scholarly journals High throughput estimation of functional cell activities reveals disease mechanisms and predicts relevant clinical outcomes

2016 ◽  
Author(s):  
Marta R. Hidalgo ◽  
Cankut Cubuk ◽  
Alicia Amadoz ◽  
Francisco Salavert ◽  
José Carbonell-Caballero ◽  
...  
Keyword(s):  
High Throughput ◽  
Gene Mutations ◽  
Diagnostic Value ◽  
Therapeutic Interventions ◽  
Biological Knowledge ◽  
Individual Gene ◽  
Main Challenge ◽  
Activity Measurements ◽  
Cancer Types ◽  
Or Gene

AbstractUnderstanding the aspects of the cell functionality that account for disease or drug action mechanisms is a main challenge for precision medicine. Here we propose a new method that models cell signaling using biological knowledge on signal transduction. The method recodes individual gene expression values (and/or gene mutations) into accurate measurements of changes in the activity of signaling circuits, which ultimately constitute high-throughput estimations of cell functionalities caused by gene activity within the pathway. Moreover, such estimations can be obtained either at cohort-level, in case/control comparisons, or personalized for individual patients. The accuracy of the method is demonstrated in an extensive analysis involving 5640 patients from 12 different cancer types. Circuit activity measurements not only have a high diagnostic value but also can be related to relevant disease outcomes such as survival, and can be used to assess therapeutic interventions.

Abstract 026: Optical Recording of Cardiac Action Potentials from Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
B Alexander Yi ◽  
Joel M Kralj ◽  
Adam E Cohen
Keyword(s):  
Stem Cell ◽  
High Throughput ◽  
Action Potentials ◽  
Pluripotent Stem Cell ◽  
Gene Mutations ◽  
Induced Pluripotent Stem Cell ◽  
Patch Clamping ◽  
Cardiac Action ◽  
Or Gene ◽  
Induced Pluripotent

The electrically excitable properties of cardiomyocytes stem from the activity of ion channels that allow the coordinated entry of ions to generate cardiac action potentials. Disruptions in ion channel function either by drugs or gene mutations can lead to cardiac arrhythmias. The ability to screen drugs or gene mutations rapidly for effects on the cardiac action potential would be of interest for both drug discovery as well as for studies of ion channel function; however, the time-consuming and technically challenging nature of conventional patch clamping can limit the ability to perform high throughput screens. Archaerhodopsin3, or Arch, is an Archaebacterial variant of the membrane protein bacteriorhodopsin that binds a retinal fluorophore whose signal is rapidly responsive to changes in membrane potential. Here, we report the use of Arch to optically record action potentials from human induced pluripotent stem cell-derived cardiomyocytes. Human induced pluripotent stem cells that stably express Arch were generated and then differentiated into cardiomyocytes. As compared to patch clamping, Arch faithfully reproduces many of the key features of cardiac action potentials and may be a tool to be used for high throughput electrophysiological screens of cardiomyocytes.

scholarly journals Microplate Orbital Mixing Improves High-Throughput Cell-Based Reporter Assay Readouts

2006 ◽  
Vol 12 (1) ◽  
pp. 140-144 ◽  
Author(s):  
Michael K. Hancock ◽  
Myleen N. Medina ◽  
Brendan M. Smith ◽  
Anthony P. Orth
Keyword(s):  
High Throughput ◽  
Dynamic Range ◽  
Cell Lysis ◽  
Single Step ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Activity Measurements ◽  
Simple Detection ◽  
Reporter Assays ◽  
Orbital Mixing

Reporter assays are commonly used for high-throughput cell-based screening of compounds, cDNAs, and siRNAs due to robust signal, ease of miniaturization, and simple detection and analysis. Among the most widely used reporter genes is the bioluminescent enzyme luciferase, which, when exposed to its substrate luciferin upon cell lysis, yields linear signal over a dynamic range of several orders of magnitude. Commercially available luciferase assay formulations have been developed permitting homogeneous, single-step cell lysis and reporter activity measurements. Assay conditions employed with these formulations are typically designed to minimize well-to-well luminescence variability due to variability in dispensing, evaporation, and incomplete sample mixing. The authors demonstrate that incorporating a microplate orbital mixing step into 96- and 384-well microplate cell-based luciferase reporter assays can greatly improve reporter readouts. They have found that orbital mixing using commercially available mixers facilitates maximal luciferase signal generation from high cell density–containing samples while minimizing variability due to partial cell lysis, thereby improving assay precision. The authors fully expect that widespread availability of mixers with sufficiently small orbits and higher speed settings will permit gains in signal and precision in the 1536-well format as well.

