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Background: Alzheimer's disease (AD) cannot currently be diagnosed by a blood test. One reason may be gender differences. Another may be the statistical methods used. The authors evaluate these possibilities. Objective: The authors applied serum lipidomics to find AD biomarkers in men and women. They hypothesized that AD biomarkers would differ between genders and that machine-learning algorithms would improve diagnostic performance. Methods: Serum lipids were analyzed by mass spectrometry for a training set of AD cases and controls and in a blinded test set. Statistical analyses considered gender differences. Results: Lipids best classifying AD subjects differed significantly between men and women. Robust statistical algorithms did not improve diagnostic performance. Conclusion: Poor performance of AD biomarkers appears to be due primarily to inherent variability in AD patients.

Imaging FlowCytobot, a submersible instrument that measures optical properties and captures images of nano- and microplankton-sized particles, has proved useful in plankton studies, but its sampling rate is limited by the ability of hydrodynamic focusing to accurately position flowing sample particles. We show that IFCB's sampling rate can be increased at least several-fold by implementing in-line acoustic focusing upstream of the flow cell. Particles are forced to the center of flow by acoustic standing waves created by a piezo-electric transducer bonded to the sample capillary and driven at the appropriate frequency. With the particles of interest confined to the center of the sample flow, the increased size of the sample core that accompanies increased sample flow rate no longer degrades image and signal quality as it otherwise would. Temperature affects the optimum frequency (through its effect on the speed of sound in water), so a relationship between sample temperature and optimum frequency for acoustic focusing was determined and utilized to control the transducer. The modified instrument's performance was evaluated through analyses of artificial particles, phytoplankton cultures, and natural seawater samples and through deployments in coastal waters. The results show that large cells, especially dinoflagellates, are acoustically focused extremely effectively (which could enable, for example, > 10-fold increased sampling rate of harmful algal bloom species, if smaller cells are ignored), while for nearly all cell types typically monitored by IFCB, threefold faster data accumulation was achieved without any compromises. Further increases are possible with more sophisticated software and/or a faster camera.

Flow cytometry provides highly sensitive multiparameter analysis of cells and particles but has been largely limited to the use of a single focused sample stream. This limits the analytical rate to ∼50K particles/s and the volumetric rate to ∼250 μL/min. Despite the analytical prowess of flow cytometry, there are applications where these rates are insufficient, such as rare cell analysis in high cellular backgrounds (e.g., circulating tumor cells and fetal cells in maternal blood), detection of cells/particles in large dilute samples (e.g., water quality, urine analysis), or high-throughput screening applications. Here we report a highly parallel acoustic flow cytometer that uses an acoustic standing wave to focus particles into 16 parallel analysis points across a 2.3 mm wide optical flow cell. A line-focused laser and wide-field collection optics are used to excite and collect the fluorescence emission of these parallel streams onto a high-speed camera for analysis. With this instrument format and fluorescent microsphere standards, we obtain analysis rates of 100K/s and flow rates of 10 mL/min, while maintaining optical performance comparable to that of a commercial flow cytometer. The results with our initial prototype instrument demonstrate that the integration of key parallelizable components, including the line-focused laser, particle focusing using multinode acoustic standing waves, and a spatially arrayed detector, can increase analytical and volumetric throughputs by orders of magnitude in a compact, simple, and cost-effective platform. Such instruments will be of great value to applications in need of high-throughput yet sensitive flow cytometry analysis.

Physical isolation of molecular computing elements holds the potential for increasing system complexity by enabling the reuse of standardized components and by protecting the components from environmental degradation. However, once elements have been compartmentalized, methods for communicating into these compartments are needed. We report the compartmentalization of steroid-responsive DNA aptamers within giant unilamellar vesicles (GUVs) that are permeable to steroid inputs. Monodisperse GUVs are loaded with aptamers using a microfluidic platform. We demonstrate the target-specific activation of individual aptamers within the GUVs and then load two noninterfering aptamers into the same GUV and demonstrate specific responses to all possible combinations of the two input steroids. Crucially, GUVs prevent the degradation of DNA components by nucleases, providing a potential mechanism for deploying nucleic acid components in vivo. Importantly, our compartments also prevent nonspecific cross-talk between complementary strands, thereby providing a method for parallel execution of cross-reacting molecular logic components. Thus, we provide a mechanism for spatially organizing molecular computing elements, which will increase system modularity by allowing standardized components to be reused.

Despite substantial research, the early diagnosis of preeclampsia remains elusive. Lipids are now recognized to be involved in regulation and pathophysiology of some disease. Shotgun lipidomic studies were undertaken to determine whether serum lipid biomarkers exist that predict preeclampsia later in the same in pregnancy. A discovery study was performed using sera collected at 12-14 weeks pregnancy from 27 controls with uncomplicated pregnancies and 29 cases that later developed preeclampsia. Lipids were extracted and analyzed by direct infusion into a TOF mass spectrometer. MS signals, demonstrating apparent differences were selected, their abundances determined, and statistical differences tested. Statistically significant lipid markers were reevaluated in a second confirmatory study having 43 controls and 37 preeclampsia cases. Multi-marker combinations were developed using those lipid biomarkers confirmed in the second study. The initial study detected 45 potential preeclampsia markers. Of these, 23 markers continued to be statistically significant in the second confirmatory set. Most of these markers, representing several lipid classes, were chemically characterized, typically providing lipid class and potential molecular components using MS(2) Several multi-marker panels with areas under the curve >0.85 and high predictive values were developed. Developed panels of serum lipidomic biomarkers appear to be able to identify most women at risk for preeclampsia in a given pregnancy at 12-14 weeks gestation.

