tbuckley/cabot-search huggingface dataset:
375. Direct Reprogramming of Fibroblast Allows Live-Cell Imaging of Autophagic Buildup in Pompe Disease Skeletal Myoblast
Authors: Yohei Sato, Hiroshi Kobayashi, Yohta Shimada, Hiroyuki Ida, Yoshikatsu Eto, Toya Ohashi
Journal: Molecular Therapy (2015)

Pompe disease is an autosomal recessive inherited metabolic disease caused by deficiency of acid alpha glucosidase (GAA). Glycogen accumulation is seen in the affected organ such as skeletal muscle, heart and liver. Autophagic buildup is associated to skeletal muscle pathology and known to inhibit the efficacy of enzyme replacement therapy. Myogenic conversion by MyoD forced expression is one of the feasible approaches towards disease modeling of neuromascular disorders. We have generated skeletal myocyte from Pompe disease fibroblast by direct reprogramming by lentiviral MyoD transfer. We have cloned human MYOD1 by RT-PCR from normal control fibroblast (AG08498). EcoR1 site was added and then cloned into 3rd generation lentiviral vector (CSII-EF1α-MCS). High titer lentiviral vector was produced by ultracentrifuge. Next we have infected CS-EF1α-MYOD1 at MOI50 to healthy control fibroblast. 48 hours after transfection, we have confirmed MYOD1 expression in cytoplasm and nucleus of control fibroblast by immunofluorescence. Other than MyoD, other myogenic factors are expressed and confirmed by immunofluorescence (MyoD, MyoG, Myf5, Pax7 and MHC). We have also infected CS-EF1α-MYOD1 to patient derived fibroblast (GM20124) and confirmed the expressions of myogenic factors, MyoD, MyoG, Myf5, Pax7 and MHC by immunofluorescence. Moreover, we have checked gene expression by RT-PCR and confirmed myogenic markers (Pax7, Pax3, Myf5, MyoD, AchR and MHC) are positive both in healthy control and patient derived fibroblasts. To visualize autophagic accumulation, we have infected RFP-GFP-LC3B vector (Promo™ Autophagy Sensor Kit) to induced myoblasts. It allows us to detect LC-3 accumulation which is suggesting autophagic buildup in pompe disease patient compared to healthy control. We have investigated autophagic buildup seen in skeletal myoblasts of Pompe disease at the live-cell imaging. Moreover, we have cloned lentiviral vector which expresses TFEB, a master gene for lysosomal biogenesis, to treat autophagic buildup and infected to MyoD induced myoblast. Massive accumulation of LC-3 is decreased after CSII-EF1α-TFEB transfection. High titer lentiviral MyoD transfer is useful in terms of direct reprogramming of fibroblast into skeletal myoblast. In addition, disease modeling of Pompe disease allows live-cell imaging of autophagic buildup of skeletal myocyote.
Pharmacokinetics and Pharmacodynamics of IPI-145, a Potent Inhibitor of Phosphoinositide-3-Kinase-δ,γ, Following Single- and Multiple-Dose Administration in Healthy Subjects and Patients with Advanced Hematologic Malignancies
Authors: Joi Dunbar, Jylle Nevejans, Charlotte McKee, Kerrie Faia, Jan Jaap van Lier, Peter Pruim, Janet Stegehuis, Sue Zhao, Brad S. Kahl, Steven M. Horwitz, Manish Patel, Anas Younes, Ian W. Flinn
Journal: Blood (2012)
Abstract 5112: In vivo molecular imaging of HIF-1α and septin 9 interaction by bimolecular fluorescence complementation
Authors: Maya Golan, Nicola J. Mabjeesh
Journal: Cancer Research (2015)

Abstract Hypoxia is a common feature of many solid tumors that promotes tumor progression and leads to radiation and chemotherapy resistance. Hypoxia-inducible factor 1 (HIF-1), as one of the major mediators of the hypoxic response, has been shown to activate hypoxia-responsive genes, which are involved in multiple aspects of tumorigenesis and cancer progression. Previously, we have described an interaction between septin 9 isoform 1 (SEPT9_i1) protein and HIF-1α, the oxygen regulated subunit of HIF-1. SEPT9_i1 is a member of the conserved family of septins, which are GTP-binding proteins that form cytoskeleton-like filaments essential for many functions in eukaryotic organisms including neoplasia. The interaction of SEPT9_i1 with HIF-1α increases protein stability and HIF-1 transcriptional activity in vitro and promotes proliferation, tumor growth and angiogenesis in vivo. Recent data indicated that SEPT9_i1 was also involved in the trafficking of cytoplasmic HIF-1α into the nucleus by direct interaction to the nuclear transporter importin-α. Herein, we utilized split YFP bimolecular fluorescence complementation (BiFC) methodology