scholarly journals Deep Mutational Scan of a cardiac sodium channel voltage sensor

2019 ◽  
Author(s):  
Andrew M. Glazer ◽  
Brett M. Kroncke ◽  
Kenneth A. Matreyek ◽  
Tao Yang ◽  
Yuko Wada ◽  
...  
Keyword(s):  
Ion Channel ◽  
Sodium Channel ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Voltage Sensor ◽  
Patch Clamping ◽  
Loss Of Function ◽  
Pathogenic Variants ◽  
Cardiac Sodium Channel

AbstractVariants in ion channel genes have classically been studied in low-throughput by patch clamping. Deep Mutational Scanning (DMS) is a complementary approach that can simultaneously assess function of thousands of variants. We have developed and validated a method to perform a DMS of variants in SCN5A, which encodes the major voltage-gated sodium channel in the heart. We created a library of nearly all possible variants in a 36 base region of SCN5A in the S4 voltage sensor of domain IV and stably integrated the library into HEK293T cells. In preliminary experiments, challenge with three drugs (veratridine, brevetoxin, and ouabain) could discriminate wildtype channels from gain and loss of function pathogenic variants. High-throughput sequencing of the pre- and post-drug challenge pools was used to count the prevalence of each variant and identify variants with abnormal function. The DMS scores identified 40 putative gain of function and 33 putative loss of function variants. For 8/9 variants, patch clamping data was consistent with the scores. These experiments demonstrate the accuracy of a high-throughput in vitro scan of SCN5A variant function, which can be used to identify deleterious variants in SCN5A and other ion channel genes.

scholarly journals Selection of aptamers against triple negative breast cancer cells using high throughput sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Débora Ferreira ◽  
Joaquim Barbosa ◽  
Diana A. Sousa ◽  
Cátia Silva ◽  
Luís D. R. Melo ◽  
...  
Keyword(s):  
Breast Cancer ◽  
High Throughput ◽  
Triple Negative Breast Cancer ◽  
High Throughput Sequencing ◽  
Triple Negative ◽  
Metastatic Breast ◽  
Target Cells ◽  
Cancer Tissue ◽  
Surface Receptors

AbstractTriple-negative breast cancer is the most aggressive subtype of invasive breast cancer with a poor prognosis and no approved targeted therapy. Hence, the identification of new and specific ligands is essential to develop novel targeted therapies. In this study, we aimed to identify new aptamers that bind to highly metastatic breast cancer MDA-MB-231 cells using the cell-SELEX technology aided by high throughput sequencing. After 8 cycles of selection, the aptamer pool was sequenced and the 25 most frequent sequences were aligned for homology within their variable core region, plotted according to their free energy and the key nucleotides possibly involved in the target binding site were analyzed. Two aptamer candidates, Apt1 and Apt2, binding specifically to the target cells with $$K_{d}$$ K d values of 44.3 ± 13.3 nM and 17.7 ± 2.7 nM, respectively, were further validated. The binding analysis clearly showed their specificity to MDA-MB-231 cells and suggested the targeting of cell surface receptors. Additionally, Apt2 revealed no toxicity in vitro and showed potential translational application due to its affinity to breast cancer tissue sections. Overall, the results suggest that Apt2 is a promising candidate to be used in triple-negative breast cancer treatment and/or diagnosis.

scholarly journals Loss-of-function and missense variants in NSD2 cause decreased methylation activity and are associated with a distinct developmental phenotype

2021 ◽  
Author(s):  
Paolo Zanoni ◽  
Katharina Steindl ◽  
Deepanwita Sengupta ◽  
Pascal Joset ◽  
Angela Bahr ◽  
...  
Keyword(s):  
Loss Of Function ◽  
Mild Phenotype ◽  
Psychological Issues ◽  
Missense Variants ◽  
Mild Developmental Delay ◽  
Pathogenic Variants ◽  
In Silico Modeling ◽  
And Function ◽  
Methylation Activity

