To meet stringent limit-of-detection specifications for low abundance target molecules, a relatively large volume of plasma is needed for many blood-based clinical diagnostics. Conventional centrifugation methods for plasma separation are not suitable for on-site testing or bedside diagnostics. Here, we report a simple, yet high-efficiency, clamshell-style, superhydrophobic plasma separator that is capable of separating a relatively large volume of plasma from several hundred microliters of whole blood (finger-prick blood volume). The plasma separator consists of a superhydrophobic top cover with a separation membrane and a superhydrophobic bottom substrate. Unlike previously reported membrane-based plasma separators, the separation membrane in our device is positioned at the top of the sandwiched whole blood film to increase the membrane separation capacity and plasma yield. In addition, the device’s superhydrophobic characteristics (i) facilitates the formation of well-defined, contracted, thin blood film with a high contact angle; (ii) minimizes biomolecular adhesion to surfaces; (iii) increases blood clotting time; and (iv) reduces blood cell hemolysis. The device demonstrated a “blood in-plasma out” capability, consistently extracting 65±21.5 μL of plasma from 200 μL of whole blood in less than 10 min without electrical power. The device was used to separate plasma from Schistosoma mansoni genomic DNA-spiked whole blood with a recovery efficiency of > 84.5 ± 25.8 %. The S. mansoni genomic DNA in the separated plasma was successfully tested on our custom-made microfluidic chip by using loop mediated isothermal amplification (LAMP) method.

To comprehensively identify proteins of the rat liver plasma membrane (PM), we have adopted a proteomics strategy that utilizes sucrose density centrifugation in conjunction with aqueous two-phase partition for plasma membrane isolation, followed by SDS-PAGE, mass spectrometry and bioinformatics. Western blot analysis showed that this method results in highly purified plasma membrane fractions, which is a key to successful plasma membrane proteomics. The PM proteins were separated by SDS-PAGE and digested with trypsin. Through nano-ESI-LC MS/MS analysis we identified 428 rat liver membrane proteins, of which 304 had a gene ontology (GO) annotation indicating a cellular component, and 204 (67%) of the latter were known integral membrane proteins or membrane-associated proteins. In addition to proteins known to be associated with the plasma membrane, several hypothetical proteins have also been identified. This study not only provides a tool to study plasma membrane proteins with low levels of contamination, but also provides a data set for proteins of high to moderate abundance in rat liver plasma membranes, thus allowing for more comprehensive characterization of membrane proteins and a better understanding of membrane dynamics.

The clinical cardiovascular utility of a diet rich in fi sh oils, particularly EPA and DHA, has been debated over the past 50 years ( 1-3 ). Large clinical outcome trials, such as the open-labeled GISSI-Prevenzione ( 4 ) or JELIS ( 5 ) studies, supported the notion that fi sh oil supplements confer therapeutic benefi t on patients with cardiovascular disease. However, an overview analysis of results of more than 50 randomized controlled trials and cohort studies addressing this question yielded equivocal results ( 6, 7 ). Consequently, the adoption of the dietary interventions with fi sh oil into clinical guidelines has been limited ( 8 ). Fish oil supplementation does infl uence a series of cardiovascular biomarkers: it decreases blood levels of triglycerides in patients with hypertriglyceridemia, an effect primarily driven by lowering the production of triglycerides from nonesterifi ed fatty acids ( 9 ); high doses reduce blood pressure in patients with essential hypertension ( 10, 11 ) and modestly inhibit indices of platelet activation ( 12 ). The mechanisms involved are unclear, but may involve a shift in formation of enzymatic and free radical-catalyzed prostanoids, refl ecting utilization of EPA and DHA rather than arachidonic acid (AA) as a substrate. It has been speculated also that cardiovascular benefi t might derive, in part, from anti-infl ammatory actions of fi sh oils: families of bioactive lipids which favor resolution of infl ammation have been suggested to be of particular importance ( 13 ). Synthetic versions of such specialized pro-resolving mediators (SPMs), products of transcellular metabolism of fi sh oils, exert anti-infl ammatory effects in vitro and when administered in vivo in several animal models ( 14-17 ). Quantities Abstract Resolvins, maresins, and protectins can be formed from fi sh oils. These specialized pro-resolving mediators (SPMs) have been implicated in the resolution of infl ammation. Synthetic versions of such SPMs exert anti-infl ammatory effects in vitro and when administered to animal models. However, their importance as endogenous products formed in suffi cient amounts to exert anti-infl ammatory actions in vivo remains speculative. We biased our ability to detect SPMs formed in healthy volunteers by supplementing fi sh oil in doses shown previously to infl uence blood pressure and platelet aggregation under placebo-controlled conditions. Additionally, we sought to determine the relative formation of SPMs during an acute infl ammatory response and its resolution, evoked in healthy volunteers by bacterial lipopolysaccharide (LPS). Bioactive lipids, enzymatic epoxyeicosatrienoic acids (EETs), and free radical-catalyzed prostanoids [isoprostanes (iPs)] formed from arachidonic acid and the fi sh oils, served as comparators. Despite the clear shift from -6 to -3 EETs and iPs, we failed to detect a consistent signal, in most cases, of SPM formation in urine or plasma in response to fi sh oil, and in all cases in response to LPS on a background of fi sh oil. Our results que...

