Cell-wide analysis of secretory granule dynamics in three dimensions in living pancreatic beta-cells: evidence against a role for AMPK-dependent phosphorylation of KLC1 at Ser517/Ser520 in glucose-stimulated insulin granule movement.
Glucose-stimulated insulin secretion from pancreatic beta-cells requires the kinesin-1/Kif5B-mediated transport of insulin granules along microtubules. 5'-AMPK (5'-AMP-activated protein kinase) is a heterotrimeric serine/threonine kinase which is activated in beta-cells at low glucose concentrations, but inhibited as glucose levels increase. Active AMPK blocks glucose-stimulated insulin secretion and the recruitment of insulin granules to the cell surface, suggesting motor proteins may be targets for this kinase. While both kinesin-1/Kif5B and KLC1 (kinesin light chain-1) contain consensus AMPK phosphorylation sites (Thr(693) and Ser(520), respectively) only recombinant GST (glutathione transferase)-KLC1 was phosphorylated by purified AMPK in vitro. To test the hypothesis that phosphorylation at this site may modulate kinesin-1-mediated granule movement, we developed an approach to study the dynamics of all the resolvable granules within a cell in three dimensions. This cell-wide approach revealed that the number of longer excursions (>10 mum) increased significantly in response to elevated glucose concentration (30 versus 3 mM) in control MIN6 beta-cells. However, similar changes were seen in cells overexpressing wild-type KLC1, phosphomimetic (S517D/S520D) or non-phosphorylatable (S517A/S520A) mutants of KLC1. Thus, changes in the phosphorylation state of KLC1 at Ser(517)/Ser(520) seem unlikely to affect motor function.


Salmonella contamination in a network of 10 pig farms interconnected within the same cooperative.
To evaluate whether pig farms interconnected within the same cooperative share similar Salmonella contamination patterns. Ten finishing pig farms within a 100 km radius of a common slaughterhouse were selected. Their inclusion was based on their association to the same cooperative and the sharing of common resources: piglets, feed, swine transporters, slaughterhouse, technicians and veterinarians. Each farm was visited three times over a 10-month period. Pig faeces, the barn front door handle, the feed pipeline, mobile objects (shovel, balance and pig board), the landing stage, the concrete slab of the feed bins, the tire tracks left on the pathways by the animal feed truck, the pig delivery truck and the carcase knacker truck and the mudguards and cabin carpets of the veterinarian and technician vehicles on their arrival at the farm were all analysed for the presence of Salmonella. All farms were not equally contaminated with Salmonella. Whereas some farms yielded up to 12 Salmonella isolates, other farms were Salmonella free. Some locations, most notably the landing stage, were more contaminated than others. Salmonella contamination was dynamic in time. Some contaminations seen on farms, on specific locations on the first visit, had disappeared on the second and third visits, but new contaminations were detected on different locations. Contamination with Salmonella was not disseminated through the network of the 10 pig farms interconnected within the same cooperative but was rather most often restricted in time to specific locations on specific farms.


Making predictions of mangrove deforestation: a comparison of two methods in Kenya.
Deforestation of mangroves is of global concern given their importance for carbon storage, biogeochemical cycling and the provision of other ecosystem services, but the links between rates of loss and potential drivers or risk factors are rarely evaluated. Here, we identified key drivers of mangrove loss in Kenya and compared two different approaches to predicting risk. Risk factors tested included various possible predictors of anthropogenic deforestation, related to population, suitability for land use change and accessibility. Two approaches were taken to modelling risk; a quantitative statistical approach and a qualitative categorical ranking approach. A quantitative model linking rates of loss to risk factors was constructed based on generalized least squares regression and using mangrove loss data from 1992 to 2000. Population density, soil type and proximity to roads were the most important predictors. In order to validate this model it was used to generate a map of losses of Kenyan mangroves predicted to have occurred between 2000 and 2010. The qualitative categorical model was constructed using data from the same selection of variables, with the coincidence of different risk factors in particular mangrove areas used in an additive manner to create a relative risk index which was then mapped. Quantitative predictions of loss were significantly correlated with the actual loss of mangroves between 2000 and 2010 and the categorical risk index values were also highly correlated with the quantitative predictions. Hence, in this case the relatively simple categorical modelling approach was of similar predictive value to the more complex quantitative model of mangrove deforestation. The advantages and disadvantages of each approach are discussed, and the implications for mangroves are outlined.


Treatment-responsive pandysautonomia in an adolescent with ganglionic α3-AChR antibodies.
Autoimmune autonomic ganglionopathy (AAG) is a rare disorder that presents with pandysautonomia typically in middle age and elderly patients. AAG is typically associated with serum autoantibodies that bind to the alpha-3 subunit of the ganglionic acetylcholine receptor (α3-AChR Ab). We report a 13 year old girl who presented with gut pseudo-obstruction, bladder dysfunction and dilated pupils unresponsive to pilocarpine. She had positive α3-AChR Ab plus other autoantibodies suggesting an autoimmune diathesis. Our patient was initially resistant to steroid therapy but responded to the addition of azathioprine resulting in a near complete clinical remission. We conclude that pandysautonomia associated with α3-AChR Ab can occur in children and has multi-organ involvement.


Assessing Patient-Specific Mechanical Properties of Aortic Wall and Peri-Aortic Structures From In Vivo DENSE Magnetic Resonance Imaging Using an Inverse Finite Element Method and Elastic Foundation Boundary Conditions.
The establishment of in vivo, noninvasive patient-specific, and regionally resolved techniques to quantify aortic properties is key to improving clinical risk assessment and scientific understanding of vascular growth and remodeling. A promising and novel technique to reach this goal is an inverse finite element method (FEM) approach that utilizes magnetic resonance imaging (MRI)-derived displacement fields from displacement encoding with stimulated echoes (DENSE). Previous studies using DENSE MRI suggested that the infrarenal abdominal aorta (IAA) deforms heterogeneously during the cardiac cycle. We hypothesize that this heterogeneity is driven in healthy aortas by regional adventitial tethering and interaction with perivascular tissues, which can be modeled with elastic foundation boundary conditions (EFBCs) using a collection of radially oriented springs with varying stiffness with circumferential distribution. Nine healthy IAAs were modeled using previously acquired patient-specific imaging and displacement fields from steady-state free procession (SSFP) and DENSE MRI, followed by assessment of aortic wall properties and heterogeneous EFBC parameters using inverse FEM. In contrast to traction-free boundary condition, prescription of EFBC reduced the nodal displacement error by 60% and reproduced the DENSE-derived heterogeneous strain distribution. Estimated aortic wall properties were in reasonable agreement with previously reported experimental biaxial testing data. The distribution of normalized EFBC stiffness was consistent among all patients and spatially correlated to standard peri-aortic anatomical features, suggesting that EFBC could be generalized for human adults with normal anatomy. This approach is computationally inexpensive, making it ideal for clinical research and future incorporation into cardiovascular fluid-structure analyses.


Renal responses during a dry saturation dive to 450 msw.
Four subjects were compressed to a simulated depth of 450 msw (46 bar) for 37 days in the main research chamber of the German underwater simulator diving facility at the GKSS Research Center, Geesthacht. The ambient gas was trimix. Urine was collected at 0700, 1300, and 1900 h each day for analysis of Na+, K+, volume, osmolality, and creatinine. Urine, antidiuretic hormone (ADH), and aldosterone were analyzed separately. Daily fluid, Na+, and K+ intake were analyzed throughout the dive. The aim of the investigation was to confirm the existence of a diuresis and natriuresis which had been observed in earlier saturation dives to 31 atm abs using He-O2. A significant diuresis was observed during compression despite a decrease in fluid intake. After compression the diuresis decreased somewhat but remained significantly above precompression control levels during the entire hyperbaric exposure. No significant change in fluid intake was observed. Daily Na+ and K+ excretion increased significantly during compression, which was accompanied by a significant increase in nocturnal excretion of Na+ and K+. Daily intake of Na+ and K+ decreased throughout the dive. Daily urine ADH decreased immediately upon compression and was associated with a parallel decrease in urine osmolality. In contrast, urinary aldosterone excretion exhibited no change during the dive despite the increase in Na+ and K+ excretion and decrease in Na+ intake.


The InsP7 phosphatase Siw14 regulates inositol pyrophosphate levels to control localization of the general stress response transcription factor Msn2.
The environmental stress response (ESR) is critical for cell survival. Yeast cells unable to synthesize inositol pyrophosphates (PP-InsPs) are unable to induce the ESR. We recently discovered a diphosphoinositol pentakisphosphate (PP-InsP5) phosphatase in Saccharomyces cerevisiae encoded by SIW14 Yeast strains deleted for SIW14 have increased levels of PP-InsPs. We hypothesized that strains with high inositol pyrophosphate levels will have an increased stress response. We examined the response of the siw14Δ mutant to heat shock, nutrient limitation, osmotic stress, and oxidative treatment using cell growth assays and found increased resistance to each. Transcriptional responses to oxidative and osmotic stresses were assessed using microarray and reverse transcriptase quantitative PCR. The ESR was partially induced in the siw14Δ mutant strain, consistent with the increased stress resistance, and the mutant strain further induced the ESR in response to oxidative and osmotic stresses. The levels of PP-InsPs increased in WT cells under oxidative stress but not under hyperosmotic stress, and they were high and unchanging in the mutant. Phosphatase activity of Siw14 was inhibited by oxidation that was reversible. To determine how altered PP-InsP levels affect the ESR, we performed epistasis experiments with mutations in rpd3 and msn2/4 combined with siw14Δ. We show that mutations in msn2Δ and msn4Δ, but not rpd3, are epistatic to siw14Δ by assessing growth under oxidative stress conditions and expression of CTT1 Msn2-GFP nuclear localization was increased in the siw14Δ. These data support a model in which the modulation of PP-InsPs influence the ESR through general stress response transcription factors Msn2/4.


