Placental HTRA1 cleaves α1-antitrypsin to generate a NET-inhibitory peptide.

Neutrophil extracellular traps (NETs) are important components of innate immunity. Neonatal neutrophils (polymorphonuclear leukocytes [PMNs]) fail to form NETs due to circulating NET-inhibitory peptides (NIPs), cleavage fragments of α1-antitrypsin (A1AT). How fetal and neonatal blood NIPs are generated remains unknown, however. The placenta expresses high-temperature requirement serine protease A1 (HTRA1) during fetal development, which can cleave A1AT. We hypothesized that placentally expressed HTRA1 regulates the formation of NIPs and that NET competency changed in PMNs isolated from neonatal HTRA1 knockout mice (HTRA1-/-). We found that umbilical cord blood plasma has elevated HTRA1 levels compared with adult plasma and that recombinant and placenta-eluted HTRA1 cleaves A1AT to generate an A1AT cleavage fragment (A1ATM383S-CF) of molecular weight similar to previously identified NIPs that block NET formation by adult neutrophils. We showed that neonatal mouse pup plasma contains A1AT fragments that inhibit NET formation by PMNs isolated from adult mice, indicating that NIP generation during gestation is conserved across species. Lipopolysaccharide-stimulated PMNs isolated from HTRA1+/+ littermate control pups exhibit delayed NET formation after birth. However, plasma from HTRA1-/- pups had no detectable NIPs, and PMNs from HTRA1-/- pups became NET competent earlier after birth compared with HTRA1+/+ littermate controls. Finally, in the cecal slurry model of neonatal sepsis, A1ATM383S-CF improved survival in C57BL/6 pups by preventing pathogenic NET formation. Our data indicate that placentally expressed HTRA1 is a serine protease that cleaves A1AT in utero to generate NIPs that regulate NET formation by human and mouse PMNs.

Identification of a Distinct Platelet Phenotype in the Elderly: ADP Hypersensitivity Coexists With Platelet PAR (Protease-Activated Receptor)-1 and PAR-4-Mediated Thrombin Resistance.

BACKGROUND: Thrombin (via PAR [protease-activated receptor]-1 and PAR-4) and ADP (via P2Y12 receptors) are potent endogenous platelet activators implicated in the development of cardiovascular disease. We aimed to assess whether platelet pathways alter with aging. METHODS: We characterized platelet activity in community-dwelling volunteers (n=174) in the following age groups: (1) 20 to 30 (young); (2) 40 to 55 (middle-aged); (3) ≥70 years (elderly). Platelet activity was assessed by aggregometry; flow cytometry (surface markers [P-selectin: alpha granule release, CD63: dense granule release, PAC-1: measure of conformationally active GPIIb/IIIa at the fibrinogen binding site]) measured under basal conditions and after agonist stimulation [ADP, thrombin, PAR-1 agonist or PAR-4 agonist]); receptor cleavage and quantification; fluorometry; calcium flux; ELISA. RESULTS: The elderly had higher basal platelet activation than the young, evidenced by increased expression of P-selectin, CD63, and PAC-1, which correlated with increasing inflammation (IL [interleukin]-1ß/IL-6). The elderly demonstrated higher P2Y12 receptor density, with greater ADP-induced platelet aggregation (P<0.05). However, elderly subjects were resistant to thrombin, achieving less activation in response to thrombin (higher EC50) and to selective stimulation of both PAR-1 and PAR-4, with higher basal PAR-1/PAR-4 cleavage and less inducible PAR-1/PAR-4 cleavage (all P<0.05). Thrombin resistance was attributable to a combination of reduced thrombin orienting receptor GPIbα (glycoprotein Ibα), reduced secondary ADP contribution to thrombin-mediated activation, and blunted calcium flux. D-Dimer, a marker of in situ thrombin generation, correlated with platelet activation in the circulation, ex vivo thrombin resistance, and circulating inflammatory mediators (TNF [tumor necrosis factor]-α/IL-6). CONCLUSIONS: Aging is associated with a distinctive platelet phenotype of increased basal activation, ADP hyperreactivity, and thrombin resistance. In situ thrombin generation associated with systemic inflammation may be novel target to prevent cardiovascular disease in the elderly.

