A disease-associated gene desert directs macrophage inflammation through ETS2.

Increasing rates of autoimmune and inflammatory disease present a burgeoning threat to human health1. This is compounded by the limited efficacy of available treatments1 and high failure rates during drug development2, highlighting an urgent need to better understand disease mechanisms. Here we show how functional genomics could address this challenge. By investigating an intergenic haplotype on chr21q22-which has been independently linked to inflammatory bowel disease, ankylosing spondylitis, primary sclerosing cholangitis and Takayasu's arteritis3-6-we identify that the causal gene, ETS2, is a central regulator of human inflammatory macrophages and delineate the shared disease mechanism that amplifies ETS2 expression. Genes regulated by ETS2 were prominently expressed in diseased tissues and more enriched for inflammatory bowel disease GWAS hits than most previously described pathways. Overexpressing ETS2 in resting macrophages reproduced the inflammatory state observed in chr21q22-associated diseases, with upregulation of multiple drug targets, including TNF and IL-23. Using a database of cellular signatures7, we identified drugs that might modulate this pathway and validated the potent anti-inflammatory activity of one class of small molecules in vitro and ex vivo. Together, this illustrates the power of functional genomics, applied directly in primary human cells, to identify immune-mediated disease mechanisms and potential therapeutic opportunities.

Manipulating twisted electrons in strong-field ionization.

We investigate the discrete orbital angular momentum (OAM) of photoelectrons freed in strong-field ionization. We use these 'twisted' electrons to provide an alternative interpretation on existing experimental work of vortex interferences caused by strong field ionization mediated by two counter-rotating circularly polarized pulses separated by a delay. Using the strong field approximation, we derive an interference condition for the vortices. In computations for a neon target we find very good agreement of the vortex condition with photoelectron momentum distributions computed with the strong field approximation, as well as with the time-dependent methods Qprop and R-Matrix. For each of these approaches we examine the OAM of the photoelectrons, finding a small number of vortex states localized in separate energy regions. We demonstrate that the vortices arise from the interference of pairs of twisted electron states. The OAM of each twisted electron state can be directly related to the number of arms of the spiral in that region. We gain further understanding by recreating the vortices with pairs of twisted electrons and use this to determine a semiclassical relation for the OAM. A discussion is included on measuring the OAM in strong field ionization directly or by employing specific laser pulse schemes as well as utilizing the OAM in time-resolved imaging of photo-induced dynamics.

Antimicrobial resistance in Salmonella that caused foodborne disease outbreaks: United States, 2003-2012.

Although most non-typhoidal Salmonella illnesses are self-limiting, antimicrobial treatment is critical for invasive infections. To describe resistance in Salmonella that caused foodborne outbreaks in the United States, we linked outbreaks submitted to the Foodborne Disease Outbreak Surveillance System to isolate susceptibility data in the National Antimicrobial Resistance Monitoring System. Resistant outbreaks were defined as those linked to one or more isolates with resistance to at least one antimicrobial drug. Multidrug resistant (MDR) outbreaks had at least one isolate resistant to three or more antimicrobial classes. Twenty-one per cent (37/176) of linked outbreaks were resistant. In outbreaks attributed to a single food group, 73% (16/22) of resistant outbreaks and 46% (31/68) of non-resistant outbreaks were attributed to foods from land animals (P < 0·05). MDR Salmonella with clinically important resistance caused 29% (14/48) of outbreaks from land animals and 8% (3/40) of outbreaks from plant products (P < 0·01). In our study, resistant Salmonella infections were more common in outbreaks attributed to foods from land animals than outbreaks from foods from plants or aquatic animals. Antimicrobial susceptibility data on isolates from foodborne Salmonella outbreaks can help determine which foods are associated with resistant infections.

Extreme-Ultraviolet-Initated High-Order Harmonic Generation: Driving Inner-Valence Electrons Using Below-Threshold-Energy Extreme-Ultraviolet Light.

We propose a novel scheme for resolving the contribution of inner- and outer-valence electrons in extreme-ultraviolet (XUV)-initiated high-harmonic generation in neon. By probing the atom with a low-energy (below the 2s ionization threshold) ultrashort XUV pulse, the 2p electron is steered away from the core, while the 2s electron is enabled to describe recollision trajectories. By selectively suppressing the 2p recollision trajectories, we can resolve the contribution of the 2s electron to the high-harmonic spectrum. We apply the classical trajectory model to account for the contribution of the 2s electron, which allows for an intuitive understanding of the process.

The effects of temperature and dissolved oxygen on antioxidant defences and oxidative damage in the fathead minnow Pimephales promelas.

