tbuckley/cabot-search huggingface dataset:
669. Standard Free Droplet Digital PCR as a New Tool for More Precise and Reliable Quality Control of Gene Therapy Vectors
Authors: Philip Boehme, Thorsten Stellberger, Wenli Zhang, Johannes Doerner, Liu Jing, Eric Schulz, Thorsten Bergmann, Manish Solanki, Armin Baiker, Anja Ehrhardt
Journal: Molecular Therapy (2015)

In case using viral vectors as therapeutics in context of gene therapy small changes in vector dose can have fatal outcome for the patient. It has been shown in earlier clinical studies that there is a non-linear dose response for most of the vectors, so the therpeutical window is narrow. Precise vector titration is of extraordinary high importance. The widely distributed standard real time PCR (qPCR) may not provide this in a satisfactory way. In this study we were aiming to address this challenge by implementing a more reliable and accurate strategy for vector quantification. We implemented a new digital PCR (ddPCR) strategy based on droplet formation as tool for vector quantification. By forming around 20.000 individual PCR reactions using sample partitioning in a water oil emulsion the system is able to detect after end point PCR if every single droplet is negative or positive for the target DNA. Importantly, providing a standard is not necessary since quantification is absolute whereas normal qPCR provides standard dependent relative quantification. By choosing primers in the region of adenoviral ITR and fiber, we were able to quantify in one single reaction high capacity adenoviral vector (HCAdV) and the helper virus, which is needed during production of the viral gene deleted HCAdV. We applied this new strategy to quantify HCAdV produced with a shortened protocol using column purification instead of traditional caesium chloride centrifugation. The virus was purified from the same characterized stock using different columns, which were also compared in this study. Once the virus was purified we infected various cell lines with different volumes of the final preparation, after 3h total DNA was isolated and different PCR strategies were performed. First our data showed that this protocol leads to sufficient HCAdV for preclinical in vitro studies. Besides the transducing units calculated from ddPCR vs. qPCR data (≈10^6 transducing units per ml), this was also confirmed by EGFP expression analysis and the number of physical particles measured via UV-vis. Also the helper virus concentration was within an acceptable range (<10%). Second we could demonstrate that our ddPCR was less time consuming, less expensive and more reliable compared to qPCR. Representative primary data is shown in figure 1a. Channel 1 represents the droplets, which were positive for the fiber gene whereas channel 2 represents the adenoviral ITR. Figure 1b shows the control plasmid where we subcloned both loci as control. We think by applying ddPCR for gene therapy, patient safety can be increased in clinical trials while scientific results become more reliable.
Abstract 3258: Breast Metastasis Suppressor 1 (BRMS1) phosphorylation appears necessary for metastasis suppressor activity
Authors: Lellys M. Contreras, Boris Sarcevic, Keke M. Pounds, Danny R. Welch
Journal: Cancer Research (2015)

Abstract Breast cancer is the most common cancer among women worldwide. BRMS1 is a metastasis suppressor that suppresses metastasis without significantly affecting the growth of a primary tumor. BRMS1 contains two coiled-coil motifs important for protein-protein interactions and two putative nuclear localization sequences (NLS - aa198-205 (NLS1) and aa239-245 (NLS2)) and one nuclear export signal which appear to mediate nuclear-cytoplasmic shuttling. Although NLS2 is not necessary for active transport into the nucleus, it is required for metastasis suppression (PMC3563580). A serine immediately upstream of NLS2 (S237) can be phosphorylated by cdk2 (PMC3037622). Therefore, we hypothesized that NLS2 and adjacent amino acids are critical mediators of metastasis suppression. To test this hypothesis, NLS2 (aa231-248) epitope-tagged with HA and GST was transduced into metastatic MDA-MB-231 and -435 breast carcinoma cells expressing green fluorescent protein and stable colonies were selected. BRMS1S237A-expressing MDA-MB-231 cells were no longer metastasis suppressed, suggesting a critical role for phosphorylation of BRMS1 in its metastasis suppressor activity. Studies are underway with full-length BRMS1S237D (phosphomimetic) as well as expression with truncated wild-type NLS2 and mutant (NLS2wt/S237A/S237D) to test whether the BRMS1 phosphorylation or the NLS2 domain itself is critical for metastasis suppressor activity. Support: CA87728, CA134981, P30-CA168524, Natl Fndn Cancer Res, Komen SAC11037, KS Bioscience Auth, NHMRC Citation Format: Lellys M. Contreras, Boris Sarcevic, Keke M. Pounds, Danny R. Welch. Breast Metastasis Suppressor 1 (BRMS1) phosphorylation appears necessary for metastasis suppressor activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3258. doi:10.1158/1538-7445.AM2015-3258
Abstract CT311: Results of a phase I/IIa dose-escalation study of the combination of decitabine and genistein against advanced solid tumors
Authors: Noël J. Raynal, Elie Kassouf, Luc Daigneault, Patrick Colin, Isabelle Plante, Guylaine Lassonde, Richard Momparler, Michel Charbonneau, Normand Blais
Journal: Cancer Research (2015)