Main effect and epistatic QTL affecting spike shattering and association with plant height revealed in two spring wheat (Triticum aestivum L.) populations.

KEY MESSAGE: A major QTL on chromosome arm 4BS was associated with reduced spike shattering and reduced plant height in coupling phase, and a second major QTL associated with reduced spike shattering was detected on chromosome arm 5AL in the same wheat variety Carberry. Spike shattering can cause severe grain yield loss in wheat. Development of cultivars with reduced shattering but having easy mechanical threshability is the target of wheat breeding programs. This study was conducted to determine quantitative trait loci (QTL) associated with shattering resistance, and epistasis among QTL in the populations Carberry/AC Cadillac and Carberry/Thatcher. Response of the populations to spike shattering was evaluated near Swift Current, SK, in four to five environments. Plant height data recorded in different locations and years were used to determine the relationship of the trait with spike shattering. Each population was genotyped and mapped with the wheat 90 K Illumina iSelect SNP array. Main effect QTL were analyzed by MapQTL 6, and epistatic interactions between main effect QTL were determined by QTLNetwork 2.0. Correlations between height and shattering ranged from 0.15 to 0.49. Carberry contributed two major QTL associated with spike shattering on chromosome arms 4BS and 5AL, detected in both populations. Carberry also contributed two minor QTL on 7AS and 7AL. AC Cadillac contributed five minor QTL on 1AL, 2DL, 3AL, 3DL and 7DS. Nine epistatic QTL interactions were identified, out of which the most consistent and synergistic interaction, that reduced the expression of shattering, occurred between 4BS and 5AL QTL. The 4BS QTL was consistently associated with reduced shattering and reduced plant height in the coupling phase. The present findings shed light on the inheritance of shattering resistance and provide genetic markers for manipulating the trait to develop wheat cultivars.

Building platelet phenotypes: Diaphanous-related formin 1 (DIAPH1)-related disorder.

Variants of the Diaphanous-Related Formin 1 (DIAPH-1) gene have recently been reported causing inherited macrothrombocytopenia. The essential/"diagnostic" characteristics associated with the disorder are emerging; however, robust and complete criteria are not established. Here, we report the first cases of DIAPH1-related disorder in Australia caused by the autosomal dominant gain-of-function DIAPH1 R1213X variant formed by truncation of the protein within the diaphanous auto-regulatory domain (DAD) with loss of regulatory motifs responsible for autoinhibitory interactions within the DIAPH1 protein. We affirm phenotypic changes induced by the DIAPH1 R1213X variant to include macrothrombocytopenia, early-onset progressive sensorineural hearing loss, and mild asymptomatic neutropenia. High-resolution microscopy confirms perturbations of cytoskeletal dynamics caused by the DIAPH1 variant and we extend the repertoire of changes generated by this variant to include alteration of procoagulant platelet formation and possible dental anomalies.

Neonatal Cranial Ultrasound Findings among Infants Born Extremely Preterm: Associations with Neurodevelopmental Outcomes at 10 Years of Age.

