The investigation of sonocatalytic activity of Er3+:YAlO3/TiO2-ZnO composite in azo dyes degradation. In this work, the emphasis was mainly placed on investigating the sonocatalytic activity of TiO(2)-ZnO mixed with Er(3+):YAlO(3), namely, Er(3+):YAlO(3)/TiO(2)-ZnO composite. It is able to utilize the sonoluminescence light to improve the sonocatalytic degradation of organic dyes. The Er(3+):YAlO(3) as up-conversion luminescence agent was synthesized by sol-gel and auto-combustion method, and then Er(3+):YAlO(3)/TiO(2)-ZnO composite as sonocatalyst were prepared by ultrasonic dispersion and liquids boil method. The prepared up-conversion luminescence agent and composites were characterized by X-ray diffraction (XRD) and scanning electron microscope (SEM). Acid Red B dye was selected to examine the sonocatalytic activity of Er(3+):YAlO(3)/TiO(2)-ZnO composite. The degradation reaction processes were monitored by UV-vis spectrophotometer and ion chromatogram. The influences on the activity of the Er(3+):YAlO(3)/TiO(2)-ZnO such as Ti/Zn molar ratio, heat-treated temperature and heat-treated time were studied. The results showed that the Er(3+):YAlO(3)/TiO(2)-ZnO composite exhibited a significantly high sonocatalytic activity compared with other catalysts in the degradation of Acid Red B. And the sonocatalyst with 1:1 Ti/Zn molar ratio heat-treated at 550°C for 60min showed the highest sonocatalytic activity. At last, the experiment also indicated that it has a good sonocatalytic activity to degrade other organic dyes.


Ultrasound-assisted extraction of phillyrin from Forsythia suspensa. Forsythia suspensa (Thunb.) Vahl is one of the most widely used traditional Chinese medicines, and possesses important biological activities, such as antibacterial, antiviral, anti-inflammatory and antioxidant activities. Phillyrin is the main bioactive component of Forsythia suspensa. In this paper, ultrasound-assisted extraction of phillyrin from Forsythia suspensa was studied with HPLC-photodiode array detection. Effects of several experimental parameters, such as type and concentration of extracting solvent, ratio of liquid to material, extraction temperature, and time of sonication on extraction efficiencies of phillyrin from Forsythia suspensa were evaluated. The optimal extraction conditions were 1g plant sample with 10 ml of 20% methanol and the extraction for 60 min at 60°C under ultrasonic irradiation. Under the optimum conditions, the yield of phillyrin was 0.713±0.009 mg/g. The results indicated that the ultrasound-assisted extraction is a very useful method for the extraction of important phytochemicals from plant materials.


Comparison of pulse rate variability with heart rate variability during obstructive sleep apnea. We investigate whether pulse rate variability (PRV) extracted from finger photo-plethysmography (Pleth) waveforms can be the substitute of heart rate variability (HRV) from RR intervals of ECG signals during obstructive sleep apnea (OSA). Simultaneous measurements (ECG and Pleth) were taken from 29 healthy subjects during normal (undisturbed sleep) breathing and 22 patients with OSA during OSA events. Highly significant (p<0.01) correlations (1.0>r>0.95) were found between heart rate (HR) and pulse rate (PR). Bland-Altman plot of HR and PR shows good agreement (<5% difference). Comparison of 2 min recording epochs demonstrated significant differences (p<0.01) in time, frequency domains and complexity analysis, between normal and OSA events using PRV as well as HRV measures. Results suggest that both HRV and PRV indices could be used to distinguish OSA events from normal breathing during sleep. However, several variability measures (SDNN, RMSSD, HF power, LF/HF and sample entropy) of PR and HR were found to be significantly (p<0.01) different during OSA events. Therefore, we conclude that PRV provides accurate inter-pulse variability to measure heart rate variability under normal breathing in sleep but does not precisely reflect HRV in sleep disordered breathing.


Development of a novel method for surgical implant design optimization through noninvasive assessment of local bone properties. A method was developed to improve the design of locking implants by finding the optimal paths for the anchoring elements, based on a high resolution pQCT assessment of local bone mineral density (BMD) distribution and bone micro-architecture (BMA). The method consists of three steps: (1) partial fixation of the implant to the bone and creation of a reference system, (2) implant removal and pQCT scan of the bone, and (3) determination of BMD and BMA of all implant-anchoring locations along the actual and alternative directions. Using a PHILOS plate, the method uncertainty was tested on an artificial humerus bone model. A cadaveric humerus was used to quantify how the uncertainty of the method affects the assessment of bone parameters. BMD and BMA were determined along four possible alternative screw paths as possible criteria for implant optimization. The method is biased by a 0.87 ± 0.12 mm systematic uncertainty and by a 0.44 ± 0.09 mm random uncertainty in locating the virtual screw position. This study shows that this method can be used to find alternative directions for the anchoring elements, which may possess better bone properties. This modification will thus produce an optimized implant design.


A complete, non-lumped, and verifiable set of upper body segment parameters for three-dimensional dynamic modeling. Dynamic models of the human trunk have been extensively used to investigate the biomechanics of lower back pain and postural instability in different populations. Despite their diverse applications, previous models rely on intrinsic upper body segment parameters (UBSP), e.g., each segment's mass-inertia characteristics. However, a comprehensive UBSP set allowing state-of-the-art, three-dimensional (3D) dynamic modeling does not exist to date. Therefore, our objective was to establish a UBSP set of all vertebral trunk segments that is accurate and complete. Based on high-resolution, transverse color images, anatomical structures of the Male Visible Human (MVH) were digitally reconstructed via commercial software. Subsequently, we identified the 3D spinal joint and 3D center of mass coordinates, the mass, and the moment of inertia tensor for 24 vertebral trunk segments and 4 upper limb segments (two segments per arm). Since the MVH images are public domain, the parameters are uniquely verifiable and expandable to also include lower limb parameters. To demonstrate the UBSP set's practicality, the parameters were finally implemented in a previously proposed inverse dynamics model of the upper body. Our findings reveal that an accurate and complete UBSP set has been obtained that will be beneficial to (1) systemize thinking in postural control studies; (2) quantify the effect of impact forces on the head and trunk (e.g., during whiplash); (3) suggest population-specific experiments based on theoretical insights into trunk dynamics (e.g., regarding lower back pain); or (4) assess the feasibility of new surgical techniques (e.g., spinal fusion) and neuroprostheses (e.g., after spinal cord injury).


Inequalities in smoking in the Czech Republic: societal or individual effects? Smoking constitutes one of the main public health problems worldwide. In the Czech Republic, one of the post-communist countries undergoing societal transition, there was a significant decrease in smoking prevalence during 1985-1997, followed by certain stagnation in prevalence of smokers. The most serious problem is the smoking among young population and socially disadvantaged groups. This paper examines social inequalities in smoking in the Czech population using multilevel approach. Data were analysed by multilevel modelling using smoking in the past, current smoking and current moderate/heavy smoking as outcomes of interest. Men were significantly more likely to be smokers than women. Further, the analysis confirmed that current smoking is the most common among young people. Education was strongly inversely related to all smoking outcomes. Smoking was also significantly more reported by divorced and unemployed individuals. While the association between small-area characteristics and smoking was limited, smoking was more common in the areas with higher unemployment and higher proportion of non-Czech nationals.


The influence of contrast agent injection on physiological flow in the circle of Willis. X-ray videodensitometry allows in vivo flow measurements from gradients in contrast agent concentration. However, the injection of contrast agent alters the flow to be measured. Here, the temporal, spatial, and inter-patient variability of the response to injection are examined. To this purpose, an injection is prescribed in the internal carotid in a 1D wave propagation model of the arterial circulation. Although the resulting effect of injection is constant over a cardiac cycle, the response does vary with the location within the cerebral circulation and the geometry of the circle of Willis. At the injection site, the injection partly suppresses the incoming blood flow, such that the distal flow is increased by approximately 10%. This corresponds to approximately 20% of the injection rate added to the blood flow during injection, depending on the vascular geometry. In the communicating arteries, the flow direction is reversed during injection. Since the measured flow is not equal to the physiological blood flow, the effect of injection should be taken into account when deriving the flow from travelling contrast agent.


Reconnecting plants and pollinators: challenges in the restoration of pollination mutualisms. Ecological restoration of plant-pollinator interactions has received surprisingly little attention, despite animal-mediated pollination underpinning reproduction of the majority of higher plants. Here, we offer a conceptual and practical framework for the ecological restoration of pollination mutualisms. Through the use of targeted restoration plantings to attract and sustain pollinators and increased knowledge of the ecological requirements of pollinators, we propose that pollination could be successfully restored in degraded ecosystems. The challenge for pollination biologists is to integrate their findings with those of plant restoration ecologists to ensure sustainable pollination in restored ecosystems.


Space, time, and number: a Kantian research program.


Crystal and molecular structure of N-(4-nitrophenyl)-β-alanine--its vibrational spectra and theoretical calculations. The N-(4-nitrophenyl)-β-alanine in crystalline form directly by the addition of 4-nitroaniline to the acrylic acid in aqueous solution has been obtained. The title β-alanine derivative crystallizes in the P2(1)/c space group of monoclinic system with four molecules per unit cell. The X-ray geometry of β-alanine derivative molecule has been compared with those obtained by molecular orbital calculations corresponding to the gas phase. In the crystal the molecules related by an inversion center interact via symmetrically equivalent O-H···O hydrogen bonds with O···O distance of 2.656(2) Å forming a dimeric structure. The dimers of β-alanine derivative weakly interact via N-H···O hydrogen bonds between the H atom of β-amine groups and one of O atom of nitro groups. The room temperature powder vibrational (infrared and Raman) measurements are in accordance with the X-ray analysis. In aqueous solution of 4-nitroaniline and acrylic acid, the double CC bond of vinyl group of acrylic acid breaks as result of 4-nitroaniline addition.


Temporal lobe seizure prediction based on a complex Gaussian wavelet. Abnormal synchronisation change is closely associated with the process of seizure generation. The immediate and accurate detection of the changes in synchronisation may offer advantages in seizure prediction. Thus, we develop a phase synchronisation detection method for this purpose. An analysis of phase synchronisation based on the complex Gaussian wavelet transform (PSW) was conducted to detect synchronised phases of long-lasting scalp electroencephalograph (EEG) recordings from eight epilepsy patients with intractable temporal lobe epilepsy. Four assessment indicators, namely sensitivity, maximum false prediction rate, seizure occurrence period and seizure prediction horizon were used to assess and compare PSW with the analysis of phase synchronisation, based on the Hilbert transform (PSH) and a random predictor Poisson process. An obvious decrease was found upon phase synchronisation prior to visual detection of electroencephalograph seizure onset, which was consistent with the EEG mechanism in the ictal events. The results suggest that PSW is the most effective among the three prediction methods. The results confirm that the analysis of phase synchronisation based on the complex Gaussian wavelet transform can be used for seizure prediction. Phase synchronisation analysis may be a useful algorithm for clinical application in epileptic prediction.