NSAID-induced small bowel diaphragms and strictures diagnosed with intraoperative enteroscopy.
Nonsteroidal anti-inflammatory drugs are known to cause mucosal damage in the stomach and duodenum, which may lead to hemorrhage and perforation. However, these medications may also cause damage in the more distal small bowel. Due to the location of these lesions, currently available diagnostic testing may yield false negative results. Two cases of nonsteroidal anti-inflammatory drug-induced small bowel diaphragms presenting as obscure gastrointestinal hemorrhage and recurrent small bowel obstruction, respectively, are discussed. Intraoperative enteroscopy was used to confirm this diagnosis after other diagnostic tests failed to identify the etiology. This procedure may increase the accuracy of exploratory laparotomy in these challenging cases.


Questionnaire survey of cardiologists' knowledge, attitudes, and guideline application of implantable cardioverter defibrillator therapy.
There are little data on cardiologists' knowledge and application of current implantable cardioverter defibrillator (ICD) guidelines, attitudes to risk, and how these may influence ICD prescription. A questionnaire survey was sent to UK cardiologists to test their knowledge and application of ICD guidelines and their estimate of the clinical benefits gained in different clinical scenarios. They were questioned on the minimum absolute risk reduction (ARR) required to justify an ICD implant and factors that influenced their decision making. Sixty responses from 23 implanters and 37 nonimplanters were obtained. Eighty-three percent implanters and 43% nonimplanters were fully aware of UK ICD National Institute of Clinical Excellence guidelines. Only 7% responders had a screening program to identify primary prevention (PP) candidates. Although the mean estimate of ARR in PP scenarios was similar to trial data, the range of estimates was very wide. The benefit in secondary prevention (SP) scenarios was overestimated by both implanters and nonimplanters. Three-year ARR believed to justify PP and SP ICDs were heavily influenced by patients' age but in patients <80 years, age was compatible with trial results. Implanters and nonimplanters correctly applied guidelines in SP scenarios with younger patients but often withheld an ICD in elderly patients. Correct application did not correlate with full awareness of guidelines. Lack of knowledge of guidelines (particularly in nonimplanters), failure of nonimplanters to offer ICDs to appropriate PP patients, age bias, and a lack of screening programs appear to be the greatest barriers to uptake of ICDs in the United Kingdom, rather than financial concerns.


Laboratory Observation of, Astrochemical Search for, and Structure of Elusive Erythrulose in the Interstellar Medium.
Rotational spectroscopy provides the most powerful means of identifying molecules of biological interest in the interstellar medium (ISM), but despite their importance, the detection of carbohydrates has remained rather elusive. Here, we present a comprehensive Fourier transform rotational spectroscopic study of elusive erythrulose, a sugar building block likely to be present in the ISM, employing a novel method of transferring the hygroscopic oily carbohydrate into the gas phase. The high sensitivity of the experiment allowed the rotational spectra of all monosubstituted isotopologue species of 13C-12C3H8O4 to be recorded, which, together with quantum chemical calculations, enabled us to determine their equilibrium geometries (reSE) with great precision. Searches employing the new experimental data for erythrulose have been undertaken in different ISM regions, so far including the cold areas Barnard 1, the pre-stellar core TMC-1, Sagittarius B2. Although no lines of erythrulose were found, this data will serve to enable future searches and possible detections in other ISM regions.


Legal aspects of telepathology.
In some legal surroundings telepathology is considered a breach of registrational barriers. The recommendation of the G 8 states in Europe for required legislation in telemedicine suggests to recognise that the localization of the remote health care professional defines the site not only of licensure but also of liability. This approach must be considered helpful, since it can solve many problems brought about by the doubtful results of private international law and conventions like the European Union (EU) and Lugano Convention. Under today's conditions in private international law it must be considered essential to agree upon a choice of law and stipulate a court of jurisdiction when doing telepathology. However, the opposing aims of insuring the patients claims and avoiding jurisdictions that exceed the local expectations of the medical professional must be reconciled. Data protection and data security are other crucial topics that require attention. Generally speaking, the principles of minimum data exchange, anonymity, pseudonymity and cryptography must be established as a basis for all telepathology procedures. Only when personal data is needed, its use can be legitimated. Written consent of the patient is advised. To guarantee a cross-border security level the regulations of the EU-Data Protection Directive need to be transformed into national law. In practise, cross-border dataflow shall only take place where the security level can be maintained even within the other country. Finally, reimbursement questions must be answered to establish a sound economical basis for telepathology. The spatial distance between the participants may yield the question, whether the service has been rendered to an extent necessary and sufficient for reimbursement. If reimbursement takes place on a cross-border or cross-regional level, severe disturbances of the health systems can occur. Regulation schemes or treaties need therefore to be developed to avoid such disturbances and encompass mutual standards of care as well as methods to balance reimbursement.


Investigation of 2 Types of Self-administered Acupressure for Persistent Cancer-Related Fatigue in Breast Cancer Survivors: A Randomized Clinical Trial.
Fatigue is a common and debilitating late-term effect of breast cancer that is associated with poor sleep and decreased quality of life, yet therapies remain limited. Acupressure has reduced fatigue in previous small studies, but rigorous clinical trials are needed. To investigate if 6 weeks of 2 types of self-administered acupressure improved fatigue, sleep, and quality of life vs usual care in breast cancer survivors and to determine if changes were sustained during a 4-week washout period. Phase 3 randomized, single-blind, clinical trial conducted from March 1, 2011, through October 31, 2014. Women were recruited from the Michigan Tumor Registry. Randomization (1:1:1) to 6 weeks of daily self-administered relaxing acupressure, stimulating acupressure, or usual care. The primary outcome was change in the Brief Fatigue Inventory score from baseline to weeks 6 and 10. Secondary analyses were sleep (Pittsburgh Sleep Quality Index) and quality of life (Long-Term Quality of Life Instrument). A total of 424 survivors of stages 0 to III breast cancer who had completed cancer treatments at least 12 months previously were screened, and 288 were randomized, with 270 receiving relaxing acupressure (n = 94), stimulating acupressure (n = 90), or usual care (n = 86). One woman withdrew owing to bruising at the acupoints. At week 6, the percentages of participants who achieved normal fatigue levels (Brief Fatigue Inventory score <4) were 66.2% (49 of 74) in relaxing acupressure, 60.9% (42 of 70) in stimulating acupressure, and 31.3% (26 of 84) in usual care. At week 10, a total of 56.3% (40 of 71) in relaxing acupressure, 60.9% (42 of 69) in stimulating acupressure, and 30.1% (25 of 83) in usual care continued to have normal fatigue. At neither time point were the 2 acupressure groups significantly different. Relaxing acupressure, but not stimulating acupressure, showed significant improvements in sleep quality compared with usual care at week 6, but not at week 10. Only relaxing acupressure significantly improved quality of life vs usual care at weeks 6 and 10. Both acupressure arms significantly reduced persistent fatigue compared with usual care, but only relaxing acupressure had significant effects on sleep quality and quality of life. Relaxing acupressure offers a possible low-cost option for managing symptoms. clinicaltrials.gov Identifier: NCT01281904.


A retrospective analysis of a societal experiment among the Danish population suggests that exposure to extra doses of vitamin A during fetal development may lower type 2 diabetes mellitus (T2DM) risk later in life.
Vitamin A deficiency has been associated with impaired fetal pancreatic development and increased risk of developing type 2 diabetes mellitus (T2DM). In 1962, mandatory margarine fortification with vitamin A was increased by 25 % in Denmark. We aimed to determine whether offspring of mothers who had been exposed to the extra vitamin A from fortification during pregnancy had a lower risk of developing T2DM in adult life, compared with offspring of mothers exposed to less vitamin A. Individuals from birth cohorts with the higher prenatal vitamin A exposure (born 1 December 1962-31 March 1964) and those with lower prenatal exposure (born 1 September 1959-31 December 1960) were followed up with regard to development of T2DM before 31 December 2012 in the Danish National Diabetes Registry and National Patient Register. Logistic and Cox regression analyses were performed to determine the risk of T2DM by vitamin A exposure level. A total of 193 803 individuals were followed up until midlife. Our results showed that individuals exposed prenatally to extra vitamin A from fortified margarine had a lower risk of developing T2DM than those exposed to lower levels: OR 0·88; 95 % CI 0·81, 0·95, P=0·001, after adjustment for sex. Fetal exposure to small, extra amounts of vitamin A from food fortification may reduce the risk of T2DM. These results may have public health relevance, as they demonstrate that one of the most costly chronic diseases may be prevented by food fortification - a simple and affordable public health nutrition intervention.


Atrial Fibrillation and the Weekend Effect Regarding Cardioversion, Length of Stay, Readmission, and Mortality.
The weekend effect is a phenomenon in which worse outcomes have been found to occur over the weekend. This has been investigated in the context of stroke, ST-elevation myocardial infarction, and pulmonary embolism among others. Atrial fibrillation (AF) is the most common sustained arrhythmia, and admissions for AF have been increasing. However, few studies exist investigating the existence of a weekend effect regarding AF. Previous studies have been limited by a pragmatic but unrealistic definition of the weekend starting at midnight on Friday and ending midnight on Sunday. In addition, the studies that exist have conflicting data regarding outcomes of mortality and length of stay (LOS). Over a 5-year period, 3233 patients with a primary diagnosis of AF were admitted to an academic center. A retrospective analysis was performed to determine rates of cardioversion, 30-day readmission, 30-day mortality, LOS, and time to cardioversion among patients admitted over the weekend compared with those admitted during the work week. Weekend was defined as the 48-hour period, including Saturday and Sunday. Baseline demographics and common risk factors were found to be equivalent in weekend admissions compared with weekday admissions. These characteristics were found to be equivalent in those who underwent cardioversion and those who did not. There was no statistically significant difference between groups in odds of cardioversion, 30-day readmission, or 30-day mortality. Difference in mean LOS and mean time to cardioversion was not statistically significant between groups. In conclusion, a weekend effect was not identified regarding AF in an academic hospital.