OBJECTIVE: To examine the association between neonatal cranial ultrasound (CUS) abnormalities among infants born extremely preterm and neurodevelopmental outcomes at 10 years of age. STUDY DESIGN: In a multicenter birth cohort of infants born at <28 weeks of gestation, 889 of 1198 survivors were evaluated for neurologic, cognitive, and behavioral outcomes at 10 years of age. Sonographic markers of white matter damage (WMD) included echolucencies in the brain parenchyma and moderate to severe ventricular enlargement. Neonatal CUS findings were classified as intraventricular hemorrhage (IVH) without WMD, IVH with WMD, WMD without IVH, and neither IVH nor WMD. RESULTS: WMD without IVH was associated with an increased risk of cognitive impairment (OR 3.5, 95% CI 1.7, 7.4), cerebral palsy (OR 14.3, 95% CI 6.5, 31.5), and epilepsy (OR 6.9; 95% CI 2.9, 16.8). Similar associations were found for WMD accompanied by IVH. Isolated IVH was not significantly associated these outcomes. CONCLUSIONS: Among children born extremely preterm, CUS abnormalities, particularly those indicative of WMD, are predictive of neurodevelopmental impairments at 10 years of age. The strongest associations were found with cerebral palsy.

Mechanistic Investigation of Drug Supersaturation in the Presence of Polysorbates as Solubilizing Additives by Solution Nuclear Magnetic Resonance Spectroscopy.

The introduction of solubilizing additives has historically been an attractive approach to address the ever-growing proportion of poorly water-soluble drug (PWSD) compounds within the modern drug discovery pipeline. Lipid-formulations, and more specifically micelle formulations, have garnered particular interest because of their simplicity, size, scalability, and avoidance of solid-state limitations. Although micelle formulations have been widely utilized, the molecular mechanism of drug solubilization in surfactant micelles is still poorly understood. In this study, a series of modern nuclear magnetic resonance (NMR) methods are utilized to gain a molecular-level understanding of intermolecular interactions and kinetics in a model system. This approach enabled the understanding of how a PWSD, 17ß-Estradiol (E2), solubilizes within a nonionic micelle system composed of polysorbate 80 (PS80). Based on one-dimensional (1D) 1H chemical shift differences of E2 in PS80 solutions, as well as intermolecular correlations established from 1D selective nuclear Overhauser effect (NOE) and two-dimensional NOE spectroscopy experiments, E2 was found to accumulate within the palisade layer of PS80 micelles. A potential hydrogen-bonding interaction between a hydroxyl group of E2 and a carbonyl group of PS80 alkane chains may allow for stabilizing E2-PS80 mixed micelles. Diffusion and relaxation NMR analysis and particle size measurements using dynamic light scattering indicate a slight increase in the micellar size with increasing degrees of supersaturation, resulting in slower mobility of the drug molecule. Based on these structural findings, a theoretical orientation model of E2 molecules with PS80 molecules was developed and validated by computational docking simulations.

Genetic analysis of loose smut (Ustilago tritici) resistance in Sonop spring wheat.

BACKGROUND: The genetics of resistance to loose smut of wheat (Triticum aestivum L.) caused by the fungus Ustilago tritici (Pers.) Rostr. is not well understood. This study examines loose smut resistance in Sonop (TD-14), a South African spring wheat variety. A doubled haploid (DH) population of 163 lines derived from the cross Diamont/TD-14 was studied. The parents and progenies were inoculated with U. tritici races T2, T9, and T39 individually in growth facilities at Morden and Swift Current, Canada. Loose smut incidence (LSI) and partial loose smut resistance (PLSR) were assessed. RESULTS: A whole genome linkage map was developed consisting of 11,519 SNP loci found on 31 linkage groups spanning 2845 cM. A new major resistance gene Ut11 was located to the distal end of chromosome arm 7BS. Ut11 conferred resistance to U. tritici race T2, but not races T9 and T39. Quantitative trait locus (QTL) mapping identified four QTL controlling LSI in the Diamont/TD-14 DH population on chromosomes 3B, 4B, 5B, and 7B (at Ut11) with TD-14 contributing the resistance alleles at three of these loci. The major QTL QUt.mrc-5B was effective against all three races and explained up to 81% of the phenotypic variation. The only QTL identified for PLSR coincided with the LSI QTL QUt.mrc-5B indicating that this locus affected both loose smut incidence and partial smutting of spikes. CONCLUSIONS: A race-specific resistance gene Ut11 and a broadly effective resistance QTL QUt.mrc-5B were the main loci controlling loose smut resistance in the differential line TD-14 (cultivar Sonop). This study provides insight into the genetics of loose smut resistance in spring wheat Sonop and the single nucleotide polymorphism (SNP) markers linked to the resistance gene Ut11 and QTL QUt.mrc-5B will be useful for selecting loose smut resistance in breeding programs.

