Anti-glucocorticoid gene therapy reverses the impairing effects of elevated corticosterone on spatial memory, hippocampal neuronal excitability, and synaptic plasticity.
Moderate release of the major stress hormones, glucocorticoids (GCs), improves hippocampal function and memory. In contrast, excessive or prolonged elevations produce impairments. Enzymatic degradation and reformation of GCs help to maintain optimal levels within target tissues, including the brain. We hypothesized that expressing a GC-degrading enzyme in hippocampal neurons would attenuate the negative impact of an excessive elevation in GC levels on synaptic physiology and spatial memory. We tested this by expressing 11-beta-hydroxysteroid dehydrogenase (type II) in dentate gyrus granule cells during a 3 d GC treatment followed by examination of synaptic responses in hippocampal slices or spatial performance in the Morris water maze. In adrenalectomized rats with basal GC replacement, additional GC treatments for 3 d reduced synaptic strength and promoted the expression of long-term depression at medial perforant path synapses, increased granule cell and CA1 pyramidal cell excitability, and impaired spatial reference memory (without influencing learning). Expression of 11-beta-hydroxysteroid dehydrogenase (type II), mostly in mature dentate gyrus granule cells, reversed the effects of high GC levels on granule cell and pyramidal cell excitability, perforant path synaptic plasticity, and spatial memory. These data demonstrate the ability of neuroprotective gene expression limited to a specific cell population to both locally and trans-synaptically offset neurophysiological disruptions produced by prolonged increases in circulating stress hormones. This report supplies the first physiological explanation for previously demonstrated cognitive sparing by anti-stress gene therapy approaches and lends additional insight into the hippocampal processes that are important for memory.


Low hemoglobin levels are independently associated with neonatal acute kidney injury: a report from the AWAKEN Study Group.
Studies in adults showed a relationship between low hemoglobin (Hb) and acute kidney injury (AKI). We performed this study to evaluate this association in newborns. We evaluated 1891 newborns from the Assessment of Worldwide AKI Epidemiology in Neonates (AWAKEN) database. We evaluated the associations for the entire cohort and 3 gestational age (GA) groups: <29, 29-<36, and ≥36 weeks' GA. Minimum Hb in the first postnatal week was significantly lower in neonates with AKI after the first postnatal week (late AKI). After controlling for multiple potential confounders, compared to neonates with a minimum Hb ≥17.0 g/dL, both those with minimum Hb ≤12.6 and 12.7-14.8 g/dL had an adjusted increased odds of late AKI (aOR 3.16, 95% CI 1.44-6.96, p = 0.04) and (aOR 2.03, 95% CI 1.05-3.93; p = 0.04), respectively. This association was no longer evident after controlling for fluid balance. The ability of minimum Hb to predict late AKI was moderate (c-statistic 0.68, 95% CI 0.64-0.72) with a sensitivity of 65.9%, a specificity of 69.7%, and a PPV of 20.8%. Lower Hb in the first postnatal week was associated with late AKI, though the association no longer remained after fluid balance was included. The current study suggests a possible novel association between low serum hemoglobin (Hb) and neonatal acute kidney injury (AKI). The study shows that low serum Hb levels in the first postnatal week are associated with increased risk of AKI after the first postnatal week. This study is the first to show this relationship in neonates. Because this study is retrospective, our observations cannot be considered proof of a causative role but do raise important questions and deserve further investigation. Whether the correction of low Hb levels might confer short- and/or long-term renal benefits in neonates was beyond the scope of this study.


The use of complementary and alternative medicine by pediatric food-allergic patients in Japan.
In developed countries, increasing food allergy prevalence and concern regarding food allergies have been reported. Although the use of complementary and alternative medicine (CAM) for the treatment of allergic diseases has increased in some Western countries, the actual proportion and patterns of CAM use for pediatric food allergies in Japan are still unknown. Fourteen allergy centers in Japan participated in the study using a questionnaire survey regarding the use of CAM by pediatric patients. A diagnosis of food allergy was made at each hospital by pediatric allergists. Surveys were completed by parents/guardians, and data were collected for a total of 962 pediatric food-allergic patients. Overall, 8.4% of the participants used CAM to treat a food allergy. The major CAM therapies used were herbal teas (22.2%), including several Japanese herbal teas, Chinese herbal medicine (18.5%) and lactic acid bacteria (16%). Among the participants using CAM to treat food allergy, 13.6% thought that the CAM being used was very effective, while 11.1% of participants thought that CAM caused some type of side effect. Our study is the first large-scale national survey regarding the use of CAM in pediatric patients with food allergies in Japan. Unlike in the USA, which has a higher rate of CAM use (17%), approximately 8.4% of food-allergic patients used CAM in Japan. Interestingly, the major types of CAM used in Japan differed from those used in the USA. Cultural differences and food customs may affect the use of CAM.


Effects of mental stress on myocardial ischemia during daily life.
To determine the relative risk of myocardial ischemia triggered by specific emotions during daily life. Relative risk was calculated by the recently developed case-crossover method, in which the frequency of a presumed trigger during nonischemic, or control, hours is compared with the trigger's frequency during ischemic, or case, hours. Outpatients at Duke University Medical Center, Durham, NC, underwent 48 hours of ambulatory electrocardiographic (ECG) monitoring with concurrent self-report measures of activities and emotions. Occurrences of negative emotions in the hour before the onset of myocardial ischemia were compared with their usual frequency based on all hours in which ischemia did not occur. From a sample of 132 patients with coronary artery disease and recent evidence of exercise-induced ischemia who underwent 48 hours of ambulatory ECG monitoring, 58 patients exhibited ambulatory ischemia and were included in the analysis. Myocardial ischemia during 48-hour ECG monitoring was defined as horizontal or downsloping ST-segment depression of 1 mm (0.1 mV) or more for 1 minute or longer compared with resting baseline. The ECG data were cross-tabulated with subjects' concurrent diary ratings of 3 negative emotions-tension, sadness, and frustration-and 2 positive emotions-happiness and feeling in contro-on a 5-point scale of intensity. The unadjusted relative risk of occurrence of myocardial ischemia in the hour following high levels of negative emotions was 3.0 (95% confidence interval [CI], 1.5-5.9; P<.01) for tension, 2.9 (95% CI, 1.0-8.0; P<.05) for sadness, and 2.6 (95% CI, 1.3-5.1; P<.01) for frustration. The corresponding risk ratios adjusted for physical activity and time of day were 2.2 (95% CI, 1.1 -4.5; P<.05) for tension, 2.2 (95% CI, 0.7-6.4; P=.16) for sadness, and 2.2 (95% CI, 1.1-4.3; P<.05) for frustration. Mental stress during daily life, including reported feelings of tension, frustration, and sadness, can more than double the risk of myocardial ischemia in the subsequent hour. The clinical significance of mental stress-induced ischemia during daily life needs to be further evaluated.


Cardiac response to acute coronary artery occlusion in exercise-trained dogs.
Exercise training is thought to exert a beneficial effect on cardiovascular function, but its effect in the normal heart following acute coronary artery occlusion is still uncertain. Studies were performed in 12 untrained (UT) and 14 endurance-trained (ET) pentobarbital anesthetized dogs. Left ventricular pressure (LVP), heart rate (HR), percent regional myocardial segmental shortening (%SL), and peripheral coronary pressure (PCP) distal to the occlusion were measured during control conditions and during a 2-min circumflex artery occlusion (CAO). During CAO, LVP, dP/dtmax, and %SL in the ischemic region were significantly reduced in both UT and ET dogs. There was no significant difference between the two groups. In addition, PCP decreased to 27 +/- 5 mm Hg and 26 +/- 9 mm Hg in the UT and ET groups, respectively, during CAO indicating no difference in coronary collateral perfusion between the groups. Regional myocardial blood flow was measured using tracer microspheres in eight of the UT and six of the ET dogs, and the decrease in blood flow to the ischemic zone during CAO was similar in both groups. These results indicate that 12-wk of endurance training does not exert a protective effect on myocardial contractile function or on myocardial perfusion in the central ischemic region during CAO in the anesthetized dog.


The Hmr and Lhr hybrid incompatibility genes suppress a broad range of heterochromatic repeats.
Hybrid incompatibilities (HIs) cause reproductive isolation between species and thus contribute to speciation. Several HI genes encode adaptively evolving proteins that localize to or interact with heterochromatin, suggesting that HIs may result from co-evolution with rapidly evolving heterochromatic DNA. Little is known, however, about the intraspecific function of these HI genes, the specific sequences they interact with, or the evolutionary forces that drive their divergence. The genes Hmr and Lhr genetically interact to cause hybrid lethality between Drosophila melanogaster and D. simulans, yet mutations in both genes are viable. Here, we report that Hmr and Lhr encode proteins that form a heterochromatic complex with Heterochromatin Protein 1 (HP1a). Using RNA-Seq analyses we discovered that Hmr and Lhr are required to repress transcripts from satellite DNAs and many families of transposable elements (TEs). By comparing Hmr and Lhr function between D. melanogaster and D. simulans we identify several satellite DNAs and TEs that are differentially regulated between the species. Hmr and Lhr mutations also cause massive overexpression of telomeric TEs and significant telomere lengthening. Hmr and Lhr therefore regulate three types of heterochromatic sequences that are responsible for the significant differences in genome size and structure between D. melanogaster and D. simulans and have high potential to cause genetic conflicts with host fitness. We further find that many TEs are overexpressed in hybrids but that those specifically mis-expressed in lethal hybrids do not closely correlate with Hmr function. Our results therefore argue that adaptive divergence of heterochromatin proteins in response to repetitive DNAs is an important underlying force driving the evolution of hybrid incompatibility genes, but that hybrid lethality likely results from novel epistatic genetic interactions that are distinct to the hybrid background.


