Improvement in Fitted Filtration Efficiency of N95 Respirators With Escalating Instruction of the Wearer.

Introduction: Fitted filtration performance of an N95 respirator may benefit from differing levels of instructions. Methods: Using a modified Occupational Safety and Health Administration fit test protocol, we measured fitted filtration efficiency for an N95 respirator in 21 screened, healthy participants given 4 levels of escalating instruction: (1) uninstructed (baseline), (2) written/pictorial manufacturer instructions, (3) step-by-step video demonstration, and (4) staff instruction (visual inspection of respirator fit and verbal suggestions to adjust when applicable). Results: Baseline fitted filtration efficiency was not significantly different between participants with or without previous experience of N95 use. Clear improvements in fitted filtration efficiency were observed progressing from baseline (average=86.1%) to manufacturer paper instructions (93.3%), video instructions (97.5%), and post staff intervention (98.3%). Baseline fitted filtration efficiency values were significantly lower than those after video instruction (p<0.037) and staff intervention (p<0.033) sessions. Conclusions: Beyond uninstructed wear or provision of manufacturer instructions, efforts to train and instruct users in proper respirator fit principles with visual feedback are likely to yield benefits to public health outcomes in reducing respiratory exposure during air quality emergencies such as airborne viral outbreaks and wildland fires.

Exposures to low-levels of fine particulate matter are associated with acute changes in heart rate variability, cardiac repolarization, and circulating blood lipids in coronary artery disease patients.

Exposure to air pollution is a major risk factor for cardiovascular disease, disease risk factors, and mortality. Specifically, particulate matter (PM), and to some extent ozone, are contributors to these effects. In addition, exposures to these pollutants may be especially dangerous for susceptible populations. In this repeated-visit panel study, cardiovascular markers were collected from thirteen male participants with stable coronary artery disease. For 0-4 days prior to the health measurement collections, daily concentrations of fine PM (PM2.5) and ozone were obtained from local central monitoring stations located near the participant's homes. Then, single (PM2.5) and two-pollutant (PM2.5 and ozone) models were used to assess whether there were short-term changes in cardiovascular health markers. Per interquartile range increase in PM2.5, there were decrements in several heart rate variability metrics, including the standard deviation of the normal-to-normal intervals (lag 3, -5.8%, 95% confidence interval (CI) = -11.5, 0.3) and root-mean squared of successive differences (five day moving average, -8.1%, 95% CI = -15.0, -0.7). In addition, increases in PM2.5 were also associated with changes in P complexity (lag 1, 4.4%, 95% CI = 0.5, 8.5), QRS complexity (lag 1, 4.9%, 95% CI = 1.4, 8.5), total cholesterol (five day moving average, -2.1%, 95% CI = -4.1, -0.1), and high-density lipoprotein cholesterol (lag 2, -1.6%, 95% CI = -3.1, -0.1). Comparisons to our previously published work on ozone were conducted. We found that ozone affected inflammation and endothelial function, whereas PM2.5 influenced heart rate variability, repolarization, and lipids. All the health changes from these two studies were found at concentrations below the United States Environmental Protection Agency's National Ambient Air Quality Standards. Our results imply clear differences in the cardiovascular outcomes observed with exposure to the two ubiquitous air pollutants PM2.5 and ozone; this observation suggests different mechanisms of toxicity for these exposures.

Fish oil blunts lung function decrements induced by acute exposure to ozone in young healthy adults: A randomized trial.

