Sample Processor Research Articles

Utilization of EBV DNA Copy Number Monitoring in Extranodal NK Lymphoma, Nasal Type in Non Asian Patients

AbstractAbstract 5088 Introduction.Extranodal NK/T-cell lymphoma, nasal type (ENKL) is a common type of non-Hodgkin's lymphoma (NHL) in Asia and Latin America but is rare in the West. Epstein-Barr Virus (EBV)-encoded RNA (EBER) is detected in most cases of ENKL. Measurement of EBV-DNA copy number (EDCN) in peripheral blood (PB) via polymerase chain reaction (PCR) has been shown in Asian patients to be a useful marker for monitoring tumor burden, response to treatment and disease recurrence (Suzuki, 2011; Ito, 2012), but data in Western patients are lacking. We conducted a pilot, retrospective study of EDCN measurements in Western patients with ENKL, nasal type. Methods.A retrospective review of an IRB-approved patient database identified 13 consecutive patients with ENKL, nasal type, diagnosed between July 1999 and August 2011. Inclusion criteria were based on WHO criteria. Six patients were diagnosed at outside institutions, received initial therapy locally and presented to OSU for evaluation after treatment or at relapse. All pathology specimens were reviewed at OSU. In situ hybridization was used to evaluate EBER expression. PB samples were serially obtained from patients at multiple time points (first visit, longitudinal, last follow up) and analyzed in real time. The Artus™ EBV PCR assay was used for EBV-DNA, extracted from EDTA plasma. 200 uL of patient plasma and 2. 5uL of internal control were processed using the EasyMAG™ sample processor. Quantification of EDCN was done using the EBV TM Master which specifically amplifies a 97bp region of the EBNA-1 gene and the ABI PRISM™ 7500 system. The amplicon was detected by measuring the FAM fluorescence. Quantification of EBV-DNA in the patient’s sample was compared to five known standards supplied by Acrometrix. The standards range from 500 to 10, 000, 000 copies/mL. Outcomes including complete response (CR), partial response (PR) and relapse/progression of disease (PD) were assessed according to the Cheson criteria. Categorical data, such as gender, race, stage of disease at diagnosis, number of treatments received and best response were summarized as frequency counts and percentages. Quantitative data, such as age, LDH, WBC, hemoglobin, platelet, were summarized as medians and ranges. Estimates of progression- free survival and overall survival were obtained using the Kaplan-Meier method. Results.Demographics, pathology, treatment, and outcome data will be provided. For all patients, involved field radiation therapy (IFRT) was part of front line therapy. Figure 1 shows EDCN for 12 evaluable patients. Three patients (1a, 2a, 3a) had elevated EDCN at diagnosis, which normalized with treatment, and remained undetectable at last follow up. All 3 patients achieved CR, 2 patients after initial therapy and 1 patient after allogeneic hematopoietic stem cell transplant (HSCT). Three patients (1b, 2b, 3b) had undetectable EBV-DNA at diagnosis, throughout treatment, and at last follow up. Three patients (1c, 2c, 3c) had no EBV-DNA measurements initially, but all 3 had rapidly increasing EDCN prior to progression and death. Three patients (1d, 2d, 3d), whose disease status and EDCN was documented at multiple time points on long term follow up are analyzed in detail. In patient 1d EBV-DNA was undetectable at diagnosis and at first and second progression, but increased rapidly at third progression and prior to death. Patient 2d had undetectable EBV-DNA at diagnosis, but after front line therapy failure EDCN increased and then closely paralleled clinical response. Following allogeneic HSCT, the EDCN increased rapidly to >10E6 copies/mL, prior to radiographic or clinical evidence of relapse. Patient 3d had elevated EDCN at diagnosis, which normalized with therapy, rose prior to clinically overt first relapse, and normalized again with SMILE chemotherapy. Conclusions.Elevated EDCN was found in the PB of many non Asian patients with ENKL, nasal type at diagnosis and should be studied prospectively as an adjuvant diagnostic marker, particularly in cases where biopsy is inconclusive or delayed. Serial changes in PB EDCN closely paralleled clinical response in many of the patients. EDCN elevation preceded clinical or radiological evidence of active disease in some patients, prompting further diagnostic work up. The value of serial monitoring of EDCN should be studied prospectively and be included in clinical trials of new therapies for ENKL, nasal type in the U. S. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Determination of the novel melatonin agonist Neu-P11 in plasma samples by liquid chromatography–tandem mass spectrometry