scholarly journals Non-invasive and high-throughput interrogation of exon-specific isoform expression

2021 ◽  
Author(s):  
Dong-Jiunn Jeffery Truong ◽  
Teeradon Phlairaharn ◽  
Bianca Eßwein ◽  
Christoph Gruber ◽  
Deniz Tümen ◽  
...  
Keyword(s):  
Stem Cells ◽  
High Throughput ◽  
High Throughput Screening ◽  
Embryonic Stem ◽  
High Sensitivity ◽  
Therapeutic Interventions ◽  
Dominant Factor ◽  
Reporter System ◽  
Isoform Expression ◽  
Induced Pluripotent

AbstractExpression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions.

scholarly journals A Review of Recent Advancement in Integrating Omics Data with Literature Mining towards Biomedical Discoveries

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Kalpana Raja ◽  
Matthew Patrick ◽  
Yilin Gao ◽  
Desmond Madu ◽  
Yuyang Yang ◽  
...  
Keyword(s):  
Text Mining ◽  
High Throughput ◽  
Literature Mining ◽  
Biological Knowledge ◽  
Omics Data ◽  
Huge Number ◽  
Automated Generation ◽  
The Past ◽  
Independent Information ◽  
Recent Advancement

In the past decade, the volume of “omics” data generated by the different high-throughput technologies has expanded exponentially. The managing, storing, and analyzing of this big data have been a great challenge for the researchers, especially when moving towards the goal of generating testable data-driven hypotheses, which has been the promise of the high-throughput experimental techniques. Different bioinformatics approaches have been developed to streamline the downstream analyzes by providing independent information to interpret and provide biological inference. Text mining (also known as literature mining) is one of the commonly used approaches for automated generation of biological knowledge from the huge number of published articles. In this review paper, we discuss the recent advancement in approaches that integrate results from omics data and information generated from text mining approaches to uncover novel biomedical information.

Longitudinal Brain Size Measurements in App/Ps1 Transgenic Mice

2010 ◽  
Vol 4 ◽  
pp. MRI.S5885 ◽  
Author(s):  
Trevor J. Vincent ◽  
Jonathan D. Thiessen ◽  
Laryssa M. Kurjewicz ◽  
Shelley L. Germscheid ◽  
Allan J. Turner ◽  
...  
Keyword(s):  
Brain Size ◽  
Three Dimensional ◽  
Gene Mutations ◽  
Mutant Form ◽  
Swedish Mutation ◽  
Significant Difference ◽  
Lateral Width ◽  
Resolution Imaging ◽  
Or Gene ◽  
The Brain

There appear to be species differences among the effects of gene mutations related to familial Alzheimer's disease on the brain during aging. To gain a better understanding of the effects of the Swedish mutation of amyloid precursor protein and the mutant form of human presenilin 1 on mice, commercially available mice from Jackson Laboratory were studied. Three dimensional T2*-weighted imaging was used to monitor the size of brains of APP/PS1 mice monthly, from 6 to 13 months of age. No significant difference was measured in the size of the medial-lateral width, dorsal-ventral height, rostral-caudal length or the volume of the APPSwe/ PS1 mouse brain. Faster and higher-resolution imaging methods are needed to accurately determine if small volume or shape changes occur in mouse brains with age or gene mutations.

scholarly journals MBNL1 alternative splicing isoforms play opposing roles in cancer

2018 ◽  
Vol 1 (5) ◽  
pp. e201800157 ◽  
Author(s):  
Tommaso Tabaglio ◽  
Diana HP Low ◽  
Winnie Koon Lay Teo ◽  
Pierre Alexis Goy ◽  
Piotr Cywoniuk ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Cancer Cells ◽  
Cancer Progression ◽  
Gene Function ◽  
Dominant Negative ◽  
Individual Gene ◽  
Cancer Types ◽  
Splicing Isoforms ◽  
Isoform Switching ◽  
And Migration

