Negative feedback control of neuronal activity by microglia.

Microglia, the brain's resident macrophages, help to regulate brain function by removing dying neurons, pruning non-functional synapses, and producing ligands that support neuronal survival1. Here we show that microglia are also critical modulators of neuronal activity and associated behavioural responses in mice. Microglia respond to neuronal activation by suppressing neuronal activity, and ablation of microglia amplifies and synchronizes the activity of neurons, leading to seizures. Suppression of neuronal activation by microglia occurs in a highly region-specific fashion and depends on the ability of microglia to sense and catabolize extracellular ATP, which is released upon neuronal activation by neurons and astrocytes. ATP triggers the recruitment of microglial protrusions and is converted by the microglial ATP/ADP hydrolysing ectoenzyme CD39 into AMP; AMP is then converted into adenosine by CD73, which is expressed on microglia as well as other brain cells. Microglial sensing of ATP, the ensuing microglia-dependent production of adenosine, and the adenosine-mediated suppression of neuronal responses via the adenosine receptor A1R are essential for the regulation of neuronal activity and animal behaviour. Our findings suggest that this microglia-driven negative feedback mechanism operates similarly to inhibitory neurons and is essential for protecting the brain from excessive activation in health and disease.

Repeated Methamphetamine Administration Results in Axon Loss Prior to Somatic Loss of Substantia Nigra Pars Compacta and Locus Coeruleus Neurons in Male but Not Female Mice.

Methamphetamine (meth) is a neurotoxic psychostimulant that increases monoamine oxidase (MAO)-dependent mitochondrial oxidant stress in axonal but not somatic compartments of substantia nigra pars compacta (SNc) and locus coeruleus (LC) neurons. Chronic meth administration results in the degeneration of SNc and LC neurons in male mice, and MAO inhibition is neuroprotective, suggesting that the deleterious effects of chronic meth begin in axons before advancing to the soma of SNc and LC neurons. To test this hypothesis, mice were administered meth (5 mg/kg) for 14, 21, or 28 days, and SNc and LC axonal lengths and numbers of neurons were quantified. In male mice, the SNc and LC axon lengths decreased with 14, 21, and 28 days of meth, whereas somatic loss was only observed after 28 days of meth; MAO inhibition (phenelzine; 20 mg/kg) prevented axonal and somatic loss of SNc and LC neurons. In contrast, chronic (28-day) meth had no effect on the axon length or numbers of SNc or LC neurons in female mice. The results demonstrate that repeated exposure to meth produces SNc and LC axonal deficits prior to somatic loss in male subjects, consistent with a dying-back pattern of degeneration, whereas female mice are resistant to chronic meth-induced degeneration.

Mitochondrial oxidant stress mediates methamphetamine neurotoxicity in substantia nigra dopaminergic neurons.

Methamphetamine abuse is associated with an increased risk of developing Parkinson's disease (PD). Recently, it was found that methamphetamine increases mitochondrial oxidant stress in substantia nigra pars compacta (SNc) dopaminergic neurons by releasing vesicular dopamine (DA) and stimulating mitochondrially-anchored monoamine oxidase (MAO). As mitochondrial oxidant stress is widely thought to be a driver of SNc degeneration in PD, these observations provide a potential explanation for the epidemiological linkage. To test this hypothesis, mice were administered methamphetamine (5 mg/kg) for 28 consecutive days with or without pretreatment with an irreversible MAO inhibitor. Chronic methamphetamine administration resulted in the degeneration of SNc dopaminergic neurons and this insult was blocked by pretreatment with a MAO inhibitor - confirming the linkage between methamphetamine, MAO and SNc degeneration. To determine if shorter bouts of consumption were as damaging, mice were given methamphetamine for two weeks and then studied. Methamphetamine treatment elevated both axonal and somatic mitochondrial oxidant stress in SNc dopaminergic neurons, was associated with a modest but significant increase in firing frequency, and caused degeneration after drug cessation. While axonal stress was sensitive to MAO inhibition, somatic stress was sensitive to Cav1 Ca2+ channel inhibition. Inhibiting either MAO or Cav1 Ca2+ channels after methamphetamine treatment attenuated subsequent SNc degeneration. Our results not only establish a mechanistic link between methamphetamine abuse and PD, they point to pharmacological strategies that could lessen PD risk for patients with a methamphetamine use disorder.

WAVE1 in neurons expressing the D1 dopamine receptor regulates cellular and behavioral actions of cocaine.

