Cell Line DNA Research Articles

Phytosphingosine inhibits cell proliferation by damaging DNA in human cell lines

Harmful cyanobacterial blooms have caused numerous biosecurity incidents owing to the production of hazardous secondary metabolites such as microcystin. Additionally, cyanobacteria also release many other components that have not been explored. We identified compounds of a toxic mixture exudated from a dominant, blooming species, Microcystis aeruginosa, and found that phytosphingosine (PHS) was one of the bioactive components. Since PHS exhibited toxicity and is deemed a hazardous substance by the European Chemicals Agency, we hypothesized that PHS is a potentially toxic compound in M. aeruginosa exudates. However, the mechanisms of PHS ecotoxicity remain unclear. We assessed the cytotoxicity of PHS using an in vitro cell model in eight human cell lines and observed that the nasopharyngeal carcinoma cell line CNE2 was the most sensitive. We exposed CNE2 cells to 0–25 µmol/L PHS for 24 hr to explore its toxicity and mechanism. PHS exposure resulted in abnormal nuclear morphology, micronuclei, and DNA damage. Moreover, PHS significantly inhibited cell proliferation and arrested cell cycle at S phase. The results of Western blot suggested that PHS increased the expression of DNA damage-related proteins (ATM, p-P53 and P21) and decreased the expression of S phase-related proteins (CDK2, CyclinA2 and CyclinE1), indicating the toxicological mechanism of PHS on CNE2 cells. These data provide evidence that PHS has genetic toxicity and inhibits cell proliferation by damaging DNA. Our study provides evidence that PHS inhibits cell proliferation by damaging DNA. While additional work is required, we propose that PHS been considered as a potentially toxic component in MaE in addition to other well-characterized secondary compounds.

Variation of extrachromosomal circular DNA in cancer cell lines

The presence of oncogene carrying eccDNAs is strongly associated with carcinogenesis and poor patient survival. Tumour biopsies and in vitro cancer cell lines are frequently utilized as models to investigate the role of eccDNA in cancer. However, eccDNAs are often lost during the in vitro growth of cancer cell lines, questioning the reproducibility of studies utilizing cancer cell line models. Here, we conducted a comprehensive analysis of eccDNA variability in seven cancer cell lines (MCA3D, PDV, HaCa4, CarC, MIA-PaCa-2, AsPC-1, and PC-3). We compared the content of unique eccDNAs between triplicates of each cell line and found that the number of unique eccDNA is specific to each cell line, while the eccDNA sequence content varied greatly among triplicates (∼ 0–1% eccDNA coordinate commonality). In the PC-3 cell line, we found that the large eccDNA (ecDNA) with MYC is present in high-copy number in an NCI cell line isolate but not present in ATCC isolates. Together, these results reveal that the sequence content of eccDNA is highly variable in cancer cell lines. This highlights the importance of testing cancer cell lines before use, and to enrich for subclones in cell lines with the desired eccDNA to get relatively pure population for studying the role of eccDNA in cancer.

Systematic multi-omics cell line profiling uncovers principles of Ewing sarcoma fusion oncogene-mediated gene regulation.

Ewing sarcoma (EwS) is characterized by EWSR1-ETS fusion transcription factors converting polymorphic GGAA microsatellites (mSats) into potent neo-enhancers. Although the paucity of additional mutations makes EwS a genuine model to study principles of cooperation between dominant fusion oncogenes and neo-enhancers, this is impeded by the limited number of well-characterized models. Here we present the Ewing Sarcoma Cell Line Atlas (ESCLA), comprising whole-genome, DNA methylation, transcriptome, proteome, and chromatin immunoprecipitation sequencing (ChIP-seq) data of 18 cell lines with inducible EWSR1-ETS knockdown. The ESCLA shows hundreds of EWSR1-ETS-targets, the nature of EWSR1-ETS-preferred GGAA mSats, and putative indirect modes of EWSR1-ETS-mediated gene regulation, converging in the duality of a specific but plastic EwS signature. We identify heterogeneously regulated EWSR1-ETS-targets as potential prognostic EwS biomarkers. Our freely available ESCLA (http://r2platform.com/escla/) is a rich resource for EwS research and highlights the power of comprehensive datasets to unravel principles of heterogeneous gene regulation by chimeric transcription factors.