BACKGROUND: Over one-third of the U.S. population is exposed to unsafe levels of ozone (O3). Dietary supplementation with fish oil (FO) or olive oil (OO) has shown protection against other air pollutants. This study evaluates potential cardiopulmonary benefits of FO or OO supplementation against acute O3 exposure in young healthy adults. METHODS: Forty-three participants (26 ± 4 years old; 47% female) were randomized to receive 3 g/day of FO, 3 g/day OO, or no supplementation (CTL) for 4 weeks prior to undergoing 2-hour exposures to filtered air and 300 ppb O3 with intermittent exercise on two consecutive days. Outcome measurements included spirometry, sputum neutrophil percentage, blood markers of inflammation, tissue injury and coagulation, vascular function, and heart rate variability. The effects of dietary supplementation and O3 on these outcomes were evaluated with linear mixed-effect models. RESULTS: Compared with filtered air, O3 exposure decreased FVC, FEV1, and FEV1/FVC immediately post exposure regardless of supplementation status. Relative to that in the CTL group, the lung function response to O3 exposure in the FO group was blunted, as evidenced by O3-induced decreases in FEV1 (Normalized CTL -0.40 ± 0.34 L, Normalized FO -0.21 ± 0.27 L) and FEV1/FVC (Normalized CTL -4.67 ± 5.0 %, Normalized FO -1.4 ± 3.18 %) values that were on average 48% and 70% smaller, respectively. Inflammatory responses measured in the sputum immediately post O3 exposure were not different among the three supplementation groups. Systolic blood pressure elevations 20-h post O3 exposure were blunted by OO supplementation. CONCLUSION: FO supplementation appears to offer protective effects against lung function decrements caused by acute O3 exposure in healthy adults.

Translational PK/PD and model-informed development of JNJ-67842125, a Fab reversal agent for JNJ-64179375, a long-acting thrombin inhibitor.

BACKGROUND AND PURPOSE: Antigen-binding fragment (Fab ) reversal agents were developed to reverse, in bleeding emergency, the long-acting anticoagulant effect of JNJ-64179375 (JNJ-9375), a monoclonal antibody that binds exosite-1 on thrombin. EXPERIMENTAL APPROACH: The pharmacokinetic and pharmacodynamic (PK/PD) activities of three reversal agents of varying in vitro binding affinities to JNJ-9375 were characterised in cynomolgus monkeys. The time course of JNJ-9375 anticoagulant activity and reversal effects of each agent were evaluated. A mechanism-based PK/PD model, which integrated free serum concentrations of reversal agent, total and free serum concentrations of JNJ-9375, and thrombin time, was developed to quantitatively relate JNJ-9375 neutralisation to reversal of induced thrombin time prolongation. Model-based allometric scale-up of the lead reversal agent and the PK/PD relationship of JNJ-9375 in healthy volunteers were utilised to predict clinical dosing regimens. KEY RESULTS: Lowering of free JNJ-9375 by the reversal agents corresponded with reversal of thrombin time prolongation. Total JNJ-9375 displayed typical mAb clearance at 2.75 ml·day-1 ·kg-1 , whereas reversal agents cleared faster between 1400 and 2400 ml·day-1 ·kg-1 . The model-estimated in vivo KD values for JNJ-9375 reversal agents were 9 nM (ICHB-256), 0.4 nM (ICHB-281) and 13.7 pM (ICHB-164), in rank-ordered agreement of their KD values determined in vitro. The three reversal agents exhibited different neutralisation characteristics in vivo, governed primarily by their binding kinetics to JNJ-9375. The model predicted a priori free JNJ-9375 kinetics after dosing ICHB-164 (JNJ-67842125) and JNJ-9375 under a different regimen. CONCLUSION AND IMPLICATIONS: The results enabled selection of JNJ-67842125 as the reversal agent for JNJ-9375.

Low levels of fine particulate matter increase vascular damage and reduce pulmonary function in young healthy adults.