Abstract Purpose Despite a few recent reports of patients harboring truncating variants in NSD2, a gene considered critical for the Wolf–Hirschhorn syndrome (WHS) phenotype, the clinical spectrum associated with NSD2 pathogenic variants remains poorly understood. Methods We collected a comprehensive series of 18 unpublished patients carrying heterozygous missense, elongating, or truncating NSD2 variants; compared their clinical data to the typical WHS phenotype after pooling them with ten previously described patients; and assessed the underlying molecular mechanism by structural modeling and measuring methylation activity in vitro. Results The core NSD2-associated phenotype includes mostly mild developmental delay, prenatal-onset growth retardation, low body mass index, and characteristic facial features distinct from WHS. Patients carrying missense variants were significantly taller and had more frequent behavioral/psychological issues compared with those harboring truncating variants. Structural in silico modeling suggested interference with NSD2’s folding and function for all missense variants in known structures. In vitro testing showed reduced methylation activity and failure to reconstitute H3K36me2 in NSD2 knockout cells for most missense variants. Conclusion NSD2 loss-of-function variants lead to a distinct, rather mild phenotype partially overlapping with WHS. To avoid confusion for patients, NSD2 deficiency may be named Rauch–Steindl syndrome after the delineators of this phenotype.

scholarly journals Application of High-Throughput Sequencing in the Diagnosis of Inherited Thrombocytopenia

2018 ◽  
Vol 24 (9_suppl) ◽  
pp. 94S-103S ◽  
Author(s):  
Qi Wang ◽  
Lijuan Cao ◽  
Guangying Sheng ◽  
Hongjie Shen ◽  
Jing Ling ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Gene Sequencing ◽  
Hereditary Diseases ◽  
Sequencing Platform ◽  
Pathogenic Variants ◽  
Diagnosis Method ◽  
Inherited Thrombocytopenia ◽  
Thrombotic Diseases ◽  
Next Generation Sequencing Ngs

Inherited thrombocytopenia is a group of hereditary diseases with a reduction in platelet count as the main clinical manifestation. Clinically, there is an urgent need for a convenient and rapid diagnosis method. We introduced a high-throughput, next-generation sequencing (NGS) platform into the routine diagnosis of patients with unexplained thrombocytopenia and analyzed the gene sequencing results to evaluate the value of NGS technology in the screening and diagnosis of inherited thrombocytopenia. From a cohort of 112 patients with thrombocytopenia, we screened 43 patients with hereditary features. For the blood samples of these 43 patients, a gene sequencing platform for hemorrhagic and thrombotic diseases comprising 89 genes was used to perform gene detection using NGS technology. When we combined the screening results with clinical features and other findings, 15 (34.9%) of 43patients were diagnosed with inherited thrombocytopenia. In addition, 19 pathogenic variants, including 8 previously unreported variants, were identified in these patients. Through the use of this detection platform, we expect to establish a more effective diagnostic approach to such disorders.

scholarly journals CDK10 Regulates Gastric Cancer Metastasis By Inhibiting lncRNA-C5ORF42-5 Via Phosphorylation Level of AKT/ERK

2021 ◽  
Author(s):  
Zi-Jian Deng ◽  
Dong-Wen Chen ◽  
Xi-Jie Chen ◽  
Jia-Ming Fang ◽  
Liang Xv ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cancer Cells ◽  
Cell Line ◽  
High Throughput ◽  
Cancer Metastasis ◽  
High Throughput Sequencing ◽  
Gastric Cancer Cells ◽  
Related Proteins