Melatonin is a major chronobiological regulator involved in circadian phasing, sleep, and numerous other functions. The novel melatonin agonist Neu-P11 is used in treatment of physiological insomnia. In animal studies Neu-P11 showed sleep-promoting effect. In a phase 1 study Neu-P11 was administered to cohorts of healthy young male volunteers in an ascending single dose study. Up to now no method for measurement of the new drug in human plasma was described and validated. The aim of this study was to develop a suitable analytical procedure. With solid phase extraction (SPE) for sample preparation and liquid chromatography tandem mass spectrometry (LC/MS/MS) a sensitive method with a lower limit of quantification of 0.39ng/ml was found. For SPE samples were put into Strada C18 cartridges and automatically extracted by a Gilson Automatic Sample Processor ASPEC XL. The HPLC was carried out with a Purospher C18 column and mobile phase gradient with acetonitrile, and ammonium acetate. The precision ranged from 0.4% to 9.5%. The deviation of the measured from the true value, of the standard samples and QCSs was lower than 10%. The method was specific, precise and accurate and the analyte was stable under the conditions of measurement. Results show that the method is suitable for the quantification of NeuP11 in human plasma after a single dose of 5, 20, 50 or 200mg.

Robotic sampling, in situ monitoring and molecular detection of marine zooplankton

Recent advances in robotic technologies provide new opportunities to conduct high-resolution sampling of patchily distributed zooplankton and associated environmental variables. We used two robots and molecular probes to assess the temporal and spatial variability of zooplankton in water samples obtained from Monterey Bay, California. The Autonomous Underwater Vehicle (AUV) Dorado is a mobile platform that carries ten, 1.8-L bottles ("Gulpers") capable of rapidly acquiring discrete seawater samples, and an extensive sensor suite for gathering contextual environmental data during day-long expeditions. Molecular assays were conducted ex situ at a shore-based laboratory. In contrast, the Environmental Sample Processor (ESP) was deployed as a stationary (moored) device capable of repeatedly “sipping” water to conduct in situ molecular assays and record environmental data during month-long deployments. Molecular analyses were conducted with the sandwich hybridization assay (SHA), which employed 18S ribosomal RNA oligonucleotide probes designed to detect calanoid and podoplean copepods, and the larvae of barnacles, mussels, polychaete worms, brachyuran crabs, and invasive green crabs (Carcinus maenas). Both the stationary and mobile sampling protocols revealed the greatest zooplankton diversity and abundance in relatively warm waters, higher in chlorophyll and lower in salinity and nitrate. Diversity and abundance were least in recently upwelled waters with the inverse conditions. High-resolution sampling revealed that while calanoid copepods were generally associated with elevated chlorophyll, they were most abundant in upwelling fronts, in some cases. These narrow features appear to provide favorable conditions for the growth and aggregation of certain zooplankton.

Seasonal Synechococcus and Thaumarchaeal population dynamics examined with high resolution with remote in situ instrumentation