in order to monitor HIF-1α/SEPT9_i1 interactions in vivo. First, we generated split YFP protein chimeras fused to HIF-1α and to SEPT9_i1 on both their amino and carboxyl termini. Different pairs of HIF-1α and SEPT9_i1 chimeras were tested to functional complementation of the split YFP fragments using transient transfection in HEK293 cells. The complemented proteins capable of fluorescence were identified as SEPT9_i1-YN (the amino terminus of YFP fused to the carboxyl terminal of SEPT9_i1) and YC-HIF-1α (the carboxyl terminus of YFP fused to the N-terminal of HIF-1α). This pair was selected for further studies in PC-3 human prostate cancer cells. YFP complementation fluorescence derived from these chimeras was increased in the presence of hypoxia mimicking agents, such as CoCl2 and the iron chelators deferoxamine (DFO) and dibenzoylmethane (DBM). The chimera, YCΔHLH lacking the HLH domain that is essential for the interaction with SEPT9_i1 gave a significant reduction in YFP complementation fluorescence. Furthermore, expression of SEPT9_i1 252-379aa fragment, the domain essential for the interaction with HIF-1α, abolished almost completely the YFP complementation fluorescence. These results reconfirm our previous studies on HIF-1α/SEPT9_i1 interactions and make this system attractive for identifying new compounds capable of disrupting this complex in order to inhibit tumor growth and angiogenesis. Citation Format: Maya Golan, Nicola J. Mabjeesh. In vivo molecular imaging of HIF-1α and septin 9 interaction by bimolecular fluorescence complementation. [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 5112. doi:10.1158/1538-7445.AM2015-5112
Abstract LB-153: In vivo visualization of the targeted DNA methylation
Authors: Yao-Li Chen, Shou-Tung Chen, Chun-Chun Chung, Chia-Chen Hsu, Ping-Yi Lin, Wan-Ling Chuang, Yi-Ju Chu, Yu-Wei Leu, Shu-Huei Hsiao
Journal: Cancer Research (2015)

Abstract DNA methylation is a dominant silencing epigenetic modification that is inheritable in somatic cells. The recruitment of DNA methylation was proven to be able to reshape the cell physiology and cause tumorigenesis. The reversible nature of the methylation modification also makes the DNA methylation a therapeutic target and the monitoring of the methylation/demethylation dynamics in vivo is thus important for the development of demethylation agents. A targeted DNA methylation method and a two-component reporter system were combined to monitor the increase and demolishment of DNA methylation in vivo. The two constructs of the reporter systems are: target gene promoter (like HIC1 and ENSA) linked with the Tet repressor gene; the other is the Tet operator linked with the reporter like EGFP (in cell), IFP and iRFP. Both constructs were co-transfected into cells like mesenchymal stem cells, MDA-MB-231 and HCT116, and stable clones with both constructs were isolated and validated. Targeted DNA methylation method was used to transfect the isolated cell and, as the increase of DNA methylation, the reporters expressed. The increased methylation was then quenched after the adding of demethylation agents. To test this system in vivo, cells with both constructs were injected into the immune-deficient mice and the tumors grew afterward. Targeted DNA methylation was performed in vivo and the increase of methylation/expression of reporter genes were observed by fluorescence-based tomography. This combined system is also effective to monitor the demethylation effects of the demethylation agents. (Supported by: NSC- 102-2320-B-194-003-MY3 and NSC-102-2314-B-371- 003-MY3, MOST Taiwan) Citation Format: Yao-Li Chen, Shou-Tung Chen, Chun-Chun Chung, Chia-Chen Hsu, Ping-Yi Lin, Wan-Ling Chuang, Yi-Ju Chu, Yu-Wei Leu, Shu-Huei Hsiao. In vivo visualization of the targeted DNA methylation. [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 LB-153. doi:10.1158/1538-7445.AM2015-LB-153
Abstract 4920: The detection of circulating tumor DNA using Quantstudio 3D digital PCR
Authors: Adam H. Greer, Glenn Mills, Hong Yin
Journal: Cancer Research (2015)