Comparing cutaneous perception induced by electrical stimulation using rectangular and round shaped electrodes. We have investigated the cutaneous perception differences for anodal and cathodal transcranial direct current stimulation (tDCS) and transcranial random noise stimulation (tRNS) between two electrode configurations: a standard, rectangle-shaped, and a circle-shaped, round geometry with the same surface area, and thus, same nominal current distribution. We have aimed to find whether a smaller perimeter length and the absence of corners in the case of the round configuration would lead to altered skin perception characteristics when compared to the rectangular geometry. Twelve subjects were tested for tDCS and tRNS skin perception characteristics in the intensity range of 200-2000 μA using round and rectangular electrode configurations. We have not found any substantial differences between detection thresholds, detection rates, false positive rates or consistent alterations in the sites of perceived stimulation. We conclude that there is no difference between the round and the rectangular electrode configurations regarding their blinding potentials. The results of this investigation indicate that the altering of the electrode geometry to a round configuration is unwarranted for better blinding purposes in future studies using tDCS and tRNS.


Nanotechnology in molecular medicine.

Nanotechnology in molecular medicine.


TubercuList--10 years after.

TubercuList--10 years after. TubercuList (http://tuberculist.epfl.ch/), the relational database that presents genome-derived information about H37Rv, the paradigm strain of Mycobacterium tuberculosis, has been active for ten years and now presents its twentieth release. Here, we describe some of the recent changes that have resulted from manual annotation with information from the scientific literature. Through manual curation, TubercuList strives to provide current gene-based information and is thus distinguished from other online sources of genome sequence data for M. tuberculosis. New, mostly small, genes have been discovered and the coordinates of some existing coding sequences have been changed when bioinformatics or experimental data suggest that this is required. Nucleotides that are polymorphic between different sources of H37Rv are annotated and gene essentiality data have been updated. A host of functional information has been gleaned from the literature and many new activities of proteins and RNAs have been included. To facilitate basic and translational research, TubercuList also provides links to other specialized databases that present diverse datasets such as 3D-structures, expression profiles, drug development criteria and drug resistance information, in addition to direct access to PubMed articles pertinent to particular genes. TubercuList has been and remains a highly valuable tool for the tuberculosis research community with >75,000 visitors per month.


The MycoBrowser portal: a comprehensive and manually annotated resource for mycobacterial genomes. In this paper, we present the MycoBrowser portal (http://mycobrowser.epfl.ch/), a resource that provides both in silico generated and manually reviewed information within databases dedicated to the complete genomes of Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium marinum and Mycobacterium smegmatis. A central component of MycoBrowser is TubercuList (http://tuberculist.epfl.ch), which has recently benefited from a new data management system and web interface. These improvements were extended to all MycoBrowser databases. We provide an overview of the functionalities available and the different ways of interrogating the data then discuss how both the new information and the latest features are helping the mycobacterial research communities.


Quick path finding--quick algorithmic solution for unambiguous labeling of phylogenetic tree nodes. Ever increasing amounts of genetic information stored in sequential databases require efficient methods to automatically reveal their phylogenetic relationships. A framework for in silico unambiguous analysis of phylogenetic trees, based on information contained in tree's topology, together with its branches length, is proposed. The resulting, translated tree has all nodes labeled, with no constraints on nodes' degree, and the subsequent finding of evolutionary pathways from the QPF-translated tree is robust and straightforward. Main features of the method are: small demands on computational time, and the ability to analyze phylogenies obtained prior to the proposed QPF analysis by any traditional tree-building technique.


Renin-angiotensin system blockade and cognitive function in patients at high risk of cardiovascular disease: analysis of data from the ONTARGET and TRANSCEND studies. cardiovascular risk factors are associated with dementia and cognitive decline. We investigated the effects of renin-angiotensin system blockade on cognitive function in patients aged 55 years and older with established atherosclerotic cardiovascular disease or diabetes with end-organ damage in two clinical trials. in the main study, ONTARGET, a double-blind, double-dummy, randomised controlled trial, the effects on cardiovascular outcomes of standard doses of an angiotensin-converting enzyme (ACE) inhibitor (ramipril), an angiotensin-receptor blocker (telmisartan), and a combination of the drugs were evaluated in 25 620 participants. In the parallel TRANSCEND trial, the effects of telmisartan were compared with those of placebo in 5926 participants intolerant to ACE inhibitors. Secondary outcomes included cognitive impairment (defined by investigator-reported diagnosis of dementia or significant cognitive dysfunction, or a score of ≤ 23 on the Mini-Mental State Examination [MMSE]) and cognitive decline (a decrease of ≤ 3 points on the MMSE from baseline during follow-up). Analyses were by intention to treat. We pooled data from these studies to identify baseline predictors of cognitive impairment and its frequency according to mean systolic blood pressure during follow-up. These studies were registered with ClinicalTrials.gov, number NCT00153101. During a median duration of 56 months (IQR 51-64) of follow-up in ONTARGET, cognitive impairment occurred in 652 (8%) of 7865 patients allocated ramipril, 584 (7%) of 7797 allocated telmisartan, and 618 (8%) of 7807 allocated combination treatment (combination vs ramipril, odds ratio [OR] 0·95, 95% CI 0·85-1·07, p= 0·39; telmisartan vs ramipril, OR 0·90, 0·80-1·01, p = 0·06). Corresponding figures for cognitive decline were 1314 (17%), 1279 (17%), and 1240 (17%) in each of the groups, respectively (telmisartan vs ramipril, OR 0·97, 0·89-1·06, p= 0·53; combination vs ramipril, OR 0·95, 0·88-1·04, p=0·28). In TRANSCEND, cognitive impairment occurred in 239 (9%) of 2694 participants allocated telmisartan compared with 245 (9%) of 2689 allocated placebo (OR 0·97, 0·81-1·17, p= 0·76). The corresponding figures for cognitive decline were 454 (17%) and 412 (16%; OR 1·10, 0·95-1·27, p= 0·22). In patients with cardiovascular disease or diabetes, different approaches to blocking of the renin-angiotensin system had no clear effects on cognitive outcomes. Although patients with the lowest systolic blood pressure had the greatest preservation of cognitive function, meta-regression analyses did not show any benefits of blood-pressure lowering on cognition over several years of treatment.


Comparison of knowledge on travel related health risks and their prevention among humanitarian aid workers and other travellers consulting at the Institut Pasteur travel clinic in Paris, France. Non-Governmental Organization (NGO) workers are a group of travellers highly exposed to infectious diseases due to the conditions and duration of their stay. Yet little is known about their knowledge concerning travel related health risks and their prevention. From July to September 2008 a self-administered questionnaire was given to all NGO workers coming for pre-travel consultation and to a comparison group of non-NGO travellers. It examined traveller's knowledge about malaria, traveller's diarrhea, transmission routes of infectious diseases and vaccine availability in both groups. 249 NGO employees and 304 non-NGO travellers were surveyed. Mean age (33.7 vs 34.7 years) and sex ratio (M/F 1.16 vs 1.29) were comparable in both groups. Travel destination was more often Africa (75% vs 39%) and duration of travel was longer in the NGO group (75% vs 15% more than 1 month). NGO travellers had significantly better knowledge about the transmission routes of infectious diseases : percentages of accurate answers varied from 20% for Japanese encephalitis, 31% for yellow fever and 41% for hepatitis A to 70% for dengue and 96% for malaria, versus respectively 9%, 17%, 40%, 54% and 94% for the comparison group. However no differences were observed between the two groups concerning the means to prevent malaria or traveller's diarrhea, or the symptoms necessitating medical consultation. In the sub-group analyses medical professionals (23% of the NGO group) performed better than other NGO workers. Even though the knowledge was slightly better in the NGO group, there are still important gaps and a combined effort of all actors is needed to improve the security of expatriated humanitarian aid workers, in particular for the non-medical staff.


Tailored sutureless meatoplasty: a new technique for correcting meatal stenosis. Standard practice for meatoplasty includes the use of sutures to evert the mucosa. We present a novel technique that is equivalent to standard meatoplasty without the need for suturing. A retrospective chart review was performed of patients who presented with primary meatal stenosis in 2007-2009. One group underwent a tailored, sutureless meatoplasty performed by one surgeon. The second group underwent a standard meatoplasty with mucosal advancement and suturing of the mucosal edges by another surgeon. Data were collected regarding demographics, presenting symptoms, symptom resolution, and postoperative complications. These were then compared between the two groups. Preoperative and postoperative uroflowmetry data were collected for the sutureless group to demonstrate objective improvement. Of 85 patients, aged 2-17 years, 60 underwent the tailored, sutureless procedure while 25 had a standard meatoplasty. The majority of patients presented with a deviated and/or narrowed stream of urine. There was no statistical difference between the two groups in regard to symptom resolution. Uroflowmetry data analyzed for 20 patients in the sutureless group demonstrated a significant improvement in Q(max) and flow pattern; there was no significant change in % post-void residual. No patient had a complication in either group, or presented with recurrence. Tailored sutureless meatoplasty is comparable to meatoplasty with mucosal advancement in resolving symptoms, improving uroflowmetry parameters, and complication rate, but is more efficient and cost effective.


The European cystic fibrosis patient registry: the power of sharing data.


Rapid screening of monoamine oxidase B inhibitors in natural extracts by capillary electrophoresis after enzymatic reaction at capillary inlet. A facile capillary electrophoresis (CE) method was developed for the screening of monoamine oxidase B (MAO-B) inhibitors in natural extracts. In this method, the enzymatic reaction occurred at the capillary inlet during a predetermined waiting period, followed by the electrophoretic separation of the reaction compounds, and detected by their UV absorbance at 280 nm. Conditions for the separation of substrates, products and enzyme were optimized. The optimal buffer composition was 50 mM N-2-hydroxyethyl-piperazine-N'-2-ethane sulphonic acid (HEPES) solution containing 10 mM SDS (pH=7.4). Under the optimal condition, the baseline separation of substrates, products and enzyme was achieved within 2 min. The present method was used to determine MAO-B kinetic constants, K(i), K(m) and IC₅₀ based on quantitative of the substrate peak area compared with the reference electropherogram obtained from without the inhibitor. A validation study shows good reproducibility for both migration time (RSD=1.8%) and peak area (RSD=3.9%). Finally, the screening of 16 natural extracts was performed, and 2 natural extracts from Fructus crataegi and Radix polygoni multiflori were identified to be positive for MAO-B inhibition.