Abstract Background: Epigenetic aberrations, including DNA hypermethylation of tumor suppressor genes are a common feature in solid tumors. Thus, targeting epigenetic pathways is a promising approach for cancer chemotherapy. However, only occasional proof-of-principle responses can be seen in solid tumors. The activity of hypomethylating drugs, such as decitabine could be improved by a treatment combination. Preclinical studies have suggested that adding genistein, a natural isofavone may synergize with demethylating drugs and improve treatment results. Methods: Here, we describe a phase I/IIa dose-escalation study of decitabine with a fixed dose of genistein. Primary objective of the phase I trial was to determine the Maximum Tolerated Dose (MTD) and to evaluate pharmacokinetic parameters in patients with advanced solid tumor. The objective of the phase IIa was to assess its efficacy in lung cancer. Decitabine was administered over 10-hours at increasing doses (60, 120, 240 mg/m2) with continuous administration of genistein 300 mg/day orally. Treatment was repeated every 4 to 6 weeks. Results: For the phase I, ten patients with advanced solid tumors were recruited. The recommended dose of decitabine (for the phase IIa) was 120 mg/m2 with neutropenia as limiting side effect. Decitabine at 120 mg/m2 and genistein produced plasma levels of 0.62 ± 0.06 μM and 8.5 ± 5.6 μM, respectively. The drug combination was well tolerated and produced stable disease for more than 7 months (7-14 months) in 50% of the patients. One patient had a 50% reduction in tumor burden after 6 months of therapy. In the phase IIa trial against advanced lung cancer, patients progressed rapidly, within 2 months of treatment mainly because of the apparition of new lesions in the liver. Conclusion: The combination of decitabine and genistein is well-tolerated in advanced solid tumors and shows interesting anticancer effects. This study on decitabine and genistein warrants future investigations to address this issue of liver metastasis in advanced lung cancer. Citation Format: Noël J. Raynal, Elie Kassouf, Luc Daigneault, Patrick Colin, Isabelle Plante, Guylaine Lassonde, Richard Momparler, Michel Charbonneau, Normand Blais. Results of a phase I/IIa dose-escalation study of the combination of decitabine and genistein against advanced solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT311. doi:10.1158/1538-7445.AM2015-CT311
T5 Towards human lung regeneration in end-stage respiratory failure: genetically-modifiable 3d organoid culture of human embryonic lung stem cells enables for the first time the study of human lung development in vitro
Authors: MZ Nikolic, JA Johnson, D Sun, O Caritg, U Laresgoiti, J Brady, G Allen, A Giangreco, EL Rawlins
Journal: Thorax (2016)