We introduce a new method to construct microfluidic devices especially useful for bulk acoustic wave (BAW)-based manipulation of cells and microparticles. To obtain efficient acoustic focusing, BAW devices require materials that have high acoustic impedance mismatch relative to the medium in which the cells/microparticles are suspended and materials with a high-quality factor. To date, silicon and glass have been the materials of choice for BAW-based acoustofluidic channel fabrication. Silicon- and glass-based fabrication is typically performed in clean room facilities, generates hazardous waste, and can take several hours to complete the microfabrication. To address some of the drawbacks in fabricating conventional BAW devices, we explored a new approach by micromachining microfluidic channels in aluminum substrates. Additionally, we demonstrate plasma bonding of poly(dimethylsiloxane) (PDMS) onto micromachined aluminum substrates. Our goal was to achieve an approach that is both low cost and effective in BAW applications. To this end, we micromachined aluminum 6061 plates and enclosed the systems with a thin PDMS cover layer. These aluminum/PDMS hybrid microfluidic devices use inexpensive materials and are simply constructed outside a clean room environment. Moreover, these devices demonstrate effectiveness in BAW applications as demonstrated by efficient acoustic focusing of polystyrene microspheres, bovine red blood cells, and Jurkat cells and the generation of multiple focused streams in flow-through systems. Graphical abstract The aluminum acoustofluidic device and the generation of multinode focusing of particles.

Recognition of Pathogen-associated Molecular Patterns (PAMPs) by Toll-like receptors is central to innate immunity. Many bacterial PAMPs such as lipopolysaccharide (LPS) and lipoteichoic acid have amphiphilic properties. The hydrophobicity of amphiphilic PAMPs contributes to increasing entropy and causes these molecules to self-aggregate or bind host carrier proteins in aqueous physiological environments. The goal of this work was to determine how innate immune signaling is impacted by physical presentation and association of amphiphilic PAMPs with serum carrier proteins, using LPS as an example molecule. Specifically, we measured LPS-induced cytokine profiles in murine macrophages when the antigen was presented associated with the various serum carrier proteins in serum versus a serum-depleted system. Our study demonstrates that the observed cytokine profiles are dramatically different when LPS is presented in buffer, versus in serum when it is associated with proteins, specifically with respect to inhibition of pro-inflammatory cytokines in the latter. These studies suggest that LPS-mediated cytokine expression is dependent on its presentation in physiological systems. The amphiphilicity of bacterial PAMPs and consequent association with lipoproteins is a feature, which should be taken into account in the design of in vitro experiments. Further studies of the interdependencies of different serum carriers can identify pathways for drug delivery and diagnostics.

Background: Alzheimer’s disease (AD) remains challenging to diagnose, especially early disease. Having serum AD biomarkers would be of significant interest both in the clinical setting and in drug development efforts. Objective: We applied a novel serum proteomic approach to interrogate the low-molecular weight proteome for serum AD biomarkers. Methods: A discovery study used sera from 58 any-stage AD cases and 55 matched controls analyzed by capillary liquid chromatography-electrospray ionization-tandem mass spectrometry. Candidate biomarkers were statistically modeled and promising biomarkers were retested in a second, blinded confirmatory study (AD cases = 68, controls = 57). Biomarkers that replicated in the second study were modeled for the diagnosis of any-stage and very early stage AD. Further, they were chemically identified by tandem MS. Results: The initial discovery study found 59 novel potential AD biomarkers. Thirteen recurred in more than one multi-marker panel. In a second, blinded, confirmatory study, these same biomarkers were retested in separate specimens. In that study, four markers validated comparing controls to patients with any-stage AD and also with very early AD. The four biomarkers with replicable ability to diagnose AD were then chemically identified. Conclusion: These results suggest novel serum AD diagnostic biomarkers can be found using this approach.

Tissue proteomics has relied heavily on two-dimensional gel electrophoresis, for protein separation and quantification, then single protein isolation, trypsin digestion, and mass spectrometric protein identification. Such methods are predominantly used for study of high-abundance, full-length proteins. Tissue peptidomics has recently been developed but is still used to study the most highly abundant species, often resulting in observation and identification of dozens of peptides only. Tissue lipidomics is likewise new, and reported studies are limited. We have developed an "omics" approach that enables over 7,000 low-molecular-weight, low-abundance species to be surveyed and have applied this to human placental tissue. Because the placenta is believed to be involved in complications of pregnancy, its proteomic evaluation is of substantial interest. In previous research on the placental proteome, abundant, high-molecular-weight proteins have been studied. Application of large-scale, global proteomics or peptidomics to the placenta have been limited, and would be challenging owing to the anatomic complexity and broad concentration range of proteins in this tissue. In our approach, involving protein depletion, capillary liquid chromatography, and tandem mass spectrometry, we attempted to identify molecular differences between two regions of the same placenta with only slightly different cellular composition. Our analysis revealed 16 species with statistically significant differences between the two regions. Tandem mass spectrometry enabled successful sequencing, or otherwise enabled chemical characterization, of twelve of these. The successful discovery and identification of regional differences between the expression of low-abundance, low-molecular weight biomolecules reveals the potential of our approach.

Aim: We sought serum biomarkers predictive of pre-eclampsia (PE). Materials & methods: Sera obtained at 12–14 weeks of pregnancy from 24 cases who later developed PE and 24 controls with uncomplicated pregnancies were processed and analyzed using a serum proteomic approach. Results: Many statistically significant serum PE biomarker candidates (n > 60) were found comparing cases and controls. In addition, logistic regression analysis modeled biomarker data resulted in 14 different multimarker combinations having high detection sensitivity and specificity (AUC >0.9). Conclusions: Developed panels of serum biomarkers appeared effective in identifying pregnant women at 12–14 weeks gestation at risk of PE later in their pregnancy.

Pathogen detection is an important problem in many areas of medicine and agriculture, which can involve genomic or transcriptomic signatures or small-molecule metabolites. We report a unified, DNA-based sensor architecture capable of isothermal detection of double-stranded DNA targets, single-stranded oligonucleotides, and small molecules. Each sensor contains independent target detection and reporter modules, enabling rapid design. We detected gene variants on plasmids by using a straightforward isothermal denaturation protocol. The sensors were highly specific, even with a randomized DNA background. We achieved a limit of detection of ∼15 pM for single-stranded targets and ∼5 nM for targets on denatured plasmids. By incorporating a blocked aptamer sequence, we also detected small molecules using the same sensor architecture. This work provides a starting point for multiplexed detection of multi-strain pathogens, and disease states caused by genetic variants (e.g., sickle cell anemia).