Fathead minnows Pimephales promelas maintained at 25° C for 6 h had significantly higher superoxide dismutase (SOD) activity than fish maintained at 7 or 32° C, but hypoxic conditions (3 mg l(-1) O2 ) over the same time period did not affect SOD activity. Fish in better body condition (length-adjusted mass) had higher SOD activity. In a separate experiment, P. promelas maintained at three water temperatures (7, 23 and 32° C) for 31 days did not differ in liver acrolein, a biomarker of oxidative stress.

Interference between competing pathways in atomic harmonic generation.

We investigate the influence of the autoionizing 3s3p(6)nℓ resonances on the fifth harmonic generated by 200-240 nm laser fields interacting with Ar. To determine the influence of a multielectron response we develop the capability within time-dependent R-matrix theory to determine the harmonic spectra generated. The fifth harmonic is affected by interference between the response of a 3s electron and the response of a 3p electron, as demonstrated by the asymmetric profiles in the harmonic yields as functions of wavelength.

Restoring Carboxylates on Highly Modified Alginates Improves Gelation, Tissue Retention and Systemic Capture.

Alginate hydrogels are gaining traction for use in drug delivery, regenerative medicine, and as tissue engineered scaffolds due to their physiological gelation conditions, high tissue biocompatibility, and wide chemical versatility. Traditionally, alginate is decorated at the carboxyl group to carry drug payloads, peptides, or proteins. While low degrees of substitution do not cause noticeable mechanical changes, high degrees of substitution can cause significant losses to alginate properties including complete loss of calcium cross-linking. While most modifications used to decorate alginate deplete the carboxyl groups, we propose that alginate modifications that replenish the carboxyl groups could overcome the loss in gel integrity and mechanics. In this report, we demonstrate that restoring carboxyl groups during functionalization maintains calcium cross-links as well as hydrogel shear-thinning and self-healing properties. In addition, we demonstrate that alginate hydrogels modified to a high degree with azide modifications that restore the carboxyl groups have improved tissue retention at intramuscular injection sites and capture blood-circulating cyclooctynes better than alginate hydrogels modified with azide modifications that deplete the carboxyl groups. Taken together, alginate modifications that restore carboxyl groups could significantly improve alginate hydrogel mechanics for clinical applications. STATEMENT OF SIGNIFICANCE: Chemical modification of hydrogels provides a powerful tool to regulate cellular adhesion, immune response, and biocompatibility with local tissues. Alginate, due to its biocompatibility and easy chemical modification, is being explored for tissue engineering and drug delivery. Unfortunately, modifying alginate to a high degree of substitution consumes carboxyl group, which are necessary for ionic gelation, leading to poor hydrogel crosslinking. We introduce alginate modifications that restore the alginate's carboxyl groups. We demonstrate that modifications that reintroduce carboxyl groups restore gelation and improve gel mechanics and tissue retention. In addition to contributing to a basic science understanding of hydrogel properties, we anticipate our approach will be useful to create tissue engineered scaffolds and drug delivery platforms.

Ultrasonic Microplotting of Microgel Bioinks.

Material scaffolds that mimic the structure, function, and bioactivity of native biological tissues are in constant development. Recently, material scaffolds composed of microgel particles have shown promise for applications ranging from bone regeneration to spheroid cell growth. Previous studies with poly N-isopropylacrylamide microgel scaffolds utilized a layer-by-layer (LBL) technique where individual, uniform microgel layers are built on top of each other resulting in a multilayer scaffold. However, this technique is limited in its applications due to the inability to control microscale deposition or patterning of multiple particle types within a microgel layer. In this study, an ultrasonic microplotting technique is used to address the limitations of LBL fabrication to create patterned microgel films. Printing parameters, such as bioink formulation, surface contact angle, and print head diameter, are optimized to identify the ideal parameters needed to successfully print microgel films. It was found that bioinks composed of 2 mg/mL of microgels and 20% polyethylene glycol by volume (v/v), on bovine serum albumin-coated glass, with a print head diameter of 50 µm resulted in the highest quality prints. Patterned films were created with a maximum resolution of 50 µm with the potential for finer resolutions to be achieved with alternative bioink compositions and printing parameters. Overall, ultrasonic microplotting can be used to create more complex microgel films than is possible with LBL techniques and offers the possibility of greater printing resolution in 3D with further technology development.

Insulin, glucagon, and glucose exhibit synchronous, sustained oscillations in fasting monkeys.