Parasite Proximity Drives the Expansion of Regulatory T Cells in Peyer's Patches following Intestinal Helminth Infection.
Helminth infections are typically chronic in nature; however, the exact molecular mechanisms by which these parasites promote or thwart host immunity remain unclear. Worm expulsion requires the differentiation of CD4(+) T cells into Th2 cells, while regulatory T cells (Tregs) act to dampen the extent of the Th2 response. Priming of T cells requires drainage or capture of antigens within lymphoid tissues, and in the case of intestinal helminths, such sites include the mucosa-associated Peyer's patches (PPs) and the draining mesenteric lymph nodes (MLN). To gain insight into when and where the activation of the adaptive T cell response takes place following intestinal helminth infection, we analyzed Th2 and Treg responses in the PPs and MLN following infection with the murine intestinal helminth Heligmosomoides polygyrus bakeri. Protective Th2 responses were observed to be largely restricted to the MLN, while a greater expansion of Tregs occurred within the PPs. Interestingly, those PPs that formed a contact with the parasite showed the greatest degree of Treg expansion and no evidence of type 2 cytokine production, indicating that the parasite may secrete products that act in a local manner to selectively promote Treg expansion. This view was supported by the finding that H. polygyrus bakeri larvae could promote Treg proliferation in vitro. Taken together, these data indicate that different degrees of Treg expansion and type 2 cytokine production occur within the PPs and MLN following infection with the intestinal helminth H. polygyrus bakeri and indicate that these organs exhibit differential responses following infection with intestinal helminths.


Stand-up magnetic metamaterials at terahertz frequencies.
We present a detailed study of non-planar or 'stand-up' split ring resonators operating at terahertz frequencies. Based on a facile multilayer electroplating fabrication, this technique can create large area split ring resonators on both rigid substrates and conformally compliant structures. In agreement with simulation results, the characterization of these metamaterials shows a strong response induced purely by the magnetic field. The retrieved parameters also exhibit negative permeability values over a broad frequency span. The extracted parameters exhibit bianisotropy due to the symmetry breaking of the substrate, and this effect is investigated for both single and broad side coupled split rings. Our 3D metamaterial examples pave the way towards numerous potential applications in the terahertz region of the spectrum.


Activity of antifolates against Pneumocystis carinii dihydrofolate reductase and identification of a potent new agent.
The therapy of Pneumocystis carinii (PC) pneumonia is often unsuccessful, particularly in patients with acquired immune deficiency syndrome (AIDS). Because of difficulties in growing the organism in vitro or obtaining purified organisms, current treatment choices have been made with little information on the metabolic effects of therapeutic agents on PC. This report quantitates the effects of the commonly used antifolates as well as the classic antineoplastic antifolate methotrexate and a lipid-soluble analogue, trimetrexate, on the target enzyme, dihydrofolate reductase (DHFR), in the PC organisms. Trimethoprim and pyrimethamine were found to be weak inhibitors (ID50 = 39,600 and 2,800 nM, respectively), while methotrexate and trimetrexate were potent reductase inhibitors (ID50 = 1.4 and 26.1 nM, respectively). transport studies with radiolabeled compounds showed that compounds with the classic folate structure (methotrexate and leucovorin) were not taken up by the intact PC organisms. In contrast, trimetrexate exhibited rapid uptake. These results suggest a major therapeutic advantage may be gained by combining a potent, readily transported PC DHFR inhibitor such as trimetrexate with the reduced folate leucovorin to achieve a highly potent antiprotozoan effect while preventing toxicity to mammalian cells.


On the Potential, Feasibility, and Effectiveness of Chat Bots in Public Health Research Going Forward.
This paper will discuss whether bots, particularly chat bots, can be useful in public health research and health or pharmacy systems operations. Bots have been discussed for many years; particularly when coupled with artificial intelligence, they offer the opportunity of automating mundane or error-ridden processes and tasks by replacing human involvement. This paper will discuss areas where there are greater advances in the use of bots, as well as areas that may benefit from the use of bots, and will offer practical ways to get started with bot technology. Several popular bot applications and bot development tools along with practical security considerations will be discussed, and a toolbox that one can begin to use to implement bots will be presented.


Skin reaction with Eosinophilia and Systemic Symptoms after lenalidomide in peritoneal Dialysis.
The Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS) syndrome is a fatal and immune-mediated idiosyncratic drug reaction, with symptoms of fever, skin eruptions (that involves more than half of the body surface), facial oedema and hematological disorders, all presenting within the latent period following drug intake. Effects can also be seen on multiple organs, most notably hepatitis in liver and acute interstitial nephritis in kidney, generally post-administration of allopurinol. The European Registry of Severe Cutaneous Adverse Reactions (RegiSCAR) classifies the DRESS Syndrome cases as "definite", "probable" or "possible", based on clinical and laboratory features. Different pathogenetic mechanisms have been involved in this disease, including immunological reactions and HHV-6 reactivation. In our experience, a 72-year-old male, affected by myeloma in peritoneal dialysis, developed a rare case of DRESS syndrome after lenalidomide administration (less than ten cases are known) with HHV-6 reactivation. According to literature, we withdrew the drug and gave methylprednisolone 0,8 mg/kg orally and IVIG 1 gr/kg for two days. Despite this therapy, DRESS syndrome relapsed during steroid taper with rash, thrombocytopenia, hepatitis and high troponin level. A single cycle of intravenous immunoglobulin 0,5 g/kg for four days was enough for syndrome remission. Only few cases are reported in literature, but because of the increasing use of lenalidomide and the autoimmune sequelae of DRESS syndrome, a broad workup and a multidisciplinar careful approach could help in diagnosis, treatment and follow-up.


A discordant monozygotic-twin approach to potential risk factors for chronic widespread pain in females.
Chronic widespread pain (CWP) is a common disorder affecting up to 15% of the general population. The objective of the present study was to explore the role of previously reported psychosocial and interpersonal risk factors on variation in CWP by investigating CWP discordant monozygotic (MZ) twins. This approach allows separation of cause and effect relationships, albeit imperfectly, as well the control for critical confounding variables such as common environment or genetics. In a total sample of N = 3,266 female twins aged 18-89 years, MZ (113 full pairs) and DZ twins (180 full pairs) discordant for CWP were selected. Items from the London fibromyalgia symptom screening questionnaire were used to discriminate cases from controls. To assess potential risk factors, including body mass index, anxiety sensitivity (AS), emotional intelligence, personality, obsessive-compulsive behavior, and coping, validated questionnaires were used. A set of univariate and multivariate logistic regression analyses were conducted. Of the variables showing significant links with CWP in the univariate individual-level analyses, including age, AS, and emotional intelligence, only emotional intelligence turned out to an independent predictor to the pathogenesis of CWP in both the individual level and discordant MZ analyses. These data indicate that in women having identical genetic risk, emotional intelligence seems to play a key role, although of small effect, in the development and/or maintenance of CWP. It further seems that many of the previously reported risk factors for CWP suffer from genetic confounding.


The Sensitivity and Specificity of Loop-Mediated Isothermal Amplification (LAMP) Assay for Tuberculosis Diagnosis in Adults with Chronic Cough in Malawi.
Current tuberculosis diagnostics lack sensitivity, and are expensive. Highly accurate, rapid and cheaper diagnostic tests are required for point of care use in low resource settings with high HIV prevalence. To investigate the sensitivity and specificity, and cost of loop-mediated isothermal amplification (LAMP) assay for tuberculosis diagnosis in adults with chronic cough compared to Xpert® MTB/RIF, fluorescence smear microscopy. Between October 2013 and March 2014, consecutive adults at a primary care clinic were screened for cough, offered HIV testing and assessed for tuberculosis using LAMP, Xpert® MTB/RIF and fluorescence smear microscopy. Sensitivity and specificity (with culture as reference standard), and costs were estimated. Of 273 adults recruited, 44.3% (121/273) were HIV-positive and 19.4% (53/273) had bacteriogically confirmed tuberculosis. The sensitivity of LAMP compared to culture was 65.0% (95% CI: 48.3% to 79.4%) with 100% (95% CI: 98.0% to 100%) specificity. The sensitivity of Xpert® MTB/RIF (77.5%, 95% CI: 61.5% to 89.2%) was similar to that of LAMP, p = 0.132. The sensitivity of concentrated fluorescence smear microscopy with routine double reading (87.5%, 95% CI: 73.2% to 95.8%) was higher than that of LAMP, p = 0.020. All three tests had high specificity. The lowest cost per test of LAMP was at batch size of 14 samples (US$ 9.98); this was lower than Xpert® MTB/RIF (US$ 13.38) but higher than fluorescence smear microscopy (US$ 0.65). The sensitivity of LAMP was similar to Xpert® MTB/RIF but lower than fluorescence smear microscopy; all three tests had high specificity. These findings support the Malawi policy that recommends a combination of fluorescence smear microscopy and Xpert® MTB/RIF prioritised for people living with HIV, already found to be smear-negative, or being considered for retreatment of tuberculosis.


Infection of Mytilus edulis by the trematode Echinostephilla patellae (Digenea: Philophthalmidae).
The blue mussel Mytilus edulis is described as second intermediate host for Echinostephilla patellae from the common limpet Patella vulgata. Mussels were infected with metacercariae of E. patellae under laboratory conditions. Average infection rates increased with increasing temperature, whereas numbers of cercariae, to which individual mussels were exposed, had no effect on relative infection success. The round to slightly oval metacercariae with an average cyst diameter of 208 microm (range 186-243 microm) encysted exclusively in the foot tissue of M. edulis. Morphologically similar metacercariae were found in naturally infected mussels at sites where parasitized P. vulgata and M. edulis are sympatric. This is the first report of E. patellae in blue mussels. The detection of M. edulis being a second intermediate host is of particular interest with regard to the abundance of the parasite and host organisms in intertidal rocky shore ecosystems. The potential role of the common limpet P. vulgata as an alternative secondary host is discussed.


The care-planning conference: Exploring aspects of person-centred interactions.
The aim of this study was to describe the care-planning conference from the participants' and researchers' perspectives, focusing on exploring aspects of person-centred interactions. A single-instrumental, qualitative case study design was used describing a care-planning conference taking place in the home of an older woman and her daughter. Data collection consisted of observation and digital recording of the care-planning conference and individual interviews with all the participants before and after the conference. Data were analysed in several phases: first, a narrative description followed by a general description and, thereafter, qualitative content analysis. The findings revealed that the care-planning conference conducted had no clear purpose and did not fulfil all parts of the planning process. Three themes emerged related to aspects of person-centred interactions. The theme "expectations meet reality" showed different expectations, and participants could not really connect during the conference. The theme "navigate without a map" revealed health professionals' lack of knowledge about the care-planning process. The theme "lose the forest for the trees" described that the conference was conducted only as part of the health professionals' duties. Management and healthcare professionals cannot automatically assume that they are delivering person-centred care. Healthcare professionals need to be sensitive to the context, use the knowledge and tools available and continuously evaluate and reassess the work carried out.