Synergistic neuroprotection by coffee components eicosanoyl-5-hydroxytryptamide and caffeine in models of Parkinson's disease and DLB.
Hyperphosphorylated α-synuclein in Lewy bodies and Lewy neurites is a characteristic neuropathological feature of Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). The catalytic subunit of the specific phosphatase, protein phosphatase 2A (PP2A) that dephosphorylates α-synuclein, is hypomethylated in these brains, thereby impeding the assembly of the active trimeric holoenzyme and reducing phosphatase activity. This phosphatase deficiency contributes to the accumulation of hyperphosphorylated α-synuclein, which tends to fibrillize more than unmodified α-synuclein. Eicosanoyl-5-hydroxytryptamide (EHT), a fatty acid derivative of serotonin found in coffee, inhibits the PP2A methylesterase so as to maintain PP2A in a highly active methylated state and mitigates the phenotype of α-synuclein transgenic (SynTg) mice. Considering epidemiologic and experimental evidence suggesting protective effects of caffeine in PD, we sought, in the present study, to test whether there is synergy between EHT and caffeine in models of α-synucleinopathy. Coadministration of these two compounds orally for 6 mo at doses that were individually ineffective in SynTg mice and in a striatal α-synuclein preformed fibril inoculation model resulted in reduced accumulation of phosphorylated α-synuclein, preserved neuronal integrity and function, diminished neuroinflammation, and improved behavioral performance. These indices were associated with increased levels of methylated PP2A in brain tissue. A similar profile of greater PP2A methylation and cytoprotection was found in SH-SY5Y cells cotreated with EHT and caffeine, but not with each compound alone. These findings suggest that these two components of coffee have synergistic effects in protecting the brain against α-synuclein-mediated toxicity through maintenance of PP2A in an active state.


Mechanism of activation of latent recombinant transforming growth factor beta 1 by plasmin.
Medium conditioned by Chinese hamster ovary (CHO) cells transfected with the simian pre-pro-TGF beta 1 cDNA contains high levels of latent TGF beta 1. The amino-terminal region of the TGF beta 1 precursor is secreted and can be detected in the conditioned medium by immunoblotting using peptide antibodies specific for amino-terminal peptides. Chemical cross-linking of CHO-conditioned medium using bis-(sulfosuccinimidyl)-suberate (BS3) followed by immunoblot analyses indicates that latent recombinant TGF beta 1 contains both the cleaved amino-terminal glycopeptide and mature TGF beta 1 polypeptide in a noncovalent association and that this association confers latency. The data presented here do not support the involvement of a unique TGF beta binding protein(s) in latent recombinant TGF beta 1. Plasmin treatment of CHO-conditioned medium resulted in the appearance of TGF beta competing activity. In addition, immunoblot analysis of plasmin-treated CHO-conditioned medium indicates that the amino-terminal glycopeptide is partially degraded and that mature TGF beta 1 is released. Thus, activation of latent TGF beta 1 may occur by proteolytic nicking within the amino-terminal glycopeptide thereby causing a disruption of tertiary structure and noncovalent bonds, which results in the release of active, mature TGF beta 1. Acid activation of latent TGF beta, in comparison, appears to be due to dissociation of the amino-terminal glycopeptide from the mature polypeptide.


Transbranchial ammonia gradients and acid-base responses to high external ammonia concentration in rainbow trout (Oncorhynchus mykiss) acclimated to different salinities.
Transbranchial ammonia gradients and blood acid-base status have been examined in rainbow trout acclimated to fresh water (FW), 33% sea water (33% SW) and sea water (SW) and exposed to 1.0 mmol l-1 total ammonia (TAmm) at pH 7.9 for 24 h. At all three salinities trout maintained large negative (inwardly directed) NH3 and NH4+ gradients throughout the exposure, presumably by active excretion of NH4+ to counteract the passive inward diffusion of ammonia. Analysis of blood non-respiratory acid-base status (delta H+m) revealed an acid load in FW trout and a base load in SW trout following 24 h of exposure. This indicates that active NH4+/H+ exchange predominates in FW whereas NH4+/Na+ is the principal exchange utilised in SW under these experimental conditions. The plasma TAmm load incurred during ammonia exposure increased with salinity. Compared to FW trout, plasma TAmm values were 34 and 73% higher in the 33% SW and SW trout, respectively, after 24 h. This cannot be explained by differences in the prevailing transbranchial PNH3 gradient because ambient PNH3 was substantially lower at the higher salinities (due to higher pK' and solubility values). We interpret the difference between FW and SW trout as an increased permeability to NH4+ in fish acclimated to the higher-salinity environments. Transbranchial diffusion of NH4+ is, therefore, probably more important as a route for ammonia excretion in SW than in FW trout, especially considering the favourable transepithelial potentials normally found in SW teleosts. In addition, increased NH4+ permeability implies that the toxicity of ammonia will be greater in seawater than in freshwater teleosts and should not simply be measured as a function of the unionised ammonia concentration when considering seawater-adapted species.


Comparison of pesticide concentrations in streams at low flow in six metropolitan areas of the United States.
To examine the effect of urban development on pesticide concentrations in streams under low-flow conditions, water samples were collected at stream sites along an urban land use gradient in six environmentally heterogeneous metropolitan areas of the United States. In all six metropolitan areas, total insecticide concentrations generally increased significantly as urban land cover in the basin increased, regardless of whether the background land cover in the basins was agricultural, forested, or shrub land. In contrast, the response of total herbicide concentrations to urbanization varied with the environmental setting. In the three metropolitan areas with predominantly forested background land cover (Raleigh-Durham, NC, USA; Atlanta, GA, USA; Portland, OR, USA), total herbicide concentrations increased significantly with increasing urban land cover. In contrast, total herbicide concentrations were not significantly related to urban land cover in the three remaining metropolitan areas, where total herbicide concentrations appeared to be strongly influenced by agricultural as well as urban sources (Milwaukee-Green Bay, WI, USA; Dallas-Fort Worth, TX, USA), or by factors not measured in the present study, such as water management (Denver, CO, USA). Pesticide concentrations rarely exceeded benchmarks for protection of aquatic life, although these low-flow concentrations are likely to be lower than at other times, such as during peak pesticide-use periods, storm events, or irrigation discharge. Normalization of pesticide concentrations by the pesticide toxicity index -- an index of relative potential toxicity -- for fish and cladocerans indicated that the pesticides detected at the highest concentrations (herbicides in five of the six metropolitan areas) were not necessarily the pesticides with the greatest potential to adversely affect aquatic life (typically insecticides such as carbaryl, chlorpyrifos, diazinon, and fipronil).


Structure of pyruvate kinase and similarities with other enzymes: possible implications for protein taxonomy and evolution.
The structure determination of pyruvate kinase shows that each subunit of the tetrameric molecule consists of three domains. The largest of these domains has a remarkable similarity to the structure of triosephosphate isomerase. Another domain shows similarities to many other nucleotide binding proteins. We discuss these similarities and their implications for current arguments on protein taxonomy and evolution.


Learnings from deaths - the Epilepsy Deaths Register.
The Epilepsy Deaths Register (EDR) differs from typical registries which concentrate primarily on clinical information. It is completed by bereaved relatives and focuses on the circumstances immediately before, and the support following, a death. It can be augmented by copies of death certificates from the families of the deceased, and all epilepsy associated deaths can be entered. The EDR is underpinned by the research and experience of the SUDEP Action team and the clinical advisors who helped design the methodology and the web-based platform. The EDR has been open since 2013 and currently has over 750 entries from over 20 different countries, the majority from the United Kingdom and the Republic of Ireland. The bereaved have shown that they place their trust in the register as a vehicle to be involved in research, even under the most difficult of circumstances. As the EDR matures, we hope to identify the common and rarer patterns of epilepsy-associated death; maintaining our dual ambitions to remain committed to listen, and to make every death count. This paper is for the Special Issue: Prevent 21: SUDEP Summit - Time to Listen.


Inhibition of bacterial perchlorate reduction by zero-valent iron.
Perchlorate was reduced by a mixed bacterial culture over a pH range of 7.0-8.9. Similar rates of perchlorate reduction were observed between pH 7.0 and 8.5, whereas significantly slower reduction occurred at pH 8.9. Addition of iron metal, Fe(0), to the mixed bacterial culture resulted in slower rates of perchlorate reduction. Negligible perchlorate reduction was observed under abiotic conditions with Fe(0) alone in a reduced anaerobic medium. The inhibition of perchlorate reduction observed in the presence of Fe(0) is in contrast to previous studies that have shown faster rates of contaminant reduction when bacteria and Fe(0) were combined compared to bacteria alone. The addition of Fe(0) resulted in a rise in pH, as well as precipitation of Fe minerals that appeared to encapsulate the bacterial cells. In experiments where pH was kept constant, the addition of Fe(0) still resulted in slower rates of perchlorate reduction suggesting that encapsulation of bacteria by Fe precipitates contributed to the inhibition of the bacterial activity independent of the effect of pH on bacteria. These results provide the first evidence linking accumulation of iron precipitates at the cell surface to inhibition of environmental contaminant degradation. Fe(0) was not a suitable amendment to stimulate perchlorate-degrading bacteria and the bacterial inhibition caused by precipitation of reduced Fe species may be important in other combined anaerobic bacterial-Fe(0) systems. Furthermore, the inhibition of bacterial activity by iron precipitation may have significant implications for the design of in situ bioremediation technologies for treatment of perchlorate plumes.