In overnight fasted rhesus monkeys, synchronous, regular oscillations occurred in the plasma concentrations of glucose, insulin, and glucagon. The oscillations displayed a period averaging 9 minutes. The amplitudes for insulin and glucagon were ten and five times greater than for glucose. Insulin cycled in and glucagon out of phase with glucose. In baboons, oscillations of glucose and insulin were smaller than in rhesus monkeys, while in man, regular oscillations were not observed.

Analysis of the structural and mechanical effects of procoagulant agents on neonatal fibrin networks following cardiopulmonary bypass.

Essentials The standard of care (SOC) for treating neonatal bleeding is transfusion of adult blood products. We compared neonatal clots formed with cryoprecipitate (SOC) to two procoagulant therapies. The current SOC resulted in clots with increased stiffness and decreased fibrinolytic properties. Procoagulant therapies may be a viable alternative to SOC treatment for neonatal bleeding. SUMMARY: Background Bleeding is a serious complication of neonates undergoing cardiopulmonary bypass (CPB) and associated with substantial morbidity and mortality. Bleeding is addressed through the transfusion of adult blood products, including platelets and cryoprecipitate. However, significant differences exist between neonatal and adult clotting components, specifically fibrinogen. Our recent ex vivo studies have shown that neonatal fibrinogen does not fully integrate with adult fibrinogen, leading to decreased susceptibility to fibrinolysis. These differences may contribute to ineffective clot formation and/or an increased risk of thrombosis. A need exists to identify more effective and safer methods to promote clotting in neonates. Objectives Procoagulant agents, such as prothrombin complex concentrates (PCCs) and recombinant activated factor VII (rFVIIa), are being used off-label to treat excessive bleeding in neonates after CPB. Because these agents stimulate endogenous fibrin formation, we hypothesize that their addition to post-CPB neonatal plasma will better recapitulate native clot properties than cryoprecipitate. Methods We analyze the structural, mechanical and degradation properties of fibrin matrices formed by neonatal plasma collected after CPB in the presence of an activated four-factor (F) PCC (FEIBA), rFVIIa, or cryoprecipitate using confocal microscopy, atomic force microscopy and a fluidics-based degradation assay. Results The ex vivo addition of FEIBA and rFVIIa to post-CPB neonatal plasma resulted in enhanced clot networks with differences in fibrin alignment, mechanics and degradation properties. Conclusions Our results suggest that these procoagulant agents could be used as an alternative to the transfusion of adult fibrinogen for the treatment of bleeding after CPB in neonates.

Absorption of calcium measured by intubation and perfusion of the intact hyman small intestine.

Absorption of calcium was measured by direct intubation and perfusion of the small intestine in 10 volunteer normal adult subjects, two adults with celiac-sprue, and one with a parathyroid adenoma. A total of 60 studies were completed using one of two different levels, duodenojejunum or ileum. Solutions containing stable calcium, radiocalcium(47), and a nonabsorbable dilution-concentration marker, polyethylene glycol, were infused at a uniform rate via the proximal lumen of a triple-lumen polyvinyl tube. The mixed intraluminal contents were continuously sampled by siphonage from two distal sites, 10 and 60 cm below the point of infusion. Unidirectional flux rates, lumen to blood and blood to lumen, and net absorption of calcium for the 50 cm segment of small intestine between the two collection sites were calculated from the measured changes in concentration of stable calcium, calcium-47, and polyethylene glycol.Flux of calcium from lumen to blood in the duodenojejunum of normal subjects was appreciable even when the concentration of calcium in the perfusate was below that of extracellular fluid and, as the intraluminal concentration of calcium was increased through a range of 0.5-3.5 mumoles/ml, was positively correlated, ranging from 1.9 to 7.0 mumoles/min per 50 cm. Repeated studies of individual subjects demonstrated a consistent pattern of absorptive efficiency in each, but significant variability from person to person. Flux from lumen to blood in the ileal segment occurred at a much lower rate than that found in the proximal intestine, and there was not a significant dependence upon intraluminal calcium concentration. The opposite flux, from blood to lumen, was low both in the duodenojejunum and ileum (average 0.76 mumoles/min per 50 cm) and was independent of the intraluminal calcium concentration. Unidirectional flux, lumen to blood, from the duodenojejunum was not altered by parathyroid extract administered at the time of the infusion, but was accelerated in the subject with a parathyroid adenoma and markedly reduced in the two subjects with celiac-sprue.

Inhibition of LtxA toxicity by blocking cholesterol binding with peptides.