LEDGF and HDGF2 relieve the nucleosome-induced barrier to transcription in differentiated cells.
FACT (facilitates chromatin transcription) is a protein complex that allows RNA polymerase II (RNAPII) to overcome the nucleosome-induced barrier to transcription. While abundant in undifferentiated cells and many cancers, FACT is not abundant or is absent in most tissues. Therefore, we screened for additional proteins that might replace FACT upon differentiation. We identified two proteins, lens epithelium-derived growth factor (LEDGF) and hepatoma-derived growth factor 2 (HDGF2), each containing two high mobility group A (HMGA)-like AT-hooks and a methyl-lysine reading Pro-Trp-Trp-Pro (PWWP) domain that binds to H3K36me2 and H3K36me3.LEDGF and HDGF2 colocalize with H3K36me2/3 at genomic regions containing active genes. In myoblasts, LEDGF and HDGF2 are enriched on most active genes. Upon differentiation to myotubes, LEDGF levels decrease, while HDGF2 levels are maintained. Moreover, HDGF2 is required for their proper expression. HDGF2 knockout myoblasts exhibit an accumulation of paused RNAPII within the transcribed region of many HDGF2 target genes, indicating a defect in early elongation.


Spectral analysis of all-night human sleep EEG in narcoleptic patients and normal subjects.
To investigate the pathophysiology of narcoleptic patients' sleep in detail, we analysed and compared the whole-night polysomnograms of narcoleptic patients and normal human subjects. Eight drug-naive narcoleptic patients and eight age-matched normal volunteers underwent polysomnography (PSG) on two consecutive nights. In addition to conventional visual scoring of the polysomnograms, rapid eye movement (REM)-density and electroencephalograph (EEG) power spectra analyses were also performed. Sleep onset REM periods and fragmented nocturnal sleep were observed as expected in our narcoleptic patients. In the narcoleptic patients, REM period duration across the night did not show the significant increasing trend that is usually observed in normal subjects. In all narcoleptic patient REM periods, eye movement densities were significantly increased. The power spectra of narcoleptic REM sleep significantly increased between 0.3 and 0.9 Hz and decreased between 1.0 and 5.4 Hz. Further analysis revealed that non-rapid eye movement (NREM) period duration and the declining trend of delta power density in the narcoleptic patients were not significantly different from the normal subjects. Compared with normal subjects, the power spectra of narcoleptic NREM sleep increased in the 1.0-1.4 Hz and 11.0-11.9 Hz frequency bands, and decreased in a 24.0-26.9 Hz frequency band. Thus, increased EEG delta and decreased beta power densities were commonly observed in both the NREM and REM sleep of the narcoleptic patients, although the decrease in beta power during REM sleep was not statistically significant. Our visual analysis revealed fragmented nocturnal sleep and increased phasic REM components in the narcoleptic patients, which suggest the disturbance of sleep maintenance mechanism(s) and excessive effects of the mechanism(s) underlying eye movement activities during REM sleep in narcolepsy. Spectral analysis revealed significant increases in delta components and decreases in beta components, which suggest decreased activity in central arousal mechanisms. These characteristics lead us to hypothesize that two countervailing mechanisms underlie narcoleptic sleep pathology.


Antipsychotic drug effects in a model of schizophrenic attentional disorder: a randomized controlled trial of the effects of haloperidol on latent inhibition in healthy people.
We studied the effects of haloperidol (0.5 mg, intravenously) on latent inhibition in an auditory paradigm and two visual paradigms in healthy subjects. Haloperidol increased latent inhibition in one visual paradigm and tended to increase latent inhibition in an auditory task, compared to saline-injected controls. These results indicate that haloperidol can enhance the selectivity of attention. In contrast, previous studies have reported that acute schizophrenics show reduced latent inhibition.


Metabolic Profiling Reveals a Dependency of Human Metastatic Breast Cancer on Mitochondrial Serine and One-Carbon Unit Metabolism.
Breast cancer is the most common cancer among American women and a major cause of mortality. To identify metabolic pathways as potential targets to treat metastatic breast cancer, we performed metabolomics profiling on the breast cancer cell line MDA-MB-231 and its tissue-tropic metastatic subclones. Here, we report that these subclones with increased metastatic potential display an altered metabolic profile compared with the parental population. In particular, the mitochondrial serine and one-carbon (1C) unit pathway is upregulated in metastatic subclones. Mechanistically, the mitochondrial serine and 1C unit pathway drives the faster proliferation of subclones through enhanced de novo purine biosynthesis. Inhibition of the first rate-limiting enzyme of the mitochondrial serine and 1C unit pathway, serine hydroxymethyltransferase (SHMT2), potently suppresses proliferation of metastatic subclones in culture and impairs growth of lung metastatic subclones at both primary and metastatic sites in mice. Some human breast cancers exhibit a significant association between the expression of genes in the mitochondrial serine and 1C unit pathway with disease outcome and higher expression of SHMT2 in metastatic tumor tissue compared with primary tumors. In addition to breast cancer, a few other cancer types, such as adrenocortical carcinoma and kidney chromophobe cell carcinoma, also display increased SHMT2 expression during disease progression. Together, these results suggest that mitochondrial serine and 1C unit metabolism plays an important role in promoting cancer progression, particularly in late-stage cancer. IMPLICATIONS: This study identifies mitochondrial serine and 1C unit metabolism as an important pathway during the progression of a subset of human breast cancers.


NovaSil clay intervention in Ghanaians at high risk for aflatoxicosis: II. Reduction in biomarkers of aflatoxin exposure in blood and urine.
The efficacy of NovaSil clay (NS) to reduce aflatoxin (AF) biomarkers of exposure was evaluated in 656 blood samples and 624 urine samples collected from study participants during a 3-month phase IIa clinical intervention trial in Ghana. NS was delivered before meals via capsules. Serum AFB (1)-albumin adduct was measured by radioimmunoassay and urinary AFM (1) metabolites were quantified by immunoaffinity-high-performance liquid chromatography (HPLC)-fluorescence methods. Levels of AFB (1) -albumin adduct in serum samples collected at baseline and at 1 month were similar (p = 0.2354 and p = 0.3645, respectively) among the placebo (PL), low dose (LD, 1.5 g NS day (-1)), and high dose (HD, 3.0 g NS day (-1)) groups. However, the levels of AFB (1)-albumin adduct at 3 months were significantly decreased in both the LD group (p < 0.0001) and the HD group (p < 0.0001) compared with levels in the PL group. Levels of AFM(1) in urine samples collected at baseline and at 1 month were not statistically different among the three study groups. However, a significant decrease (up to 58%) in the median level of AFM (1) in samples collected at 3 months was found in the HD group when compared with the median level in the PL group (p < 0.0391). In addition, significant effects were found for dose, time, and dose-time interaction with serum AFB(1)-albumin adduct and dose-time interaction with urinary AFM (1) metabolites. The results suggest that capsules containing NS clay can be used to reduce effectively the bioavailability of dietary AF based on a reduction of AF-specific biomarkers.


An explorative study of metabolic responses to mental stress and yoga practices in yoga practitioners, non-yoga practitioners and individuals with metabolic syndrome.
Stress places a metabolic burden on homeostasis and is linked to heightened sympathetic activity, increased energy expenditure and pathology. The yogic state is a hypometabolic state that corresponds with mind-body coherence and reduced stress. This study aimed to investigate metabolic responses to stress and different yoga practices in regular yoga practitioners (YP), non-yoga practitioners (NY) and metabolic syndrome patients (MS). YP (n = 16), NY (n = 15) and MS (n = 15) subjects underwent an experimental protocol that comprised of different 5-minute interventions including mental arithmetic stress test (MAST), alternate nostril breathing (ANB), Kapabhati breathing (KB) and meditation (Med) interspersed with 5 minutes of quiet resting (neutral condition (NC)). During the intervention periods continuous body weight adjusted oxygen consumption (VO2ml/min/kg) was measured using open circuit indirect calorimetry with a canopy hood. This is the first study to report oxygen consumption (OC) in yoga practitioners during and after MAST and the first to report both within and between different populations. The results were analysed with SPSS 16 using 3X9 mixed factorial ANOVAs. The single between-subject factor was group (YP, NY and MS), the single within-subject factor was made up of the nine intervention phases (NC1, MAST, NC2, ANB, NC3, KB, NC4, Med, NC5). The results demonstrated that the regular YP group had significantly less OC and greater variability in their OC across all phases compared to the MS group (p = .003) and NY group (p = .01). All groups significantly raised their OC during the mental arithmetic stress, however the MS group had a significantly blunted post-stress recovery whereas the YP group rapidly recovered back to baseline levels with post stress recovery being greater than either the NY group or MS group. Yoga practitioners have greater metabolic variability compared to non-yoga practitioners and metabolic syndrome patients with reduced oxygen requirements during resting conditions and more rapid post-stress recovery. OC in metabolic syndrome patients displays significantly blunted post-stress recovery demonstrating reduced metabolic resilience. Our results support the findings of previous randomised trials that suggest regular yoga practice may mitigate against the effects of metabolic syndrome. ACTRN12614001075673; Date of Registration: 07/10/2014.


Characterization of a distinct population of circulating human non-adherent endothelial forming cells and their recruitment via intercellular adhesion molecule-3.
Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133(+) population of non-adherent endothelial forming cells (naEFCs) which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from 'early' endothelial progenitor cells (EPCs). Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin, (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs). Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM)-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis.