The extent of and the oncogenic role played by alternative splicing (AS) in cancer are well documented. Nonetheless, only few studies have attempted to dissect individual gene function at an isoform level. Here, we focus on the AS of splicing factors during prostate cancer progression, as these factors are known to undergo extensive AS and have the potential to affect hundreds of downstream genes. We identified exon 7 (ex7) in the MBNL1 (Muscleblind-like 1) transcript as being the most differentially included exon in cancer, both in cell lines and in patients' samples. In contrast, MBNL1 overall expression was down-regulated, consistently with its described role as a tumor suppressor. This observation holds true in the majority of cancer types analyzed. We first identified components associated to the U2 splicing complex (SF3B1, SF3A1, and PHF5A) as required for efficient ex7 inclusion and we confirmed that this exon is fundamental for MBNL1 protein homodimerization. We next used splice-switching antisense oligonucleotides (AONs) or siRNAs to compare the effect of MBNL1 splicing isoform switching with knockdown. We report that whereas the absence of MBNL1 is tolerated in cancer cells, the expression of isoforms lacking ex7 (MBNL1 Δex7) induces DNA damage and inhibits cell viability and migration, acting as dominant negative proteins. Our data demonstrate the importance of studying gene function at the level of alternative spliced isoforms and support our conclusion that MBNL1 Δex7 proteins are antisurvival factors with a defined tumor suppressive role that cancer cells tend to down-regulate in favor of MBNL +ex7 isoforms.

scholarly journals Therapeutic Targeting of the Gas6/Axl Signaling Pathway in Cancer

2021 ◽  
Vol 22 (18) ◽  
pp. 9953
Author(s):  
Mai Tanaka ◽  
Dietmar W. Siemann
Keyword(s):  
Signaling Pathway ◽  
Receptor Tyrosine Kinase ◽  
Therapeutic Agents ◽  
Therapeutic Interventions ◽  
Therapeutic Resistance ◽  
Clinical Settings ◽  
Affinity Ligand ◽  
Current State ◽  
Cancer Types ◽  
High Affinity Ligand

Many signaling pathways are dysregulated in cancer cells and the host tumor microenvironment. Aberrant receptor tyrosine kinase (RTK) pathways promote cancer development, progression, and metastasis. Hence, numerous therapeutic interventions targeting RTKs have been actively pursued. Axl is an RTK that belongs to the Tyro3, Axl, MerTK (TAM) subfamily. Axl binds to a high affinity ligand growth arrest specific 6 (Gas6) that belongs to the vitamin K-dependent family of proteins. The Gas6/Axl signaling pathway has been implicated to promote progression, metastasis, immune evasion, and therapeutic resistance in many cancer types. Therapeutic agents targeting Gas6 and Axl have been developed, and promising results have been observed in both preclinical and clinical settings when such agents are used alone or in combination therapy. This review examines the current state of therapeutics targeting the Gas6/Axl pathway in cancer and discusses Gas6- and Axl-targeting agents that have been evaluated preclinically and clinically.

scholarly journals Estimating Clinically Relevant Cut-Off Values for a High-Throughput Quantitative Real-Time PCR Detecting Bacterial Respiratory Pathogens in Cattle

2021 ◽  
Vol 8 ◽  
Author(s):  
Alicia F. Klompmaker ◽  
Maria Brydensholt ◽  
Anne Marie Michelsen ◽  
Matthew J. Denwood ◽  
Carsten T. Kirkeby ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Pasteurella Multocida ◽  
Age Groups ◽  
Therapeutic Interventions ◽  
Respiratory Pathogens ◽  
Logistic Regression Models ◽  
Histophilus Somni ◽  
Nasal Swabs

Bovine respiratory disease (BRD) results from interactions between pathogens, environmental stressors, and host factors. Obtaining a diagnosis of the causal pathogens is challenging but the use of high-throughput real-time PCR (rtPCR) may help target preventive and therapeutic interventions. The aim of this study was to improve the interpretation of rtPCR results by analysing their associations with clinical observations. The objective was to develop and illustrate a field-data driven statistical method to guide the selection of relevant quantification cycle cut-off values for pathogens associated with BRD for the high-throughput rtPCR system “Fluidigm BioMark HD” based on nasal swabs from calves. We used data from 36 herds enrolled in a Danish field study where 340 calves within pre-determined age-groups were subject to clinical examination and nasal swabs up to four times. The samples were analysed with the rtPCR system. Each of the 1,025 observation units were classified as sick with BRD or healthy, based on clinical scores. The optimal rtPCR results to predict BRD were investigated for Pasteurella multocida, Mycoplasma bovis, Histophilus somni, Mannheimia haemolytica, and Trueperella pyogenes by interpreting scatterplots and results of mixed effects logistic regression models. The clinically relevant rtPCR cut-off suggested for P. multocida and M. bovis was ≤ 21.3. For H. somni it was ≤ 17.4, while no cut-off could be determined for M. haemolytica and T. pyogenes. The demonstrated approach can provide objective support in the choice of clinically relevant cut-offs. However, for robust performance of the regression model sufficient amounts of suitable data are required.