Monterey Bay, CA is an Eastern boundary upwelling system that is nitrogen limited much of the year. In order to resolve population dynamics of microorganisms important for nutrient cycling in this region, we deployed the Environmental Sample Processor with quantitative PCR assays targeting both ribosomal RNA genes and functional genes for subclades of cyanobacteria (Synechococcus) and ammonia-oxidizing Archaea (Thaumarchaeota) populations. Results showed a strong correlation between Thaumarchaea abundances and nitrate during the spring upwelling but not the fall sampling period. In relatively stratified fall waters, the Thaumarchaeota community reached higher numbers than in the spring, and an unexpected positive correlation with chlorophyll concentration was observed. Further, we detected drops in Synechococcus abundance that occurred on short (that is, daily) time scales. Upwelling intensity and blooms of eukaryotic phytoplankton strongly influenced Synechococcus distributions in the spring and fall, revealing what appear to be the environmental limitations of Synechococcus populations in this region. Each of these findings has implications for Monterey Bay biogeochemistry. High-resolution sampling provides a better-resolved framework within which to observe changes in the plankton community. We conclude that controls on these ecosystems change on smaller scales than are routinely assessed, and that more predictable trends will be uncovered if they are evaluated within seasonal (monthly), rather than on annual or interannual scales.

Underwater application of quantitative PCR on an ocean mooring.

The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions.

Metatranscriptomic analysis of autonomously collected and preserved marine bacterioplankton

Planktonic microbial activity and community structure is dynamic, and can change dramatically on time scales of hours to days. Yet for logistical reasons, this temporal scale is typically under-sampled in the marine environment. In order to facilitate higher-resolution, long-term observation of microbial diversity and activity, we developed a protocol for automated collection and fixation of marine microbes using the Environmental Sample Processor (ESP) platform. The protocol applies a preservative (RNALater) to cells collected on filters, for long-term storage and preservation of total cellular RNA. Microbial samples preserved using this protocol yielded high-quality RNA after 30 days of storage at room temperature, or onboard the ESP at in situ temperatures. Pyrosequencing of complementary DNA libraries generated from ESP-collected and preserved samples yielded transcript abundance profiles nearly indistinguishable from those derived from conventionally treated replicate samples. To demonstrate the utility of the method, we used a moored ESP to remotely and autonomously collect Monterey Bay seawater for metatranscriptomic analysis. Community RNA was extracted and pyrosequenced from samples collected at four time points over the course of a single day. In all four samples, the oxygenic photoautotrophs were predominantly eukaryotic, while the bacterial community was dominated by Polaribacter-like Flavobacteria and a Rhodobacterales bacterium sharing high similarity with Rhodobacterales sp. HTCC2255. However, each time point was associated with distinct species abundance and gene transcript profiles. These laboratory and field tests confirmed that autonomous collection and preservation is a feasible and useful approach for characterizing the expressed genes and environmental responses of marine microbial communities.

Harmful phytoplankton ecology studies using an autonomous molecular analytical and ocean observing network

Using autonomous molecular analytical devices embedded within an ocean observatory, we studied harmful algal bloom (HAB) ecology in the dynamic coastal waters of Monterey Bay, California. During studies in 2007 and 2008, HAB species abundance and toxin concentrations were quantified periodically at two locations by Environmental Sample Processor (ESP) robotic biochemistry systems. Concurrently, environmental variability and processes were characterized by sensors co‐located with ESP network nodes, regional ocean moorings, autonomous underwater vehicle surveys, and satellite remote sensing. The two locations differed in their long‐term average physical and biological conditions and in their degree of exposure to episodic wind‐forced variability. While anomalously weak upwelling and strong stratification during the 2007 study favored toxigenic dinoflagellates (Alexandrium catenella), anomalously strong upwelling during the 2008 study favored toxigenic diatoms (Pseudo‐nitzschia spp.). During both studies, raphidophytes (Heterosigma akashiwo) were detected within a similar range of concentrations, and they reached higher abundances at the relatively sheltered, stratified site. During 2008, cellular domoic acid reached higher concentrations and was far more variable at the shallower ESP node, where phytoplankton populations were influenced by resuspended sediments. Episodic variability caused by wind forcing, lateral mixing, internal waves, and subsurface phytoplankton layers influenced ESP detection patterns. The results illustrate the importance of mobilizing HAB detection on autonomous platforms that can intelligently target sample acquisition as a function of environmental conditions and biological patch encounter.