Effect of annual influenza vaccination on reducing lung cancer in patients with chronic obstructive pulmonary disease from a population-based cohort study.
Chronic obstructive pulmonary disease (COPD) patients are at a higher risk of development of lung cancer. Frequent exacerbations of COPD trigger the disease course to chronic inflammation which likely plays a role in the pathogenesis of lung cancer. Previous studies showed influenza virus infection is one of important causes for exacerbations of COPD. Therefore, the aim of this study was to know whether influenza vaccination could reduce the incidence of lung cancer in patients with COPD.This cohort study enrolled patients (≥55 years old) with a recorded diagnosis of COPD between January 1, 2000 and December 31, 2012 by using the Taiwan Health Insurance Database. A propensity score was calculated to reduce vaccine therapy selection bias. Cox proportional hazard regressions were used to investigate the association between the influenza vaccination and lung cancer incidence after adjusting for known confounding factors. Besides, we categorized the patients into 4 groups according to vaccination status (unvaccinated, total number of vaccinations: 1, 2-3, ≥4) to evaluate the dose-dependent effect on reducing lung cancer occurrence of lung cancer in COPD patients.Our study comprised of 28,752 eligible individuals from the COPD cohort database. Among them, 51% (14,630) received influenza vaccination; the rest (49%) of the COPD patients did not receive influenza vaccination. We observed that COPD patients receiving influenza vaccination had a lower risk of lung cancer (adjusted HR = 0.40, 95% CI (0.35-0.45), P < .001). We also founded comparable protective effect in both sexes and all age groups (55-64, 65-74, ≥75) regardless of influenza seasonality. Furthermore, dose-dependent protective effect could be seen after stratifying patients according to the total number vaccinations, the adjusted HRs for lung cancer risk were 0.48 (0.40-0.54) and 0.24 (0.20-0.29) for patients who received 2 to 3 and ≥4 vaccinations during the follow-up period.This population-based cohort study demonstrated that annual influenza vaccination administration could reduce incidence of lung cancer in COPD patients.


Equity and child-survival strategies.
Recent advances in child survival have often been at the expense of increasing inequity. Successive interventions are applied to the same population sectors, while the same children in other sectors consistently miss out, leading to a trend towards increasing inequity in child survival. This is particularly important in the case of pneumonia, the leading cause of child death, which is closely linked to poverty and malnutrition, and for which effective community-based case management is more difficult to achieve than for other causes of child death. The key strategies for the prevention of childhood pneumonia are case management, mainly through Integrated Management of Childhood Illness (IMCI), and immunization, particularly the newer vaccines against Haemophilus influenzae type b (Hib) and pneumococcus. There is a tendency to introduce both interventions into communities that already have access to basic health care and preventive services, thereby increasing the relative disadvantage experienced by those children without such access. Both strategies can be implemented in such a way as to decrease rather than increase inequity. It is important to monitor equity when introducing child-survival interventions. Economic poverty, as measured by analyses based on wealth quintiles, is an important determinant of inequity in health outcomes but in some settings other factors may be of greater importance. Geography and ethnicity can both lead to failed access to health care, and therefore inequity in child survival. Poorly functioning health facilities are also of major importance. Countries need to be aware of the main determinants of inequity in their communities so that measures can be taken to ensure that IMCI, new vaccine implementation and other child-survival strategies are introduced in an equitable manner.


Glycemic management in diabetes and the associated cardiovascular risk: are we helping or hurting our patients?
Diabetes, which is a metabolic disorder with multiple comorbidities, increases the risk of cardiovascular disease. Although it was once assumed that controlling plasma glucose levels would reduce diabetes-related morbidity and mortality, recent trials have demonstrated that this is not consistently the case. Data from large, well-designed trials suggest that intensive glycemic therapy may be useful in preventing cardiovascular events if initiated early in the disease course, but may be harmful or not useful if applied to high-risk patients with a longer history of diabetes. Furthermore, the cardiovascular safety of existing individual antihyperglycemic agents remains largely unknown. We review the relationship between glycemic control targets and cardiovascular outcomes, as well as the current understanding of the cardiovascular effects of existing glucose-lowering therapies. This information has affected recommendations for diabetes care in Japan and the United States differently, and supports a more comprehensive and prospective approach to cardiovascular safety assessments of diabetes therapies in the future. Results from ongoing cardiovascular outcomes trials of diabetes medications may help to define optimal glucose-lowering strategies for patients at high risk of cardiovascular complications. Until then, glycemic control targets and the medications used to achieve them should be individualized according to each patient's age, duration of diabetes, risk of hypoglycemia, risk of cardiovascular complications, and life expectancy.


A simple and rapid technique for the detection of rifampin resistance in Mycobacterium leprae.
The rifampin resistance of Mycobacterium leprae is due to missense mutations in the rpoB gene encoding the beta-subunit of the essential enzyme RNA polymerase. A rapid and very simple method has been developed to detect rifampin resistance in small numbers of M. leprae present in biopsies. It involves polymerase chain reaction amplification of a defined region of the rpoB gene followed by single-strand conformational polymorphism analysis (PCR-SSCP). The reliability of the method has been tested on a sample of known drug-resistant and susceptible isolates of M. leprae.


The use of human mesenchymal stem cells encapsulated in RGD modified alginate microspheres in the repair of myocardial infarction in the rat.
The combination of scaffold material and cell transplantation therapy has been extensively investigated in cardiac tissue engineering. However, many polymers are difficult to administer or lack the structural integrity to restore LV function. Additionally, polymers need to be biological friendly, favorably influence the microenvironment and increase stem cell retention and survival. This study determined whether human mesenchymal stem cells (hMSCs) encapsulated in RGD modified alginate microspheres are capable of facilitating myocardial repair. The in vitro study of hMSCs demonstrated that the RGD modified alginate can improve cell attachment, growth and increase angiogenic growth factor expression. Alginate microbeads and hMSCs encapsulated in microbeads successfully maintained LV shape and prevented negative LV remodeling after an MI. Cell survival was significantly increased in the encapsulated hMSC group compared with PBS control or cells alone. Microspheres, hMSCs, and hMSCs in microspheres groups reduced infarct area and enhanced arteriole formation. In summary, surface modification and microencapsulation techniques can be combined with cell transplantation leading to the maintenance of LV geometry, preservation of LV function, increase of angiogenesis and improvement of cell survival.


The Development of Ovine Gastric and Intestinal Organoids for Studying Ruminant Host-Pathogen Interactions.
Gastrointestinal (GI) infections in sheep have significant implications for animal health, welfare and productivity, as well as being a source of zoonotic pathogens. Interactions between pathogens and epithelial cells at the mucosal surface play a key role in determining the outcome of GI infections; however, the inaccessibility of the GI tract in vivo significantly limits the ability to study such interactions in detail. We therefore developed ovine epithelial organoids representing physiologically important gastric and intestinal sites of infection, specifically the abomasum (analogous to the stomach in monogastrics) and ileum. We show that both abomasal and ileal organoids form self-organising three-dimensional structures with a single epithelial layer and a central lumen that are stable in culture over serial passage. We performed RNA-seq analysis on abomasal and ileal tissue from multiple animals and on organoids across multiple passages and show the transcript profile of both abomasal and ileal organoids cultured under identical conditions are reflective of the tissue from which they were derived and that the transcript profile in organoids is stable over at least five serial passages. In addition, we demonstrate that the organoids can be successfully cryopreserved and resuscitated, allowing long-term storage of organoid lines, thereby reducing the number of animals required as a source of tissue. We also report the first published observations of a helminth infecting gastric and intestinal organoids by challenge with the sheep parasitic nematode Teladorsagia circumcincta, demonstrating the utility of these organoids for pathogen co-culture experiments. Finally, the polarity in the abomasal and ileal organoids can be inverted to make the apical surface directly accessible to pathogens or their products, here shown by infection of apical-out organoids with the zoonotic enteric bacterial pathogen Salmonella enterica serovar Typhimurium. In summary, we report a simple and reliable in vitro culture system for generation and maintenance of small ruminant intestinal and gastric organoids. In line with 3Rs principals, use of such organoids will reduce and replace animals in host-pathogen research.


Cost-effectiveness of nucleoside analog therapy for hepatitis B in China: a Markov analysis.
The aim of this study was to investigate the economic consequences of nucleoside analog therapy for hepatitis B treatment in China. A cost-utility analysis of treatments for HBeAg-positive and HBeAg-negative chronic hepatitis B (CHB) was conducted using a Markov model, in which patients' yearly transitions between different health states were tracked. Patients were tracked as they moved between the following health states: CHB, HBeAg seroconversion (HBeAg-positive CHB patients can have this special health state), virologic resistance, virologic response, compensated cirrhosis, decompensated cirrhosis, hepatocellular carcinoma, liver transplantation, and death. The transition parameters were derived either from systematic reviews of the literature or from previous economic studies. Cost and utility data came from studies based on a Chinese CHB cohort. One-way sensitivity analyses as well as second-order Monte Carlo and probabilistic sensitivity analyses were performed. The entecavir strategy yielded the most quality-adjusted life years (QALYs) for both HBeAg-positive and HBeAg-negative patients when compared with the "no treatment," the lamivudine, the adefovir, and the telbivudine strategies. The risks of complications and mortality also decreased. In the economic analysis, the "no treatment" strategy was the least effective, whereas the entecavir strategy was both the least expensive and the most cost-effective option, followed by telbivudine and lamivudine. The probabilistic sensitivity analysis showed that the entecavir strategy would result in improved cost-effectiveness in >90% of cases at a threshold of $20,000 per QALY. In a one-way sensitivity analysis, the most influential parameters impacting the model's robustness were the utilities of the CHB and virologic response health states. In China, when treating both HBeAg-positive and HBeAg-negative CHB populations, entecavir is the most cost-effective option when compared with lamivudine, adefovir, and telbivudine.


Isotropic non-ideal cloaks providing improved invisibility by adaptive segmentation and optimal refractive index profile from ordering isotropic materials.
Mimicking the ideal cloak, which is anisotropic and inhomogeneous, can be achieved by alternating homogeneous isotropic materials, whose permittivity and permeability of each isotropic coating can be determined from effective medium theory. An improved two-fold method is proposed by optimally discretizing the cloak and re-ordering the combination of the effective parameters of each layer to form a smooth step-index profile. The roles of impedance matching and index matching are investigated for cloaking effects. Smoothing the index profile leads to better invisibility than that obtained by smoothing the impedance profile, since the forward scattering can be further diminished. Nonlinear-transformationbased spherical ideal cloaks are studied, and improved design method is explored together with different segmentation schemes. Significant improvement in invisibility is always observed for the optimal segmentation in virtual space with the proposed two-fold design method no matter how nonlinear the coordinate transformation is.