Development and validation of a sensitive and selective UHPLC-MS/MS method for simultaneous determination of both free and total eicosapentaeonic acid and docosahexenoic acid in human plasma. A sensitive, selective, and quantitative method for the simultaneous determination of free and total eicosapentaeonic acid (EPA) and docosahexenoic acid (DHA) has been developed and validated in human plasma using fatty acid free human serum albumin as a surrogate matrix. Clean-up for free EPA and DHA employs a liquid-liquid extraction with hexane to remove plasma interferences and provide for cleaner chromatography. The method for total EPA and DHA requires a digestion of the triglycerides followed by liquid-liquid extraction with hexane. Ultra high performance liquid chromatography (UHPLC) technology on a BEH C18 stationary phase column with 1.7 μm particle size was used for chromatographic separation, coupled to tandem mass spectrometry (UHPLC-MS/MS). The method for free EPA and DHA was validated over the concentration range of 0.05-25 μg/mL, while total EPA and DHA concentration range was 0.5-250 μg/mL. The results from assay validation show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies. To our knowledge, this work represents the first UHPLC-MS/MS based method that combines both free and total EPA and DHA with a relatively small sample volume (25 μL aliquot) and a run time of 1.5 min, facilitating automation and high throughput analysis.


Challenges and opportunities in oncoproteomics.


Forthcoming challenges in the management of direct-acting antiviral agents (DAAs) for hepatitis C. Agents that specifically target the replication cycle of the virus direct-acting antiviral agents (DAAs) by directly inhibiting the NS3/4A serine protease, the NS5B polymerase and NS5A are currently in clinical development. The need to achieve serum drug concentrations able to suppress viral replication is a key factor for a successful antiviral therapy and the prevention of resistance. Thus pharmacokinetics parameters became important issues for drugs used in the therapy of hepatitis C. The ratio of C(min)/IC(50) (inhibitory quotient or IQ) can provide a surrogate measure of a drug's ability to suppress HCV replication, by taking into account the relationship between plasma drug levels and viral susceptibility to the drug. Ritonavir boosting may be a useful strategy to improve pharmacokinetic parameters. Characterising resistance to DAAs in clinical trials is essential for the management of a drug regimen to reduce the development of resistance and thereby maximise SVR rate. The lesson of HIV therapy, provide a compelling case for the exploration of combinations of direct-acting antiviral agents.


Hmong food helps us remember who we are: perspectives of food culture and health among Hmong women with young children. To examine perspectives on food habits, acculturation, and health among Hmong women with young children in northern California. Qualitative interviews were conducted with 15 Hmong mothers with young children in a low-income community of northern California. The interviews were transcribed and coded based on the principles of grounded theory. The main themes that emerged from the interviews included comparisons between American and Hmong food cultures, food culture preservation, obesity issues, diverse acculturation processes, and intergenerational food habits. Hmong food culture has been identified as the representation of their healthful lifestyle, self-identity, and social support. At the same time, mothers are facing enormous challenges in bridging 2 extreme generations; the less acculturated, immigrant adults and the highly acculturated, US-born children in their households. Addressing the healthful aspects of Hmong food culture and healthful options of American food should be a focus of nutrition-related programs.


Employment of mid-level providers in primary care and control of diabetes. Examine potential associations between inclusion of mid-level providers in United States Veterans Affairs (VA) primary care programs and diabetes control. We established a cohort of diabetes patients (alive October 1, 1999) using the VA Diabetes Registry and VA corporate databases. 1999 VA Survey of Primary Care Practices data were combined with individual-patient information. We used a two-level hierarchical model to determine the relationship between staffing characteristics and hemoglobin A1c (HbA1c), among 88,682 patients from 198 clinics. Inclusion of nurse practitioners (NPs) at relatively limited levels (% of all providers who are NPs) in the primary care program was significantly associated with HbA1c lower by 0.31 percentage points (95% CI, -0.50% to -0.12%) compared to programs that did not include NPs. Having some level of NP staffing vs. no NP staffing was associated HbA1c lower by 0.25%. Inclusion of physician assistants (PAs) in primary care programs was generally not associated with a statistically significant difference in HbA1c. The exception is that moderate levels of PA staffing were associated with slightly higher HbA1c [0.18%, 95% CI, 0.02-0.34)]. Diabetes control among primary care patients appeared to benefit from inclusion of NPs, while an analogous association was not found for PAs.


Predictors of low-income, obese mothers' use of healthful weight management behaviors. To examine the influence of personal and environmental factors on healthful weight management behaviors mediated through self-efficacy among low-income obese mothers. Cross-sectional design. Special Supplemental Nutrition Program for Women, Infants, and Children in Wisconsin. Two hundred eighty-four obese mothers aged 18-34 years. Personal factors (eg, eating low-fat/low-calorie food for weight management), environmental factors, self-efficacy, and weight management behaviors. Data were collected via self-administered written questionnaires. Latent class cluster analysis and structural logistic regression. Women who perceived more importance of eating low-fat/low-calorie food for weight management were more likely to practice multiple behavioral strategies to manage their weight (odds ratio = 0.37) than women who perceived less importance of eating low-fat/low-calorie food for weight management. Women who reported higher positive mood self-efficacy were more likely (odds ratio = 1.63) to practice multiple strategies to manage their weight than women who reported lower positive mood self-efficacy. Environmental factors did not predict use of multiple healthful weight management behaviors. No mediation effect was found. Stressing the importance of food choices and reinforcing learners' self-confidence to make healthful choices at parties and celebrations may be helpful ways to address common barriers to healthful weight management.


The Indian Ocean dipole and cholera incidence in Bangladesh: a time-series analysis. It has been reported that the El Niño-Southern Oscillation (ENSO) influences the interannual variation of endemic cholera in Bangladesh. There is increased interest in the influence of the Indian Ocean dipole (IOD), a climate mode of coupled ocean-atmosphere variability, on regional ocean climate in the Bay of Bengal and on Indian monsoon rainfall. We explored the relationship between the IOD and the number of cholera patients in Bangladesh, controlling for the effects of ENSO. Time-series regression was performed. Negative binomial models were used to estimate associations between the monthly number of hospital visits for cholera in Dhaka and Matlab (1993-2007) and the dipole mode index (DMI) controlling for ENSO index [NINO3, a measure of the average sea surface temperature (SST) in the Niño 3 region], seasonal, and interannual variations. Associations between cholera cases and SST and sea surface height (SSH) of the northern Bay of Bengal were also examined. A 0.1-unit increase in average DMI during the current month through 3 months before was associated with an increase in cholera incidence of 2.6% [(95% confidence interval (CI), 0.0-5.2; p = 0.05] in Dhaka and 6.9% (95% CI, 3.2-10.8; p < 0.01) in Matlab. Cholera incidence in Dhaka increased by 2.4% (95% CI, 0.0-5.0; p = 0.06) after a 0.1-unit decrease in DMI 4-7 months before. Hospital visits for cholera in both areas were positively associated with SST 0-3 months before, after adjusting for SSH (p < 0.01). These findings suggest that both negative and positive dipole events are associated with an increased incidence of cholera in Bangladesh with varying time lags.


Association between circulating specific leukocyte types and blood pressure: the atherosclerosis risk in communities (ARIC) study. Although total white blood cell (WBC) count has been associated with hypertension, the association between specific WBC types and blood pressure (BP) levels has not been studied. In a cohort of 5746 middle-age African-American and white adults free of clinical cardiovascular disease and cancer and not taking hypertension or anti-inflammatory medications, BP was measured at baseline and 3, 6, and 9 years later. Levels of circulating neutrophils, lymphocytes, and monocytes were measured at baseline. In African-Americans, but much less so in whites, increased neutrophil levels and decreased lymphocyte levels were significantly associated with elevation of BP but did not influence the rate of change of BP over time. The mean BP difference between the highest and lowest quartiles of neutrophils was approximately 8 mm Hg for systolic BP (SBP), 4 mm Hg for mean arterial pressure (MAP), and 5 mm Hg for pulse pressure (PP). The mean BP difference between the lowest and highest quartiles of lymphocytes was approximately 6 mm Hg for SBP, 2 mm Hg for diastolic BP (DBP), 3 mm Hg for MAP, and 4 mm Hg for PP. Increased neutrophils and decreased lymphocytes are significantly correlated with the regulation of BP and the development of hypertension, especially in African-Americans.


Fish consumption and mercury exposure among Louisiana recreational anglers. Methylmercury (MeHg) exposure assessments among average fish consumers in the United States may underestimate exposures among U.S. subpopulations with high intakes of regionally specific fish. We examined relationships among fish consumption, estimated mercury (Hg) intake, and measured Hg exposure within one such potentially highly exposed group, recreational anglers in the state of Louisiana, USA. We surveyed 534 anglers in 2006 using interviews at boat launches and fishing tournaments combined with an Internet-based survey method. Hair samples from 402 of these anglers were collected and analyzed for total Hg. Questionnaires provided information on species-specific fish consumption during the 3 months before the survey. Anglers' median hair Hg concentration was 0.81 µg/g (n = 398; range, 0.02-10.7 µg/g); 40% of participants had levels >1 µg/g, which approximately corresponds to the U.S. Environmental Protection Agency's reference dose. Fish consumption and Hg intake were significantly positively associated with hair Hg. Participants reported consuming nearly 80 different fish types, many of which are specific to the region. Unlike the general U.S. population, which acquires most of its Hg from commercial seafood sources, approximately 64% of participants' fish meals and 74% of their estimated Hg intake came from recreationally caught seafood. Study participants had relatively elevated hair Hg concentrations and reported consumption of a wide variety of fish, particularly locally caught fish. This group represents a highly exposed subpopulation with an exposure profile that differs from fish consumers in other regions of the United States, suggesting a need for more regionally specific exposure estimates and public health advisories.