Abstract Purpose: The purpose of this study is to employ the Quantstudio 3D (Q3D) system to detect circulating tumor DNA in cancer patients’ plasma. Background: Circulating tumor DNA (ctDNA) as a non-invasive biomarker plays roles in monitoring solid tumor progression, metastasis, and therapeutic effects. The identification of ctDNA depends on mutant determination. The digital PCR technology is a powerful tool to identify ctDNA. The Q3D platform is a relatively new digital PCR systemwhcih has not been extensively tested in the detection of ctDNA. In this study, we tested the capacity of Q3D in detecting ctDNA. Methods: Genomic DNA was extracted from tumor tissue of patients. Variants were identified in tumor tissue DNA by Illumina's TruSeq Amplicon Cancer Panel. To detect the circulating tumor DNA, DNA was extracted from the plasma of the patient with known variants identified by targeted-sequencing. The circulating DNA was isolated from 400μl of plasma from 12 patients using QIAamp DNA Blood kit. The DNA concentration was measured using the Qubit 2.0. The quality and size of the circulating DNA was determined using the Agilent 2100. The circulating DNA size was approximately 170-180 nucleotides in length, as reported in literatures. The variant determination by Q3D was tested with custom TagMan SNP Genotyping Assays. The absolute copy number of variant was determined with QuantStudio® 3D AnalysisSuite™ Software. Results: We first tested several assays with known-variant tumor tissue DNA samples in Q3D. The mutants identified by assays are BRAF V600E, KRAS G12D, KRAS G12V, and PIK3CA E542K. The results showed that data points of mutant call, wild type call were well separated from non-signal data points. We examined the sensitivity of these assays using serial dilution of tumor DNA sample in non-tumor human tissue DNA. 0.1% to 1% of mutants were detectable depending specific assays. We collected circulating DNA from XX Patients’ plasma. The DNA concentrations ranged from 5 ng/ml to 30 ng/ml of plasma. KRAS G12D and G12V was detected with circulating DNA from patients carrying KRAS mutant. The mutant frequency of KRAS G12D and G12V determined by Q3D is 3.2% and 3.7%, respectively. The absolute mutant copy number for KRAS G12D is 9 and for KRAS G12V is 8. Conclusions: Q3D with validated custom TagMan SNP Genotyping Assays is applicable in detecting ctDNA. We will expect to utilize the technique to evaluate ctDNA. Citation Format: Adam H. Greer, Glenn Mills, Hong Yin. The detection of circulating tumor DNA using Quantstudio 3D digital PCR. [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 4920. doi:10.1158/1538-7445.AM2015-4920
Abstract 920: Mechanism of the transferrin receptor 1 dysregulation in hepatocarcinogenesis
Authors: Iryna Kindrat, Volodymyr Tryndyak, Aline de Conti, Svitlana Shpyleva, Anna Erstenyuk, Frederick A. Beland, Igor Pogribny
Journal: Cancer Research (2015)

Abstract Hepatocellular carcinoma (HCC) is one of the most prevalent life-threatening human cancers, with a rapidly increasing incidence worldwide. The pathogenesis of HCC is complex and consists of multiple interconnected molecular alterations. Current evidence indicates that altered iron metabolism plays an important role in the development and progression of HCC; however, there is a lack of conclusive information regarding the mechanisms of this dysregulation. In previous studies using different models of rodent carcinogenesis, we demonstrated prominent alterations of intracellular iron metabolism in preneoplastic livers, including changes in gene expression and the level of related proteins involved in the regulation of intracellular hepatic iron metabolism, primarily up-regulation of transferrin receptor 1 (TFRC). In the present study, we investigated the role and underlying mechanisms of the aberrant expression of TFRC, the main receptor controlling cellular iron uptake, in liver carcinogenesis. In both preneoplastic liver tissue from a relevant rat model of human HCC and in human HCC cells, there was a significant increase in the levels of TFRC mRNA and protein. This resulted in a substantial increase in TFRC/ferroportin (FPN) ratio, favoring an accumulation of iron in the HCC cells. The increase in levels of TFRC was attributed to a post-transcriptional microRNA-mediated mechanism. This was demonstrated by a computational analysis and confirmed experimentally by in vitro transfection experiments showing that transfection of human HCC cell lines with has-miR-152 effectively suppressed the expression of TFRC. Additionally, there was a strong inverse correlation between the level of TRFC and the expression of miR-152 in human HCC cells (r = -0.99, p = 4.7×10-9). Finally, the expression of TFRC and miR-152 in human HCC tissues was evaluated using the TCGA database. We observed an over-expression