Toward Developing Discriminating Dissolution Methods for Formulations Containing Nanoparticulates in Solution: The Impact of Particle Drift and Drug Activity in Solution.

Enabling formulations are an attractive approach to increase the dissolution rate, solubility, and oral bioavailability of poorly soluble compounds. With the growing prevalence of poorly soluble drug compounds in the pharmaceutical pipeline, supersaturating drug delivery systems (SDDS), a subset of enabling formulations, have grown in popularity due to their properties allowing for drug concentrations greater than the corresponding crystalline solubility. However, the extent of supersaturation generated as the enabling formulation traverses the gastrointestinal (GI) tract is dynamic and poorly understood. The dynamic nature of supersaturation is a result of several competing kinetic processes such as dissolution, solubilization by formulation and endogenous surfactants, crystallization, and absorption. Ultimately, the free drug concentration, which is equivalent to the drug's inherent thermodynamic activity amid these kinetic processes, defines the true driving force for drug absorption. However, in cases where solubilizing agents are present (i.e., surfactants and bile salts), drug molecules may associate with colloidal nanoscale species, complicating drug activity determination. These nanoscale species can drift into the aqueous boundary layer (ABL), increasing the local API activity at the membrane surface, resulting in increased bioavailability. Herein, a novel approach was developed to accurately measure thermodynamic drug activity in complex media containing drug distributed in nanoparticulate species. This approach captures the influence of the ABL on the observed flux and, ultimately, the predicted unbound drug concentration. The results demonstrate that this approach can help to (1) measure the true extent of local supersaturation in complex systems containing solubilizing excipients and (2) elucidate the mechanisms by which colloidal aggregates can modulate the drug activity in solution and potentially enhance the flux observed across a membrane. The utilization of these techniques may provide development scientists with a strategy to evaluate formulation sensitivity to nanospeciation and allow formulators to maximize the driving force for absorption in a complex environment, perhaps enabling the development of dissolution methods with greater discrimination and correlation to pre-clinical and clinical data sets.

Platelet activation and placenta-mediated adverse pregnancy outcomes: an ancillary study to the Effects of Aspirin in Gestation and Reproduction trial.

