In situ 10-cell RNA sequencing in tissue and tumor biopsy samples.

Single-cell transcriptomic methods classify new and existing cell types very effectively, but alternative approaches are needed to quantify the individual regulatory states of cells in their native tissue context. We combined the tissue preservation and single-cell resolution of laser capture with an improved preamplification procedure enabling RNA sequencing of 10 microdissected cells. This in situ 10-cell RNA sequencing (10cRNA-seq) can exploit fluorescent reporters of cell type in genetically engineered mice and is compatible with freshly cryoembedded clinical biopsies from patients. Through recombinant RNA spike-ins, we estimate dropout-free technical reliability as low as ~250 copies and a 50% detection sensitivity of ~45 copies per 10-cell reaction. By using small pools of microdissected cells, 10cRNA-seq improves technical per-cell reliability and sensitivity beyond existing approaches for single-cell RNA sequencing (scRNA-seq). Detection of low-abundance transcripts by 10cRNA-seq is comparable to random 10-cell groups of scRNA-seq data, suggesting no loss of gene recovery when cells are isolated in situ. Combined with existing approaches to deconvolve small pools of cells, 10cRNA-seq offers a reliable, unbiased, and sensitive way to measure cell-state heterogeneity in tissues and tumors.

Differential gene expression during placentation in pregnancies conceived with different fertility treatments compared with spontaneous pregnancies.

OBJECTIVE: To identify differences in the transcriptomic profiles during placentation from pregnancies conceived spontaneously vs. those with infertility using non-in vitro fertilization (IVF) fertility treatment (NIFT) or IVF. DESIGN: Cohort study. SETTING: Academic medical center. PATIENT(S): Women undergoing chorionic villus sampling at gestational age 11-13 weeks (n = 141), with pregnancies that were conceived spontaneously (n = 74), with NIFT (n = 33), or with IVF (n = 34), resulting in the delivery of viable offspring. INTERVENTION(S): Collection of chorionic villus samples from women who conceived spontaneously, with NIFT, or with IVF for gene expression analysis using RNA sequencing. MAIN OUTCOME MEASURE(S): Baseline maternal, paternal, and fetal demographics, maternal medical conditions, pregnancy complications, and outcomes. Differential gene expression of first-trimester placenta. RESULT(S): There were few differences in the transcriptome of first-trimester placenta from NIFT, IVF, and spontaneous pregnancies. There was one protein-coding differentially expressed gene (DEG) between the spontaneous and infertility groups, CACNA1I, one protein-coding DEG between the spontaneous and IVF groups, CACNA1I, and five protein-coding DEGs between the NIFT and IVF groups, SLC18A2, CCL21, FXYD2, PAEP, and DNER. CONCLUSION(S): This is the first and largest study looking at transcriptomic profiles of first-trimester placenta demonstrating similar transcriptomic profiles in pregnancies conceived using NIFT or IVF and spontaneous conceptions. Gene expression differences found to be highest in the NIFT group suggest that the underlying infertility, in addition to treatment-related factors, may contribute to the observed gene expression profiles.

Systems Analysis of Adaptive Responses to MAP Kinase Pathway Blockade in BRAF Mutant Melanoma.

Fifty percent of cutaneous melanomas are driven by activated BRAFV600E, but tumors treated with RAF inhibitors, even when they respond dramatically, rapidly adapt and develop resistance. Thus, there is a pressing need to identify the major mechanisms of intrinsic and adaptive resistance and develop drug combinations that target these resistance mechanisms. In a combinatorial drug screen on a panel of 12 treatment-naïve BRAFV600E mutant melanoma cell lines of varying levels of resistance to mitogen-activated protein kinase (MAPK) pathway inhibition, we identified the combination of PLX4720, a targeted inhibitor of mutated BRaf, and lapatinib, an inhibitor of the ErbB family of receptor tyrosine kinases, as synergistically cytotoxic in the subset of cell lines that displayed the most resistance to PLX4720. To identify potential mechanisms of resistance to PLX4720 treatment and synergy with lapatinib treatment, we performed a multi-platform functional genomics analysis to profile the genome as well as the transcriptional and proteomic responses of these cell lines to treatment with PLX4720. We found modest levels of resistance correlated with the zygosity of the BRAF V600E allele and receptor tyrosine kinase (RTK) mutational status. Layered over base-line resistance was substantial upregulation of many ErbB pathway genes in response to BRaf inhibition, thus generating the vulnerability to combination with lapatinib. The transcriptional responses of ErbB pathway genes are associated with a number of transcription factors, including ETS2 and its associated cofactors that represent a convergent regulatory mechanism conferring synergistic drug susceptibility in the context of diverse mutational landscapes.

Characterization of the nasopharyngeal microbiota in health and during rhinovirus challenge.

BACKGROUND: The bacterial communities of the nasopharynx play an important role in upper respiratory tract infections (URTIs). Our study represents the first survey of the nasopharynx during a known, controlled viral challenge. We aimed to gain a better understanding of the composition and dynamics of the nasopharyngeal microbiome during viral infection. METHODS: Rhinovirus illnesses were induced by self-inoculation using the finger to nose or eye natural transmission route in ten otherwise healthy young adults. Nasal lavage fluid samples (NLF) samples were collected at specific time points before, during, and following experimental rhinovirus inoculation. Bacterial DNA from each sample (N = 97 from 10 subjects) was subjected to 16S rRNA sequencing by amplifying the V1-V2 hypervariable region followed by sequencing using the 454-FLX platform. RESULTS: This survey of the nasopharyngeal microbiota revealed a highly complex microbial ecosystem. Taxonomic composition varied widely between subjects and between time points of the same subject. We also observed significantly higher diversity in not infected individuals compared to infected individuals. Two genera - Neisseria and Propionibacterium - differed significantly between infected and not infected individuals. Certain phyla, including Firmicutes, Actinobacteria, and Proteobacteria, were detected in all samples. CONCLUSIONS: Our results reveal the complex and diverse nature of the nasopharyngeal microbiota in both healthy and viral-challenged adults. Although some phyla were common to all samples, differences in levels of diversity and selected phyla were detected between infected and uninfected participants. Deeper, species-level metagenomic sequencing in a larger sample is warranted.