Insertion of an immunodominant T helper cell epitope within the Group A Streptococcus M protein promotes an IFN-γ-dependent shift from a non-protective to a protective immune response.

The common pathogen Group A Streptococcus (GAS, Streptococcus pyogenes) is an extracellular bacterium that is associated with a multitude of infectious syndromes spanning a wide range of severity. The surface-exposed M protein is a major GAS virulence factor that is also target for protective antibody responses. In this study, we use a murine immunization model to investigate aspects of the cellular and molecular foundation for protective adaptive immune responses generated against GAS. We show that a wild type M1 GAS strain induces a non-protective antibody response, while an isogenic strain carrying the immunodominant 2W T helper cell epitope within the M protein elicits an immune response that is protective against the parental non-recombinant M1 GAS strain. Although the two strains induce total anti-GAS IgG levels of similar magnitude, only the 2W-carrying strain promotes elevated titers of the complement-fixing IgG2c subclass. Protection is dependent on IFN-γ, and IFN-γ-deficient mice show a specific reduction in IgG2c levels. Our findings suggest that inclusion of the 2W T cell epitope in the M protein confers essential qualitative alterations in the adaptive immune response against GAS, and that sparsity in IFN-γ-promoting Th cell epitopes in the M protein may constitute an immune evasion mechanism, evolved to allow the pathogen to avoid attack by complement-fixing antibodies.

Effects of carotenoid pigmentation in salmon on antibiotic extraction recovery, matrix effects and accuracy of quantification by ultrahigh performance liquid chromatography coupled to tandem mass spectrometry.

Carotenoid pigmentation in salmon may interfere with the accuracy of antibiotic analysis with ultra-high pressure liquid chromatography coupled to tandem spectrometry (UPLC-MS/MS) by causing matrix effects or affecting the recovery of compounds during extraction. In the present study, we used both pigmented and non-pigmented salmon to understand the role pigments play on antibiotic analysis, and tested whether clean-up of the extract with dispersive solid phase extraction (dSPE) or hydrophilic-lipophilic balance (HLB) SPE clean-up reduces matrix effects. Thirty antibiotics and their respective class-specific surrogate standards were measured in Sockeye (pigmented), King (pigmented) and Ivory King (non-pigmented) salmon extracted using the QUEChERS method, or a modified QUEChERS method involving dSPE or HLB SPE clean-up (for Sockeye salmon only). Significant matrix effects and lower percent recoveries of spiked antibiotics were observed in pigmented versus non-pigmented salmon extracted with the QUEChERS method. Dispersive SPE clean-up did not improve extraction recoveries or matrix effects. However, SPE clean-up with HLB columns improved matrix effects for several antibiotics but reduced the percent recovery to < 30%. Across all types of salmon analyzed, the accuracy of quantitation was minimally impacted, likely due to similar behavior of the surrogate standards tagged to each antibiotic class during extraction. Our results demonstrate that carotenoids in salmon are associated with significant matrix effects and low extraction recoveries, but do not impact accuracy.

Chronic exposure to traffic-related air pollution reduces lipid mediators of linoleic acid and soluble epoxide hydrolase in serum of female rats.

Chronic exposure to traffic-related air pollution (TRAP) is known to promote systemic inflammation, which is thought to underlie respiratory, cardiovascular, metabolic and neurological disorders. It is not known whether chronic TRAP exposure dampens inflammation resolution, the homeostatic process for stopping inflammation and repairing damaged cells. In vivo, inflammation resolution is facilitated by bioactive lipid mediators known as oxylipins, which are derived from the oxidation of polyunsaturated fatty acids. To understand the effects of chronic TRAP exposure on lipid-mediated inflammation resolution pathways, we measured total (i.e. free+bound) pro-inflammatory and pro-resolving lipid mediators in serum of female rats exposed to TRAP or filtered air (FA) for 14 months. Compared to rats exposed to FA, TRAP-exposed rats showed a significant 36-48% reduction in fatty acid alcohols, specifically, 9-hydroxyoctadecadienoic acid (9-HODE), 11,12-dihydroxyeicosatetraenoic acid (11,12-DiHETE) and 16,17-dihydroxydocosapentaenoic acid (16, 17-DiHDPA). The decrease in fatty acid diols (11,12-DiHETE and 16, 17-DiHDPA) corresponded to a significant 34-39% reduction in the diol to epoxide ratio, a marker of soluble epoxide hydrolase activity; this enzyme is typically upregulated during inflammation. The findings demonstrate that 14 months exposure to TRAP reduced pro-inflammatory 9-HODE concentration and dampened soluble epoxide hydrolase activation, suggesting adaptive immune changes in lipid mediator pathways involved in inflammation resolution.