BACKGROUND: Fine particulate matter (PM2.5) related mild inflammation, altered autonomic control of cardiovascular function, and changes to cell function have been observed in controlled human exposure studies. METHODS: To measure the systemic and cardiopulmonary impacts of low-level PM exposure, we exposed 20 healthy, young volunteers to PM2.5, in the form of concentrated ambient particles (mean: 37.8 µg/m3, SD 6.5), and filtered air (mean: 2.1 µg/m3, SD 2.6). In this double-blind, crossover study the exposure order was randomized. During the 4 h exposure, volunteers (7 females and 13 males) underwent light intensity exercise to regulate ventilation rate. We measured pulmonary, cardiac, and hematologic end points before exposure, 1 h after exposure, and again 20 h after exposure. RESULTS: Low-level PM2.5 resulted in both pulmonary and extra-pulmonary changes characterized by alterations in systematic inflammation markers, cardiac repolarization, and decreased pulmonary function. A mean increase in PM2.5 concentration (37.8 µg/m3) significantly increased serum amyloid A (SAA), C-reactive protein (CRP), soluble intercellular adhesion molecule-1 (sICAM-1), and soluble vascular cell adhesion molecule-1 (sVCAM-1), 1 h after exposure by 8.7, 9.1, 10.7, and 6.6%, respectively, relative to the filtered air control. SAA remained significantly elevated (34.6%) 20 h after PM2.5 exposure which was accompanied by a 5.7% decrease in percent neutrophils. Decreased pulmonary function was observed 1 h after exposure through a 0.8 and 1.2% decrease in forced expiratory volume in 1 s (FEV1) and FEV1/ forced vital capacity (FEV1/FVC) respectively. Additionally, sex specific changes were observed in repolarization outcomes following PM2.5 exposure. In males, P-wave and QRS complex were increased by 15.4 and 5.4% 1 h after exposure. CONCLUSIONS: This study is the first controlled human exposure study to demonstrate biological effects in response to exposure to concentrated ambient air PM2.5 particles at levels near the PM2.5 US NAAQS standard. CLINICAL TRIAL REGISTRATION INFORMATION: clinicaltrials.gov ; Identifier: NCT03232086 . The study was registered retrospectively on July 25, 2017, prior to final data collection on October 25, 2017 and data analysis.

Conjugation of a peptide to an antibody engineered with free cysteines dramatically improves half-life and activity.

The long circulating half-life and inherently bivalent architecture of IgGs provide an ideal vehicle for presenting otherwise short-lived G-protein-coupled receptor agonists in a format that enables avidity-driven enhancement of potency. Here, we describe the site-specific conjugation of a dual agonist peptide (an oxyntomodulin variant engineered for potency and in vivo stability) to the complementarity-determining regions (CDRs) of an immunologically silent IgG4. A cysteine-containing heavy chain CDR3 variant was identified that provided clean conjugation to a bromoacetylated peptide without interference from any of the endogenous mAb cysteine residues. The resulting mAb-peptide homodimer has high potency at both target receptors (glucagon receptor, GCGR, and glucagon-like peptide 1 receptor, GLP-1R) driven by an increase in receptor avidity provided by the spatially defined presentation of the peptides. Interestingly, the avidity effects are different at the two target receptors. A single dose of the long-acting peptide conjugate robustly inhibited food intake and decreased body weight in insulin resistant diet-induced obese mice, in addition to ameliorating glucose intolerance. Inhibition of food intake and decrease in body weight was also seen in overweight cynomolgus monkeys. The weight loss resulting from dosing with the bivalently conjugated dual agonist was significantly greater than for the monomeric analog, clearly demonstrating translation of the measured in vitro avidity to in vivo pharmacology.

A Long-Acting PYY3-36 Analog Mediates Robust Anorectic Efficacy with Minimal Emesis in Nonhuman Primates.

The gut hormone PYY3-36 reduces food intake in humans and exhibits at least additive efficacy in combination with GLP-1. However, the utility of PYY analogs as anti-obesity agents has been severely limited by emesis and rapid proteolysis, a profile similarly observed with native PYY3-36 in obese rhesus macaques. Here, we found that antibody conjugation of a cyclized PYY3-36 analog achieved high NPY2R selectivity, unprecedented in vivo stability, and gradual infusion-like exposure. These properties permitted profound reduction of food intake when administered to macaques for 23 days without a single emetic event in any animal. Co-administration with the GLP-1 receptor agonist liraglutide for an additional 5 days further reduced food intake with only one animal experiencing a single bout of emesis. This antibody-conjugated PYY analog therefore may enable the long-sought potential of GLP-1/PYY-based combination treatment to achieve robust, well-tolerated weight reduction in obese patients.

