

bioRxiv2025

Oncogene SETDB1s Dual Role in Endometrial Cancer: Driving Tumor Progression and Immune Escape

Oncogene SETDB1, an H3K9 methyltransferase, drives tumorigenesis in various cancers. Using endometrial cancer (EC) as a model, we discovered SETDB1s dual mechanisms in driving EC tumorigenesis and mediating immune evasion. SETDB1 knockout (SETDB1-/-) tumor-bearing mice exhibited prolonged survival of up to 100 days. Transcriptomic profiling of SETDB1-/- EC cells revealed decreased expression of oncogenes (POLR2A, MSH6, FLNA) and increased expression of tumor suppressor genes (PGR, RERG, ZNF582), which indicates that SETDB1 intrinsically promotes EC tumor proliferation by regulating these downstream genes. SETDB1 repressed repeat elements and the interferon pathway, mediating immune evasion extrinsically by inhibiting anti-tumor macrophage infiltration. ChIP-seq analysis showed SETDB1 binding at pericentromeric regions on many chromosomes and numerous ZNFs. Loss of SETDB1 resulted in abnormal cell division. SETDB1-/- tumors displayed reduced proliferation markers (Ki67, pHH3) and increased macrophage infiltration. Mechanistically, SETDB1 promotes CD47 (a dont-eat-me signal) and represses CCL5 and CXCL9 (macrophage and T-cell recruiting chemokines), contributing to immune evasion. M1-like macrophages killed more SETDB1-/- cells in co-culture. Additionally, SETDB1 knockout in mouse EC cells reduced tumor growth in C57BL/6 mice, with increased macrophage and CD4+ T-cell infiltration. Our results indicates that elevated SETDB1 and its target genes can predict higher tumor grade and worse survival, suggesting that targeting SETDB1 could be a promising therapeutic strategy for EC.

cancer biology↗

bioRxiv2025

Ebola's Hidden Target: Virus Transmission to and Accumulation within Skin

Ebola virus (EBOV), the causative agent of Ebola virus disease (EVD), remains one of WHOs top ten threats to global health. Infectious EBOV virions can be found on the surface of skin late during systemic infection and passed from the deceased through skin-to-skin contact. Here, we assess viral load and antigen expression in the skin of EBOV-infected non-human primates (NHP) and mouse adapted-EBOV (ma-EBOV) - infected mice and use the low containment viral model, rVSV/EBOV GP, to mechanistically define skin infection in mice. Viral RNA peaked within the skin proximal to the site of injection in EBOV-infected NHPs on day 6. In contrast, mouse skin sites distal to the site of ma-EBOV injection achieved maximal viral loads by day 3. At late times of infection, viral antigen-positive cells co-localized with markers for endothelial, stromal, and immune cells in the dermis. Epidermal cells within and surrounding hair follicles also harbored viral antigen, suggesting a potential mechanism of virus trafficking to the epidermal surface. Despite robust viral infection, distal skin sites of ma-EBOV-infected mice had low expression of proinflammatory stimulated genes. A similar cellular tropism was observed in the skin of mice infected with rVSV/EBOV GP, with discrete focal areas of intense infection. When virus was applied to the surface of gently abraded skin to remove the stratum corneum, epidermal keratinocytes were robustly infected, followed by systemic viral dissemination. To define cell surface receptors critical for virus trafficking to and replication within the skin, mice lacking the phosphatidylserine receptors were infected intraperitoneally with rVSV/EBOV GP. At day 3 of infection, skin distal to the site of infection of TIM-1 knock out (KO) mice had significantly lower levels of infectious virus than the control mice, suggesting that TIM-1 is essential for efficient distribution of virus to the skin. Our findings reveal that EBOV targets specific skin cell populations at late times of viral infection and that the host receptor TIM-1 is required for optimal viral dissemination.

microbiology↗

bioRxiv2024

IL-13 decreases susceptibility to airway epithelial SARS-CoV-2 infection but increases disease severity in vivo