The leukotoxin (LtxA) produced by Aggregatibacter actinomycetemcomitans kills host immune cells, allowing the bacterium to establish an ecological niche in the upper aerodigestive tract of its human host. The interaction of LtxA with human immune cells is both complex and multifaceted, involving membrane lipids as well as cell-surface proteins. In the initial encounter with the host cell, LtxA associates with lymphocyte function-associated antigen-1, a cell surface adhesion glycoprotein. However, we have also demonstrated that the toxin associates strongly with the plasma membrane lipids, specifically cholesterol. This association with cholesterol is regulated by a cholesterol recognition amino acid consensus (CRAC) motif, with a sequence of (334) LEEYSKR(340), in the N-terminal region of the toxin. Here, we have demonstrated that removal of cholesterol from the plasma membrane or mutation of the LtxA CRAC motif inhibits the activity of the toxin in THP-1 cells. To inhibit LtxA activity, we designed a short peptide corresponding to the CRAC(336) motif of LtxA (CRAC(336WT)). This peptide binds to cholesterol and thereby inhibits the toxicity of LtxA in THP-1 cells. Previously, we showed that this peptide inhibits LtxA toxicity against Jn.9 (Jurkat) cells, indicating that peptides derived from the cholesterol-binding site of LtxA may have a potential clinical applicability in controlling infections of repeats-in-toxin-producing organisms.

Mucosal production of uric acid by airway epithelial cells contributes to particulate matter-induced allergic sensitization.

Exposure to particulate matter (PM), a major component of air pollution, contributes to increased morbidity and mortality worldwide. PM induces innate immune responses and contributes to allergic sensitization, although the mechanisms governing this process remain unclear. Lung mucosal uric acid has also been linked to allergic sensitization. The links among PM exposure, uric acid, and allergic sensitization remain unexplored. We therefore investigated the mechanisms behind PM-induced allergic sensitization in the context of lung mucosal uric acid. PM10 and house dust mite exposure selectively induced lung mucosal uric acid production and secretion in vivo, which did not occur with other challenges (lipopolysaccharide, virus, bacteria, or inflammatory/fibrotic stimuli). PM10-induced uric acid mediates allergic sensitization and augments antigen-specific T-cell proliferation, which is inhibited by uricase. We then demonstrate that human airway epithelial cells secrete uric acid basally and after stimulation through a previously unidentified mucosal secretion system. Our work discovers a previously unknown mechanism of air pollution-induced, uric acid-mediated, allergic sensitization that may be important in the pathogenesis of asthma.

Effect of castration timing and oral meloxicam administration on growth performance, inflammation, behavior, and carcass quality of beef calves.

Beef bull calves (n = 62) were assigned randomly, within sire breed, to 1 of 4 treatments at birth. Treatments were 1) surgical castration near birth, 2) surgical castration near birth with oral administration of meloxicam (1 mg/kg BW), 3) surgical castration at weaning (WNG), or 4) surgical castration at weaning with oral administration of meloxicam (1 mg/kg BW; WMX). A subset of calves (n = 7/treatment group) were selected randomly near birth for blood collection, behavioral analyses, and rectal temperature (RT) records for a 7-d postcastration period on d 0 (birth), 1, 3, and 7, and on d 214 (weaning), 214 + 6 h, 215, 217, 221, and 228. Calf standing and lying activity were monitored from the same subsets by recording x- and y-axis positions of an accelerometer attached to the right metatarsus for 7 d postcastration. Calf BW was recorded throughout the entire production cycle, and carcass data were collected at slaughter. For statistical analyses, bulls left intact at birth were considered a positive control (BUL) for observations that occurred before their treatment application at weaning; likewise, bulls castrated at birth were considered a negative control (STR) during postweaning observations. No difference (P > 0.88) occurred in ADG between treatments throughout the preweaning period (d 0 to 214); however, 56-d postweaning ADG was greatest ( P= 0.02) in STR, intermediate in WMX, and least in WNG. At weaning, haptoglobin (Hp) was greater (P ≤ 0.005) for WNG and WMX compared to STR on d 214+6 h, 215, and 217, and Hp was greater (P = 0.05) in WNG compared to WMX on d 217. Neutrophils increased (P < 0.001) and red blood cells decreased (P ≤ 0.03) for WNG and WMX on d 214+6 h and 217, respectively. Postweaning behavior observations indicated that STR calves spent the least proportion of time standing (P = 0.002) when compared to WNG and WMX. Furthermore, WMX calves exhibited a greater proportion of time spent standing (P = 0.03) compared to WNG. Grazing and finishing phase ADG and carcass measurements did not differ (P ≥ 0.24) across treatments. In this study, surgical castration at weaning, but not near birth, altered the acute phase response, behavior, and growth performance. Oral meloxicam reduced serum Hp and improved ADG briefly when administered to calves castrated at weaning. Oral administration of meloxicam may be efficacious for mitigating some of the stress and inflammation associated with castration of weaning-age bull calves.

