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Directional export of messenger RNA (mRNA) protein particles (mRNPs) through nuclear pore complexes (NPCs) requires multiple factors. In Saccharomyces cerevisiae, the NPC proteins Nup159 and Nup42 are asymmetrically localized to the cytoplasmic face and have distinct functional domains: a phenylalanine-glycine (FG) repeat domain that docks mRNP transport receptors and domains that bind the DEAD-box ATPase Dbp5 and its activating cofactor Gle1, respectively. We speculated that the Nup42 and Nup159 FG domains play a role in positioning mRNPs for the terminal mRNP-remodeling steps carried out by Dbp5. Here we find that deletion (Δ) of both the Nup42 and Nup159 FG domains results in a cold-sensitive poly(A)+ mRNA export defect. The nup42ΔFG nup159ΔFG mutant also has synthetic lethal genetic interactions with dbp5 and gle1 mutants. RNA cross-linking experiments further indicate that the nup42ΔFG nup159ΔFG mutant has a reduced capacity for mRNP remodeling during export. To further analyze the role of these FG domains, we replaced the Nup159 or Nup42 FG domains with FG domains from other Nups. These FG "swaps" demonstrate that only certain FG domains are functional at the NPC cytoplasmic face. Strikingly, fusing the Nup42 FG domain to the carboxy-terminus of Gle1 bypasses the need for the endogenous Nup42 FG domain, highlighting the importance of proximal positioning for these factors. We conclude that the Nup42 and Nup159 FG domains target the mRNP to Gle1 and Dbp5 for mRNP remodeling at the NPC. Moreover, these results provide key evidence that character and context play a direct role in FG domain function and mRNA export.
Copyright © 2014 by the Genetics Society of America.
Cross-species transmission of zoonotic coronaviruses (CoVs) can result in pandemic disease outbreaks. Middle East respiratory syndrome CoV (MERS-CoV), identified in 2012, has caused 182 cases to date, with ~43% mortality, and no small animal model has been reported. MERS-CoV and Pipistrellus bat coronavirus (BtCoV) strain HKU5 of Betacoronavirus (β-CoV) subgroup 2c share >65% identity at the amino acid level in several regions, including nonstructural protein 5 (nsp5) and the nucleocapsid (N) protein, which are significant drug and vaccine targets. BtCoV HKU5 has been described in silico but has not been shown to replicate in culture, thus hampering drug and vaccine studies against subgroup 2c β-CoVs. We report the synthetic reconstruction and testing of BtCoV HKU5 containing the severe acute respiratory syndrome (SARS)-CoV spike (S) glycoprotein ectodomain (BtCoV HKU5-SE). This virus replicates efficiently in cell culture and in young and aged mice, where the virus targets airway and alveolar epithelial cells. Unlike some subgroup 2b SARS-CoV vaccines that elicit a strong eosinophilia following challenge, we demonstrate that BtCoV HKU5 and MERS-CoV N-expressing Venezuelan equine encephalitis virus replicon particle (VRP) vaccines do not cause extensive eosinophilia following BtCoV HKU5-SE challenge. Passage of BtCoV HKU5-SE in young mice resulted in enhanced virulence, causing 20% weight loss, diffuse alveolar damage, and hyaline membrane formation in aged mice. Passaged virus was characterized by mutations in the nsp13, nsp14, open reading frame 5 (ORF5) and M genes. Finally, we identified an inhibitor active against the nsp5 proteases of subgroup 2c β-CoVs. Synthetic-genome platforms capable of reconstituting emerging zoonotic viral pathogens or their phylogenetic relatives provide new strategies for identifying broad-based therapeutics, evaluating vaccine outcomes, and studying viral pathogenesis. IMPORTANCE The 2012 outbreak of MERS-CoV raises the specter of another global epidemic, similar to the 2003 SARS-CoV epidemic. MERS-CoV is related to BtCoV HKU5 in target regions that are essential for drug and vaccine testing. Because no small animal model exists to evaluate MERS-CoV pathogenesis or to test vaccines, we constructed a recombinant BtCoV HKU5 that expressed a region of the SARS-CoV spike (S) glycoprotein, thereby allowing the recombinant virus to grow in cell culture and in mice. We show that this recombinant virus targets airway epithelial cells and causes disease in aged mice. We use this platform to (i) identify a broad-spectrum antiviral that can potentially inhibit viruses closely related to MERS-CoV, (ii) demonstrate the absence of increased eosinophilic immune pathology for MERS-CoV N protein-based vaccines, and (iii) mouse adapt this virus to identify viral genetic determinants of cross-species transmission and virulence. This study holds significance as a strategy to control newly emerging viruses.