Quantitation of intact monoclonal antibody in biological samples: comparison of different data processing strategies.

AIM: Sample extraction using immuno-affinity capture coupled with LC-high-resolution mass spectrometer has recently emerged as a novel approach for the determination of concentrations of large molecules at intact level in biological matrix. METHODOLOGY: In the current work, different data processing strategies for intact protein bioanalysis, deconvoluted mass spectra or extracted ion chromatogram, were applied to quantitate monoclonal antibody in biological samples for comparison of assay performance. CONCLUSION: Both deconvolution and extracted ion chromatogram strategies showed similar selectivity, sensitivity, accuracy and precision. The monkey pharmacokinetics data obtained from both approaches agreed well with each other, and agreed with data obtained from surrogate peptide approach. The pros and cons, and optimal parameters of each approach were discussed.

Heme oxygenase activity increases after exercise in healthy volunteers.

Heme oxygenase (HO) is an essential, rate-limiting protein which catalyses the breakdown of heme to iron, carbon monoxide (CO), and biliverdin. The alpha methene bridge of the heme is eliminated as CO which can be measured as blood carboxyhaemoglobin (COHb). Using blood concentrations of COHb as a measure reflecting HO activity, we tested the postulate that the activity of HO changes with exercise. Ten healthy, nonsmoking volunteers (5 females and 5 males with a mean age ± standard deviation of 25.7 ± 3.2 years), lifetime nonsmokers with no history of respiratory diseases and not taking any medication, were included in the study. Subjects were exposed to filtered air for 2 hrs while alternating exercise for 15 minutes on a cycle ergometer with rest for 15 minutes. Workload was adjusted so that subjects breathed at a ventilatory rate, normalised for body surface area, of 25 L/m2/minute. Immediately before, immediately after, and the day following exercise, blood was drawn by standard venipuncture technique. COHb was determined using the interleukin (IL) 682 Co-Oximeter (Instrumentation Laboratory, Bedford, MA). COHb increased in each participant during the exercise session with the mean value (± standard deviation) almost doubling (1.1 ± 1.6 to 2.1 ± 1.6%) and returned to baseline by the following day (1.3 ± 1.3%). We conclude that exercise increases HO activity.

Ozone exposure is associated with acute changes in inflammation, fibrinolysis, and endothelial cell function in coronary artery disease patients.

BACKGROUND: Air pollution is a major risk factor for cardiovascular disease, of which ozone is a major contributor. Several studies have found associations between ozone and cardiovascular morbidity, but the results have been inconclusive. We investigated associations between ozone and changes across biological pathways associated with cardiovascular disease. METHODS: Using a panel study design, 13 participants with coronary artery disease were assessed for markers of systemic inflammation, heart rate variability and repolarization, lipids, blood pressure, and endothelial function. Daily measurements of ozone and particulate matter (PM2.5) were obtained from central monitoring stations. Single (ozone) and two-pollutant (ozone and PM2.5) models were used to assess percent changes in measurements per interquartile ranges of pollutants. RESULTS: Per interquartile increase in ozone, changes in tissue plasminogen factor (6.6%, 95% confidence intervals (CI) = 0.4, 13.2), plasminogen activator inhibitor-1 (40.5%, 95% CI = 8.7, 81.6), neutrophils (8.7% 95% CI = 1.5, 16.4), monocytes (10.2%, 95% CI = 1.0, 20.1), interleukin-6 (15.9%, 95% CI = 3.6, 29.6), large-artery elasticity index (-19.5%, 95% CI = -34.0, -1.7), and the baseline diameter of the brachial artery (-2.5%, 95% CI = -5.0, 0.1) were observed. These associations were robust in the two-pollutant model. CONCLUSIONS: We observed alterations across several pathways associated with cardiovascular disease in 13 coronary artery disease patients following ozone exposures, independent of PM2.5. The results support the biological plausibility of ozone-induced cardiovascular effects. The effects were found at concentrations below the EPA National Ambient Air Quality Standards for both ozone and PM2.5.