Treatments available to prevent progression of virus-induced lung diseases, including coronavirus disease 2019 (COVID-19) are of limited benefit once respiratory failure occurs. The efficacy of approved and emerging cytokine signaling-modulating antibodies is variable and is affected by disease course and patient-specific inflammation patterns. Therefore, understanding the role of inflammation on the viral infectious cycle is critical for effective use of cytokine-modulating agents. We investigated the role of the type 2 cytokine IL-13 on SARS-CoV-2 binding/entry, replication, and host response in primary HAE cells in vitro and in a model of mouse-adapted SARS-CoV-2 infection in vivo. IL-13 protected airway epithelial cells from SARS-CoV-2 infection in vitro by decreasing the abundance of ACE2- expressing ciliated cells rather than by neutralization in the airway surface liquid or by interferon-mediated antiviral effects. In contrast, IL-13 worsened disease severity in mice; the effects were mediated by eicosanoid signaling and were abolished in mice deficient in the phospholipase A2 enzyme PLA2G2D. We conclude that IL-13-induced inflammation differentially affects multiple steps of COVID-19 pathogenesis. IL-13-induced inflammation may be protective against initial SARS-CoV-2 airway epithelial infection; however, it enhances disease progression in vivo. Blockade of IL-13 and/or eicosanoid signaling may be protective against progression to severe respiratory virus-induced lung disease. RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSPrior to this study, various pieces of evidence indicated the significant role of cytokines in the pathogenesis and progression of COVID-19. Severe COVID-19 cases were marked by cytokine storm syndrome, leading to immune activation and hyperinflammation. Treatments aimed at modulating cytokine signaling, such as IL-6 receptor antagonists, had shown moderate effects in managing severe COVID-19 cases. Studies also revealed an excessive production of type 2 cytokines, particularly IL-13 and IL-4, in the plasma and lungs of COVID-19 patients, which was associated with adverse outcomes. Treatment with anti-IL-13 monoclonal antibodies improved survival following SARS-CoV-2 infection, suggesting that IL-13 plays a role in disease severity. Type 2 cytokines were observed to potentially suppress type 1 responses, essential for viral clearance, and imbalances between these cytokine types were linked to negative COVID-19 outcomes. These findings highlighted the complex interactions between cytokines and the immune response during viral infections, underscoring the importance of understanding IL-13s role in COVID-19 and related lung diseases for developing effective therapeutic interventions. Added value of this studyIn this study, we explored the impact of IL-13-induced inflammation on various stages of the SARS-CoV-2 infection cycle using both murine (in vivo) and primary human airway epithelial (in vitro) culture models. Our findings indicated that IL-13 provided protection to airway epithelial cells against SARS-CoV-2 infection in vitro, partly by reducing the number of ACE2- expressing ciliated cells. Conversely, IL-13 exacerbated the severity of SARS2-N501YMA30-induced disease in mice, primarily through Pla2g2d-mediated eicosanoid biosynthesis. Implications of the available evidenceCurrent evidence indicates that PLA2G2D plays a crucial role in the IL-13-driven exacerbation of COVID-19 in mice, suggesting that targeting the IL-13-PLA2G2D axis could help protect against SARS-CoV-2 infection. These insights are important for clinical research, especially for studies focusing on drugs that modify IL-13 signaling or modulate eicosanoids in the treatment of asthma and respiratory virus-induced lung diseases.

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↗

bioRxiv2023

Enhanced ISGylation reduces respiratory distress following Francisella novicida infection

The Interferon-Stimulated Gene 15 (ISG15) is a ubiquitin-like protein induced by viral and bacterial infection. ISG15 covalently modifies host and pathogenic proteins in a process called ISGylation. Yet, the consequences of ISGylation on protein fate and function remain to be determined. Here we sought to assess whether ISGylation would be protective following bacterial pneumonia caused by Francisella novicida. We found that infection with F. novicida induces ISGylation both in vitro in macrophages and in vivo in the lung, liver, and spleen of mice infected intranasally. Surprisingly, ISG15 and ISGylation do not affect bacterial burden in the lung in vivo, but in a model of enhanced ISGylation (usp18C61A/C61A) mice have decreased respiratory distress relative to Isg15-/- animals. In order to understand the mechanism which underlies this phenotype, we mapped the ISGylome of F. novicida-infected mouse lungs using label-free quantitative mass spectrometry and identified enrichment in ISGylation of proteins involved in the innate immune response and cytosolic nucleotide signaling. We validated ISGylation of the sterile alpha motif and HD-containing protein 1 (SAMHD1) via immunoprecipitation. SAMHD1 depletes cytosolic dinucleotide stores critical for retroviral replication but it is unknown how its activity could affect bacterial infection. Structure-function analysis indicates that ISG15 modification sites in usp18C61A/C61A mice could prevent SAMHD1 dimerization and therefore abrogate function. Accordingly, deletion of SAMHD1 in fibroblasts with enhanced ISGylation reduces bacterial load. Taken together, unchecked ISGylation plays a protective role in F. novicida infection in vivo through improved respiratory function. Thus, inhibiting USP18 may be a promising therapeutic strategy for both viral and bacterial pneumonia. Author summaryFrancisella tularensis is a bacterial pathogen responsible for the disease tularemia, which can result in severe respiratory infection if as few as ten bacteria are inhaled. Our cells have many ways of managing infections, including the production of proteins designed to fight off foreign pathogens. One protein produced following infection is the interferon-stimulated gene 15 (ISG15). ISG15 is a ubiquitin-like molecule, meaning that it can be chemically attached to other proteins. When bound ISG15 changes the stability, interacting partners, or function of its target in a process termed ISGylation. Here we show that ISG15 is produced following infection with Francisella. We found that enhanced ISGylation led to less severe respiratory symptoms. To better understand the mechanism by which ISGylation protects from infection we identified the ISG15-modified proteins in the lung using mass-spectrometry-based proteomics. We found protein targets that are involved in the control of immune signaling pathways including sterile alpha motif and HD-containing protein 1 (SAMHD1) which, when deleted in cells with enhanced ISGylation, leads to better bacterial clearance. Together, we show that enhanced ISGylation plays a protective role following bacterial pneumonia, indicating that targeting this pathway could prove a beneficial therapeutic in both bacterial and viral respiratory diseases.