Methylome Analysis of Epstein-Barr Virus with Nanopore Sequencing Reveals Viral State Dynamics

Epstein Barr Virus (EBV) is a gammaherpesvirus responsible for a broad spectrum of human diseases, including hematologic malignancies and autoimmune disorders. EBV maintains lifelong infection by entering an endosomal state of latency inside its host cell. In response to external stimuli EBV undergoes lytic activation, resulting in vast gene expression changes, DNA replication, and eventually release of infectious viral particles. DNA methylation of the EBV genome is essential to regulating its life cycle. Both lytic and latent life stages are key determinants of EBV-driven disease, but current clinical assays for EBV are limited to detecting the presence of EBV and do not provide important information on EBV lytic state. Thus, making clinical decisions based on EBV detection alone has remained challenging. To address these limitations, we have developed an EBV analysis pipeline utilizing nanopore sequencing from amplification-free, native DNA that directly interrogates CpG dinucleotide DNA methylation on the EBV genome. Our workflow generates highly accurate EBV methylation calls for the majority of CpGs across the EBV genome. We also deployed a quantitative mass-spectrometry-based methylation assay (EpiTYPER) to validate our data and provide broader analysis of a representative subset of methylation sites (n=123 methylation sites from n=25 EBV loci). To initially investigate the relationship between locus-specific EBV DNA methylation and EBV gene expression, we compared DNA methylation and RNA sequencing profiles in EBV-positive cell lines (n=7). We correlated global EBV DNA methylation levels and the overall patterns of latent versus lytic EBV gene expression. We found that average methylation levels are related to the ratio of latent versus lytic gene expression, establishing nanopore-derived DNA methylation as a method to quantify latent and lytic states in a given sample. We next shifted our approach to bin nanopore reads based on the average methylation of all CpGs per read, hypothesizing that latent/lytic viral states derive from distinct subpopulations of EBV endosomes that contain methylated versus unmethylated DNA. We uncovered widespread co-occurrence of high and low methylated viral DNA in cell lines, indicating that EBV methylation states exist in defined subfractions with either present or absent DNA methylation. In cell models of latent EBV infection (Akata, Kem, MEC1, Mutu, Rael) this approach showed that methylation states dynamically responded to lytic induction and selective pressure via antiviral treatment with ganciclovir. Of note, we observed a shift in methylation without the appearance of partially methylated DNA, indicating that EBV lytic activation/replication is associated with the appearance of unmethylated EBV DNA, and not gradual demethylation of endosomes in the latent viral state. Finally, we aimed to obtain measurements of overall EBV status in patients, using nanopore sequencing of cell-free DNA (cfDNA) obtained from patient plasma. We analyzed patients diagnosed with different EBV-positive diseases from The Ohio State University Medical Center, including n=3 patients with lymphomas (Burkitt, plasmablastic and diffuse large B cell lymphoma) and n=2 patients with post-transplant lymphoproliferative disorder (PTLD). The patient samples with lymphoma displayed a near uniformly high methylation profile, with little evidence of unmethylated DNA/lytic replication. One of the PTLD patients analyzed showed a distinct bi-modal distribution of per read EBV methylation values, indicating the presence of lytic replication in a significant proportion of cells. In conclusion, nanopore sequencing analysis of methylated versus unmethylated EBV DNA subfractions is a quantitative method for determining viral states. In patient samples, nanopore sequencing revealed methylation heterogeneity in cfDNA and may be a useful advancement compared to standard clinical EBV detection. Quantifying the relative ratio of latent versus lytic virus in a liquid biopsy sample could be used to guide therapy selection for patients displaying EBV-positivity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Downregulation of the Mutated WT1 Alleles in Malignant Hematopoietic Cells Via Nonsense-Mediated mRNA Decay Contradicts Their Prognostic Impact

