Shaken baby syndrome often occurs after shaking in response to crying bouts. We questioned whether the use of the educational materials from the Period of PURPLE Crying program would change maternal knowledge and behaviour related to shaking.
We performed a randomized controlled trial in which 1279 mothers received materials from the Period of PURPLE Crying program or control materials during a home visit by a nurse by 2 weeks after the birth of their child. At 5 weeks, the mothers completed a diary to record their behaviour and their infants' behaviour. Two months after giving birth, the mothers completed a telephone survey to assess their knowledge and behaviour.
The mean score (range 0–100 points) for knowledge about infant crying was greater among mothers who received the PURPLE materials (63.8 points) than among mothers who received the control materials (58.4 points) (difference 5.4 points, 95% confidence interval [CI] 4.1 to 6.5 points). The mean scores were similar for both groups for shaking knowledge and reported maternal responses to crying, inconsolable crying and self-talk responses. Compared with mothers who received control materials, mothers who received the PURPLE materials reported sharing information about walking away if frustrated more often (51.5% v. 38.5%, difference 13.0%, 95% CI 6.9% to 19.2%), the dangers of shaking (49.3% v. 36.4%, difference 12.9%, 95% CI 6.8% to 19.0%), and infant crying (67.6% v. 60.0%, difference 7.6%, 95% CI 1.7% to 13.5%). Walking away during inconsolable crying was significantly higher among mothers who received the PURPLE materials than among those who received control materials (0.067 v. 0.039 events per day, rate ratio 1.7, 95% CI 1.1 to 2.6).
The receipt of the Period of PURPLE Crying materials led to higher maternal scores for knowledge about infant crying and for some behaviours considered to be important for the prevention of shaking. (ClinicalTrials.gov trial register no. NCT00175422.)
Under a mandate from the U.S. Congress, the National Toxicology Program (NTP) of the U.S. Department of Health and Human Services conducts animal bioassays for carcinogenicity of potentially toxic chemicals to which the U.S. population might be exposed. Methyleugenol, a natural as well as synthesized substance, was nominated for study because it is structurally similar to safrole, a known animal carcinogen. Methyleugenol was found to be a very potent multisite carcinogen in male and female F344/N rats and B6C3F1 mice at all doses tested in 2-year NTP bioassays using gavage dosing. For this reason, human toxicokinetic studies were added to the traditional NTP protocol. A commercial brand of gingersnaps was found by chemists at the Centers for Disease Control and Prevention to contain a relatively high concentration of methyleugenol. After thorough scientific and clinical review, and approval by a National Institutes of Health institutional review board for the protection of human subjects, a study was conducted with nine healthy adult male and female human volunteers. The volunteers were given 12 gingersnaps for breakfast. Blood was drawn immediately before the meal and at 15, 30, 60, and 120 min afterward. The mean +/- SD fasting level of methyleugenol in serum was 16.2 +/- 4.0 pg/g wet weight. Peak blood levels were found at 15 min (mean +/- SD, 53.9 +/- 7.3 pg/g wet weight), followed by a rapid decline; the half-life of elimination was about 90 min. The peak levels were within the range of methyleugenol blood levels in the U.S. population, as measured concurrently in a subset of nonfasting participants in the Third National Health and Nutrition Examination Survey (NHANES III).
We have measured 29 pesticides in plasma samples collected at birth between 1998 and 2001 from 230 mother and newborn pairs enrolled in the Columbia Center for Children's Environmental Health prospective cohort study. Our prior research has shown widespread pesticide use during pregnancy among this urban minority cohort from New York City. We also measured eight pesticides in 48-hr personal air samples collected from the mothers during pregnancy. The following seven pesticides were detected in 48-83% of plasma samples (range, 1-270 pg/g): the organophosphates chlorpyrifos and diazinon, the carbamates bendiocarb and 2-isopropoxyphenol (metabolite of propoxur), and the fungicides dicloran, phthalimide (metabolite of folpet and captan), and tetrahydrophthalimide (metabolite of captan and captafol). Maternal and cord plasma levels were similar and, except for phthalimide, were highly correlated (p < 0.001). Chlorpyrifos, diazinon, and propoxur were detected in 100% of personal air samples (range, 0.7-6,010 ng/m(3)). Diazinon and propoxur levels were significantly higher in the personal air of women reporting use of an exterminator, can sprays, and/or pest bombs during pregnancy compared with women reporting no pesticide use or use of lower toxicity methods only. A significant correlation was seen between personal air level of chlorpyrifos, diazinon, and propoxur and levels of these insecticides or their metabolites in plasma samples (maternal and/or cord, p < 0.05). The fungicide ortho-phenylphenol was also detected in 100% of air samples but was not measured in plasma. The remaining 22 pesticides were detected in 0-45% of air or plasma samples. Chlorpyrifos, diazinon, propoxur, and bendiocarb levels in air and/or plasma decreased significantly between 1998 and 2001. Findings indicate that pesticide exposures are frequent but decreasing and that the pesticides are readily transferred to the developing fetus during pregnancy.