Migration selection, protection, and acculturation in health: a binational perspective on older adults.
In this article, we test for four potential explanations of the Hispanic Health Paradox (HHP): the "salmon bias," emigration selection, and sociocultural protection originating in either destination or sending country. To reduce biases related to attrition by return migration typical of most U.S.-based surveys, we combine data from the Mexican Health and Aging Study in Mexico and the U.S. National Health Interview Survey to compare self-reported diabetes, hypertension, current smoking, obesity, and self-rated health among Mexican-born men ages 50 and older according to their previous U.S. migration experience, and U.S.-born Mexican Americans and non-Hispanic whites. We also use height, a measure of health during childhood, to bolster some of our tests. We find an immigrant advantage relative to non-Hispanic whites in hypertension and, to a lesser extent, obesity. We find evidence consistent with emigration selection and the salmon bias in height, hypertension, and self-rated health among immigrants with less than 15 years of experience in the United States; we do not find conclusive evidence consistent with sociocultural protection mechanisms. Finally, we illustrate that although ignoring return migrants when testing for the HHP and its mechanisms, as well as for the association between U.S. experience and health, exaggerates these associations, they are not fully driven by return migration-related attrition.


Si1-xGex nanoantennas with a tailored Raman response and light-to-heat conversion for advanced sensing applications.
Active light-emitting all-dielectric nanoantennas recently have demonstrated great potential as highly efficient nanoscale light sources owing to their strong luminescent and Raman responses. However, their large-scale fabrication faces a number of problems related to productivity limits of existing lithography techniques. Thus, high-throughput fabrication strategies allowing in a facile way to tailor of the nanoantenna emission and thermal properties in the process of their fabrication are highly desirable for various applications. Here, we propose a cost-effective approach to large-scale fabrication of Si1-xGex alloyed Mie nanoresonators possessing an enhanced inherent Raman response which can be simply tailored via tuning the Ge concentration. Moreover, by tailoring the relative Ge composition one can gradually change a complex refractive index of the produced Si1-xGex alloy, which affects the ratio between radiative and nonradiative losses in Si1-xGex nanoantennas, which is crucial for optimization of their optical heating efficiency. Composition-tunable Si1-xGex nanoantennas with an optimized size, light-to-heat conversion and Raman response are implemented for non-invasive sensing of 4-aminothiophenol molecules with a temperature feedback modality and high subwavelength spatial resolution. The results are important for advanced multichannel optical sensing, providing information on analyte's composition, analyte-nanoantenna temperature response and spatial position.


Condition index monitoring supports conservation priorities for the protection of threatened grass-finch populations.
Conservation agencies are often faced with the difficult task of prioritizing what recovery actions receive support. With the number of species under threat of decline growing globally, research that informs conservation priorities is greatly needed. The relative vulnerability of cryptic or nomadic species is often uncertain, because populations are difficult to monitor and local populations often seem stable in the short term. This uncertainty can lead to inaction when populations are in need of protection. We tested the feasibility of using differences in condition indices as an indication of population vulnerability to decline for related threatened Australian finch sub-species. The Gouldian finch represents a relatively well-studied endangered species, which has a seasonal and site-specific pattern of condition index variation that differs from the closely related non-declining long-tailed finch. We used Gouldian and long-tailed finch condition variation as a model to compare with lesser studied, threatened star and black-throated finches. We compared body condition (fat and muscle scores), haematocrit and stress levels (corticosterone) among populations, seasons and years to determine whether lesser studied finch populations matched the model of an endangered species or a non-declining species. While vulnerable finch populations often had lower muscle and higher fat and corticosterone concentrations during moult (seasonal pattern similar to Gouldian finches), haematocrit values did not differ among populations in a predictable way. Star and black-throated finch populations, which were predicted to be vulnerable to decline, showed evidence of poor condition during moult, supporting their status as vulnerable. Our findings highlight how measures of condition can provide insight into the relative vulnerability of animal and plant populations to decline and will allow the prioritization of efforts towards the populations most likely to be in jeopardy of extinction.


Histone deacetylase inhibitors and pancreatic cancer: are there any promising clinical trials?
Pancreatic cancer, although not very frequent, has an exceptionally high mortality rate, making it one of the most common causes of cancer mortality in developed countries. Pancreatic cancer is difficult to diagnose, allowing few patients to have the necessary treatment at a relatively early stage. Despite a marginal benefit in survival, the overall response of pancreatic cancer to current systemic therapy continues to be poor, and new therapies are desperately needed. Histone deacetylase (HDAC) enzymes play an important role in the development and progression of cancer and HDAC inhibitors (HDACIs) have been shown to induce differentiation and cell cycle arrest, activate the extrinsic or intrinsic pathways of apoptosis, and inhibit invasion, migration and angiogenesis in different cancer cell lines. As a result of promising preclinical data, various HDACIs are being tested as either monotherapeutic agents or in combination regimens for both solid and hematological malignancies. Vorinostat was the first HDACI approved by the Food and Drug Administration for patients with cutaneous T-cell lymphoma. The use of HDACIs in clinical trials, in pretreated and relapsed patients suffering from advanced pancreatic cancer is discussed. Unfortunately, clinical data for HDACIs in patients with pancreatic cancer are inadequate, because only a few studies have included patients suffering from this type of neoplasm and the number of pancreatic cancer patients that entered HDACIs phase II/III trials, among others with advanced solid tumors, is very limited. More studies recruiting patients with pancreatic cancer remain to determine the efficiency of these therapies.


Recent developments in microfluidics-based chemotaxis studies.
Microfluidic devices can better control cellular microenvironments compared to conventional cell migration assays. Over the past few years, microfluidics-based chemotaxis studies showed a rapid growth. New strategies were developed to explore cell migration in manipulated chemical gradients. In addition to expanding the use of microfluidic devices for a broader range of cell types, microfluidic devices were used to study cell migration and chemotaxis in complex environments. Furthermore, high-throughput microfluidic chemotaxis devices and integrated microfluidic chemotaxis systems were developed for medical and commercial applications. In this article, we review recent developments in microfluidics-based chemotaxis studies and discuss the new trends in this field observed over the past few years.


Complete genome sequence of Truepera radiovictrix type strain (RQ-24).
Truepera radiovictrix Albuquerque et al. 2005 is the type species of the genus Truepera within the phylum "Deinococcus/Thermus". T. radiovictrix is of special interest not only because of its isolated phylogenetic location in the order Deinococcales, but also because of its ability to grow under multiple extreme conditions in alkaline, moderately saline, and high temperature habitats. Of particular interest is the fact that, T. radiovictrix is also remarkably resistant to ionizing radiation, a feature it shares with members of the genus Deinococcus. This is the first completed genome sequence of a member of the family Trueperaceae and the fourth type strain genome sequence from a member of the order Deinococcales. The 3,260,398 bp long genome with its 2,994 protein-coding and 52 RNA genes consists of one circular chromosome and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Mixotrophic Iron-Oxidizing Thiomonas Isolates from an Acid Mine Drainage-Affected Creek.
Natural attenuation of heavy metals occurs via coupled microbial iron cycling and metal precipitation in creeks impacted by acid mine drainage (AMD). Here, we describe the isolation, characterization, and genomic sequencing of two iron-oxidizing bacteria (FeOB) species: Thiomonas ferrovorans FB-6 and Thiomonas metallidurans FB-Cd, isolated from slightly acidic (pH 6.3), Fe-rich, AMD-impacted creek sediments. These strains precipitated amorphous iron oxides, lepidocrocite, goethite, and magnetite or maghemite and grew at a pH optimum of 5.5. While Thiomonas spp. are known as mixotrophic sulfur oxidizers and As oxidizers, the FB strains oxidized Fe, which suggests they can efficiently remove Fe and other metals via coprecipitation. Previous evidence for Thiomonas sp. Fe oxidation is largely ambiguous, possibly because of difficulty demonstrating Fe oxidation in heterotrophic/mixotrophic organisms. Therefore, we also conducted a genomic analysis to identify genetic mechanisms of Fe oxidation, other metal transformations, and additional adaptations, comparing the two FB strain genomes with 12 other Thiomonas genomes. The FB strains fall within a relatively novel group of Thiomonas strains that includes another strain (b6) with solid evidence of Fe oxidation. Most Thiomonas isolates, including the FB strains, have the putative iron oxidation gene cyc2, but only the two FB strains possess the putative Fe oxidase genes mtoAB The two FB strain genomes contain the highest numbers of strain-specific gene clusters, greatly increasing the known Thiomonas genetic potential. Our results revealed that the FB strains are two distinct novel species of Thiomonas with the genetic potential for bioremediation of AMD via iron oxidation.IMPORTANCE As AMD moves through the environment, it impacts aquatic ecosystems, but at the same time, these ecosystems can naturally attenuate contaminated waters via acid neutralization and catalyzing metal precipitation. This is the case in the former Ronneburg uranium-mining district, where AMD impacts creek sediments. We isolated and characterized two iron-oxidizing Thiomonas species that are mildly acidophilic to neutrophilic and that have two genetic pathways for iron oxidation. These Thiomonas species are well positioned to naturally attenuate AMD as it discharges across the landscape.


A rare combined injury of dorsal fracture-dislocation of four carpometacarpal joints and trapezium, trapezoid and distal radius bone fractures.
Incorrect or delayed diagnosis and treatment of the carpometacarpal fracture-dislocations is often associated with poor prognosis. We present a rare case of unusual pattern of injury, involving dorsal dislocation of four ulnar carpometacarpal joints, associated with fracture of the trapezium, a burst fracture of the trapezoid bone and an extra-articular fracture of the third distal of the radius. The first surgical intervention was followed by unsatisfactory results, confirmed by the CT scans. A second surgery followed and an open reduction and pinning with K wires performed. Post-operative follow up lasting for nine months revealed a very good surgical outcome.


