Quantitative Synthetic Molecular Standard for Coxiella burnetii

Development and Validation of a Quantitative Synthetic Molecular Standard for Coxiella burnetii

1/28/2020 — 1/30/2020

Q fever is a highly infectious zoonotic disease caused by Coxiella burnetii, a group B biological warfare agent found worldwide that infects humans and a wide range of domestic and wild animals. This disease is associated with reproductive disorders in animals; in humans, symptoms can be mild or severe and may progress to pneumonia or chronic complications such as endocarditis, meningoencephalitis, and osteomyelitis. To control zoonotic transmission, accurate and sensitive detection is critical. Currently, real-time PCR assays are routinely used to ensure rapid detection and quantification; however, the accuracy and reproducibility of these assays are limited by the lack of precisely quantified controls. While genomic DNA can be used as a standard for these assays, C. burnetii is difficult to cultivate as it is a slow growing obligate intracellular pathogen that requires BSL-3 facilities. To address this need, ATCC developed a quantitative synthetic molecular standard for C. burnetti (ATCC® BAA-4000SD™). As a proof-of-concept, we tested the functionality of the standard via qPCR with a published primer and probe set, and protocol. To make high-containment pathogens more accessible, this approach was also extended to Nipah virus and Lassa virus.

The objective of this study is to use advanced technologies including CRISPR-Cas9 and bioluminescence to generate novel human cell lines for use as both in vitro and in vivo models in cancer research. CRISPR-Cas9 was used to knock-in the KRASG13D point mutation into the A375 malignant melanoma cell line, which also contains the BRAFV600E mutation. The resulting KRASG13D mutant isogenic line A375 exhibits significant resistance to BRAF inhibitors when studied both in traditional 2D and 3D cell culture. We developed additional models for use in live-animal bioluminescence imaging by introducing a stable luciferase reporter into the isogenic A375 and KRASG13D A375 cell lines. A xenograft model was utilized in this study and the live bioluminescence signal was quantified to correlate tumor growth with luciferase expression. Both A375-Luc2 and KRASG13D A375-Luc2 grew as tumors with increasing levels of bioluminescence when injected into nude mice. In conclusion, the combination of CRISPR-Cas9 technology and stable luciferase expression allows for the generation of isogenic luciferase-expressing cell lines, which are valuable tools for elucidating mechanisms involved in tumorigenesis and for studying drug responses in vitro and in vivo.

Technologies for the targeted disruption of gene expression are powerful tools for studying gene function. The RNA-guided CRISPR-associated nuclease Cas9 provides an effective means of introducing targeted loss of function (LOF) mutations in the genome. Cas9 can be programmed to induce DNA double strand breaks (DSBs) at specific genomic loci through guide RNAs (gRNA), which when targeted to coding regions of genes can create frame shift indel mutations resulting in a LOF allele. In addition to the use of the nuclease activity of Cas9, the CRISPR-Cas9 technology can also be repurposed as a sequence-specific, non-mutagenic gene regulation tool. Coupling of the engineered nuclease-deficient Cas9 (dCas9) to a transcriptional repressor domain can robustly silence expression of endogenous genes with high specificity, resulting in ‘CRISPR interference’ (CRISPRi). Here we report the creation of Cas9-expressing HEK-293 and CRISPRi A549 cell lines, in which the Cas9 or KRAB-dCas9 expression cassette was integrated into AAVS1 safe harbor locus. These CRISPR tool cell lines are valuable tools that greatly simplify the study of human gene function and provide potential applications for precise gene knockout and knockdown in human cells.

A key feature of organoid culture involves embedding cells within an extracellular matrix that permits the cells to grow in three dimensions into large, complex structures with varying morphologies. However, these features can also make quantification of culture health and proliferation challenging. Unlike 2D monolayer cell cultures, organoids do not proliferate as single cells, which can make cell counting and viability quantification approaches difficult. Here, approaches including commercially available kits to quantify metabolism or ATP and the trypan blue dye exclusion assay were utilized. The results were compared with label-free imaging approaches from multiple instrument platforms. Additionally, a small-scale toxicity assay was performed with various chemotherapy drugs to assess the assays. Results varied between models, donors, tissues, and cancer types. Overall, to accurately assess the growth of such complex organoid cultures, significant optimization and validation may be required. Depending on the specific application, either imaging based or cell-based assay approaches may be suitable.