Complexes between nascent polypeptides and their molecular chaperones in the cytosol of mammalian cells.
Folding of newly synthesized proteins in vivo is believed to be facilitated by the cooperative interaction of a defined group of proteins known as molecular chaperones. We investigated the direct interaction of chaperones with nascent polypeptides in the cytosol of mammalian cells by multiple methods. A new approach using a polyclonal antibody to puromycin allowed us to tag and capture a population of truncated nascent polypeptides with no bias as to the identity of the bound chaperones. In addition, antibodies that recognize the cytosolic chaperones hsp70, CCT (TRiC), hsp40, p48 (Hip), and hsp90 were compared on the basis of their ability to coprecipitate nascent polypeptides, both before and after chemical cross-linking. By all three approaches, hsp70 was found to be the predominant chaperone bound to nascent polypeptides. The interaction between hsp70 and nascent polypeptides is apparently dynamic under physiological conditions but can be stabilized by depletion of ATP or by cross-linking. The cytosolic chaperonin CCT was found to bind primarily to full-length, newly synthesized actin, and tubulin. We demonstrate and caution that nascent polypeptides have a propensity for binding many proteins nonspecifically in cell lysates. Although current models of protein folding in vivo have described additional components in contact with nascent polypeptides, our data indicate that the hsp70 and, perhaps, the hsp90 families are the predominant classes of molecular chaperones that interact with the general population of cytosolic nascent polypeptides.


Accuracy of different reconstruction intervals to quantify left ventricular function and mass in cardiac computed tomography examinations.
To compare the accuracy of cardiac dual-source CT (DSCT) reconstructions obtained at 5% and 10% of the cardiac cycle and MRI for quantifying global left ventricular (LV) function and mass in heart transplant recipients. We prospectively included 23 heart transplant recipients (21 male, mean age 60±11.7 years) who underwent cardiac DSCT and MRI examinations. We compared LV parameters on cardiac DSCT reconstructions obtained at 5% (0%-95%) and 10% (0%-90%) intervals of the cardiac cycle and on double-oblique short-axis MR images. We determined ejection fraction (EF), end-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV), and myocardial mass using commercially available semiautomated segmentation analysis software for DSCT datasets and conventional manual contour tracing for MR studies. Using different reconstruction intervals to quantify LV parameters at DSCT resulted in non-significant differences (P>.05). Compared to MRI, DSCT slightly overestimated LV-EDV, ESV, and mass when both 5% (11.5±25.1ml, 6.8±10.9ml, and 28.3±21.6g, respectively) and 10% (mean difference 15.3±26.3ml, 7.4±11.5ml, and 29.3±18.7g, respectively) reconstruction intervals were used. DSCT and MRI estimates of EF and SV were not significantly different. In heart transplant recipients, DSCT allows reliable quantification of LV function and mass compared with MRI, even using 10% interval reconstructions.


Computer generated holograms for carbon nanotube arrays.
Multiwalled carbon nanotubes are highly diffractive structures in the optical regime. Their metallic character and large scattering cross-section allow their usage as diffractive elements in Fraunhofer holograms. This work elaborates some important features of the far field diffraction patterns produced from periodic arrays of nanotubes. A theoretical approach for the interaction of arrays of nanotubes with light is presented and a computer generated hologram is calculated by means of periodical patterns. Based on the results, fabrication of carbon nanotube arrays (in holographic patterns) was performed. Experimentally measured diffraction patterns were in good agreement with the calculations.


Ecosystem properties and forest decline in contrasting long-term chronosequences.
During succession, ecosystem development occurs; but in the long-term absence of catastrophic disturbance, a decline phase eventually follows. We studied six long-term chronosequences, in Australia, Sweden, Alaska, Hawaii, and New Zealand; for each, the decline phase was associated with a reduction in tree basal area and an increase in the substrate nitrogen-to-phosphorus ratio, indicating increasing phosphorus limitation over time. These changes were often associated with reductions in litter decomposition rates, phosphorus release from litter, and biomass and activity of decomposer microbes. Our findings suggest that the maximal biomass phase reached during succession cannot be maintained in the long-term absence of major disturbance, and that similar patterns of decline occur in forested ecosystems spanning the tropical, temperate, and boreal zones.


Impact of sonochemical synthesis condition on the structural and physical properties of MnFe2O4 spinel ferrite nanoparticles.
Herein, we report sonochemical synthesis of MnFe2O4 spinel ferrite nanoparticles using UZ SONOPULS HD 2070 Ultrasonic homogenizer (frequency: 20 kHz and power: 70 W). The sonication time and percentage amplitude of ultrasonic power input cause appreciable changes in the structural, cation distribution and physical properties of MnFe2O4 nanoparticles. The average crystallite size of synthesized MnFe2O4 nanoparticles was increased with increase of sonication time and percentage amplitude of ultrasonic power input. The occupational formula by X-ray photoelectron spectroscopy for prepared spinel ferrite nanoparticles was (Mn0.29Fe0.42)[Mn0.71Fe1.58]O4 and (Mn0.28Fe0.54) [Mn0.72Fe1.46]O4 at sonication time 20 min and 80 min, respectively. The value of the saturation magnetization was increased from 1.9 emu/g to 52.5 emu/g with increase of sonication time 20 min to 80 min at constant 50% amplitude of ultrasonic power input, whereas, it was increased from 30.2 emu/g to 59.4 emu/g with increase of the percentage amplitude of ultrasonic power input at constant sonication time 60 min. The highest value of dielectric constant (ε') was 499 at 1 kHz for nanoparticles at sonication time 20 min, whereas, ac conductivity was 368 × 10-9 S/cm at 1 kHz for spinel ferrite nanoparticles at sonication time 20 min. The demonstrated controllable physical characteristics over sonication time and percentage amplitude of ultrasonic power input are a key step to design spinel ferrite material of desired properties for specific application. The investigation of microwave operating frequency suggest that these prepared spinel ferrite nanoparticles are potential candidate for fabrication of devices at high frequency applications.


Automated quantification and integrative analysis of 2D and 3D mitochondrial shape and network properties.
Mitochondrial morphology and function are coupled in healthy cells, during pathological conditions and (adaptation to) endogenous and exogenous stress. In this sense mitochondrial shape can range from small globular compartments to complex filamentous networks, even within the same cell. Understanding how mitochondrial morphological changes (i.e. "mitochondrial dynamics") are linked to cellular (patho) physiology is currently the subject of intense study and requires detailed quantitative information. During the last decade, various computational approaches have been developed for automated 2-dimensional (2D) analysis of mitochondrial morphology and number in microscopy images. Although these strategies are well suited for analysis of adhering cells with a flat morphology they are not applicable for thicker cells, which require a three-dimensional (3D) image acquisition and analysis procedure. Here we developed and validated an automated image analysis algorithm allowing simultaneous 3D quantification of mitochondrial morphology and network properties in human endothelial cells (HUVECs). Cells expressing a mitochondria-targeted green fluorescence protein (mitoGFP) were visualized by 3D confocal microscopy and mitochondrial morphology was quantified using both the established 2D method and the new 3D strategy. We demonstrate that both analyses can be used to characterize and discriminate between various mitochondrial morphologies and network properties. However, the results from 2D and 3D analysis were not equivalent when filamentous mitochondria in normal HUVECs were compared with circular/spherical mitochondria in metabolically stressed HUVECs treated with rotenone (ROT). 2D quantification suggested that metabolic stress induced mitochondrial fragmentation and loss of biomass. In contrast, 3D analysis revealed that the mitochondrial network structure was dissolved without affecting the amount and size of the organelles. Thus, our results demonstrate that 3D imaging and quantification are crucial for proper understanding of mitochondrial shape and topology in non-flat cells. In summary, we here present an integrative method for unbiased 3D quantification of mitochondrial shape and network properties in mammalian cells.


Mass spectrometry reveals the missing links in the assembly pathway of the bacterial 20 S proteasome.
The 20 S proteasome is an essential proteolytic particle, responsible for degrading short-lived and abnormal intracellular proteins. The 700-kDa assembly is comprised of 14 alpha-type and 14 beta-type subunits, which form a cylindrical architecture composed of four stacked heptameric rings (alpha7beta7beta7alpha7). The formation of the 20 S proteasome is a complex process that involves a cascade of folding, assembly, and processing events. To date, the understanding of the assembly pathway is incomplete due to the experimental challenges of capturing short-lived intermediates. In this study, we have applied a real-time mass spectrometry approach to capture transient species along the assembly pathway of the 20 S proteasome from Rhodococcus erythropolis. In the course of assembly, we observed formation of an early alpha/beta-heterodimer as well as an unprocessed half-proteasome particle. Formation of mature holoproteasomes occurred in concert with the disappearance of half-proteasomes. We also analyzed the beta-subunits before and during assembly and reveal that those with longer propeptides are incorporated into half- and full proteasomes more rapidly than those that are heavily truncated. To characterize the preholoproteasome, formed by docking of two unprocessed half-proteasomes and not observed during assembly of wild type subunits, we trapped this intermediate using a beta-subunit mutational variant. In summary, this study provides evidence for transient intermediates in the assembly pathway and reveals detailed insight into the cleavage sites of the propeptide.