Subclinical rhythmic electrographic discharges of adults and transient global amnesia: a causal or casual association? Subclinical rhythmic electrographic discharges of adults (SREDA) is an uncommon distinctive EEG rhythmic pattern with uncertain significance. We report a patient with transient global amnesia in whom an EEG recording, performed after the acute phase of the neurological event, showed SREDA. Based on this EEG finding, we discuss about a possible correlation between SREDA and transient global amnesia. The presence of SREDA in our patient with transient global amnesia seems to be incidental. When described in association with transient global amnesia, SREDA should be recognized in order to avoid misdiagnosis. Although SREDA has been fortuitously described in association with transient global amnesia, a patho-physiological correlation with mechanisms which produce it seems unlikely.


Renal dysfunction impairs circadian variation of endothelial function in patients with essential hypertension. Some cardiovascular disorders disturb circadian variation of endothelial function. We investigated whether deterioration of renal function alters circadian variation of endothelial function in patients with hypertension. Endothelial function was assessed by the peak forearm blood flow (FBF) response to reactive hyperemia, and 24-hour ambulatory blood pressure monitoring was performed in 25 patients with essential hypertension (61 ± 17 years). Relationships among renal function, 24-hour blood pressure, and endothelial function were analyzed. The ratio of nighttime to daytime mean arterial pressure was inversely correlated with estimated glomerular filtration rate (eGFR) (r = -0.43, P = .03). The FBF response to reactive hyperemia examined at 21:00, but not at 6:30 or 11:30, was significantly correlated with eGFR (r = 0.44, P = .03). Furthermore, the ratio of FBF response measured at 21:00 to that measured at 6:30 was independently correlated with eGFR (β = 0.47, P = .02). Renal dysfunction is associated with the derangement of circadian variation of both endothelial function and blood pressure. Nocturnal blood pressure is elevated, and evening endothelial function deteriorates in parallel with a decline in renal function in hypertensive patients.


Vascular and cardiac impairments in rats inhaling ozone and diesel exhaust particles. Mechanisms of cardiovascular injuries from exposure to gas and particulate air pollutants are unknown. We sought to determine whether episodic exposure of rats to ozone or diesel exhaust particles (DEP) causes differential cardiovascular impairments that are exacerbated by ozone plus DEP. Male Wistar Kyoto rats (10-12 weeks of age) were exposed to air, ozone (0.4 ppm), DEP (2.1 mg/m(3)), or ozone (0.38 ppm) + DEP (2.2 mg/m(3)) for 5 hr/day, 1 day/week for 16 weeks, or to air, ozone (0.51 or 1.0 ppm), or DEP (1.9 mg/m(3)) for 5 hr/day for 2 days. At the end of each exposure period, we examined pulmonary and cardiovascular biomarkers of injury. In the 16-week study, we observed mild pulmonary pathology in the ozone, DEP, and ozone + DEP exposure groups, a slight decrease in circulating lymphocytes in the ozone and DEP groups, and decreased platelets in the DEP group. After 16 weeks of exposure, mRNA biomarkers of oxidative stress (hemeoxygenase-1), thrombosis (tissue factor, plasminogen activator inhibitor-1, tissue plasminogen activator, and von Willebrand factor), vasoconstriction (endothelin-1, endothelin receptors A and B, endothelial NO synthase) and proteolysis [matrix metalloprotease (MMP)-2, MMP-3, and tissue inhibitor of matrix metalloprotease-2] were increased by DEP and/or ozone in the aorta, but not in the heart. Aortic LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) mRNA and protein increased after ozone exposure, and LOX-1 protein increased after exposure to ozone + DEP. RAGE (receptor for advanced glycation end products) mRNA increased in the ozone + DEP group. Exposure to ozone or DEP depleted cardiac mitochondrial phospholipid fatty acids (DEP > ozone). The combined effect of ozone and DEP exposure was less pronounced than exposure to either pollutant alone. Exposure to ozone or DEP for 2 days (acute) caused mild changes in the aorta. In animals exposed to ozone or DEP alone for 16 weeks, we observed elevated biomarkers of vascular impairments in the aorta, with the loss of phospholipid fatty acids in myocardial mitochondria. We conclude that there is a possible role of oxidized lipids and protein through LOX-1 and/or RAGE signaling.


Use of in vitro HTS-derived concentration-response data as biological descriptors improves the accuracy of QSAR models of in vivo toxicity. Quantitative high-throughput screening (qHTS) assays are increasingly being used to inform chemical hazard identification. Hundreds of chemicals have been tested in dozens of cell lines across extensive concentration ranges by the National Toxicology Program in collaboration with the National Institutes of Health Chemical Genomics Center. Our goal was to test a hypothesis that dose-response data points of the qHTS assays can serve as biological descriptors of assayed chemicals and, when combined with conventional chemical descriptors, improve the accuracy of quantitative structure-activity relationship (QSAR) models applied to prediction of in vivo toxicity end points. We obtained cell viability qHTS concentration-response data for 1,408 substances assayed in 13 cell lines from PubChem; for a subset of these compounds, rodent acute toxicity half-maximal lethal dose (LD50) data were also available. We used the k nearest neighbor classification and random forest QSAR methods to model LD50 data using chemical descriptors either alone (conventional models) or combined with biological descriptors derived from the concentration-response qHTS data (hybrid models). Critical to our approach was the use of a novel noise-filtering algorithm to treat qHTS data. Both the external classification accuracy and coverage (i.e., fraction of compounds in the external set that fall within the applicability domain) of the hybrid QSAR models were superior to conventional models. Concentration-response qHTS data may serve as informative biological descriptors of molecules that, when combined with conventional chemical descriptors, may considerably improve the accuracy and utility of computational approaches for predicting in vivo animal toxicity end points.


Importance of blood pressure control in left ventricular mass regression. Blood pressure (BP) reduction to 140/90 mm Hg or lower using renin-angiotensin-system blockers reportedly provides the greatest left ventricular (LV) mass regression; β-blockers have less effect. This study examined whether combination antihypertensive therapy would provide greater benefit. With a double-blind, parallel-group design, the effects of 3 different combinations, carvedilol controlled-release (CR)/lisinopril, atenolol/lisinopril, and lisinopril, on left ventricular mass index (LVMI) were assessed by MRI after 12 months. Patients were treated to achieve guideline-recommended BP (<140 mm Hg/<90 mm Hg; diabetes: <130 mm Hg/<80 mm Hg). Sample size was calculated to achieve 90% power to detect a 5 g/m(2) difference in mean change from baseline in LVMI between the carvedilol CR/lisinopril group and each of the other treatment groups. Of 287 patients randomized, more than 50% were titrated to maximum dosage; 73% reached targeted BP. At month 12 (last observation carried forward ≥ month 9) for 195 evaluable subjects, mean BP was similar in all groups (carvedilol CR/lisinopril: 128.8/77.9; atenolol/lisinopril: 128.7/76.5; lisinopril: 126.3/80.3 mm Hg). Compared with baseline, mean LVMI decreased to a similar extent in all groups (carvedilol CR/lisinopril: -6.3; atenolol/lisinopril: -6.7; lisinopril: -7.9 g/m(2)). Achievement of targeted BP control is more important than treatment regimen in achieving LV mass reduction.


Aggression and sexual behavior in best-selling pornography videos: a content analysis update. This current study analyzes the content of popular pornographic videos, with the objectives of updating depictions of aggression, degradation, and sexual practices and comparing the study's results to previous content analysis studies. Findings indicate high levels of aggression in pornography in both verbal and physical forms. Of the 304 scenes analyzed, 88.2% contained physical aggression, principally spanking, gagging, and slapping, while 48.7% of scenes contained verbal aggression, primarily name-calling. Perpetrators of aggression were usually male, whereas targets of aggression were overwhelmingly female. Targets most often showed pleasure or responded neutrally to the aggression.


The impossibility of bearing witness: wartime rape and the promise of justice. Testimonies of wartime sexual violence contribute to the recognition of rape as a serious human rights violation. Although acknowledgement and justice are imperative to ending silence and impunity, this article critiques some commonly held therapeutic assumptions about disclosure through examining the way so-called "unspeakable" events are communicated through legal discourse. In this article, the author explores the inherent limitations of language for bearing witness to wartime rape, specifically focusing on international war crimes tribunals. The author argues that trials contribute to the impossibility of bearing witness through both the appropriation of trauma and the failure of law to accommodate traumatic experiences.


The relation between blood pressure changes induced by passive leg raising and arterial stiffness. Passive leg raising (PLR) elicits cardiorespiratory changes because of intra-thoracic blood pooling. We previously found PLR to lower blood pressure (BP) in healthy subjects. To determine the effects of cardiovascular (CV) risk factors on the PLR BP lowering response, we measured brachial artery, systolic (SBP), diastolic (DBP), and pulse pressure (PP) before and 1 minute after 60° PLR, in 125 subjects (58% males, age 48 ± 18 years) with/without CV risk factors/disease. Baseline carotid-radial pulse wave velocity (PWV) and augmentation index (AI) were measured by applanation tonometry. Changes (∆) in SBP and BA PP ranged from +31 to -27 and +35 to -20 mm Hg and were normally distributed. Mean BP decreases upon PLR were ΔSBP -3 ± 9 (P < .001), and ΔPP -2 ± 8 mm Hg (P = .04). On univariate analyses, ΔSBP was correlated with age (r = 0.31, P < .001), PWV (r = 0.53, P < .001), AI (r = 0.25, P < .01), hypertension (r = 0.37, P < .001), hypercholesterolemia (r = 0.25, P < .01) and renal insufficiency (r = 0.19, P = .038). ΔPP was correlated with age (r = 0.31, P < .001), PWV (r = 0.45, P < .001), AI (r = 0.23, P = .01), hypertension (r = 0.22, P = .01), hypercholesterolemia (r = 0.27, P < .001). Multivariate analysis showed PWV the only independent predictor of ΔSBP (B = 3.2, r(2) = 0.40, P < .001) and of ΔPP (B = 2.6, r(2) = 0.36, P < .001). Therefore, changes in SBP and PP induced by PLR are independently and inversely related to carotid-radial PWV.


Deconstructing self-blame following sexual assault: the critical roles of cognitive content and process. As part of a larger study, predictors of self-blame were investigated in a sample of 149 undergraduate sexual assault survivors. Each participant completed questionnaires regarding their preassault, peritraumatic, and postassault experiences and participated in an individual interview. Results confirmed the central hypothesis that, although several established correlates independently relate to self-blame, only cognitive content and process variables- negative self-cognitions and counterfactual-preventability cognitions-uniquely predict self-blame in a multivariate model.


