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Wook Chung;
Jennifer L. Goeckeler-Fried;
Viktoria Havasi;
Annette Chiang;
Steven M. Rowe;
Zackery E. Plyler;
Jeong Hong;
Marina Mazur;
Gary A Piazza;
Adam B. Keeton;
E. Lucile White;
Lynn Rasmussen;
Allan M. Weissman;
R. Aldrin Denny;
Jeffrey L. Brodsky;
Eric Sorscher
Small molecules that correct the folding defects and enhance surface localization of the F508del mutation in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) comprise an important therapeutic strategy for cystic fibrosis lung disease. However, compounds that rescue the F508del mutant protein to wild type (WT) levels have not been identified. In this report, we consider obstacles to obtaining robust and therapeutically relevant levels of F508del CFTR. For example, markedly diminished steady state amounts of F508del CFTR compared to WT CFTR are present in recombinant bronchial epithelial cell lines, even when much higher levels of mutant transcript are present. In human primary airway cells, the paucity of Band B F508del is even more pronounced, although F508del and WT mRNA concentrations are comparable. Therefore, to augment levels of "repairable" F508del CFTR and identify small molecules that then correct this pool, we developed compound library screening protocols based on automated protein detection. First, cell-based imaging measurements were used to semi-quantitatively estimate distribution of F508del CFTR by high content analysis of two-dimensional images. We evaluated ∼2,000 known bioactive compounds from the NIH Roadmap Molecular Libraries Small Molecule Repository in a pilot screen and identified agents that increase the F508del protein pool. Second, we analyzed ∼10,000 compounds representing diverse chemical scaffolds for effects on total CFTR expression using a multi-plate fluorescence protocol and describe compounds that promote F508del maturation. Together, our findings demonstrate proof of principle that agents identified in this fashion can augment the level of endoplasmic reticulum (ER) resident "Band B" F508del CFTR suitable for pharmacologic correction. As further evidence in support of this strategy, PYR-41 - a compound that inhibits the E1 ubiquitin activating enzyme - was shown to synergistically enhance F508del rescue by C18, a small molecule corrector. Our combined results indicate that increasing the levels of ER-localized CFTR available for repair provides a novel route to correct F508del CFTR.
Patch-clamp recording has enabled single-cell electrical, morphological and genetic studies at unparalleled resolution. Yet it remains a laborious and low-throughput technique, making it largely impractical for large-scale measurements such as cell type and connectivity characterization of neurons in the brain. Specifically, the technique is critically limited by the ubiquitous practice of manually replacing patch-clamp pipettes after each recording. To circumvent this limitation, we developed a simple, fast, and automated method for cleaning glass pipette electrodes that enables their reuse within one minute. By immersing pipette tips into Alconox, a commercially-available detergent, followed by rinsing, we were able to reuse pipettes 10 times with no degradation in signal fidelity, in experimental preparations ranging from human embryonic kidney cells to neurons in culture, slices, and in vivo. Undetectable trace amounts of Alconox remaining in the pipette after cleaning did not affect ion channel pharmacology. We demonstrate the utility of pipette cleaning by developing the first robot to perform sequential patch-clamp recordings in cell culture and in vivo without a human operator.
Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge—a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry.
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Maryna Kapustina;
Denis Tsygankov;
Jia Zhao;
Timothy Wessler;
Xiaofeng Yang;
Alex Chen;
Nathan Roach;
Timothy C. Elston;
Qi Wang;
Ken Jacobson;
M. Gregory Forest
Cells transition from spread to rounded morphologies in diverse physiological contexts including mitosis and mesenchymal-to-amoeboid transitions. When these drastic shape changes occur rapidly, cell volume and surface area are approximately conserved. Consequently, the rounded cells are suddenly presented with a several-fold excess of cell surface whose area far exceeds that of a smooth sphere enclosing the cell volume. This excess is stored in a population of bleb-like protrusions (BLiPs), whose size distribution is shown by electron micrographs to be skewed. We introduce three complementary models of rounded cell morphologies with a prescribed excess surface area. A 2D Hamiltonian model provides a mechanistic description of how discrete attachment points between the cell surface and cortex together with surface bending energy can generate a morphology that satisfies a prescribed excess area and BLiP number density. A 3D random seed-and-growth model simulates efficient packing of BLiPs over a primary rounded shape, demonstrating a pathway for skewed BLiP size distributions that recapitulate 3D morphologies. Finally, a phase field model (2D and 3D) posits energy-based constitutive laws for the cell membrane, nematic F-actin cortex, interior cytosol, and external aqueous medium. The cell surface is equipped with a spontaneous curvature function, a proxy for the cell surface-cortex couple, that is a priori unknown, which the model “learns” from the thin section transmission electron micrograph image (2D) or the “seed and growth” model image (3D). Converged phase field simulations predict self-consistent amplitudes and spatial localization of pressure and stress throughout the cell for any posited stationary morphology target and cell compartment constitutive properties. The models form a general framework for future studies of cell morphological dynamics in a variety of biological contexts.