Differential susceptibility to plasticity: a 'missing link' between gene-culture co-evolution and neuropsychiatric spectrum disorders?
Brüne's proposal that erstwhile 'vulnerability' genes need to be reconsidered as 'plasticity' genes, given the potential for certain environments to yield increased positive function in the same domain as potential dysfunction, has implications for psychiatric nosology as well as a more dynamic understanding of the relationship between genes and culture. In addition to validating neuropsychiatric spectrum disorder nosologies by calling for similar methodological shifts in gene-environment-interaction studies, Brüne's position elevates the importance of environmental contexts - inclusive of socio-cultural variables - as mechanisms that contribute to clinical presentation. We assert that when models of susceptibility to plasticity and neuropsychiatric spectrum disorders are concomitantly considered, a new line of inquiry emerges into the co-evolution and co-determination of socio-cultural contexts and endophenotypes. This presents potentially unique opportunities, benefits, challenges, and responsibilities for research and practice in psychiatry. Please see related manuscript: http://www.biomedcentral.com/1741-7015/10/38.


Stimulation of granulocytic cell iodination by tannins and related compounds.
Tannic acid stimulated the iodination (incorporation of radioactive iodine into an acid-insoluble fraction) of human peripheral blood polymorphonuclear cells (PMN) and human promyelocytic leukemic HL-60 cells, without affecting the iodination of 9 other cultured cell lines. The stimulation of both PMN and HL-60 cells depended on incubation time and temperature, and was significantly suppressed by myeloperoxidase inhibitors. Among chemically defined natural polyphenols, condensed tannins (epicatechin gallate oligomers) and monomeric and oligomeric hydrolyzable tannins potently stimulated PMN iodination, whereas polyphenols of lower molecular weight (gallic acid, alkyl gallates, epicatechin, epicatechin gallate, epigallocatechin, caffeic acid derivatives and licorice flavonoids) had much less activity. Various synthetic polyphenolic compounds structurally unrelated to tannins also stimulated PMN iodination depending upon their molecular weight, but to a slightly lesser extent. The results suggest that the stimulation activity of tannins and related polyphenols might depend more on their molecular weights than the number of hydroxyl groups on each benzene ring in the molecule, or the presence of sugars or hexahydroxydiphenoyl groups.


Effects of nanoparticle size on cellular uptake and liver MRI with polyvinylpyrrolidone-coated iron oxide nanoparticles.
The effect of nanoparticle size (30-120 nm) on magnetic resonance imaging (MRI) of hepatic lesions in vivo has been systematically examined using polyvinylpyrrolidone (PVP)-coated iron oxide nanoparticles (PVP-IOs). Such biocompatible PVP-IOs with different sizes were synthesized by a simple one-pot pyrolysis method. These PVP-IOs exhibited good crystallinity and high T(2) relaxivities, and the relaxivity increased with the size of the magnetic nanoparticles. It was found that cellular uptake changed with both size and surface physiochemical properties, and that PVP-IO-37 with a core size of 37 nm and hydrodynamic particle size of 100 nm exhibited higher cellular uptake rate and greater distribution than other PVP-IOs and Feridex. We systematically investigated the effect of nanoparticle size on MRI of normal liver and hepatic lesions in vivo. The physical and chemical properties of the nanoparticles influenced their pharmacokinetic behavior, which ultimately determined their ability to accumulate in the liver. The contrast enhancement of PVP-IOs within the liver was highly dependent on the overall size of the nanoparticles, and the 100 nm PVP-IO-37 nanoparticles exhibited the greatest enhancement. These results will have implications in designing engineered nanoparticles that are optimized as MR contrast agents or for use in therapeutics.


Contribution of menthofuran to the hepatotoxicity of pulegone: assessment based on matched area under the curve and on matched time course.
In recent years it has become increasingly evident that the toxicity produced by a variety of compounds can be attributed to their metabolites. Simple dose-toxicity studies of a metabolite will help to elucidate its toxic effect, but it is not possible to quantify its role in the toxicity produced by the parent compound unless the disposition of the preformed and endogenously formed metabolite is taken into account. We assessed the contribution of the metabolite, R-(+)-menthofuran (MF), to the hepatotoxicity observed after i.p. administration of R-(+)-pulegone (PUL) to rats. As the major constituent of pennyroyal oil, PUL has been linked to the deaths of several young women over the past several years. After i.p. administration of PUL and MF to separate groups of rats at doses selected to match the area under the curve of generated and synthetic MF, plasma alanine transferase was comparable between groups. Although the area under the curve was matched, the peak concentration in plasma of MF formed in vivo is 4.5 times higher and occurs much earlier than after the administration of MF itself. When the exposure of rats to preformed and synthetic MF is matched with respect to time course in plasma, PUL produces more than twice the increase in plasma alanine transferase and hepatocellular necrosis than does MF. The results suggest that events other than those associated with the disposition of MF contribute to the hepatotoxicity observed after ingestion of PUL, and they emphasize the importance of time course in assessing the role of a metabolite in the toxicity of a given compound.


Concurrent gene signatures for han chinese breast cancers.
The interplay between copy number variation (CNV) and differential gene expression may be able to shed light on molecular process underlying breast cancer and lead to the discovery of cancer-related genes. In the current study, genes concurrently identified in array comparative genomic hybridization (CGH) and gene expression microarrays were used to derive gene signatures for Han Chinese breast cancers. We performed 23 array CGHs and 81 gene expression microarrays in breast cancer samples from Taiwanese women. Genes with coherent patterns of both CNV and differential gene expression were identified from the 21 samples assayed using both platforms. We used these genes to derive signatures associated with clinical ER and HER2 status and disease-free survival. DISTRIBUTIONS OF SIGNATURE GENES WERE STRONGLY ASSOCIATED WITH CHROMOSOMAL LOCATION: chromosome 16 for ER and 17 for HER2. A breast cancer risk predictive model was built based on the first supervised principal component from 16 genes (RCAN3, MCOLN2, DENND2D, RWDD3, ZMYM6, CAPZA1, GPR18, WARS2, TRIM45, SCRN1, CSNK1E, HBXIP, CSDE1, MRPL20, IKZF1, and COL20A1), and distinct survival patterns were observed between the high- and low-risk groups from the combined dataset of 408 microarrays. The risk score was significantly higher in breast cancer patients with recurrence, metastasis, or mortality than in relapse-free individuals (0.241 versus 0, P<0.001). The concurrent gene risk predictive model remained discriminative across distinct clinical ER and HER2 statuses in subgroup analysis. Prognostic comparisons with published gene expression signatures showed a better discerning ability of concurrent genes, many of which were rarely identifiable if expression data were pre-selected by phenotype correlations or variability of individual genes. We conclude that parallel analysis of CGH and microarray data, in conjunction with known gene expression patterns, can be used to identify biomarkers with prognostic values in breast cancer.


A perspective on the proposal for neurocognitive disorder criteria in DSM-5 as applied to HIV-associated neurocognitive disorders.
HIV-associated neurocognitive disorders remain common in the current era of effective antiretroviral therapy. However, the severity at presentation of these disorders has been reduced, and the typical manifestations have changed. A revision of the American Academy of Neurology (AAN) criteria has been made on this basis, and a revision of the analogous criteria by the American Psychiatric Association will be forthcoming in the Diagnostic and Statistical Manual of Mental Disorders (DSM)-5. This article compares the relevant sets of diagnostic criteria that will be employed. It is concluded that a greater degree of integration of the revised, HIV-specific AAN criteria for HIV-associated neurocognitive disorders with the criteria proposed for the DSM-5 would prove advantageous for research, clinical, educational and administrative purposes.


Treatment of hyperactive children with monoamine oxidase inhibitors. I. Clinical efficacy.
Fourteen boys (mean age, 9.2 +/- 1.5 years) with Attention Deficit Disorder (ADD) With Hyperactivity were treated with dextroamphetamine sulfate or a monoamine oxidase inhibitor (MAOI) (six received clorgyline, eight received tranylcypromine sulfate) for four weeks each in a double-blind, cross-over study that included a two-week placebo washout between active drug periods. The MAOIs had immediate, clinically significant benefit and were clinically indistinguishable from dextroamphetamine. Most children responded to both stimulant and MAOI. These findings of equivalent efficacy of MAOIs in ADD are in contrast to our previous studies with neurotransmitter system selective agents, which showed only weak effects, and suggest that multiple neurotransmitter alterations may be required for stimulant drug effects in ADD. The immediate response to MAOIs indicates a different mechanism from that mediating antidepressant effect. The MAOIs may be useful alternate treatments in selected cases of ADD.