During the last several years, illegal commercial application of methyl parathion (MP) in domestic settings in several U.S. Southeastern and Midwestern States has affected largely inner-city residents. As part of a multiagency response involving the U.S. Environmental Protection Agency (U.S. EPA), the Agency for Toxic Substances and Disease Registry (ATSDR), and state and local health departments, our laboratory developed a rapid, high-throughput, selective method for quantifying p-nitrophenol (PNP), a biomarker of MP exposure, using isotope dilution high-performance liquid chromatography-tandem mass spectrometry. We measured PNP in approximately 16,000 samples collected from residents of seven different states. Using this method, we were able to receive sample batches from each state; prepare, analyze, and quantify the samples for PNP; verify the results; and report the data to the health departments and ATSDR in about 48 hr. These data indicate that many residents had urinary PNP concentrations well in excess of those of the general U.S. population. In fact, their urinary PNP concentrations were more consistent with those seen in occupational settings or in poisoning cases. Although these data, when coupled with other MP metabolite data, suggest that many residents with the highest concentrations of urinary PNP had significant exposure to MP, they do not unequivocally rule out exposure to PNP resulting from environmental degradation of MP. Even with their limitations, these data were used with the assumption that all PNP was derived from MP exposure, which enabled the U.S. EPA and ATSDR to develop a comprehensive, biologically driven response that was protective of human health, especially susceptible populations, and included clinical evaluations, outreach activities, community education, integrated pest management, and decontamination of homes.
We developed a sensitive and accurate analytical method for quantifying methyleugenol (ME) in human serum. Our method uses a simple solid-phase extraction followed by a highly specific analysis using isotope dilution gas chromatography-high resolution mass spectrometry. Our method is very accurate; its limit of detection is 3.1 pg/g and its average coefficient of variation is 14% over a 200-pg/g range. We applied this method to measure serum ME concentrations in adults in the general U.S. population. ME was detected in 98% of our samples, with a mean ME concentration of 24 pg/g (range < 3.1-390 pg/g). Lipid adjustment of the data did not alter the distribution. Bivariate and multivariate analyses using selected demographic variables showed only marginal relationships between race/ethnicity and sex/fasting status with serum ME concentrations. Although no demographic variable was a good predictor of ME exposure or dose, our data indicate prevalent exposure of U.S. adults to ME. Detailed pharmacokinetic studies are required to determine the relationship between ME intake and human serum ME concentrations.
Alternating bands of acid and base formation have been detected along the length of the internodal cell of Nitella clavata when it is illuminated, while in the dark this phenomenon is minimal. Chloride influx occurs only or largely in the acid-extruding regions, and this is also a light-dependent ion movement. Chloride efflux is slightly dependent on illumination and is not localized as are H+ efflux and Cl- influx. The results obtained support Kitasato's (1968) proposal that a large passive H+ influx is balanced by an active efflux of this ion. Transport mechanisms suggested by the correlations of Cl- and HCO3- influxes with H+ extrusion are discussed.
ESCRT filaments wrap helically around liposomes and assemble into various helical structures in vitro. Dividing Sulfolobus cells further exhibit a thin, dynamic belt coating division furrows. Together these data suggest that spiraling filaments are involved in membrane scission.