scholarly journals High Dose Ifosfamide in Relapsed and Unresectable High-Grade Osteosarcoma Patients: A Retrospective Series

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2389 ◽  
Author(s):  
Emanuela Palmerini ◽  
Elisabetta Setola ◽  
Giovanni Grignani ◽  
Lorenzo D’Ambrosio ◽  
Alessandro Comandone ◽  
...  
Keyword(s):  
Gene Mutations ◽  
Progression Free Survival ◽  
Complete Response ◽  
High Dose ◽  
Free Survival ◽  
Duration Of Response ◽  
Or Gene ◽  
High Grade Osteosarcoma ◽  
And Control ◽  
Generation Sequencing

Background: The evidence on high-dose ifosfamide (HD-IFO) use in patients with relapsed osteosarcoma is limited. We performed a retrospective study to analyze HD-IFO activity. Methods: Patients with osteosarcoma relapsed after standard treatment [methotrexate, doxorubicin, cisplatin +/− ifosfamide (MAP+/−I)] with measurable disease according to RECIST1.1 were eligible to ifosfamide (3 g/m2/day) continuous infusion (c.i.) days 1–5 q21d. RECIST1.1 overall response rate (ORR) (complete response (CR) + partial response (PR)), progression-free survival at 6-month (6m-PFS), duration of response (DOR), and 2-year overall survival (2y-OS) were assessed. PARP1 expression and gene mutations were tested by immunohistochemistry and next-generation sequencing. Results: 51 patients were included. ORR was 20% (1 CR + 9 PR). Median DOR was 5 months (95%CI 2–7). Median PFS, 6m-PFS, OS, and 2y-OS were 6 months (95%CI 4–9), 51%, 15 months (10–19), and 30%, respectively. A second surgical complete remission (CR2) was achieved in 26 (51%) patients. After multivariate analysis, previous use of ifosfamide (HR 2.007, p = 0.034) and CR2 (HR 0.126, p < 0.001) showed a significant correlation with PFS and OS, respectively. No significant correlation was found between outcomes and PARP1 or gene mutations. Conclusions: HD-IFO should be considered as the standard first-line treatment option in relapsed osteosarcoma and control arm of future trial in this setting.

scholarly journals Frequent Occurrence of NRAS and BRAF Mutations in Human Acral Naevi

Cancers ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 546 ◽  
Author(s):  
Philipp Jansen ◽  
Ioana Cosgarea ◽  
Rajmohan Murali ◽  
Inga Möller ◽  
Antje Sucker ◽  
...  
Keyword(s):  
Malignant Tumors ◽  
Gene Mutations ◽  
Diagnostic Value ◽  
Clinicopathological Parameters ◽  
Braf Mutations ◽  
Acral Melanoma ◽  
Targeted Next Generation Sequencing ◽  
Melanocytic Tumors ◽  
Frequent Mutations ◽  
Melanoma Subtypes

Acral naevi are benign melanocytic tumors occurring at acral sites. Occasionally they can progress to become malignant tumors (melanomas). The genetics of acral naevi have not been assessed in larger studies. In our study, a large cohort of 130 acral naevi was screened for gene mutations known to be important in other naevi and melanoma subtypes by targeted next-generation sequencing. Mutation status was correlated with clinicopathological parameters. Frequent mutations in genes activating the MAP kinase pathway were identified, including n = 87 (67%) BRAF, n = 24 (18%) NRAS, and one (1%) MAP2K1 mutations. BRAF mutations were almost exclusively V600E (n = 86, 99%) and primarily found in junctional and compound naevi. NRAS mutations were either Q61K or Q61R and frequently identified in dermal naevi. Recurrent non-V600E BRAF, KIT, NF1, and TERT promoter mutations, present in acral melanoma, were not identified. Our study identifies BRAF and NRAS mutations as the primary pathogenic event in acral naevi, however, distributed differently to those in non-acral naevi. The mutational profile of acral naevi is distinct from acral melanoma, which may be of diagnostic value in distinguishing these entities.

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