Citations

Abstract The development of pancreatic cancer requires both obtain of oncogenic Kras mutations and up-regulation of growth factor signaling. However, how oncogenic Kras regulates growth factor signaling in pancreatic cancer development is still unclear. Using a transgenic mouse model harboring an oncogenic Kras mutation and a 3D organoid explant culture of primary pancreatic acini, we here show that expression of mutant Kras in pancreatic acinar cells alters their mitochondrial metabolism, resulting in increased generation of mitochondrial reactive oxygen species (mROS). We further demonstrate that mROS induced by oncogenic Kras is required for transdifferentiating acinar cells to a duct-like phenotype and eventually progression to pancreatic intraepithelial neoplasia (PanIN), the most common precursor lesions of pancreatic ductal adenocarcinoma (PDAC). This transdiffentiation process is mediated by increased levels of epidermal growth factor receptor (EGFR) as well as its ligands, EGF and TGFα, through activation of the ROS-receptive transcription factors, NF-κB1 and NF-κB2. In Kas transgenic mice, the interception of KrasG12D-mediated generation of mROS significantly reduced the formation of pre-neoplastic lesions. Hence, our data provide mechanistic insight of how oncogenic Kras mutant crosstalks with growth factor signaling to cause the formation of pancreatic cancer. Citation Format: Geou-Yarh Liou, Heike Doeppler, Kathleen E. DelGirono, Howard C. Crawford, Michael P. Murphy, Peter Storz. Oncogenic Kras-induced mitochondrial oxidative stress in pancreatic acini modulates the formation of precancerous lesions through autocrine EGFR signaling. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr PR01.

OBJECTIVE Improvement in treatment outcome for patients with glioblastoma multiforme (GBM) requires a multifaceted approach due to dysregulation of numerous signaling pathways. The murine double minute 2 (MDM2) protein may fulfill this requirement because it is involved in the regulation of growth, survival, and invasion. The objective of this study was to investigate the impact of modulating MDM2 function in combination with front-line temozolomide (TMZ) therapy in GBM. METHODS The combination of TMZ with the MDM2 protein–protein interaction inhibitor nutlin3a was evaluated for effects on cell growth, p53 pathway activation, expression of DNA repair proteins, and invasive properties. In vivo efficacy was assessed in xenograft models of human GBM. RESULTS In combination, TMZ/nutlin3a was additive to synergistic in decreasing growth of wild-type p53 GBM cells. Pharmacodynamic studies demonstrated that inhibition of cell growth following exposure to TMZ/nutlin3a correlated with: 1) activation of the p53 pathway, 2) downregulation of DNA repair proteins, 3) persistence of DNA damage, and 4) decreased invasion. Pharmacokinetic studies indicated that nutlin3a was detected in human intracranial tumor xenografts. To assess therapeutic potential, efficacy studies were conducted in a xenograft model of intracranial GBM by using GBM cells derived from a recurrent wild-type p53 GBM that is highly TMZ resistant (GBM10). Three 5-day cycles of TMZ/nutlin3a resulted in a significant increase in the survival of mice with GBM10 intracranial tumors compared with single-agent therapy. CONCLUSIONS Modulation of MDM2/p53-associated signaling pathways is a novel approach for decreasing TMZ resistance in GBM. To the authors' knowledge, this is the first study in a humanized intracranial patient-derived xenograft model to demonstrate the efficacy of combining front-line TMZ therapy and an inhibitor of MDM2 protein–protein interactions.

Context: Psychosocial factors can impact lung transplant outcomes. However, it is currently unknown whether abuse survivorship influences lung transplant survival. Objective: To characterize the abuse history of adult lung transplant patients and determine whether such history is associated with mortality. Patients and Other Participants: Adult lung transplant recipients evaluated from 2000 to 2004. Main Outcome Measures: The main outcome was post-lung transplantation survival. The secondary outcomes included demographic, transplantation, or psychological assessment differences between those with a history of abuse survivorship and those without. Results: Thirty-three lung transplant recipients (35.5% male, median age: 55 years) were included. A history of abuse survivorship was common (24.2%) and was associated with decreased survival following lung transplantation ( P = .003). There was no difference in sex, marital status, or smoking history between abuse survivors and those who denied being the victim of abuse. Abuse survivors had a higher Personality Assessment Screener total score, a measure of maladaptive personality traits ( P = .02). Conclusion: Abuse survivorship is common in lung transplant patients and associated with increased posttransplant mortality and increased maladaptive personality traits. This preliminary evidence suggests that lung transplant patients should be screened for abuse history and provided with appropriate treatment of survivorship issues to potentially improve their health outcomes from transplantation.

Abstract The zebrafish ( Danio rerio ) is a premier nonmammalian vertebrate model organism. This small aquatic fish is utilized in multiple disciplines in the Mayo Clinic community and by many laboratories around the world because of its biological similarity to humans, its advanced molecular genetics, the elucidation of its genome sequence, and the ever-expanding and outstanding new biological tools now available to the zebrafish researcher. The Mayo Clinic Zebrafish Facility (MCZF) houses ∼2,000 tanks annotated using an in-house, Internet cloud-based bar-coding system tied to our established zfishbook.org web infrastructure. Paramecia are the primary food source for larval fish rearing, using a simplified culture protocol described herein. The MCZF supports the specific ongoing research in a variety of laboratories, while also serving as a local hub for new scientists as they learn to tap into the potential of this model system for understanding normal development, disease, and as models of health.

Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (C t ) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98–100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score genotypes. ASQ is cost-effective because universal fluorescent probes negate the necessity of designing expensive probes for each locus.

AbstractBackgroundThe optimal treatment for respiratory syncytial virus (RSV) infection in adult immunocompromised patients is unknown. We assessed the management of RSV and other non‐influenza respiratory viruses in Midwestern transplant centers.MethodsA survey assessing strategies for RSV and other non‐influenza respiratory viral infections was sent to 13 centers.ResultsMultiplex polymerase chain reaction assay was used for diagnosis in 11/12 centers. Eight of 12 centers used inhaled ribavirin (RBV) in some patient populations. Barriers included cost, safety, lack of evidence, and inconvenience. Six of 12 used intravenous immunoglobulin (IVIG), mostly in combination with RBV. Inhaled RBV was used more than oral, and in the post‐stem cell transplant population, patients with lower respiratory tract infection (LRTI), graft‐versus‐host disease, and more recent transplantation were treated at higher rates. Ten centers had experience with lung transplant patients; all used either oral or inhaled RBV for LRTI, 6/10 treated upper respiratory tract infection (URTI). No center treated non‐lung solid organ transplant (SOT) recipients with URTI; 7/11 would use oral or inhaled RBV in the same group with LRTI. Patients with hematologic malignancy without hematopoietic stem cell transplantation were treated with RBV at a similar frequency to non‐lung SOT recipients. Three of 12 centers, in severe cases, treated parainfluenza and metapneumovirus, and 1/12 treated coronavirus.ConclusionsTreatment of RSV in immunocompromised patients varied greatly. While most centers treat LRTI, treatment of URTI was variable. No consensus was found regarding the use of oral versus inhaled RBV, or the use of IVIG. The presence of such heterogeneity demonstrates the need for further studies defining optimal treatment of RSV in immunocompromised hosts.

Postmenopausal women with nonalcoholic steatohepatitis are at an increased risk of hepatic fibrosis compared with premenopausal women. Whether duration of estrogen deficiency in postmenopausal state dictates an individual's fibrosis risk remains uninvestigated. We assessed the associations of age at menopause and time from menopause with fibrosis severity in postmenopausal women with nonalcoholic fatty liver disease. Data from 488 postmenopausal women with (1) histologic diagnosis of nonalcoholic fatty liver disease and (2) self‐reported information on age at menopause were analyzed. The associations of premature menopause (age at menopause of <40 years) and time from menopause (age at study enrollment ‐ age at menopause, years) with fibrosis severity (stage 0‐4) were assessed using multiple ordinal logistic regression models with and without adjusting for clinical confounders. Among the participants (age at menopause 43.7 ± 8.6 years), women with premature menopause (29.3%) were younger at enrollment ( P < 0.001) and used hormone replacement therapy more often ( P < 0.003). After adjusting for age at enrollment, race, waist circumference standardized by body mass index, current smoking, current alcohol use, hypertension, diabetes/impaired fasting glucose, homeostatic model assessment of insulin resistance, and hormone replacement therapy, premature menopause was associated with an increased likelihood of having more severe fibrosis (adjusted cumulative odds ratio = 1.9, 95% confidence interval 1.3‐2.7, P = 0.001), while time from menopause was directly associated with an increased likelihood of having more severe fibrosis (adjusted cumulative odds ratio for 5‐year unit = 1.2, 95% confidence interval 1.1‐1.3, P = 0.002). Conclusion : Duration of estrogen deficiency in postmenopausal state confers fibrosis risk among postmenopausal women with nonalcoholic fatty liver disease. (H epatology 2016;64:85–91)

Introduction: Our study assesses the utility of telemetry in identifying decompensation in patients with documented cardiopulmonary arrest. Methods: A retrospective review of inpatients who experienced a cardiopulmonary arrest from May 1, 2008, until June 30, 2014, was performed. Telemetry records 24 hours prior to and immediately preceding cardiopulmonary arrest were reviewed. Patient subanalyses based on clinical demographics were made as well as analyses of survival comparing patients with identifiable rhythm changes in telemetry to those without. Results: Of 242 patients included in the study, 75 (31.0%) and 110 (45.5%) experienced telemetry changes at the 24-hour and immediately preceding time periods, respectively. Of the telemetry changes, the majority were classified as nonmalignant (n = 50, 66.7% and n = 66, 55.5% at 24 hours prior and immediately preceding, respectively). There was no difference in telemetry changes between intensive care unit (ICU) and non-ICU patients and among patients stratified according to the American Heart Association telemetry indications. There was no difference in survival when comparing patients with telemetry changes immediately preceding and at 24 hours prior to an event (n = 30, 27.3% and n = 15, 20.0%) to those without telemetry changes during the same periods (n = 27, 20.5% and n = 42, 25.2%; P = .22 and .39). Conclusion: Telemetry has limited utility in predicting clinical decompensation in the inpatient setting.