The endosomal-sorting complex required for transport (ESCRT) is evolutionarily conserved from Archaea to eukaryotes. The complex drives membrane scission events in a range of processes, including cytokinesis in Metazoa and some Archaea. CdvA is the protein in Archaea that recruits ESCRT-III to the membrane. Using electron cryotomography (ECT), we find that CdvA polymerizes into helical filaments wrapped around liposomes. ESCRT-III proteins are responsible for the cinching of membranes and have been shown to assemble into helical tubes in vitro, but here we show that they also can form nested tubes and nested cones, which reveal surprisingly numerous and versatile contacts. To observe the ESCRT–CdvA complex in a physiological context, we used ECT to image the archaeon Sulfolobus acidocaldarius and observed a distinct protein belt at the leading edge of constriction furrows in dividing cells. The known dimensions of ESCRT-III proteins constrain their possible orientations within each of these structures and point to the involvement of spiraling filaments in membrane scission.
Few data exist in Latin America concerning the association between organophosphate (OP) urinary metabolites and the consumption of fruits and vegetables and other exposure risk variables in schoolchildren.
We collected samples of urine from 190 Chilean children aged 6-12 years, fruits and vegetables, water and soil from schools and homes, and sociodemographic data through a questionnaire. We measured urinary dialkylphosphate (DAP) OP metabolites and OP pesticide residues in food consumed by these 190 children during two seasons: December 2010 (summer) and May 2011(fall). We analyzed the relationship between urinary DAP concentrations and pesticide residues in food, home pesticide use, and residential location.
Diethylalkylphosphates (DEAP) and dimethylalkylphosphates (DMAP) were detected in urine in 76% and 27% of samples, respectively. Factors associated with urinary DEAP included chlorpyrifos in consumed fruits (p<0.0001), urinary creatinine (p<0.0001), rural residence (p=0.02) and age less than 9 years (p=0.004). Factors associated with urinary DMAP included the presence of phosmet residues in fruits (p<0.0001), close proximity to a farm (p=0.002), home fenitrothion use (p=0.009), and season (p<0.0001).
Urinary DAP levels in Chilean school children were high compared previously reported studies. The presence of chlorpyrifos and phosmet residues in fruits was the major factor predicting urinary DAP metabolite concentrations in children.
Dialkylphosphate metabolites; organophosphate pesticides residues; schoolchildren
Concerns regarding the safety of cosmetically tinted contact lenses have been reported in the literature. The purpose of this paper is to evaluate the safety of cosmetically tinted contact lenses in a large number of patients across six clinical trials that varied from 1 week to 3 months in duration.
Lenses tested included: Naturelle limbal ring daily disposable, Lacelle limbal ring daily disposable, Lacelle colored cosmetic daily disposable, Lacelle limbal ring planned replacement at 2 weeks, and Alamode traditional/annual colored cosmetic lens. The primary safety outcome was slit-lamp examination, including epithelial edema, epithelial microcysts, corneal staining, bulbar injection, limbal injection, upper lid tarsal conjunctival abnormalities, corneal neovascularization, and corneal infiltrates. High contrast logMAR visual acuity with lenses, and lens wearing time, movement, and centration, are also presented.
A total of 871 subjects (1,742 eyes) and 23 clinical investigators participated in the six studies, with an average completion rate of 96.4% across all studies. The mean age of the patients was 26.8 ± 6.6 years, and 86.7% of participants were female. The total number of slit-lamp examinations across the six studies was 2,456 visits by eye (1,228 visits by patient). There were no slit-lamp signs > grade 2 for any finding, with the exception of corneal staining in one study. In this study, grade 3 corneal staining was noted in one eye (0.1%) at follow-up visit 1 and four (0.6%) of all eligible dispensed eyes at follow-up visit 2, with no eyes requiring medical treatment. No adverse events were reported during any of the trials.
The cosmetically tinted lenses evaluated in this meta-analysis appear to be safe when properly prescribed by an eye care professional and used in a compliant manner.
contact lens; hydrogel; cosmetically tinted contact lens; compliance
Changes in right ventricular (RV) morphology are associated with morbidity and mortality in heart and lung disease. We examined the association of abnormal RV structure and function with the risk of heart failure (HF) or cardiovascular death in a population-based multiethnic sample free of clinical cardiovascular disease at baseline.
