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Abstract
Significant loss of pigmentation can increase visual disability, skin cancer risk, and psychosocial stress. Tyrosinase (TYR) catalyzes the first and rate-limiting step of melanin synthesis. Inhibitors of TYR are well established and are currently used in clinical settings; however, there is a dearth of direct activators of TYR. Here, using a human TYR construct, we developed high-throughput screening methods, in cell confirmatory assays employing 13C-tyrosine tracing, and computational analysis techniques, and identified ampyrone (4-aminoantipyrine) as a TYR activator. Ampyrone increased the in vitro catalytic activity of the human recombinant intra-melanosomal domain of TYR (hTYR) and its hypomorphic variant, Pro406Leu (P406L), a cause of oculocutaneous albinism type 1B (OCA1B). Moreover, ampyrone induced melanin synthesis in both wild-type and OCA1B human melanocytes, mouse OCA2 melanocytes, as well as 3-dimensional (3D) human skin cultures. Computational studies provided additional insight into the effects of direct TYR agonists on enzyme activity. Our results identified ampyrone as a lead candidate for TYR activation, potentially supporting the development of therapies for patients with genetic and acquired diseases of hypopigmentation.
Authors
Monika B. Dolinska, Yuhong A. Wang, Nathan P. Coussens, Vijay K. Kalaskar, Zuhal Eraslan, Samuel J. Grondin, Joseph Bonica, Sarah Toay, Matthew D. Hall, Min Shen, Matthew Boxer, Qiuying Chen, Steven S. Gross, Nabeel Attarwala, Yingyos Jittayasothorn, Ramakrisha P. Alur, Dhyanam Shukla, Robin Kee, Charles DeYoung, Cuilee Sha, David R. Adams, Stacie K. Loftus, Tiziana Cogliati, Yuri V. Sergeev, Jonathan H. Zippin, Brian P. Brooks
Abstract
Several genes guide inner ear development, and mutations in these genes can cause malformations that result in congenital hearing loss. However, the contribution of noncoding regulatory elements remains largely unclear. This study investigates the function of distal enhancer elements in the transcriptional regulation of GDF6, a gene implicated in cochlear development. Using mouse models with targeted deletions, human inner ear organoids, and CRISPR interference (CRISPRi), we identified a downstream regulatory interval harboring a developmental enhancer required to maintain GDF6 expression during otic epithelial maturation and cochlear morphogenesis. Deletion of this regulatory region or targeting of CRISPRi-based repressors to these regions resulted in decreased GDF6 expression, failure of otic-epithelium development, and prevention of hair cell-like differentiation, reflecting cochlear aplasia observed in patients with corresponding genomic deletions. These findings highlight the contribution of long-range regulatory elements to auditory development and illustrate how their disruption contributes to human deafness.
Authors
Mohammad Faraz Zafeer, Clemer Abad, Havva Ortabozkoyun, Memoona Ramzan, Guney Bademci, Maria C. Robayo, Duygu Duman, Rolen M. Quadros, Shengru Guo, Juan I. Young, Anthony J. Griswold, Channabasavaiah B. Gurumurthy, Derek M. Dykxhoorn, Katherina Walz, Mustafa Tekin
Abstract
Pulmonary arterial hypertension (PAH) is a progressive vascular syndrome characterized by aberrant signaling, severe pulmonary artery remodeling, and right ventricular (RV) failure, a major driver of morbidity and mortality. Dysregulation of the apelinergic pathway has been implicated in pulmonary vascular remodeling in PAH. Using a sugen-hypoxia rat model of PAH, we assessed the ability of a novel apelin analog, resistant to native peptidase degradation, to reverse the pathological hallmarks of PAH and RV dysfunction. Apelin analog therapy corrected the vascular lesions in the lungs and nearly normalized pulmonary arterial pressures. Early cardiorenal syndrome, RV dilation and dysfunction as well as RV cardiomyocyte and fibroblast activation induced by pressure overload, were also reversed by apelin analog treatment. Single-nucleus RNA sequencing of the lungs and RV revealed apelin-analog treatment activated several protective pathways, including rebalancing protective BMPR2 (bone morphogenetic protein receptor type 2) signaling to counteract excessive pathogenic TGFBR2 (transforming growth factor β receptor 2) activity in PAH. These findings highlight the therapeutic potential of exogenous apelin in reversing pulmonary vascular and cardiac pathologies in PAH and support further investigation to evaluate the clinical benefits of apelin analog treatment in patients with PAH and RV failure.