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Omar Niss;
Satheesh Chonat;
Neha Dagaonkar;
Marya O. Almansoori;
Karol Kerr;
Zora R. Rogers;
Patrick T. McGann;
Maa-Ohui Quarmyne;
Mary Risinger;
Kejian Zhang;
Theodosia A. Kalfa
Hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) are heterogeneous red blood cell (RBC) membrane disorders that result from mutations in the genes encoding α-spectrin (SPTA1), β-spectrin (SPTB), or protein 4.1R (EPB41). The resulting defects alter the horizontal cytoskeletal associations and affect RBC membrane stability and deformability causing shortened RBC survival. The clinical diagnosis of HE and HPP relies on identifying characteristic RBC morphology on peripheral blood smear and specific membrane biomechanical properties using osmotic gradient ektacytometry. However, this phenotypic diagnosis may not be readily available in patients requiring frequent transfusions, and does not predict disease course or severity. Using Next-Generation sequencing, we identified the causative genetic mutations in fifteen patients with clinically suspected HE or HPP and correlated the identified mutations with the clinical phenotype and ektacytometry profile. In addition to identifying three novel mutations, gene sequencing confirmed and, when the RBC morphology was not evaluable, identified the diagnosis. Moreover, genotypic differences justified the phenotypic differences within families with HE/HPP.
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Carleen M. Sabusap;
Wei Wang;
Carmel M. McNicholas;
Wook Joon Chung;
Lianwu Fu;
Hui Wen;
Marina Mazur;
Kevin L. Kirk;
James F. Collawn;
Jeong Hong;
Eric Sorscher
Emerging knowledge indicates the difficulty in categorizing unusual cystic fibrosis (CF) mutations, with regard to both pathogenic mechanism and theratype. As case in point, we present data concerning P67L mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), a defect carried by a small number of individuals with CF and sometimes attributed to a channel conductance abnormality. Findings from our laboratory and others establish that P67L causes protein misfolding, disrupts maturation, confers gating defects, is thermally stable, and exhibits near normal conductance. These results provide one framework by which rare CF alleles such as P67L can be more comprehensively profiled vis-à-vis molecular pathogenesis. We also demonstrate that emerging CF treatments - ivacaftor and lumacaftor - can mediate pronounced pharmacologic activation of P67L CFTR. Infrequent CF alleles are often improperly characterized, in part, due to the small numbers of patients involved. Moreover, access to new personalized treatments among patients with ultra-orphan genotypes has been limited by difficulty arranging phase III clinical trials, and off-label prescribing has been impaired by high drug cost and difficulty arranging third party reimbursement. Rare CFTR mutations such as P67L are emblematic of the challenges to "precision" medicine, including use of the best available mechanistic knowledge to treat patients with unusual forms of disease.
Purpose: To develop and validate the Pediatric Risk Estimation Score for Children Using Extracorporeal Respiratory Support (Ped-RESCUERS). Ped-RESCUERS is designed to estimate the in-hospital mortality risk for children prior to receiving respiratory extracorporeal membrane oxygenation (ECMO) support. Methods: This study used data from an international registry of patients aged 29 days to less than 18 years who received ECMO support from 2009 to 2014. We divided the registry into development and validation datasets by calendar date. Candidate variables were selected for model inclusion if the variable independently changed the mortality risk by at least 2 % in a Bayesian logistic regression model with in-hospital mortality as the outcome. We characterized the model’s ability to discriminate mortality with the area under curve (AUC) of the receiver operating characteristic. Results: From 2009 to 2014, 2458 non-neonatal children received ECMO for respiratory support, with a mortality rate of 39.8 %. The development dataset contained 1611 children receiving ECMO support from 2009 to 2012. The model included the following variables: pre-ECMO pH, pre-ECMO arterial partial pressure of carbon dioxide, hours of intubation prior to ECMO support, hours of admission at ECMO center prior to ECMO support, ventilator type, mean airway pressure, pre-ECMO use of milrinone, and a diagnosis of pertussis, asthma, bronchiolitis, or malignancy. The validation dataset included 438 children receiving ECMO support from 2013 to 2014. The Ped-RESCUERS model from the development dataset had an AUC of 0.690, and the validation dataset had an AUC of 0.634. Conclusions: Ped-RESCUERS provides a novel measure of pre-ECMO mortality risk. Future studies should seek external validation and improved discrimination of this mortality prediction tool.
