12 research outputs found
Microscopy-Based Automated Live Cell Screening for Small Molecules That Affect Ciliation
The primary monocilium, or cilium, is a single antenna-like organelle that protrudes from the surface of most mammalian cell types, and serves as a signaling hub. Mutations of cilia-associated genes result in severe genetic disorders termed ciliopathies. Among these, the most common is autosomal dominant polycystic kidney disease (ADPKD); less common genetic diseases include Bardet–Biedl syndrome, Joubert syndrome, nephronophthisis, and others. Important signaling cascades with receptor systems localized exclusively or in part at cilia include Sonic Hedgehog (SHH), platelet derived growth factor alpha (PDGFRα), WNTs, polycystins, and others. Changes in ciliation during development or in pathological conditions such as cancer impacts signaling by these proteins. Notably, ciliation status of cells is coupled closely to the cell cycle, with cilia protruding in quiescent (G0) or early G1 cells, declining in S/G2, and absent in M phase, and has been proposed to contribute to cell cycle regulation. Because of this complex biology, the elaborate machinery regulating ciliary assembly and disassembly receives input from many cellular proteins relevant to cell cycle control, development, and oncogenic transformation, making study of genetic factors and drugs influencing ciliation of high interest. One of the most effective tools to investigate the dynamics of the cilia under different conditions is the imaging of live cells. However, developing assays to observe the primary cilium in real time can be challenging, and requires a consideration of multiple details related to the cilia biology. With the dual goals of identifying small molecules that may have beneficial activity through action on human diseases, and of identifying ciliary activities of existing agents that are in common use or development, we here describe creation and evaluation of three autofluorescent cell lines derived from the immortalized retinal pigmented epithelium parental cell line hTERT-RPE1. These cell lines stably express the ciliary-targeted fluorescent proteins L13-Arl13bGFP, pEGFP-mSmo, and tdTomato-MCHR1-N-10. We then describe methods for use of these cell lines in high throughput screening of libraries of small molecule compounds to identify positive and negative regulators of ciliary disassembly
Primary central nervous system post-transplant lymphoproliferative disorder after allogeneic stem cell transplantation: a case report
PurposePrimary central nervous system, diffuse large B-cell lymphoma, post-transplant lymphoproliferative disorder in the cerebellopontine angle after an allogeneic stem cell transplantation has never been reported in the literature. Typically, diffuse large B-cell lymphoma is non-polyploid. We report the first case of a patient with polyploid post-transplant lymphoproliferative disorder in the cerebellopontine angle who presented with back pain.Case presentationA 45-year-old man with a history of nodular sclerosing classic Hodgkin lymphoma stage IIB treated with systemic chemotherapy, external radiation and autologous stem cell transplant and double umbilical cord allogeneic transplant presented with several weeks of back pain. He was found to have a small right cerebellopontine angle mass thought to be consistent with a meningioma. Patient presented again two weeks later with acute onset of severe headache, right sided ptosis, right facial numbness, weakness and possible seizure event. Repeat MRI scans showed an interval and significant increase of the right cerebellopontine angle lesion. Biopsy of the cerebellopontine angle lesion was planned with suspicion of lymphoma. Intraoperative pathology consultation findings were not consistent with an acoustic neuroma, meningioma, or epidermoid cyst. Lymphoma could not be definitively identified by intra-operative frozen section. However, it was suspected, and a portion of fresh specimen was submitted for flow cytometry analysis. A near total resection of the tumor and decompression of the brainstem was achieved. Final pathologic analysis was positive for post-transplant lymphoproliferative disorder, monomorphic type, diffuse large B-cell lymphoma, non-germinal center B-cell type, EBV+, post-transplant (allogeneic stem cell) setting (post-transplant lymphoproliferative disorder (PTLD), monomorphic type, diffuse large B-cell lymphoma, non-germinal center B-cell type (non-GCB), EBV-positive under pre-2022 WHO terminology). The patient began a high-dose methotrexate-based regimen (the MATRIX regimen).ConclusionsOur case illustrates an unusual presentation of post-transplant lymphoproliferative disorder in the cerebellopontine angle in a patient with a remote history of allogeneic stem cell transplantation. It demonstrates the importance of keeping primary central nervous system post-transplant lymphoproliferative disorder on the differential for patients who present with back pain or headache that have a history of allogeneic stem cell transplant
Advanced Sintering Techniques in Design of Planar IT SOFC and Supported Oxygen Separation Membranes
Trimethylammonium Sn(IV) and Pb(IV) Chlorometalate Complexes with Incorporated Dichlorine
Supramolecular dichloro-chlorostannate(IV) and -plumbate(IV) complexes (Me3NH)2{[MCl6]Cl2} (M = Sn (1), Pb (2)) feature dichlorine units incorporated into a halometalate framework. Both compounds were characterized by X-ray diffractometry and Raman spectroscopy
Structural and Transport Properties of E-Beam Sintered Lanthanide Tungstates and Tungstates-Molybdates