Perpetrators of intimate partner sexual violence: are there unique characteristics associated with making partners have sex without a condom? This study examined correlates of making an intimate partner engage in unprotected sex among perpetrators of sexual violence. Based on the Confluence Model, we hypothesized that power and impersonal sex motives would be higher among perpetrators who made a dating partner have unprotected sex. Among a subsample of 78 male college students, significant differences were found for acceptance of verbal pressure, positive attitudes about casual sex, frequency of sexual intercourse, and physical injuries to dating partners. These findings highlight the importance of integrating theories and interventions directed at sexual assault and sexual risk reduction.


Domestic violence and sexual assault agency directors' perspectives on services that help survivors. Community-based domestic violence and sexual assault service providers need sound knowledge regarding services that work well to improve the lives of survivors. This exploratory, qualitative research aimed to help provide such knowledge by investigating domestic violence and sexual assault agency executive directors' ( n = 14) opinions regarding what services are most helpful for survivors. In-depth interviews with directors provided findings about (a) critical services for survivors; (b) essential service delivery practices; (c) ideal services that are challenging to deliver because of funding and other barriers; and (d) areas of service delivery practice uncertainty due to a lack of best practices.


Telling our stories: the importance of women's narratives of resistance. While more research is emerging on women's capacity for successful resistance and the benefits of self-defense training for women, "resistance" to the idea of women's resistance remains high. In this note, I describe the importance of bringing true accounts of successful resistance to both academic and lay communities, and the benefits to women of recounting their own experiences of successful resistance to violence and harassment. Such accounts provide new scripts for women's options in responding to violence, mark attempted but thwarted rape as a visible event, and remind women of their own agency in the face of assault.


The importance of epistemology: a response to Rosenstein's commentary.


Commentary on Jamie L. Callahan's "Manifestations of power and control: training as the catalyst for scandal at the United States Air Force Academy".


Trans-centromere effects on meiotic recombination in the zebrafish. We report that lack of crossover along one chromosome arm is associated with high-frequency occurrence of recombination close to the opposing arm's centromere during zebrafish meiotic recombination. Our data indicate that recombination behavior on the two arms of a chromosome is linked. These results inform mapping strategies for telomeric mutants.


The DNA polymerase activity of Saccharomyces cerevisiae Rev1 is biologically significant. A cell's ability to tolerate DNA damage is directly connected to the human development of diseases and cancer. To better understand the processes underlying mutagenesis, we studied the cell's reliance on the potentially error-prone translesion synthesis (TLS), and an error-free, template-switching pathway in Saccharomyces cerevisiae. The primary proteins mediating S. cerevisiae TLS are three DNA polymerases (Pols): Rev1, Pol ζ (Rev3/7), and Pol η (Rad30), all with human homologs. Rev1's noncatalytic role in recruiting other DNA polymerases is known to be important for TLS. However, the biological significance of Rev1's unusual conserved DNA polymerase activity, which inserts dC, is much less well understood. Here, we demonstrate that inactivating Rev1's DNA polymerase function sensitizes cells to both chronic and acute exposure to 4-nitroquinoline-1-oxide (4-NQO) but not to UV or cisplatin. Full Rev1-dependent resistance to 4-NQO, however, also requires the additional Rev1 functions. When error-free tolerance is disrupted through deletion of MMS2, Rev1's catalytic activity is more vital for 4-NQO resistance, possibly explaining why the biological significance of Rev1's catalytic activity has been elusive. In the presence or absence of Mms2-dependent error-free tolerance, the catalytic dead strain of Rev1 exhibits a lower 4-NQO-induced mutation frequency than wild type. Furthermore, Pol ζ, but not Pol η, also contributes to 4-NQO resistance. These results show that Rev1's catalytic activity is important in vivo when the cell has to cope with specific DNA lesions, such as N(2)-dG.


The developmental timing regulator HBL-1 modulates the dauer formation decision in Caenorhabditis elegans. Animals developing in the wild encounter a range of environmental conditions, and so developmental mechanisms have evolved that can accommodate different environmental contingencies. Harsh environmental conditions cause Caenorhabditis elegans larvae to arrest as stress-resistant "dauer" larvae after the second larval stage (L2), thereby indefinitely postponing L3 cell fates. HBL-1 is a key transcriptional regulator of L2 vs. L3 cell fate. Through the analysis of genetic interactions between mutations of hbl-1 and of genes encoding regulators of dauer larva formation, we find that hbl-1 can also modulate the dauer formation decision in a complex manner. We propose that dynamic interactions between genes that regulate stage-specific cell fate decisions and those that regulate dauer formation promote the robustness of developmental outcomes to changing environmental conditions.


Socially-responsive gene expression in male Drosophila melanogaster is influenced by the sex of the interacting partner. Behavior is influenced by an organism's genes and environment, including its interactions with same or opposite sex individuals. Drosophila melanogaster perform innate, yet socially modifiable, courtship behaviors that are sex specific and require rapid integration and response to multiple sensory cues. Furthermore, males must recognize and distinguish other males from female courtship objects. It is likely that perception, integration, and response to sex-specific cues is partially mediated by changes in gene expression. Reasoning that social interactions with members of either sex would impact gene expression, we compared expression profiles in heads of males that courted females, males that interacted with other males, or males that did not interact with another fly. Expression of 281 loci changes when males interact with females, whereas 505 changes occur in response to male-male interactions. Of these genes, 265 are responsive to encounters with either sex and 240 respond specifically to male-male interactions. Interestingly, 16 genes change expression only when a male courts a female, suggesting that these changes are a specific response to male-female courtship interactions. We supported our hypothesis that socially-responsive genes can function in behavior by showing that egghead (egh) expression, which increases during social interactions, is required for robust male-to-female courtship. We predict that analyzing additional socially-responsive genes will give us insight into genes and neural signaling pathways that influence reproductive and other behavioral interactions.


Glycosylation genes expressed in seam cells determine complex surface properties and bacterial adhesion to the cuticle of Caenorhabditis elegans. The surface of the nematode Caenorhabditis elegans is poorly understood but critical for its interactions with the environment and with pathogens. We show here that six genes (bus-2, bus-4, and bus-12, together with the previously cloned srf-3, bus-8, and bus-17) encode proteins predicted to act in surface glycosylation, thereby affecting disease susceptibility, locomotory competence, and sexual recognition. Mutations in all six genes cause resistance to the bacterial pathogen Microbacterium nematophilum, and most of these mutations also affect bacterial adhesion and biofilm formation by Yersinia species, demonstrating that both infection and biofilm formation depend on interaction with complex surface carbohydrates. A new bacterial interaction, involving locomotory inhibition by a strain of Bacillus pumilus, reveals diversity in the surface properties of these mutants. Another biological property--contact recognition of hermaphrodites by males during mating--was also found to be impaired in mutants of all six genes. An important common feature is that all are expressed most strongly in seam cells, rather than in the main hypodermal syncytium, indicating that seam cells play the major role in secreting surface coat and consequently in determining environmental interactions. To test for possible redundancies in gene action, the 15 double mutants for this set of genes were constructed and examined, but no synthetic phenotypes were observed. Comparison of the six genes shows that each has distinctive properties, suggesting that they do not act in a linear pathway.


H2A.Z (Htz1) controls the cell-cycle-dependent establishment of transcriptional silencing at Saccharomyces cerevisiae telomeres. The establishment of transcriptional silencing in Saccharomyces cerevisiae requires progression through the cell cycle. We have previously found that transit through M-phase is necessary and sufficient to establish silencing at telomeres following induction of the Sir3 silencing factor. In this study we find that halting cell-cycle progression in either G(1) or at the beginning of M-phase limits the ability of Sir3 to associate with a telomere-linked reporter gene and prevents the changes in histone modifications associated with gene repression. Deletion of genes coding for the histone variant H2A.Z (Htz1 in yeast) and histone acetyltransferase Sas2 abolish the cell-cycle progression requirement for the establishment of silencing. Cells blocked in telophase (but not at metaphase) are also able to establish silencing. We show that H2A.Z binds to the promoter of our telomere-linked reporter gene and that this binding diminishes in silenced cells. Finally, we observe a specific displacement of H2A.Z from chromatin in telophase-blocked cells, regardless of the silencing status of the reporter gene. These results suggest that the requirement for M-phase in the establishment of silencing may reflect a cell-cycle regulated relaxation of heterochromatin barriers.


Escherichia coli rnlA and rnlB compose a novel toxin-antitoxin system. RNase LS was originally identified as a potential antagonist of bacteriophage T4 infection. When T4 dmd is defective, RNase LS activity rapidly increases after T4 infection and cleaves T4 mRNAs to antagonize T4 reproduction. Here we show that rnlA, a structural gene of RNase LS, encodes a novel toxin, and that rnlB (formally yfjO), located immediately downstream of rnlA, encodes an antitoxin against RnlA. Ectopic expression of RnlA caused inhibition of cell growth and rapid degradation of mRNAs in ΔrnlAB cells. On the other hand, RnlB neutralized these RnlA effects. Furthermore, overexpression of RnlB in wild-type cells could completely suppress the growth defect of a T4 dmd mutant, that is, excess RnlB inhibited RNase LS activity. Pull-down analysis showed a specific interaction between RnlA and RnlB. Compared to RnlA, RnlB was extremely unstable, being degraded by ClpXP and Lon proteases, and this instability may increase RNase LS activity after T4 infection. All of these results suggested that rnlA-rnlB define a new toxin-antitoxin (TA) system.


Guanine nucleotide pool imbalance impairs multiple steps of protein synthesis and disrupts GCN4 translational control in Saccharomyces cerevisiae. Purine nucleotides are structural components of the genetic material, function as phosphate donors, participate in cellular signaling, are cofactors in enzymatic reactions, and constitute the main carriers of cellular energy. Thus, imbalances in A/G nucleotide biosynthesis affect nearly the whole cellular metabolism and must be tightly regulated. We have identified a substitution mutation (G388D) that reduces the activity of the GMP synthase Gua1 in budding yeast and the total G-nucleotide pool, leading to precipitous reductions in the GDP/GTP ratio and ATP level in vivo. gua1-G388D strongly reduces the rate of growth, impairs general protein synthesis, and derepresses translation of GCN4 mRNA, encoding a transcriptional activator of diverse amino acid biosynthetic enzymes. Although processing of pre-tRNA(i)(Met) and other tRNA precursors, and the aminoacylation of tRNA(i)(Met) are also strongly impaired in gua1-G388D cells, tRNA(i)(Met)-containing complexes with the macromolecular composition of the eIF2·tRNA(i)(Met.)GTP complex (TC) and the multifactor complex (MFC) required for translation initiation accumulate ∼10-fold in gua1-G388D cells and, to a lesser extent, in wild-type (WT) cells treated with 6-azauracil (6AU). Consistently, addition of an external supply of guanine reverts all the phenotypes of gua1-G388D cells, but not those of gua1-G388D Δhpt1 mutants unable to refill the internal GMP pool through the salvage pathway. These and other findings suggest that a defect in guanine nucleotide biosynthesis evokes a reduction in the rate of general protein synthesis by impairing multiple steps of the process, disrupts the gene-specific reinitiation mechanism for translation of GCN4 mRNA and has far-reaching effects in cell biology and metabolism.