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Introduction: Lupus nephritis (LN) is a serious organ manifestation of systemic lupus erythematosus. Histologic overlap is relatively common in the six pathologic classes (I to VI) of LN. For example, mixed proliferative LN (MPLN) often includes features of classes III & V or classes IV & V combined. We performed a comparative evaluation of renal outcomes in patients with MPLN to patients with pure proliferative LN (PPLN) against pre-specified renal outcomes, and we also identified predictor of clinical outcomes among those with PPLN and MPLN. Hypothesis: Individuals with MPLN will have worse short-term renal outcomes compared to those with PPLN. Methods: We retrospectively reviewed 278 adult LN patients (≥18 years old) identified from an Emory University Hospital registry of native renal biopsies performed between January 2000 and December 2011. The final analytic sample consisted of individuals with a diagnosis of PPLN (n = 60) and MPLN (n = 96). We analyzed differences in clinical and laboratory characteristics at baseline. We also assessed associations between LN category and renal outcomes (complete remission and time to ESRD) with logistic and Cox proportional hazards models within two years of baseline. Results: The study population was predominantly female (83.97%) and African American (71.8%) with a mean age of 33.4 years at baseline. Over a median follow up of 1.02 years, we did not find any statistically significant associations between MPLN and the development of ESRD or remission when compared to patients with PPLN (adjusted HR = 0.30, 95% CI = 0.07, 1.26). Conclusion: There was no association between mixed or pure histopathologic features of LN at presentation and rate of complete or partial remission but higher baseline eGFR was associated with a lower probability of complete remission among patients with lupus nephritis.
Oxidized low-density lipoprotein (LDL) has an important role in atherogenesis; however, the mechanisms underlying cell-mediated LDL oxidation remain to be elucidated. The present study investigated whether native-LDL induced lipid raft formation, in order to gain further insight into LDL oxidation. Confocal microscopic analysis revealed that lipid rafts were aggregated or clustered in the membrane, which were colocalized with myeloperoxidase (MPO) upon native LDL stimulation; however, in the presence of methyl-β-cyclodextrin (MβCD), LDL-stimulated aggregation, translocation, and colocalization of lipid rafts components was abolished.. In addition, lipid raft disruptors MβCD and filipin decreased malondialdehyde expression levels. Density gradient centrifugation coupled to label-free quantitative proteomic analysis identified 1,449 individual proteins, of which 203 were significantly upregulated following native-LDL stimulation. Functional classification of the proteins identified in the lipid rafts revealed that the expression levels of translocation proteins were upregulated. In conclusion, the results of the present study indicated that native-LDL induced lipid raft clustering in macrophages, and the expression levels of several proteins were altered in the stimulated macrophages, which provided novel insights into the mechanism underlying LDL oxidation.
Desmosomes are prominent adhesive junctions present between many epithelial cells as well as cardiomyocytes. The mechanisms controlling desmosome assembly and remodeling in epithelial and cardiac tissue are poorly understood. We recently identified protein palmitoylation as a mechanism regulating desmosome dynamics. In this study, we have focused on the palmitoylation of the desmosomal cadherin desmoglein-2 (Dsg2) and characterized the role that palmitoylation of Dsg2 plays in its localization and stability in cultured cells. We identified two cysteine residues in the juxtamembrane (intracellular anchor) domain of Dsg2 that, when mutated, eliminate its palmitoylation. These cysteine residues are conserved in all four desmoglein family members. Although mutant Dsg2 localizes to endogenous desmosomes, there is a significant delay in its incorporation into junctions, and the mutant is also present in a cytoplasmic pool. Triton X-100 solubility assays demonstrate that mutant Dsg2 is more soluble than wild-type protein. Interestingly, trafficking of the mutant Dsg2 to the cell surface was delayed, and a pool of the non-palmitoylated Dsg2 co-localized with lysosomal markers. Taken together, these data suggest that palmitoylation of Dsg2 regulates protein transport to the plasma membrane. Modulation of the palmitoylation status of desmosomal cadherins can affect desmosome dynamics.