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ATCC Corporate Workshop

Association of Molecular Pathology (AMP) Annual Meeting

11/16/2017 — 11/18/2017

The complexities involved in 16S rRNA and shotgun metagenomic analysis methods pose significant challenges for microbiome research and frequently result in the introduction of biases. One of the primary obstacles in assay standardization is the limited availability of reference materials and robust analytical tools. To support this need, ATCC has developed mock microbial communities from fully sequenced and characterized ATCC strains, selected based on their phenotypic and genotypic attributes or relevance in disease-specific research. These mock communities mimic mixed metagenomics samples and offer a universal control for microbiome analyses and assay development.

Abstract: According to the World Health Organization, antimicrobial resistance (AMR) among Gram-negative bacteria continues to increase on a global scale. It is estimated that more than 23,000 people in the U.S. alone die each year from infections with multidrug-resistant (MDR) bacteria. New therapeutic agents are critical to stem this trend, but new technologies are required for shortening the time from discovery to production. To support this effort, ATCC has developed a collection of 33 fully characterized Gram-negative isolates representing current MDR disease strains from around the globe. Strains were evaluated using whole genome sequencing (WGS) and a novel annotation program to identify AMR genes and protein targets. Using public databases, ATCC developed and validated an accurate and efficient bioinformatics pipeline for the automated assembly and annotation of microbial genomes. Next-generation sequencing (NGS) data from all 33 individual isolates in combination with the novel bioinformatics pipeline were used to identify AMR genes and predictive targets that could be associated with the observed phenotype. Using our proprietary bioinformatics pipeline, we created a searchable database of AMR determinants containing the WGS information as well as a list of known AMR genes with their corresponding nucleotide sequences.

Abstract: The emergence and spread of antibacterial resistance among Gram-negative bacteria has become a global challenge for health care. Multiple programs have been implemented to reduce exposure and spread, but new therapeutics are necessary to combat these challenges. For the discovery and development of novel therapeutic agents to become a reality, multidrug-resistant (MDR) clinical isolates that represent current disease strains from around the globe are required. To support this effort, ATCC has collected and characterized 33 Gram-negative isolates using standard and new technologies.

Abstract: Complex behavior within eukaryotic cells manifest from layered regulatory networks changing the transcription of many genes. To systematically study these pathways by modulating individual components—or in the case of synthetic biology, building new network architectures by creating DNA circuit—it is critical to control multiple genes simultaneously under tightly regulated or inducible expression. In the case of network construction in Saccharomyces cerevisiae, there has been a lack of both suitable, well-characterized parts (promoters and regulators) as well as a standardized platform for DNA assembly and delivery of gene circuits. Here, we present a framework for building gene circuits as well as a set of fully characterized DNA parts for use in Saccharomyces cerevisiae. The entire procedure of building a gene circuit from more than 10 basic parts took less than 5 days with only a workload of 1-3 hours per day. A diverse promoter collection comprising five different types was generated: constitutive, yeast native inducible, synthetic inducible, synthetic promoters regulated by activators, and synthetic promoters regulated by repressors. Altogether, the range of promoters span 2-fold to 105-fold expression above the background, the new inducible systems allow 11-fold change in expression, and the activators/repressors show a maximum 35-fold and 45-fold change of expression. This study demonstrates the feasibility for the quick and easy construction of gene circuits for delivery into S. cerevisiae and the utility of a fully characterized set of diverse promoters, activators, and repressors. This assembly system combined with DNA parts will be useful for constructing large-scale gene circuit libraries with reliable gene expression and for designing logic operations for a complex network in S. cerevisiae. Moreover, we anticipate that our system will allow for the controlled study of multi-step pathways by enabling manipulation of single protein expression.

Abstract: Complexities involved in 16S rRNA and shotgun metagenomic analysis methods pose significant challenges in microbiome research as various biases can be introduced during PCR amplification, library preparation, sequencing, and analysis. One of the primary obstacles in assay standardization is the limited availability of reference materials. To support this need, we developed microbiome reference standards from fully sequenced and characterized ATCC® strains and evaluated their use in a proof-of-concept study. Here, four standards were created comprising mixtures of 10 or 20 genomic DNAs in equal or staggered quantities prepared from a diverse set of bacteria that were selected based on relevant attributes such as Gram stain, genome size, GC content, and other special characteristics. Initially, we performed an inter-laboratory comparison of the 16S rRNA V4 region from three different commercial laboratories. Analysis of the resulting sequencing data using One Codex revealed variability in the number of true positives and false positives as well as the relative abundances. A subsequent comparison of three different regions of the 16S rRNA gene—V1V2 (27f-YM+3 338R), V3V4 (341F & 806R), and V4 (515F & 806R)—revealed that only the analysis of the V1/V2 region using One Codex was able to profile the bacteria to the species level. Following this analysis, we evaluated a shotgun metagenomics approach and compared it to the 16S rRNA V1/V2 results. Here, the shotgun metagenomics approach showed a high correlation between the expected vs. observed ratios as compared to the 16S rRNA results, which had variable correlation for three standards with equal and staggered ratios. These results demonstrate that 16S rRNA community profiling of the V1V2 region and the shotgun metagenomic approach could identify bacterial strains to the species level, with the latter method generating relative abundances statistically close to the expected. Taken together, this proof-of-concept study demonstrates the potential use of ATCC® Microbiome Standards in the identification of potential biases and methodology drawbacks associated with microbiome studies.

