Browsing by Subject "Molecular medicine"

Now showing 1 - 8 of 8
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    Cilia-associated wound repair mediated by IFT88 in retinal pigment epithelium
    (Springer Nature, 2023-05-21) Ning, Ke; Bhuckory, Mohajeet B.; Lo, Chien‑Hui; Sendayen, Brent E.; Kowal, Tia J.; Chen, Ming; Bansal, Ruchi; Chang, Kun‑Che; Vollrath, Douglas; Berbari, Nicolas F.; Mahajan, Vinit B.; Hu, Yang; Sun, Yang; Biology, School of Science
    Primary cilia are conserved organelles that integrate extracellular cues into intracellular signals and are critical for diverse processes, including cellular development and repair responses. Deficits in ciliary function cause multisystemic human diseases known as ciliopathies. In the eye, atrophy of the retinal pigment epithelium (RPE) is a common feature of many ciliopathies. However, the roles of RPE cilia in vivo remain poorly understood. In this study, we first found that mouse RPE cells only transiently form primary cilia. We then examined the RPE in the mouse model of Bardet-Biedl Syndrome 4 (BBS4), a ciliopathy associated with retinal degeneration in humans, and found that ciliation in BBS4 mutant RPE cells is disrupted early during development. Next, using a laser-induced injury model in vivo, we found that primary cilia in RPE reassemble in response to laser injury during RPE wound healing and then rapidly disassemble after the repair is completed. Finally, we demonstrated that RPE-specific depletion of primary cilia in a conditional mouse model of cilia loss promoted wound healing and enhanced cell proliferation. In summary, our data suggest that RPE cilia contribute to both retinal development and repair and provide insights into potential therapeutic targets for more common RPE degenerative diseases.
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    Establishment and characterization of patient-derived xenograft of a rare pediatric anaplastic pleomorphic xanthoastrocytoma (PXA) bearing a CDC42SE2-BRAF fusion
    (Springer Nature, 2023-06-06) Damayanti, Nur P.; Saadatzadeh, M. Reza; Dobrota, Erika; Ordaz, Josue D.; Bailey, Barbara J.; Pandya, Pankita H.; Bijangi-Vishehsaraei, Khadijeh; Shannon, Harlan E.; Alfonso, Anthony; Coy, Kathy; Trowbridge, Melissa; Sinn, Anthony L.; Zhang, Zhong-Yin; Gallagher, Rosa I.; Wulfkuhle, Julia; Petricoin, Emanuel; Richardson, Angela M.; Marshall, Mark S.; Lion, Alex; Ferguson, Michael J.; Balsara, Karl E.; Pollok, Karen E.; Neurological Surgery, School of Medicine
    Pleomorphic xanthoastrocytoma (PXA) is a rare subset of primary pediatric glioma with 70% 5-year disease free survival. However, up to 20% of cases present with local recurrence and malignant transformation into more aggressive type anaplastic PXA (AXPA) or glioblastoma. The understanding of disease etiology and mechanisms driving PXA and APXA are limited, and there is no standard of care. Therefore, development of relevant preclinical models to investigate molecular underpinnings of disease and to guide novel therapeutic approaches are of interest. Here, for the first time we established, and characterized a patient-derived xenograft (PDX) from a leptomeningeal spread of a patient with recurrent APXA bearing a novel CDC42SE2-BRAF fusion. An integrated -omics analysis was conducted to assess model fidelity of the genomic, transcriptomic, and proteomic/phosphoproteomic landscapes. A stable xenoline was derived directly from the patient recurrent tumor and maintained in 2D and 3D culture systems. Conserved histology features between the PDX and matched APXA specimen were maintained through serial passages. Whole exome sequencing (WES) demonstrated a high degree