Overexpression of ribonucleotide reductase in transfected human KB cells increases their resistance to hydroxyurea: M2 but not M1 is sufficient to increase resistance to hydroxyurea in transfected cells.
Ribonucleotide reductase (RR) is a rate-limiting enzyme in DNA synthesis. The enzyme consists of two subunits, M1 and M2. Hydroxyurea (HU) is an M2-specific inhibitor. It has been shown that a HU-resistant clone derived from stepwise exposure to HU overexpresses the M2 mRNA and the RR protein (Y. Yen et al., Cancer Res., 54: 3868-3691, 1994). In this study, we established stable clones by transfecting human KB cells with the cDNA of human wild-type RR in which each subunit was overexpressed by a SV40 promoter. The mammalian cell expression vector ph beta APr-1 was used for constructing M1, M2, and M1/M2 subunit cDNA. The transfected cells were selected with G418. The clones designated M2-D, M1-D, X-D, and KB-V represent transfectant clones which contain M2 cDNA, M1 cDNA, M1/M2 cDNA, and vector alone, respectively. The parental KB cells and clones containing vector plasmid KB-V express equally low amounts of M2 and M1 mRNA from the endogenous genes. The expression of M2 mRNA and M1 mRNA is elevated 2-3 fold in the X-D transfectants. M2-D clone demonstrated a 6-fold higher M2 mRNA level although the M1 mRNA expression remains the same as parental cells. M1-D transfectants have a 3-fold increase in M1 mRNA expression relative to parental cells, but reveal no alteration of M2 mRNA. Southern analysis of genomic DNA suggested the incorporation of the plasmid into the genome. The X-D clone revealed both integration of the M2 and M1 gene while the M2-D clone only showed M2 gene integration. The M1-D clone revealed M1 gene integration relative to the parental cells. The Western blot of M2 protein showed a 3-fold increase in the X-D and M2-D clones whereas the M2 protein level in M1-D was the same as it was in parental cells. The M1 protein was increased 3-fold in X-D and 1.5-fold in M1-D over that of parental cells. However, lower M1 protein levels were identified in the M2-D clone. The specific activity of the RR enzyme from each transfectant showed a 3-fold increase in both the X-D and M2-D clones and slightly increased in M1-D clone over that of parental cells. However, X-D and M2-D both demonstrated a 3-fold increase in resistance to HU as compared to M1-D which showed the same sensitivity as the parental enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)


Histochemical changes of brain dopamine in an acute stage of cerebral ischemia in gerbils.
The fluorescence histochemical method of Falck et al. was applied to 40 gerbil brains after ligation of a unilateral common carotid artery to investigate alterations of brain dopamine in the acute stage of cerebral ischemia. The distribution of dopaminergic terminals and cell bodies in gerbils is the same as in other mammals. On the ligated side after one hour of ischemia, diffuse green fluorescence of dopaminergic terminals showed only a slight decrease in intensity when compared to the nonligated side. But white matter and bundles of myelinated fibers adjacent to and in the dopamine-rich regions had an intense green fluorescence in contrast to the non-ligated side where they are normally non-fluorescent. This is considered to indicate the extraneuronal leakage and diffusion of dopamine. The intensity of extraneuronal green fluorescence was especially high in glial cells. Occasionally, there was also an unusual green fluorescence in the lumen of small vessels in dopamine-rich regions on the ligated side. Dopaminergic cell bodies in the substantia nigra on the ligated side revealed a conspicuous reduction in the fluorescence intensity in severely affected cases. After 2 or 3 hours of ischemia, there was a marked reduction or disappearance of the diffuse green fluorescence on the ligated side. This may be attributed in part to further diffusion of leaked dopamine.


Assessment of Marginal Bone Loss around Platform-Matched and Platform-Switched Implants - A Prospective Study.
The aim of the present study was to perform a software-assisted radiographic assessment of the effect of platform-switching on marginal bone loss (MBL) around dental implants. Forty patients requiring a dental implant in non-grafted partially edentulous mandibles were enrolled and categorized into implants receiving a platform-matched abutment (control group) or implants with a platform-switched abutment (test group). Standardized digital periapical radiographs were taken at the time of implant placement (T0), at implant loading (T1) and 1-year after functional loading (T2). Software-assisted radiographic assessment of the MBL horizontal, vertical and area changes was performed and compared between time intervals (T1-T0, T2-T1 and T2-T0). Mean radiographic horizontal MBL (hMBL) and vertical MBL (vMBL) from implant placement to 1-year after implant loading (T2-T0) were significantly increased around platform-matched when compared to platform-switched abutments (1.04 mm vs 0.84 mm, p<0.05) and (0.99 mm vs 0.82 mm, p<0.05), respectively. Additionally, bone loss area (BLa) was greater (0.77 mm2 vs 0.63 mm2; p<0.05) for platform-matched compared to platform-switched abutments. Platform-switching has a positive impact upon the amount of bone modeling after loading implants with internal hexagon connection.


Evidence for two classes of nociceptive modulating neurons in the periaqueductal gray.
The midbrain periaqueductal gray (PAG) and the rostral ventromedial medulla (RVM) are important links in a neuronal network that modulates nociceptive transmission. In the RVM, 2 classes of cells have been identified that show changes in activity at the time of the tail-flick response (TF) elicited by noxious heat (Fields et al., 1983a). We now report that neurons in the PAG region also show changes in activity related to TF. Extracellular recordings were made from the PAG and the ventrally adjacent tegmentum at sites from which it was possible to inhibit TF using stimulating currents of 10 microA or less. Cell activity, time of TF occurrence, and tail temperature were recorded during 5 repetitions of the heat stimulus. Periresponse and peristimulus histograms were plotted with reference to the TF and tail temperature, respectively. A significant number of neurons in the PAG region showed changes in activity that preceded the TF. "Midbrain On-cells" (13.6% of the sample) displayed an abrupt increase in firing just prior to the TF. "Midbrain Off-cells" (4.4%) paused just prior to the TF. The remaining neurons (241 of 294, or 82%) did not exhibit changes in firing prior to the TF. Thus, cells with changes in activity related to the TF are present in the PAG region as well as in the RVM. The PAG has a large projection to the RVM, and microinjection of morphine in the PAG increases activity of RVM Off-cells and decreases that of RVM On-cells.(ABSTRACT TRUNCATED AT 250 WORDS)


An improved sequence-aided T-RFLP analysis of bacterial succession during oyster mushroom substrate preparation.
While oyster mushroom (Pleurotus spp.) is one of the most popular cultivated edible mushrooms, there is scanty information about the microbial community taking part in mushroom substrate production. In this study, an improved sequence-aided terminal restriction fragment length polymorphism (T-RFLP) was used to identify and (semi-)quantify the dominant bacteria of oyster mushroom substrate preparation. The main features of the improved T-RFLP data analysis were the alignment of chromatograms with variable clustering thresholds, the visualization of data matrix with principal component analysis ordination superimposed with cluster analysis, and the search for stage-specific peaks (bacterial taxa) with similarity percentage (analysis of similarity) analysis, followed by identification with clone libraries. By applying this method, the dominance of the following bacterial genera was revealed during oyster mushroom substrate preparation: Pseudomonas and Sphingomonas at startup, Bacillus, Geobacillus, Ureibacillus, Pseudoxanthomonas, and Thermobispora at the end of partial composting, and finally several genera of Actinobacteria, Thermus, Bacillus, Geobacillus, Thermobacillus, and Ureibacillus in the mature substrate. As the proportion of uncultured bacteria increased during the process, it is worth establishing strain collections from partial composting and from mature substrate for searching new species.


Study protocol for a randomized controlled trial: the feasibility and impact of cardiovascular absolute risk assessment in Australian general practice.
Although cardiovascular absolute risk (CVAR) assessment has been recommended for use in Australian general practice for a number of years, there is continuing uncertainty about its implementation and impact. Our previous work has developed a multifaceted implementation model. This study aims to investigate both the feasibility of using this model and the impact of CVAR assessment and management on general practice clinical processes and patient care. This cluster randomized controlled trial will be conducted in general practices in Sydney, involving general practitioners (GPs), other practice staff, and patients aged 45 to 69 years without existing cardiovascular disease. A total of 32 practices (40 GPs) and 1,320 patients will be recruited. Randomization will be conducted at the practice level. The intervention group of GPs will be trained to use a CVAR implementation model, whereas the control group of GPs will continue usual care. Study outcomes include clinical processes, patient risk, use of lifestyle intervention, and prescription of antihypertensive and lipid-lowering medications. Data will be collected and analyzed using mixed methods. Study outcomes before and after the intervention will be compared, and the 2 groups will also be compared after adjusting for baseline difference and clustering factors. This trial will be the first study in Australian general practice and one of few international studies to evaluate the impact of implementing CVAR assessment and management. Results of this study will help improve the primary prevention of cardiovascular disease and inform guidelines for clinical practice and the implementation of other health initiatives.


Lamins: the structure and protein complexes.
Lamins are nuclear intermediate filament (IF) proteins. They assemble to fibrous structures that are positioned between the inner nuclear membrane and the peripheral chromatin. A small fraction of lamins is also present in the nucleoplasm. Lamins are required to maintain the nuclear structure and, together with their associated proteins, are involved in most nuclear activities. Mutations in lamins cause >14 distinct diseases, called laminopathies, that include heart, muscle, fat and early aging diseases. However, it is not clear how lamins are organized in vivo and how the disease mutations affect lamin organization and functions. Here, we will review structural aspects of lamin assembly, discuss differences between peripheral and nucleoplasmic lamins and describe the protein complexes that lamins form.


Continuum of the United States military's traumatic brain injury care: adjusting to the changing battlefield.
Over the past 8 years, advances in the US Military Health System (MHS) have led to extensive changes in the way combat casualty care is provided to deployed service members with a traumatic brain injury (TBI). Changes include the application of cutting-edge Clinical Practice Guidelines, use of pioneering technologies, and advances in evacuation procedures. Compared with previous engagements, current operations occur on a much smaller scale, and more frequently in austere environments, such that effective medical support is increasingly challenging. In this paper, the authors describe key aspects of the current continuum of TBI care in the US military, from the point of injury through rehabilitation, with an emphasis on how emerging technologies and evidence-based Clinical Practice Guidelines assist MHS clinicians with providing the best clinical care possible in the changing battlefield.