BACKGROUND: Platelet activation may play a role in the pathophysiology of placenta-mediated obstetrical complications, as evidenced by the efficacy of aspirin in preventing preeclampsia, but published data regarding the relationship between biomarkers for platelet activation and adverse obstetrical outcomes are sparse. In particular, it is unknown whether prepregnancy biomarkers of platelet activation are associated with adverse pregnancy outcomes. OBJECTIVE: This study aimed to determine the following: (1) whether maternal plasma concentrations of platelet factor 4 are associated with risk of placenta-mediated adverse obstetrical outcomes, and (2) whether these associations are modified by low-dose aspirin. STUDY DESIGN: This ancillary study included measurement of platelet factor 4 among 1185 of 1228 women of reproductive age enrolled in the Effects of Aspirin in Gestation and Reproduction trial with available plasma samples, with relevant outcomes assessed among 584 women with pregnancies lasting at least 20 weeks' gestation. We measured platelet factor 4 in plasma samples obtained at the prepregnancy study visit (before randomization to low-dose aspirin or placebo), 12 weeks' gestation, and 28 weeks' gestation. The primary outcome was a composite of hypertensive disorders of pregnancy, placental abruption, and small-for-gestational-age infant. We estimated the relative risks (RRs) and 95% confidence intervals (CIs) for the association between platelet factor 4 and the composite and individual outcomes at each time point using log-binomial regression that was weighted to account for potential selection bias and adjusted for age, body mass index, education, income, and smoking. To evaluate the potential effect modification of aspirin, we stratified the analyses by aspirin treatment assignment. RESULTS: During follow-up, 95 women experienced the composite adverse obstetrical outcome, with 57 cases of hypertensive disorders of pregnancy, 35 of small for gestational age, and 6 of placental abruption. Overall, prepregnancy platelet factor 4 was positively associated with the composite outcome (third tertile vs first tertile; relative risk, 2.36; 95% confidence interval, 1.38-4.03) and with hypertensive disorders of pregnancy (third tertile vs first tertile; relative risk, 2.14; 95% confidence interval, 1.08-4.23). In analyses stratified by treatment group, associations were stronger in the placebo group (third tertile vs first tertile; relative risk, 3.36; 95% confidence interval, 1.42-7.93) than in the aspirin group (third tertile vs first tertile; relative risk, 1.78; 95% confidence interval, 0.90-3.50). CONCLUSION: High concentrations of platelet factor 4 before pregnancy are associated with increased risk of placenta-mediated adverse pregnancy outcomes, particularly for hypertensive disorders of pregnancy. Aspirin may mitigate the increased risk of these outcomes among women with higher plasma concentrations of preconception platelet factor 4, but low-dose aspirin nonresponders may require higher doses of aspirin or alternate therapies to achieve obstetrical risk reduction.

Mapping quantitative trait loci associated with common bunt resistance in a spring wheat (Triticum aestivum L.) variety Lillian.

KEY MESSAGE: Based on their consistency over environments, two QTL identified in Lillian on chromosomes 5A and 7A could be useful targets for marker assisted breeding of common bunt resistance. Common bunt of wheat (Triticum aestivum L.) caused by Tilletia tritici and T. laevis is an economically important disease because of losses in grain yield and reduced grain quality. Resistance can be quantitative, under the control of multiple small effect genes. The Canada Western Red Spring wheat variety Lillian is moderately resistant to common bunt races found on the Canadian prairies. This study was conducted to identify and map quantitative trait loci (QTL) conferring resistance against common bunt in Lillian. A doubled haploid population comprising 280 lines was developed from F1 plants of the cross of Lillian by Vesper. The lines were inoculated at seeding with the two races L16 (T. laevis) and T19 (T. tritici), grown in field near Swift Current, SK, in 2014, 2015 and 2016 and assessed for disease incidence. The lines were genotyped with the 90 K iSelect SNP genotyping assay, and a high-density genetic map was constructed. Quantitative trait locus analysis was performed with MapQTL.6® software. Two relatively stable common bunt resistance QTL, detected in two of the 3 years, were identified on chromosomes 5A and 7A from Lillian. In addition, three less stable QTL, appearing in one out of 3 years, were identified: one was contributed by Lillian on chromosome 3D and two were contributed by Vesper on chromosomes 1D and 2A. Epistatic interaction was identified for the bunt incidence between 3D and 7A resulting in greater bunt resistance. Future bunt resistance breeding will benefit from combining these QTL through gene pyramiding.

Human CD19-Targeted Mouse T Cells Induce B Cell Aplasia and Toxicity in Human CD19 Transgenic Mice.