Integrated microfluidic bioprocessor for solid phase capture immunoassays

A microfluidic device for solid-phase immunoassays based on microparticle labeling is developed using microvalve-control structures for automated sample processing. Programmable microvalve control in a multilayer structure provides automated sample delivery, adjustable hydrodynamic washing and compatibility with a wide range of substrates. Capture antibodies are derivatized on glass surfaces within the processor using an APTES patterning method, and magnetic microspheres conjugated with a secondary detection antibody are used as labels in a capture-sandwich format. In this microfluidic processor, washing force can be precisely controlled to remove the nonspecifically bound microparticles. Automated microfluidic immunoassays are demonstrated for mouse immunoglobulin (IgG) and human prostate specific antigen (PSA) with limits of detection of 1.8 and 3 pM, respectively. The sample processor architecture is easily parallelized for high-throughput analysis and easily interfaced with various assay substrates.

Abstract 2187: Optimization free oncogene mutation screening work flow

Abstract BRCA1 and BRCA2 are the two primary breast cancer susceptibility genes in which identifying mutations is important to access cancer risk and decision for treatment. Prescreening of exons by High Resolution Melt (HRM) prior to sequencing can reduce effort in mutation screening in large transcripts such as the BRCA genes. Published methods for BRCA1 and 2 HRM screening have relied on complicated touchdown cycling protocols and custom reagent mixes that can lead to variable result. We have validated a streamlined workflow for adaptability in a clinical diagnostic environment. First, genomic DNA template was extracted on an automated sample processor using magnetic bead technology to purify genomic DNA directly from blood sample. Then, the PCR was performed using a PCR master mix optimized for HRM coupled with universal cycling conditions. We have successfully amplified all 120 amplicons covering exons of both BRCA genes using universal PCR condition. Potential variations identified by HRM screening were confirmed by direct sequencing of HRM PCR amplicons with M13 universal tags. By using unified protocol for PCR and universal sequencing for all amplicons the risk of incorrect procedure is greatly reduced. This simplified workflow for oncogene mutation screening using automated sample extraction and universal protocol for PCR and sequencing provides significant advantages for application in diagnostic settings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2187.

What are "ecogenomic sensors?" A review and thoughts for the future

Abstract. The OceanSensors08 workshop held 31 March–4 April 2008 in Warnemünde, Germany, brought together an international group of marine scientists, sensor developers and technologists with a common interest in shaping the future of ocean sensing. In preparation for that meeting a series of review papers was commissioned, one of which was meant to cover Sensors for Ocean-omics. The "ocean-omics" topic was cast very broadly. The notion was to review use of genetic techniques for assessing presence and diversity of organisms, their genomic capacity and gene expression, and to provide a prospectus of how such methods could be applied in an autonomous capacity. I chose "ecogenomic sensor" as a descriptor to convey the essence of such a system – a device that integrates genetic level sensing with larger scale environmental characterization. This phrase is derived from workshops refining the US's Ocean Observatories Initiative (OOI) and visions for instrument systems that could be deployed on such a network. But what exactly are ecogenomic sensors? A clear definition is lacking and conceptualizations far outweigh actual hardware that can be deployed in the ocean. This prospectus builds from that point. I advance a definition of "ecogenomic sensor" and outline the opportunities and challenges associated with developing such instruments. Suggestions as to how this technology may be further refined and applied are offered against the backdrop of the Autonomous Microbial Genosensor (AMG) and Environmental Sample Processor (ESP). Applications that center on detection of DNA and RNA are emphasized. The word "review" appears in the title at the request of the editors.