Authors
Jennie Vu, Pavel Zhabyeyev, Kemar J. Brown, Joshua M. Gorham, Daniel M. DeLaughter, Huachen Chen, Thilina U. Jayawardena, Ander Vergara, Maria Alexiou, Anjalee Wijewardane, Conrad Fischer, Charlotte Avet, Abby Ewasiuk, Faqi Wang, Mark C. Chappell, Yuri Kim, Michel Bouvier, John C. Vederas, Christine E. Seidman, Jonathan G. Seidman, Gavin Y. Oudit
Abstract
Circulating fatty acids (FA) are constitutively taken up by basolateral kidney proximal tubule transporters and are the preferred metabolic substrate. In many chronic kidney diseases, the damaged glomerular filtration barrier permits passage of albumin-bound FA, which are reabsorbed by apical FA transport protein-2 (FATP2). Bilateral FA uptake leads to lipotoxicity and progressive renal function decline, but the relative apical versus basolateral contribution and intracellular mechanisms are not established. Apical or bilateral (but not basolateral) palmitate incubation with human proximal tubule cells stimulated endoplasmic reticulum (ER) stress gene expression, ER stress pathway activation, and ER fragmentation. Apical or bilateral palmitate was associated with reduced lipid droplets, and decreased expression of ER-localized lipid droplet biogenesis transcripts. Inhibition of lipid droplet formation also precipitated ER stress, suggesting diminished sequestration of FA metabolites as the cause. Indeed, C16:0 ceramide was increased in bilateral palmitate-treated cells, and in kidneys from mice that phenocopy progressive diabetic kidney disease. Ceramide synthesis inhibition abrogated ER stress, and transfection with C16:0 ceramide decreased ER membrane fluidity and caused ER stress. We conclude that aberrant filtration and uptake of FA by apical FATP2 exceeded the capacity for lipid droplet incorporation, and led to cytotoxicity from ceramide-induced ER lipid bilayer stress.
Authors
Zhiyu Liu, Robert J. Gaivin, Shenaz Khan, Vincent Li, Amal Chaba, Fraser J. Moss, Usman Sabir, Takhar Kasumov, Tingwei Mu, Jeffrey R. Schelling
Abstract
Recent evidence suggests a role for biological factors to explain increased risk for active pulmonary tuberculosis (PTB) among men. We conducted a prospective cohort study in Mali of treatment naive males and females with laboratory-confirmed PTB and latent TB infection (LTBI) and healthy controls of similar ages to determine the relationship between alterations in gonadal steroids, tuberculosis (TB) disease status, and treatment outcomes. Prior to treatment, males with PTB had lower testosterone concentrations compared to males with LTBI or healthy males. Reduced testosterone concentrations in males with PTB were transient, returning healthy ranges by month 2 of treatment, which corresponded to the end of intensive TB treatment. Estradiol concentrations in females were not altered by PTB or infection status yet increased at month 6 of treatment. Testosterone, but not estradiol, was a strong predictor of cure during treatment. Testosterone, but not estradiol, concentrations in PTB cases were inversely correlated with IFN-γ, IL-6, and IL-2. Concentrations of IL-17 and IL-10 were lower in males than females at the end of TB treatment. Our results suggest that TB-induced changes in testosterone concentrations during PTB and in response to treatment occur in males and could contribute to sex differences in TB pathogenesis.