of conservation in the genomic landscape between PDX and matched human tumor, including small variants (Pearson's r = 0.794-0.839) and tumor mutational burden (~ 3 mutations/MB). Large chromosomal variations including chromosomal gains and losses were preserved in PDX. Notably, chromosomal gain in chromosomes 4-9, 17 and 18 and loss in the short arm of chromosome 9 associated with homozygous 9p21.3 deletion involving CDKN2A/B locus were identified in both patient tumor and PDX sample. Moreover, chromosomal rearrangement involving 7q34 fusion; CDC42SE-BRAF t (5;7) (q31.1, q34) (5:130,721,239, 7:140,482,820) was identified in the PDX tumor, xenoline and matched human tumor. Transcriptomic profile of the patient's tumor was retained in PDX (Pearson r = 0.88) and in xenoline (Pearson r = 0.63) as well as preservation of enriched signaling pathways (FDR Adjusted P < 0.05) including MAPK, EGFR and PI3K/AKT pathways. The multi-omics data of (WES, transcriptome, and reverse phase protein array (RPPA) was integrated to deduce potential actionable pathways for treatment (FDR < 0.05) including KEGG01521, KEGG05202, and KEGG05200. Both xenoline and PDX were resistant to the MEK inhibitors trametinib or mirdametinib at clinically relevant doses, recapitulating the patient's resistance to such treatment in the clinic. This set of APXA models will serve as a preclinical resource for developing novel therapeutic regimens for rare anaplastic PXAs and pediatric high-grade gliomas bearing BRAF fusions.
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    High-content image-based analysis and proteomic profiling identifies Tau phosphorylation inhibitors in a human iPSC-derived glutamatergic neuronal model of tauopathy
    (Springer Nature, 2021-08-23) Cheng, Chialin; Reis, Surya A.; Adams, Emily T.; Fass, Daniel M.; Angus, Steven P.; Stuhlmiller, Timothy J.; Richardson, Jared; Olafson, Hailey; Wang, Eric T.; Patnaik, Debasis; Beauchamp, Roberta L.; Feldman, Danielle A.; Silva, M. Catarina; Sur, Mriganka; Johnson, Gary L.; Ramesh, Vijaya; Miller, Bruce L.; Temple, Sally; Kosik, Kenneth S.; Dickerson, Bradford C.; Haggarty, Stephen J.; Pediatrics, School of Medicine
    Mutations in MAPT (microtubule-associated protein tau) cause frontotemporal dementia (FTD). MAPT mutations are associated with abnormal tau phosphorylation levels and accumulation of misfolded tau protein that can propagate between neurons ultimately leading to cell death (tauopathy). Recently, a p.A152T tau variant was identified as a risk factor for FTD, Alzheimer's disease, and synucleinopathies. Here we used induced pluripotent stem cells (iPSC) from a patient carrying this p.A152T variant to create a robust, functional cellular assay system for probing pathophysiological tau accumulation and phosphorylation. Using stably transduced iPSC-derived neural progenitor cells engineered to enable inducible expression of the pro-neural transcription factor Neurogenin 2 (Ngn2), we generated disease-relevant, cortical-like glutamatergic neurons in a scalable, high-throughput screening compatible format. Utilizing automated confocal microscopy, and an advanced image-processing pipeline optimized for analysis of morphologically complex human neuronal cultures, we report quantitative, subcellular localization-specific effects of multiple kinase inhibitors on tau, including ones under clinical investigation not previously reported to affect tau phosphorylation. These results demonstrate the potential for using patient iPSC-derived ex vivo models of tauopathy as genetically accurate, disease-relevant systems to probe tau biochemistry and support the discovery of novel therapeutics for tauopathies.