Remote Detection of Marine Microbes, Small Invertebrates, Harmful Algae, and Biotoxins using the Environmental Sample Processor (ESP)

The advent of ocean observatories is creating unique opportunities for deploying novel sensor systems. We are exploring that potential through the development and application of the Environmental Sample Processor (ESP). ESP is an electromechanical/fluidic system designed to collect discrete water samples, concentrate microorganisms, and automate application of molecular probe technologies. Development and application of ESP grew from extensive partnerships galvanized by the National Oceanographic Partnership Program. Near-real-time observations are currently achieved using low-density DNA probe and protein arrays. Filter-based sandwich hybridization methodology enables direct detection of ribosomal RNA sequences diagnostic for groups of bacteria and archaea, as well as a variety of invertebrates and harmful algal species. An antibody-based technique is used for detecting domoic acid, an algal biotoxin. To date, ESP has been deployed in ocean waters from the near surface to 1000 m. Shallow-water deployments demonstrated application of all four types of assays in single deployments lasting up to 30 days and provided the first remote detection of such phylogenetically diverse organisms and metabolites on one platform. Deep-water applications focused on detection of invertebrates associated with whale falls, using remotely operated vehicle-based operations lasting several days. Current work emphasizes incorporating a four-channel, real-time polymerase chain reaction module, extending operations to 4000-m water depth, and increasing deployment duration.

Remote, subsurface detection of the algal toxin domoic acid onboard the Environmental Sample Processor: Assay development and field trials

The ability to detect harmful algal bloom (HAB) species and their toxins in real- or near real-time is a critical need for researchers studying HAB/toxin dynamics, as well as for coastal resource managers charged with monitoring bloom populations in order to mitigate their wide ranging impacts. The Environmental Sample Processor (ESP), a robotic electromechanical/fluidic system, was developed for the autonomous, subsurface application of molecular diagnostic tests and has successfully detected several HAB species using DNA probe arrays during field deployments. Since toxin production and thus the potential for public health and ecosystem effects varies considerably in natural phytoplankton populations, the concurrent detection of HAB species and their toxins onboard the ESP is essential. We describe herein the development of methods for extracting the algal toxin domoic acid (DA) from Pseudo-nitzschia cells (extraction efficiency >90%) and testing of samples using a competitive ELISA onboard the ESP. The assay detection limit is in the low ng/mL range (in extract), which corresponds to low ng/L levels of DA in seawater for a 0.5 L sample volume acquired by the ESP. We also report the first in situ detection of both a HAB organism (i.e., Pseudo-nitzschia) and its toxin, domoic acid, via the sequential (within 2–3 h) conduct of species- and toxin-specific assays during ESP deployments in Monterey Bay, CA, USA. Efforts are now underway to further refine the assay and conduct additional calibration exercises with the aim of obtaining more reliable, accurate estimates of bloom toxicity and thus their potential impacts.

Near real‐time, autonomous detection of marine bacterioplankton on a coastal mooring in Monterey Bay, California, using rRNA‐targeted DNA probes

A sandwich hybridization assay (SHA) was developed to detect 16S rRNAs indicative of phylogenetically distinct groups of marine bacterioplankton in a 96-well plate format as well as low-density arrays printed on a membrane support. The arrays were used in a field-deployable instrument, the Environmental Sample Processor (ESP). The SHA employs a chaotropic buffer for both cell homogenization and hybridization, thus target sequences are captured directly from crude homogenates. Capture probes for seven of nine different bacterioplankton clades examined reacted specifically when challenged with target and non-target 16S rRNAs derived from in vitro transcribed 16S rRNA genes cloned from natural samples. Detection limits were between 0.10-1.98 and 4.43- 12.54 fmole ml(-1) homogenate for the 96-well plate and array SHA respectively. Arrays printed with five of the bacterioplankton-specific capture probes were deployed on the ESP in Monterey Bay, CA, twice in 2006 for a total of 25 days and also utilized in a laboratory time series study. Groups detected included marine alphaproteobacteria, SAR11, marine cyanobacteria, marine group I crenarchaea, and marine group II euryarchaea. To our knowledge this represents the first report of remote in situ DNA probe-based detection of marine bacterioplankton.