Dietary Supplementation with Olive Oil or Fish Oil and Vascular Effects of Concentrated Ambient Particulate Matter Exposure in Human Volunteers.

BACKGROUND: Exposure to ambient particulate matter (PM) induces endothelial dysfunction, a risk factor for cardiovascular disease. Olive oil (OO) and fish oil (FO) supplements have beneficial effects on endothelial function. OBJECTIVE: In this study we evaluated the potential efficacy of OO and FO in mitigating endothelial dysfunction and disruption of hemostasis caused by exposure to particulate matter (PM). METHODS AND RESULTS: Forty-two participants (58 ± 1 years of age) received either 3 g/day of OO or FO, or no supplements (naive) for 4 weeks prior to undergoing 2-hr exposures to filtered air and concentrated ambient particulate matter (CAP; mean, 253 ± 16 µg/m3). Endothelial function was assessed by flow-mediated dilation (FMD) of the brachial artery preexposure, immediately postexposure, and 20 hr postexposure. Levels of endothelin-1 and markers of fibrinolysis and inflammation were also measured. The FMD was significantly lower after CAP exposure in the naive (-19.4%; 95% CI: -36.4, -2.3 per 100 µg/m3 CAP relative to baseline; p = 0.03) and FO groups (-13.7%; 95% CI: -24.5, -2.9; p = 0.01), but not in the OO group (-7.6%; 95% CI: -21.5, 6.3; p = 0.27). Tissue plasminogen activator levels were significantly increased immediately after (11.6%; 95% CI: 0.8, 22.2; p = 0.04) and 20 hr after CAP exposure in the OO group. Endothelin-1 levels were significantly increased 20 hr after CAP exposure in the naive group only (17.1%; 95% CI: 2.2, 32.0; p = 0.03). CONCLUSIONS: Short-term exposure to CAP induced vascular endothelial dysfunction. OO supplementation attenuated CAP-induced reduction of FMD and changes in blood markers associated with vasoconstriction and fibrinolysis, suggesting that OO supplementation may be an efficacious intervention to protect against vascular effects of exposure to PM. CITATION: Tong H, Rappold AG, Caughey M, Hinderliter AL, Bassett M, Montilla T, Case MW, Berntsen J, Bromberg PA, Cascio WE, Diaz-Sanchez D, Devlin RB, Samet JM. 2015. Dietary supplementation with olive oil or fish oil and vascular effects of concentrated ambient particulate matter exposure in human volunteers. Environ Health Perspect 123:1173-1179; http://dx.doi.org/10.1289/ehp.1408988.

Interaction effects of temperature and ozone on lung function and markers of systemic inflammation, coagulation, and fibrinolysis: a crossover study of healthy young volunteers.

