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121.
Scott M. Robinson Ginger Tsueng Jon Sin Vrushali Mangale Shahad Rahawi Laura L. McIntyre Wesley Williams Nelson Kha Casey Cruz Bryan M. Hancock David P. Nguyen M. Richard Sayen Brett J. Hilton Kelly S. Doran Anca M. Segall Roland Wolkowicz Christopher T. Cornell J. Lindsay Whitton Roberta A. Gottlieb Ralph Feuer 《PLoS pathogens》2014,10(4)
Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, and pancreatitis in humans. We genetically engineered a unique molecular marker, “fluorescent timer” protein, within our infectious CVB3 clone and isolated a high-titer recombinant viral stock (Timer-CVB3) following transfection in HeLa cells. “Fluorescent timer” protein undergoes slow conversion of fluorescence from green to red over time, and Timer-CVB3 can be utilized to track virus infection and dissemination in real time. Upon infection with Timer-CVB3, HeLa cells, neural progenitor and stem cells (NPSCs), and C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours as determined by fluorescence microscopy or flow cytometric analysis. The conversion of “fluorescent timer” protein in HeLa cells infected with Timer-CVB3 could be interrupted by fixation, suggesting that the fluorophore was stabilized by formaldehyde cross-linking reactions. Induction of a type I interferon response or ribavirin treatment reduced the progression of cell-to-cell virus spread in HeLa cells or NPSCs infected with Timer-CVB3. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discrete virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. “Fluorescent timer” protein colocalized closely with viral 3A protein within virus replication organelles. Intriguingly, infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of abundant extracellular microvesicles (EMVs) containing matured “fluorescent timer” protein and infectious virus representing a novel route of virus dissemination. CVB3 virions were readily observed within purified EMVs by transmission electron microscopy, and infectious virus was identified within low-density isopycnic iodixanol gradient fractions consistent with membrane association. The preferential detection of the lipidated form of LC3 protein (LC3 II) in released EMVs harboring infectious virus suggests that the autophagy pathway plays a crucial role in microvesicle shedding and virus release, similar to a process previously described as autophagosome-mediated exit without lysis (AWOL) observed during poliovirus replication. Through the use of this novel recombinant virus which provides more dynamic information from static fluorescent images, we hope to gain a better understanding of CVB3 tropism, intracellular membrane reorganization, and virus-associated microvesicle dissemination within the host. 相似文献
122.
James C. Stegen Amy L. Freestone Thomas O. Crist Marti J. Anderson Jonathan M. Chase Liza S. Comita Howard V. Cornell Kendi F. Davies Susan P. Harrison Allen H. Hurlbert Brian D. Inouye Nathan J. B. Kraft Jonathan A. Myers Nathan J. Sanders Nathan G. Swenson Mark Vellend 《Global Ecology and Biogeography》2013,22(2):202-212
123.
Julie Brault Ronald J. Meis Linhong Li Ezekiel Bello Taylor Liu Colin L. Sweeney Sherry M. Koontz Kennichi Dowdell Narda Theobald Janet Lee Cornell Allen Aaron B. Clark Juan C. Ravell Michael J. Lenardo Gary A. Dahl Harry L. Malech Suk See De Ravin 《Cytotherapy》2021,23(3):203-210
Background aimX-linked MAGT1 deficiency with increased susceptibility to EBV-infection and N-linked glycosylation defect' (XMEN) disease is caused by mutations in the magnesium transporter 1 (MAGT1) gene. Loss of MAGT1 function results in a glycosylation defect that abrogates expression of key immune proteins such as the NKG2D receptor on CD8+ T and NK cells, which is critical for the recognition and killing of virus-infected and transformed cells, a biomarker for MAGT1 function. Patients with XMEN disease frequently have increased susceptibility to EBV infections and EBV-associated B cell malignancies, for which no specific treatment options are currently available. Experimental transfer of donor EBV-specific cytotoxic T cells may be beneficial but carries the risks of eliciting alloimmune responses. An approach for cell therapy to address viral infections and associated complications that avoids the risks of alloimmunity is needed.MethodsHere the authors assess the feasibility and efficiency of correcting autologous lymphocytes from XMEN patients by MAGT1 mRNA electroporation (EP) that avoids genomic integration and can be scaled for clinical application.Results and conclusionsRestoration of NKG2D expression was demonstrated in XMEN patient lymphocytes after MAGT1 mRNA electroporation that reach healthy donor levels in CD8+ T and NK cells at 1-2 days after EP. NKG2D expression persisted at ~50% for 2 weeks after EP. Functionally, mRNA-correction of XMEN NK cells rescued cytotoxic activity also to healthy donor NK cell level. The restored NKG2D receptor expression and function were unaffected by cryopreservation, which will make feasible repeat infusions of MAGT1 mRNA-corrected autologous XMEN CD8+ T and NK cells for potential short term therapy for XMEN patients without the risks of alloimmunization. 相似文献
124.