Effects of acute tachycardia on left ventricular adenine nucleotide levels and subsequent tolerance to ischemia.
Electrophysiologically guided surgical procedures for the ablation of supraventricular and ventricular dysrhythmias often require prolonged periods of tachycardia to complete intraoperative mapping studies. It is unknown whether tachycardia depletes the myocardium of high-energy compounds or alters subsequent tolerance to ischemia. In the present study, 12 anesthetized dogs were paced from the right ventricle at a cycle length of 250 msec for 60 minutes. In seven animals, drill biopsy specimens were taken from the left ventricular free wall for analysis of adenine nucleotide levels and their breakdown products before and after pacing and after 20 minutes of recovery from pacing. In the remaining five animals, the heart was made totally ischemic immediately after tachycardia and the time to the onset of ischemic contracture was determined and compared to that of five nonpaced control dogs. Acute tachycardia resulted in no significant reduction in adenine nucleotide levels compared to control values. Furthermore, in hearts rendered totally ischemic after tachycardia, the mean time to ischemic contracture was 65.6 +/- 1.3 minutes versus 63.6 +/- 2 minutes in nonpaced control animals (no significant difference). These data show that pacing-induced tachycardia in the normal heart does not decrease adenine nucleotide levels or affect subsequent tolerance to ischemia. These results may be clinically relevant to patients without coronary artery disease who undergo operative procedures necessitating prolonged periods of tachycardia for intraoperative mapping to identify the site of arrhythmogenesis.


Prebiotic chemicals-amino acid and phosphorus-in the coma of comet 67P/Churyumov-Gerasimenko.
The importance of comets for the origin of life on Earth has been advocated for many decades. Amino acids are key ingredients in chemistry, leading to life as we know it. Many primitive meteorites contain amino acids, and it is generally believed that these are formed by aqueous alterations. In the collector aerogel and foil samples of the Stardust mission after the flyby at comet Wild 2, the simplest form of amino acids, glycine, has been found together with precursor molecules methylamine and ethylamine. Because of contamination issues of the samples, a cometary origin was deduced from the (13)C isotopic signature. We report the presence of volatile glycine accompanied by methylamine and ethylamine in the coma of 67P/Churyumov-Gerasimenko measured by the ROSINA (Rosetta Orbiter Spectrometer for Ion and Neutral Analysis) mass spectrometer, confirming the Stardust results. Together with the detection of phosphorus and a multitude of organic molecules, this result demonstrates that comets could have played a crucial role in the emergence of life on Earth.


Effects of nonequilibrium growth, nonstoichiometry, and film orientation on the metal-to-insulator transition in NdNiO₃ thin films.
Next-generation devices will rely on exotic functional properties not found in traditional systems. One class of materials of particular interest for applications are those possessing metal-to-insulator transitions (MITs). In this work, we probe the relationship between variations in the growth process, subsequent variations in cation stoichiometry, and the MIT in NdNiO3 thin films. Slight variations in the growth conditions, in particular the laser fluence, during pulsed-laser deposition growth of NdNiO3 produces films that are both single-phase and coherently strained to a range of substrates despite possessing as much as 15% Nd-excess. Subsequent study of the temperature-dependence of the electronic transport reveals dramatic changes in both the onset and magnitude of the resistivity change at the MIT with increasing cation nonstoichiometry giving rise to a decrease (and ultimately a suppression) of the transition and the magnitude of the resistivity change. From there, the electronic transport of nearly ideal NdNiO3 thin films are studied as a function of epitaxial strain, thickness, and orientation. Overall, transitioning from tensile to compressive strain results in a systematic reduction of the onset and magnitude of the resistivity change across the MIT, thinner films are found to possess sharper MITs with larger changes in the resistivity at the transition, and (001)-oriented films exhibit sharper and larger MITs as compared to (110)- and (111)-oriented films as a result of highly anisotropic in-plane transport in the latter.


Acoustic Radiation Force Impulse (ARFI) elastography quantification of muscle stiffness over a course of gradual isometric contractions: a preliminary study.
To evaluate the feasibility of quantitative analysis of muscle stiffness by Acoustic Radiation Forced Impulse (ARFI) elastography over a course of graduate isometric voluntary contractions. The stiffness of the bilateral biceps muscle of 13 healthy volunteers was measured in real time by ARFI elastography, while the forearm was in neutral-extended position, 90 degree self-flexed positions and 90 degree self-flexed position, with altered weights ranging from 1 to 8 kg placed on flattened palmar surfaces consecutively. The determined increases in biceps muscle stiffness were measured for both arms and correlated with the loadings weights adopted at progressive trial stages. The mean shear wave velocity (SWV) values of biceps muscles in a neutral position, in 90 degree flex position and 90 degree flex position with 1 to 8 kg weights on palmar surfaces were 2.162+/-0.302 m/sec, 3.382+/-0.581 m/sec, and 3.897+/-0.585 to 5.562+/-0.587 m/sec, respectively. Significant correlations between the muscle SWV values and related palmar weights and between the SWV values of right and left sides at different trial stages were identified (r=0.951 and r=0.954, respectively). A mutual propagation path of deep regions to entire areas was described to account for the distribution of increase in stiffness with increases in palmar weights. The confidence of method regarding inter-observer difference was confirmed by the correlation analyses of the results (r=0.998). ARFI elastography is a feasible imaging modality for quantifying the stiffness of isometrically voluntarily contracting muscles.


Continuous table acquisition MRI for radiotherapy treatment planning: distortion assessment with a new extended 3D volumetric phantom.
Accurate geometry is required for radiotherapy treatment planning (RTP). When considering the use of magnetic resonance imaging (MRI) for RTP, geometric distortions observed in the acquired images should be considered. While scanner technology and vendor supplied correction algorithms provide some correction, large distortions are still present in images, even when considering considerably smaller scan lengths than those typically acquired with CT in conventional RTP. This study investigates MRI acquisition with a moving table compared with static scans for potential geometric benefits for RTP. A full field of view (FOV) phantom (diameter 500 mm; length 513 mm) was developed for measuring geometric distortions in MR images over volumes pertinent to RTP. The phantom consisted of layers of refined plastic within which vitamin E capsules were inserted. The phantom was scanned on CT to provide the geometric gold standard and on MRI, with differences in capsule location determining the distortion. MRI images were acquired with two techniques. For the first method, standard static table acquisitions were considered. Both 2D and 3D acquisition techniques were investigated. With the second technique, images were acquired with a moving table. The same sequence was acquired with a static table and then with table speeds of 1.1 mm/s and 2 mm/s. All of the MR images acquired were registered to the CT dataset using a deformable B-spline registration with the resulting deformation fields providing the distortion information for each acquisition. MR images acquired with the moving table enabled imaging of the whole phantom length while images acquired with a static table were only able to image 50%-70% of the phantom length of 513 mm. Maximum distortion values were reduced across a larger volume when imaging with a moving table. Increased table speed resulted in a larger contribution of distortion from gradient nonlinearities in the through-plane direction and an increased blurring of capsule images, resulting in an apparent capsule volume increase by up to 170% in extreme axial FOV regions. Blurring increased with table speed and in the central regions of the phantom, geometric distortion was less for static table acquisitions compared to a table speed of 2 mm/s over the same volume. Overall, the best geometric accuracy was achieved with a table speed of 1.1 mm/s. The phantom designed enables full FOV imaging for distortion assessment for the purposes of RTP. MRI acquisition with a moving table extends the imaging volume in the z direction with reduced distortions which could be useful particularly if considering MR-only planning. If utilizing MR images to provide additional soft tissue information to the planning CT, standard acquisition sequences over a smaller volume would avoid introducing additional blurring or distortions from the through-plane table movement.


Pseudomonas aeruginosa lasI/rhlI quorum sensing genes promote phagocytosis and aquaporin 9 redistribution to the leading and trailing regions in macrophages.
Pseudomonas aeruginosa controls production of its multiple virulence factors and biofilm development via the quorum sensing (QS) system. QS signals also interact with and affect the behavior of eukaryotic cells. Host water homeostasis and aquaporins (AQP) are essential during pathological conditions since they interfere with the cell cytoskeleton and signaling, and hereby affect cell morphology and functions. We investigated the contribution of P. aeruginosa QS genes lasI/rhlI to phagocytosis, cell morphology, AQP9 expression, and distribution in human macrophages, using immunoblotting, confocal, and nanoscale imaging. Wild type P. aeruginosa with a functional QS system was a more attractive prey for macrophages than the lasI/rhlI mutant lacking the production of QS molecules, 3O-C12-HSL, and C4-HSL, and associated virulence factors. The P. aeruginosa infections resulted in elevated AQP9 expression and relocalization to the leading and trailing regions in macrophages, increased cell area and length; bacteria with a functional QS system lasI/rhlI achieved stronger responses. We present evidence for a new role of water fluxes via AQP9 during bacteria-macrophage interaction and for the QS system as an important stimulus in this process. These novel events in the interplay between P. aeruginosa and macrophages may influence on the outcome of infection, inflammation, and development of disease.


A pill for the partner via the chlamydia patient? Results from a mixed method study among sexual health care providers in the Netherlands.
Chlamydia prevalence in the Netherlands remains high despite targeted efforts. Effective Partner Notification (PN) and Partner Treatment (PT) can interrupt transmission and prevent re-infections. Patient Initiated Partner Treatment (PIPT) may strengthen chlamydia control. This study explores the current practice of PN and PT, and benefits of, and barriers and facilitators for PIPT among professionals in sexual health care in the Netherlands. A qualitative study was performed among GPs, GP-assistants (GPAs), physicians and nurses working at Sexual Health Clinics (SHC) and key-informants on ethnical diversity using topic lists in focus groups (N = 40) and semi-structured questionnaires in individual interviews (N = 9). Topics included current practices regarding PN and PT, attitude regarding PIPT, and perceived barriers and facilitators for PIPT. Interviews were taped, transcribed verbatim, and coded using ATLAS.ti. A quantitative online questionnaire on the same topics was sent to all physicians and nurses employed at Dutch SHC (complete response rate 26% (84/321)). The qualitative study showed that all professionals support the need for more attention to PN, and that they saw advantages in PIPT. Mentioned barriers included unwilling PN-behaviour, Dutch legislation, several medical considerations and inadequate skills of GPs. Also, concerns about limited knowledge of cultural sensitivity around PN and PT were raised. Mentioned facilitators of PIPT were reliable home based test-kits, phone-contact between professionals and notified partners, more consultation time for GPs or GPAs and additional training. The online questionnaire showed that SHC employees agreed that partners should be treated as soon as possible, but also that they were reluctant towards PIPT without counselling and testing. Professionals saw advantages in PIPT, but they also identified barriers hampering the potential introduction of PIPT. Improving PN and counselling skills with specific focus on cultural sensitivity is needed. PIPT could be considered for specific partners. PIPT in combination with home based testing and using e-healthcare should be further explored and developed.