of TFRC in human HCC tissue samples and a markedly decreased level of miR-152 when compared to non-tumor liver tissue. These results demonstrate that dysregulation of cellular iron metabolism in liver carcinogenesis is driven by an up-regulation of TFRC, and indicate that miR-152-specific targeting of TFRC to decrease its levels may provide a selective anticancer therapeutic approach for the treatment of HCC. Citation Format: Iryna Kindrat, Volodymyr Tryndyak, Aline de Conti, Svitlana Shpyleva, Anna Erstenyuk, Frederick A. Beland, Igor Pogribny. Mechanism of the transferrin receptor 1 dysregulation in hepatocarcinogenesis. [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 920. doi:10.1158/1538-7445.AM2015-920
Abstract 1544: Association of tumor location with the breast and clinicopathological characteristics
Authors: Seth Rummel, Nick Costantino, Matthew T. Hueman, Craig D. Shriver, Rachel E. Ellsworth
Journal: Cancer Research (2015)

Abstract Introduction Tumor location within the breast varies, with the highest frequency in the upper outer quadrant (UOQ) and lowest in the lower inner quadrant (LIQ). What is not well understood is whether tumor location is prognostic. To determine whether tumor location is prognostic, associations between tumor site and clinicopathological characteristics were evaluated. Methods The Clinical Breast Care Project database was queried to identify all patients whose tumor site- UOQ, upper inner quadrant (UIQ), central, (LIQ), lower outer quadrant (LOQ) - was determined by a single, dedicated breast pathologist between 2001 and 2013. Patients with multicentric disease (n = 122) or tumors spanning multiple quadrants (n = 381) were excluded from further analysis. Clinicopathological characteristics evaluated included age at diagnosis, ethnicity, BMI, tumor stage, grade, and size, and hormone receptor, HER2 and lymph node status. Data was analyzed using chi-square and Mann-Whitney tests for univariate analysis with multivariate analysis performed using principal components analysis and multiple logistic regression. Significance was defined as P&amp;lt;0.05. Results Of the 980 patients with defined tumor location, 30 had bilateral disease. Tumor location in the UOQ (51.5%) was higher than in the UIQ (15.6%), LOQ (14.2%), central (10.6%) or LIQ (8.1%). Tumors in the central quadrant were significantly more likely to have higher tumor stage (P = 0.003) and size (P&amp;lt;0.001), positive lymph node status (P&amp;lt;0.001), and mortality rates (P = 0.011) compared to other quadrants. Analysis within each tumor size, however, found no difference in lymph node status by tumor location. After multivariate analysis, only tumor size and lymph node status remained significantly associated with mortality. Discussion Although tumors in the central region are associated with less favorable outcome, this association was driven by significantly larger tumor size, which has been associated with positive lymph node status. Tumors in the central region are known to be more difficult to detect mammographically and the majority of tumors in this region are palpable, thus, tumor location is not an independent prognostic factor, but rather less favorable prognosis, including larger tumor size, higher grade and more lymph node involvement in the central region can be attributed to delayed diagnosis. Citation Format: Seth Rummel, Nick Costantino, Matthew T. Hueman, Craig D. Shriver, Rachel E. Ellsworth. Association of tumor location with the breast and clinicopathological characteristics. [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 1544. doi:10.1158/1538-7445.AM2015-1544
Comparison of <sup>68</sup>Ga-HBED-CC PSMA-PET/CT and multiparametric MRI for gross tumour volume detection in patients with primary prostate cancer based on slice by slice comparison with histopathology
Authors: Constantinos Zamboglou, Vanessa Drendel, Cordula A. Jilg, Hans C. Rischke, Teresa I. Beck, Wolfgang Schultze-Seemann, Tobias Krauss, Michael Mix, Florian Schiller, Ulrich Wetterauer, Martin Werner, Mathias Langer, Michael Bock, Philipp T. Meyer, Anca L. Grosu
Journal: Theranostics (2016)