Authors
Djeneba Dabitao, Bocar Baya, Ibrahim Sanogo, Amadou Somboro, Mamadou Wague, Mamadou D. Coulibaly, Isaac Koloma, Mahamadou Kone, Mohamed Nantoume, Nadie Coulibaly, Behinan Stephane, Mariam Coulibaly, Mamadou Perou, Moumine Sanogo, Ayouba Diarra, Seydou Samake, Bassirou Diarra, Mahamadou Diakite, Souleymane Diallo, Yacouba Toloba, Chad J. Achenbach, Jane L. Holl, Seydou Doumbia, Robert L. Murphy, William R. Bishai, Sabra L. Klein
Abstract
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease caused by the selective loss of upper and lower motor neurons. There is a considerable variability in the disease progression of sporadic ALS, but the molecular basis for phenotypic heterogeneity remains largely unknown. ALS patients often manifest systemic metabolic abnormalities such as glucose intolerance and hypermetabolic state. We conducted reverse translational research to explore therapeutic targets in ALS based on the systemic metabolic alterations in patients and identified several metabolites associated with the disease progression, including metabolites involved in the expanded endocannabinoid system (ECS). In particular, the levels of N-acyl taurines (NATs) were correlated with the longitudinal change in the revised ALS functional rating scale and survival. Experiments with ALS cellular models, iPS cells derived from ALS patients and SOD1G93A transgenic mice revealed that PF-04457845, a fatty acid amide hydrolase inhibitor, upregulated the expanded ECS, particularly the levels of NATs and ameliorated motor neuron degeneration through the regulation of microglial environment, synapse plasticity, and neuronal development. These results collectively indicate that dysregulation of NATs is associated with ALS progression and PF-04457845 may represent a potential disease-modifying therapy for ALS.
Authors
Daisuke Ito, Madoka Iida, Yohei Iguchi, Atsushi Hashizume, Shinichiro Yamada, Yoshiyuki Kishimoto, Shota Komori, Kazuki Obara, Shuto Nishisaki, Satoshi Yokoi, Teppei Shimamura, Yuto Takemoto, Masahiro Nakatochi, Tomohiro Akashi, Kunihiko Hinohara, Hyeon-Cheol Lee-Okada, Yohei Okada, Junichi Niwa, Gen Sobue, Shinji Tanaka, Ken Takashina, Takehiko Yokomizo, Masahisa Katsuno
Abstract
While glucagon-like peptide-1 receptor agonists (GLP-1RAs) like semaglutide are effective in treating obesity, up to 45% of the resulting weight loss can be attributed to skeletal muscle loss. Given the critical role of skeletal muscle in health and mobility, this may have long-term adverse consequences. Herein we investigated whether oral ketone ester supplementation could prevent semaglutide-induced muscle loss and explored the underlying molecular mechanisms. Obese, glucose-intolerant mice received vehicle, semaglutide, or semaglutide plus a β-hydroxybutyrate–generating ketone ester for three weeks. Body composition, muscle strength, and endurance were assessed longitudinally. Semaglutide monotherapy reduced lean mass, impaired muscle strength, and suppressed mitochondrial gene expression while elevating atrophy-related genes in skeletal muscle samples. Co-administration with ketone ester preserved skeletal muscle mass and function without compromising fat loss. Mechanistically, ketone ester co-treatment prevented semaglutide-induced changes in mitochondrial and atrophy-related gene expression, suggesting mitochondrial defects and impaired ketone metabolism contribute to GLP-1RA-induced muscle loss. Together, these findings demonstrate that ketone ester supplementation can maintain muscle mass and performance during semaglutide-driven weight loss. These preclinical findings support ketone therapy as a promising strategy to counteract the sarcopenia-promoting effects of GLP-1RAs and warrant clinical evaluation to assess its translational potential.
Authors
Yasser Abuetabh, Mya A. Schmidt, Masaaki Naganuma, Ramana Vaka, Mahmoud A. El-Ghiaty, Shelly Braun, Ethan A. Kwan, Matthieu C.P. Zolondek, Darius Sahid, Laibah Khan, Rajat K. Shandal, Ashley L. Trudeau, Yaning Li, Sufyan O. Malik, Qiuyu Sun, Danica K. Roth, Daniela Y. Morales-Llamas, Jody L. Levasseur, Mourad Ferdaoussi, Richard P. Fahlman, Jason R.B. Dyck
Abstract
Inflammatory bowel disease (IBD), encompassing ulcerative colitis (UC) and Crohn’s disease (CD), is marked by chronic intestinal inflammation and dysregulated immunity. Although UC and CD affect different areas of the gastrointestinal tract, both diseases share aberrant CD4+ memory T cell responses, with HLA-DRB1 as a major genetic risk factor. HLA-DRB1 encodes MHC class II molecules that influence the CD4+ T cell receptor (TCR) repertoire, yet how these genotypes shape TCR specificity in IBD remains unclear. Here, we genotyped HLA-DRB1 and profiled 3.13 million TCRb sequences from circulating memory CD4+ T cells in 33 IBD patients (20 UC, 13 CD) and 14 healthy controls. Using the GLIPH2 algorithm, we distilled 468,441 candidates based on CDR3 amino acid motifs into 440 high-confidence TCR specificity groups significantly enriched among individuals sharing HLA-DRB1 alleles. Notably, five specificity groups were IBD-enriched and shared between UC and CD, suggesting common antigen targets in both diseases. We also observed increased frequencies of clonally expanded cytotoxic GZMB+PRF1+ memory CD4+ T cells and KIRs+CD8+ T cells in a subset of risk-allele carriers with IBD. These findings elucidate distinct, HLA-linked TCR specificity groups in IBD and provide mechanistic insights that may advance antigen discovery and personalized medicine.