Wiskott-Aldrich syndrome protein (WASP) family verprolin homologous protein 1 (WAVE1) regulates actin-related protein 2/3 (Arp2/3) complex-mediated actin polymerization. Our previous studies have found WAVE1 to be inhibited by Cdk5-mediated phosphorylation in brain and to play a role in the regulation of dendritic spine morphology. Here we report that mice in which WAVE1 was knocked out (KO) in neurons expressing the D1 dopamine receptor (D1-KO), but not mice where WAVE1 was knocked out in neurons expressing the D2 dopamine receptor (D2-KO), exhibited a significant decrease in place preference associated with cocaine. In contrast to wild-type (WT) and WAVE1 D2-KO mice, cocaine-induced sensitized locomotor behavior was not maintained in WAVE1 D1-KO mice. After chronic cocaine administration and following withdrawal, an acute cocaine challenge induced WAVE1 activation in striatum, which was assessed by dephosphorylation. The cocaine-induced WAVE1 dephosphorylation was attenuated by coadministration of either a D1 dopamine receptor or NMDA glutamate receptor antagonist. Upon an acute challenge of cocaine following chronic cocaine exposure and withdrawal, we also observed in WT, but not in WAVE1 D1-KO mice, a decrease in dendritic spine density and a decrease in the frequency of excitatory postsynaptic AMPA receptor currents in medium spiny projection neurons expressing the D1 dopamine receptor (D1-MSNs) in the nucleus accumbens. These results suggest that WAVE1 is involved selectively in D1-MSNs in cocaine-evoked neuronal activity-mediated feedback regulation of glutamatergic synapses.

Dopaminergic modulation of striatal function and Parkinson's disease.

The striatum is richly innervated by mesencephalic dopaminergic neurons that modulate a diverse array of cellular and synaptic functions that control goal-directed actions and habits. The loss of this innervation has long been thought to be the principal cause of the cardinal motor symptoms of Parkinson's disease (PD). Moreover, chronic, pharmacological overstimulation of striatal dopamine (DA) receptors is generally viewed as the trigger for levodopa-induced dyskinesia (LID) in late-stage PD patients. Here, we discuss recent advances in our understanding of the relationship between the striatum and DA, particularly as it relates to PD and LID. First, it has become clear that chronic perturbations of DA levels in PD and LID bring about cell type-specific, homeostatic changes in spiny projection neurons (SPNs) that tend to normalize striatal activity. Second, perturbations in DA signaling also bring about non-homeostatic aberrations in synaptic plasticity that contribute to disease symptoms. Third, it has become evident that striatal interneurons are major determinants of network activity and behavior in PD and LID. Finally, recent work examining the activity of SPNs in freely moving animals has revealed that the pathophysiology induced by altered DA signaling is not limited to imbalance in the average spiking in direct and indirect pathways, but involves more nuanced disruptions of neuronal ensemble activity.

Simple and inexpensive micromachined aluminum microfluidic devices for acoustic focusing of particles and cells.

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.

Line-Focused Optical Excitation of Parallel Acoustic Focused Sample Streams for High Volumetric and Analytical Rate Flow Cytometry.

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.

Multiplexed Lipid Bilayers on Silica Microspheres for Analytical Screening Applications.

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.

Detection and confirmation of serum lipid biomarkers for preeclampsia using direct infusion mass spectrometry.

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.

Global "omics" evaluation of human placental responses to preeclamptic conditions.

BACKGROUND: Preeclampsia (PE) is a leading cause of maternal death. Its cause is still debated but there is general agreement that the placenta plays a central role. Perhaps the most commonly proposed contributors to PE include placental hypoxia, oxidative stress, and increased proinflammatory cytokines. How the placenta responds to these abnormalities has been considered but not as part of a comprehensive analysis of low-molecular-weight biomolecules and their responses to these accepted PE conditions. OBJECTIVE: Using a peptidomic approach, we sought to identify a set of molecules exhibiting differential expression in consequence of provocative agents/chemical mediators of PE applied to healthy human placental tissue. STUDY DESIGN: Known PE conditions were imposed on normal placental tissue from 13 uncomplicated pregnancies and changes in the low-molecular-weight peptidome were evaluated. A t test was used to identify potential markers for each imposed stress. These markers were then submitted to a least absolute shrinkage and selection operator multinomial logistic regression model to identify signatures specific to each stressor. Estimates of model performance on external data were obtained through internal validation. RESULTS: A total of 146 markers were increased/decreased as a consequence of exposure to proposed mediators of PE. Of these 75 changed with hypoxia; 23 with hypoxia-reoxygenation/oxidative stress and 48 from exposure to tumor necrosis factor-α. These markers were chemically characterized using tandem mass spectrometry. Identification rates were: hypoxia, 34%; hypoxia-reoxygenation, 60%; and tumor necrosis factor-α, 50%. Least absolute shrinkage and selection operator modeling specified 16 markers that effectively distinguished all groups, ie, the 3 abnormal conditions and control. Bootstrap estimates of misclassification rates, multiclass area under the curve, and Brier score were 0.108, 0.944, and 0.160, respectively. CONCLUSION: Using this approach we found previously unknown molecular changes in response to individual PE conditions that allowed development biomolecular signatures for exposure to each accepted pathogenic condition.