microbiology↗

bioRxiv2023

Characterization of the First Turtle Organoids: A Model for Investigating Unique Adaptations with Biomedical Potential

Painted turtles are remarkable for their well-developed freeze tolerance and associated resilience to hypoxia/anoxia, oxidative stress, and ability to supercool. They are, therefore, an ideal model for biomedical research on hypoxia-induced injuries (including strokes), tissue cooling during extensive surgeries, and organ cryopreservation. Yet, the seasonal reproduction and slow maturation of turtles hinder basic and applied biomedical research. To overcome these limitations, we developed the first adult stem cell-derived turtle hepatic organoids, which provide 3D self-assembled structures that mimic their original tissue and allow for in vitro testing and experimentation without constantly harvesting donor tissue and screening offspring. Our pioneering work with turtles represents the first for this vertebrate Order and complements the only other organoid lines from non-avian reptiles, derived from snake venom glands. Here we report the isolation and characterization of hepatic organoids derived from painted, snapping, and spiny softshell turtles spanning [~]175 million years of evolution, with a subset being preserved in a biobank. Morphological and transcriptomics revealed organoid cells resembling cholangiocytes, which was then compared to the tissue of origin. Deriving turtle organoids from multiple species and life stages demonstrates that our techniques are broadly applicable to chelonians, permitting the development of functional genomic tools currently missing in most herpetological research. When combined with genetic editing, this platform will further support studies of genome-to-phenome mapping, gene function, genome architecture, and adaptive responses to climate change, among others. We discuss the unique abilities of turtles, including their overwintering potential, which has implications for ecological, evolutionary, and biomedical research. SIGNIFICANCEHere we developed the first turtle-derived organoid biobank from the liver of multiple chelonians with a subset characterized via histology, RNA sequencing transcriptomics, single-nuclei RNA sequencing, and transmission electron microscopy. Furthermore, we discuss the potential of the 3D organoid model to investigate unique physiological adaptations of turtles which could unravel the molecular mechanisms underlying their overwintering capacity, opening the door for in vitro biomedical studies relevant to hepatic ischemia-reperfusion injury to organ cryopreservation, beyond fundamental ecology and evolution. This organoid biobank represents a novel resource for the scientific community to support research regarding the unique adaptations of this understudied Order of vertebrates.

genetics↗

bioRxiv2022

ISG15-modification of the Arp2/3 complex restricts pathogen spread

The ubiquitin-like protein, ISG15, can act as a cytokine or can covalently modify host and pathogen-derived proteins. The consequences of ISG15 modification on substrate fate remain unknown. Here we reveal that ISGylation of the Arp2/3 complex slows actin filament formation and stabilizes Arp2/3 dependent structures including cortical actin and lamella. When properly controlled, this serves as an antibacterial and antiviral host defense strategy to directly restrict actin-mediated pathogen spread. However, Listeria monocytogenes takes advantage in models of dysregulated ISGylation, leading to increased mortality due to augmented spread. The underlying molecular mechanism responsible for the ISG15-dependent impact on actin-based motility is due to failed bacterial separation after division. This promotes spread by enabling the formation of multi-headed bacterial "bazookas" with stabilized comet tails that can disseminate deeper into tissues. A bacterial mutant that cannot recruit Arp2/3 or a non-ISGylatable mutant of Arp3 is sufficient to rescue slowed comet tail speed and restrict spread. Importantly, ISG15-deficient neonatal mice have aberrant epidermal epithelia characterized by keratinocytes with diffuse cortical actin, which could underlie observed defects in wound healing in human patients who lack ISG15. Ultimately, our discovery links host innate immune responses to cytoskeletal dynamics with therapeutic implications for viral infection and metastasis.