Methods and Results
The Multi-Ethnic Study of Atherosclerosis (MESA) performed cardiac magnetic resonance imaging (MRI) on 5098 participants between 2000–2002 with follow-up for incident heart failure and cardiovascular death (“death”) until January 2008. RV volumes and mass were available for 4204 participants. The study sample (N = 4,144) was 61.4 ± 10.1 years old and 47.6 % male. The presence of RV hypertrophy (increased RV mass) was associated with a more than twice the risk of heart failure or death after adjustment for demographics, body mass index, education, C-reactive protein level, hypertension, and smoking status (HR = 2.52, 95%CI 1.55–4.10, p < 0.001) and a doubling of risk (or more) with left ventricular mass at the mean value or lower (p for interaction = 0.05).
RV hypertrophy was associated with the risk of heart failure or death in a multi-ethnic population free of clinical cardiovascular disease at baseline.
right ventricle; pulmonary heart disease; magnetic resonance imaging; pulmonary hypertension; survival
Background The extent to which adult height, a biomarker of the interplay of genetic endowment and early-life experiences, is related to risk of chronic diseases in adulthood is uncertain.
Methods We calculated hazard ratios (HRs) for height, assessed in increments of 6.5 cm, using individual–participant data on 174 374 deaths or major non-fatal vascular outcomes recorded among 1 085 949 people in 121 prospective studies.
Results For people born between 1900 and 1960, mean adult height increased 0.5–1 cm with each successive decade of birth. After adjustment for age, sex, smoking and year of birth, HRs per 6.5 cm greater height were 0.97 (95% confidence interval: 0.96–0.99) for death from any cause, 0.94 (0.93–0.96) for death from vascular causes, 1.04 (1.03–1.06) for death from cancer and 0.92 (0.90–0.94) for death from other causes. Height was negatively associated with death from coronary disease, stroke subtypes, heart failure, stomach and oral cancers, chronic obstructive pulmonary disease, mental disorders, liver disease and external causes. In contrast, height was positively associated with death from ruptured aortic aneurysm, pulmonary embolism, melanoma and cancers of the pancreas, endocrine and nervous systems, ovary, breast, prostate, colorectum, blood and lung. HRs per 6.5 cm greater height ranged from 1.26 (1.12–1.42) for risk of melanoma death to 0.84 (0.80–0.89) for risk of death from chronic obstructive pulmonary disease. HRs were not appreciably altered after further adjustment for adiposity, blood pressure, lipids, inflammation biomarkers, diabetes mellitus, alcohol consumption or socio-economic indicators.
Conclusion Adult height has directionally opposing relationships with risk of death from several different major causes of chronic diseases.
Height; cardiovascular disease; cancer; cause-specific mortality; epidemiological study; meta-analysis
Restrictions on smoking in public places have become increasingly widespread in the United States, particularly since the year 2005. National-scale studies in Europe and local-scale studies in the United States have found decreases in hospital admissions for acute myocardial infarction (AMI) following smoking bans. The authors analyzed AMI admission rates for the years 1999–2008 in 387 US counties that enacted comprehensive smoking bans across 9 US states, using a study population of approximately 6 million Medicare enrollees aged 65 years or older. Effects of smoking bans on AMI admissions were estimated by using Poisson regression with linear and nonlinear adjustment for secular trend and random effects at the county level. Under the assumption of linearity in the secular trend of declining AMI, smoking bans were associated with a statistically significant ban-associated decrease in admissions for AMI in the 12 months following the ban. However, the estimated effect was attenuated to nearly zero when the assumption of linearity in the underlying trend was relaxed. This analysis demonstrates that estimation of potential health benefits associated with comprehensive smoking bans is challenged by the need to adjust for nonlinearity in secular trend.
environmental tobacco smoke; mixed-effects models; secondhand smoke; smoking bans
Oxidative stress is caused predominantly by accumulation of hydrogen peroxide and distinguishes inflamed tissue from healthy tissue. Hydrogen peroxide could potentially be useful as a stimulus for targeted drug delivery to diseased tissue. However, current polymeric systems are not sensitive to biologically relevant concentrations of H2O2 (50-100 μM). Here we report a new biocompatible polymeric capsule capable of undergoing backbone degradation and thus release upon exposure to such concentrations of hydrogen peroxide. Two polymeric structures were developed differing with respect to the linkage between the boronic ester group and the polymeric backbone: either direct (1) or via an ether linkage (2). Both polymers are stable in aqueous solution at normal pH, and exposure to peroxide induces the removal of the boronic ester protecting groups at physiological pH and temperature, revealing phenols along the backbone, which undergo quinone methide rearrangement to lead to polymer degradation. Considerably faster backbone degradation was observed for polymer 2 over polymer 1 by NMR and GPC. Nanoparticles were formulated from these novel materials to analyze their oxidation triggered release properties. While nanoparticles formulated from polymer 1 only released 50% of the reporter dye after exposure to 1 mM H2O2 for 26 h, nanoparticles formulated from polymer 2 did so within 10 h and were able to release their cargo selectively in biologically relevant concentrations of H2O2. Nanoparticles formulated from polymer 2 showed a two fold enhancement of release upon incubation with activated neutrophils while controls showed non specific response to ROS producing cells. These polymers represent a novel, biologically relevant and biocompatible approach to biodegradable H2O2-triggered release systems that can degrade into small molecules, release their cargo, and should be easily cleared by the body.