Authors
Joshua E. Chan, Azam Mohsin, Jens Krijgsman, Ciska Lindelauf, Qinghui Mu, Brianna Cavalla, Xuhuai Ji, Sarah E. Streett, Vincent van Unen, Mark M. Davis
Abstract
B cells contribute to the pathogenesis of food allergies as they induce allergen-specific antibody production. Clinically-used allergen-specific immunotherapies have shown to induce regulatory B cell (Bregs) subsets as well as target and reduce allergy-driving B cell functions. This report aims to elucidate the contribution of regulatory B cells to an allergen-encapsulating nanoparticle (aeNP) immunotherapy in a murine model of food allergy. In this model, B cells directly associated with aeNPs. CD20+ B cell depletion after aeNP treatment increased the number of mice with severe allergic reactions during oral food challenges and reduced the expansion of regulatory immune cells including CD103+ dendritic cells (DCs) and CCR9+ gut-homing regulatory T cells, indicating that B cells are a component of aeNP immunomodulation. B cell communication in the gastrointestinal tract of aeNP-treated mice identified CD23 signaling as a potential inducer of regulatory CD103+ DC functions and disrupter of allergy-driving B cell-T cell communication. These tolerogenic signaling patterns were also identified in IL-10+ B cells, which have been known to impart regulatory immune effects in both murine and human disease. Ultimately, B cells are a component of the complex immunomodulation leading to aeNP efficacy at reducing allergic reactivity.
Authors
Laila M. Rad, Michael N. Saunders, Laura A. Williams, Katarzyna W. Janczak, Chris L. Dorsett, Kate V. Griffin, Elizabeth J. Bealer, Jeffrey A. Ma, Sayre A. Tillery, Jyotirmoy Roy, Stephen D. Miller, Jessica J. O'Konek, Lonnie D. Shea
Abstract
YAP/TAZ signaling is required for initiation of lung alveolar repair, yet previous studies in idiopathic pulmonary fibrosis (IPF) predicted increased YAP/TAZ signaling in alveolar epithelial cells (AECs). We investigated whether persistent YAP/TAZ AEC signaling contributes to failed epithelial repair and persistent fibrotic remodeling. In IPF lungs, we identified increased YAP+/TAZ+ AECs and increased transcriptional target expression. Pharmacological YAP/TAZ activation in human AEC organoids and in murine AT2 cell organoids generated with genetic YAP/TAZ activation (YTactive) (via deletion of Hippo-kinases Stk3/4), resulted in phenotype shifts into aberrant transitional and airway-like states. Bleomycin injury of YTactive mice resulted in persistent fibrotic remodeling at 28- and 56-days post-bleomycin injury. Gene promoter activity associated with transitional cell markers (Krt19, Hopx, and Runx2) was increased in YTactive AT2 cells. Immunofluorescent staining showed a loss of AT2 associated Cebpa and increased Krt19 in YTactive lineage traced AT2 cells 28 days post-injury. Inhibition of YAP/TAZ using Verteporfin resulted in improved lung repair in YTactive mouse lungs, including restored Cebpa and decreased Krt19+ transitional cells. These findings demonstrate sustained YAP/TAZ activation drives abnormal alveolar repair and persistent fibrotic remodeling. Blocking aberrant persistent YAP/TAZ activity promotes adaptive repair and has potential as a therapeutic strategy for pulmonary fibrosis.
Authors
Isabella P. Gaona, A. Scott McCall, Natalie M. Geis, Arlo C. Colvard, Gianluca T. DiGiovanni, Taylor P. Sherrill, Ujjal K. Singha, David S. Nichols, Ana P. Serezani, Holly E. David, Jean-Philippe Cartailler, Shristi Shrestha, Sergey S. Gutor, Timothy S. Blackwell, Jonathan A. Kropski, Jason J. Gokey
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