<h3>Introduction and objectives</h3> Regeneration of healthy lung tissue in patients with end-stage respiratory disease (ESRD) would cure disease, rather than treating symptoms. For this a detailed understanding of lung development is needed and the mouse has been used extensively as an <i>in vivo</i> genetically-modifiable model. Differentiation and validation of human induced pluripotent stem cells (hiPSCs) is entirely based on mouse literature. The most important epithelial stem cell population in developing lungs is found in distal branching tips, and these Sox9+ lung epithelial stem cells (LESCs) generate all epithelial lineages. Our objective was to develop a self-renewing, genetically-modifiable epithelial <i>in vitro</i> culture system from human embryonic LESCs and differentiate them into alveolar and bronchiolar cells. <h3>Methods</h3> Human embryonic LESCs were characterised using genome-wide transcriptional analysis (RNAseq) and immunohistochemistry (5–20 post-conceptional weeks). LESCs were microdissected and self-renewing expansion in 3D organoid culture was established empirically. Using RNAseq and immunohistochemistry, we assessed the similarities between cultured and fresh LESCs. Genetic stability was evaluated by karyotyping. Organoids were differentiated <i>in vitro,</i> or <i>in vivo</i> using xenotransplantation into bleomycin-injured adult mouse lungs or kidney capsule. Gene editing was done using CRISPR-Cas9 to delete <i>SOX9</i>. <h3>Results</h3> RNAseq of LESCs identified broad-scale transcriptional differences between mouse and human embryonic lung stem cells. Human LESCs were successfully expanded for over 10 months as karyotypically-stable 3D organoids using a combination of 7 signalling molecules. The LESC stem cell markers, transcriptome and organoid morphology were maintained throughout the culture period. Bronchiolar and alveolar differentiation was achieved <i>in vitro</i> and <i>in vivo</i>. Moreover, xenotransplantation of organoids into bleomycin-injured adult mouse lungs was extremely efficient. Knocking out <i>SOX9</i> led to a loss-of-self-renewing phenotype. <h3>Conclusions</h3> Our novel genetically-modifiable human embryonic lung culture system enables for the first time the<i> in vitro</i> study of human lung development and disease modelling. We anticipate that this work will transform lung regenerative medicine by guiding the development of improved protocols for hiPSC differentiation and manipulation of adult stem cells <i>in vivo</i>, with benefits for patients with ESRD. Additionally, our protocol for xenotransplantation following lung injury provides the first method for efficient future cell therapy.
Downregulation of Graft-Versus-Host-Disease (GVHD) by Treatment with Histone Deacetylase (HDAC) Inhibitors Involves Modulation of the JAK/STAT Signaling Pathway.
Authors: Corinna Leng, Margathe Gries, Suzanne Lentzsch, Simone Lusatis, Paolo Mascagni, Markus Y. Mapara
Journal: Blood (2004)
Impaired Activity of Mitochondrial Respiratory Chain Enzyme Complexes and Mitochondrial Genomic Aberrations in Leukemia Cells from Patients with Acute Myeloid Leukemia.
Authors: Myung-Geun Shin, Hyeoung Joon Kim, Hye-Ran Kim, Il-Kwon Lee, Duck Cho, Seung-Jung Kee, Hoon Kook, Tai-Ju Hwang, Jong-Hee Shin, Soon-Pal Suh, Dong-Wook Ryang
Journal: Blood (2006)
Mitochondrial Phylogenomics Resolves the Global Spread of Higher Termites, Ecosystem Engineers of the Tropics
Authors: Thomas Bourguignon, Nathan Lo, Jan Šobotník, Simon Y. W. Ho, Naeem Iqbal, Eric Coissac, Maria Lee, Martin M. Jendryka, David Sillam-Dussès, Barbora Křížková, Yves Roisin, Theodore A. Evans
Journal: Molecular Biology and Evolution (2016)

The higher termites (Termitidae) are keystone species and ecosystem engineers. They have exceptional biomass and play important roles in decomposition of dead plant matter, in soil manipulation, and as the primary food for many animals, especially in the tropics. Higher termites are most diverse in rainforests, with estimated origins in the late Eocene (∼54 Ma), postdating the breakup of Pangaea and Gondwana when most continents became separated. Since termites are poor fliers, their origin and spread across the globe requires alternative explanation. Here, we show that higher termites originated 42–54 Ma in Africa and subsequently underwent at least 24 dispersal events between the continents in two main periods. Using phylogenetic analyses of mitochondrial genomes from 415 species, including all higher termite taxonomic and feeding groups, we inferred 10 dispersal events to South America and Asia 35–23 Ma, coinciding with the sharp decrease in global temperature, sea level, and rainforest cover in the Oligocene. After global temperatures increased, 23–5 Ma, there was only one more dispersal to South America but 11 to Asia and Australia, and one dispersal back to Africa. Most of these dispersal events were transoceanic and might have occurred via floating logs. The spread of higher termites across oceans was helped by the novel ecological opportunities brought about by environmental and ecosystem change, and led termites to become one of the few insect groups with specialized mammal predators. This has parallels with modern invasive species that have been able to thrive in human-impacted ecosystems.
Prognosis of HER2 expression in cholangiocarcinoma when evaluated using gastric cancer methodology of immunohistochemistry.
Authors: Viviane Teodoro Oliveira Fernandes, Milton Jose De Barros E. Silva, Maria D. Begnami, Augusto Saito
Journal: Journal of Clinical Oncology (2015)