Oxidative stress; Reactive oxygen species; bioresponsive materials; polymer degradation; hydrogen peroxide; nanoparticles; biocompatible delivery
Whole genome sequencing is a powerful tool in the discovery of single nucleotide polymorphisms (SNPs) and small insertions/deletions (indels) among mutant strains, which simplifies forward genetics approaches. However, identification of the causative mutation among a large number of non-causative SNPs in a mutant strain remains a big challenge. In the unicellular biflagellate green alga Chlamydomonas reinhardtii, we generated a SNP/indel library that contains over 2 million polymorphisms from four wild-type strains, one highly polymorphic strain that is frequently used in meiotic mapping, ten mutant strains that have flagellar assembly or motility defects, and one mutant strain, imp3, which has a mating defect. A comparison of polymorphisms in the imp3 strain and the other 15 strains allowed us to identify a deletion of the last three amino acids, Y313F314L315, in a protein phosphatase 2A catalytic subunit (PP2A3) in the imp3 strain. Introduction of a wild-type HA-tagged PP2A3 rescues the mutant phenotype, but mutant HA-PP2A3 at Y313 or L315 fail to rescue. Our immunoprecipitation results indicate that the Y313, L315, or YFLΔ mutations do not affect the binding of PP2A3 to the scaffold subunit, PP2A-2r. In contrast, the Y313, L315, or YFLΔ mutations affect both the stability and the localization of PP2A3. The PP2A3 protein is less abundant in these mutants and fails to accumulate in the basal body area as observed in transformants with either wild-type HA-PP2A3 or a HA-PP2A3 with a V310T change. The accumulation of HA-PP2A3 in the basal body region disappears in mated dikaryons, which suggests that the localization of PP2A3 may be essential to the mating process. Overall, our results demonstrate that the terminal YFL tail of PP2A3 is important in the regulation on Chlamydomonas mating.
Whole genome sequencing is a powerful tool to detect changes in genomic DNA. However, how to identify a causative mutation from over 20,000 changes remains a big challenge. For the unicellular green alga Chlamydomonas, we built a library that consists of over 2 million changes from 16 strains. A comparison of changes found in a mutant strain with a mating defect, imp3, to 15 other strains, leads to the identification of a three amino acid deletion in the catalytic subunit of a protein phosphatase 2A (PP2A3). The mating defect of imp3 is rescued by an HA-tagged PP2A3 gene. Introduction of the HA-tagged PP2A3 gene with various mutations in these three amino acids reveals that they play a key role in stabilizing and ensuring the proper localization of PP2A3. The ubiquitous enzyme PP2A is involved in diverse cellular processes. Our discovery that PP2A3 is involved in the Chlamydomonas mating signaling pathway, which also contains the polycystin2 homolog (PKD2), makes Chlamydomonas mating an excellent model to study ciliary/flagellar signaling. Since both PP2A and PKD2 play important roles in human health, further investigation of the relationship between these two proteins in Chlamydomonas will facilitate better understanding of their functions.
Numerous longitudinal studies have identified risk factors for the onset of most eating disorders (EDs). Identifying women at highest risk within a high-risk sample would allow for focusing of preventive resources and also suggests different etiologies.
A longitudinal cohort study over 3 years in a high-risk sample of 236 college-age women randomized to the control group of a prevention trial for EDs. Potential risk factors and interactions between risk factors were assessed using the methods developed previously. Main outcome measures were time to onset of a subthreshold or full ED.
