Diversity of metals and metal-interactive bacterial populations in different types of Arctic snow and frost flowers: Implications on snow freeze-melt processes in a changing climate.

Arctic snow has been shown to be a reactive interface for key physical, chemical, and microbiological processes, affecting the Arctic's oxidation, biodiversity, radiation, and climate. To explore the potential links between snow-borne metal contaminants and metal-interactive bacteria, to freezing/melting processes, we performed concurrent chemical characterization, genomic, and morphological analysis of five different Arctic snowpack (accumulated, blowing, fresh falling, surface hoar, and wind pack snow) and frost flower in Utqiagvik (Barrow), Alaska, using Montreal urban snow as reference. Several complementary analytical techniques, including triple quad ICP-MS/MS along with various chromatography techniques, thermal ionization mass spectrometer (TIMS), high-resolution transition electron microscopy with electron dispersive X-ray spectroscopy (HR-TEM/EDS), and next generation sequencing (NGS), were deployed. Distinct metal composition and bacterial distribution among samples were observed. The concentration of 27 different transition, post-transition, rare, and radioactive metals were determined in molten snow and frost flower, as well as filtered samples. The range of three highest detected metal concentrations among samples were: Hg (3.294-134.485 µg/L), Fe (0.719-34.469 µg/L), and Sr (1.676-19,297.000 µg/L). NGS analysis led to the identification of metal interacting bacteria in all types of snow and frost flowers in the Arctic (blowing snow (1239), surface hoar snow (2243), windpack (2431), frost flowers (1440)), and Montreal urban snow (5498)) with specific bacterial genera such as: Acinetobacter, Arcenicella, Azospirillum (surface hoar snow), Arthrobacter, Paenibacillus (blowing snow), and Cycloclasticus, OM182 clade (frost flower). Several types of bacteria with confirmed or associated ice nucleation activity were observed in different types of snow, and frost flower including Pseudomonas genera (e.g., Pseudomonas fluorescens), Flavobacterium, Corynebacterium, and Pseudoxanthomonas. The implications of the above findings to snow-air interactions including nanoparticles, namely during melting and freezing cycles, and to probe the impact of various natural and anthropogenic activities are herein discussed.

Expression Concordance of 325 Novel RNA Biomarkers between Data Generated by NanoString nCounter and Affymetrix GeneChip.

BACKGROUND: With the development of new drug combinations and targeted treatments for multiple types of cancer, the ability to stratify categories of patient populations and to develop companion diagnostics has become increasingly important. A panel of 325 RNA biomarkers was selected based on cancer-related biological processes of healthy cells and gene expression changes over time during nonmalignant epithelial cell organization. This "cancer in reverse" approach resulted in a panel of biomarkers relevant for at least 7 cancer types, providing gene expression profiles representing key cellular signaling pathways beyond mutations in "driver genes." Objective. To further investigate this biomarker panel, the objective of the current study is to (1) validate the assay reproducibility for the 325 RNA biomarkers and (2) compare gene expression profiles side by side using two technology platforms. METHODS AND RESULTS: We have mapped the 325 RNA transcripts and in a custom NanoString nCounter expression panel to be compared to all potential probe sets in the Affymetrix Human Genome U133 Plus 2.0. The experiments were conducted with 10 unique biological formalin-fixed paraffin-embedded (FFPE) breast tumor samples. Each site extracted RNA from four sections of 10-micron thick FFPE tissue over three different days by two different operators using an optimized standard operating procedure and quality control criteria. Samples were analyzed using mas5 in BioConductor and NanoStringNorm in R. Pearson correlation showed reproducibility between sites for all 60 samples with r = 0.995 for Affymetrix and r = 0.999 for NanoString. Correlation in multiple days and multiple users was for Affymetrix r = (0.962 - 0.999) and for NanoString r = (0.982 - 0.991). CONCLUSION: The 325 RNA biomarkers showed reproducibility in two technology platforms with moderate to high concordance. Future directions include performing clinical validation studies and generating rationale for patient selection in clinical trials using the technically validated assay.

Genome sequencing in microfabricated high-density picolitre reactors.

The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.

Affinity protection chromatography for efficient labeling of antibodies for use in affinity capillary electrophoresis.

Capillary electrophoresis immunoassay (CEIA) is shown to be substantially more sensitive to the antibody (Ab) reagent quality than are immunosorbent methods such as enzyme-linked immunosorbent assays (ELISA). Cyanine 5 (Cy5)-labeled monoclonal anti-ovalbumin (mAb*) was inactive for CEIA of ovalbumin (Ov), yet was functional in ELISA for Ov. ELISA showed the mAb* was at least ten times less active, accounting for the poor CEIA performance. Labeled polyclonal Ab was inactive for a dye to protein ratio greater than 1.6. An affinity protection chromatography procedure (APC) was developed for Ab labeling, which avoided degradation of the Ab binding site. Ov was covalently bound to cyanogen bromide activated cellulose gel in a column, and used to capture the Ab. The coupling efficiency for Ov to the gel was 74-97%, Ab could then be bound with 95-100% efficiency, and Ab* was recovered in 50% yield following labeling on the column. This procedure was performed successfully in three different laboratories, indicating the robustness of the optimized APC synthetic method. No inactive Ab* could be detected in the APC product. The CEIA detection limit for ovalbumin using APC labeled mAb was 173 nM, when [Ab*] was fixed at 163 nM. The association constants of mAb and mAb* were determined by CEIA.