Alzheimer's disease (AD) is a neurodegenerative disorder lacking early biochemical diagnosis and treatment. Lipids have been implicated in neurodegenerative disorders including AD. A shotgun lipidomic approach was undertaken to determine if lipid biomarkers exist that can discriminate AD cases from controls. The discovery study involved sera from 29 different stage AD cases and 32 controls. Lipid extraction was performed using organic solvent and the samples were directly infused into a time-of-flight mass spectrometer. Differences between AD cases and controls were detected with 87 statistically significant lipid candidate markers found. These potential lipid markers were reevaluated in a second confirmatory study involving 27 cases and 30 controls. Of the 87 candidates from the first study, 35 continued to be statistically significant in the second confirmatory set. Tandem MS studies were performed and almost all confirmed markers were characterized and classified. Using a Bayesian lasso probit regression model on the confirmed markers, a multi-marker set with AUC = 0.886 was developed comparing all stages of AD with controls. Additionally, using confirmed biomarkers, multi-marker sets with AUCs 0.90 were developed for each specific AD Clinical Dementia Rating versus controls, including the earliest stage of AD. More conservative and likely more realistic statistical analyses still found multi-marker sets that appeared useful in diagnosing AD. Finally, using ordinal modeling a set of markers was developed that staged AD accurately 70% of the time, p = 0.0079. These results suggest that these serum lipidomic biomarkers may help diagnose and perhaps even stage AD.

Most druggable targets are membrane components, including membrane proteins and soluble proteins that interact with ligands or receptors embedded in membranes. Current target-based screening and intermolecular interaction assays generally do not include the lipid membrane environment in presenting these targets, possibly altering their native structure and leading to misleading or incorrect results. To address this issue, an ideal assay involving membrane components would (1) mimic the natural membrane environment, (2) be amenable to high-throughput implementation, and (3) be easily multiplexed. In a step toward developing such an ideal target-based analytical assay for membrane components, we present fluorescently indexed multiplexed biomimetic membrane assays amenable to high-throughput flow cytometric detection. We build fluorescently multiplexed biomimetic membrane assays by using varying amounts of a fluorescently labeled lipid, NBD-DOPE [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)], incorporated into a phospholipid membrane bilayer supported on 3 μm silica microspheres. Using flow cytometry, we demonstrate this multiplexed approach by measuring specific affinity of two well-characterized systems, the fluorescently labeled soluble proteins cholera toxin B subunit-Alexa 647 and streptavidin-PE/Cy5, to membranes containing different amounts of ligand targets of these proteins, GM1 and biotin-DOPE, respectively. This work will enable future efforts in developing highly efficient biomimetic assays for interaction analysis and drug screening involving membrane components.

Lipopolysaccharide (LPS) is an amphiphilic lipoglycan that is the primary component of the outer membrane of Gramnegative bacteria. Classified as a pathogen associated molecular pattern (PAMPs), LPS is an essential biomarker for identifying pathogen serogroups. Structurally, LPS is comprised of a hydrophobic lipophilic domain that partitions into the outer membrane of Gram-negative bacteria. Previous work by our team explored biophysical interactions of LPS in supported lipid bilayer assemblies (sLBAs), and demonstrated LPS-induced hole formation in DOPC lipid bilayers. Here, we have incorporated cholesterol and sphingomyelin into sLBAs to evaluate the interaction of LPS in a more physiologically relevant system. The goal of this work was to determine whether increasing membrane complexity of sLBAs, and changing physiological parameters such as temperature, affects LPS-induced hole formation. Integrating cholesterol and sphingomyelin into sLBAs decreased LPS-induced hole formation at lower concentrations of LPS, and bacterial serotype contributed to differences in hole formation as a response to changes in temperature. We also investigated the possibility of LPS-induced hole formation in cellular systems using the cytokine response in both TLR4 (+)/(-) murine macrophages. LPS was presented to each cell line in murine serum, delipidated serum, and buffer (i.e. no serum), and the resulting cytokine levels were measured. Results indicate that the method of LPS presentation directly affects cellular cytokine expression. The two model systems presented in this study provide preliminary insight into the interactions of LPS in the host, and suggest the significance of amphiphile-carrier interactions in regulating host-pathogen biology during infection.

A modern flow cytometer can analyze and sort particles on a one by one basis at rates of 50,000 particles per second. Flow cytometers can also measure as many as 17 channels of fluorescence, several angles of scattered light, and other non-optical parameters such as particle impedance. More specialized flow cytometers can provide even greater analysis power, such as single molecule detection, imaging, and full spectral collection, at reduced rates. These capabilities have made flow cytometers an invaluable tool for numerous applications including cellular immunophenotyping, CD4+ T-cell counting, multiplex microsphere analysis, high-throughput screening, and rare cell analysis and sorting. Many bio-analytical techniques have been influenced by the advent of microfluidics as a component in analytical tools and flow cytometry is no exception. Here we detail the functions and uses of a modern flow cytometer, review the recent and historical contributions of microfluidics and microfabricated devices to field of flow cytometry, examine current application areas, and suggest opportunities for the synergistic application of microfabrication approaches to modern flow cytometry.

1. Isoeugenol (IEG) has been tested for toxicity and carcinogenicity due to high potential for human exposure and the structural resemblance to known carcinogenic allylbenzenes. In order to support the interpretation of toxicity and carcinogenecity study outcomes, a toxicokinetic study was performed in which both sexes of F344 rats and B6C3F1 mice were given IEG as a single intravenous (IV) or gavage administration. 2. Following IV administration, IEG was rapidly eliminated from systemic circulation in both species and sexes. Gavage administration revealed a rapid absorption of IEG with tmax values ≤20 min for both species and sexes. In rats, AUC increased in a greater than dose-proportional manner and Clapp values decreased with increasing dose in both sexes suggesting saturation of IEG metabolism. On the other hand, Clapp values in male mice increased with increasing dose suggesting induction of IEG metabolism although this was not evident in the females. 3. Absolute bioavailability was greater in female rats (19%) than male rats (10%) (p < 0.0001), but was not different between the sexes for mice (28% males; 31% females) (p = 0.2437). The collective toxicokinetic data supported that low bioavailability following administration of IEG was the result of extensive first-pass metabolism.