The clinical success of chimeric antigen receptor (CAR) T cell therapy for CD19+ B cell malignancies can be limited by acute toxicities and immunoglobulin replacement needs due to B cell aplasia from persistent CAR T cells. Life-threatening complications include cytokine release syndrome and neurologic adverse events, the exact etiologies of which are unclear. To elucidate the underlying toxicity mechanisms and test potentially safer CAR T cells, we developed a mouse model in which human CD19 (hCD19)-specific mouse CAR T cells were adoptively transferred into mice whose normal B cells express a hCD19 transgene at hemizygous levels. Compared to homozygous hCD19 transgenic mice that have ∼75% fewer circulating B cells, hemizygous mice had hCD19 frequencies and antigen density more closely simulating human B cells. Hemizygous mice given a lethal dose of hCD19 transgene-expressing lymphoma cells and treated with CAR T cells had undetectable tumor levels. Recipients experienced B cell aplasia and antigen- and dose-dependent acute toxicities mirroring patient complications. Interleukin-6 (IL-6), interferon γ (IFN-γ), and inflammatory pathway transcripts were enriched in affected tissues. As in patients, antibody-mediated neutralization of IL-6 (and IFN-γ) blunted toxicity. Apparent behavioral abnormalities associated with decreased microglial cells point to CAR-T-cell-induced neurotoxicity. This model will prove useful in testing strategies designed to improve hCD19-specific CAR T cell safety.

Innovation in Ontario's Family Health Teams: How Palliative and Coordinated Care Programs Are Filling Service Gaps and Keeping Patients at Home.

Collaborative interprofessional primary care (PC) teams are widely seen as an essential attribute of high-performing PC systems (Aggarwal and Hutchinson 2012). Effective PC teams play a key role in the mobilization of healthcare resources and navigation of the health and social care system for their patients. In Ontario, the establishment of Family Health Teams has resulted in the implementation of unique programs that deliver services to palliative and elderly patients with a focus on keeping them at home and out of hospital. Case studies cited in this article highlight two innovative programs in Family Health Teams and provide perspectives on lessons for successful implementation.

Autonomic Dysfunction in Neonates with Hypoxic Ischemic Encephalopathy Undergoing Therapeutic Hypothermia Impairs Physiological Responses to Routine Care Events.

OBJECTIVE: To evaluate whether infants with hypoxic-ischemic encephalopathy and evidence of autonomic dysfunction have aberrant physiological responses to care events that could contribute to evolving brain injury. STUDY DESIGN: Continuous tracings of heart rate (HR), blood pressure (BP), cerebral near infrared spectroscopy, and video electroencephalogram data were recorded from newborn infants with hypoxic-ischemic encephalopathy who were treated with hypothermia. Videos between 16 and 24 hours of age identified 99 distinct care events, including stimulating events (diaper changes, painful procedures), and vagal stimuli (endotracheal tube manipulations, pupil examinations). Pre-event HR variability was used to stratify patients into groups with impaired versus intact autonomic nervous system (ANS) function. Postevent physiological responses were compared between groups with the nearest mean classification approach. RESULTS: Infants with intact ANS had increases in HR/BP after stimulating events, whereas those with impaired ANS showed no change or decreased HR/BP. With vagal stimuli, the HR decreased in infants with intact ANS but changed minimally in those with impaired ANS. A pupil examination in infants with an intact ANS led to a stable or increased BP, whereas the BP decreased in the group with an impaired ANS. Near infrared spectroscopy measures of cerebral blood flow/blood volume increased after diaper changes in infants with an impaired ANS, but were stable or decreased in those with an intact ANS. CONCLUSION: HR variability metrics identified infants with impaired ANS function at risk for maladaptive responses to care events. These data support the potential use of HR variability as a real-time, continuous physiological biomarker to guide neuroprotective care in high-risk newborns.

Deriving individualised /r/ targets from the acoustics of children's non-rhotic vowels.

In visual-acoustic biofeedback for rhotic errors, learners are guided to match the third formant (F3) location to a visual target on a real-time acoustic spectrum. As the acoustic properties of correct English /r/differ across speakers, this study aimed to improve target selection by investigating the validity of individualised targets derived from children's non-rhotic vowels. A previously proposed prediction formula was adjusted using data from a child normative sample and tested in two groups of children. Study 1 found that predicted values were unexpectedly higher than actual F3 values in children whose /r/ errors had been remediated. To understand this discrepancy, Study 2 applied the formula to typically developing children and found that predicted values were also higher than actual F3 values, suggesting that different normative data might better represent the current samples. An updated formula is proposed, which can be used to generate individualised targets within acoustic biofeedback applications.