cell biology↗

bioRxiv2022

Selective Androgen Receptor Modulator Microparticle Formulation Reverses Muscle Hyperalgesia in Mouse Model of Widespread Muscle Pain

Currently, there is a need for the generation of non-opioid analgesics for treating chronic pain. Preclinical and clinical studies demonstrate the analgesic effects of testosterone. However, treatment with testosterone is not feasible due to adverse effects. Selective androgen receptor modulators (SARMs) were developed to overcome these limitations by minimizing activation of androgenic side effects. First, we demonstrate SARM administration alleviates widespread muscle pain in male and female mice. We then developed a SARM-loaded PLGA microparticle formulation that reverses widespread muscle pain in two injections. In vitro and in vivo release kinetics demonstrate the microparticle formulation had sustained SARM release for 4 weeks. Antagonism of androgen receptors blocked the analgesic effects of the SARM microparticles. SARM treatment had no effect on cardiac or liver enzymes, cardiac histology, and did not produce rewarding behavior. These studies demonstrate SARM microparticles as a potential therapeutic for chronic muscle pain. One Sentence SummaryA selective androgen receptor modulator microparticle formulation alleviates widespread muscle pain in male and female mice while being non-toxic.

neuroscience↗

bioRxiv2022

Mice expressing P301S mutant human tau have deficits in interval timing

Interval timing is a key executive process that involves estimating the duration of an interval over several seconds or minutes. Patients with Alzheimers disease (AD) have deficits in interval timing. Since temporal control of action is highly conserved across mammalian species, studying interval timing tasks in animal AD models may be relevant to human disease. Amyloid plaques and tau neurofibrillary tangles are hallmark features of AD. While rodent models of amyloid pathology are known to have interval timing impairments, to our knowledge, interval timing has not been studied in models of tauopathy. Here, we evaluate interval timing performance of P301S transgenic mice, a widely studied model of tauopathy that overexpresses human tau with the P301S mutation. We employed the switch interval timing task, and found that P301S mice consistently underestimated temporal intervals compared to wild-type controls, responding early in anticipation of the target interval. Our study indicating timing deficits in a mouse tauopathy model could have relevance to human tauopathies such as AD. HIGHLIGHTSO_LIWe examined interval timing behavior in mice expressing P301S mutant tau C_LIO_LIP301S mice responded earler than littermate controls C_LIO_LIThese data provide insight into animal models of tauopathy C_LI

neuroscience↗

bioRxiv2021

Oncogenic RABL6A promotes NF1-associated MPNST progression in vivo

BackgroundMalignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas that display complex molecular and genetic alterations. Powerful tumor suppressors CDKN2A and TP53 are commonly disrupted in these lesions along with NF1, a gene that encodes a negative regulator of Ras. Many additional factors have been implicated in MPNST pathogenesis. A greater understanding of critical drivers of the disease is needed to guide more informed targeted therapies for patients. RABL6A is a newly identified driver of MPNST cell survival and proliferation whose in vivo role in the disease is unknown. MethodsUsing CRISPR-Cas9 targeting of Nf1+Cdkn2a or Nf1+Tp53 in the sciatic nerve to form de novo MPNSTs, we investigated the biological significance of RABL6A in MPNST development. Molecular evaluation of terminal tumors (western blot, qRT-PCR, immunohistochemistry) yielded several insights. ResultsMice lacking Rabl6 displayed slower tumor growth and extended survival relative to wildtype animals in both genetic contexts. YAP oncogenic activity was selectively downregulated in RABL6A-null, Nf1+Cdkn2a lesions but not in RABL6A-null, Nf1+Tp53 tumors. Regardless of genetic context, loss of RABL6A caused upregulation of the CDK inhibitor, p27 in tumors. Paradoxically, both models displayed elevated Myc protein expression and Ki67 staining in terminal tumors lacking RABL6A. ConclusionsThese findings demonstrate RABL6A is required for optimal tumor progression of NF1 mutant MPNSTs in vivo in both Cdkn2a and p53 inactivated settings. However, sustained RABL6A loss may provide selective pressure for molecular alterations, such as Myc upregulation, that ultimately promote an unwanted, hyper-proliferative tumor phenotype akin to drug resistant lesions. Importance of the StudyMPNSTs are aggressive, deadly, and challenging to treat tumors due to location around nerves and high mutational burden. Many factors implicated in MPNST genesis have yet to be fully tested for biological significance in disease formation. We establish a critical physiological role for a new oncoprotein, RABL6A, in promoting NF1-associated MPNST progression. We identify novel RABL6A-regulated pathways that likely contribute to tumor growth, specifically YAP and Myc signaling, and found that sustained RABL6A loss eventually yields more proliferative tumors. We liken RABL6A deficient tumors to those being treated with therapies targeting RABL6A effectors, such as CDKs. Therefore, those lesions should provide a powerful platform to uncover key mediators of drug resistance. Our data suggest oncogenic YAP and Myc could be such mediators of resistance. This study provides a novel system to examine one of the most pressing clinical challenges, drug resistant tumor growth and relapse, in cancer therapy.