Abstract: A shortcoming in microbiome research is the lack of reference standards to control biases introduced by differential DNA extractability, interference with amplification, library preparation, next-generation sequencing platforms, and data analysis. The aim of this research is to develop and test novel molecular barcodes for use as spike-in reference standards in the form of oligonucleotides or recombinants in microbiome studies.

Abstract: The complexities involved in 16S rRNA-based and metagenomics analysis methods pose significant challenges for standardization as bias can be introduced during DNA extraction, amplification, library preparation, sequencing, and bioinformatics analysis. One of the primary challenges in assay standardization is the limited availability of reference materials. To address this issue, we evaluated the use of two mock microbial communities in the form of lyophilized, whole-cell standards as full-process controls.

Abstract: A mutant BRAF gene can lead to uncontrolled cell growth through overactivation of the RAS-RAF-MAPK signaling pathway. The BRAFV600E mutation occurs in approximately 40% to 50% of melanomas. Although current BRAF inhibitors have been used to successfully treat melanomas containing the BRAFV600E mutation, patients often become resistant to BRAF inhibitors within a few months. A number of clinical studies have indicated that secondary mutations in RAS or NF1 are associated with BRAF resistance. However, due to the genetic heterogeneity commonly observed in tumors, it is unclear if those secondary mutations already existed within low percentage subclones, or if they were acquired through drug treatment. Further, it has yet to be determined whether such genetic variants are only associated with resistance, or whether they actually cause the BRAF inhibitor resistance. In this study, we used genome editing CRISPR technology to generate two drug-resistant melanoma cell lines (ATCC® CRL-1619IG-2™ and CRL-1619IG-1™) that contain NRASQ61K or KRASG13D mutations. These isogenic lines were derived from the parental A375 (ATCC® CRL-1619™) melanoma cell line, which naturally contains BRAFV600E. When compared to the parental line, the isogenic cell models demonstrated that genetically modified NRAS or KRAS genes at the endogenous level directly leads to significant resistance to BRAF inhibitors.

Single guide RNAs (sgRNAs) were designed and built to guide Cas9 to bind and cut desired regions in the NRAS or KRAS gene targets. The parental cell line A375 was co-transfected with the single guide and CRISPR all-in-one plasmid alongside a donor plasmid. Transfected cells were sorted into single cells and expanded for subsequent screening of desired gene mutation events. The introduction of the NRASQ61K or KRASG13D mutation in the cells was then confirmed via Sanger sequencing and NGS at the genetic and transcriptional levels. Drug responses to BRAF-specific inhibitors and non-specific chemotherapy drugs were compared between RAS isogenic A375 cell lines and parental A375 cell line in 2D and 3D culture environments. Testing results demonstrated that the isogenic cell lines created by CRISPR showed significant resistance to BRAF inhibitors in comparison to the parental control in both 2D and 3D culture environments. These two novel in vitro cell models with endogenous level RAS mutants provide direct biofunctional evidence that acquiring a drug-resistant gene drives tumor cell survival under targeted therapeutic treatment.

Abstract: In vitro angiogenesis models are crucial to the study of blood vessel growth. In this study an assay-ready, pre-mixed co-culture of human telomerase-immortalized (hTERT) human aortic endothelial cells that express green fluorescent protein and hTERT mesenchymal stem cells produce a vascular network reminiscent of in vivo angiogenesis.

Characterization of hTERT-immortalized Prostate-derived Stromal and Epithelial Cells

An Authentic in vitro Model for Tumor Microenvironment Studies

4/1/2017 — 4/5/2017

Abstract: Tumor development begins with mutational changes to the genetic makeup of a cell; tumor progression is not solely determined by the mutated cell, but also by the tumor's microenvironment. Prostate cancer, a leading cancer diagnosed in men, has been determined to be highly influenced by its surrounding stroma, particularly fibroblasts. It has been demonstrated that cancer-associated prostate fibroblasts (CAFs) differ from normal-associated prostate fibroblasts (NAFs). However, human prostate cancer model systems have focused largely on prostate cancer epithelial cells. Currently, a need exists for a more physiologically relevant human cell model system to study prostate cancer progression within the context of its tumor microenvironment. In this study, we characterized three prostate-derived cells: CAFs, NAFs, and prostate epithelial cells (PrEs); all three lines were immortalized by(human telomerase reverse transcriptase (hTERT) alone, and have been continuously passaged for more than 40 PDL in our hands. Our data shows that the hTERT-immortalized CAFs proliferate faster than the NAFs; in addition, both CAFs and NAFs express fibroblast markers such as TE7 and alpha smooth muscle actin (α-SMA), while neither cell line expresses epithelial markers such as CK14. Both CAFs and NAFs also express elevated levels of α-SMA upon TGF-β stimulation. All three prostate-derived cells weakly express the prostate specific marker AR, and show similar markers staining after long time passaging. Importantly, conditioned media collected from CAFs promotes tumor cell growth better than NAF conditioned media. In conclusion, CAFs, NAFs, and immortalized PrEs may provide a very valuable model system for the study of prostate cancer cell progression and tumor microenvironment studies.