BACKGROUND: Trends in climate suggest that extreme weather events such as heat waves will become more common. High levels of the gaseous pollutant ozone are associated with elevated temperatures. Ozone has been associated with respiratory diseases as well as cardiovascular morbidity and mortality and can reduce lung function and alter systemic markers of fibrinolysis. The interaction between ozone and temperature is unclear. METHODS: Sixteen healthy volunteers were exposed in a randomized crossover study to 0.3 ppm ozone and clean air for 2 hr at moderate (22°C) temperature and again at an elevated temperature (32.5°C). In each case lung function was performed and blood taken before and immediately after exposure and the next morning. RESULTS: Ozone exposure at 22°C resulted in a decrease in markers of fibrinolysis the next day. There was a 51.8% net decrease in PAI-1 (plasminogen activator inhibitor-1), a 12.1% net decrease in plasminogen, and a 17.8% net increase in D-dimer. These significantly differed from the response at 32.5°C, where there was a 44.9% (p = 0.002) and a 27.9% (p = 0.001) increase in PAI-1 and plasminogen, respectively, and a 12.5% (p = 0.042) decrease in D-dimer. In contrast, decrements in lung function following ozone exposure were comparable at both moderate and elevated temperatures (forced expiratory volume in 1 sec, -12.4% vs. -7.5%, p > 0.05). No changes in systemic markers of inflammation were observed for either temperature. CONCLUSION: Ozone-induced systemic but not respiratory effects varied according to temperature. Our study suggests that at moderate temperature ozone may activate the fibrinolytic pathway, while at elevated temperature ozone may impair it. These findings provide a biological basis for the interaction between temperature and ozone on mortality observed in some epidemiologic studies.

Diesel exhaust modulates ozone-induced lung function decrements in healthy human volunteers.

The potential effects of combinations of dilute whole diesel exhaust (DE) and ozone (O3), each a common component of ambient airborne pollutant mixtures, on lung function were examined. Healthy young human volunteers were exposed for 2 hr to pollutants while exercising (~50 L/min) intermittently on two consecutive days. Day 1 exposures were either to filtered air, DE (300 µg/m³), O3 (0.300 ppm), or the combination of both pollutants. On Day 2 all exposures were to O3 (0.300 ppm), and Day 3 served as a followup observation day. Lung function was assessed by spirometry just prior to, immediately after, and up to 4 hr post-exposure on each exposure day. Functional pulmonary responses to the pollutants were also characterized based on stratification by glutathione S-transferase mu 1 (GSTM1) genotype. On Day 1, exposure to air or DE did not change FEV1 or FVC in the subject population (n = 15). The co-exposure to O3 and DE decreased FEV1 (17.6%) to a greater extent than O3 alone (9.9%). To test for synergistic exposure effects, i.e., in a greater than additive fashion, FEV1 changes post individual O3 and DE exposures were summed together and compared to the combined DE and O3 exposure; the p value was 0.057. On Day 2, subjects who received DE exposure on Day 1 had a larger FEV1 decrement (14.7%) immediately after the O3 exposure than the individuals' matched response following a Day 1 air exposure (10.9%). GSTM1 genotype did not affect the magnitude of lung function changes in a significant fashion. These data suggest that altered respiratory responses to the combination of O3 and DE exposure can be observed showing a greater than additive manner. In addition, O3-induced lung function decrements are greater with a prior exposure to DE compared to a prior exposure to filtered air. Based on the joint occurrence of these pollutants in the ambient environment, the potential exists for interactions in more than an additive fashion affecting lung physiological processes.

Exposure to wood smoke particles produces inflammation in healthy volunteers.

OBJECTIVES: Human exposure to wood smoke particles (WSP) impacts on human health through changes in indoor air quality, exposures from wild fires, burning of biomass and air pollution. This investigation tested the postulate that healthy volunteers exposed to WSP would demonstrate evidence of both pulmonary and systemic inflammation. METHODS: Ten volunteers were exposed to filtered air and, 3 weeks or more later, WSP. Each exposure included alternating 15 min of exercise and 15 min of rest for a total duration of 2 h. Wood smoke was generated by heating an oak log on an electric element and then delivered to the exposure chamber. Endpoints measured in the volunteers included symptoms, pulmonary function tests, measures of heart rate variability and repolarisation, blood indices and analysis of cells and fluid obtained during bronchoalveolar lavage. RESULTS: Mean particle mass for the 10 exposures to air and WSP was measured using the mass of particles collected on filters and found to be below the detectable limit and 485±84 µg/m(3), respectively (mean±SD). There was no change in either symptom prevalence or pulmonary function with exposure to WSP. At 20 h after wood smoke exposure, blood tests demonstrated an increased percentage of neutrophils, and bronchial and bronchoalveolar lavage revealed a neutrophilic influx. CONCLUSIONS: We conclude that exposure of healthy volunteers to WSP may be associated with evidence of both systemic and pulmonary inflammation.

