Skin pigmentation is a complex process; melanocytes produce melanin and package it into melanosomes that are in turn exocytosed into the surrounding extracellular matrix and adjacent cells. Numerous genes play a role in controlling pigmentation at various levels of melanin production. Mutations in these genes are characteristic of multiple skin disorders, including hyperpigmentation, hypopigmentation, and mixed hyper/hypopigmentation. Additionally, extrinsic factors secreted by the surrounding resident cell types also regulate the melanin expression in adult melanocytes. Human primary cells can be a useful model for elucidating melanocyte biology. However, primary cells have their limitations such as donor variability, a limited lifespan and loss of melanin. Therefore, there is a need for a more robust human cell model system for studying skin pigmentation.

Making Sense Out of Microbiome Data

The Importance of Standardized Reference Materials

11/1/2018 — 11/3/2018

Next-generation sequencing technologies have influenced microbiome analyses in tremendous ways, opening up applications in the areas of clinical, diagnostic, therapeutic, and environmental research. However, the complexities involved in 16S rRNA community profiling and shotgun metagenomics methods often result in the introduction of biases throughout the workflow, ultimately impacting data interpretation and reproducibility. To address this, ATCC has developed fully sequenced, authenticated microbiome standards that can be used in assay standardization or as daily run controls. This workshop will highlight the utility of these standards and will focus on the assays and sequencing tools from Illumina that facilitate microbiome research.

The scientific community is currently experiencing an outpouring of research surrounding extracellular vesicles (EVs) and, more specifically, exosomes. This is due not only to their critical role in intercellular communication, but also to their potential to be used as diagnostic tools and/or therapeutic agents in a wide range of pathological conditions1. The rising interest in exosomes, coupled with the immense volume of research, underlies significant needs for both the isolation of high quality exosomes from large-scale batches and the development of industry standards for the characterization and quality control testing of exosomes. While traditional methods, which include the use of ultra-centrifugation and density gradients, are suitable for small-scale studies, the development of scalable and robust processes for the isolation of functional exosomes is essential to meet the growing needs of the scientific community. Here, we report the use of tangential flow filtration (TFF) for the isolation and concentration of functional exosomes from large batches of conditioned culture medium to include lung carcinoma cells, human mesenchymal stem cells (MSCs), and human induced pluripotent stem cells (iPSCs).

Though there is an abundance of studies, applications, and publications on the human bacterial microbiome, there are a limited number of reagents and publications focused specifically on the “virome”. Next-generation sequencing (NGS) has enabled virus sequencing on a large scale at an affordable cost. However, the complexities involved in the NGS methodology and the diversity of viral genomes pose a significant challenge to assay standardization. Therefore, there is a critical need for standardized reference materials across the research and diagnostics communities to serve as controls in assay development. To support this need, we are developing a viral panel comprising both quantified virus and virus nucleic acids prepared from diverse RNA and DNA virus families.

Complex sophisticated behavior within cells manifests from multiple regulatory networks, in which transcriptional factors (TFs) regulate gene expression, while binding to their cognate operator sequences. Here, we present a framework for building gene circuits and present a set of well-characterized DNA parts for use in Saccharomyces cerevisiae. This study demonstrates the feasibility of quickly and easily constructing gene circuits for delivery into S. cerevisiae; the utility of a fully characterized set of diverse promoters, activators, and repressors; and the applicability of this system in constructing large-scale gene circuit libraries with reliable gene expression and designing logic operations for a complex network in S. cerevisiae.

Previously described environmental, animal, and human Escherichia isolates were found to be monophyletic but were referred to as “cryptic” because they were found to be phenotypically indistinguishable from representative E. coli strains. The whole-genome sequence of these strains was obtained, de novo assembled, and compared to type/reference strains using digital DNA-DNA hybridization (dDDH). A phylogenomic analysis shows the existence of distinct clades that indicate both multiple novel subspecies of E. coli and novel species of Escherichia. At the genomic level, these strains form cohesive phylogenomic clusters and are sufficiently distinguishable from existing taxa that they warrant possible formal reclassification into novel species and/or subspecies.

Metagenomic analyses have provided insight into the abundance and taxonomic profiles of microbiomes. As the clinical and biotechnological applications of microbiome research continue to expand, researchers are now leveraging metatranscriptomics to explore organism-level function in microbiome samples via RNA-Seq technology. To facilitate this research, we have developed whole cell mock community standards representing complex mixtures of diverse bacterial species. In the following study, we used these mock community standards to create metagenomics and metatranscriptomic profiles to validate the bioinformatics analysis of whole genome shotgun sequencing and RNA-Seq data. 

Human parechovirus 3 (HPeV3) has been increasingly identified in cases of aseptic meningitis among neonates and young infants less than 1 year of age, and is associated with paralysis, viral-like sepsis, central nervous system (CNS) infection, and sudden death. Because these clinical manifestations are similar to those associated with enterovirus infections, HPeV3 infections are often misdiagnosed, which in turn results in poor patient outcome. Therefore, molecular detection assays that provide a rapid and accurate diagnosis of HPeV3 are critical for ensuring prompt and appropriate treatment. In the following proof-of-concept study, we designed and developed a HPeV3 synthetic molecular standard and validated it using qRT-PCR.

In this study, we utilized prostate cancer-associated fibroblasts (CAFs), prostate normal-associated fibroblast (NAFs), and normal prostate epithelial (PrE) cells; all three lines were immortalized by human telomerase reverse transcriptase (hTERT) alone and then were cryopreserved, thawed, and continuously passaged without any indication of a decrease in growth rate. Cell proliferation was measured under the influence of CAFs and NAFs for various prostate-derived epithelial cells. The effects of stromal cells on prostate cell proliferation was cell line-dependent. CAF cells promoted the growth of PrE, DU145, and A549, while NAF cells inhibited their growth. Meanwhile, CAF cells inhibited the growth of the viral transduced RWPE1 cell line, while NAF cells promoted the growth of the prostate epithelial cell LNCap. This study demonstrates that these hTERT-immortalized cells from human prostate are a valuable model system for the study of prostate cancer cell progression and tumor microenvironment studies.

In this study, we use the CRISPR/Cas9 system to generate isogenic drug-resistant 2D or 3D cancer models, or used in studies designed to further our understanding of the mechanisms of acquired drug resistance. Two different models were generated from the BRAF inhibitor sensitive A375 melanoma cell line. We introduced either the NRAS Q61K or the KRAS G13D point mutations, both of which are known to confer BRAF inhibitor resistance and are commonly encountered in BRAF resistant tumor samples. We then assessed the susceptibility of these new isogenic cell lines to traditional BRAF inhibitors in 2D and 3D model systems. We also determined the specific effect of these point mutations on the RAS-RAF-MAPK signaling pathway, a key component of cell-cycle escape and tumor proliferation. Furthermore, we assessed the impact of these mutations on the expression of programmed death-ligand 1 (PD-L1).