A Next Generation Connectivity Map: L1000 Platform and the First 1,000,000 Profiles.

We previously piloted the concept of a Connectivity Map (CMap), whereby genes, drugs, and disease states are connected by virtue of common gene-expression signatures. Here, we report more than a 1,000-fold scale-up of the CMap as part of the NIH LINCS Consortium, made possible by a new, low-cost, high-throughput reduced representation expression profiling method that we term L1000. We show that L1000 is highly reproducible, comparable to RNA sequencing, and suitable for computational inference of the expression levels of 81% of non-measured transcripts. We further show that the expanded CMap can be used to discover mechanism of action of small molecules, functionally annotate genetic variants of disease genes, and inform clinical trials. The 1.3 million L1000 profiles described here, as well as tools for their analysis, are available at https://clue.io.

Simultaneous quantitative analysis of 39 common toxicological drugs for increased efficiency in an ante- and postmortem laboratory.

BACKGROUND: A novel forensic method was developed to quantitate 39 drugs of toxicological interest for ante-mortem and postmortem analysis. This method was created to combine and replace four existing quantitation methods as well as add three additional compounds of interest and serves to drastically increase the efficiency of the criminalists and reduce the case backlog. The method is currently applied to ante-mortem blood, postmortem blood, urine, liver, brain, and gastric contents. METHODS: The extraction was performed by using a protein precipitation and DPX WAX-S tips with analysis on a Waters® i-class Acquity ultra-performance liquid chromatography with a Phenomenex Kinetex® Column (1.7 µm Biphenyl Å, 2.1 ×100 mm) followed by a Waters® XeVo-TQS tandem mass spectrometer using positive electrospray ionization in multiple reaction monitor mode. The sample volume required for analysis was 0.5 mL, or 0.5 g, an improvement from 4 mL when performing previous methods utilized in the laboratory. RESULTS: The improved method incorporated the 2017 recommended cut-offs for toxicological investigation of driving under the influence of drugs and was validated following the SWGTOX and ANSI/ASB guidelines of method validation. The advantages of analyzing low volume cases and/or detecting drugs previously outside the laboratory's scope of analysis, (such as gabapentin, pregabalin and baclofen) will be presented in two case studies. CONCLUSION: The multi-drug quantitation method allowed for the analysis of 39 drugs including a hydrolysis step, if needed, with only 0.5 mL or 0.5 g of sample. The method condensed two previously un-validated quantitative methods and two additional qualitative methods, which detected many commonly seen drugs, all into a single method. Three additional analytes of interest, gabapentin, pregabalin and baclofen, which it had previously been unable to detect, were added to the new method. The added benefit of these new drugs added both the coroner's investigators in cause of death determination and driving under the influence of drugs investigation especially with the high prevalence of gabapentin.

A Library of Phosphoproteomic and Chromatin Signatures for Characterizing Cellular Responses to Drug Perturbations.

Although the value of proteomics has been demonstrated, cost and scale are typically prohibitive, and gene expression profiling remains dominant for characterizing cellular responses to perturbations. However, high-throughput sentinel assays provide an opportunity for proteomics to contribute at a meaningful scale. We present a systematic library resource (90 drugs × 6 cell lines) of proteomic signatures that measure changes in the reduced-representation phosphoproteome (P100) and changes in epigenetic marks on histones (GCP). A majority of these drugs elicited reproducible signatures, but notable cell line- and assay-specific differences were observed. Using the "connectivity" framework, we compared signatures across cell types and integrated data across assays, including a transcriptional assay (L1000). Consistent connectivity among cell types revealed cellular responses that transcended lineage, and consistent connectivity among assays revealed unexpected associations between drugs. We further leveraged the resource against public data to formulate hypotheses for treatment of multiple myeloma and acute lymphocytic leukemia. This resource is publicly available at https://clue.io/proteomics.

Ultra Performance Liquid Chromatography with Tandem Mass Spectrometry for the Quantitation of Seventeen Sedative Hypnotics in Six Common Toxicological Matrices.

An ultra performance liquid chromatography triple quadrupole mass spectrometry (LC-MS-MS) method for the quantification of 14 benzodiazepines and three sedative hypnotics is presented. The fast and inexpensive assay was developed for California's Orange County Crime Lab for use in antemortem (AM) and postmortem casework. The drugs were rapidly cleaned up from AM blood, postmortem blood, urine, liver, brain and stomach contents using DPX(®) Weak Anion Exchange (DPX WAX) tips fitted on a pneumatic extractor, which can process up to 48 samples at one time. Assay performance was determined for validation based on recommendations by the Scientific Working Group for Forensic Toxicology for linearity, limit of quantitation, limit of detection, bias, precision (within run and between run), dilution integrity, carry-over, selectivity, recovery, ion suppression and extracted sample stability. Linearity was verified using the therapeutic and toxic ranges of all 17 analytes. Final verification of the method was confirmed by four analysts using 20 blind matrix matched samples. All results were within 20% of each other and the expected value.

Factors affecting photosynthetic rates of periphyton in shallow streams of southeastern Pennsylvania.

Photosynthetic rates during low-flow summer conditions were examined for eight reaches of the Christina Watershed in southeastern Pennsylvania with respect to light and nutrient availability. Photosynthetic rates in these shallow streams ranged from 0.9 to 25.4 g O2/m2 d. Soluble orthophosphorus concentrations ranged from 0.004 to 0.63 mg/L, and phosphorus was considered the limiting nutrient for periphyton growth based on a nitrogen/phosphorus ratio exceeding 10 (by weight). Light availability varied among reaches from densely wooded canopies to open pasture. Nutrient availability was examined in terms of concentration and advective flux, which was calculated as the product of stream velocity and nutrient concentration. Cluster analyses were applied to group the nutrient and light availability factors and the photosynthetic rate responses into low, moderate, and high levels. The most consistent predictor of high photosynthetic rates was the joint occurrence of moderate-to-high levels of light and nutrient flux.