Lanthanide tungstates and molybdates are promising materials for hydrogen separation membranes due to their high protonic conductivity. A promising approach to fabricating ceramics based on these materials is radiation thermal sintering. The current work aims at studying the effect of radiation thermal sintering on the structural morphological and transport properties of (Nd,Ln)5.5(W,Mo)O11.25–δ as promising materials for hydrogen separation membranes. The defect fluorite structure was shown to be preserved during radiation thermal sintering at 1100 °C. The presence of protons in hydrated samples was confirmed by TGA. According to four-electrode studies and the isotope exchange of oxygen with C18O2, the samples demonstrate a high proton conductivity and oxygen mobility. Residual porosity (up to 29%) observed for these samples can be dealt with during membrane preparation by adding sintering aids and/or metal alloys nanoparticles. Hence, sintering by e-beams can be applied to the manufacturing of hydrogen separation membranes based on these materials
Ganetespib limits ciliation and cystogenesis in autosomal-dominant polycystic kidney disease (ADPKD)
Autosomal-dominant polycystic kidney disease (ADPKD) is associated with progressive formation of renal cysts, kidney enlargement, hypertension, and typically end-stage renal disease. In ADPKD, inherited mutations disrupt function of the polycystins (encoded by PKD1 and PKD2), thus causing loss of a cyst-repressive signal emanating from the renal cilium. Genetic studies have suggested ciliary maintenance is essential for ADPKD pathogenesis. Heat shock protein 90 (HSP90) clients include multiple proteins linked to ciliary maintenance. We determined that ganetespib, a clinical HSP90 inhibitor, inhibited proteasomal repression of NEK8 and the Aurora-A activator trichoplein, rapidly activating Aurora-A kinase and causing ciliary loss in vitro. Using conditional mouse models for ADPKD, we performed long-term (10 or 50 wk) dosing experiments that demonstrated HSP90 inhibition caused durable in vivo loss of cilia, controlled cystic growth, and ameliorated symptoms induced by loss of Pkd1 or Pkd2. Ganetespib efficacy was not increased by combination with 2-deoxy-n-glucose, a glycolysis inhibitor showing some promise for ADPKD. These studies identify a new biologic activity for HSP90 and support a cilia-based mechanism for cyst repression
Anti-MĂĽllerian Hormone Signaling Regulates Epithelial Plasticity and Chemoresistance in Lung Cancer
Anti-Müllerian hormone (AMH) and its type II receptor AMHR2, both previously thought to primarily function in gonadal tissue, were unexpectedly identified as potent regulators of transforming growth factor (TGF-β)/bone morphogenetic protein (BMP) signaling and epithelial-mesenchymal transition (EMT) in lung cancer. AMH is a TGF-β/BMP superfamily member, and AMHR2 heterodimerizes with type I receptors (ALK2, ALK3) also used by the type II receptor for BMP (BMPR2). AMH signaling regulates expression of BMPR2, ALK2, and ALK3, supports protein kinase B-nuclear factor κB (AKT-NF-κB) and SMAD survival signaling, and influences BMP-dependent signaling in non-small cell lung cancer (NSCLC). AMH and AMHR2 are selectively expressed in epithelial versus mesenchymal cells, and loss of AMH/AMHR2 induces EMT. Independent induction of EMT reduces expression of AMH and AMHR2. Importantly, EMT associated with depletion of AMH or AMHR2 results in chemoresistance but sensitizes cells to the heat shock protein 90 (HSP90) inhibitor ganetespib. Recognition of this AMH/AMHR2 axis helps to further elucidate TGF-β/BMP resistance-associated signaling and suggests new strategies for therapeutic targeting of EMT
Unexpected Activities in Regulating Ciliation Contribute to Off-target Effects of Targeted Drugs
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Author Correction: Clonally expanded CD8 T cells characterize amyotrophic lateral sclerosis-4
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Clonally expanded CD8 T cells characterize amyotrophic lateral sclerosis-4
Amyotrophic lateral sclerosis (ALS) is a heterogenous neurodegenerative disorder that affects motor neurons and voluntary muscle control1. ALS heterogeneity includes the age of manifestation, the rate of progression and the anatomical sites of symptom onset. Disease-causing mutations in specific genes have been identified and define different subtypes of ALS1. Although several ALS-associated genes have been shown to affect immune functions2, whether specific immune features account for ALS heterogeneity is poorly understood. Amyotrophic lateral sclerosis-4 (ALS4) is characterized by juvenile onset and slow progression3. Patients with ALS4 show motor difficulties by the time that they are in their thirties, and most of them require devices to assist with walking by their fifties. ALS4 is caused by mutations in the senataxin gene (SETX). Here, using Setx knock-in mice that carry the ALS4-causative L389S mutation, we describe an immunological signature that consists of clonally expanded, terminally differentiated effector memory (TEMRA) CD8 T cells in the central nervous system and the blood of knock-in mice. Increased frequencies of antigen-specific CD8 T cells in knock-in mice mirror the progression of motor neuron disease and correlate with anti-glioma immunity. Furthermore, bone marrow transplantation experiments indicate that the immune system has a key role in ALS4 neurodegeneration. In patients with ALS4, clonally expanded TEMRA CD8 T cells circulate in the peripheral blood. Our results provide evidence of an antigen-specific CD8 T cell response in ALS4, which could be used to unravel disease mechanisms and as a potential biomarker of disease state