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    A Modified Collagen Dressing Induces Transition of Inflammatory to Reparative Phenotype of Wound Macrophages
    (Nature Research, 2019-10-04) Das, Amitava; Abas, Motaz; Biswas, Nirupam; Banerjee, Pradipta; Ghosh, Nandini; Rawat, Atul; Khanna, Savita; Roy, Sashwati; Sen, Chandan K.; Surgery, School of Medicine
    Collagen containing wound-care dressings are extensively used. However, the mechanism of action of these dressings remain unclear. Earlier studies utilizing a modified collagen gel (MCG) dressing demonstrated improved vascularization of ischemic wounds and better healing outcomes. Wound macrophages are pivotal in facilitating wound angiogenesis and timely healing. The current study was designed to investigate the effect of MCG on wound macrophage phenotype and function. MCG augmented recruitment of macrophage at the wound-site, attenuated pro-inflammatory and promoted anti-inflammatory macrophage polarization. Additionally, MCG increased anti-inflammatory IL-10, IL-4 and pro-angiogenic VEGF production, indicating a direct role of MCG in resolving wound inflammation and improving angiogenesis. At the wound-site, impairment in clearance of apoptotic cell bioburden enables chronic inflammation. Engulfment of apoptotic cells by macrophages (efferocytosis) resolves inflammation via a miR-21-PDCD4-IL-10 pathway. MCG-treated wound macrophages exhibited a significantly bolstered efferocytosis index. Such favorable outcome significantly induced miR-21 expression. MCG-mediated IL-10 production was dampened under conditions of miR-21 knockdown pointing towards miR-21 as a causative factor. Pharmacological inhibition of JNK attenuated IL-10 production by MCG, implicating miR-21-JNK pathway in MCG-mediated IL-10 production by macrophages. This work provides direct evidence demonstrating that a collagen-based wound-care dressing may influence wound macrophage function and therefore modify wound inflammation outcomes.
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    Preterm birth buccal cell epigenetic biomarkers to facilitate preventative medicine
    (Springer Nature, 2022-03-01) Winchester, Paul; Nilsson, Eric; Beck, Daniel; Skinner, Michael K.; Pediatrics, School of Medicine
    Preterm birth is the major cause of newborn and infant mortality affecting nearly one in every ten live births. The current study was designed to develop an epigenetic biomarker for susceptibility of preterm birth using buccal cells from the mother, father, and child (triads). An epigenome-wide association study (EWAS) was used to identify differential DNA methylation regions (DMRs) using a comparison of control term birth versus preterm birth triads. Epigenetic DMR associations with preterm birth were identified for both the mother and father that were distinct and suggest potential epigenetic contributions from both parents. The mother (165 DMRs) and female child (136 DMRs) at p < 1e-04 had the highest number of DMRs and were highly similar suggesting potential epigenetic inheritance of the epimutations. The male child had negligible DMR associations. The DMR associated genes for each group involve previously identified preterm birth associated genes. Observations identify a potential paternal germline contribution for preterm birth and identify the potential epigenetic inheritance of preterm birth susceptibility for the female child later in life. Although expanded clinical trials and preconception trials are required to optimize the potential epigenetic biomarkers, such epigenetic biomarkers may allow preventative medicine strategies to reduce the incidence of preterm birth.
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    Somatic cell hemoglobin modulates nitrogen oxide metabolism in the human airway epithelium
    (Springer Nature, 2021-07-29) Marozkina, Nadzeya; Smith, Laura; Zhao, Yi; Zein, Joe; Chmiel, James F.; Kim, Jeeho; Kiselar, Janna; Davis, Michael D.; Cunningham, Rebekah S.; Randell, Scott H.; Gaston, Benjamin; Pediatrics, School of Medicine
    Endothelial hemoglobin (Hb)α regulates endothelial nitric oxide synthase (eNOS) biochemistry. We hypothesized that Hb could also be expressed and biochemically active in the ciliated human airway epithelium. Primary human airway epithelial cells, cultured at air–liquid interface (ALI), were obtained by clinical airway brushings or from explanted lungs. Human airway Hb mRNA data were from publically available databases; or from RT-PCR. Hb proteins were identified by immunoprecipitation, immunoblot, immunohistochemistry, immunofluorescence and liquid chromatography- mass spectrometry. Viral vectors were used to alter Hbβ expression. Heme and nitrogen oxides were measured colorimetrically. Hb mRNA was expressed in human ciliated epithelial cells. Heme proteins (Hbα, β, and δ) were detected in ALI cultures by several methods. Higher levels of airway epithelial Hbβ gene expression were associated with lower FEV1 in asthma. Both Hbβ knockdown and overexpression affected cell morphology. Hbβ and eNOS were apically colocalized. Binding heme with CO decreased extracellular accumulation of nitrogen oxides. Human airway epithelial cells express Hb. Higher levels of Hbβ gene expression were associated with airflow obstruction. Hbβ and eNOS were colocalized in ciliated cells, and heme affected oxidation of the NOS product. Epithelial Hb expression may be relevant to human airways diseases.