At the 3-year follow-up, 11.2% of participants had developed a full or partial ED. Seven of 88 potential risk factors could be classified as independent risk factors, seven as proxies, and two as overlapping factors. Critical comments about eating from teacher/coach/siblings and a history of depression were the most potent risk factors. The incidence for participants with either or both of these risk factors was 34.8% (16/46) compared to 4.2% (6/144) for participants without these risk factors, with a sensitivity of 0.75 and a specificity of 0.82.
Targeting preventive interventions at women with high weight and shape concerns, a history of critical comments about eating weight and shape, and a history of depression may reduce the risk for EDs.
To validate a diagnostic instrument for pediatric delirium in critically ill children, both ventilated and nonventilated, that uses standardized, developmentally appropriate measurements.
Design and Setting
A prospective observational cohort study investigating the Pediatric Confusion Assessment Method for Intensive Care Unit (pCAM-ICU) patients in the pediatric medical, surgical, and cardiac intensive care unit of a university-based medical center.
A total of 68 pediatric critically ill patients, at least 5 years of age, were enrolled from July 1, 2008, to March 30, 2009.
Criterion validity including sensitivity and specificity and interrater reliability were determined using daily delirium assessments with the pCAM-ICU by two critical care clinicians compared with delirium diagnosis by pediatric psychiatrists using Diagnostic and Statistical Manual, 4th Edition, Text Revision criteria.
A total of 146 paired assessments were completed among 68 enrolled patients with a mean age of 12.2 yrs. Compared with the reference standard for diagnosing delirium, the pCAM-ICU demonstrated a sensitivity of 83% (95% confidence interval, 66–93%), a specificity of 99% (95% confidence interval, 95–100%), and a high interrater reliability (κ = 0.96; 95% confidence interval, 0.74–1.0).
The pCAM-ICU is a highly valid reliable instrument for the diagnosis of pediatric delirium in critically ill children chronologically and developmentally at least 5 yrs of age. Use of the pCAM-ICU may expedite diagnosis and consultation with neuropsychiatry specialists for treatment of pediatric delirium. In addition, the pCAM-ICU may provide a means for delirium monitoring in future epidemiologic and interventional studies in critically ill children. (Crit Care Med 2011; 39:150–157)
pediatrics; delirium; critical care; consciousness disorders; critical illness
The purpose of this study was to confirm initial reports of elevated behavior problems in children with single-suture craniosynostosis (SSC), using multiple informants, longitudinal analyses and a control group. We hypothesized higher levels of maladjustment for children with SSC than comparison children, particularly at the older age and in selected areas of previously observed vulnerability: attention and social adjustment.
A Child Behavior Checklist (CBCL) was completed when children were ~19 months by 436 mothers (219 with SSC) and 371 fathers (177 with SSC); and at ~37 months by 361 mothers (175 with SSC) and 303 fathers (142 with SSC). A minimum of one caregiver/teacher report was available for 169 of these children (74 with SSC) using the Caregiver-Teacher Report Form (CTRF).
Average CBCL/CTRF externalizing, internalizing and total scores for all informants were consistently higher (worse) for children with SSC than control group children, but most differences were small and statistically non-significant. No differences associated with suture site were found. At the oldest age point, both mothers and fathers (but not teachers) generated higher average scores for patients than for controls on scales measuring attention and social problems, with small to medium effects sizes (0.20 to 0.32).
On average toddlers/preschoolers with SSC show behavioral development that is largely indistinguishable from same-aged peers of similar socioeconomic background. The predictive significance of small group differences in attention and social adjustment will be assessed in a follow-up of this cohort at age 7.
Hair bundles of the inner ear have a unique structure and protein composition that underlies their sensitivity to mechanical stimulation. Using mass spectrometry, we identified and quantified >1100 proteins, present from a few to 400,000 copies per stereocilium, from purified chick bundles; 336 of these were significantly enriched in bundles. Bundle proteins that we detected have been shown to regulate cytoskeleton structure and dynamics, energy metabolism, phospholipid synthesis, and cell signaling. Three-dimensional imaging using electron tomography allowed us to count the number of actin-actin crosslinkers and actin-membrane connectors; these values compared well to those obtained from mass spectrometry. Network analysis revealed several hub proteins, including RDX (radixin) and SLC9A3R2 (NHERF2), which interact with many bundle proteins and may perform functions essential for bundle structure and function. The quantitative mass spectrometry of bundle proteins reported here establishes a framework for future characterization of dynamic processes that shape bundle structure and function.