Background The Wilms Tumor 1 (WT1) gene is highly expressed in almost all types of malignancies including acute myeloid leukemia (AML). It is also mutated in up to 20% of AML blasts in pediatric and adult patients. Both, the increased expression and also the presence of mutations are correlated negatively with chance of survival in different AML patient cohorts. We and others have previously showed devastating survival outcomes in children with AML blasts harboring WT1 mutations in combination FLT3-ITD and NUP98-NSD1 (Niktoreh et al., 2019). As WT1 can act as both, oncogene and tumor suppressor gene, this study was designed to analyze the WT1 genomic locus mutations in acute leukemia blasts. Methods and Results Screening for WT1 sequence alterations. Using the TruSight Myeloid Sequencing PanelTM (Illumina), we initially screened 35 leukemia and lymphoma cell lines for genomic alterations in 54 genes. This panel allowed us to detect even low variant-allele-frequency (VAF) alterations in the mutational hotspots of WT1, exons 7 and 9. In addition to a known WT1 mutation in CTS cells (AML M1), we discovered novel alterations in three other malignant cell lines. KASUMI-6 cells (AML M2) harbored the identical mutation as CTS cells, a 2-bp deletion in WT1 exon 7 leading to frameshift with a premature termination codon (c.1202_1203delGA, p.Arg401IlefsTer3). The U937 histiocytic lymphoma cells carried the known TP53 mutation and also an unknown heterozygous nonsense mutation in WT1 exon 7, changing the arginine 369 into a premature stop codon (c.1105C>T, p.Arg369Ter). Interestingly, MOLM-1 cells (CML in blast crisis) harbored a p.Arg462Gln alteration. Next, we performed Sanger sequencing for the complete coding region of WT1, including the upstream promoter as well as the 5'UTR and 3'UTR regions, all 10 exons and the adjacent 150 bp intronic sequences at the exon-intron boundaries. Sanger sequencing confirmed the mutations discovered via NGS and revealed single bp alterations in the WT1 promoter region of CTS cells and in the 5'UTR of K-562 cells. We also detected a recurring variation in intron 3 of WT1 (c.872+16G>A) in the cell lines K-562, NB-4, M07e and UOC-M1; the functional implications of the alterations are yet to be determined. Functional impact of WT1 mutations. To understand the potential impact of the WT1 sequence alterations, we studied the WT1 mRNA expression in these cells in the presence of cycloheximide (CHX), an inhibitor of the nonsense-mediated mRNA decay (NMD) machinery. As shown in figure 1A, U937 cells were used as negative control cells, as they are known to have no expression of the WT1 mRNA. Despite the presence of mutations in CTS, KASUMI-6 and MOLM-1 cells, all three cell lines expressed elevated levels of WT1, well within the range of 30 other malignant cell lines without any alterations/mutations in the WT1 locus (data not shown). To differentiate the WT1 expression between the mutated and the wildtype allele, we performed a fragment length analysis on a capillary electrophoresis system using the complimentary DNA of CHX-treated cell lines. For these analyses, we employed a multiplex PCR strategy using ABL1 as housekeeping gene and a primer pair binding in exons 4 and 10 of WT1. As delineated in figure 1B, the mutated alleles in CTS and KASUMI-6 cells, which are 2-bps shorter, could be easily separated from the wildtypes (data for KASUMI-6 not shown). Additionally, since our primer pair also amplified the alternatively spliced exon 5 and the alternative splice donor at the end of exon 9, leading to inclusion or exclusion of three amino acids (KTS), we were able to separately analyze the four major WT1 isoforms from both alleles. As shown in figure 1B, the expression levels of the WT1 mutated alleles of CHX-treated cells increased 2- to 3-fold compared to DMSO-treated cells in CTS and 3- to 4-fold in KASUMI-6 (data not shown). Conclusion Careful analyses of the WT1 genomic locus in 35 malignant hematological cell lines revealed previously unknown heterozygous loss-of-function WT1 mutations in KASUMI-6, MOLM-1 and U-937 cells. mRNA expression analysis demonstrated that these mutations are strongly regulated by NMD machinery, leading to (almost) complete downregulation of the mutated WT1 allele. These results strongly question any direct effect of WT1 mutations in the leukemic blasts and therefore cannot explain the role of mutations in WT1 as a prognostic factor in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CRISPR and iPSCs: Recent Developments and Future Perspectives in Neurodegenerative Disease Modelling, Research, and Therapeutics.

Neurodegenerative diseases are prominent causes of pain, suffering, and death worldwide. Traditional approaches modelling neurodegenerative diseases are deficient, and therefore, improved strategies that effectively recapitulate the pathophysiological conditions of neurodegenerative diseases are the need of the hour. The generation of human-induced pluripotent stem cells (iPSCs) has transformed our ability to model neurodegenerative diseases in vitro and provide an unlimited source of cells (including desired neuronal cell types) for cell replacement therapy. Recently, CRISPR/Cas9-based genome editing has also been gaining popularity because of the flexibility they provide to generate and ablate disease phenotypes. In addition, the recent advancements in CRISPR/Cas9 technology enables researchers to seamlessly target and introduce precise modifications in the genomic DNA of different human cell lines, including iPSCs. CRISPR-iPSC-based disease modelling, therefore, allows scientists to recapitulate the pathological aspects of most neurodegenerative processes and investigate the role of pathological gene variants in healthy non-patient cell lines. This review outlines how iPSCs, CRISPR/Cas9, and CRISPR-iPSC-based approaches accelerate research on neurodegenerative diseases and take us closer to a cure for neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Amyotrophic Lateral Sclerosis, and so forth.

Abstract 539: Development of a novel 5-plex copy number ddPCR assay for ctDNA analysis