The exact detection and delineation of the intraprostatic tumour burden is crucial for treatment planning in primary prostate cancer (PCa). We compared 68Ga-HBED-CC-PSMA PET/CT with multiparametric MRI (mpMRI) for diagnosis and tumour delineation in patients with primary PCa based on slice by slice correlation with histopathological reference material.Seven patients with histopathologically proven primary PCa underwent 68Ga-HBED-CC-PSMA PET/CT and MRI followed by radical prostatectomy. Resected prostates were scanned by ex-vivo CT in a special localizer and prepared for histopathology. Invasive PCa was delineated on a HE stained histologic tissue slide and matched to ex-vivo CT to obtain gross tumor volume (GTV-)histo. Ex-vivo CT including GTV-histo and MRI data were matched to in-vivo CT(PET). Consensus contours based on MRI (GTV-MRI), PSMA PET (GTV-PET) or the combination of both (GTV-union/-intersection) were created. In each in-vivo CT slice the prostate was separated into 4 equal segments and sensitivity and specificity for PSMA PET and mpMRI were assessed by comparison with histological reference material. Furthermore, the spatial overlap between GTV-histo and GTV-PET/-MRI and the Sørensen-Dice coefficient (DSC) were calculated. In the case of multifocal PCa (4/7 patients), SUV values (PSMA PET) and ADC-values (diffusion weighted MRI) were obtained for each lesion.PSMA PET and mpMRI detected PCa in all patients. GTV-histo was detected in 225 of 340 segments (66.2%). Sensitivity and specificity for GTV-PET, GTV-MRI, GTV-union and GTV-intersection were 75% and 87%, 70% and 82%, 82% and 67%, 55% and 99%, respectively. GTV-histo had on average the highest overlap with GTV-union (57±22%), which was significantly higher than overlap with GTV-MRI (p=0.016) and GTV-PET (p=0.016), respectively. The mean DSC for GTV-union, GTV-PET and GTV-MRI was 0.51 (±0.18), 0.45 (±0.17) and 0.48 (±0.19), respectively. In every patient with multifocal PCa there was one lesion which had both the highest SUV and the lowest ADC-value (mean and max).In a slice by slice analysis with histopathology, 68Ga-HBED-CC-PSMA PET/CT and mpMRI showed high sensitivity and specificity in detection of primary PCa. A combination of both methods performed even better in terms of sensitivity (GTV-union) and specificity (GTV-intersection). A moderate to good spatial overlap with GTV-histo was observed for PSMA PET/CT and mpMRI alone which was significantly improved by GTV-union. Further studies are warranted to analyse the impact of these preliminary findings for diagnostic (multimodal guided TRUS biopsy) and therapeutic (focal therapy) strategies in primary PCa.
Pathological changes of adipose tissue in secondary lymphoedema
Authors: K. Tashiro, J. Feng, S.-H. Wu, T. Mashiko, K. Kanayama, M. Narushima, H. Uda, S. Miyamoto, I. Koshima, K. Yoshimura
Journal: British Journal of Dermatology (2016)

The pathophysiology of lymphoedema is poorly understood. Current treatment options include compression therapy, resection, liposuction and lymphatic microsurgery, but determining the optimal treatment approach for each patient remains challenging.We characterized skin and adipose tissue alterations in the setting of secondary lymphoedema.Morphological and histopathological evaluations were conducted for 70 specimens collected from 26 female patients with lower-extremity secondary lymphoedema following surgical intervention for gynaecological cancers. Indocyanine green lymphography was performed for each patient to assess lymphoedema severity.Macroscopic and ultrasound findings revealed that lymphoedema adipose tissue had larger lobules of adipose tissue, with these lobules surrounded by thick collagen fibres and interstitial lymphatic fluid. In lymphoedema specimens, adipocytes displayed hypertrophic changes and more collagen fibre deposits when examined using electron microscopy, whole-mount staining and immunohistochemistry. The number of capillary lymphatic channels was also found to be increased in the dermis of lymphoedema limbs. Crown-like structures (dead adipocytes surrounded by M1 macrophages) were less frequently seen in lymphoedema samples. Flow cytometry revealed that, among the cellular components of adipose tissue, adipose-derived stem/stromal cells and M2 macrophages were decreased in number in lymphoedema adipose tissue compared with normal controls.These findings suggest that long-term lymphatic volume overload can induce chronic tissue inflammation, progressive fibrosis, impaired homeostasis, altered remodelling of adipose tissue, impaired regenerative capacity and immunological dysfunction. Further elucidation of the pathophysiological mechanisms underlying lymphoedema will lead to more reliable therapeutic strategies.