Necrotic platelets provide a procoagulant surface during thrombosis.

A subpopulation of platelets fulfills a procoagulant role in hemostasis and thrombosis by enabling the thrombin burst required for fibrin formation and clot stability at the site of vascular injury. Excess procoagulant activity is linked with pathological thrombosis. The identity of the procoagulant platelet has been elusive. The cell death marker 4-[N-(S-glutathionylacetyl)amino]phenylarsonous acid (GSAO) rapidly enters a subpopulation of agonist-stimulated platelets via an organic anion-transporting polypeptide and is retained in the cytosol through covalent reaction with protein dithiols. Labeling with GSAO, together with exposure of P-selectin, distinguishes necrotic from apoptotic platelets and correlates with procoagulant potential. GSAO(+) platelets form in occluding murine thrombi after ferric chloride injury and are attenuated with megakaryocyte-directed deletion of the cyclophilin D gene. These platelets form a procoagulant surface, supporting fibrin formation, and reduction in GSAO(+) platelets is associated with reduction in platelet thrombus size and fibrin formation. Analysis of platelets from human subjects receiving aspirin therapy indicates that these procoagulant platelets form despite aspirin therapy, but are attenuated by inhibition of the necrosis pathway. These findings indicate that the major subpopulation of platelets involved in fibrin formation are formed via regulated necrosis involving cyclophilin D, and that they may be targeted independent of platelet activation.

Effect of Temperature on Heart Rate Variability in Neonatal ICU Patients With Hypoxic-Ischemic Encephalopathy.

OBJECTIVE: To determine whether measures of heart rate variability are related to changes in temperature during rewarming after therapeutic hypothermia for hypoxic-ischemic encephalopathy. DESIGN: Prospective observational study. SETTING: Level 4 neonatal ICU in a free-standing academic children's hospital. PATIENTS: Forty-four infants with moderate to severe hypoxic-ischemic encephalopathy treated with therapeutic hypothermia. INTERVENTIONS: Continuous electrocardiogram data from 2 hours prior to rewarming through 2 hours after completion of rewarming (up to 10 hr) were analyzed. MEASUREMENTS AND MAIN RESULTS: Median beat-to-beat interval and measures of heart rate variability were quantified including beat-to-beat interval SD, low and high frequency relative spectral power, detrended fluctuation analysis short and long α exponents (αS and αL), and root mean square short and long time scales. The relationships between heart rate variability measures and esophageal/axillary temperatures were evaluated. Heart rate variability measures low frequency, αS, and root mean square short and long time scales were negatively associated, whereas αL was positively associated, with temperature (p < 0.01). These findings signify an overall decrease in heart rate variability as temperature increased toward normothermia. CONCLUSIONS: Measures of heart rate variability are temperature dependent in the range of therapeutic hypothermia to normothermia. Core body temperature needs to be considered when evaluating heart rate variability metrics as potential physiologic biomarkers of illness severity in hypoxic-ischemic encephalopathy infants undergoing therapeutic hypothermia.

Enumeration of extracellular vesicles by a new improved flow cytometric method is comparable to fluorescence mode nanoparticle tracking analysis.