Forced vital capacity, airway obstruction and survival in a general population sample from the USA. Many studies show a link between forced expiratory volume in 1 s (FEV(1)) and survival in the general population and this has been interpreted as a link between airway obstruction and survival. However, the observation that vital capacity is also associated with survival weakens this interpretation. Data on spirometry and survival were taken from the Atherosclerosis Risk in Communities (ARIC) limited access dataset. Survival among 7489 participants with usable spirometry and complete data was regressed against measures of ventilatory function after controlling for many other factors likely to be associated with survival. Survival was strongly associated with forced vital capacity (FVC) after adjustment for FEV(1), but not the other way round. The fully adjusted hazard ratio (HR) associated with high FVC was 0.90 in men (95% CI 0.80 to 1.00; p=0.049) and 0.82 in women (95% CI 0.70 to 0.95; p=0.01). This compares with 0.98 for FEV(1) in men (95% CI 0.90 to 1.07; p.0.72) and 1.01 in women (95% CI 0.89 to 1.15; p=0.84). There was no association between survival and airway obstruction as measured by the FEV(1)/FVC ratio. FVC but not airway obstruction predicts survival in asymptomatic adults without chronic respiratory diagnoses or persistent respiratory symptoms. The association is not explained by age, anthropometry, smoking, income occupation or blood pressure. As FVC later in life, cardiovascular risk, type II diabetes mellitus and low-grade systemic inflammation are all associated with poor fetal growth, these other conditions may be partly responsible for the poor survival in those with low FVC.


Drosophila Mis12 complex acts as a single functional unit essential for anaphase chromosome movement and a robust spindle assembly checkpoint. The kinetochore is a dynamic multiprotein complex assembled at the centromere in mitosis. Exactly how the structure of the kinetochore changes during mitosis and how its individual components contribute to chromosome segregation is largely unknown. Here we have focused on the contribution of the Mis12 complex to kinetochore assembly and function throughout mitosis in Drosophila. We show that despite the sequential kinetochore recruitment of Mis12 complex subunits Mis12 and Nsl1, the complex acts as a single functional unit. mis12 and nsl1 mutants show strikingly similar developmental and mitotic defects in which chromosomes are able to congress at metaphase, but their anaphase movement is strongly affected. While kinetochore association of Ndc80 absolutely depends on both Mis12 and Nsl1, BubR1 localization shows only partial dependency. In the presence of residual centromeric BubR1 the checkpoint still responds to microtubule depolymerization but is significantly weaker. These observations point to a complexity of the checkpoint response that may reflect subpopulations of BubR1 associated with residual kinetochore components, the core centromere, or elsewhere in the cell. Importantly our results indicate that core structural elements of the inner plate of the kinetochore have a greater contribution to faithful chromosome segregation in anaphase than in earlier stages of mitosis.


Influence of body mass index on outcome of the mechanically ventilated patients. There are limited data on the impact of body mass index on outcomes in mechanically ventilated patients. Secondary analysis of a cohort including 4698 patients mechanically ventilated. Patients were screened daily for management of mechanical ventilation, complications (acute respiratory distress syndrome, sepsis, ventilator associated pneumonia, barotrauma), organ failure (cardiovascular, respiratory, renal, hepatic, haematological) and mortality in the intensive care unit. To estimate the impact of body mass index on acute respiratory distress syndrome and mortality, the authors constructed models using generalised estimating equations (GEE). Patients were evaluated based on their body mass index: 184 patients (3.7%) were underweight, 1995 patients (40%) normal weight, 1781 patients (35.8%) overweight, 792 patients (15.9%) obese and 216 patients (4.3%) severely obese. Severely obese patients were more likely to receive low tidal volume based on actual body weight but high volumes based on predicted body weight. In obese patients, the authors observed a higher incidence of acute respiratory distress syndrome and acute renal failure. After adjustment, the body mass index was significantly associated with the development of acute respiratory distress syndrome: compared with normal weight; OR 1.69 (95% CI 1.07 to 2.69) for obese and OR 2.38 (95% CI 1.15 to 4.89) for severely obese. There were no differences in outcomes (duration of mechanical ventilation, length of stay and mortality in intensive care unit and hospital) based on body mass index categories. In this cohort, obese patients were more likely to have significant complications but there were no associations with increased mortality.


Indices of bronchial reactivity and sensitivity.


The potential danger of a solely interferon-gamma release assay-based approach to testing for latent Mycobacterium tuberculosis infection in children.


Deeper and wider: income and mortality in the USA over three decades. Literature on the socio-economic 'gradient' in health often asserts that income is associated with better health not only for the very poor, but also across the entire income distribution. In addition, changes in the shape of the association between incomes during a period of increasing economic inequality have not been previously studied. The goal of the current study was to estimate and compare the shape of the relationship between income and mortality in the USA for the 1970s, the 1980s and the 1990s. Using income and mortality data from the Panel Study of Income Dynamics for respondents aged 35-64 years, we used a Bayesian Cox Model with regression splines to model the risk of mortality over three 10-year follow-up periods. To identify whether income was more strongly associated with mortality at different parts of the income distribution, we treated income as a linear spline with an unknown knot location. The shape of the association between income and mortality was quite non-linear, with a much stronger association at lower levels of income. The relationship between income and mortality in the USA was also not invariant across time, with the increased risk of death associated with lower income applying to an increasing proportion of the US population over time (9th percentile of income in 1970-79, 20th percentile in 1980-89 and 32nd percentile in 1990-99). Our analyses do not support the claim that income is associated with mortality throughout the income distribution, nor is the association between income and mortality the same across periods. Based on our analyses, a focus on the bottom 30% of the income distribution would seem to return the greatest benefits in reducing socio-economic inequalities in health.


Structural basis for the interaction between the growth factor-binding protein GRB10 and the E3 ubiquitin ligase NEDD4. In addition to inhibiting insulin receptor and IGF1R kinase activity by directly binding to the receptors, GRB10 can also negatively regulate insulin and IGF1 signaling by mediating insulin receptor and IGF1R degradation through ubiquitination. It has been shown that GRB10 can interact with the C2 domain of the E3 ubiquitin ligase NEDD4 through its Src homology 2 (SH2) domain. Therefore, GRB10 might act as a connector, bringing NEDD4 close to IGF1R to facilitate the ubiquitination of IGF1R by NEDD4. This is the first case in which it has been found that an SH2 domain could colocalize a ubiquitin ligase and its substrate. Here we report the crystal structure of the NEDD4 C2-GRB10 SH2 complex at 2.0 Å. The structure shows that there are three interaction interfaces between NEDD4 C2 and GRB10 SH2. The main interface centers on an antiparallel β-sheet composed of the F β-strand of GRB10 SH2 and the C β-strand of NEDD4 C2. NEDD4 C2 binds at nonclassical sites on the SH2 domain surface, far from the classical phosphotyrosine-binding pocket. Hence, this interaction is phosphotyrosine-independent, and GRB10 SH2 can bind the C2 domain of NEDD4 and the kinase domain of IGF1R simultaneously. Based on these results, a model of how NEDD4 interacts with IGF1R through GRB10 has been proposed. This report provides further evidence that SH2 domains can participate in important signaling interactions beyond the classical recognition of phosphotyrosine.


Mechanisms of autoinhibition of IRF-7 and a probable model for inactivation of IRF-7 by Kaposi's sarcoma-associated herpesvirus protein ORF45. IRF-7 is the master regulator of type I interferon-dependent immune responses controlling both innate and adaptive immunity. Given the significance of IRF-7 in the induction of immune responses, many viruses have developed strategies to inhibit its activity to evade or antagonize host antiviral responses. We previously demonstrated that ORF45, a KSHV immediate-early protein as well as a tegument protein of virions, interacts with IRF-7 and inhibits virus-mediated type I interferon induction by blocking IRF-7 phosphorylation and nuclear translocation (Zhu, F. X., King, S. M., Smith, E. J., Levy, D. E., and Yuan, Y. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 5573-5578). In this report, we sought to reveal the mechanism underlying the ORF45-mediated inactivation of IRF-7. We found that ORF45 interacts with the inhibitory domain of IRF-7. The most striking feature in the IRF-7 inhibitory domain is two α-helices H3 and H4 that contain many hydrophobic residues and two β-sheets located between the helices that are also very hydrophobic. These hydrophobic subdomains mediate intramolecular interactions that keep the molecule in a closed (inactive) form. Mutagenesis studies confirm the contribution of the hydrophobic helices and sheets to the autoinhibition of IRF-7 in the absence of viral signal. The binding of ORF45 to the critical domain of IRF-7 leads to a hypothesis that ORF45 may maintain the IRF-7 molecule in the closed form and prevent it from being activated in response to viral infection.