Field applications of the second‐generation Environmental Sample Processor (ESP) for remote detection of harmful algae: 2006‐2007

We assess the application of the second‐generation Environmental Sample Processor (ESP) for the detection of harmful algal bloom (HAB) species in field and laboratory settings using two molecular probe techniques: a sandwich hybridization assay (SHA) and fluorescent in situ hybridization (FISH). During spring 2006, the first time this new instrument was deployed, the ESP successfully automated application of DNA probe arrays for various HAB species and other planktonic taxa, but non‐specific background binding on the SHA probe array support made results interpretation problematic. Following 2006, the DNA array support membrane that we were using was replaced with a different membrane, and the SHA chemistry was adjusted. The sensitivity and dynamic range of these modifications were assessed using 96‐well plate and ESP array SHA formats for several HAB species found commonly in Monterey Bay over a range of concentrations; responses were significantly correlated (p < 0.01). Modified arrays were deployed in 2007. Compared to 2006, probe arrays showed improved signal:noise, and remote detection of various HAB species was demonstrated. We confirmed that the ESP and affiliated assays can detect HAB populations at levels below those posing human health concerns, and results can be related to prevailing environmental conditions in near real‐time.

Fast Analysis of Free Amino Acids in Tobacco by HPLC with Fluorescence Detection and Automated Derivatization

AbstractA fast, simple, and sensitive HPLC method for the determination of free amino acids in tobacco was described. A fully automated sample processor performed precolumn derivatization of both primary and secondary amino acids with o‐phthalaldehyde/3‐mercaptopropionic acid and 9‐fluorenylmethyl chloroformate (FMOC‐Cl), respectively. All reactions were fully automated by means of an injector programme and accomplished in 10 min. Sample preparation consisted of a single step of extraction with 0.1 mol/L HCl at ambient temperature (assisted by sonication) in 30 min, followed by filtration of an aliquot and derivatization. By optimization of sample preparation and HPLC conditions, separation of 20 amino acids in 30 min was achieved. Detection limits ranged from 0.50 to 1.40 μg/g; coefficients of variation ranged from 1.8% to 3.9%; recoveries ranged from 84.6% to 108.5%. The method was applied to the analysis of amino acids contents of tobacco leaves in different varieties and flue‐curing period.

A robotic molecular method for in situ detection of marine invertebrate larvae

Knowledge of the temporal and spatial abundance of invertebrate larvae is critical to understanding the dispersal capabilities and recruitment potential of marine and aquatic organisms. Traditional microscopic analyses are time-consuming and difficult given the diversity of larval species and a frequent lack of discriminating morphological characteristics. Here, we describe a sensitive rRNA targeted sandwich hybridization assay (SHA) that uses oligonucleotide probes to detect and enumerate the larvae of invasive green crabs (Carcinus maenas), native blue mussels (Mytilus), native barnacles (Balanus) and polychaetes (Osedax and Ophelia) that occur in the Monterey Bay National Marine Sanctuary, California. Laboratory-based assays demonstrate specificity, high sensitivity, and a quantitative response to cultured samples from three of the target organisms. Oligonucleotide probes were then printed in arrays on nitrocellulose membranes and deployed in our robotic Environmental Sample Processor (ESP) to detect larvae in situ and autonomously. We demonstrate that the SHA-detection method and ESP robot can be used for near real-time, in situ detection of larval species in the marine environment.

Automated, high-throughput immunofluorescence staining: a new approach

A high-throughput antibody staining and washing system for large-scale routine indirect immunofluorescence testing was developed, using a robotic sample processor. The Tecan robotic sample processor (Miniprep 75/2, Tecan Benelux BV, Giessen, The Netherlands) was introduced, adapted and evaluated for its suitability for large-scale immunofluorescence screening of potato samples for the presence of the plant pathogenic quarantine bacterium, Ralstonia solanacearum. Throughput and quality of antibody–antigen reactions were compared to manual sample preparation for the immunofluorescence test according to a standard EU testing protocol, using 216 standard potato extracts, including 204 known negative extracts and 12 known positive extracts, and 24 positive control strain samples. Throughput of the Tecan robotic sample processor easily outcompeted manual staining of immunofluorescence slides. The robot was twice as fast as manual processing and is easily cost effective. With the robot 288 samples per day can be processed as compared to 144 samples per day with manual processing. No difference between quality of antibody–antigen reactions and readability of preparations under a microscope could be detected. The new Tecan robotic sample processor was found to be useful for high-throughput fluorescent antibody staining and washing in large-scale, routine indirect immunofluorescence testing.