A unified sensor architecture for isothermal detection of double-stranded DNA, oligonucleotides, and small molecules.

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).

Dynamic Response of Mycobacterium vanbaalenii PYR-1 to BP Deepwater Horizon Crude Oil.

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 C12 and C13 being degraded at the highest rate, although alkanes up to C28 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.

Novel "omics" approach for study of low-abundance, low-molecular-weight components of a complex biological tissue: regional differences between chorionic and basal plates of the human placenta.

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.

Catalytic molecular logic devices by DNAzyme displacement.

Chemical reactions catalyzed by DNAzymes offer a route to programmable modification of biomolecules for therapeutic purposes. To this end, we have developed a new type of catalytic DNA-based logic gates in which DNAzyme catalysis is controlled via toehold-mediated strand displacement reactions. We refer to these as DNAzyme displacement gates. The use of toeholds to guide input binding provides a favorable pathway for input recognition, and the innate catalytic activity of DNAzymes allows amplification of nanomolar input concentrations. We demonstrate detection of arbitrary input sequences by rational introduction of mismatched bases into inhibitor strands. Furthermore, we illustrate the applicability of DNAzyme displacement to compute logic functions involving multiple logic gates. This work will enable sophisticated logical control of a range of biochemical modifications, with applications in pathogen detection and autonomous theranostics.

Association of cord blood digitalis-like factor and necrotizing enterocolitis.

OBJECTIVE: Endogenous digoxin-like factor (EDLF) has been linked to vasoconstriction, altered membrane transport, and apoptosis. Our objective was to determine whether increased EDLF in the cord sera of preterm infants was associated with an increased incidence of necrotizing enterocolitis (NEC). STUDY DESIGN: Cord sera from pregnant women enrolled in a randomized trial of MgSO4 for fetal neuroprotection were analyzed for EDLF using a red cell Rb(+) uptake assay in which the inhibition of sodium pump-mediated Rb(+) transport was used as a functional assay of EDLF. Specimens were assayed blinded to neonatal outcome. Cases (NEC, n = 25) and controls (neonates not developing stage 2 or 3 NEC, n = 24) were matched by study center and gestational age. None of the women had preeclampsia. Cases and controls were compared using the Wilcoxon test for continuous and the Fisher exact test for categorical variables. A conditional logistic regression analysis was used to assess the odds of case vs control by EDLF level. RESULTS: Cases and controls were not significantly different for gestational age, race, maternal steroid use, premature rupture of membranes, or MgSO4 treatment. In logistic models adjusted for treatment group, race, premature rupture of membranes, and gestational age, cord sera EDLF was significantly associated with development of NEC (P = .023). CONCLUSION: These data demonstrated an association between cord sera EDLF and NEC.

Signal propagation in multi-layer DNAzyme cascades using structured chimeric substrates.

Signal propagation through enzyme cascades is a critical component of information processing in cellular systems. Although such systems have potential as biomolecular computing tools, rational design of synthetic protein networks remains infeasible. DNA strands with catalytic activity (DNAzymes) are an attractive alternative, enabling rational cascade design through predictable base-pair hybridization principles. Multi-layered DNAzyme signaling and logic cascades are now reported. Signaling between DNAzymes was achieved using a structured chimeric substrate (SCS) that releases a downstream activator after cleavage by an upstream DNAzyme. The SCS can be activated by various upstream DNAzymes, can be coupled to DNA strand-displacement devices, and is highly resistant to interference from background DNA. This work enables the rational design of synthetic DNAzyme regulatory networks, with potential applications in biomolecular computing, biodetection, and autonomous theranostics.

Differential Serum Peptidomics Reveal Multi-Marker Models That Predict Breast Cancer Progression.