In preparing for the threat of a pandemic of avian H5N1 influenza virus, we need to consider the significant delay (4 to 6 months) necessary to produce a strain-matched vaccine. As some degree of cross-reactivity between seasonal influenza vaccines and H5N1 virus has been reported, this was further explored in the ferret model to determine the targets of protective immunity. Ferrets were vaccinated with two intramuscular inoculations of trivalent inactivated split influenza vaccine or subcomponent vaccines, with and without adjuvant, and later challenged with a lethal dose of A/Vietnam/1203/2004 (H5N1) influenza virus. We confirmed that vaccination with seasonal influenza vaccine afforded partial protection against lethal H5N1 challenge and showed that use of either AlPO4 or Iscomatrix adjuvant with the vaccine resulted in complete protection against disease and death. The protection was due exclusively to the H1N1 vaccine component, and although the hemagglutinin contributed to protection, the dominant protective response was targeted toward the neuraminidase (NA) and correlated with sialic acid cleavage-inhibiting antibody titers. Purified heterologous NA formulated with Iscomatrix adjuvant was also protective. These results suggest that adjuvanted seasonal trivalent vaccine could be used as an interim measure to decrease morbidity and mortality from H5N1 prior to the availability of a specific vaccine. The data also highlight that an inducer of cross-protective immunity is the NA, a protein whose levels are not normally monitored in vaccines and whose capacity to induce immunity in recipients is not normally assessed.
The Polo-like kinase homologue in Trypanosoma brucei (TbPLK) phosphorylates the calcium-binding protein TbCentrin2 in vitro and in vivo. Mutation of a single phosphosite on TbCentrin2 in vivo causes defects in the assembly of a suite of cytoskeletal organelles essential for correct inheritance of the new flagellum, mimicking TbPLK inactivation.
In the protist parasite Trypanosoma brucei, the single Polo-like kinase (TbPLK) controls the inheritance of a suite of organelles that help position the parasite's single flagellum. These include the basal bodies, the bilobe, and the flagellar attachment zone (FAZ). TbCentrin2 was previously shown to be a target for TbPLK in vitro, and this is extended in this study to in vivo studies, highlighting a crucial role for serine 54 in the N-terminal domain. Duplication of the bilobe correlates with the presence of TbPLK and phospho-TbCentrin2, identified using phosphospecific antiserum. Mutation of S54 leads to slow growth (S54A) or no growth (S54D), the latter suggesting that dephosphorylation is needed to complete bilobe duplication and subsequent downstream events necessary for flagellum inheritance.
Recent findings have demonstrated an indispensable role for granulocyte-emacrophage colony-stimulating factor (GM-CSF) in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). However the signaling pathways and cell populations that regulate GM-CSF production in vivo remain to be elucidated. Our work demonstrates that the interleukin-1 receptor type 1 (IL-1R) is required for GM-CSF production following both TCR and cytokine induced stimulation of immune cells in vitro. Conventional αβ and γδ T cells were both identified to be potent producers of GM-CSF. Moreover secretion of GM-CSF was dependent on IL-1R under both IL-12 and IL-23 induced stimulatory conditions. Deficiency in IL-1R conferred significant protection from EAE and this correlated with reduced production of GM-CSF and attenuated infiltration of inflammatory cells into the central nervous system (CNS). We also find that GM-CSF production in vivo is not restricted to a defined CD4+ T cell lineage, but is rather heterogeneously expressed in the effector CD4+ T cell population. In addition, inflammasome derived IL-1β upstream of IL-1R is a critical regulator of GM-CSF production by T cells during priming and the adaptor protein, MYD88, promotes GM-CSF production in both αβ and γδ T cells. These findings highlight the importance of inflammasome-derived IL-1β and the IL-1R/MYD88 signaling axis in the regulation of GM-CSF production.