Global expression analysis of fetal liver hematopoietic stem cells (FL HSCs) revealed the presence of unspliced pre-mRNA for a number of genes in normal FL HSCs. In a subset of these genes, Crebbp+/- FL HSCs had less unprocessed pre-mRNA without a corresponding reduction in total mRNA levels. Among the genes thus identified were the key regulators of HSC function Itga4, Msi2 and Tcf4. A similar but much weaker effect was apparent in Ep300+/- FL HSCs, indicating that, in this context as in others, the two paralogs are not interchangeable. As a group, the down-regulated intronic probe sets could discriminate adult HSCs from more mature cell types, suggesting that the underlying mechanism is regulated with differentiation stage and is active in both fetal and adult hematopoiesis. Consistent with increased myelopoiesis in Crebbp hemizygous mice, targeted reduction of CREBBP abundance by shRNA in the multipotent EML cell line triggered spontaneous myeloid differentiation in the absence of the normally required inductive signals. In addition, differences in protein levels between phenotypically distinct EML subpopulations were better predicted by taking into account not only the total mRNA signal but also the amount of unspliced message present. CREBBP thus appears to selectively influence the timing and degree of pre-mRNA processing of genes essential for HSC regulation and thereby has the potential to alter subsequent cell fate decisions in HSCs. 相似文献
125.
Strong negative reactions, physical symptoms, and behavioral disruptions due to environmental odors are common in the adult population. We investigated relationships among such environmental chemosensory responsivity (CR), personality traits, affective states, and odor perception. Study 1 showed that CR and neuroticism were positively correlated in a sample of young adults (n = 101), suggesting that persons high in neuroticism respond more negatively to environmental odors. Study 2 explored the relationships among CR, noise responsivity (NR), neuroticism, and odor perception (i.e., pleasantness and intensity) in a subset of participants (n = 40). High CR was associated with high NR. Regression analyses indicated that high CR predicted higher odor intensity ratings and low olfactory threshold (high sensitivity) predicted lower pleasantness ratings. However, neuroticism was not directly associated with odor ratings or thresholds. Overall, the results suggest that CR and odor thresholds predict perceptual ratings of odors and that high CR is associated with nonchemosensory affective traits. 相似文献
126.
Methylthioadenosine/S-adenosylhomocysteine nucleosidase, a critical enzyme for bacterial metabolism 总被引:1,自引:0,他引:1
The importance of methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidase in bacteria has started to be appreciated only in the past decade. A comprehensive analysis of its various roles here demonstrates that it is an integral component of the activated methyl cycle, which recycles adenine and methionine through S-adenosylmethionine (SAM)-mediated methylation reactions, and also produces the universal quorum-sensing signal, autoinducer-2 (AI-2). SAM is also essential for synthesis of polyamines, N-acylhomoserine lactone (autoinducer-1), and production of vitamins and other biomolecules formed by SAM radical reactions. MTA, SAH and 5'-deoxyadenosine (5'dADO) are product inhibitors of these reactions, and are substrates of MTA/SAH nucleosidase, underscoring its importance in a wide array of metabolic reactions. Inhibition of this enzyme by certain substrate analogues also limits synthesis of autoinducers and hence causes reduction in biofilm formation and may attenuate virulence. Interestingly, the inhibitors of MTA/SAH nucleosidase are very effective against the Lyme disease causing spirochaete, Borrelia burgdorferi, which uniquely expresses three homologous functional enzymes. These results indicate that inhibition of this enzyme can affect growth of different bacteria by affecting different mechanisms. Therefore, new inhibitors are currently being explored for development of potential novel broad-spectrum antimicrobials. 相似文献
127.
North A Cornell S Ovaskainen O 《Evolution; international journal of organic evolution》2011,65(6):1739-1751
It is generally well understood that some ecological factors select for increased and others for decreased dispersal. However, it has remained difficult to assess how the evolutionary dynamics are influenced by the spatio-temporal structure of the environment. We address this question with an individual-based model that enables habitat structure to be controlled through variables such as patch size, patch turnover rate, and patch quality. Increasing patch size at the expense of patch density can select for more or less dispersal, depending on the initial configuration. In landscapes consisting of high-quality and long-lived habitat patches, patch degradation selects for increased dispersal, yet patch loss may select for reduced dispersal. These trends do not depend on the component of life-history that is affected by habitat quality or the component of life-history through which density-dependence operates. Our results are based on a mathematical method that enables derivation of both the evolutionary stable strategy and the stationary genotype distribution that evolves in a polymorphic population. The two approaches generally lead to similar predictions. However, the evolutionary stable strategy assumes that the ecological and evolutionary time scales can be separated, and we find that violation of this assumption can critically alter the evolutionary outcome. 相似文献
128.