Here, we assess how the differential expression of low molecular weight serum peptides might predict breast cancer progression with high confidence. We apply an LC/MS-MS-based, unbiased 'omics' analysis of serum samples from breast cancer patients to identify molecules that are differentially expressed in stage I and III breast cancer. Results were generated using standard and machine learning-based analytical workflows. With standard workflow, a discovery study yielded 65 circulating biomarker candidates with statistically significant differential expression. A second study confirmed the differential expression of a subset of these markers. Models based on combinations of multiple biomarkers were generated using an exploratory algorithm designed to generate greater diagnostic power and accuracy than any individual markers. Individual biomarkers and the more complex multi-marker models were then tested in a blinded validation study. The multi-marker models retained their predictive power in the validation study, the best of which attained an AUC of 0.84, with a sensitivity of 43% and a specificity of 88%. One of the markers with m/z 761.38, which was downregulated, was identified as a fibrinogen alpha chain. Machine learning-based analysis yielded a classifier that correctly categorizes every subject in the study and demonstrates parameter constraints required for high confidence in classifier output. These results suggest that serum peptide biomarker models could be optimized to assess breast cancer stage in a clinical setting.

Is that a treatment program? Examining the physical landscape of opioid treatment programs in Los Angeles.

INTRODUCTION: Little attention has been paid to the built environment of outpatient opioid treatment programs, despite the need to increase access to medications for opioid use disorder, particularly among people of color. The aims of this study were to rate the attractiveness of publicly-funded opioid treatment programs (OTPs) in Los Angeles County and explore whether less attractive OTPs are found in disadvantaged neighborhoods. METHODS: We conducted observations of the exteriors of all OTPs located in specialty substance use disorder treatment clinics in Los Angeles County in 2021 (N = 44). We created an attractiveness index based on attractiveness of the building exteriors and the surrounding grounds, the presence of disorder such as garbage and graffiti, and the presence of bars on the windows. We tested whether less attractive facilities were more likely to be situated in disadvantaged neighborhoods with high concentrations of racial/ethnic minorities. RESULTS: Most building exteriors were found to have an ordinary level of attractiveness or rated as unattractive. The grounds were largely unattractive. We found a significant negative association between attractiveness and neighborhood disadvantage. CONCLUSION: This project was a preliminary study of the physical conditions of OTPs in Los Angeles. We found was that the physical conditions of OTPs in LA County were generally poor. Research has identified many individual and structural barriers to treatment for people with opioid use disorders. Future research should empirically test the association between the built environment of treatment clinics and access to treatment, particularly in communities of color.

Tissue proteomics of the low-molecular weight proteome using an integrated cLC-ESI-QTOFMS approach.

Analysis of the protein/peptide composition of tissue has provided meaningful insights into tissue biology and even disease mechanisms. However, little has been published regarding top down methods to investigate lower molecular weight (MW) (500-5000 Da) species in tissue. Here, we evaluate a tissue proteomics approach involving tissue homogenization followed by depletion of large proteins and then cLC-MS (where c stands for capillary) analysis to interrogate the low MW/low abundance tissue proteome. In the development of this method, sheep heart, lung, liver, kidney, and spleen were surveyed to test our ability to observe tissue differences. After categorical tissue differences were demonstrated, a detailed study of this method's reproducibility was undertaken to determine whether or not it is suitable for analyzing more subtle differences in the abundance of small proteins and peptides. Our results suggest that this method should be useful in exploring the low MW proteome of tissues.

Elastomeric negative acoustic contrast particles for affinity capture assays.

This report describes the development of elastomeric capture microparticles (ECµPs) and their use with acoustophoretic separation to perform microparticle assays via flow cytometry.We have developed simple methods to form ECµPs by cross-linking droplets of common commercially available silicone precursors in suspension followed by surface functionalization with biomolecular recognition reagents. The ECµPs are compressible particles that exhibit negative acoustic contrast in ultrasound when suspended in aqueous media, blood serum, or diluted blood. In this study, these particles have been functionalized with antibodies to bind prostate specific antigen and immunoglobulin (IgG). Specific separation of the ECµPs from blood cells is achieved by flowing them through a microfluidic acoustophoretic device that uses an ultrasonic standing wave to align the blood cells, which exhibit positive acoustic contrast, at a node in the acoustic pressure distribution while aligning the negative acoustic contrast ECµPs at the antinodes. Laminar flow of the separated particles to downstream collection ports allows for collection of the separated negative contrast (ECµPs) and positive contrast particles (cells). Separated ECµPs were analyzed via flow cytometry to demonstrate nanomolar detection for prostate specific antigen in aqueous buffer and picomolar detection for IgG in plasma and diluted blood samples. This approach has potential applications in the development of rapid assays that detect the presence of low concentrations of biomarkers in a number of biological sample types.