Abstract Background: Gastric cancer is the fourth most common malignant disease. Both CDK10 and long noncoding RNAs (lncRNAs) have been found to exert biological functions in multiple cancers. However, it is still unclear whether CDK10 represses tumor progression in gastric cancer by reducing potential targeting lncRNAs.Methods: The functions of CDK10 and lncRNA-C5ORF42-5 in proliferation, invasion and migration were assessed by MTS assays, colony formation assays, cell cycle and apoptosis assays, Transwell assays, wound healing assays and animal experiments. We used high-throughput sequencing to confirm the existence of lncRNA-C5ORF42-5 and quantitative real-time PCR was used to evaluate lncRNA expression. Then, with RNA-seq sequencing as well as GO function and KEGG enrichment analysis, we identified the signaling pathways in which lncRNA-C5ORF42-5 was involved in gastric cancer. Finally, western blotting was used to identify the genes regulated by lncRNA-C5ORF42-5.Results: Our results showed that CDK10 is expressed at relatively low levels in gastric cancer cell lines and inhibits the progression of gastric cancer cells both in vitro and in vivo. Next, based on high-throughput sequencing, we identified a novel lncRNA, lncRNA-C5ORF42-5, in the stable CDK10-overexpressing cell line compared with the CDK-knockdown cell line and their controls. Additionally, we confirmed that lncRNA-C5ORF42-5 acts as an oncogene to promote metastasis in gastric cancer in vitro and in vivo. We then ascertained that lncRNA-C5ORF42-5 is a major contributor to the function of CDK10 in gastric cancer metastasis by upregulating lncRNA-C5ORF42-5 to reverse the effects of CDK10 overexpression. Finally, we explored the mechanism by which lncRNA-C5ORF42-5 overexpression affects gastric cancer cells to elucidate whether lncRNA-C5ORF42-5 may increase the activity of the SMAD pathway of BMP signaling and promote the expression of EMT-related proteins, such as E-cadherin. Additionally, overexpression of lncRNA-C5ORF42-5 affected the phosphorylation levels of AKT and ERK.Conclusion: Our findings suggest that CDK10 overexpression represses gastric cancer tumor progression by reducing lncRNA-C5ORF42-5 and hindering activation of the related proteins in metastatic signaling pathways, which provides new insight into developing effective therapeutic strategies in the treatment of metastatic gastric cancer.

CTNND1 to mediate bone metastasis of triple-negative breast cancer via regulating CXCR4.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e13045-e13045
Author(s):  
Chang Gong ◽  
Qun Lin ◽  
Xiaolin Fang ◽  
Wenguo Jiang ◽  
Jun Li ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Bone Metastasis ◽  
High Throughput ◽  
Triple Negative Breast Cancer ◽  
High Throughput Sequencing ◽  
Triple Negative ◽  
Mtor Pathway ◽  
Tissue Samples

e13045 Background: Compared to lumial breast cancer, the proporation of triple-negative breast cancer (TNBC) with bone metastases (BMs) is relatively low and few data focusing on the mechanism of the BMs in TNBC are available, Here, we screened that CTNND1 was associated with BMs of TNBC by integrating high-throughput sequencing, and further investigated the role of CTNND1 in BMs of TNBC in vitro. Methods: TNBC tissue samples with only BMs (n = 6) and without any metastasis (n = 10) were tested using high-throughput sequencing and 11 differentially expressed relative genes were identified. We then quantified these 11 genes in normal breast tissue samples (n = 26), TNBC tissue samples with only BMs (n = 10), TNBC tissue samples without any metastasis (n = 88) as well as luminal tissue samples with BMs（n = 10）through qPCR and immunohistochemical staining (IHC). The effects of knocking down CTNND1 on the interaction between TNBC cells and osteoblasts were examined by cell adhesion, transwell migration and matrigel invasion assays. To explorethe role of CTNND1 in mediating bone metastasis in TNBC, we used RNA-sequencing to find out the relative downstream gene CXCR4 and PI3K-AKT-mTOR pathway and verified it in vitro by Western Blotting. Results: Combining our high-throughput sequencing data, qPCR and IHC in clinical tissue samples, we verified that CTNND1 was decreased in TNBC patients with bone metastasis compared to normal tissue and luminal tissue with BMs. Knocking down of CTNND1 in TNBC cells including MDA-MB-231, MDA-MB-468 and BT549 weakened cells adhesion, but facilitated cells migration and invasion. Mechanically, knocking down of CTNND1 upregulated CXCR4 via activating PI3K-AKT-mTOR pathway in TNBC but not luminal and HER2- positive breast cancer cells lines. Conclusions: CTNND1 mediates bone metastasis in triple-negative breast cancer via regulating CXCR4.CTNND1 may serve as a potential predictor of bone metastasis for TNBC patients.

