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2019

To harness the quantitative potential of next-generation sequencing for data normalization, spike-in controls are essential. We have engineered three bacterial genomes (Escherichia coli, Staphylococcus aureus, and Clostridium perfringens) to contain a unique synthetic DNA tag that can be detected via 16S rRNA profiling and whole genome sequencing assays. To demonstrate the utility of the spike-in control in microbiome studies, we mixed precise quantities of genomic DNA from the recombinant bacterial strains to create a genomic DNA spike-in standard. This quantified standard was spiked into a known mock community (ATCC® MSA-1000™) containing genomic DNA prepared from 10 different bacterial strains. The resulting data showed that the unique tag of all three bacteria was identifiable and quantifiable by shotgun and 16S rRNA amplicon sequencing using V1/V2, V3/V4, and V4 primers. Spiking these recombinant bacterial genomic DNA at an optimal concentration did not affect microbiome abundance. Further, we demonstrated that the spike-in standard was applicable as an internal control for absolute quantitation. These proof-of-concept experiments support the utility of using a spike-in control with a unique 16S rRNA tag to monitor the full process of a microbiome workflow for both 16S rRNA and shotgun metagenomics assays.

With over 2 million infections per year in the United States alone, antimicrobial resistance (AMR) among bacterial pathogens has become a serious threat to global health. To thwart the AMR threat via drug discovery and diagnostics research, accurate characterization of AMR gene clusters, mobile elements, insertions, and deletions in bacterial genomes is crucial. To that end, we developed the ATCC Global Priority Superbugs Collection, which comprises 57 fully authenticated, characterized, and sequenced strains representing critical level pathogens. Here, we discuss the phenotypic and genotypic characterization of these strains through antimicrobial susceptibility profiling and a standardized sequencing, assembly, and annotation pipeline.

The Hepatitis A (HAV), B (HBV), C (HCV), and E (HEV) viruses are the most common causes of acute and chronic liver disease, which is a major source of morbidity and mortality worldwide. Currently, real-time PCR assays are routinely used to ensure the rapid detection and quantification of these viruses; however, the accuracy and reproducibility of these assays are limited by the lack of precisely quantified control materials, which are essential for determining analytical specificity and sensitivity. To address this need, ATCC has developed synthetic quantitative molecular standards for HAV, HBV, HCV, and HEV that are compliant with ISO 13485, quantified via digital PCR, and validated with published qRT-PCR assays. In the following proof-of-concept study, the HAV, HBV, HCV, and HEV synthetic molecular standards were used to generate standard curves with published primer sets. Furthermore, the standard curves for HBV and HCV were used to quantify the 3rd WHO international standard for HBV and the 4th WHO international standard for HCV from the National Institute for Biological Standards and Control (NIBSC) via the same specific published assays.

3/30/2019 — 4/3/2019

Human and mouse cancer cell lines are used in xenograft and syngeneic models, respectively, for studying in vivo tumor formation and development, evaluating metastasis, measuring tumor burden in whole animals, and monitoring response to therapeutic treatment. Whole animal in vivo imaging has been widely applied by researchers due to the ease of operation in visualizing in vivo biological events while eliminating the requirement for animal subject sacrifice, allowing for continual monitoring/imaging of a single individual animal, and reducing the amount of inter-animal variation. Luciferase reporters provide a relatively simple, robust, and highly sensitive means to measure biological processes and to assess drug efficacy in animal models through in vivo bioluminescence imaging. Here, we report on the generation of a panel of cell lines which express high levels of luciferase and have broad applications for in vitro and in vivo studies.

3/30/2019 — 4/3/2019

Exosomes are subcellular nanoparticles (50–200 nm in size) that are released from cells through a fusion of multicellular bodies with plasma membrane. Exosomes are currently being evaluated as potential diagnostic tools in a number of diseases including cancer. Exosomes are stable carriers of cell-free cargo in the form of DNA, RNA, and protein, thereby making them an attractive candidate for early detection of cancer via liquid biopsy. Tumor exosomes have also been linked to stimulation of tumor cell growth, angiogenesis, metastasis, and suppression of the immune system. However, isolating a consistent population of exosomes can be challenging and the need exists for highly characterized exosomes for use as reference standards for research and diagnostic applications. We have developed a novel method employing tangential flow filtration for  isolation of  large quantities of pure and sterile exosomes from cell culture media.