Antibiotic standards stored as a mixture in water: methanol are unstable at various temperatures irrespective of pH and glass container silanization.

It is well-established that antibiotics stored individually at their optimal pH and in appropriate solvents are stable over time. However, limited information exists on the stability of antibiotics from multiple classes when prepared and stored as a mixture prior to multiresidue analysis by mass spectrometry. This study tested the stability of antibiotic standard mixtures from eight classes [amphenicols, tetracyclines, sulfonamides, quinolones, macrolides, ß-lactams, lincosamides and miscellaneous (i.e., trimethoprim)] in relation to the water:methanol ratio, presence of sodium hydroxide base (to solubilise quinolones), storage temperature, and container type including plain and silanized glass vials. Antibiotics were analysed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry. Several antibiotics, mainly quinolones, tetracyclines and macrolides, were unstable when stored as mixtures for one week regardless of the water:methanol ratio, storage temperature (4, -20 or -80 °C) and presence/absence of sodium hydroxide. Silanization of glassware improved the storage stability of quinolones and macrolides but reduced the stability of tetracyclines and other antibiotics including florfenicol amine, penicillin G, erythromycin and sulfadiazine. Our results show that several antibiotics in water:methanol are unstable when stored as a mixture and suggest a limited advantage of using base or silanized glass vials for the preparation and storage of antibiotic standards mixed together. Freshly prepared antibiotic standard mixtures are recommended for multi-residue quantitation of antibiotics.Abbreviations AMOX: amoxicillin; AMP: ampicillin; AZ: azithromycin; CAP: chloramphenicol; CE: collision energy; CTC: chlortetracycline; CIP: ciprofloxacin; DOX: doxycycline; ENO: enoxacin; ENRO: enrofloxacin; ERYTH: erythromycin; FF: florfenicol; FFA: florfenicol amine; FLU: flumequine; HDPE: high-density polyethylene; LC-MS/MS: liquid chromatography-tandem mass spectrometry; LIN: lincomycin; MRM: multiple reaction monitoring; NOR: norfloxacin; OFL-D3: ofloxacin-D3; OXO: oxolinic acid; OTC: oxytetracycline; PEN-G: penicillin G; PEN-V: penicillin V; ROX: roxithromycin; SDM: sulfadimethoxine; SDZ: sulfadiazine; SMX: sulfamethoxazole; SMZ-D4: sulfamethazine-D4; SSZ: sulfasalazine; TC: tetracycline; TAP: thiamphenicol; TILM: tilmicosin; TRIM: trimethoprim; TL: tolerance limit; VIRG: virginiamycin; UPLC-MS/MS: ultra-high pressure liquid chromatography-tandem mass spectrometry.

Sample-to-Answer Robotic ELISA.

Enzyme-linked immunosorbent assays (ELISA), as one of the most used immunoassays, have been conducted ubiquitously in hospitals, research laboratories, etc. However, the conventional ELISA procedure is usually laborious, occupies bulky instruments, consumes lengthy operation time, and relies considerably on the skills of technicians, and such limitations call for innovations to develop a fully automated ELISA platform. In this paper, we have presented a system incorporating a robotic-microfluidic interface (RoMI) and a modular hybrid microfluidic chip that embeds a highly sensitive nanofibrous membrane, referred to as the Robotic ELISA, to achieve human-free sample-to-answer ELISA tests in a fully programmable and automated manner. It carries out multiple bioanalytical procedures to replace the manual steps involved in classic ELISA operations, including the pneumatically driven high-precision pipetting, efficient mixing and enrichment enabled by back-and-forth flows, washing, and integrated machine vision for colorimetric readout. The Robotic ELISA platform has achieved a low limit of detection of 0.1 ng/mL in the detection of a low sample volume (15 µL) of chloramphenicol within 20 min without human intervention, which is significantly faster than that of the conventional ELISA procedure. Benefiting from its modular design and automated operations, the Robotic ELISA platform has great potential to be deployed for a broad range of detections in various resource-limited settings or high-risk environments, where human involvement needs to be minimized while the testing timeliness, consistency, and sensitivity are all desired.

Quantification of Nonpersistent Pesticides in Small Volumes of Human Breast Milk with Ultrahigh Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry.