Treatment of type 1 diabetes with gene transfer-induced cellular reprogramming requires a pancreatic transcription factor such as Neurogenin-3 (Ngn3) and as of yet unknown component of the adenoviral particle. Despite intensive study, there are many unsolved processes related to the mechanisms and physiological parameters related to diabetes correction using this approach. While we confirm that systemic delivery of adenovirus (Ad)-Ngn3 provides long-lasting correction of streptozotocin (STZ)-induced hyperglycemia and restoration of growth curves, we found that insulin levels and glucose tolerance tests are not fully restored. By altering the innate and antigen-specific immune responses, we establish that the former likely plays some role in the reprogramming process. Interestingly, Ad-hNgn3 therapy in diabetic animals appeared to protect them from secondary STZ challenge. The resistance to secondary STZ response was more pronounced at later time points, indicating that a period of cell maturation and/or expansion may be required in order to promote lasting correction. More importantly, these results suggest that the long-term reprogrammed cells are not fully reprogrammed into β-cells, which in the case of autoimmune diabetes may be advantageous in a long-term treatment strategy. Finally, we show that the prophylactic administration of Ad-hNgn3 before diabetic induction protected mice from developing hyperglycemia, demonstrating the potential for reducing or eliminating disease progression should treatment be initiated early or before onset of symptoms.
DNA based transposon systems offer a technology for active and efficient delivery of genes into human cells. An emerging field is directed at manipulating such systems to achieve site-directed integration as compared to un-targeted integration which occurs with native or unmodified transposon systems. The naturally active piggyBac transposon system is derived from insects but has been shown to be very efficient in gene-modifying human cells. Recent efforts have utilized the fusion of DNA binding domains to the piggyBac transposase enzyme with the goal of targeting integration to specific locations in the human genome. In this chapter, we describe methodology for engineering and characterizing chimeric piggyBac transposase enzymes, including experimental approaches for evaluating activity and targeting specificity in the human genome.
Zebrafish has become a widely used model for analysis of gene function. Several methods have been used to create mutations in this organism and thousands of mutant lines are available. However, all the conventional zebrafish mutations affect the gene in all cells at all time, making it difficult to determine tissue-specific functions. We have adopted a FlEx Trap approach to generate conditional mutations in zebrafish by gene-trap mutagenesis. Combined with appropriate Cre or Flp lines, the insertional mutants not only allow spatial- and temporal-specific gene inactivation but also permit spatial- and temporal-specific rescue of the disrupted gene. We provide experimental details on how to generate and use such mutations.
Malaria results in over 650,000 deaths each year; thus, there is an urgent need for an effective vaccine. Pre-clinical studies and recently reported human trials suggest that pre-erythrocytic stage vaccines can provide protection against infection. A Phase 1, randomized, placebo-controlled, dose-escalation study was conducted with a vaccine composed of a replication-deficient adenovirus-35 backbone with P. falciparum circumsporozoite (CS) surface antigen (Ad35.CS.01). Healthy adult subjects received three doses of 10 (8), 10 (9), 10 (10), or 10 (11) vp/mL Ad35.CS.01 vaccine or saline placebo intramuscularly at 0, 1, and 6-mo intervals. Adverse events were assessed and anti-CS antibody responses were determined by ELISA. Seventy-two individuals were enrolled, with age, gender, and ethnicity similar across each study arm. While the vaccine was generally well tolerated, adverse events were more frequent in the highest dose groups (10 (10) and 10 (11) vp/mL). More robust humoral responses were also noted at the highest doses, with 73% developing a positive ELISA response after the three dose series of 10 (11) vp/mL. The Ad35.CS.01 vaccine was most immunogenic at the highest dosages (10 (10) and 10 (11) vp/mL). Reactogenicity findings were more common after the 10 (11) vp/mL dose, although most were mild or moderate in nature and resolved without therapy.
PURPOSE - We conducted genome-wide miRNA-sequencing (miRNA-seq) in primary cancer tissue from patients of lung adenocarcinoma to identify markers for the presence of lymph node metastasis.
EXPERIMENTAL DESIGN - Markers for lymph node metastasis identified by sequencing were validated in a separate cohort using quantitative PCR. After additional validation in the The Cancer Genome Atlas (TCGA) dataset, functional characterization studies were conducted in vitro.