Metal ion-assembled micro-collagen heterotrimers.

Collagen mimetic peptides (CMPs) provide critical insight into the assembly, stability, and structure of the triple helical collagen protein. The majority of natural fibrous collagens are aab or abc heterotrimers, yet few examples of heterotrimeric CMPs have been reported. Previously, CMP heterotrimers have only been accessible by total syntheses or by introducing complementary interstrand electrostatic or steric interactions. Here, we describe an abc CMP heterotrimer in which each contributing CMP consists of only three amino acids: glycine, proline and 4-hydroxyproline. Assembly of the heterotrimeric triple helix is directed by a combination of metal-ion coordination to set the relative register of the CMPs, and minimization of valence frustration to direct heterotrimerization. Assembly of the four-component mixture is facile and extremely rapid, and equilibration to the abc heterotrimer occurs within a few hours at modestly elevated temperatures. The melting temperatures of the metal-assembled collagen trimers are higher by some 30°C than the apopeptide assemblies. Two iterations of the design are described, and the outcomes suggest possibilities for designing self-assembling abc and abb heterotrimers.

Recursively enriched dynamic combinatorial libraries for the self-selection of optimally stable proteins.

Even at very low template (guest) concentrations, the optimal template-assembled host from a dynamic combinatorial library (DCL) of host fragments may be unobtainable because hetero-oligomers will always be present at higher concentrations than isoenergetic homo-oligomers. Recursively enriched dynamic combinatorial libraries (REDCLs) offer a general solution to this problem that should be applicable to any self-selecting system under thermodynamic control. The utility of the REDCL strategy is demonstrated by determination of the optimal hydrophobic core packing in a template-assembled triple helical protein for which the template is a metal ion and the contributing host fragments are components of a 36-member conformationally restricted peptide library in which each peptide is augmented with a metal-binding moiety. Convergence of the 8436-member DCL to 5 optimal trimers (0.06% of the DCL) is complete after four cycles of enrichment. The core packing of the optimal sequences is shown to be native-like, and to reflect the hydrophobic amino acid preferences found in natural parallel three-stranded coiled coils. The influence of potentially critical amino acids on the outcome of the recursive enrichment is explored in a second REDCL. The same peptide sequences were returned and were shown to populate seven of the 8436 possible trimers, or 0.08% of the DCL.

Lung function and inflammatory responses in healthy young adults exposed to 0.06 ppm ozone for 6.6 hours.

RATIONALE: Exposure to ozone causes a decrease in spirometric lung function and an increase in airway inflammation in healthy young adults at concentrations as low as 0.08 ppm, close to the National Ambient Air Quality Standard for ground level ozone. OBJECTIVES: To test whether airway effects occur below the current ozone standard and if they are more pronounced in potentially susceptible individuals, such as those deficient in the antioxidant gene glutathione S-transferase mu 1 (GSTM1). METHODS: Pulmonary function and subjective symptoms were measured in 59 healthy young adults (19-35 yr) immediately before and after exposure to 0.0 (clean air, CA) and 0.06 ppm ozone for 6.6 hours in a chamber while undergoing intermittent moderate exercise. The polymorphonuclear neutrophil (PMN) influx was measured in 24 subjects 16 to 18 hours postexposure. MEASUREMENTS AND MAIN RESULTS: Subjects experienced a significantly greater (P = 0.008) change in FEV(1) (± SE) immediately after exposure to 0.06 ppm ozone compared with CA (-1.71 ± 0.50% vs. -0.002 ± 0.46%). The decrement in FVC was also greater (P = 0.02) after ozone versus CA (-2.32 ± 0.41% vs. -1.13 ± 0.34%). Similarly, changes in %PMN were greater after ozone (54.0 ± 4.6%) than CA (38.3 ± 3.7%) exposure (P < 0.001). Symptom scores were not different between ozone versus CA. There were no significant differences in changes in FEV(1), FVC, and %PMN between subjects with GSTM1-positive and GSTM1-null genotypes. CONCLUSIONS: Exposure of healthy young adults to 0.06 ppm ozone for 6.6 hours causes a significant decrement of FEV(1) and an increase in neutrophilic inflammation in the airways. GSTM1 genotype alone appears to have no significant role in modifying the effects.