Genomics and Targeted Therapies in Gastroesophageal Adenocarcinoma.
Gastroesophageal adenocarcinomas (GEA) are devastating diseases with stark global presence. Over the past 10 years, there have been minimal improvements in treatment approach despite numerous clinical trials. Here, we review recent progress toward understanding the molecular features of these cancers and the diagnostic and therapeutic challenges posed by their intrinsic genomic instability and heterogeneity. We highlight the potential of genomic heterogeneity to influence clinical trial outcomes for targeted therapies and emphasize the need for comprehensive molecular profiling to guide treatment selection and adapt treatment to resistance and genomic evolution. Revising our clinical approach to GEA by leveraging genomic advances will be integral to the success of current and future treatments, especially as novel targets become therapeutically tractable. SIGNIFICANCE: GEAs are deadly cancers with few treatment options. Characterization of the genomic landscape of these cancers has revealed considerable genetic diversity and spatial heterogeneity. Understanding these fundamental properties of GEA will be critical for overcoming barriers to the development of novel, more effective therapeutic strategies.


Induction of GDNF mRNA expression by melatonin in rat C6 glioma cells.
In order to determine the physiological effect of melatonin on glial cell line-derived neurotrophic factor (GDNF), which is reportedly up-regulated by high doses of this hormone, concentration-dependent studies were carried out in cultured cells. RT-PCR studies indicated that, in addition to GDNF, rat C6 glioma cells express both of the G protein-coupled melatonin receptor subtypes, MT1 and MT2. When C6 cells were treated with physiological (0.05-1 nM) or higher (10 and 100 nM) concentrations of melatonin for 24 h, a significant induction of relative GDNF mRNA levels (n = 4) was detected by semi-quantitative RT-PCR. These findings suggest that induction of GDNF is involved in physiological neuroprotection by melatonin. Given the potency of GDNF in maintaining nigrostriatal dopaminergic integrity, understanding the mechanisms of its induction by melatonin could provide novel therapies for Parkinson's disease.


Improving genome assemblies by sequencing PCR products with PacBio.
Advances in sequencing technologies have dramatically reduced costs in producing high-quality draft genomes. However, there are still many contigs and possible misassembled regions in those draft genomes. Improving the quality of these genomes requires an efficient and economical means to close gaps and resequence some regions. Sequencing pooled gap region PCR products with Pacific Biosciences (PacBio) provides a significantly less expensive means for this need. We have developed a genome improvement pipeline with this strategy after decreasing a loading bias against larger PCR products in the PacBio process. Compared with Sanger technology, this approach is not only cost-effective but also can close gaps greater than 2.5 kb in a single round of reactions, and sequence through high GC regions as well as difficult secondary structures such as small hairpin loops.


Exclusion of agricultural lands in spatial conservation prioritization strategies: consequences for biodiversity and ecosystem service representation.
Agroecosystems have traditionally been considered incompatible with biological conservation goals, and often been excluded from spatial conservation prioritization strategies. The consequences for the representativeness of identified priority areas have been little explored. Here, we evaluate these for biodiversity and carbon storage representation when agricultural land areas are excluded from a spatial prioritization strategy for South America. Comparing different prioritization approaches, we also assess how the spatial overlap of priority areas changes. The exclusion of agricultural lands was detrimental to biodiversity representation, indicating that priority areas for agricultural production overlap with areas of relatively high occurrence of species. By contrast, exclusion of agricultural lands benefits representation of carbon storage within priority areas, as lands of high value for agriculture and carbon storage overlap little. When agricultural lands were included and equally weighted with biodiversity and carbon storage, a balanced representation resulted. Our findings suggest that with appropriate management, South American agroecosystems can significantly contribute to biodiversity conservation.


Rv1460, a SufR homologue, is a repressor of the suf operon in Mycobacterium tuberculosis.
Iron-sulphur (Fe-S) clusters are ubiquitous co-factors which require multi-protein systems for their synthesis. In Mycobacterium tuberculosis, the Rv1460-Rv1461-Rv1462-Rv1463-csd-Rv1465-Rv1466 operon (suf operon) encodes the primary Fe-S cluster biogenesis system. The first gene in this operon, Rv1460, shares homology with the cyanobacterial SufR, which functions as a transcriptional repressor of the sufBCDS operon. Rv1460's function in M. tuberculosis has however not been determined. In this study, we demonstrate that M. tuberculosis mutants lacking a functional Rv1460 protein are impaired for growth under standard culture conditions. Elevated expression of Rv1460 and Rv1461 was observed in the mutant, implicating Rv1460 in the regulation of the suf operon. Binding of an Fe-S cluster to purified recombinant Rv1460 was confirmed by UV-visible spectroscopy and circular dichroism. Furthermore, three conserved cysteine residues, C203, C216 and C244, proposed to provide ligands for the coordination of an Fe-S cluster, were shown to be required for the function of Rv1460 in M. tuberculosis. Rv1460 therefore seems to be functionally analogous to cyanobacterial SufR.


Immunochemical and biological analysis of allergenicity with excretory-secretory products of anisakis simplex third stage larva.
Anisakis simplex third stage larvae (L3) are parasites that frequently give rise to allergic responses. The larvae molt into fourth stage larvae (L4), and at each stage they produce L3-excretory-secretory products (L3-ESP) and L4-ESP, respectively, which are different in their main protein constituents. Although the allergenicity of L4-ESP has been investigated by several research groups, research on the allergenicity of L3-ESP has not been carried out by any researcher. In this investigation, the allergenicity and antigenicity of L3-ESP were investigated in comparison with L4-ESP, using rat sera. Rat sera were produced by L3 oral infection two times with a 9-week interval. Larvae ESP prepared by culture were concentrated and fractioned using lyophilizer and a centrifugal filter device, respectively. Immunochemical analysis was performed using both indirect ELISA and immunoblot. Biological allergenicity was analyzed by RBL-2H3 exocytosis. With the indirect ELISA, the optical density (OD) value of the nonfractioned (NF)-L3ESP was only one third of that of the NF-L4ESP in both specific IgM and IgG. On measuring specific IgE, the OD of NF-L3ESP was less than one tenth of that of NF-L4ESP. In addition, neither antigen nor allergen was shown in NF-L3ESP, but it was shown in NF-L4ESP with immunoblot. However, the biological allergenicity of NF-L3ESP was comparable to that of NF-L4ESP. To demonstrate the presence of any allergen, L3-ESP was fractioned and found to carry twelve visualized allergen bands from 10 to 186 kDa by immunoblot. These results indicate that L3-ESP may include the important allergens necessary to induce the allergy by L3 oral infection, as compared to L4-ESP.


Age-Dependent TDP-43-Mediated Motor Neuron Degeneration Requires GSK3, hat-trick, and xmas-2.
The RNA-processing protein TDP-43 is central to the pathogenesis of amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron (MN) disease. TDP-43 is conserved in Drosophila, where it has been the topic of considerable study, but how TDP-43 mutations lead to age-dependent neurodegeneration is unclear and most approaches have not directly examined changes in MN morphology with age. We used a mosaic approach to study age-dependent MN loss in the adult fly leg where it is possible to resolve single motor axons, NMJs and active zones, and perform rapid forward genetic screens. We show that expression of TDP-43(Q331K) caused dying-back of NMJs and axons, which could not be suppressed by mutations that block Wallerian degeneration. We report the identification of three genes that suppress TDP-43 toxicity, including shaggy/GSK3, a known modifier of neurodegeneration. The two additional novel suppressors, hat-trick and xmas-2, function in chromatin modeling and RNA export, two processes recently implicated in human ALS. Loss of shaggy/GSK3, hat-trick, or xmas-2 does not suppress Wallerian degeneration, arguing TDP-43(Q331K)-induced and Wallerian degeneration are genetically distinct processes. In addition to delineating genetic factors that modify TDP-43 toxicity, these results establish the Drosophila adult leg as a valuable new tool for the in vivo study of adult MN phenotypes.


Cardiac-specific abrogation of NF- kappa B activation in mice by transdominant expression of a mutant I kappa B alpha.
Nuclear factor-kappaB (NF-kappa B) is a pleiotropic oxidant-sensitive transcription factor that is present in the cytosol in an inactive form complexed to an inhibitory kappaB (I kappa B) monomer. Various stimuli, including ischemia, hypoxia, free radicals, cytokines, and lipopolysaccharide (LPS), activate NF-kappa B by inducing phosphorylation of I kappa B. Phosphorylation of serine residues at positions 32 and 36 is critical for ubiquitination and degradation of I kappa B alpha with consequent migration of NF-kappa B to the nucleus. Although NF-kappa B is thought to contribute to numerous pathophysiologic processes, definitive assessment of its role has been hindered by the inability to achieve specific inhibition in vivo. Pharmacologic inhibitors of NF-kappa B are available, but their utility for in vivo studies is limited by their relative lack of specificity. Targeted ablation of genes encoding NF-kappa B subunits has not been productive in this regard because of fetal lethality in the case of p65 and functional redundancy in the Rel family of proteins. To overcome these limitations, we have created a viable transgenic mouse that expresses a phosphorylation-resistant mutant of I kappa B alpha (I kappa B alpha(S32A,S36A)) under the direction of a cardiac-specific promoter. Several transgenic lines were obtained with copy numbers ranging from one to seven. The mice exhibit normal cardiac morphology and histology. Total myocardial I kappa B alpha protein level is elevated 3.5- to 6.5-fold with a concomitant 50-60% decrease in the level of I kappa B beta. Importantly, expression of I kappa B(S32A,S36A) results in complete abrogation of myocardial NF-kappa B activation in response to tumor necrosis factor- alpha (TNF-alpha) and LPS stimulation. Thus, novel transgenic mice have been created that make it possible to achieve cardiac-specific and selective inhibition of NF-kappa B in vivo. These transgenic mice should be useful in studies of various cardiac pathophysiological phenomena that involve NF-kappa B activation, including ischemic preconditioning, heart failure, septic shock, acute coronary syndromes, cardiac allograft rejection, and apoptosis.


