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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 Utqiaġvik (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.

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.

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.

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.

Background: We developed an algorithm based on the gene expression of tumor biopsies to identify the best combination of biomarkers to reliably predict a patient’s response to relevant cancer treatments. This algorithm is derived from 325 genes whose expression showed significant changes during differentiation of non-malignant human mammary epithelial cells cultured in laminin-rich extracellular matrix. Of these 325 genes, 251 are novel and not present in 9 other cancer based gene expression panels such as FoundationOne or PAM50. These differentiated cells formed multicellular structures with defined lumens and tight junctions and with specific localizations of cadherin and integrins. In contrast, cells from breast cancers displayed a general loss of structure. Previous work showed that different sets of these 325 biomarkers accurately predicted overall breast cancer patients' survival or response to neoadjuvant therapy in multiple independent studies. Objective: Predict cell-signaling pathways, drug associations, and disease associations for the 325 biomarkers (BA325) in contrast to other cancer gene panels. Methods and Results: The Qiagen Ingenuity program was used to identify pathways and disease states containing significant overlap with BA325. Multiple cell signaling pathways including cell proliferation, migration, invasion, and metabolism were found in BA325, while most other cancer biomarker panels were highly concentrated in cell proliferation. Examples of significant pathway associations relevant for oncology drug discovery and targeted treatments include Cell Cycle Control of Chromosomal Replication (p=8.1E-14), Polo-like-kinase and HSP90 complex (p=6.3E-07), G2/M DNA Damage Checkpoint (p=6.4E-07), Integrin Signaling (p=3E-05), Integrin Linked Kinase Signaling (p=4.51E-05), BRCA1 DNA Damage Response (p=1.75E-04), Estrogen Mediated S-phase entry (p=4.24E-04), Regulation of Actin Based Motility by RHO (p=4.55E-04), Adherins Junction Remodeling (p=5.6E-04), Actin Cytoskeleton Signaling (p=7.25E-04), Mismatch Repair (p=1.6E-03), RAN Signaling (p=1.64E-03), Protein Ubiquitination (p=1.78E-03) and Cholesterol Biosynthesis (p=4.8E-03). This wide coverage of cellular signaling pathways provides an advantage in developing predictive tests for treatments targeting highly complex cell signaling networks. Surprisingly, even though the BA325 panel was obtained during non-malignant differentiation, the most significant disease state associated with these genes was mammary tumor, demonstrating the validity of this approach to probe breast cancer biology. Conclusion: This analysis demonstrates that the BA325 panel is useful both in understanding non-malignant mammary epithelial differentiation and breast cancer tumors. Citation Format: Edward C. Goodwin, Said Attiya, Marcia Fournier. A novel panel of 325 biomarkers is part of a large interconnected network representing multiple cell signaling pathways and allowing development of predictive tests for oncology drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 405. doi:10.1158/1538-7445.AM2017-405

Background: With new developments of oncology drug combinations and targeted treatments, 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 non-malignant epithelial cell organization. This "cancer in reverse" approach in combination with empirically derived algorithms resulted in a panel of biomarkers having little overlap with 9 other widely-used commercial gene panels analyzed to date (e.g., overlap with FoundationOne was 2% (lowest) and with OncotypeDX was 14% (highest)), providing a more in-depth and comprehensive view of gene expression profiles and key cellular signaling pathways beyond mutations in "driver-genes", and drug associations including chemotherapies, immunotherapies, and targeted-therapies. Objective: In order to technically validate an assay for the 325 RNA-biomarkers we compared gene expression profiles side-by-side using two technology platforms to address the reproducibility of the assay. Methods and Results: We have mapped the 325 RNA transcripts and 7 housekeeping genes in a custom NanoString n-Counter expression panel to be compared to all potential probe sets in the Affymetrix Human Genome U133 Plus 2.0 microarray. 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-microns thick FFPE tissue over three different days by three 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 were for Affymetrix r=(0.962-0.999) and for NanoString r=(0.982-0.991). The platforms were compared using relative expression fold changes using linear regression (lm). Concordant genes were defined to have gene expression levels within +/- 2 standard deviation (sd) of each other at 95% confidence interval, or to have greater than +/- 2 sd but have changes in the same direction in both platforms. The discordant genes were defined to have opposite direction of changes. By this definition, approximately 90% of the genes fell into the concordant category. Conclusion: The 325 RNA-biomarkers showed reproducibility in two technology platforms with high concordance. With five predictive tests under development for breast (3), lung and pancreatic cancers future directions include performing clinical validation studies and generate rationale for patient selection in clinical trials using the technically validated assays. Citation Format: Marcia V. Fournier, Said Attiya, Joan Chen, John Obenauer, Anup Madan, Patricia Smith. Technical validation of novel 325 RNA predictive biomarkers using gene expression data generated by Nanostring n-counter and Affymetrix microarray [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1785. doi:10.1158/1538-7445.AM2017-1785

An integrated microsystem providing rapid analyses of trace-level tryptic digests for proteomics application is presented. This modular microsystem includes an autosampler and a microfabricated device comprising a sample introduction port and an array of separation channels together with a low dead-volume facility enabling an interface to nanoelectrospray mass spectrometry. Sequential injection and separation of peptide standards and tryptic digests was achieved with a throughput of up to 30 samples per hour with less than 3% sample carryover. Replicate injections of peptide mixtures indicated that reproducibility of migration time was typically better than 2.3% relative standard deviation (RSD), whereas RSD values of 3.7–11.8% were observed on peak height. Mass spectral detection of sub-micromolar protein digests (< 7 fmol per injection) was achieved using a quadrupole/time-of-flight instrument in less than 2 min per sample with peak widths of 1.8–7.0 s. The analytical potential of this integrated device for the identification of gel-isolated proteins from Neisseria meningitidis immunotype L3 has been demonstrated using both peptide mass-fingerprint database searching and on-line tandem mass spectrometry.