Tetravalent (VIV) and pentavalent (VV) forms of vanadium were selected for testing by the National Toxicology Program via drinking water exposure due to potential human exposure. To aid in the test article selection, drinking water formulations (125–2000 mg/L) of vanadyl sulfate (VIV), sodium orthovanadate, and sodium metavanadate (VV) were characterized by ultraviolet/visible (UV/VIS) spectroscopy, mass spectrometry (MS), or 51V nuclear magnetic resonance (NMR) spectroscopy. Aqueous formulations of orthovanadate, metavanadate, and vanadyl sulfate in general were basic, neutral, and acidic, respectively. Changes in vanadium speciation were investigated by adjusting formulation pH to acidic, neutral, or basic. There was no visible difference in UV/VIS spectra of pentavalent forms. NMR and MS analyses showed that the predominant oxidovanadate species in both ortho- and metavanadate formulations at basic and acidic pH, respectively, were the monomer and decamer, while, a mixture of oxidovanadates were present at neutral pH. Oxidovanadate species were not observed in vanadyl sulfate formulations at acidic pH but were observed at basic pH suggesting conversion of VIV to VV. These data suggest that formulations of both ortho- and metavanadate form similar oxidovanadate species in acidic, neutral and basic pH and exist mainly in the VV form while vanadyl sulfate exists mainly as VIV in acidic pH. Therefore, the formulation stability overtime was investigated only for sodium metavanadate and vanadyl sulfate. Drinking water formulations (50 and 2000 mg/L) of metavanadate (~pH 7) and vanadyl sulfate (~pH 3.5) were ≥92 % of target concentration up to 42 days at ~5 °C and ambient temperature demonstrating the utility in toxicology studies.

1. Methyleugenol (MEG) has been used as a flavouring agent in food, as a fragrance in cosmetic products, and as an insect attractant. MEG was carcinogenic in both rats and mice following gavage administration. In this study we investigated plasma toxicokinetics of MEG in F344 rats and B6C3F1 mice of both sexes following single gavage (37, 75, or 150 mg/kg) and intravenous (IV) (37 mg/kg) administration.2. Following IV administration, MEG was rapidly distributed and cleared from the systemic circulation in both species and sexes. Absorption of MEG was rapid following gavage administration with secondary peaks in the plasma MEG concentration-versus-time profiles. Cmax and AUCT increased and the clearance decreased greater than proportional to the dose in rats and mice of both sexes. In general, rats had higher internal exposure to MEG than mice.3. The results for AUCT and clearance suggest that perhaps the metabolism of MEG is saturated at higher doses tested in this study. Absolute bioavailability followin...

A general strategy for controlling particle movement across streams would enable new capabilities in single-cell analysis, solid-phase reaction control, and biophysics research. Transferring cells across streams is difficult to achieve in a well-controlled manner, since it requires precise control of fluid flow along with external force fields or precisely manufactured mechanical structures. Herein a strategy is introduced for particle transfer based on passive inertial lift forces and shifts in the distribution of these forces for channels with shifting aspect ratios. Uniquely, use of the dominant wall-effect lift parallel to the particle rotation direction is explored and utilized to achieve controllable cross-stream motion. In this way, particles are positioned to migrate across laminar streams and enter a new solution without significant disturbance of the interface at rates exceeding 1000 particles per second and sub-millisecond transfer times. The capabilities of rapid inertial solution exchange (RInSE) for preparation of hematological samples and other cellular assays are demonstrated. Lastly, improvements to inline flow cytometry after RInSE of excess fluorescent dye and focusing for downstream analysis are characterized. The described approach is simply applied to manipulating cells and particles and quickly exposing them to or removing them from a reacting solution, with broader applications in control and analysis of low affinity interactions on cells or particles.

Flow cytometry can simultaneously measure and analyze multiple properties of single cells or particles with high sensitivity and precision. Yet, conventional flow cytometers have fundamental limitations with regards to analyzing particles larger than about 70 microns, analyzing at flow rates greater than a few hundred microliters per minute, and providing analysis rates greater than 50,000 per second. To overcome these limits, we have developed multi-node acoustic focusing flow cells that can position particles (as small as a red blood cell and as large as 107 microns in diameter) into as many as 37 parallel flow streams. We demonstrate the potential of such flow cells for the development of high throughput, parallel flow cytometers by precision focusing of flow cytometry alignment microspheres, red blood cells, and the analysis of CD4+ cellular immunophenotyping assay. This approach will have significant impact towards the creation of high throughput flow cytometers for rare cell detection applications (e.g. circulating tumor cells), applications requiring large particle analysis, and high volume flow cytometry.

Anthrax lethal toxin (LT) is a major virulence factor of Bacillus anthracis. The vast majority of the anthrax toxin-related literature describes the assembly of LT as a cell-dependent process. However, some reports have provided evidence for the existence of a fully assembled LT, either in vitro or in the bloodstream of anthrax-infected animals. To follow up on this work, we present studies on fully-assembled LT. We first demonstrate facile and cell-free assembly and purification of LT. We then show that fully assembled LT binds an anthrax toxin receptor with almost 100-fold higher affinity than the protective antigen (PA) alone. Quantitative cell intoxication assays were used to determine the LD(50) (lethal dose 50) for LT. The cell-binding studies revealed that LT binds mammalian cells using a different mode from PA. Even when PA-specific receptors were blocked, fully assembled LT was able to bind the cell surface. Our studies support the existing evidence that LT fully assembles in the blood stream and can bind and intoxicate mammalian cells with very high affinity and efficacy. More importantly, the data presented here invoke the possibility that LT may bind cells in a receptor-independent fashion, or recognize receptors that do not interact with PA. Hence, blood borne LT may emerge as a novel therapeutic target for combating anthrax.