Existing methods for the analysis of pesticides in human breast milk involve multiple extraction steps requiring large sample and solvent volumes, which can be a major obstacle in large epidemiologic studies. Here, we developed a simple, low-volume method for extracting organophosphates, pyrethroids, carbamates, atrazine, and imidacloprid from 100 to 200 µL of human breast milk. Multiple extraction protocols were tested including microwave-assisted acid/base digestion and double-solvent extraction with 2 or 20 mL of 2:1 (v/v) dichloromethane/hexane, with or without subsequent solid-phase extraction (SPE) cleanup. Samples were analyzed by liquid chromatography tandem mass spectrometry. Analyte recoveries and reproducibility were highest when 100-200 µL of milk were extracted with 2 mL of dichloromethane/hexane without subsequent SPE steps. Analysis of 79 breast milk samples using this method revealed the presence of carbamates, organophosphates, pyrethroids, and imidacloprid at detection frequencies of 79-96, 53-90, 1-7, and 61%, respectively. This study demonstrates the feasibility of a simple low-volume extraction method for measuring pesticides in human breast milk.

Triacylglycerols are preferentially oxidized over free fatty acids in heated soybean oil.

In oil, free fatty acids (FFAs) are thought compared the efficiency of hydrolysis wto be the preferred substrate for lipid oxidation although triacylglycerols (TAGs) are the predominant lipid class. We determined the preferential oxidation substrate (TAGs versus FFAs) in soybean oil heated at 100 °C for 24 h, after validating a method for quantifying esterified and free lipid oxidation products (i.e., oxylipins) with mass-spectrometry. Reaction velocities and turnover (velocity per unit substrate) of FFA, and free and TAG-bound (esterified) oxylipins were determined. FFA hydrolysis rate and turnover were orders of magnitude greater (16-4217 fold) than that of esterified and free oxylipin formation. The velocity and turnover of TAG-bound oxylipins was significantly greater than free oxylipins by 282- and 3-fold, respectively. The results suggest that during heating, TAGs are preferentially oxidized over FFAs, despite the rapid hydrolysis and availability of individual FFAs as substrates for oxidation. TAG-bound oxylipins may serve as better markers of lipid oxidation.

Linoleic acid-derived 13-hydroxyoctadecadienoic acid is absorbed and incorporated into rat tissues.

Linoleic acid (LNA)-derived 13-hydroxyoctadecadienoic acid (13-HODE) is a bioactive lipid mediator that regulates multiple signaling processes in vivo. 13-HODE is also produced when LNA is oxidized during food processing. However, the absorption and incorporation kinetics of dietary 13-HODE into tissues is not known. The present study measured unesterified d4-13-HODE plasma bioavailability and incorporation into rat liver, adipose, heart and brain following gavage or intravenous (IV) injection (n = 3 per group). Mass spectrometry analysis revealed that d4-13-HODE was absorbed within 20 min of gavage, and continued to incorporate into plasma esterified lipid fractions throughout the 90 min monitoring period (incorporation half-life of 71 min). Following IV injection, unesterified d4-13-HODE was rapidly eliminated from plasma with a half-life of 1 min. Analysis of tracer incorporation kinetics into rat tissues following IV injection or gavage revealed that the esterified tracer preferentially incorporated into liver, adipose and heart compared to unesterified d4-13-HODE. No tracer was detected in the brain. This study demonstrates that dietary 13-HODE is absorbed, and incorporated into peripheral tissues from esterified plasma lipid pools. Understanding the chronic effects of dietary 13-HODE exposure on peripheral tissue physiology and metabolism merits future investigation.

Quantitation of Oxylipins in Fish and Algae Oil Supplements Using Optimized Hydrolysis Procedures and Ultra-High Performance Liquid Chromatography Coupled to Tandem Mass-Spectrometry.

Fish and algae oil supplements are enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are precursors to oxidized fatty acids, known as oxylipins. Here, we optimized a base hydrolysis method for measuring oxylipins in oil with ultrahigh-performance liquid chromatography coupled to tandem mass-spectrometry (UPLC-MS/MS) and quantified them in fish and algae oil supplements. Hydrolysis of 2 µL of oil with sodium carbonate resulted in greater oxylipin concentrations and minimal matrix effects, compared to higher oil volumes (10, 20, and 30 µL). Oxylipin yield was higher when oil was hydrolyzed in methanol containing 0.1% acetic acid and 0.1% butylated hydroxytoluene, compared to no methanol, and using sodium hydroxide versus sodium carbonate. Oxylipins extracted from 2 µL of oil using sodium hydroxide in solvent showed that EPA-derived oxylipins were most abundant in fish oil (84-87%), whereas DHA-oxylipins were abundant in algae oil (83%). This study shows that fish and algae oils are direct sources of EPA- and DHA-derived oxylipins.

Permeability and Line-Tension-Dependent Response of Polyunsaturated Membranes to Osmotic Stresses.