scholarly journals T cell neoepitope discovery in colorectal cancer by high throughput profiling of somatic mutations in expressed genes

Gut ◽  
2015 ◽  
Vol 66 (3) ◽  
pp. 454-463 ◽  
Author(s):  
Daniele Mennonna ◽  
Cristina Maccalli ◽  
Michele C Romano ◽  
Claudio Garavaglia ◽  
Filippo Capocefalo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Cell Epitope ◽  
Patient Specific ◽  
Cancer Genes ◽  
Healthy Donors

ObjectivePatient-specific (unique) tumour antigens, encoded by somatically mutated cancer genes, generate neoepitopes that are implicated in the induction of tumour-controlling T cell responses. Recent advancements in massive DNA sequencing combined with robust T cell epitope predictions have allowed their systematic identification in several malignancies.DesignWe undertook the identification of unique neoepitopes in colorectal cancers (CRCs) by using high-throughput sequencing of cDNAs expressed by standard cancer cell cultures, and by related cancer stem/initiating cells (CSCs) cultures, coupled with a reverse immunology approach not requiring human leukocyte antigen (HLA) allele-specific epitope predictions.ResultsSeveral unique mutated antigens of CRC, shared by standard cancer and related CSC cultures, were identified by this strategy. CD8+and CD4+T cells, either autologous to the patient or derived from HLA-matched healthy donors, were readily expanded in vitro by peptides spanning different cancer mutations and specifically recognised differentiated cancer cells and CSC cultures, expressing the mutations. Neoepitope-specific CD8+T cell frequency was also increased in a patient, compared with healthy donors, supporting the occurrence of clonal expansion in vivo.ConclusionsThese results provide a proof-of-concept approach for the identification of unique neoepitopes that are immunogenic in patients with CRC and can also target T cells against the most aggressive CSC component.

scholarly journals Elucidation of Insertion Elements Carried on Plasmids andIn VitroConstruction of Shuttle Vectors from the Toxic Cyanobacterium Planktothrix

2014 ◽  
Vol 80 (16) ◽  
pp. 4887-4897 ◽  
Author(s):  
Guntram Christiansen ◽  
Alexander Goesmann ◽  
Rainer Kurmayer
Keyword(s):  
Homologous Recombination ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Gene Clusters ◽  
Content Type ◽  
Shuttle Vectors ◽  
Is Element ◽  
Toxic Cyanobacterium ◽  
Is Elements

ABSTRACTSeveral gene clusters that are responsible for toxin synthesis in bloom-forming cyanobacteria have been found to be associated with transposable elements (TEs). In particular, insertion sequence (IS) elements were shown to play a role in the inactivation or recombination of the genes responsible for cyanotoxin synthesis. Plasmids have been considered important vectors of IS element distribution to the host. In this study, we aimed to elucidate the IS elements propagated on the plasmids and the chromosome of the toxic cyanobacteriumPlanktothrix agardhiiNIVA-CYA126/8 by means of high-throughput sequencing. In total, five plasmids (pPA5.5, pPA14, pPA50, pPA79, and pPA115, of 5, 6, 50, 79, and 120 kbp, respectively) were elucidated, and two plasmids (pPA5.5, pPA115) were found to propagate full IS element copies. Large stretches of shared DNA information between plasmids were constituted of TEs. Two plasmids (pPA5.5, pPA14) were used as candidates to engineer shuttle vectors (named pPA5.5SV and pPA14SV, respectively)in vitroby PCR amplification and the subsequent transposition of the Tn5 cattransposon containing the R6Kγ origin of replication ofEscherichia coli. While pPA5.5SV was found to be fully segregated, pPA14SV consistently co-occurred with its wild-type plasmid even under the highest selective pressure. Interestingly, the Tn5 cattransposon became transferred by homologous recombination into another plasmid, pPA50. The availability of shuttle vectors is considered to be of relevance in investigating genome plasticity as a consequence of homologous recombination events. Combining the potential of high-throughput sequencing andin vitroproduction of shuttle vectors makes it simple to produce species-specific shuttle vectors for many cultivable prokaryotes.