Synchronous, sustained oscillation of C-peptide and insulin in the plasma of fasting monkeys.

The previously reported synchronous oscillations in plasma glucose and insulin levels have been further studied to determine whether the phenomenon can be attributed to cyclic secretion or degradation of the hormone. Plasma C-peptide concentrations were measured and found to cycle with the same period as plasma insulin suggesting that the oscillations of insulin arise from changing secretion rather than degradation. The amplitude of C-peptide oscillation was 50 percent that of insulin. Assuming first order kinetics and the same relative rates of disappearance as in humans, this difference in amplitudes in consistent with equal secretion rates for the two peptides.

Facial frequency manipulation normalizes face discrimination in AD.

People with AD have deficient contrast sensitivity and impaired face discrimination. The authors presented photographs of unfamiliar faces of three different sizes to enhance the low, middle, or high facial frequency information (cycles per face). Patients with AD demonstrated normal discrimination of small faces only, indicating that impaired contrast sensitivity at low facial frequencies contributes to their poor face discrimination.

Tooth pulp-driven neurons in somatosensory cortex of primates: role in pain mechanisms including a review of the literature.

The tooth pulp of primates was stimulated electrically while searching for evoked unit potentials in the cerebral cortex. Control procedures were employed to assure that the electrical stimuli reached only tooth pulp fibers but no extrapulpal sensory fibers. In addition, an electrode was inserted in soft tissue surrounding the tooth for separate excitation of extrapulpal axons. A tooth pulp projection area was identified in the "face area" of primary somatosensory cortex. Two major neuron groups were encountered, one excited only by the extrapulpal soft tissue stimulus, the other by tooth pulp stimuli. Within the pulp-projection area, soft tissue-driven neurons were most numerous in superficial cortex of the postcentral gyrus, pulp-driven neurons dominated in deep cortex in the base of the central sulcus. The pulp-driven population divided into several functional subsets: those excited from one pulp only (conceivably capable of localizing pulpal stimuli), those excited from more than one pulp and those excited from both pulp and extrapulpal soft tissue. Within each of these 3 pulp-driven subsets, some units responded to single shock, others only to a train of shocks. Mean discharge latency was shortest for the population excited only from soft tissue, intermediate for pulp-driven units excited by single shock and longest for pulp-driven units excited only by trains of shocks. Both soft tissue and pulp stimuli evoked extensive inhibitory effects. In the Discussion, the possible role of pulp-driven neurons in pain is considered. The functional properties of some neurons are consistent with a role in stimulus localization but those of the remaining neurons suggest other roles in pain. An examination of the literature on cortex and pain suggests that normally somatosensory cortex is important for localizing painful stimuli and that it contributes to other pain mechanisms as well. After certain lesions, somatosensory cortex has the capacity for generating "central" pain just like other structures in the nociceptive pathway.

Spatial summation of pre-pain and pain in human teeth.

The purpose of this work was to investigate the relation between the sensations of pain and 'pre-pain' evoked by stimulation of teeth in human subjects. Electrical pulses of progressively increasing amplitude, generated by a computer-controlled stimulator, were applied to 1 or 2 teeth, and the subjects responded by indicating the nature of the resulting sensation. Pre-pain and pain could be readily and rapidly distinguished by all 11 subjects (response latency about 0.4 sec). Both sensations had stable thresholds with relatively small variance (S.D. 10-15% of threshold value) for a given subject. Subjects characterized the stimuli as indifferent or unpleasant, localized, and brief. By using special stimulation strategies (termed 'optimal trajectories') for exciting 2 teeth simultaneously, spatial summation for pre-pain was demonstrated in most subjects and for pain in almost all subjects. Spatial summation of pre-pain resulted in pain rather than in more intense pre-pain. These results are consistent with both the dual modality (separate afferent fibers for pre-pain and pain) and the single modality hypotheses (single type of afferent fibers) of tooth pulp sensibility, but favor single modality innervation.

A method based on PCR for the construction of cDNA libraries and probes from small amounts of tissue.

A PCR-based method is described for the production of cDNA libraries and total cDNA probes from a few milligrams of tissue. Using a model system, we show how a PCR library and PCR probes can be used to identify genes expressed at different levels in two tissues. Small amounts of tissue derived from two plants, one infected with arabis mosaic virus and the other uninfected, were used to make a library and probes. This library and the probes were used to identify viral genes expressed only in the infected plant.