Monitoring of International Diabetes Federation-recommended Clinical Diabetes Indicators in a Public Health Centre in Southwest Trinidad.
To examine availability of International Diabetes Federation (IDF)-recommended diabetes indicators in the medical charts of patients in active care at a public health centre in southwest Trinidad and Tobago, and to determine clinical status of the patient population according to Caribbean Health Research Council/Pan American Health Organization (CHRC/PAHO) guidelines for disease control. Data were extracted from the medical records of consecutive patients with diagnosed diabetes who presented for routine care at the health centre over a seven-month period. The three most recent dates and results for the following clinical indicators were extracted: glycated haemoglobin (HbA1c), blood pressure, lipid panel, random blood sugar and weight. Data were extracted from 486 patient medical records (91% of patients who presented for care). The majority of records, 366 (76%), had one of three recommended IDF indicators of HbA1c, blood pressure or low-density lipoprotein (LDL) in the past year, 58 (12%) had two, 55 (11%) had three and seven (1%) had no indicators recorded. Random blood sugar and blood pressure were recorded in 93% of records, while only 20% had an HbA1c reported in the past year. The vast majority of patients did not meet guidelines for control of blood sugar, blood pressure or cholesterol. Due to a non-standardized HbA1c assay, rate of controlled HbA1c, based on CHRC/PAHO clinical guidelines, could not be determined. Although availability of indicators suggests an increase from prior audits reported in the literature, current reporting patterns challenge optimal patient management and future systematic evaluation of trends in diabetes care and outcomes.


Exploring the relationship between hope and burnout in competitive sport.
Researchers have postulated that hope may be an important factor associated with burnout. Consistent with hope theory contentions, low-hope individuals may be susceptible to burnout because they are prone to experience goal blockage, frustration, and negative affect, all of which likely increase the risk of burnout. We examined the relationship between hope and athlete burnout among 178 competitive athletes (63 females and 115 males) aged 15-20 years. Hope was significantly and negatively correlated with all three burnout subscales: emotional/physical exhaustion, a reduced sense of accomplishment, and sport devaluation. Moreover, results of a multivariate analysis of variance showed that low-hope athletes scored significantly higher than medium- and high-hope athletes on all three burnout dimensions. Finally, results revealed that agency thinking was a significant predictor of all burnout dimensions. Frustration over unmet goals and a perceived lack of agency, a characteristic of low-hope athletes, might pose a risk factor in athlete burnout, whereas being able to maintain hope appears to be associated with health and well-being.


Autoantibodies to ion channels at the neuromuscular junction.
Autoantibodies directed against voltage- or ligand-gated ion channels and their associated proteins at the neuromuscular junction give rise to a family of neurological autoimmune diseases. Antibodies to acetylcholine receptors or muscle-specific kinase present on the postsynaptic muscle membrane are associated with different forms of myasthenia gravis (MG). Antibodies to the presynaptic voltage-gated potassium and calcium channels are responsible for acquired neuromyotonia and Lambert-Eaton myasthenic syndrome (LEMS), respectively. The patients respond to immunotherapies and their plasma can transfer defects in neuromuscular transmission to mice, indicating that these are antibody-mediated conditions. In a small proportion of cases, ion channel antibodies have also been implicated in neurological dysfunction in the central nervous system. In these conditions, a proportion of the patients have an underlying tumour, thymoma in both MG and neuromyotonia and small cell lung carcinoma in LEMS, emphasising the putative role of autoimmunity to tumour antigens as a cause of neurological disease.


Economic considerations for the hepatologist.
This is an era of rapid change in health care systems and clinical practice. In the face of increasing national health care expenditures, physicians are confronted with an increased demand to justify practices and to show the value of their services. Hepatologists are being required to show that their practices are both effective and cost-effective. This has led to an expanding body of literature examining the cost-effectiveness of medical practices. To evaluate these economic analyses the reader must be familiar with the concepts used in economic analysis and have a clear understanding of both how these analyses are performed and how the results can be applied to clinical practice. The purpose of this article is to provide the reader with the essential concepts for evaluating economic analyses in the medical literature and to provide published criteria for performing and critiquing an economic analysis. The terms used in economic analysis are outlined and defined. The criteria for performing an economic analysis are listed. Examples are given to emphasize the key points.


Serum level of interleukin-17A in patients with alopecia areata and its relationship to age.
There is strong evidence for an autoimmune etiology of alopecia areata (AA). Interleukin-17A (IL-17A) is a Th17 proinflammatory cytokine that has been linked to the pathogeneses of diverse autoimmune and inflammatory diseases. This study aimed to measure serum IL-17A in AA patients and to study associations between IL-17A levels and AA severity, duration, and age of onset, and patient gender and age. The study enrolled 39 AA patients and 37 healthy control subjects. Scalp involvement was assessed using the Severity of Alopecia Tool (SALT), and clinical disease severity was determined. Serum IL-17A was measured using ELISAs. Serum IL-17A was significantly higher in AA patients than in control subjects (P < 0.001). Correlations between serum IL-17A and gender, disease duration, SALT score, and disease severity were non-significant. Serum IL-17A was significantly higher in patients aged ≤30 years than in patients aged >30 years (P = 0.045). Age and serum IL-17A were significantly negatively correlated in patients with AA (rs = -0.363, P = 0.023) but not in control subjects (rs = -0.294, P = 0.077). Patients with juvenile-onset AA had significantly higher IL-17A levels than those with maturity-onset disease (P = 0.034). There was a significant negative correlation between age at disease onset and serum IL-17A (rs = -0.349, P = 0.029). It is possible that IL-17A plays a role in the pathogenesis of AA. Serum IL-17A may be influenced by patient age and age of onset of AA but does not seem to influence disease severity.


Allicin attenuates pathological cardiac hypertrophy by inhibiting autophagy via activation of PI3K/Akt/mTOR and MAPK/ERK/mTOR signaling pathways.
Cardiac hypertrophy is an adaptive response of the myocardium to pressure or volume overload. Recent evidences indicate that allicin can prevent cardiac hypertrophy. However, it is not clear whether allicin alleviates cardiac hypertrophy by inhibiting autophagy. We aimed to investigate the effects of allicin on pressure overload-induced cardiac hypertrophy, and further to clarify the related mechanism. Cardiac hypertrophy was successfully established by abdominal aortic constriction (AAC) in rats, and cardiomyocytes hypertrophy was simulated by angiotensin II (Ang II) in vitro. Hemodynamic parameters were monitored by organism function experiment system in vivo. The changes of cell surface area were observed using HE and immunofluorescence staining in vivoand in vitro, respectively. The expressions of cardiac hypertrophy relative protein (BNP and β-MHC), autophagy marker protein (LC3-II and Beclin-1), Akt, PI3K and ERK were detected by western blot. Allicin could improve cardiac function, and reduce cardiomyocytes size, and decrease BNP and β-MHC protein expressions. Further results showed that allicin could lower LC3-II and Beclin-1 protein expressions both in vivo and in vitro experiments. And pharmacological inhibitor of mTOR, rapamycin could antagonize the effects of allicin on Ang II-induced cardiac hypertrophy and autophagy. Simultaneously, allicin could promote the expressions of p-Akt, p-PI3K and p-ERK protein. These findings reveal a novel mechanism of allicin attenuating cardiac hypertrophy which allicin could inhibit excessive autophagy via activating PI3K/Akt/mTOR and MAPK/ERK/mTOR signaling pathways.


A Topology Potential-Based Method for Identifying Essential Proteins from PPI Networks.
Essential proteins are indispensable for cellular life. It is of great significance to identify essential proteins that can help us understand the minimal requirements for cellular life and is also very important for drug design. However, identification of essential proteins based on experimental approaches are typically time-consuming and expensive. With the development of high-throughput technology in the post-genomic era, more and more protein-protein interaction data can be obtained, which make it possible to study essential proteins from the network level. There have been a series of computational approaches proposed for predicting essential proteins based on network topologies. Most of these topology based essential protein discovery methods were to use network centralities. In this paper, we investigate the essential proteins' topological characters from a completely new perspective. To our knowledge it is the first time that topology potential is used to identify essential proteins from a protein-protein interaction (PPI) network. The basic idea is that each protein in the network can be viewed as a material particle which creates a potential field around itself and the interaction of all proteins forms a topological field over the network. By defining and computing the value of each protein's topology potential, we can obtain a more precise ranking which reflects the importance of proteins from the PPI network. The experimental results show that topology potential-based methods TP and TP-NC outperform traditional topology measures: degree centrality (DC), betweenness centrality (BC), closeness centrality (CC), subgraph centrality (SC), eigenvector centrality (EC), information centrality (IC), and network centrality (NC) for predicting essential proteins. In addition, these centrality measures are improved on their performance for identifying essential proteins in biological network when controlled by topology potential.


