A microRNA expression and regulatory element activity atlas of the mouse immune system.

To better define the control of immune system regulation, we generated an atlas of microRNA (miRNA) expression from 63 mouse immune cell populations and connected these signatures with assay for transposase-accessible chromatin using sequencing (ATAC-seq), chromatin immunoprecipitation followed by sequencing (ChIP-seq) and nascent RNA profiles to establish a map of miRNA promoter and enhancer usage in immune cells. miRNA complexity was relatively low, with >90% of the miRNA compartment of each population comprising <75 miRNAs; however, each cell type had a unique miRNA signature. Integration of miRNA expression with chromatin accessibility revealed putative regulatory elements for differentially expressed miRNAs, including miR-21a, miR-146a and miR-223. The integrated maps suggest that many miRNAs utilize multiple promoters to reach high abundance and identified dominant and divergent miRNA regulatory elements between lineages and during development that may be used by clustered miRNAs, such as miR-99a/let-7c/miR-125b, to achieve distinct expression. These studies, with web-accessible data, help delineate the cis-regulatory elements controlling miRNA signatures of the immune system.

Streamlined Protocol for Deep Proteomic Profiling of FAC-sorted Cells and Its Application to Freshly Isolated Murine Immune Cells.

Proteomic profiling describes the molecular landscape of proteins in cells immediately available to sense, transduce, and enact the appropriate responses to extracellular queues. Transcriptional profiling has proven invaluable to our understanding of cellular responses; however, insights may be lost as mounting evidence suggests transcript levels only moderately correlate with protein levels in steady state cells. Mass spectrometry-based quantitative proteomics is a well-suited and widely used analytical tool for studying global protein abundances. Typical proteomic workflows are often limited by the amount of sample input that is required for deep and quantitative proteome profiling. This is especially true if the cells of interest need to be purified by fluorescence-activated cell sorting (FACS) and one wants to avoid ex vivo culturing. To address this need, we developed an easy to implement, streamlined workflow that enables quantitative proteome profiling from roughly 2 µg of protein input per experimental condition. Utilizing a combination of facile cell collection from cell sorting, solid-state isobaric labeling and multiplexing of peptides, and small-scale fractionation, we profiled the proteomes of 12 freshly isolated, primary murine immune cell types. Analyzing half of the 3e5 cells collected per cell type, we quantified over 7000 proteins across 12 key immune cell populations directly from their resident tissues. We show that low input proteomics is precise, and the data generated accurately reflects many aspects of known immunology, while expanding the list of cell-type specific proteins across the cell types profiled. The low input proteomics methods we developed are readily adaptable and broadly applicable to any cell or sample types and should enable proteome profiling in systems previously unattainable.

The cis-Regulatory Atlas of the Mouse Immune System.

A complete chart of cis-regulatory elements and their dynamic activity is necessary to understand the transcriptional basis of differentiation and function of an organ system. We generated matched epigenome and transcriptome measurements in 86 primary cell types that span the mouse immune system and its differentiation cascades. This breadth of data enable variance components analysis that suggests that genes fall into two distinct classes, controlled by either enhancer- or promoter-driven logic, and multiple regression that connects genes to the enhancers that regulate them. Relating transcription factor (TF) expression to the genome-wide accessibility of their binding motifs classifies them as predominantly openers or closers of local chromatin accessibility, pinpointing specific cis-regulatory elements where binding of given TFs is likely functionally relevant, validated by chromatin immunoprecipitation sequencing (ChIP-seq). Overall, this cis-regulatory atlas provides a trove of information on transcriptional regulation through immune differentiation and a foundational scaffold to define key regulatory events throughout the immunological genome.

BEVACIZUMAB LEVELS IN BREAST MILK AFTER LONG-TERM INTRAVITREAL INJECTIONS.

PURPOSE: The purpose of this study is to determine whether bevacizumab is detectable in the breast milk of nursing mothers. METHODS: Breast milk samples were collected from 2 patients receiving monthly intravitreal bevacizumab injections for choroidal neovascularization over the course of 16 months. Enzyme-linked immunosorbent assay and Western blot analysis was used to determine the levels of bevacizumab in the milk samples. RESULTS: An enzyme-linked immunosorbent assay was developed using antibodies specific to bevacizumab in which the sensitivity threshold was 3 ng/mL. All breast milk samples assayed from the two patients actively undergoing treatment did not have detectable levels of bevacizumab. Samples collected 1.5 hours and 7 hours after an injection and 2 randomly chosen samples were negative by Western blot analysis. CONCLUSION: A sensitive assay to detect bevacizumab in breast milk samples assayed suggests that intravitreal injections do not result in detectable bevacizumab in breast milk.