Abstract Background: Analysis of cell-free circulating tumor DNA (ctDNA) from blood provides an opportunity to detect potential mechanisms of resistance to targeted therapies in melanoma patients. Approximately 20% and 17% of patients undergoing BRAF targeted therapy develop resistance associated with copy number increases in either BRAF or cylinD1 (CCND1), respectively. Detection of BRAF or CCND1 copy number gains prior to radiographic tumor progression could potentially prompt a therapeutic switch aimed at treating patients with smaller tumors burdens, which may improve survival. Conventional droplet digital PCR (ddPCR) for copy number analysis uses two probes, one target and one reference gene (2-plex). To improve the accuracy and sensitivity of the copy number analysis and account for possible genetic alterations in the reference gene, we performed a 5-probe multiplex (5-plex) reaction using three reference genes and two targets. Methods: We used cell lines with known copy number gains for the BRAF gene (M395-R) and CCND1 gene (SkMel-187) to identify copy number increases using ddPCR. To estimate the limit of detection of the copy number gains in plasma, high and low concentrations of the cell line DNAs were spiked into healthy plasma and divided into three replicate samples of 3ml each. DNA was extracted from plasma using the Circulating DSP NA Kit (Qiagen). The ddPCR reaction mix used 4X Supermix and five different copy number specific probes targeting: two genes of interest, BRAF (7q34) and CCND1 (11q13.3); and three control genes: FOXI3 (2p11.2), AGO1 (1p34.3) and TTC5 (14q11.2). Each probe was titrated to display a unique cluster location on the 2D plot. Probe concentrations were: BRAF: FAM 2X; CCND1: FAM 1X; FOXI3: HEX 2X; AGO1: HEX 1X; TTC5: FAM 0.7X/HEX 0.4X. Each extraction was divided into 4 replicate wells. Following droplet generation, gene copies were analyzed using the QX Manager 1.2 Standard Edition for advanced analysis. Results: Probe titration resulted in 32 quantifiably distinct ddPCR clusters on a single 2D plot. Copies/ul of the reference genes were used as a benchmark for diploid status. We determined that the M395-R cell line had 45 copies of the BRAF gene and that SKMel-187 had eight copies of the CCND1 gene compared to diploid PBMC DNA (Promega). Based on the spike-in experiments, we estimate that we can detect copy number gains in plasmas with 10% or 20% tumor fraction when tumor cells have approximately 45 or 8 copies of the target gene, respectively. Conclusion: We successfully multiplexed five copy number assays in a single ddPCR reaction, and showed that copy number changes could be identifiable with as little as 10% tumor fraction within plasma when the tumor-associated copy number gains were relatively high. Overall, the 5-plex assay has the potential to be excellent tool for copy number detection in blood, and can be customized as needed for other targets. Citation Format: Jennifer M. Wiggins, Mahrukh Syeda, David Polsky. Development of a novel 5-plex copy number ddPCR assay for ctDNA analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 539.

Lab validation of an ultrasensitive ctDNA pan-cancer MRD assay using whole-genome sequencing.

e13582 Background: Minimal residual disease (MRD) monitoring using liquid biopsy for solid tumors requires a highly sensitive and specific assay that can overcome the limitation of low abundance cfDNA in a standard blood draw. We developed a whole-genome sequencing (WGS)-based assay to detect the presence of circulating tumor DNA (ctDNA) in plasma. The C2i assay is a tumor-informed assay that uses personalized tumor signature, advanced noise models, and artificial intelligence (AI) modalities to interrogate plasma for the presence of ctDNA longitudinally. Methods: The C2i test was developed in accordance with CAP/CLIA and New York state validation principles. We used contrived samples to establish analytical validation of the assay performance, which was then validated with a large clinical cohort of early-stage patients across various cancer types. Briefly, aggregated tumor signatures derived from cancer cell lines were fragmented and spiked into a contrived healthy plasma pool; the mixed samples were used to assess the presence of tumor DNA signature down to tumor fractions of 10e-4. Positive samples are identified by tumor-derived variants detected above the noise levels. Noise modeling was established using a panel of normal (PON) approach. We assessed the analytical sensitivity, specificity, and accuracy using 348 contrived samples derived from five different cancer cell lines. Reproducibility and precision were assessed with multiple replicates, and statistical concordance was reported. This validation was complemented by a cohort of 200 patients and ̃1000 plasma samples across a variety of cancer types including, NSCLC, MIBC, CRC, GBM, Breast Cancer, and a mixture of other cancer types. Results: Cancer cell lines, representing the five most prevalent disease indications, used for determining analytical sensitivity are as follows: CRC HT-29, Breast SK-BR3, Bladder HT-1376, Lung HCI-H526, and Prostate LNCaP. The cell line DNA was enzymatically fragmented and size-selected to mimic ctDNA. This ctDNA was spiked into cfDNA extracted from healthy volunteers at various dilution levels, varying from 10e0 to 10e-4. The 95% probability of detecting ctDNA was established at 10e-4. The reproducibility of tumor signature between replicates was assessed to be greater than 90%. The assay was performed using both normal and maximum input amounts. These performance estimates were then validated on a cohort of plasma collected from early-stage (stage I-III) patients across various cancer types. Conclusions: C2i MRD test is an ultrasensitive pan-cancer MRD monitoring assay used in several clinical trials across the world. We present an extensive analytical and clinical validation of the assay supporting its high performance.