The cardiovascular safety of nonsteroidal antiinflammatory drugs (NSAIDs) may be influenced by interactions with antiplatelet doses of aspirin. We sought to quantitate precisely the propensity of commonly consumed NSAIDs-ibuprofen, naproxen, and celecoxib-to cause a drug-drug interaction with aspirin in vivo by measuring the target engagement of aspirin directly by MS. We developed a novel assay of cyclooxygenase-1 (COX-1) acetylation in platelets isolated from volunteers who were administered aspirin and used conventional and microfluidic assays to evaluate platelet function. Although ibuprofen, naproxen, and celecoxib all had the potential to compete with the access of aspirin to the substrate binding channel of COX-1 in vitro, exposure of volunteers to a single therapeutic dose of each NSAID followed by 325 mg aspirin revealed a potent drug-drug interaction between ibuprofen and aspirin and between naproxen and aspirin but not between celecoxib and aspirin. The imprecision of estimates of aspirin consumption and the differential impact on the ability of aspirin to inactivate platelet COX-1 will confound head-to-head comparisons of distinct NSAIDs in ongoing clinical studies designed to measure their cardiovascular risk.aspirin | acetylation | cyclooxygenase | MS | nonsteroidal antiinflammatory drugs C hronic pain, most commonly inflammatory musculoskeletal pain, afflicts hundreds of millions worldwide (1). Nonsteroidal antiinflammatory drugs (NSAIDs) consumed chronically or intermittently remain the mainstay of therapy for inflammationassociated pain. These agents inhibit cyclooxygenase (COX)-1 and COX-2, thereby reducing the production of inflammatory prostanoids, lipid mediators that lower the activation threshold of nociceptors and sensory neurons. The prevalence of chronic pain rises in the elderly, coinciding with an increase in concomitant disease (1), which complicates drug treatment. Pain management in patients with preexisting cardiovascular disease is a particular challenge because of the cardiovascular adverse effects of NSAIDs and the risk of drug-drug interactions that might undermine the antiplatelet effects of aspirin prescribed for cardioprotection (2).Although NSAIDs relieve pain effectively, they can cause serious renal and cardiovascular complications by inhibiting COXdependent prostanoids with homeostatic functions (3, 4). All NSAIDs have the potential to elevate blood pressure and may cause heart failure. COX-2-selective NSAIDs, which were developed to reduce gastrointestinal toxicity, additionally raise the rate of myocardial infraction and stroke (5, 6), affecting ∼1-2% of patients exposed per year (3,4). This adverse drug reaction may have caused thousands of deaths in the general population. Traditional NSAIDs (tNSAIDs) have also been associated with cardiovascular events, but although pharmacoepidemiological studies and metaanalyses of randomized, controlled trials suggest that not all tNSAIDs carry the same risk, there is considerable heterogeneity across studies in the comparative ...