Extracellular vesicles (EVs) play a role in a variety of physiological and pathological processes. However, use of EVs as biomarkers has been hampered by limitations of current detection and enumeration methods. We compared fluorescence-threshold flow cytometry (FT-FC) to nanoparticle tracking analysis (NTA) for enumeration of cell culture-derived EVs. FT-FC and NTA utilising fluorescence mode (F-NTA) enumerated similar numbers of EVs stained with a membrane dye PKH67. Both methods were sufficiently sensitive to detect cell-derived EVs above the background of culture medium. Light scatter NTA (LS-NTA) detected 10-100× more particles than either fluorescence-based method but demonstrated poor specificity. F-NTA appeared to have better sensitivity for <100nm vesicles, however, the FT-FC method combined direct enumeration of EVs with high sensitivity and specificity in the >100nm range. Due to wider availability and higher degree of automation and standardisation, FT-FC is a reasonable surrogate to F-NTA for quantification of EVs. FROM THE CLINICAL EDITOR: Extracellular vesicles are small particles, which can act as tools for intercellular communication. One recent area of interest in EVs is their potentials as biomarkers. In this article, the authors investigated and compared fluorescence-threshold flow cytometry (FT-FC) to nanoparticle tracking analysis (NTA) for the detection of EVs and showed that FT- FC method could be more advantageous. This technique should provide a new alternative for the future.

The Curious Case of the Camelthorn: Competition, Coexistence, and Nest-Site Limitation in a Multispecies Mutualism.

Myrmecophyte plants house ants within domatia in exchange for protection against herbivores. Ant-myrmecophyte mutualisms exhibit two general patterns due to competition between ants for plant occupancy: (i) domatia nest sites are a limiting resource and (ii) each individual plant hosts one ant species at a time. However, individual camelthorn trees (Vachellia erioloba) typically host two to four ant species simultaneously, often coexisting in adjacent domatia on the same branch. Such fine-grain spatial coexistence brings into question the conventional wisdom on ant-myrmecophyte mutualisms. Camelthorn ants appear not to be nest-site limited, despite low abundance of suitable domatia, and have random distributions of nest sites within and across trees. These patterns suggest a lack of competition between ants for domatia and contrast strongly with other ant-myrmecophyte systems. Comparison of this unusual case with others suggests that spatial scale is crucial to coexistence or competitive exclusion involving multiple ant species. Furthermore, coexistence may be facilitated when co-occurring ant species diverge strongly on at least one niche axis. Our conclusions provide recommendations for future ant-myrmecophyte research, particularly in utilizing multispecies systems to further our understanding of mutualism biology.

Evaluation of peroxisome proliferator-activated receptor agonists on interleukin-5-induced eosinophil differentiation.

Peroxisome proliferator-activated receptor (PPAR) agonists have been suggested as novel therapeutics for the treatment of inflammatory lung disease, such as allergic asthma. Treatment with PPAR agonists has been shown to inhibit airway eosinophilia in murine models of allergic asthma, which can occur through several mechanisms including attenuated generation of chemoattractants (e.g. eotaxin) and decreased eosinophil migrational responses. In addition, studies report that PPAR agonists can inhibit the differentiation of several cell types. To date, no studies have examined the effects of PPAR agonists on interleukin-5 (IL-5) -induced eosinophil differentiation from haemopoietic progenitor cells. Non-adherent mononuclear cells or CD34(+) cells isolated from the peripheral blood of allergic subjects were grown for 2 weeks in Methocult(®) cultures with IL-5 (10 ng/ml) and IL-3 (25 ng/ml) in the presence of 1-1000 nm PPARα agonist (GW9578), PPARß/δ agonist (GW501516), PPARγ agonist (rosiglitazone) or diluent. The number of eosinophil/basophil colony-forming units (Eo/B CFU) was quantified by light microscopy. The signalling mechanism involved was assessed by phosphoflow. Blood-extracted CD34(+) cells cultured with IL-5 or IL-5 + IL-3 formed Eo/B CFU, which were significantly inhibited by rosiglitazone (100 nm, P < 0·01) but not GW9578 or GW501516. In addition, rosglitazone significantly inhibited IL-5-induced phosphorylation of extracellular signal-regulated kinase 1/2. We observed an inhibitory effect of rosiglitazone on eosinophil differentiation in vitro, mediated by attenuation of the extracellular signal-regulated kinase 1/2 signalling pathway. These findings indicate that the PPARγ agonist can attenuate tissue eosinophilia by interfering with local differentiative responses.