A high-throughput real-time PCR tissue-of-origin test to distinguish blood from lymphoblastoid cell line DNA for (epi)genomic studies

Lymphoblastoid cell lines (LCLs) derive from blood infected in vitro by Epstein–Barr virus and were used in several genetic, transcriptomic and epigenomic studies. Although few changes were shown between LCL and blood genotypes (SNPs) validating their use in genetics, more were highlighted for other genomic features and/or in their transcriptome and epigenome. This could render them less appropriate for these studies, notably when blood DNA could still be available. Here we developed a simple, high-throughput and cost-effective real-time PCR approach allowing to distinguish blood from LCL DNA samples based on the presence of EBV relative load and rearranged T-cell receptors γ and β. Our approach was able to achieve 98.5% sensitivity and 100% specificity on DNA of known origin (458 blood and 316 LCL DNA). It was further applied to 1957 DNA samples from the CEPH Aging cohort comprising DNA of uncertain origin, identifying 784 blood and 1016 LCL DNA. A subset of these DNA was further analyzed with an epigenetic clock indicating that DNA extracted from blood should be preferred to LCL for DNA methylation-based age prediction analysis. Our approach could thereby be a powerful tool to ascertain the origin of DNA in old collections prior to (epi)genomic studies.

Abstract P5-13-29: Analytical and clinical validation of a ctDNA assay for detecting copy number loss and structural rearrangement variants contributing to homologous recombination and repair (HRR) deficiency

Abstract Background: Inactivating HRR gene mutations can lead to HRR deficiency (HRD) and predict response to PARPi therapy in patients with breast cancer. Copy number loss and large genomic rearrangements (LGR) can result in HRD but are challenging to detect in ctDNA. Here, we present the analytical validation of homozygous deletions, loss of heterozygosity (LoH) and LGR detection on the Guardant360 (G360) liquid biopsy panel, previously validated for detection of small variants, copy number amplifications, and fusions. We present real-world outcomes of BRCA1/2-mutant PARPi-treated patients to demonstrate the clinical validity of the detected variants. Methods: Analytical validation was performed using the G360 83-gene ctDNA panel. Cell line DNA and clinical patient cfDNA were titrated into matched normal cell line DNA or healthy donor cfDNA to establish the limit of detection (LoD) and precision for copy number loss and LGRs, respectively. Accuracy results were compared to those from an orthogonal, externally validated tissue and ctDNA panel. De-identified, longitudinal, claims data were linked to the cancer genomic profiles in Guardant INFORM, a clinical-genomics database. Advanced PARPi treated breast cancer patients with an inactivating or reversion BRCA1/2 mutation detected by G360 were assessed. Results: The analytical sensitivity (95% LoD) for detecting homozygous and LoH deletions for deletion sizes >10MB was established at tumor fractions (TF) of 12.5% and 25%, respectively. The 95% LoD for LGRs was 0.2% variant allele fraction (VAF). The per-sample false positive rate for copy number loss and LGRs was <0.5%. Prevalence of BRCA1/2 homozygous deletions, LoH and LGRs in >1000 advanced breast cancer patients was 1.8%, 16.6% and 0.25% respectively, compared to 2.4%, 56.7% and 0.3% in TCGA. To verify the clinical impact of cfDNA-detected HRR alterations, overall survival was determined for PARPi-treated patients with >1 BRCA1/2 germline or somatic SNV, indel or LGR reversion mutation to be 23.2 months [16.4, 30, CI, n=75] compared to 54.4 [28, NA, CI, n=14] months for BRCA1/2-mutant patients without a reversion (p-value=0.049). Conclusion:. This analytical validation demonstrates that G360 detection of inactivating mutations, including copy number loss and LGRs, is highly sensitive, reliable and robust. Real-world evidence analysis confirmed worse survival outcomes in PARPi treated patients harboring a BRCA1/2 reversion compared to BRCA1/2-mutant patients with no reversion. This data further supports ctDNA as a compelling non-invasive means to identify potential PARPi sensitizing and resistance mutations in patients with advanced breast cancer. Citation Format: Jennifer Yen, Leylah Drusbosky, Caroline Weipert, Nicole Zhang, David Hanna, Catalin Barbacioru, Hao Wang, Alex Artyomenko, Arielle Yablonovitch, Yu Fu, Aaron Hardin, Nagesh Alla, Robert Foley, Max Maligska, Bhargavi Panchangam, Phil Yen, Jane Meisel, Neelima Vidula, Massimo Cristofanilli, Jeremy Force, Michael Dorschner, Martina Lefterova, Elena Helman, Becky Nagy, Darya Chudova, AmirAli Talasaz. Analytical and clinical validation of a ctDNA assay for detecting copy number loss and structural rearrangement variants contributing to homologous recombination and repair (HRR) deficiency [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-29.

Evaluation of a D-Octaarginine-linked polymer as a transfection tool for transient and stable transgene expression in human and murine cell lines.