Arginine 104 is a key catalytic residue in leukotriene C4 synthase. Human leukotriene C(4) synthase (hLTC(4)S) is an integral membrane enzyme that conjugates leukotriene (LT) A(4) with glutathione to form LTC(4), a precursor to the cysteinyl leukotrienes (LTC(4), LTD(4), and LTE(4)) that are involved in the pathogenesis of human bronchial asthma. From the crystal structure of hLTC(4)S, Arg-104 and Arg-31 have been implicated in the conjugation reaction. Here, we used site-directed mutagenesis, UV spectroscopy, and x-ray crystallography to examine the catalytic role of Arg-104 and Arg-31. Exchange of Arg-104 with Ala, Ser, Thr, or Lys abolished 94.3-99.9% of the specific activity against LTA(4). Steady-state kinetics of R104A and R104S revealed that the K(m) for GSH was not significantly affected. UV difference spectra of the binary enzyme-GSH complex indicated that GSH ionization depends on the presence of Arg-104 because no thiolate signal, with λ(max) at 239 nm, could be detected using R104A or R104S hLTC(4)S. Apparently, the interaction of Arg-104 with the thiol group of GSH reduces its pK(a) to allow formation of a thiolate anion and subsequent nucleophilic attack at C6 of LTA(4). On the other hand, exchange of Arg-31 with Ala or Glu reduced the catalytic activity of hLTC(4)S by 88 and 70%, respectively, without significantly affecting the k(cat)/K(m) values for GSH, and a crystal structure of R31Q hLTC(4)S (2.1 Å) revealed a Gln-31 side chain pointing away from the active site. We conclude that Arg-104 plays a critical role in the catalytic mechanism of hLTC(4)S, whereas a functional role of Arg-31 seems more elusive. Because Arg-104 is a conserved residue, our results pertain to other homologous membrane proteins and represent a structure-function paradigm probably common to all microsomal GSH transferases.


Biochemical and structural studies of a HECT-like ubiquitin ligase from Escherichia coli O157:H7. Many microbial pathogens deliver effector proteins via the type III secretion system into infected host cells. Elucidating the function of these effectors is essential for our understanding of pathogenesis. Here, we describe biochemical and structural characterization of an effector protein (NleL) from Escherichia coli O157:H7, a widespread pathogen causing severe foodborne diseases. We show that NleL functionally and structurally mimics eukaryotic HECT E3 ligases and catalyzes formation of unanchored polyubiquitin chains using Lys(6) and Lys(48) linkage. The catalytic cysteine residue forms a thioester intermediate with ubiquitin. The structure of NleL contains two domains, a β-helix domain formed by pentapeptide repeats and a bilobed catalytic domain reminiscent of the N- and C-lobe architecture of HECT E3s. Six structures of NleL observed in two crystal forms revealed a large range of different positions of the C-lobe relative to the N-lobe, indicating that the helix linking the two lobes is extremely flexible. Comparing the structure of NleL with that of the Salmonella homolog SopA showed that the orientation of the C-lobes differ by as much as 108°, suggesting that large movements of the C-lobe may be required to facilitate the transfer of ubiquitin from E2 to the substrate. These results provide critical knowledge toward understanding the molecular mechanism by which pathogens utilize the host ubiquitination system during infection.


Engineered external guide sequences effectively block viral gene expression and replication in cultured cells. Ribonuclease P (RNase P) complexed with external guide sequence (EGS) represents a novel nucleic acid-based gene interference approach to modulate gene expression. We have previously used an in vitro selection procedure to generate EGS variants that efficiently direct human RNase P to cleave a target mRNA in vitro. In this study, a variant was used to target the mRNA encoding the protease of human cytomegalovirus (HCMV), which is essential for viral capsid formation and replication. The EGS variant was about 35-fold more active in inducing human RNase P to cleave the mRNA in vitro than the EGS derived from a natural tRNA. Moreover, a reduction of 95% in the expression of the protease and a reduction of 4,000-fold in viral growth were observed in HCMV-infected cells that expressed the EGS variant, whereas a reduction of 80% in the protease expression and an inhibition of 150-fold in viral growth were detected in cells that expressed the EGS derived from a natural tRNA sequence. No significant reduction in viral protease expression or viral growth was observed in cells that either did not express an EGS or produced a "disabled" EGS, which carried nucleotide mutations that precluded RNase P recognition. Our results provide direct evidence that engineered EGS variant is highly effective in blocking HCMV expression and growth by targeting the viral protease. Furthermore, these results demonstrate the utility of engineered EGS RNAs in gene targeting applications, including the inhibition of HCMV infection by blocking the expression of virus-encoded essential proteins.


Prostaglandin E2 activates and utilizes mTORC2 as a central signaling locus for the regulation of mast cell chemotaxis and mediator release. Prostaglandin (PG) E(2), a potent mediator produced in inflamed tissues, can substantially influence mast cell responses including adhesion to basement membrane proteins, chemotaxis, and chemokine production. However, the signaling pathways by which PGE(2) induces mast cell chemotaxis and chemokine production remains undefined. In this study, we identified the downstream target of phosphatidylinositol 3-kinase, mammalian target of rapamycin (mTOR), as a key regulator of these responses. In mouse bone marrow-derived mast cells, PGE(2) was found to induce activation of mTORC1 (mTOR complexed to raptor) as indicated by increased p70S6K and 4E-BP1 phosphorylation, and activation of mTORC2 (mTOR complexed to rictor), as indicated by increased phosphorylation of AKT at position Ser(473). Selective inhibition of the mTORC1 cascade by rapamycin or by the use of raptor-targeted shRNA failed to decrease PGE(2)-mediated chemotaxis or chemokine generation. However, inhibition of the mTORC2 cascade through the dual mTORC1/mTORC2 inhibitor Torin, or through rictor-targeted shRNA, resulted in a significant attenuation in PGE(2)-mediated chemotaxis, which was associated with a comparable decrease in actin polymerization. Furthermore, mTORC2 down-regulation decreased PGE(2)-induced production of the chemokine monocyte chemoattractant protein-1 (CCL2), which was linked to a significant reduction in ROS production. These findings are consistent with the conclusion that activation of mTORC2, downstream of PI3K, represents a critical signaling locus for chemotaxis and chemokine release from PGE(2)-activated mast cells.


The DNA damage response pathway regulates the alternative splicing of the apoptotic mediator Bcl-x. Alternative splicing often produces effectors with opposite functions in apoptosis. Splicing decisions must therefore be tightly connected to stresses, stimuli, and pathways that control cell survival and cell growth. We have shown previously that PKC signaling prevents the production of proapoptotic Bcl-x(S) to favor the accumulation of the larger antiapoptotic Bcl-x(L) splice variant in 293 cells. Here we show that the genotoxic stress induced by oxaliplatin elicits an ATM-, CHK2-, and p53-dependent splicing switch that favors the production of the proapoptotic Bcl-x(S) variant. This DNA damage-induced splicing shift requires the activity of protein-tyrosine phosphatases. Interestingly, the ATM/CHK2/p53/tyrosine phosphatases pathway activated by oxaliplatin regulates Bcl-x splicing through the same regulatory sequence element (SB1) that receives signals from the PKC pathway. Convergence of the PKC and DNA damage signaling routes may control the abundance of a key splicing repressor because SB1-mediated repression is lost when protein synthesis is impaired but is rescued by blocking proteasome-mediated protein degradation. The SB1 splicing regulatory module therefore receives antagonistic signals from the PKC and the p53-dependent DNA damage response pathways to control the balance of pro- and antiapoptotic Bcl-x splice variants.


Directed evolution of a thermostable quorum-quenching lactonase from the amidohydrolase superfamily. A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-L-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh β-strands. This E101N/R230I mutant had an increased value of k(cat)/K(m) of 72-fold toward 3-oxo-N-dodecanoyl-L-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-L-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the β-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis.


Molecular basis of reduced glucosylceramidase activity in the most common Gaucher disease mutant, N370S. Gaucher disease is caused by the defective activity of the lysosomal hydrolase, glucosylceramidase. Although the x-ray structure of wild type glucosylceramidase has been resolved, little is known about the structural features of any of the >200 mutations. Various treatments for Gaucher disease are available, including enzyme replacement and chaperone therapies. The latter involves binding of competitive inhibitors at the active site to enable correct folding and transport of the mutant enzyme to the lysosome. We now use molecular dynamics, a set of structural analysis tools, and several statistical methods to determine the flexible behavior of the N370S Gaucher mutant at various pH values, with and without binding the chaperone, N-butyl-deoxynojirimycin. We focus on the effect of the chaperone on the whole protein, on the active site, and on three important structural loops, and we demonstrate how the chaperone modifies the behavior of N370S in such a way that it becomes more active at lysosomal pH. Our results suggest a mechanism whereby the binding of N-butyl-deoxynojirimycin helps target correctly folded glucosylceramidase to the lysosome, contributes to binding with saposin C, and explains the initiation of the substrate-enzyme complex. Such analysis provides a new framework for determination of the structure of other Gaucher disease mutants and suggests new approaches for rational drug design.


Up-regulation of adiponectin by resveratrol: the essential roles of the Akt/FOXO1 and AMP-activated protein kinase signaling pathways and DsbA-L. The natural polyphenol resveratrol (RSV) displays a wide spectrum of health beneficial activities, yet the precise mechanisms remain to be fully elucidated. Here we show that RSV promotes the multimerization and cellular levels of adiponectin in 3T3-L1 adipocytes. The stimulatory effect of RSV was not affected by knocking out Sirt1, but was diminished by suppressing the expression levels of DsbA-L, a recently identified adiponectin-interactive protein that promotes adiponectin multimerization. Suppression of the Akt signaling pathway resulted in an increase in the expression levels of DsbA-L and adiponectin. On the other hand, knocking out FOXO1 or suppressing the activity or expression levels of the AMP-activated protein kinase (AMPK) down-regulated DsbA-L and adiponectin. The stimulatory effect of RSV on adiponectin and DsbA-L expression was completely diminished in FOXO1-suppressed and AMPK-inactivated 3T3-L1 adipocytes. Taken together, our results demonstrate that RSV promotes adiponectin multimerization in 3T3-L1 adipocytes via a Sirt1-independent mechanism. In addition, we show that the stimulatory effect of RSV is regulated by both the Akt/FOXO1 and the AMPK signaling pathways. Last, we show that DsbA-L plays a critical role in the promoting effect of RSV on adiponectin multimerization and cellular levels.


STAT1 is a master regulator of pancreatic {beta}-cell apoptosis and islet inflammation. Cytokines produced by islet-infiltrating immune cells induce β-cell apoptosis in type 1 diabetes. The IFN-γ-regulated transcription factors STAT1/IRF-1 have apparently divergent effects on β-cells. Thus, STAT1 promotes apoptosis and inflammation, whereas IRF-1 down-regulates inflammatory mediators. To understand the molecular basis for these differential outcomes within a single signal transduction pathway, we presently characterized the gene networks regulated by STAT1 and IRF-1 in β-cells. This was done by using siRNA approaches coupled to microarray analysis of insulin-producing cells exposed or not to IL-1β and IFN-γ. Relevant microarray findings were further studied in INS-1E cells and primary rat β-cells. STAT1, but not IRF-1, mediates the cytokine-induced loss of the differentiated β-cell phenotype, as indicated by decreased insulin, Pdx1, MafA, and Glut2. Furthermore, STAT1 regulates cytokine-induced apoptosis via up-regulation of the proapoptotic protein DP5. STAT1 and IRF-1 have opposite effects on cytokine-induced chemokine production, with IRF-1 exerting negative feedback inhibition on STAT1 and downstream chemokine expression. The present study elucidates the transcriptional networks through which the IFN-γ/STAT1/IRF-1 axis controls β-cell function/differentiation, demise, and islet inflammation.