e15203 Background: The tumors of the biliary tract can arise anywhere in the tree biliary or less frequent from transdifferentiation of hepatocytes. This tumor is lethal with heterogeneous behavior and complex molecular biology representing 0.6-1% of tumors among adults. Several studies highlight the role of HER2 in cholangiocarcinoma pathogenesis and its expression seems to be present from the onset of carcinogenesis. The prognostic value of HER2 in this population is matter of debate and the pattern of expression of HER2 is heterogeneous as in gastric cancer. The aim of this study was to evaluate the prognostic role of HER2 by immunohistochemistry using the methodology validated for gastric cancer in western population with cholangiocarcinoma. Methods: We have done a retrospective review of clinical and pathological features on eletronic medical charts of patients with cholangiocarcinoma and HER2 immunohistochemistry testing using gastric cancer methodology. Results: Among the 46 patients evaluated, 38 were evaluated for the expression of HER2. The expression of Her (2 or 3+) was found in (30%). In multivariate analysis, the expression of HER2 (2 or 3 +) was a independent prognostic factor with HR for mortality of 3.08 (1.23 to 7.28) with p: 0.01. This prognostic factor was observed in both localized and metastatic disease. Conclusions: HER2 immunohistochemistry using gastric cancer methodology was positive (2 or 3+) in 30% of our western population of cholangiocarcinoma and was a independent prognostic factor. Prospective studies are needed to validate the best method for assessing HER2 expression and if there is a predictive value for using anti-HER therapy in this selected population.
IMGui—A Desktop GUI Application for Isolation with Migration Analyses
Authors: Jared Knoblauch, Arun Sethuraman, Jody Hey
Journal: Molecular Biology and Evolution (2016)

The Isolation with Migration (IM) programs (e.g., IMa2) have been utilized extensively by evolutionary biologists for model-based inference of demographic parameters including effective population sizes, migration rates, and divergence times. Here, we describe a graphical user interface for the latest IM program. IMGui provides a comprehensive set of tools for performing demographic analyses, tracking progress of runs, and visualizing results. Developed using node. js and the Electron framework, IMGui is an application that runs on any desktop operating system, and is available for download at https://github.com/jaredgk/IMgui-electron-packages.
A Review of Mutation Analysis In the TOPS Trial of Standard Dose Versus High Dose IM In CML Suggests That Refinements to the ELN Recommendations for Mutation Screening May Be Appropriate
Authors: Susan Branford, Hyun-Gyung Goh, Barbara Izzo, Lan Beppu, Christine-Elke Ortmann, Kamila Duniec, Yu Jin, Richard C. Woodman, Fabrizio Pane, Dong-Wook Kim, Jerald P. Radich, Timothy P. Hughes
Journal: Blood (2010)
Abstract 4529: Pharmacokinetics (PK) of LOXO-101 during the first-in-human Phase I study in patients with advanced solid tumors: Interim update
Authors: Howard A. Burris, Alice T. Shaw, Todd M. Bauer, Anna F. Farago, Robert C. Doebele, Steven Smith, Nisha Nanda, Scott Cruickshank, Jennifer A. Low, Marcia S. Brose
Journal: Cancer Research (2015)