Poly(N-vinylacetamide-co-acrylic acid) coupled with d-octaarginine (VP-R8) promotes the cellular uptake of peptides/proteins in vitro; however, details of the transfection efficacy of VP-R8, such as the cell types possessing high gene transfer, are not known. Herein, we compared the ability of VP-R8 to induce the cellular uptake of plasmid DNA in mouse and human cell lines from different tissues and organs. A green fluorescent protein (GFP)-expression plasmid was used as model genetic material, and fluorescence as an indicator of uptake and plasmid-derived protein expression. Three mouse and three human cell lines were incubated with a mixture of plasmid and VP-R8, and fluorescence analysis were performed two days after transfection. To confirm stable transgene expression, we performed drug selection three days after transfection. A commercially available polymer-based DNA transfection reagent (PTR) was used as the transfection control and standard for comparing transgene expression efficiency. In the case of transient transgene expression, slight-to-moderate GFP expression was observed in all cell lines transfected with plasmid via VP-R8; however, transfection efficiency was lower than using the PTR for gene delivery. In the case of stable transgene expression, VP-R8 promoted drug-resistance acquisition more efficiently than the PTR did. Cells that developed drug resistance after VP-R8-mediated gene transfection expressed GFP more efficiently than cells that developed drug resistance after transfection with the PTR. Thus, VP-R8 shows potential as an in vitro or ex vivo nonviral transfection tool for generating cell lines with stable transgene expression.

Virtual-Partition Digital PCR for High-Precision Chromosomal Counting Applications.

Digital PCR (dPCR) is the gold-standard analytical platform for rapid high-precision quantification of genomic fragments. However, current dPCR assays are generally limited to monitoring 1-2 analytes per sample, thereby limiting the platform's ability to address some clinical applications that require the simultaneous monitoring of 20-50 analytes per sample. Here, we present virtual-partition dPCR (VPdPCR), a novel analysis methodology enabling the detection of 10 or more target regions per color channel using conventional dPCR hardware and workflow. Furthermore, VPdPCR enables dPCR instruments to overcome upper quantitation limits caused by partitioning error. While traditional dPCR analysis establishes a single threshold to separate negative and positive partitions, VPdPCR establishes multiple thresholds to identify the number of unique targets present in each positive droplet based on fluorescence intensity. Each physical partition is then divided into a series of virtual partitions, and the resulting increase in partition count substantially decreases partitioning error. We present both a theoretical analysis of the advantages of VPdPCR and an experimental demonstration in the form of a 20-plex assay for noninvasive fetal aneuploidy testing. This demonstration assay─tested on 432 samples contrived from sheared cell-line DNA at multiple input concentrations and simulated fractions of euploid or trisomy-21 "fetal" DNA─is analyzed using both traditional dPCR thresholding and VPdPCR. VPdPCR analysis significantly lowers the variance of the chromosomal ratio across replicates and increases the accuracy of trisomy identification when compared to traditional dPCR, yielding > 98% single-well sensitivity and specificity. VPdPCR has substantial promise for increasing the utility of dPCR in applications requiring ultrahigh-precision quantitation.

ADSP follow‐up study: NCRAD biospecimens

The National Centralized Repository for Alzheimer's Disease and Related Dementias (NCRAD) is collaborating with the Alzheimer's Disease Sequencing Project (ADSP) to ensure that subjects with sequencing have available biospecimens. The latest phase of the ADSP, the Follow-Up Study (ADSP-FUS), is generating sequencing on additional subjects from diverse populations and ethnic groups; many of these subjects have samples available at NCRAD for validation and extension studies.NCRAD collects, stores, and distributes biospecimens from subjects in studies included in the ADSP-FUS, such as the National Institute on Aging Late Onset Alzheimer's Disease (NIA-LOAD) Family Based Study, the Alzheimer's Disease Neuroimaging Initiative (ADNI), and subjects accrued by Alzheimer's Disease Research Centers (ADRCs). DNA for subjects in these studies is shipped to The American Genome Center (TAGC), under the direction of Dr. Clifton Dalgard, at the Uniformed Services University of the Health Sciences (USUHS), for whole genome sequencing. NCRAD has online catalogs for many of these cohorts, including information on biospecimens as well as demographic and clinical data, to help investigators identify and request biospecimens.NCRAD has shipped >6,000 DNA samples to USUHS for sequencing and inclusion in ADSP-FUS from subjects with additional biospecimens housed at NCRAD. Many of these subjects have unique data sets or unique phenotypes, including the >1,000 early onset Alzheimer's disease (AD) samples, 773 samples from ADNI, and >3,000 AD cases/controls with clinical and/or autopsy data. The remainder were samples from cohorts focused on diverse populations and ethnic groups. For ADNI, genomic and cell line DNA, RNA, and lymphoblastoid cell lines (LCLs) are available to approved investigators; for NIA-LOAD, genomic and cell line DNA and LCLs are available, with a subset also having peripheral blood mononuclear cells (PBMCs). For ADRC subjects, in addition to genomic or cell line DNA, RNA, plasma, serum, and PBMCs are available for select subjects. Currently, there are also iPSCs or fibroblasts available for 18 ADSP-FUS subjects. NCRAD catalogs are currently available for the ADRCs and for NIA-LOAD.NCRAD has extensive biospecimens available for investigators interested in validation or extension studies leveraging the whole genome sequencing data available through the ADSP-FUS.