Inhibition of suppressive T cell factor 1 (TCF-1) isoforms in naive CD4+ T cells is mediated by IL-4/STAT6 signaling. The Wnt pathway transcription factor T cell factor 1 (TCF-1) plays essential roles in the control of several developmental processes, including T cell development in the thymus. Although previously regarded as being required only during early T cell development, recent studies demonstrate an important role for TCF-1 in T helper 2 (Th2) cell polarization. TCF-1 was shown to activate expression of the Th2 transcription factor GATA-binding protein 3 (GATA3) and thus to promote the development of IL-4-producing Th2 cells independent of STAT6 signaling. In this study, we show that TCF-1 is down-regulated in human naive CD4(+) T cells cultured under Th2-polarizing conditions. The down-regulation is largely due to the polarizing cytokine IL-4 because IL-4 alone is sufficient to substantially inhibit TCF-1 expression. The IL-4-induced suppression of TCF-1 is mediated by STAT6, as shown by electrophoretic mobility shift assays, chromatin immunoprecipitation, and STAT6 knockdown experiments. Moreover, we found that IL-4/STAT6 predominantly inhibits the shorter, dominant-negative TCF-1 isoforms, which were reported to inhibit IL-4 transcription. Thus, this study provides a model for an IL-4/STAT6-dependent fine tuning mechanism of TCF-1-driven T helper cell polarization.


X-ray and biochemical analysis of N370S mutant human acid β-glucosidase. Gaucher disease is caused by mutations in the enzyme acid β-glucosidase (GCase), the most common of which is the substitution of serine for asparagine at residue 370 (N370S). To characterize the nature of this mutation, we expressed human N370S GCase in insect cells and compared the x-ray structure and biochemical properties of the purified protein with that of the recombinant human GCase (imiglucerase, Cerezyme®). The x-ray structure of N370S mutant acid β-glucosidase at acidic and neutral pH values indicates that the overall folding of the N370S mutant is identical to that of recombinant GCase. Subtle differences were observed in the conformation of a flexible loop at the active site and in the hydrogen bonding ability of aromatic residues on this loop with residue 370 and the catalytic residues Glu-235 and Glu-340. Circular dichroism spectroscopy showed a pH-dependent change in the environment of tryptophan residues in imiglucerase that is absent in N370S GCase. The mutant protein was catalytically deficient with reduced V(max) and increased K(m) values for the substrate p-nitrophenyl-β-D-glucopyranoside and reduced sensitivity to competitive inhibitors. N370S GCase was more stable to thermal denaturation and had an increased lysosomal half-life compared with imiglucerase following uptake into macrophages. The competitive inhibitor N-(n-nonyl)deoxynojirimycin increased lysosomal levels of both N370S and imiglucerase 2-3-fold by reducing lysosomal degradation. Overall, these data indicate that the N370S mutation results in a normally folded but less flexible protein with reduced catalytic activity compared with imiglucerase.


Single domain intracellular antibodies from diverse libraries: emphasizing dual functions of LMO2 protein interactions using a single VH domain. Interfering intracellular antibodies are valuable for biological studies as drug surrogates and as potential macromolecular drugs per se. Their application is still limited because of the difficulty of acquisition of functional intracellular antibodies. We describe the use of the new intracellular antibody capture procedure (IAC(3)) to facilitate direct isolation of functional single domain antibody fragments using four independent target molecules (LMO2, TP53, CRAF1, and Hoxa9) from a set of diverse libraries. Initially, these have variability in only one of the three antigen-binding CDR regions of VH or VL and first round single domains are affinity matured by iterative randomization of the two other CDRs and reselection. We highlight the approach using a single domain binding to LMO2 protein. Our results show that interfering with LMO2 protein function demonstrates a role specifically in erythroid differentiation, confirm a necessary and sufficient function for LMO2 as a cancer therapy target in T-cell neoplasia and allowed for the first time production of soluble recombinant LMO2 protein by co-expression with intracellular domain antibodies. Co-crystallization of LMO2 and the anti-LMO2 VH protein was successful. These results demonstrate that this third generation IAC(3) offers a robust toolbox for various biomedical applications and consolidates functional features of the LMO2 protein complex, which includes the importance of Lmo2-Ldb1 protein interaction.


Surprising substrate versatility in SLC5A6: Na+-coupled I- transport by the human Na+/multivitamin transporter (hSMVT). Iodide (I(-)) is an essential constituent of the thyroid hormones triiodothyronine and thyroxine, which are required for the development of the central nervous system in the fetus and newborn. I(-) uptake in the thyroid is mediated by the Na(+)/I(-) symporter (NIS). NIS has gained particular medical interest due to its sensitivity to the environmental pollutant perchlorate (ClO(4)(-)) and its implication in radioiodide cancer treatment. Recently, others have shown that I(-) absorption in the intestine is mediated by NIS (Nicola, J. P., Basquin, C., Portulano, C., Reyna-Neyra, A., Paroder, M., and Carrasco, N. (2009) Am. J. Physiol. Cell Physiol. 296, C654-662). However, their data suggest the participation of other systems in the homeostasis of I(-), in particular because in vivo uptake studies revealed a ClO(4)(-)-insensitive transport component. Here, we describe Na(+)-coupled I(-) uptake by the human Na(+)/multivitamin transporter (hSMVT), a related protein isolated from the placenta, where it was suggested to supply the fetus with the water-soluble vitamins biotin and pantothenic acid, and α-lipoic acid. hSMVT-mediated Na(+)/I(-) symport is inhibited by the other three organic hSMVT substrates but not by NIS substrates; notably, hSMVT is insensitive to ClO(4)(-). Because hSMVT is found in the intestine and in many other tissues, we propose that hSMVT may play an important role in the homeostasis of I(-) in the body.


TNF-alpha induces macroautophagy and regulates MHC class II expression in human skeletal muscle cells. Macroautophagy, a homeostatic process that shuttles cytoplasmic constituents into endosomal and lysosomal compartments, has recently been shown to deliver antigens for presentation on major histocompatibility complex (MHC) class II molecules. Skeletal muscle fibers show a high level of constitutive macroautophagy and express MHC class II molecules upon immune activation. We found that tumor necrosis factor-α (TNF-α), a monokine overexpressed in inflammatory myopathies, led to a marked up-regulation of macroautophagy in skeletal myocytes. Furthermore, TNF-α augmented surface expression of MHC class II molecules in interferon-γ (IFN-γ)-treated myoblasts. The synergistic effect of TNF-α and IFN-γ on the induction of MHC class II surface expression was not reflected by higher intracellular human leukocyte antigen (HLA)-DR levels and was reversed by macroautophagy inhibition, suggesting that TNF-α facilitates antigen processing via macroautophagy for more efficient MHC class II loading. Muscle biopsies from patients with sporadic inclusion body myositis, a well defined myopathy with chronic inflammation, showed that over 20% of fibers that contained autophagosomes costained for MHC class II molecules and that more than 40% of double-positive muscle fibers had contact with CD4(+) and CD8(+) immune cells. These findings establish a mechanism through which TNF-α regulates both macroautophagy and MHC class II expression and suggest that macroautophagy-mediated antigen presentation contributes to the immunological environment of the inflamed human skeletal muscle.


Bacillus subtilis CwlP of the SP-{beta} prophage has two novel peptidoglycan hydrolase domains, muramidase and cross-linkage digesting DD-endopeptidase. For bacteria and bacteriophages, cell wall digestion by hydrolases is a very important event. We investigated one of the proteins involved in cell wall digestion, the yomI gene product (renamed CwlP). The gene is located in the SP-β prophage region of the Bacillus subtilis chromosome. Inspection of the Pfam database indicates that CwlP contains soluble lytic transglycosylase (SLT) and peptidase M23 domains, which are similar to Escherichia coli lytic transglycosylase Slt70, and the Staphylococcus aureus Gly-Gly endopeptidase LytM, respectively. The SLT domain of CwlP exhibits hydrolytic activity toward the B. subtilis cell wall; however, reverse phase (RP)-HPLC and mass spectrometry revealed that the CwlP-SLT domain has only muramidase activity. In addition, the peptidase M23 domain of CwlP exhibited hydrolytic activity and could cleave d-Ala-diaminopimelic acid cross-linkage, a property associated with dd-endopeptidases. Remarkably, the M23 domain of CwlP possessed a unique Zn(2+)-independent endopeptidase activity; this contrasts with all other characterized M23 peptidases (and enzymes similar to CwlP), which are Zn(2+) dependent. Both domains of CwlP could hydrolyze the peptidoglycan and cell wall of B. subtilis. However, the M23 domain digested neither the peptidoglycans nor the cell walls of S. aureus or Streptococcus thermophilus. The effect of defined point mutations in conserved amino acid residues of CwlP is also determined.


Decoding toxicity: deducing the sequence requirements of IbsC, a type I toxin in Escherichia coli. Bacterial genomes encode a collection of small peptides that are deleterious to their hosts when overexpressed. The physiological relevance of the majority of these peptides is unknown at present, although many of them have been implicated in regulatory processes important for cell survival and adaptability. One peptide that is of particular interest to us is a 19-amino acid proteic toxin, coined IbsC, whose production is repressed by SibC, an RNA antitoxin. Together, IbsC and SibC constitute a type I toxin-antitoxin (TA) pair. To better understand the function of IbsC and to decipher the sequence determinants for its toxic phenotype, we carried out extensive sequence analyses of the peptide. We generated a series of truncation and single amino acid deletion mutants to determine the minimal sequence required for toxicity. We further probed into functionally relevant amino acids with a comprehensive set of IbsC mutants produced using a systematic sequence randomization strategy. We found that IbsC remained toxic in the presence of multiple deletions and single amino acid substitutions, despite being well-conserved in Escherichia coli and across other Gram-negative bacteria. The toxicity of this peptide was determined to be dependent on a stretch of highly hydrophobic residues near its center. Our results defined sequence-function relationship of IbsC and offered additional insights into properties common to membrane-targeting type I toxins in E. coli and related species.