Objective. To test utility of cystatin-C as a marker of glomerular filtration rate during pregnancy, we performed serial correlations with inulin clearance during pregnancy and postpartum. Methods. Twelve subjects received inulin infusions and serum cystatin-C at three time points. Pearson's correlation coefficient was calculated. Results. Cystatin-C levels ranged 0.66–1.48 mg/L during pregnancy, and 0.72–1.26 mg/L postpartum. Inulin clearance ranged 130–188 mL/min during pregnancy, and 110–167 mL/min postpartum. Cystatin-C did not correlate with inulin clearance at any time point. Conclusion. Serum cystatin-C did not correlate with inulin clearance during pregnancy or postpartum.

In the National Toxicology Program’s toxicity studies, rats were more sensitive than mice to Bis(2-chloroethoxy)methane (CEM) – induced cardiac toxicity following dermal application to male and female F344/N rats and B6C3F1 mice. Thiodiglycolic acid (TDGA) is a major metabolite of CEM in rats. It has been implicated that chemicals metabolized to TDGA cause cardiac toxicity in humans. Therefore, the toxicokinetics of CEM and TDGA were investigated in male and female F344/N rats and B6C3F1 mice following a single intravenous administration or dermal application of CEM to aid in the interpretation of the toxicity data. Absorption of CEM following dermal application was rapid in both species and genders. Bioavailability following dermal application was low but was higher in rats than in mice with females of both species showing higher bioavailability than males. CEM was rapidly distributed to the heart, thymus, and liver following both routes of administration. Plasma CEM C(max) and AUC(∞) increased proportionally with dose, although at the dermal dose of 400 mg/kg in rats and 600 mg/kg in mice non-linear kinetics were apparent. Following dermal application, dose-normalized plasma CEM C(max) and AUC(∞) was significantly higher in rats than in mice (p-value < 0.0001 for all comparisons except for C(max) in the highest dose groups where p-value = 0.053). In rats, dose-normalized plasma CEM C(max) and AUC(∞) was higher in females than in males: however, the difference was significant only at the lowest dose (p-value = 0.009 for C(max) and 0.056 for AUC(∞)). Similar to rats, female mice also showed higher C(max) and AUC(∞) in females than in male: the difference was significant only for C(max) at the lowest dose (p-value = 0.002). Dose-normalized heart CEM C(max) was higher in rats than in mice and in females than their male counterparts. The liver CEM C(max) was lower compared to that of heart and thymus in both rats and mice following intravenous administration and in rats following dermal application. This is likely due to the rapid metabolism of CEM in the liver as evidenced by the high concentration of TDGA measured in the liver. Dose-normalized plasma and heart TDGA C(max) values were higher in rats compared to mice. In rats, females had higher plasma and heart TDGA C(max) than males; however, there was no gender difference in plasma or heart TDGA C(max) in mice. These findings support the increased sensitivity of rats compared to mice to CEM-induced cardiac toxicity. Data also suggest that, either CEM C(max) or AUC can be used to predict the CEM-induced cardiac toxicity. Although, both plasma and heart TDGA C(max) was consistent with the observed species difference and the gender difference in rats, the gender difference in mice to cardiac toxicity could not be explained based on the TDGA data. This animal study suggests that toxicologically significant concentrations of CEM and TDGA could possibly be achieved in the systemic circulation and/or target tissues in humans as a result of dermal exposure to CEM.

Mechanisms mediating preeclampsia (PE) are unclear. Endogenous digitalis-like factors (EDLFs) are sodium pump (SP) inhibitors implicated in essential hypertension, but not fully explored in PE. This study asks whether EDLFs are present and increased in PE and considers their source.EDLF in sera and placentas from third trimester women with uncomplicated pregnancies or PE was assessed by a Rb(+) uptake assay. A digoxin antibody Fab fragment (Digibind) known to inactivate EDLFs was also used to assess EDLFs.PE serum caused significantly more SP inhibition than serum from uncomplicated pregnancies. This inhibition was concentration-dependent and reversed by Digibind. Serum from uncomplicated pregnancies showed no concentration-dependence or reversal with Digibind. Placental homogenates from control women showed little SP inhibition, but homogenates from PE women showed marked SP inhibition reversed by Digibind.These studies evidence EDLF in PE serum. Additionally, PE placentas have high EDLF and may represent a source.

Acoustic standing waves can precisely focus flowing particles or cells into tightly positioned streams for interrogation or downstream separations. The efficiency of an acoustic standing wave device is dependent upon operating at a resonance frequency. Small changes in a system's temperature and sample salinity can shift the device's resonance condition, leading to poor focusing. Practical implementation of an acoustic standing wave system requires an automated resonance control system to adjust the standing wave frequency in response to environmental changes. Here we have developed a rigorous approach for quantifying the optimal acoustic focusing frequency at any given environmental condition. We have demonstrated our approach across a wide range of temperature and salinity conditions to provide a robust characterization of how the optimal acoustic focusing resonance frequency shifts across these conditions. To generalize these results, two microfluidic bulk acoustic standing wave systems (a steel capillary and an etched silicon wafer) were examined. Models of these temperature and salinity effects suggest that it is the speed of sound within the liquid sample that dominates the resonance frequency shift. Using these results, a simple reference table can be generated to predict the optimal resonance condition as a function of temperature and salinity. Additionally, we show that there is a local impedance minimum associated with the optimal system resonance. The integration of the environmental results for coarse frequency tuning followed by a local impedance characterization for fine frequency adjustments, yields a highly accurate method of resonance control. Such an approach works across a wide range of environmental conditions, is easy to automate, and could have a significant impact across a wide range of microfluidic acoustic standing wave systems.