The ENmix DNA methylation analysis pipeline for Illumina BeadChip and comparisons with seven other preprocessing pipelines

BackgroundIllumina DNA methylation arrays are high-throughput platforms for cost-effective genome-wide profiling of individual CpGs. Experimental and technical factors introduce appreciable measurement variation, some of which can be mitigated by careful “preprocessing” of raw data.MethodsHere we describe the ENmix preprocessing pipeline and compare it to a set of seven published alternative pipelines (ChAMP, Illumina, SWAN, Funnorm, Noob, wateRmelon, and RnBeads). We use two large sets of duplicate sample measurements with 450 K and EPIC arrays, along with mixtures of isogenic methylated and unmethylated cell line DNA to compare raw data and that preprocessed via different pipelines.ResultsOur evaluations show that the ENmix pipeline performs the best with significantly higher correlation and lower absolute difference between duplicate pairs, higher intraclass correlation coefficients (ICC) and smaller deviations from expected methylation level in mixture experiments. In addition to the pipeline function, ENmix software provides an integrated set of functions for reading in raw data files from mouse and human arrays, quality control, data preprocessing, visualization, detection of differentially methylated regions (DMRs), estimation of cell type proportions, and calculation of methylation age clocks. ENmix is computationally efficient, flexible and allows parallel computing. To facilitate further evaluations, we make all datasets and evaluation code publicly available.ConclusionCareful selection of robust data preprocessing methods is critical for DNA methylation array studies. ENmix outperformed other pipelines in our evaluations to minimize experimental variation and to improve data quality and study power.

Molecular Genetics of Pre-B Acute Lymphoblastic Leukemia Sister Cell Lines during Disease Progression.

For many years, immortalized tumor cell lines have been used as reliable tools to understand the function of oncogenes and tumor suppressor genes. Today, we know that tumors can comprise subclones with common and with subclone-specific genetic alterations. We sequenced DNA and RNA of sequential sister cell lines obtained from patients with pre-B acute lymphoblastic leukemia at different phases of the disease. All five pairs of cell lines carry alterations that are typical for this disease: loss of tumor suppressors (CDKN2A, CDKN2B), expression of fusion genes (ETV6-RUNX1, BCR-ABL1, MEF2D-BCL9) or of genes targeted by point mutations (KRAS A146T, NRAS G12C, PAX5 R38H). MEF2D-BCL9 and PAX R38H mutations in cell lines have hitherto been undescribed, suggesting that YCUB-4 (MEF2D-BCL9), PC-53 (PAX R38H) and their sister cell lines will be useful models to elucidate the function of these genes. All aberrations mentioned above occur in both sister cell lines, demonstrating that the sisters derive from a common ancestor. However, we also found mutations that are specific for one sister cell line only, pointing to individual subclones of the primary tumor as originating cells. Our data show that sequential sister cell lines can be used to study the clonal development of tumors and to elucidate the function of common and clone-specific mutations.

Enhanced cytotoxic activity of beta carotene conjugated liposomes towards breast cancer cell line: comparative studies with cyclophosphamide.

This work aims to evaluate cyclophosphamide (Cyclo) cytotoxic efficacy combined with liposomes in the presence or absence of beta carotene (beta) by detecting the effects of these compounds on the breast cancer cell line (MCF-7) DNA damage. The IC50 value for beta in cytotoxic assay with MCF-7 treated cells was 21.15 μg/ml, while with liposomal beta (LipoBeta) being 121 μg/ml. The free Cyclo IC50 value was 719.86 μg/ml, its liposomal form (LipoCyclo) was 172 μg/ml. The results indicated that in contrast with Cyclo and control values, all comet assay parameters for the LipoBeta were significantly increased (P < 0.05). In MCF-7 cells treated with beta, the findings show a higher intensity of comet tail than those treated with LipoBeta. The presence of several double-strand breaks suggests this high intensity relative to the head. The molecular combination between Cyclo and liposomes in the presence or absence of beta was characterized. Dynamic light scattering measurements confirmed the mono-dispersity of all samples. The incorporation of Cyclo or beta into liposomes exhibited a slight shift to higher temperature compared to the main peak of empty liposomes that exists at 101.5°C which creates a conformational disorder within the phospholipids. The FTIR study showed structural alterations in vesicles after liposome encapsulation.

Validation of a Large Custom-Designed Pharmacogenomics Panel on an Array Genotyping Platform.