Controlling a Robotic Marine Environmental Sampler with the Ruby Scripting Language

The Environmental Sample Processor (ESP) is an autonomous robotic instrument developed at the Monterey Bay Research Aquarium Institute (MBARI) that operates below the ocean's surface, sampling raw seawater and executing a variety of sample manipulation and analytical protocols, in situ. It uses DNA and antibody probes to identify marine planktonic organisms and substances they produce. Initial prototypes of the ESP were hosted on an Intel i486 CPU running a commercial real-time operating system (OS). The application, coded in C++, included a custom ‘macro’ language interpreter to direct biochemical analyses. To achieve greater flexibility and minimize the development effort for the 2nd generation of the ESP (2G ESP), MBARI replaced its ‘macro’ language with a general purpose, open-source scripting language, selecting Ruby for its unique combination of a succinct, English-like syntax with a seamless underlying object-oriented paradigm. The 2G ESP application, aside from custom servo control firmware, is coded entirely in Ruby, hosted on a low-power ARM9 CPU running Linux. Servo control was distributed onto a network of dedicated microcontrollers to cope with the nondeterministic delays inherent in the Linux operating system and Ruby interpreter.

Distinctive T Cell-suppressive Signals from Nuclearized Type 1 Sphingosine 1-Phosphate G Protein-coupled Receptors

Sphingosine 1-phosphate (S1P) generated by cells of innate immunity and the type 1 S1P G protein-coupled receptor (S1P(1)) on mobile T cells constitute a major system for control of lymphoid organ traffic and tissue migration of T cells. Now we show that T cell activation mediated by the T cell antigen receptor translocates plasma membrane S1P(1) to nuclear envelope membranes for association there with G(i/o), Erk (1/2), and other proteins that plasma membrane S1P(1) uses to signal T cell proliferation. However, nuclear S1P(1) and plasma membrane S1P(1) transduce opposite effects of S1P on T cell proliferation and relevant signaling as exemplified by respective decreases and increases in T cell nuclear concentrations of both phospho-Erk and active (phosphorylated) c-Jun. T cell antigen receptor-mediated activation of T cells therefore both eliminates migration responses to S1P by down-regulation of plasma membrane S1P(1) and translocates the S1P-S1P(1) axis into the nuclear domain where signals are directed to transcriptional control of immune functions other than migration.

Application of environmental sample processor (ESP) methodology for quantifying Pseudo‐nitzschia australis using ribosomal RNA‐targeted probes in sandwich and fluorescent in situ hybridization formats

In this contribution, we assess the application of methodology used in a novel in situ remote sampling platform, the environmental sample processor (ESP), for the identification and enumeration of a harmful algal species, the domoic acid‐producing diatom Pseudo‐nitzschia australis, using 2 molecular assays: a sandwich hybridization assay (SHA) and fluorescent in situ hybridization (FISH). Both the SHA and FISH assays were initially designed as laboratory‐based methods that are now emulated in the ESP. Response of the SHA to a range of concentrations of P. australis using the laboratory (96‐well plate) and ESP (DNA probe array) formats showed that the two were highly correlated. Enumeration of cells filtered and archived for FISH using a manifold designed for laboratory applications agreed well with counts of cells filtered and archived in the ESP at ≥2.5 × 104 cells L−1. At lower concentrations, it becomes statistically unlikely to derive a reliable abundance estimate, suggesting that FISH is better suited for qualitative analyses unless the target organism is very abundant. We also assessed the suitability of an oligonucleotide as synthetic target sequence to act as a SHA reagent quality control and internal standard for the plate assay. This was successful, but the probe and/or associated reagents were stable for only ~60 days. Our results show that the ESP is capable of detecting P. australis in near real‐time and also supports whole‐cell archival samples, making it a potentially useful tool for future research and monitoring initiatives.