12/5/2019 — 12/6/2019

New York City New York

The advancement and accessibility of next-generation sequencing (NGS) technologies have rapidly transformed microbiological research by providing the ability to analyze and profile microbial communities via metagenomics analyses. These sequencing-based applications have relied on the availability of fully assembled reference genomes for bioinformatics analyses, particularly for variant calling in diagnostic and clinical microbiology. However, despite the availability of existing genome sequences in public databases, the quality, completeness, authenticity, accuracy, and traceability of genomic data are inadequate; the lack of standards for genome quality exacerbates these underlying problems. To address this, ATCC has implemented a robust NGS and genome assembly workflow to advance authentication of bacterial strains in the ATCC collection. Our workflow is accompanied by rigorous quality control methods and criteria to ensure that the data proceeding to the next step are the highest quality. Only data that pass all quality control criteria are published to the ATCC Genome Portal, an online database of reference-grade bacterial genomes.

Kidneys are the major organs in the body responsible for the elimination of many xenobiotics and prescription drugs; having relevant models for drug interaction and toxicity studies is a necessity. Primary cells and continuous cell lines have traditionally been used in these studies. We have generated human telomerase reverse transcriptase (hTERT) immortalized renal proximal tubule epithelial cells (hTERT-RPTEC) that can overcome the limitations of donor variability and senescence of primary cells, yet show key primary cell functionality. 

11/17/2019 — 11/20/2019

Phoenix Arizona

Skin pigmentation is a complex process mediated by melanocytes; mutations in the multiple genes that regulate this process are characteristic of numerous skin disorders, including hyperpigmentation, hypopigmentation, and mixed hyperpigmentation/hypopigmentation. Melanin expression in adult melanocytes is also influenced by additional extrinsic and intrinsic factors such as hormonal changes, inflammation, age, and exposure to UV light. In order to better understand melanocyte biology, there is a need for relevant biological models. The human telomerase reverse transcriptase (hTERT)-immortalized melanocytes described here are a robust model for studying melanocyte function by providing primary melanocyte functionality but exhibiting ‘immortalized’ characteristics for more than 40 population doublings (PDL) without detectable signs of replicative senescence. 

To date, a significant amount of work has been performed on the human microbiome to evaluate its composition and influence on physiology; this research has led to additional studies on microbiomes localized at specific sites of the human body (e.g., skin, oral, vaginal). Given that fungi are ubiquitous and live in symbiosis with the human body, researchers are now actively looking into the role of the mycobiome in human health and disease. Recent advancements in sequencing technologies have enabled the community profiling of fungi; however, the complexities associated with metagenomics sequencing analyses have posed significant challenges toward standardization. To address this need, ATCC has developed genomic DNA and whole cell mock microbial communities comprising ten medically relevant fungal species mixed in even proportions. In this proof-of-concept study, we demonstrate the use these standards in evaluating DNA extraction and sequencing methods for mycobiome analysis.

Scientific progress depends on a strong foundation of data credibility. Yet, research is frequently limited by the lack of reliable reference materials. With microbiological research entering the ‘omics era, scientists are now equipped with tools such as whole-genome sequencing (WGS), which has diverse applications in the areas of microbiome research, clinical diagnostics, public health, and therapeutics development. Translational studies in these areas necessitate the need for authenticated standards along with high-quality reference genomes. Despite the availability of publicly available genomic data, the quality, authenticity, and accuracy of the data are inadequate and the lack of official standards for determining genome quality exacerbates these underlying issues. In this workshop, we discuss the importance of credible reference materials in microbial genomics with a particular focus on microbiome and phylogenetic studies. We also preview ATCC’s genome portal of high-quality assembled genomes for advancing the authentication of ATCC strains.

Most commonly known for their probiotic properties, Bifidobacteria are gram-positive, rod-shaped, anaerobic bacteria often found in the digestive tracts of various mammals and insects. Belonging to the same family as Bifidobacterium, Gardnerella comprises a single species (G. vaginalis) implicated in bacterial vaginosis, whose taxonomic status has often been disputed. Various phylogenetic trees of the Bifidobacteriaceae family place G. vaginalis centrally within the Bifidobacterium genus on the basis of 16S rRNA sequences. Additionally, the discrimination between Gardnerella and Bifidobacterium has proven difficult in the laboratory. In this study, we aim to elucidate the taxonomic position of G. vaginalis and revise the classification of the Bifidobacterium genus through whole-genome sequencing (WGS) of 62 type strains of Bifidobacterium and the type strain of G. vaginalis