The lipidome of plant plasma membranes-enriched in cellular phospholipids containing at least one polyunsaturated fatty acid tail and a variety of phytosterols and phytosphingolipids-is adapted to significant abiotic stresses. But how mesoscale membrane properties of these membranes such as permeability and deformability, which arise from their unique molecular compositions and corresponding lateral organization, facilitate response to global mechanical stresses is largely unknown. Here, using giant vesicles reconstituting mixtures of polyunsaturated lipids (soy phosphatidylcholine), glucosylceramide, and sitosterol common to plant membranes, we find that the membranes adopt "janus-like" domain morphologies and display anomalous solute permeabilities. The former textures the membrane with a single sterol-glucosylceramide-enriched, liquid-ordered domain separated from a liquid-disordered phase consisting primarily of soy phosphatidylcholine. When subject to osmotic downshifts, the giant unilamellar vesicles (GUVs) respond by transiently producing well-known swell-burst cycles. In each cycle, the influx of water swells the GUV, rendering the membrane tense. Subsequent rupture of the membrane through transient poration, which localizes in the liquid-disordered phase or at the domain boundaries, reduces the osmotic stress by expelling some of the excess osmolytes (and solvent) before sealing. When subject to abrupt hypertonic stress, they deform by nucleating buds at the domain phase boundaries. Remarkably, this incipient vesiculation is reversed in a statistically significant fraction of GUVs because of the interplay with solute permeation timescales, which render osmotic stresses short-lived. This, then, suggests a novel control mechanism in which an interplay of permeability and deformability regulates osmotically induced membrane deformation and limits vesiculation-induced loss of membrane material. Interestingly, recapitulation of such dynamic morphological reconfigurability-switching between budded and nonbudded morphologies-due to the interplay of membrane permeability, which temporally reverses the osmotic gradient, and domain boundaries, which select modes of deformations, might prove valuable in endowing synthetic cells with novel morphological responsiveness.

Molecular dynamics simulations of ternary lipid bilayers containing plant sterol and glucosylceramide.

An atomic-level molecular dynamics simulation was carried out to study the effects of a plant sterol (sitosterol) and glucosylceramide (GlcCer) on a 1,2-dilinoleoylposphocholine (DLiPC) membrane. Initially, a membrane containing 50mol% sitosterol was compared with that containing the same ratio of cholesterol. These simulations showed differential condensing and ordering effects of sitosterol and cholesterol, with cholesterol being slightly more efficient than sitosterol in packing the membrane more tightly to a liquid ordered phase. By incorporation of 9.3% GlcCer on DLiPC/sterol membrane no notable change was observed in terms of area per lipid, bilayer thickness, order parameter and lateral diffusion. Some clusters of GlcCer/sterol were observed at higher ratio of GlcCer (15.5%), supporting the existence of GlcCer/sitosterol-enriched Lo-domains on the nanometer scale in the plant lipid mixture.

Antibiotic Resistance Pattern and Distribution of pslA Gene Among Biofilm Producing Pseudomonas aeruginosa Isolated From Waste Water of a Burn Center.

BACKGROUND: Pseudomonas aeruginosa is considered as a major cause of hospital-acquired infections due to its high antibacterial resistance. Biofilm formation is a well-known pathogenic mechanism in P. aeruginosa infections, since sessile bacteria are protected in an extracellular matrix of exopolysaccharide. The expression of polysaccharide synthesis locus (pslA gene) can be important for biofilm formation by P. aeruginosa. OBJECTIVES: The purpose of this research was to evaluate the antibiotic resistance pattern and distribution of the pslA gene among biofilm-producing P. aeruginosa isolates obtained from waste water of Burn Centre in Guilan, Iran. MATERIALS AND METHODS: Fifty isolates of P. aeruginosa were obtained from waste water of a burn center. The P. aeruginosa isolates were identified using standard bacteriological procedures. Drug susceptibility test was performed by disk diffusion method for all the isolates against nine antimicrobial agents. Biofilm formation was measured by microtiter plate assay. Polymerase chain reaction (PCR) was used to identify the presence of the pslA gene among the isolates. RESULTS: Biofilm formation was observed in 70% of the P. aeruginosa isolates. The potential formation of biofilm was significantly associated with resistance to gentamicin, imipenem, tobramycin and piperacillin. In addition, the pslA gene only existed in biofilm-producing isolates with a frequency of 42.9% (n = 15). CONCLUSIONS: The findings of the present study well demonstrated that the P. aeruginosa biofilm-producing isolates were more resistant to the tested antibiotics. Furthermore, because of wide distribution, it seems that the pslA gene is associated with biofilm formation.