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    Synaptosome microRNAs regulate synapse functions in Alzheimer's disease
    (Springer Nature, 2022-08-08) Kumar, Subodh; Orlov, Erika; Gowda, Prashanth; Bose, Chhanda; Swerdlow, Russell H.; Lahiri, Debomoy K.; Reddy, P. Hemachandra; Psychiatry, School of Medicine
    MicroRNAs (miRNAs) are found in nerve terminals, synaptic vesicles, and synaptosomes, but it is unclear whether synaptic and cytosolic miRNA populations differ in Alzheimer's disease (AD) or if synaptosomal miRNAs affect AD synapse activity. To address these questions, we generated synaptosomes and cytosolic fractions from postmortem brains of AD and unaffected control (UC) samples and analyzed them using a global Affymetrix miRNAs microarray platform. A group of miRNAs significantly differed (P < 0.0001) with high fold changes variance (+/- >200-fold) in their expressions in different comparisons: (1) UC synaptosome vs UC cytosol, (2) AD synaptosomes vs AD cytosol, (3) AD cytosol vs UC cytosol, and (4) AD synaptosomes vs UC synaptosomes. MiRNAs data analysis revealed that some potential miRNAs were consistently different across sample groups. These differentially expressed miRNAs were further validated using AD postmortem brains, brains of APP transgenic (Tg2576), Tau transgenic (P301L), and wild-type mice. The miR-501-3p, miR-502-3p, and miR-877-5p were identified as potential synaptosomal miRNAs upregulated with disease progression based on AD Braak stages. Gene Ontology Enrichment and Ingenuity Pathway Analysis of synaptosomal miRNAs showed the involvement of miRNAs in nervous system development, cell junction organization, synapse assembly formation, and function of GABAergic synapse. This is the first description of synaptic versus cytosolic miRNAs in AD and their significance in synapse function.
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    The molecular make up of smoldering myeloma highlights the evolutionary pathways leading to multiple myeloma
    (Springer Nature, 2021-01-12) Boyle, Eileen M.; Deshpande, Shayu; Tytarenko, Ruslana; Ashby, Cody; Wang, Yan; Bauer, Michael A.; Johnson, Sarah K.; Wardell, Christopher P.; Thanendrarajan, Sharmilan; Zangari, Maurizio; Facon, Thierry; Dumontet, Charles; Barlogie, Bart; Arbini, Arnaldo; Rustad, Even H.; Maura, Francesco; Landgren, Ola; Zhan, Fenghuang; van Rhee, Frits; Schinke, Carolina; Davies, Faith E.; Morgan, Gareth J.; Walker, Brian A.; Medicine, School of Medicine
    Smoldering myeloma (SMM) is associated with a high-risk of progression to myeloma (MM). We report the results of a study of 82 patients with both targeted sequencing that included a capture of the immunoglobulin and MYC regions. By comparing these results to newly diagnosed myeloma (MM) we show fewer NRAS and FAM46C mutations together with fewer adverse translocations, del(1p), del(14q), del(16q), and del(17p) in SMM consistent with their role as drivers of the transition to MM. KRAS mutations are associated with a shorter time to progression (HR 3.5 (1.5-8.1), p = 0.001). In an analysis of change in clonal structure over time we studied 53 samples from nine patients at multiple time points. Branching evolutionary patterns, novel mutations, biallelic hits in crucial tumour suppressor genes, and segmental copy number changes are key mechanisms underlying the transition to MM, which can precede progression and be used to guide early intervention strategies.