Protein core packing by dynamic combinatorial chemistry.

We describe the first example of the recursive selection of biologically relevant macromolecules from a dynamic combinatorial library (DCL). A small library of 36 peptides was allowed to undergo self-association in aqueous solution to form 8436 trimers. The stability of each of these trimers was governed by the formation of a well-packed hydrophobic core. The DCL allowed variation of the hydrophobic residues comprising this core over all combinations of glycine, alanine, valine, leucine, isoleucine, and phenylalanine at six positions. The study leads to three important conclusions: (i) fewer than 0.2% of all possible core packing arrangements have high folding stabilities; (ii) these arrangements are stabilized by intimate "jigsaw" packing, not by sequestration of maximum hydrophobic surface area; (iii) a well-defined "rule" for packing of stable cores exists, but this rule is nuanced by the presence of two unexpected amino acid sequences and the absence of one expected amino acid sequence.

Electrostatic determinants of stability in parallel 3-stranded coiled coils.

The optimal positioning of salt-bridging interactions in a parallel alpha-helical homotrimeric coiled coil has been explored in a metal ion-assembled polypeptide trimer of 60 residues; arginine-glutamate pairs are more stabilizing than the corresponding lysine-glutamate pairs, and optimal stabilization is obtained with positively charged arginine residues at the c positions of the alpha-helical heptad and negatively charged glutamate residues at the e positions.

Endothelial dysfunction: associations with exposure to ambient fine particles in diabetic individuals.

BACKGROUND: Exposure to fine airborne particulate matter [< or =2.5 microm in aerodynamic diameter (PM(2.5))] has been associated with cardiovascular and hematologic effects, especially in older people with cardiovascular disease. Some epidemiologic studies suggest that adults with diabetes also may be a particularly susceptible population. OBJECTIVES: The purpose of this study was to analyze the short-term effects of ambient PM(2.5) on markers of endothelial function in diabetic volunteers. METHODS: We conducted a prospective panel study in 22 people with type 2 diabetes mellitus in Chapel Hill, North Carolina (USA), from November 2004 to December 2005. We acquired daily measurements of PM(2.5) and meteorologic data at central monitoring sites. On 4 consecutive days, we measured endothelial function by brachial artery ultrasound in all participants and by pulsewave measurements in a subgroup. Data were analyzed using additive mixed models with a random participant effect and adjusted for season, day of the week, and meteorology. RESULTS: Flow-mediated dilatation decreased in association with PM(2.5) during the first 24 hr, whereas small-artery elasticity index decreased with a delay of 1 and 3 days. These PM(2.5)-associated decrements in endothelial function were greater among participants with a high body mass index, high glycosylated hemoglobin A1c, low adiponectin, or the null polymorphism of glutathione S-transferase M1. However, high levels of myeloperoxidase on the examination day led to strongest effects on endothelial dysfunction. CONCLUSIONS: These data demonstrate that PM(2.5) exposure may cause immediate endothelial dysfunction. Clinical characteristics associated with insulin resistance were associated with enhanced effects of PM on endothelial function. In addition, participants with greater oxidative potential seem to be more susceptible.