NLR; Inflammasome; Caspase-1; IL-1R; IL-1β; EAE
We examined the relationship between grip strength declines and muscle-tendon responses induced by long-term performance of a high-repetition, low-force (HRLF) reaching task in rats. We hypothesized that grip strength declines would correlate with inflammation, fibrosis and degradation in flexor digitorum muscles and tendons. Grip strength declined after training, and further in weeks 18 and 24, in reach limbs of HRLF rats. Flexor digitorum tissues of reach limbs showed low-grade increases in inflammatory cytokines: IL-1β after training and in week 18, IL-1α in week 18, TNF-α and IL-6 after training and in week 24, and IL-10 in week 24, with greater increases in tendons than muscles. Similar cytokine increases were detected in serum with HRLF: IL-1α and IL-10 in week 18, and TNF-α and IL-6 in week 24. Grip strength correlated inversely with IL-6 in muscles, tendons and serum, and TNF-α in muscles and serum. Four fibrogenic proteins, TGFB1, CTGF, PDGFab and PDGFbb, and hydroxyproline, a marker of collagen synthesis, increased in serum in HRLF weeks 18 or 24, concomitant with epitendon thickening, increased muscle and tendon TGFB1 and CTGF. A collagenolytic gelatinase, MMP2, increased by week 18 in serum, tendons and muscles of HRLF rats. Grip strength correlated inversely with TGFB1 in muscles, tendons and serum; with CTGF-immunoreactive fibroblasts in tendons; and with MMP2 in tendons and serum. Thus, motor declines correlated with low-grade systemic and musculotendinous inflammation throughout task performance, and increased fibrogenic and degradative proteins with prolonged task performance. Serum TNF-α, IL-6, TGFB1, CTGF and MMP2 may serve as serum biomarkers of work-related musculoskeletal disorders, although further studies in humans are needed.
Clostridium difficile is the leading cause of antibiotic-associated diarrhea in hospitals worldwide, due to hypervirulent epidemic strains with the ability to produce increased quantities of the large toxins TcdA and TcdB. Unfortunately, accurate quantification of TcdA and TcdB from different toxinotypes using small samples has not yet been reported. In the present study, we quantify C. difficile toxins in <0.1 mL of culture filtrate by quantitative label-free mass spectrometry (MS) using data-independent analysis (MSE). In addition, analyses of both purified TcdA and TcdB as well as a standard culture filtrate were performed using gel-based and gel-independent proteomic platforms. Gel-based proteomic analysis was then used to generate basic information on toxin integrity and provided sequence confirmation. Gel-independent in-solution digestion of both toxins using five different proteolytic enzymes with MS analysis generated broad amino acid sequence coverage (91% for TcdA and 95% for TcdB). Proteomic analysis of a culture filtrate identified a total of 101 proteins, among them TcdA, TcdB, and S-layer proteins.
Web-based lifestyle counselling designed to improve adherence to self-management behaviours for diet, exercise and medication has been shown to reduce blood pressure (BP). However, the long-term clinical outcome of these interventions is not established. Our aim was to establish whether an e-counselling program is independently associated with improved clinical outcomes over a 12-month period, as defined by the following criteria: (1) reduction of systolic BP, diastolic BP, pulse pressure and associated risk factors for cardiovascular events; and (2) adherence to self-management behaviour (diet, exercise, smoke-free living and prescribed medication).
Methods and analysis
Reducing risk with e-based support for adherence to lifestyle change in hypertension is a two-parallel group, double-blind randomised controlled trial that will utilise a two (Groups: e-counselling vs control) by three (assessment intervals: baseline, 4-month and 12-month outcome) design. BP, lipoprotein cholesterol, physical activity and dietary behaviours and psychological distress will be measured at each assessment. We plan to recruit 528 participants (35–74 years of age) diagnosed with stage 1 or 2 hypertension (systolic BP, 140–180 mm Hg; diastolic BP 90–110 mm Hg) from three major cities (Toronto, London, Vancouver) and one rural area (Grey Bruce region) across Canada between February 2012 and July 2015. Controls will receive general educational e-messages on heart healthy living and the e-counselling group will receive tailored e-messages that are matched to their stage of readiness for change. For both groups, e-messages will be sent proactively on a weekly basis during months 1–4, then bi-weekly during months 5–8 and then monthly during months 9–12.
Ethics and dissemination
Ethical approval has been obtained from all recruitment sites. This will be one of the first studies to evaluate the long-term efficacy of preventive e-counselling strategies for cardiovascular disease prevention in patients with hypertension. Findings from this study will be used to guide the ongoing development of e-counselling services.
Clinicaltrial.gov NCT01541540; http://clinicaltrials.gov/ct2/show/NCT01541540.