scholarly journals Implementation of High-Throughput Sequencing (HTS) in Aptamer Selection Technology

2020 ◽  
Vol 21 (22) ◽  
pp. 8774
Author(s):  
Natalia Komarova ◽  
Daria Barkova ◽  
Alexander Kuznetsov
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Combinatorial Libraries ◽  
Structure And Function ◽  
Huge Number ◽  
Systematic Evolution ◽  
And Function ◽  
Exponential Enrichment

Aptamers are nucleic acid ligands that bind specifically to a target of interest. Aptamers have gained in popularity due to their high potential for different applications in analysis, diagnostics, and therapeutics. The procedure called systematic evolution of ligands by exponential enrichment (SELEX) is used for aptamer isolation from large nucleic acid combinatorial libraries. The huge number of unique sequences implemented in the in vitro evolution in the SELEX process imposes the necessity of performing extensive sequencing of the selected nucleic acid pools. High-throughput sequencing (HTS) meets this demand of SELEX. Analysis of the data obtained from sequencing of the libraries produced during and after aptamer isolation provides an informative basis for precise aptamer identification and for examining the structure and function of nucleic acid ligands. This review discusses the technical aspects and the potential of the integration of HTS with SELEX.

scholarly journals Cardiac Sodium Channel Dysfunction and Dilated Cardiomyopathy: A Contemporary Reappraisal of Pathophysiological Concepts

2019 ◽  
Vol 8 (7) ◽  
pp. 1029 ◽  
Author(s):  
Asatryan
Keyword(s):  
Dilated Cardiomyopathy ◽  
Sodium Channel ◽  
Ventricular Remodeling ◽  
Left Ventricular Remodeling ◽  
Left Ventricular ◽  
Conduction Disturbances ◽  
Pathogenic Variants ◽  
Cardiac Sodium Channel ◽  
Precision Therapy ◽  
Bona Fide

A key emerging theme in translational cardiovascular medicine is the need to identify specific causes of arrhythmias and heart failure, defined by phenotype and/or genotype that will respond to a particular intervention. Unlike other genes implicated in hereditary arrhythmias and cardiomyopathies, pathogenic/likely pathogenic variants in the cardiac sodium channel alpha subunit gene (SCN5A) produce a remarkably diverse set of electrical and structural phenotypes, one of them being dilated cardiomyopathy. There has been debate about whether left ventricular remodeling is a bona fide phenotypic feature of cardiac sodium channel dysfunction, or a consequence of tachyarrhythmias or conduction disturbances. In light of recent findings, a critical digest of the available experimental and medical literature is necessary. This paper provides a critical appraisal of the evidence linking a dysfunctional cardiac sodium channel to ventricular dysfunction, and discusses the potential mechanisms involved in shaping this phenotype along with implications for precision therapy.

scholarly journals Molecular Fitness Landscapes from High-Coverage Sequence Profiling

2019 ◽  
Vol 48 (1) ◽  
pp. 1-18 ◽  
Author(s):  
Celia Blanco ◽  
Evan Janzen ◽  
Abe Pressman ◽  
Ranajay Saha ◽  
Irene A. Chen
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Fitness Landscape ◽  
Complete Sequence ◽  
Fitness Landscapes ◽  
Future Research ◽  
High Coverage

The function of fitness (or molecular activity) in the space of all possible sequences is known as the fitness landscape. Evolution is a random walk on the fitness landscape, with a bias toward climbing hills. Mapping the topography of real fitness landscapes is fundamental to understanding evolution, but previous efforts were hampered by the difficulty of obtaining large, quantitative data sets. The accessibility of high-throughput sequencing (HTS) has transformed this study, enabling large-scale enumeration of fitness for many mutants and even complete sequence spaces in some cases. We review the progress of high-throughput studies in mapping molecular fitness landscapes, both in vitro and in vivo, as well as opportunities for future research. Such studies are rapidly growing in number. HTS is expected to have a profound effect on the understanding of real molecular fitness landscapes.

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