Abstract Background LOXO-101 is the first highly selective and potent small-molecule inhibitor of TRKA, TRKB, and TRKC to be dosed clinically. It has approximately 100-fold selectivity over other kinase and non-kinase targets. We report here the interim results of the first-in-man Phase I study which evaluates the safety, maximum-tolerated dose (MTD), and PK of LOXO-101 in patients with advanced solid tumors. Methods This is an ongoing open-label, multicenter, 3+3 dose escalation Phase I study of LOXO-101. Patients with solid tumors refractory to standard therapy, with normal hematopoietic and major organ function were eligible for study. LOXO-101 was administered orally as a single dose, followed by QD or BID doses for continuous 28-day cycles. Receptor occupancy was determined with an Emax model based on unbound LOXO-101 plasma concentrations. Results To date, data are available for 8 patients including patients treated at each of the first two dose levels, 50 mg QD and 100 mg QD. LOXO-101 has been well tolerated; the MTD has not been reached and no adverse events have been reported more than once other than fatigue (two patients; related but not serious) and syncope (two patients; unrelated and serious). Maximum plasma concentrations of LOXO-101 were reached 30-60 minutes following dosing and exposure increased in approximate proportion with dose. Mean values of Cl/F, V/F, and half-life were 0.5 L/h/kg, 2 L/kg, and 3.5 h, respectively, and were independent of dose. Exposure was similar following the single dose and on Day 1 and Day 8 of repeated dosing in all subjects. The unbound drug levels of LOXO-101 appear sufficient for approximately 98% inhibition of TRKA/B/C at peak concentrations with once-daily dosing of 50 or 100 mg. Conclusions LOXO-101 has been well tolerated and has sufficient systemic exposure for robust inhibition of the TRKA, TRKB, and TRKC kinases without inhibition of other kinases. With dose escalation continuing, additional data and follow up will be provided at time of presentation. Citation Format: Howard A. Burris, Alice T. Shaw, Todd M. Bauer, Anna F. Farago, Robert C. Doebele, Steven Smith, Nisha Nanda, Scott Cruickshank, Jennifer A. Low, Marcia S. Brose. Pharmacokinetics (PK) of LOXO-101 during the first-in-human Phase I study in patients with advanced solid tumors: Interim update. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4529. doi:10.1158/1538-7445.AM2015-4529
Association of Serum Anti-GAD Antibody and HLA Haplotypes with Type 1 Diabetes Mellitus Triggered by Nivolumab in Patients with Non–Small Cell Lung Cancer
Authors: Yuko Usui, Hibiki Udagawa, Shingo Matsumoto, Kenjiro Imai, Ken Ohashi, Masayuki Ishibashi, Keisuke Kirita, Shigeki Umemura, Kiyotaka Yoh, Seiji Niho, Keiichiro Osame, Koichi Goto
Journal: Journal of Thoracic Oncology (2016)
Abstract 3784: Regulation of chromatin condensation by mitotic checkpoint protein MAD2
Authors: Jayasha Shandilya, Stefan GE Roberts
Journal: Cancer Research (2015)

Abstract The process of cell division is stringently regulated by spindle and mitotic checkpoint components mainly MAD2, MAD1, CDC20, BUBR1, BUB3 and MAD3. These checkpoint proteins detect unattached or improperly attached kinetochore-microtubule binding sites and generate an anaphase-wait signal by inhibiting the anaphase-promoting complex (APC/C). Activation of checkpoint signaling arrests the cells at metaphase until the attachment errors are rectified and all the chromosomes are properly bi-oriented at the metaphase plate. Together with these checkpoint proteins, the expression of checkpoint-regulatory factors such as p31 (comet) and recently identified, WT1 can hypo- or hyper-activate the signaling pathway, respectively and regulate the timing of mitotic exit. Recently we found that WT1 can interact with MAD2 and regulate the mitotic checkpoint function. Furthermore our studies show that MAD2 and WT1 can also influence the chromatin condensation process required for mitotic progression. We analyzed the effect of MAD2 and WT1 perturbations on the epigenetic mark (phosphorylation of histone H3) associated with condensed chromatin state. Our results show that MAD2 regulates the recruitment of chromatin modifying enzyme Aurora kinase B during mitosis. We further show that MAD2 is an important factor required for synchronization of key events beyond the checkpoint signaling sites to ensure the fidelity of chromosome segregation. Citation Format: Jayasha Shandilya, Stefan GE Roberts. Regulation of chromatin condensation by mitotic checkpoint protein MAD2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3784. doi:10.1158/1538-7445.AM2015-3784