Pharmacogenomics has the potential to improve patient outcomes through predicting drug response. We designed and evaluated the analytical performance of a custom OpenArray® pharmacogenomics panel targeting 478 single-nucleotide variants (SNVs). Forty Coriell Institute cell line (CCL) DNA samples and DNA isolated from 28 whole-blood samples were used for accuracy evaluation. Genotyping calls were compared to at least 1 reference method: next-generation sequencing, Sequenom MassARRAY®, or Sanger sequencing. For precision evaluation, 23 CCL samples were analyzed 3 times and reproducibility of the assays was assessed. For sensitivity evaluation, 6 CCL samples and 5 whole-blood DNA samples were analyzed at DNA concentrations of 10 ng/µL and 50 ng/µL, and their reproducibility and genotyping call rates were compared. For 443 variants, all samples assayed had concordant calls with at least 1 reference genotype and also demonstrated reproducibility. However, 6 of these 443 variants showed an unsatisfactory performance, such as low PCR amplification or insufficient separation of genotypes in scatter plots. Call rates were comparable between 50 ng/µL DNA (99.6%) and 10 ng/µL (99.2%). Use of 10 ng/µL DNA resulted in an incorrect call for a single sample for a single variant. Thus, as recommended by the manufacturer, 50 ng/µL is the preferred concentration for patient genotyping. We evaluated a custom-designed pharmacogenomics panel and found that it reliably interrogated 437 variants. Clinically actionable results from selected variants on this panel are currently used in clinical studies employing pharmacogenomics for clinical decision-making.

Genetic and pharmacological interrogation of cancer vulnerability using a multiplexed cell line screening platform

The multiplexed cancer cell line screening platform PRISM demonstrated its utility in testing hundreds of cell lines in a single run, possessing the potential to speed up anti-cancer drug discovery, validation and optimization. Here we described the development and implementation of a next-generation PRISM platform combining Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated gene editing, cell line DNA barcoding and next-generation sequencing to enable genetic and/or pharmacological assessment of target addiction in hundreds of cell lines simultaneously. Both compound and CRISPR-knockout PRISM screens well recapitulated the results from individual assays and showed high consistency with a public database.

Antiviral and Virucidal Properties of Essential Oils and Isolated Compounds - A Scientific Approach.

Essential oils and isolated essential oil compounds are known to exert various pharmacological effects, such as antibacterial, antifungal, antiviral, anti-inflammatory, anti-immunomodulatory, antioxidant, and wound healing effects. Based on selected articles, this review deals with the potential antiviral and virucidal activities of essential oils and essential oil compounds together with their mechanism of action as well as in silico studies involving viral and host cell-specific target molecules that are indispensable for virus cell adsorption, penetration, and replication. The reported in vitro and in vivo studies highlight the baseline data about the latest findings of essential oils and essential oil compounds antiviral and virucidal effects on enveloped and non-enveloped viruses, taking into account available biochemical and molecular biological tests. The results of many in vitro studies revealed that several essential oils and essential oil compounds from different medicinal and aromatic plants are potent antiviral and virucidal agents that inhibit viral progeny by blocking different steps of the viral infection/replication cycle of DNA and RNA viruses in various host cell lines. Studies in mice infected with viruses causing respiratory diseases showed that different essential oils and essential oil compounds were able to prolong the life of infected animals, reduce virus titers in brain and lung tissues, and significantly inhibit the synthesis of proinflammatory cytokines and chemokines. In addition, some in vitro studies on hydrophilic nano-delivery systems encapsulating essential oils/essential oil compounds exhibited a promising way to improve the chemical stability and enhance the water solubility, bioavailabilty, and antiviral efficacy of essential oils and essential oil compounds.

Laboratory Verification of a BRCA1 and BRCA2 Massively Parallel Sequencing Assay from Wet Bench to Bioinformatics for Germline DNA Analysis.

Introduction A robust genetic test for BRCA1 and BRCA2 genes is necessary for the diagnosis, prognosis, and treatment of patients with hereditary breast and ovarian cancer. We evaluated a commercial amplicon-based massively parallel sequencing (MPS) assay, BRCA MASTR Plus on the MiSeq platform, for germline BRCA genetic testing. Methods This study was performed on 31 DNA from cell lines and proficiency testing samples to establish the accuracy of the assay. A reference cell line DNA, NA12878 was used to determine the reproducibility of the assay. Discordant MPS result was resolved orthogonally by the current gold-standard Sanger sequencing method. Results The analytical accuracy, sensitivity, and specificity for variant detection were 93.55, 92.86, and 100.00%, respectively. Both sequencing depth and variant allele frequencies were highly reproducible by comparing the NA12878 DNA tested in three separate runs. The single discordant result, later confirmed by Sanger sequencing was due to the inability of the MASTR Reporter software to identify a 40-bp deletion in BRCA1 . Conclusion The BRCA MASTR Plus assay on the MiSeq platform is accurate and reproducible for germline BRCA genetic testing, making it suitable for use in a clinical diagnostic laboratory. However, Sanger sequencing may still serve as a confirmatory method to improve diagnostic capability of the MPS assay.