Nanoparticle spectroscopic tags based on surface enhanced Raman scattering (SERS) are playing an increasingly important role in bioassay and imaging applications. The ability to rapidly characterize large populations of such tags spectroscopically in a high-throughput flow-based platform will open new areas for their application and provide new tools for advancing their development. We demonstrate here a high-resolution spectral flow cytometer capable of acquiring Raman spectra of individual SERS-tags at flow rates of hundreds of particles per second, while maintaining the spectral resolution required to make full use of the detailed information encoded in the Raman signature for advanced multiplexing needs. The approach allows multiple optical parameters to be acquired simultaneously over thousands of individual nanoparticle tags. Characteristics such as tag size, brightness, and spectral uniformity are correlated on a per-particle basis. The tags evaluated here display highly uniform spectral signatures, but with greater variability in brightness. Subpopulations in the SERS response, not apparent in ensemble measurements, are also shown to exist. Relating tag variability to synthesis parameters makes flow-based spectral characterization a powerful tool for advancing particle development through its ability to provide rapid feedback on strategies aimed at constraining desired tag properties. Evidence for single-tag signal saturation at high excitation power densities is also shown, suggesting a role for high-throughput investigation of fundamental properties of the SERS tags as well.

We have developed a rapid (under 4 hours), multiplex, nucleic acid assay, adapted to a microsphere array detection platform. We call this assay multiplex oligonucleotide ligation-PCR (MOL-PCR). Unlike other ligation-based assays that require multiple steps, our protocol consists of a single tube reaction, followed by hybridization to a Luminex microsphere array for detection. We demonstrate the ability of this assay to simultaneously detect diverse nucleic acid signatures (e.g., unique sequences, single nucleotide polymorphisms) in a single multiplex reaction. Detection probes consist of modular components that enable target detection, probe amplification, and subsequent capture onto microsphere arrays. To demonstrate the utility of our assay, we applied it to the detection of three biothreat agents, B. anthracis, Y. pestis, and F. tularensis. Combined with the ease and robustness of this assay, the results presented here show a strong potential of our assay for use in diagnostics and surveillance.

Fully digital data acquisition systems for use in flow cytometry provide excellent flexibility and precision. Here, we demonstrate the development of a low cost, small, and low power digital flow cytometry data acquisition system using a single microcontroller chip with an integrated analog to digital converter (ADC). Our demonstration system uses a commercially available evaluation board making the system simple to integrate into a flow cytometer. We have evaluated this system using calibration microspheres analyzed on commercial, slow-flow, and CCD-based flow cytometers. In our evaluations, our demonstration data system clearly resolves all eight peaks of a Rainbow microsphere set on both a slow-flow flow cytometer and a retrofitted BD FACScalibur, which indicates it has the sensitivity and resolution required for most flow cytometry applications. It is also capable of millisecond time resolution, full waveform collection, and selective triggering of data collection from a CCD camera. The capability of our demonstration system suggests that the use of microcontrollers for flow cytometry digital data-acquisition will be increasingly valuable for extending the life of older cytometers and provides a compelling data-system design approach for low-cost, portable flow cytometers.

The low-abundance, low molecular weight serum proteome has high potential for the discovery of new biomarkers using mass spectrometry (MS). Because the serum proteome is large and complex, defining relative quantitative differences for a molecular species between comparison groups requires an approach with robust separation capability, high sensitivity, as well as high mass resolution. Capillary liquid chromatography (cLC)/MS provides both the necessary separation technique and the sensitivity to observe many low-abundance peptides. Subsequent identification of potential serum peptide biomarkers observed in the cLC/MS step can in principle be accomplished by in series cLC/MS/MS without further sample preparation or additional instrumentation. In this report a novel cLC/MS/MS method for peptide sequencing is described that surpasses previously reported size limits for amino acid sequencing accomplished by collisional fragmentation using a tandem time-of-flight MS instrument. As a demonstration of the approach, two low-abundance peptides with masses of approximately 4000-5000 Da were selected for MS/MS sequencing. The multi-channel analyzer (MCA) was used in a novel way that allowed for summation of 120 fragmentation spectra for each of several customized collision energies, providing more thorough fragmentation coverage of each peptide with improved signal to noise. The peak list from this composite analysis was submitted to Mascot for identification. The two index peptides, 4279 Da and 5061 Da, were successfully identified. The peptides were a 39 amino acid immunoglobulin G heavy chain variable region fragment and a 47 amino acid fibrin alpha isoform C-terminal fragment. The method described here provides the ability both to survey thousands of serum molecules and to couple that with markedly enhanced cLC/MS/MS peptide sequencing capabilities, providing a promising technique for serum biomarker discovery.

A digital signal processing (DSP)-based digital data acquisition system has been developed to support novel flow cytometry efforts. The system flexibility includes how it detects, captures, and processes event data. Custom data capture boards utilizing analog to digital converters (ADCs) and field programmable gate arrays (FPGA) detect events and capture correlated event data. A commercial DSP board processes the captured data and sends the results over the IEEE 1394 bus to the host computer that provides a user interface for acquisition, display, analysis, and storage. The system collects list mode data, correlated pulse shapes, or streaming data from a variety of detector types using Linux, Mac OS X, and Windows host computers. It extracts pulse features not found on commercial systems with excellent sensitivity and linearity over a wide dynamic range. List mode data are saved in FCS 3.0 formatted files while streaming or correlated waveform data are saved in custom format files for postprocessing. Open, reconfigurable cytometric acquisition system is compact, scaleable, flexible, and modular. Programmable feature extraction algorithms have exciting possibilities for both new and existing applications. The recent availability of a commercial data capture board will enable general availability of similar systems. © Published 2007 Wiley-Liss, Inc.