A highly sensitive laser induced fluorescence (LIF) detection system based on a 635 nm laser diode and cyanine-5 (Cy-5) dye, is described for use with a planar, microfluidic, capillary electrophoresis (CE) chip. The CE-chip is able to determine a protein biological threat agent simulant, ovalbumin (Ov), by performing an immunoassay separation of Cy-5 labeled anti-ovalbumin from its complex with Ov, in under 30 s. A confocal, epiluminescent detection system utilizing a photomultiplier tube gave optimum results with a 400 microm pinhole, an Omega 682DF22 emission filter, a 645DRLP02 dichroic mirror, a 634.54 +/- 5 nm excitation filter, and a Power Technology ACMO8 635 nm laser operated at 11.2 mW. Using this detector, a microchip CE device with a separation efficiency of 42,000 plates and an etch depth of 20 microm, gave a concentration detection limit of 9 pM Cy-5. This limit corresponds to the determination of 4560 injected molecules and detection of 900 of these molecules, given a probe volume of 1.6 pl and a probing efficiency of 20%.

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.

A six-channel microfluidic immunoassay device with a scanned fluorescence detection system is described. Six independent mixing, reaction, and separation manifolds are integrated within one microfluidic wafer, along with two optical alignment channels. The manifolds are operated simultaneously and data are acquired using a singlepoint fluorescence detector with a galvano-scanner to step between separation channels. A detection limit of 30 pM was obtained for fluorescein with the scanning detector, using a 7.1-Hz sampling rate for each of the reaction manifolds and alignment channels (57-Hz overall sampling rate). Simultaneous direct immunoassays for ovalbumin and for anti-estradiol were performed within the microfluidic device. Mixing, reaction, and separation could be performed within 60 s in all cases and within 30 s under optimized conditions. Simultaneous calibration and analysis could be performed with calibrant in several manifolds and sample in the other manifolds, allowing a complete immunoassay to be run within 30 s. Careful chip conditioning with methanol, water, and 0.1 M NaOH resulted in peak height RSD values of 3-8% (N = 5 or 6), allowing for cross-channel calibration. The limit of detection (LOD) for an anti-estradial assay obtained in any single channel was 4.3 nM. The LOD for the cross-channel calibration was 6.4 nM. Factors influencing chip and detection system design and performance are discussed in detail.

An interface design is presented that facilitates automated sample introduction into an electrokinetic microchip, without perturbing the liquids within the microfluidic device. The design utilizes an interface flow channel with a volume flow resistance that is 0.54-4.1 x 10(6) times lower than the volume flow resistance of the electrokinetic fluid manifold used for mixing, reaction, separation, and analysis. A channel, 300 microm deep, 1 mm wide and 15-20 mm long, was etched in glass substrates to create the sample introduction channel (SIC) for a manifold of electrokinetic flow channels in the range of 10-13 microm depth and 36-275 microm width. Volume flow rates of up to 1 mL/min were pumped through the SIC without perturbing the solutions within the electrokinetic channel manifold. Calculations support this observation, suggesting a leakage flow to electroosmotic flow ratio of 0.1:1% in the electrokinetic channels, arising from 66-700 microL/min pressure-driven flow rates in the SIC. Peak heights for capillary electrophoresis separations in the electrokinetic flow manifold showed no dependence on whether the SIC pump was on or off. On-chip mixing, reaction and separation of anti-ovalbumin and ovalbumin could be performed with good quantitative results, independent of the SIC pump operation. Reproducibility of injection performance, estimated from peak height variations, ranged from 1.5-4%, depending upon the device design and the sample composition.

Two microfluidic designs are presented which increase the range of techniques available for performing recombinant DNA procedures on-chip: mRNA purification with single strand DNA synthesis, and DNA transfection in cells via electroporation.

We present here the development of elements for a fully integrated sample processing system, including a rapid-exchange, sample introduction port for the world-to-chip interface, and an integrated immunoassay reactor, which incorporates a method for performing on-chip calibration, on-chip mixing, reaction and separation of immunoreagents, sample and products.

The development of the components and subsystems of an automated microchipbased platform for bioanalysis by immunoassay and gene probe assay is described. The device uses micromachined glass plates for the fabrication of channel networks and combines electroosmotic pumping and capillary electrophoresis for fluid transport and separation. The complete system enables the on-chip integration of the key elements in analytical processing: injection, mixing, separation, detection and elimination. The analysis platform is being designed for use with an on-line aerosol collector for environmental monitoring.

Nature 437, 376–380 (2005) In this paper, the second name of Chun Heen Ho was misspelled as He. In the earlier Corrigendum (Nature 439, 502; 2006) the surnames of Stephen K. Hutchison and Brian C. Godwin were misspelled as Hutchinson and Goodwin. In addition, in the Supplementary Information of the original paper, there were errors in the sequences of the DNA capture and enrichment primers, and in the formulation of emulsion oil.