An Education Intervention to Enhance Staff Self-Efficacy to Provide Dementia Care in an Acute Care Hospital in Canada: A Nonrandomized Controlled Study.
Education is needed for enhanced capacity of acute hospitals to provide dementia care. A nonrandomized controlled, repeated-measures design was used to evaluate a dementia education program delivered to an intervention group (IG, n = 468), compared to a wait-listed group (n = 277), representing separate sites of a multisite hospital. Participants completed self-efficacy for dementia and satisfaction measures and provided written descriptions of dementia care collected at baseline, postintervention (IG only), and at 8-week follow-up. Oral narratives were gathered from IG participants 8 weeks postintervention. The IG demonstrated significant improvement in self-efficacy scores from baseline to immediately postintervention (P < .001), sustained at 8 weeks. There were no changes from baseline to 8 weeks postintervention evident in the wait-listed group (P = .21). Intervention group participants described positive impacts including implementation of person-centered care approaches. Implementation of dementia care education programs throughout hospital settings is promising for the enhancement of dementia care.


Short-term effects of ozone air pollution on ischaemic stroke occurrence: a case-crossover analysis from a 10-year population-based study in Dijon, France.
To evaluate the association between air pollutants and the occurrence of acute stroke from 10-year population-based study. The daily stroke count was obtained from Dijon Stroke Register between March 1994 and December 2004. The register recorded all first-ever strokes among residents of Dijon (150,000 inhabitants) in France, using standard diagnostic criteria. Pollutant concentrations (SO2, CO, NO2, O3 and PM10) were measured hourly. A bi-directional case-crossover design was used to examine the association between air pollutant and stroke onset. The conditional logistic regression model included the meteorological parameters (temperature, relative humidity), influenza epidemics and holidays. The authors collected 493 large artery infarcts, 397 small artery infarcts, 530 cardio-embolic infarcts, 67 undeterminate infarcts, 371 transient ischaemic attacks and 220 haemorrhagic strokes. For single-pollutant model and for a 10 mg/m(3) increase of O3 exposure, a positive association was observed only in men, over 40 years of age, between ischaemic stroke occurrence and O3 levels with 1-day lag, (OR 1.133, 95% CI 1.052 to 1.220) and 0-day lag (OR 1.058, 95% CI 0.987 to 1.134). No significant associations were found for haemorrhagic stroke. In two-pollutant models, the effects of O3 remained significant after each of the other pollutants were included in the model, in particular with PM10. A significant association was observed for ischaemic strokes of large arteries (p = 0.02) and for transient ischaemic attacks (p = 0.01). Moreover, the authors found an exposure-response relations between O3 exposure and ischaemic stroke (test for trend, p = 0.01). An increase in association in men with several cardiovascular risk factors (smoker, dyslipidemia and hypertension) was also observed. These observational data argue for an association between ischaemic stroke occurrence and O3 pollution levels; these results still need to be confirmed by other studies.


Fluorescence Imaging of Bacterial Killing by Antimicrobial Peptide Dendrimer G3KL.
We recently discovered that peptide dendrimers such as G3KL ((KL)8(KKL)4(KKL)2KKL, K = branching l-lysine) exert strong activity against Gram-negative bacteria including Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli. Herein, we report a detailed mechanistic study using fluorescence labeled analogs bearing fluorescein (G3KL-Fluo) or dansyl (G3KL-Dansyl), which show a similar bioactivity profile as G3KL. Imaging bacterial killing by super-resolution stimulated emission depletion (STED) microscopy, time-lapse imaging, and transmission electron microscopy (TEM) reveals that the dendrimer localizes at the bacterial membrane, induces membrane depolarization and permeabilization, and destroys the outer leaflet and the inner membrane. G3KL accumulates in bacteria against which it is active; however, it only weakly penetrates into eukaryotic cells without inducing significant toxicity. G3KL furthermore binds to lipopolysaccharide (LPS) and inhibits the LPS induced release of TNF-α by macrophages, similarly to polymyxin B. Taken together, these experiments show that G3KL behaves as a potent membrane disruptive antimicrobial peptide.


Build loyalty in business markets.
Companies often apply consumer marketing solutions in business markets without realizing that such strategies only hamper the acquisition and retention of profitable customers. Unlike consumers, business customers inevitably need customized products, quantities, or prices. A company in a business market must therefore manage customers individually, showing how its products or services can help solve each buyer's problems. And it must learn to reap the enormous benefits of loyalty by developing individual relationships with customers. To achieve these ends, the firm's marketers must become aware of the different types of benefits the company offers and convey their value to the appropriate executives in the customer company. It's especially important to inform customers about what the author calls nontangible nonfinancial benefits-above-and-beyond efforts, such as delivering supplies on holidays to keep customers' production lines going. The author has developed a simple set of devices-the benefit stack and the decision-maker stack-to help marketers communicate their firm's myriad benefits. The vendor lists the benefits it offers, then lists the customer's decision makers, specifying their concerns, motivations, and power bases. By linking the two stacks, the vendor can systematically communicate how it will meet each decision-maker's needs. The author has also developed a tool called a loyalty ladder, which helps a company determine how much time and money to spend on relationships with various customers. As customers become increasingly loyal, they display behaviors in a predictable sequence, from growing the relationship and providing word-of-mouth endorsements to investing in the vendor company. The author has found that customers follow the same sequence of loyalty behaviors in all business markets.


Children and the risk of fractures caused by oral corticosteroids.
Oral corticosteroids are known to increase the risk of fracture in adults, but their effects in children remain uncertain. The medical records of general practitioners in the United Kingdom (from the General Practice Research Database) were used to estimate the incidence rates of fracture of children ages 4-17 years taking oral corticosteroids (n = 37,562) and of control children taking nonsystemic corticosteroids (n = 345,748). Each child with a fracture (n = 22,846) was subsequently matched by age, sex, practice, and calendar time to one child without a fracture. The average duration of treatment was 6.4 days (median, 5 days). The risk of fracture was increased in children with a history of frequent use of oral corticosteroids; children who received four or more courses of oral corticosteroids had an adjusted odds ratio (OR) for fracture of 1.32 (95% CI, 1.03-1.69). Of the various fracture types, the risk of humerus fracture was doubled in children who received four or more courses of oral corticosteroids (adjusted OR, 2.17 [1.01-4.67]). Fracture risk was also increased among children using 30 mg prednisolone or more each day (adjusted OR for fracture, 1.24 [1.00-1.52]) and among those receiving four or more courses of oral corticosteroids (OR, 1.32 [1.03-1.69]). Children who stopped taking oral corticosteroids had a comparable risk of fracture to those in the control group. Our findings suggest that children who require more than four courses of oral corticosteroid as treatment for underlying disease are at increased risk of fracture. It is not entirely clear whether this relates directly to oral corticosteroid use or the underlying disease and its severity. Irrespective of these issues, this group of children is at increased risk of fracture.


Inhibition of T lymphocyte activation by cAMP is associated with down-regulation of two parallel mitogen-activated protein kinase pathways, the extracellular signal-related kinase and c-Jun N-terminal kinase.
The induction of T cell proliferation requires signals from the TCR and a co-receptor molecule, such as CD28, that activate parallel and partially cross-reactive signaling pathways. These pathways are disrupted by agonists that utilize adenylate cyclase and cAMP-dependent protein kinase A (PKA). We found that the adenylate cyclase activator, forskolin, inhibits anti-CD3-induced shift in Lck electrophoretic mobility, suggesting an intervention at the TCR-coupled phosphoinositide turnover that precedes the activation of PKC. The shift of Lck following direct PKC activation by 12-O-tetradecanoyl phorbol 13-acetate, which bypasses early receptor-triggered biochemical events, is insensitive to forskolin. Nevertheless, forskolin also inhibits PKC downstream events, such as c-jun expression, which is critical for the activation process of T cells. To further analyze potential cross points between positively and negatively regulating signaling pathways in T cells, we tested the effects of activators of the adenylate cyclase or PKA on two parallel mitogen-activated protein kinase signaling pathways mediated by extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase. Using a PKC-specific inhibitor, GF109203X, or PKC-depleted T cells, we found that a large part of the anti-CD3-induced ERK activation is PKC dependent. Both PKC-dependent and -independent activation of ERK were sensitive to inhibition by forskolin or a cell-permeable cAMP analogue, dbcAMP. Furthermore, the effect of 12-O-tetradecanoyl phorbol 13-acetate and ionomycin, which synergized to fully activate c-Jun N-terminal kinase, was also sensitive to inhibition by forskolin. Our results suggest that PKA inhibits T cell activation by interfering with multiple events along the two signaling pathways operating downstream of the TCR and the CD28 co-receptor molecules.


Geoengineering potential of artificially enhanced silicate weathering of olivine.
Geoengineering is a proposed action to manipulate Earth's climate in order to counteract global warming from anthropogenic greenhouse gas emissions. We investigate the potential of a specific geoengineering technique, carbon sequestration by artificially enhanced silicate weathering via the dissolution of olivine. This approach would not only operate against rising temperatures but would also oppose ocean acidification, because it influences the global climate via the carbon cycle. If important details of the marine chemistry are taken into consideration, a new mass ratio of CO(2) sequestration per olivine dissolution of about 1 is achieved, 20% smaller than previously assumed. We calculate that this approach has the potential to sequestrate up to 1 Pg of C per year directly, if olivine is distributed as fine powder over land areas of the humid tropics, but this rate is limited by the saturation concentration of silicic acid. In our calculations for the Amazon and Congo river catchments, a maximum annual dissolution of 1.8 and 0.4 Pg of olivine seems possible, corresponding to the sequestration of 0.5 and 0.1 Pg of C per year, but these upper limit sequestration rates come at the environmental cost of pH values in the rivers rising to 8.2. Open water dissolution of fine-grained olivine and an enhancement of the biological pump by the rising riverine input of silicic acid might increase our estimate of the carbon sequestration, but additional research is needed here. We finally calculate with a carbon cycle model the consequences of sequestration rates of 1-5 Pg of C per year for the 21st century by this technique.