Ku SY Yip P Cornell KA Riscoe MK Behr JB Guillerm G Howell PL 《The Journal of biological chemistry》2007,282(30):22195-22206
The methionine salvage pathway is ubiquitous in all organisms, but metabolic variations exist between bacteria and mammals. 5-Methylthioribose (MTR) kinase is a key enzyme in methionine salvage in bacteria and the absence of a mammalian homolog suggests that it is a good target for the design of novel antibiotics. The structures of the apo-form of Bacillus subtilis MTR kinase, as well as its ADP, ADP-PO(4), AMPPCP, and AMPPCP-MTR complexes have been determined. MTR kinase has a bilobal eukaryotic protein kinase fold but exhibits a number of unique features. The protein lacks the DFG motif typically found at the beginning of the activation loop and instead coordinates magnesium via a DXE motif (Asp(250)-Glu(252)). In addition, the glycine-rich loop of the protein, analogous to the "Gly triad" in protein kinases, does not interact extensively with the nucleotide. The MTR substrate-binding site consists of Asp(233) of the catalytic HGD motif, a novel twin arginine motif (Arg(340)/Arg(341)), and a semi-conserved W-loop, which appears to regulate MTR binding specificity. No lobe closure is observed for MTR kinase upon substrate binding. This is probably because the enzyme lacks the lobe closure/inducing interactions between the C-lobe of the protein and the ribosyl moiety of the nucleotide that are typically responsible for lobe closure in protein kinases. The current structures suggest that MTR kinase has a dissociative mechanism. 相似文献
129.
Xiaotian Zhong Weijun Ma Caryl L. Meade Amy S. Tam Eliza Llewellyn Richard Cornell Kaffa Cote John J. Scarcelli Jeffrey K. Marshall Boriana Tzvetkova Bruno Figueroa Dana DiNino Annette Sievers Christopher Lee Jane Guo Evan Mahan Christopher Francis Khetemenee Lam Aaron M. D'Antona Richard Zollner Hongli L. Zhu Ron Kriz Will Somers Laura Lin 《Biotechnology progress》2019,35(1):e2724
Large-scale transient expression in mammalian cells is a rapid protein production technology often used to shorten overall timelines for biotherapeutics drug discovery. In this study we demonstrate transient expression in a Chinese hamster ovary (CHO) host (ExpiCHO-S™) cell line capable of achieving high recombinant antibody expression titers, comparable to levels obtained using human embryonic kidney (HEK) 293 cells. For some antibodies, ExpiCHO-S™ cells generated protein materials with better titers and improved protein quality characteristics (i.e., less aggregation) than those from HEK293. Green fluorescent protein imaging data indicated that ExpiCHO-S™ displayed a delayed but prolonged transient protein expression process compared to HEK293. When therapeutic glycoproteins containing non-Fc N-linked glycans were expressed in transient ExpiCHO-S™, the glycan pattern was unexpectedly found to have few sialylated N-glycans, in contrast to glycans produced within a stable CHO expression system. To improve N-glycan sialylation in transient ExpiCHO-S™, we co-transfected galactosyltransferase and sialyltransferase genes along with the target genes, as well as supplemented the culture medium with glycan precursors. The authors have demonstrated that co-transfection of glycosyltransferases combined with medium addition of galactose and uridine led to increased sialylation content of N-glycans during transient ExpiCHO-S™ expression. These results have provided a scientific basis for developing a future transient CHO system with N-glycan compositions that are similar to those profiles obtained from stable CHO protein production systems. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2724, 2019 相似文献
130.
Charles?G. Cranfield Bruce?A. Cornell Stephan?L. Grage Paul Duckworth Sonia Carne Anne?S. Ulrich Boris Martinac 《Biophysical journal》2014,106(1):182-189
In this work, we present experimental data, supported by a quantitative model, on the generation and effect of potential gradients across a tethered bilayer lipid membrane (tBLM) with, to the best of our knowledge, novel architecture. A challenge to generating potential gradients across tBLMs arises from the tethering coordination chemistry requiring an inert metal such as gold, resulting in any externally applied voltage source being capacitively coupled to the tBLM. This in turn causes any potential across the tBLM assembly to decay to zero in milliseconds to seconds, depending on the level of membrane conductance. Transient voltages applied to tBLMs by pulsed or ramped direct-current amperometry can, however, provide current-voltage (I/V) data that may be used to measure the voltage dependency of the membrane conductance. We show that potential gradients >∼150 mV induce membrane defects that permit the insertion of pore-forming peptides. Further, we report here the novel (to our knowledge) use of real-time modeling of conventional low-voltage alternating-current impedance spectroscopy to identify whether the conduction arising from the insertion of a polypeptide is uniform or heterogeneous on scales of nanometers to micrometers across the membrane. The utility of this tBLM architecture and these techniques is demonstrated by characterizing the resulting conduction properties of the antimicrobial peptide PGLa. 相似文献