We investigated the response of the hydrocarbon-degrading Mycobacterium vanbaalenii PYR-1 to crude oil from the BP Deepwater Horizon (DWH) spill, using substrate depletion, genomic, and proteome analyses. M. vanbaalenii PYR-1 cultures were incubated with BP DWH crude oil, and proteomes and degradation of alkanes and polycyclic aromatic hydrocarbons (PAHs) were analyzed at four time points over 30 days. Gas chromatography-mass spectrometry (GC-MS) analysis showed a chain length-dependent pattern of alkane degradation, with C 12 and C 13 being degraded at the highest rate, although alkanes up to C 28 were degraded. Whereas phenanthrene and pyrene were completely degraded, a significantly smaller amount of fluoranthene was degraded. Proteome analysis identified 3,948 proteins, with 876 and 1,859 proteins up- and downregulated, respectively. We observed dynamic changes in protein expression during BP crude oil incubation, including transcriptional factors and transporters potentially involved in adaptation to crude oil. The proteome also provided a molecular basis for the metabolism of the aliphatic and aromatic hydrocarbon components in the BP DWH crude oil, which included upregulation of AlkB alkane hydroxylase and an expression pattern of PAH-metabolizing enzymes different from those in previous proteome expression studies of strain PYR-1 incubated with pure or mixed PAHs, particularly the ring-hydroxylating oxygenase (RHO) responsible for the initial oxidation of aromatic hydrocarbons. Based on these results, a comprehensive cellular response of M. vanbaalenii PYR-1 to BP crude oil was proposed. This study increases our fundamental understanding of the impact of crude oil on the cellular response of bacteria and provides data needed for development of practical bioremediation applications.

Certain Shiga toxin-producing Escherichia coli (STEC) are virulent human pathogens that are most often acquired through contaminated food. The United States Department of Agriculture, Food Safety and Inspection Service has declared several serogroups of STEC as adulterants in non-intact raw beef products. Hence, sensitive and specific tests for the detection of these STEC are a necessity for implementation in food safety programs. E. coli serogroups are identified by their respective O-antigen moiety on the lipopolysaccharide (LPS) macromolecule. We propose that the development of O-antigen-specific immunological assays can facilitate simple and rapid discriminatory detection of STEC in beef. However, the resources (antigens and antibodies) required for such development are not readily available. To overcome this, we extracted and characterized LPS and O-antigen from six STEC strains. Using hot phenol extraction, we isolated the LPS component from each strain and purified it using a series of steps to eliminate proteins, nucleic acids, and lipid A antigens. Antigens and crude LPS extracts were characterized using gel electrophoresis, immunoblotting, and modified Western blotting with commercially available antibodies, thus assessing the serogroup specificity and sensitivity of available ligands as well. The results indicate that, while many commercially available antibodies bind LPS, their activities and specificities are highly variable, and often not as specific as those required for serogroup discrimination. This variability could be minimized by the production of antibodies specific for the O-antigen. Additionally, the antigens generated from this study provide a source of characterized LPS and O-antigen standards for six serogroups of STEC.

Shiga toxin-producing Escherichia coli (STEC) poses a serious threat to human health through the consumption of contaminated food products, particularly beef and produce. Early detection in the food chain, and discrimination from other non-pathogenic Escherichia coli (E. coli), is critical to preventing human outbreaks, and meeting current agricultural screening standards. These pathogens often present in low concentrations in contaminated samples, making discriminatory detection difficult without the use of costly, time-consuming methods (e.g. culture). Using multiple signal transduction schemes (including novel optical methods designed for amphiphiles), specific recognition antibodies, and a waveguide-based optical biosensor developed at Los Alamos National Laboratory, we have developed ultrasensitive detection methods for lipopolysaccharides (LPS), and protein biomarkers (Shiga toxin) of STEC in complex samples (e.g. beef lysates). Waveguides functionalized with phospholipid bilayers were used to pull down amphiphilic LPS, using methods (membrane insertion) developed by our team. The assay format exploits the amphiphilic biochemistry of lipoglycans, and allows for rapid, sensitive detection with a single fluorescent reporter. We have used a combination of biophysical methods (atomic force and fluorescence microscopy) to characterize the interaction of amphiphiles with lipid bilayers, to efficiently design these assays. Sandwich immunoassays were used for detection of protein toxins. Biomarkers were spiked into homogenated ground beef samples to determine performance and limit of detection. Future work will focus on the development of discriminatory antibodies for STEC serotypes, and using quantum dots as the fluorescence reporter to enable multiplex screening of biomarkers.

We quantitatively measured protective antigen (PA) binding to human cells targeted by anthrax lethal toxin (LT). Affinities were less than 50nM for all cells, but differentiated cells (macrophages and neutrophils) had significantly increased PA binding and endothelial cells demonstrated the most binding. Combined with the function of such cells, this suggests that PA receptors interact with the extracellular matrix and that differentiation increases the number of PA-specific receptors, which supports previously observed differentiation-induced LT susceptibility. Our results quantifiably confirm that the generality of PA binding will complicate its use as a tumor targeting agent.

We present preliminary results for a magnetic flow spectrometer for magnetic microparticle separation, and a magnetic flow cytometer for particle identification. The application of the instrument is to high-throughput bioassay. Here we report on the first application of our magnetic spectrometer to the sorting of ferromagnetic and superparamagnetic microspheres in flow. The system is based on a permanent magnet quadrupole and separates the polymer coated magnetic microspheres based on their magnetic moments. The cryogenic section of our magnetic flow cytometer, which involves SQUID-based detection of the sorted magnetic microspheres based on their magnetic moments, has been re-engineered to permit a smaller standoff between the SQUID array and the flowing magnetic particles. We present preliminary results with the new experimental setup, with an emphasis on both spatial and signal resolution.