Among women, breast cancer continues to be the most common cancer, with metastasis being the leading cause of mortality in patients around the world. Epithelial to mesenchymal transition (EMT)—the process by which epithelial cells shift to the mesenchymal state—has been implicated in many aspects of breast cancer tumorigenesis, metastasis, and drug resistance. However, despite the extensive accumulation of data on the association of EMT with cancer over the years, EMT has not been an active target for therapeutic development. This is due in part to the lack of appropriate in vitro models. Here, we have exploited some of the basic biology of EMT to create an advanced in vitro metastatic breast cancer reporter cell line model for use in basic research and the discovery of new EMT inhibitors.

3/30/2019 — 4/3/2019

World-wide, metastasis continues to be the leading cause of death in cancer patients. Although epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) have been implicated in the incidence of cancer metastasis and drug resistance, their impact in cancer progression and patient survival is not fully understood. This is partly due to the lack of suitable in vitro models. Thus, to facilitate the utility of the EMT concept in therapeutic development, we have utilized some of the basic biology of EMT/MET to create a novel advanced in vitro model for use in both basic research and discovery of new anti-EMT drugs.

Epithelial-to-mesenchymal transition (EMT) describes a dynamic and reversible process where cells lose their epithelial characteristics and acquire mesenchymal properties. Accumulating evidence indicates that EMT displays an array of intermediate states, a phenotype referred to as “partial EMT”.1 EMT is executed in response to signaling pathway molecules and microRNAs (miRNAs) that induce the expression of specific EMT-associated transcription factors (EMT-TFs), including Zeb1/2, Snail1/2, and Twist. There is clinical evidence and an ever-growing body of research indicating that EMT plays an important role in cancer cell dissemination and distal metastasis.2 Therefore, targeting EMT is considered a novel opportunity in anti-cancer treatment and drug development.

Metastasis is responsible for most cancer-related deaths. One mechanism of metastasis involves epithelial-to-mesenchymal transition (EMT), a process characterized by the decrease in cell adhesion and increase in cell motility. Cells undergoing EMT often display downregulation of epithelial markers (such as E-cadherin; ECAD) and upregulation of mesenchymal markers (such as vimentin; VIM). Besides metastasis, EMT has also been reported to be associated with other pathological conditions, such as acquired therapeutic drug resistance. Given the roles that EMT plays in the pathological processes, it is of increasing interest as a target for anti-cancer treatment and drug discovery. In vitro reporter models have proven to be a valuable tool for dissecting the signaling pathways that regulate the EMT process and for screening compounds targeting EMT. In previously developed EMT reporter cell lines, the reporter gene was driven by a truncated EMT marker gene promoter. Therefore, the establishment of a more physiologically relevant reporter cell model is critical for advancing our knowledge of EMT.

Propagation of Human Organoids

Utilizing Tissue-specific Reagent Kits and Ready-to-use Wnt-3a and R-Spondin1 Conditioned Media

3/30/2019 — 4/3/2019

Three-dimensional “organoid” growth of tumors may represent a more physiologically relevant in vitro model system than traditional two-dimensional monolayer cultures of cancer cell lines. With the increased availability of cryopreserved human cancer organoids generated by academic laboratories, large-scale biobanking initiatives, and commercial sources, there is an unmet need for simplified, standardized, and cost-effective methods for preparation of the complex growth media required by these models. Human organoid culture media contains a variety of recombinant proteins, small molecules, and other growth factors that are costly to purchase in small-scale, time consuming to reconstitute and aliquot, and demonstrate varying stability and shelf life once prepared. Organoid culture media often also utilizes undefined conditioned media (CM) from one or more engineered cell lines that must be cultured separately, requiring additional time and resources to maintain. These lines secrete critical growth factors and the CM generated must be carefully prepared, collected, and stored. CM is subject to variability in activity levels due to batch-to-batch and protocol-to-protocol differences that can affect subsequent organoid culture performance.