RESULTS - MiR-31 was upregulated in lung adenocarcinoma tissues from patients with lymph node metastases compared with those without lymph node metastases. We confirmed miR-31 to be upregulated in lymph node-positive patients in a separate patient cohort (P = 0.009, t test), and to be expressed at higher levels in adenocarcinoma tissue than in matched normal adjacent lung tissues (P < 0.0001, paired t test). MiR-31 was then validated as a marker for lymph node metastasis in an external validation cohort of 233 lung adenocarcinoma cases of the TCGA (P = 0.031, t test). In vitro functional assays showed that miR-31 increases cell migration, invasion, and proliferation in an ERK1/2 signaling-dependent manner. Notably, miR-31 was a significant predictor of survival in a multivariate cox regression model even when controlling for cancer staging. Exploratory in silico analysis showed that low expression of miR-31 is associated with excellent survival for T2N0 patients.
CONCLUSIONS - We applied miRNA-seq to study microRNomes in lung adenocarcinoma tissue samples for the first time and potentially identified a miRNA predicting the presence of lymph node metastasis and survival outcomes in patients of lung adenocarcinoma.
Sterile alpha and armadillo-motif containing protein (SARM), a highly conserved and structurally unique member of the MyD88 family of Toll-like receptor adaptors, plays an important role in innate immunity signaling and apoptosis. Its exact mechanism of intracellular action remains unclear. Apoptosis is an ancient and ubiquitous process of programmed cell death that results in disruption of the nuclear lamina and, ultimately, dismantling of the nucleus. In addition to supporting the nuclear membrane, lamins serve important roles in chromatin organization, epigenetic regulation, transcription, nuclear transport, and mitosis. Mutations and other damage that destabilize nuclear lamins (laminopathies) underlie a number of intractable human diseases. Here, we report that SARM translocates to the nucleus of human embryonic kidney cells by using its amino-terminal Armadillo repeat region. Within the nucleus, SARM forms a previously unreported lattice akin to the nuclear lamina scaffold. Moreover, we show that SARM protects lamins from apoptotic degradation and reduces internucleosomal DNA fragmentation in response to signaling induced by the proinflammatory cytokine Tumor Necrosis Factor alpha. These findings indicate an important link between the innate immunity adaptor SARM and stabilization of nuclear lamins during inflammation-driven apoptosis in human cells.
Erythropoietin (EPO) can protect the retina from acute damage, but long-term systemic treatment induces polycythemia. Intraocular gene delivery of EPO is not protective despite producing high levels of EPO likely due to its bellshaped dose curve. The goal of this study was to identify a therapeutic dose of continuously produced EPO in the eye. We packaged a mutated form of EPO (EPOR76E) that has equivalent neuroprotective activity as wild-type EPO and attenuated erythropoietic activity into a recombinant adeno-associated viral vector under the control of the tetracycline inducible promoter. This vector was injected into the subretinal space of homozygous postnatal 5-7 day retinal degeneration slow mice, that express the tetracycline transactivators from a retinal pigment epithelium specific promoter. At weaning, mice received a single intraperitoneal injection of doxycycline and were then maintained on water with or without doxycycline until postnatal day 60. Intraocular EPO levels and outer nuclear layer thickness were quantified and correlated. Control eyes contained 6.1 ± 0.1 (SEM) mU/ml EPO. The eyes of mice that received an intraperitoneal injection of doxycycline contained 11.8 ± 2.0 (SEM) mU/ml EPO-R76E. Treatment with doxycycline water induced production of 35.9 ± 2.4 (SEM) mU/ml EPO-R76E in the eye. The outer nuclear layer was approximately 8 μm thicker in eyes of mice that received doxycycline water as compared to the control groups. Our data indicates that drug delivery systems should be optimized to deliver at least 36 mU/ml EPO into the eye since this dose was effective for the treatment of a progressive retinal degeneration.
INTRODUCTION - Muscle growth and regeneration are processes closely associated with proliferation, differentiation, and apoptosis of muscle cells. Death-associated protein 1 (DAP1) has been identified as a negative regulator of autophagy. Little is known about the function of DAP1 in the regulation of myogenesis and satellite cells.
METHODS - Chicken satellite cells were transfected with DAP1 cloned into the pCMS-enhanced green fluorescent protein vector or pcDNA3.1 vector, or a small interference RNA against the endogenous DAP1 gene. The cells were assayed for proliferation, differentiation, and apoptosis.
RESULTS - The overexpression of DAP1 increased proliferation, differentiation, and myotube diameter, but it had no effect on satellite cell apoptosis. In contrast, knockdown of DAP1 significantly decreased proliferation, differentiation, and number of nuclei per myotube, and it increased apoptosis of the cells.
CONCLUSION - DAP1 is required for regulating myogenesis and apoptosis of satellite cells, which may affect muscle mass accretion and regeneration, and ameliorate muscle sarcopenia.
Copyright © 2013 Wiley Periodicals, Inc.