Background: While conventional multiparameter flow cytometers have proven highly successful, there are several types of analytical measurements that would benefit from a more comprehensive and flexible approach to spectral analysis including, but certainly not limited to spectral deconvolution of overlapping emission spectra, fluorescence resonance energy transfer measurements, metachromic dye analysis, free versus bound dye resolution, and Raman spectroscopy. Methods: Our system utilizes a diffraction grating to disperse the collected fluorescence and side-scattered light from cells or microspheres passing through the interrogation region over a rectangular charge-coupled-device image sensor. The flow cell and collection optics are taken from a conventional flow cytometer with minimal modifications to assure modularity of the system. Results: Calibration of the prototype spectral analysis flow cytometer included wavelength characterization and calibration of the dispersive optics. Benchmarking of the system demonstrated a single particle/cell intensity sensitivity of 2160 MESF of R-Phycoerythrin. Single particle spectra taken with our instrument were validated against bulk solution fluorimeter and conventional flow cytometer measurements. Coefficients of variation of integrated spectral fluorescence intensity of several sets of standard fluorescent microspheres ranged from 1.4 to 4.8% on the spectral system. Spectral discrimination of free versus PI bound to cells is also demonstrated. Conclusions: It is demonstrated that the flow spectrometer has sufficient sensitivity and wavelength resolution to detect single cells and microspheres, including multi-fluorophore labeled microspheres. The capability to use both standard mathematical deconvolution techniques for data analysis, coupled with the feasibility of integration with existing flow cytometers, will improve the accuracy and precision of ratiometric measurements, enable the analysis of more discrete emission bands within a given wavelength range, and allow more precise resolution of the relative contribution of individual fluorophores in multiply-tagged samples, thereby enabling a range of new applications involving the spectral analysis of single cells and particles. © 2006 International Society for Analytical Cytology

Background General methods for accurate determination of microsphere surface protein loading are needed for applications from protein arrays to molecular assembly studies. Current methods include bulk absorption measurements of stained microspheres or use of known fluorescently tagged binding partners, which limit sensitivity and general applicability, respectively. Methods Microspheres bearing covalently coupled proteins were stained with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) using different incubation times and dye concentrations to determine optimal staining conditions. The CBQCA fluorescence of microspheres (measured by flow cytometry) bearing known amounts of protein were used to generate standard curves of CBQCA fluorescence response versus the amount of microsphere surface protein. CBQCA was also used to stain noncovalent protein interactions. Results Maximal labeling was attained within 1 h with 1 mM CBQCA. Linear fluorescence response occurred between 8 × 104 and 1 × 106 proteins/microsphere. CBQCA staining did not disturb noncovalent protein interactions. Conclusions We have developed methods using CBQCA and flow cytometry to quickly and simply quantify the amount of protein on the surface of a microsphere. Importantly, this approach could be extended to other formats (e.g., chips). Further, because it does not disturb noncovalent protein–protein interactions, it may be possible to use this approach to detect protein interactions without the use of purified prestained probes. Published 2005 Wiley-Liss, Inc.

During normal pregnancy, studies have shown increased activity of the renin-angiotensin-aldosterone system (RAAS) and a dissociation of plasma renin activity (PRA) and aldosterone (Aldo) evidenced by a greater increase in Aldo relative to PRA. The aims of this study were to examine the RAAS response to stimulation by upright posture and suppression by saline infusion and to investigate the PRA-Aldo dissociation under these two conditions.We studied 24 healthy normotensive women (mean+/-standard error of mean, ages 29+/-1 yrs) in sodium (Na) balance in the second and third trimesters and postpartum. Subjects underwent a 24-hour urine collection which was analyzed for Na, norepinephrine (NE), epinephrine (Epi), and dopamine (DA); a posture study with analysis of blood pressure (BP), PRA, Aldo, NE, Epi, DA, and cortisol; and a 0.9% NaCl infusion study (500 mL/hr for 3 hrs) with analysis of BP, PRA, Aldo, cortisol, and digitalis-like factor (DLF). Analyses included paired t tests to compare posture and saline responses, repeated measures to compare across periods, and percent change to evaluate the PRA-Aldo dissociation.During pregnancy, PRA, Aldo, BP, catecholamines, and cortisol levels were significantly greater in upright than left lateral decubitus (LLD) posture, and the percent change in Aldo was significantly greater than the percent change in PRA. During pregnancy in response to saline infusion, BP did not change; the PRA and Aldo significantly decreased; the percent change in Aldo was significantly greater than the percent change in PRA in the second trimester; and serum DLF and cortisol levels significantly decreased.In longitudinally studied normal pregnancy, PRA and Aldo levels were dissociated at baseline, with stimulation and, to a lesser degree, with suppression. Norepinephrine, adrenocorticotrophic hormone, and DLF may contribute to this dissociation, and clarification of these interactions may provide insight into the regulation of aldosterone during normal and hypertensive pregnancy.

trans-Cinnamaldehyde is a widely used natural ingredient that is added to foods and cosmetics as a flavoring and fragrance agent. Male and female F344/N rats and B6C3F(1) mice were exposed to microencapsulated trans-cinnamaldehyde in the feed for three months or two years. All studies included untreated and vehicle control groups. In the three-month studies, rats and mice were given diets containing 4100, 8200, 16,500, or 33,000 ppm trans-cinnamaldehyde. In rats, feed consumption was reduced in all exposed groups. In mice, feed consumption was reduced in the highest dose groups. Body weights of all treated males were less than controls. Body weights were reduced in female rats exposed to 16,500 or 33,000 ppm and female mice exposed to 8200 ppm or greater. All rats survived to the end of the study but some male mice in the highest dose groups died due to inanition from unpalatability of the dosed feed. The incidence of squamous epithelial hyperplasia of the forestomach was significantly increased in rats exposed to 8200 ppm or greater and female mice exposed to 33,000 ppm. In mice, the incidence of olfactory epithelial degeneration of the nasal cavity was significantly increased in males and females exposed to 16,500 ppm and females exposed to 33,000 ppm. In the two-year studies, rats and mice were exposed to 1000, 2100, or 4100 ppm trans-cinnamaldehyde. Body weights were reduced in mice exposed to 2100 ppm and in rats and mice exposed to 4100 ppm. In rats, hippuric acid excretion was dose proportional indicating that absorption, metabolism, and excretion were not saturated. No neoplasms were attributed to trans-cinnamaldehyde in rats or mice. Squamous cell papillomas and carcinomas of the forestomach were observed in male and female mice but the incidences were within the NTP historical control range and were not considered to be related to trans-cinnamaldehyde exposure.