cancer biology↗

bioRxiv2021

Topography-dependent gene expression and function of common cell archetypes in large and small porcine airways

The small airways of humans are affected early in several lung diseases. However, because they are relatively inaccessible, little is known about the epithelial cells that line these airways. We performed a single cell RNA-seq census of small and large airways of wild-type pigs and pigs with disrupted cystic fibrosis transmembrane conductance regulator (CFTR) gene. The sequencing data showed that small airway epithelia had similar major cell types as large airways but no ionocytes; moreover, lack of CFTR expression had minimal effect on the transcriptome. Small airway epithelial cells expressed a different transcriptome than large airway cells. Quantitative immunohistochemistry showed that small airway basal cells participate in epithelial barrier function. Finally, sequencing data and in vitro electrophysiologic studies suggest that small airway epithelia have a water and ion transport advantage. Our data highlight the archetypal nature of basal, secretory, and ciliated airway cells with location-dependent gene expression and function.

genomics↗

bioRxiv2021

RABL6A promotes pancreatic neuroendocrine tumor angiogenesis and progression in vivo

Pancreatic neuroendocrine tumors (pNETs) are difficult-to-treat neoplasms whose incidence is rising. Greater understanding of pNET pathogenesis is needed to identify new biomarkers and targets for improved therapy. RABL6A, a novel oncogenic GTPase, is highly expressed in patient pNETs and required for pNET cell proliferation and survival in vitro. Here, we investigated the role of RABL6A in pNET progression in vivo using a well-established model of the disease. RIP-Tag2 (RT2) mice develop functional pNETs (insulinomas) due to SV40 large T-antigen expression in pancreatic islet {beta} cells. RABL6A loss in RT2 mice significantly delayed pancreatic tumor formation, reduced tumor angiogenesis and mitoses, and extended survival. Those effects correlated with upregulation of anti-angiogenic p19ARF and downregulation of proangiogenic c-Myc in RABL6A-deficient islets and tumors. Our findings demonstrate that RABL6A is a bona fide oncogenic driver of pNET angiogenesis and development in vivo.

cancer biology↗

bioRxiv2020

K18-hACE2 Mice for Studies of COVID-19 Treatments and Pathogenesis Including Anosmia

The ongoing COVID-19 pandemic is associated with substantial morbidity and mortality. While much has been learned in the first months of the pandemic, many features of COVID-19 pathogenesis remain to be determined. For example, anosmia is a common presentation and many patients with this finding show no or only minor respiratory signs. Studies in animals experimentally infected with SARS-CoV-2, the cause of COVID-19, provide opportunities to study aspects of the disease not easily investigated in human patients. COVID-19 severity ranges from asymptomatic to lethal. Most experimental infections provide insights into mild disease. Here, using K18-hACE2 mice that we originally developed for SARS studies, we show that infection with SARS-CoV-2 causes severe disease in the lung, and in some mice, the brain. Evidence of thrombosis and vasculitis was detected in mice with severe pneumonia. Further, we show that infusion of convalescent plasma (CP) from a recovered COVID-19 patient provided protection against lethal disease. Mice developed anosmia at early times after infection. Notably, while treatment with CP prevented significant clinical disease, it did not prevent anosmia. Thus K18-hACE2 mice provide a useful model for studying the pathological underpinnings of both mild and lethal COVID-19 and for assessing therapeutic interventions.

microbiology↗