[Meta-analysis of the Italian studies on short-term effects of air pollution].
In recent years, much attention has been given to review reports on the early effects of air pollution on health, measured through daily series of deaths and/or hospital admissions. A number of large planned meta-analyses (in which methods for data retrieval and processing are commonly planned a priori for all participating centers) are on going both in the US and in Europe. The National Mortality, Morbidity and Air Pollution Study included data from 90 US cities, whereas APHEA (Air Pollution and Health, a European Approach) considers data from about 30 european cities. The present paper summarizes methods and findings of MISA, a meta-analysis of data from 8 Italian cities. It belongs to an ad hoc supplement of Epidemiologia & Prevenzione (Epidemiol Prev 2001; 25 (2) Suppl: 1-72), the official Journal of the Italian Association of Epidemiology, which contains a full description of the study. MISA was launched on March 2000, within the project "Statistics, Environment and Health" (GRASPA), funded by the Italian Ministry of Education. Additional support was given by the Authorities of the 8 participating cities (from North to South: Turin, Milan, Verona, Ravenna, Bologna, Florence, Rome and Palermo). DAILY HEALTH DATA: Deaths certificate and hospital admission data have been collected respectively from the Local Health Authority and regional files. The same programme for retrieval of data on selected hospital admissions for acute conditions was used in the 8 cities. Main data are summarized in Table 1. DAILY CONCENTRATION OF POLLUTANTS: Most data were obtained from Regional Environmental Protection Agencies, which are responsible for environmental monitoring since 1993. Verona, Palermo and Milan (1990-94) data were obtained from local sources. Monitors with more than 25% of missing data were excluded. Meteorological data were collected by the same monitors and completed with data from monitors situated in the suburbs or (in Milan and Bologna) in the airport. The monitors were selected by a group of experts to ensure comparability. For SO2 and NO2 daily averages of hourly measurements were used, whereas concentrations of ozone and CO were estimated as the maximum 8 hours moving average. Total suspended particulate or PM10 were measured as 24 hours deposition. All analyses used the whole range of observed values (Table 2). Daily data were considered as missing when more than 25% of hourly data were not available. Missing data in one monitor were imputed as average of data from the remaining monitors weighted by the ratio between the specific monitor's year average and the general year average of all the selected city monitors. Missing data in one day were imputed as average of four days (preceding and following day, the same day of the previous and following weeks). In the city of Florence and Palermo PM10 concentrations were available. For the other cities we applied a conversion factor from PTS to PM10 (0.6 for Turin and 0.8 for all the others) estimated through validation studies. Ozone concentrations were used only where background monitors were available (Turin, Verona, Bologna and Florence) and limited to the warm season (May through September). A common protocol for the city-specific analyses was defined on the basis of a structured exploratory analysis. The adopted basic model was a Generalized Additive Model for Poisson data. Effect estimates were age-adjusted (0-64, 65-74, 75+) and formal tests of interaction pollutant-age were conducted. In the first two age groups, indicator variables for seasonality were specified, and cubic splines with fixed number of degree of freedom were specified for the last age group and for all age groups for the morbidity data. Model adequacy was checked by residual analysis and inspection of the partial autocorrelation function. In a sensitivity analysis non linear pollutant effects were considered and overdispersed [table: see text] transitional models were fitted; the analysis was conducted for all lags 0-3 and some distributed lags (0-1, 1-2, 0-3); no multipollutant models were fitted. The same model was fitted to the city data. No model selection was done: Table 3 describes the steps in model building. In the meta-analysis, for each outcome, the estimates for each pollutant and for each city were combined using fixed and random effects models. Heterogeneity of effects was tested according to DerSimonian and Laird. Results were checked using a hierarchical bayesian model, which was used to investigate heterogeneity across cities in a meta-regression phase. Non informative priors were used. Posterior distributions of parameters of interest have been obtained with WinBUGS. 10,000 iterations (excluding [table: see text] the first 2000) were retained, while for the meta-regression 100,000 iterations (excluding the first 4000) were stored. To approximate the marginal posteriors only one sample out of five were used. Achieved convergence was assessed using the Gelman and Rubin approach. In the meta-regression the models specified were the following: [formula: see text] i denotes city, j calendar period (1990-1994; 1995-1999). The first model includes only period as effect modifier, while the second model other potential variables. The ui terms (which do not vary with j) represent city specific random effects. For each pollutant, the meta-analysis detected a statistically significant association with mortality for natural causes. But for ozone, positive associations were commonly found for death and hospital admissions for both cardiovascular and respiratory diseases. Indeed, the only estimates whose lower 95% confidence limit bore a negative sign regarded the association between PM10 and mortality from respiratory diseases. Ozone in the warm season was positively and significantly associated with daily mortality and mortality for cardiovascular diseases whereas other estimates did not reach statistical significance and some were negative (only lag 0-1 for external comparability are reported in Table 4). Risks were highest (up to 4%) for respiratory conditions (Table 4). They were more pronounced at lag 1-2 for mortality, and at lag 0-3 for hospital admissions. Age was an effect modifier for mortality, the elderly being more susceptible. In the random effect meta-analysis, at lag 1-2, excess risks for unit increase of the pollutants at age 75+ and at age 0-64 were respectively: 4.9% and -0.4% for SO2, 1.7% and 0.6% for NO2; 2.3% and 0.2% for CO. Corresponding figures for PM10 at lag 0-1 were 1.1% and 0.2%. The effect of PM10 on mortality [table: see text] was greater during the warm season (2.8% vs 0.8%). A complete analysis is reported in the Italian text. Here we provide some details on the effects of PM10, about which the residual heterogeneity across cities was highest (Table 4). In addition, the epidemiological evidence on the hazards from this fraction of particulate matter is more controversial. Table 5 reports the excess risk estimated through the meta-analysis in 1995-99 for a 10 micrograms/m3 increase of PM10 for some outcomes. Proper prior distributions (overdispersed normal and inverse gamma) were adopted in the final bayesian analyses. The sensitivity of results to the choice of the priors were investigated (we defined proper and improper uniform, student's t), obtaining comparable results. Total natural mortality was significantly heterogeneous across cities (Q = 18.96, 5 df, p < 0.001). City-specific estimates are represented graphically in Fig. 1. As expected, the confidence (credibility) intervals are widest [table: see text] for bayesian estimates, intermediate for those obtained under a random effects model, and narrowest for those found under a fixed effects model. Nevertheless, differences in point estimates are negligible. A North-South gradient in risk is obvious. Table 6 shows, for the cities for which mortality data were available, the improvement in precision and the shrinkage of effect estimates toward the overall mean introduced by the bayesian modelling. In the meta-regression, total mortality and a deprivation score were associated with greater effects. The excess risks on hospital admission were modified by the deprivation score and by the NO2/PM10 ratio. Overall, the risk estimates were greater in the calendar period 1995-99 and there was a North-South gradient, with larger effects in cities located in Central and Southern Italy (Florence, Rome, Palermo). The meta-analysis of the Italian studies on short-term effects of air pollution in 8 cities, MISA, exhibits the following features: With the exception of Naples, all greatest Italian cities were included; overall a population of 7 million was enrolled. The study protocol was accurate with regard to the selection of hospital admissions for acute conditions. Monitored data of concentration of pollutant were carefully evaluated before their inclusion in the meta-analysis. City specific analyses were carried out according to a common protocol controlling for seasonality, influenza epidemics, age and meterological variables; [table: see text] the protocol derived from a structured exploratory analysis. The meta-analysis was done using fixed and random effects models; a hierarchical bayesian model was fitted in a sensitivity analysis. The heterogeneity of effects across cities was investigated using a hierarchical bayesian model for meta-regression. While mortality data are of good quality, hospital admission data are more problematic. Since the filing criteria for the latter changed around 1995, comparability of results before and after such date is limited. Moreover, hospital admissions rely on availability of beds, the offer of which may be restricted during the warm season. Comparability of pollutant concentration estimates among cities may have been influenced by differences in monitor characteristics. (ABSTRACT TRUNCATED)


Substrate selectivity of high-activity mutants of human butyrylcholinesterase.
Cocaine is one of the most addictive drugs, and there is still no FDA (Food and Drug Administration)-approved medication specific for cocaine abuse. A promising therapeutic strategy is to accelerate cocaine metabolism, producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e. cocaine hydrolysis catalyzed by butyrylcholinesterase (BChE) in plasma. However, the native BChE has a low catalytic efficiency against the abused cocaine, i.e. (-)-cocaine. Our recently designed and discovered A199S/F227A/S287G/A328W/Y332G mutant and other mutants of human BChE have a considerably improved catalytic efficiency against (-)-cocaine. In the present study, we carried out both computational modeling and experimental kinetic analysis on the catalytic activities of these promising new BChE mutants against other known substrates, including neurotransmitter acetylcholine (ACh), acetylthiocholine (ATC), butyrylthiocholine (BTC), and (+)-cocaine, in comparison with the corresponding catalytic activity against (-)-cocaine. Both the computational modeling and kinetic analysis have consistently revealed that all the examined amino acid mutations only considerably improve the catalytic efficiency of human BChE against (-)-cocaine, without significantly improving the catalytic efficiency of the enzyme against any of the other substrates examined. In particular, all the examined BChE mutants have a slightly lower catalytic efficiency against neurotransmitter ACh compared to the wild-type BChE. This observation gives us confidence in developing an anti-cocaine enzyme therapy by using one of these BChE mutants, particularly the A199S/F227A/S287G/A328W/Y332G mutant.


Phosphatidylinositol 3-kinase/Akt induced by erythropoietin renders the erythroid differentiation factor GATA-1 competent for TIMP-1 gene transactivation.
The contribution of erythropoietin to the differentiation of the red blood cell lineage remains elusive, and the demonstration of a molecular link between erythropoietin and the transcription of genes associated with erythroid differentiation is lacking. In erythroid cells, expression of the tissue inhibitor of matrix metalloproteinase (TIMP-1) is strictly dependent on erythropoietin. We report here that erythropoietin regulates the transcription of the TIMP-1 gene upon binding to its receptor in erythroid cells by triggering the activation of phosphatidylinositol 3-kinase (PI3K)/Akt. We found that Akt directly phosphorylates the transcription factor GATA-1 at serine 310 and that this site-specific phosphorylation is required for the transcriptional activation of the TIMP-1 promoter. This chain of events can be recapitulated in nonerythroid cells by transfection of the implicated molecular partners, resulting in the expression of the normally silent endogenous TIMP-1 gene. Conversely, TIMP-1 secretion is profoundly decreased in erythroid cells from fetal livers of transgenic knock-in mice homozygous for a GATA(S310A) gene, which encodes a GATA-1 mutant that cannot be phosphorylated at Ser(310). Furthermore, retrovirus-mediated expression of GATA(S310A) into GATA-1(null)-derived embryonic stem cells decreases the rate of hemoglobinization by more than 50% compared to expressed wild-type GATA-1. These findings provide the first example of a chain of coupling mechanisms between the binding of erythropoietin to its receptor and GATA-1-dependent gene expression.


Severe asthma: how can we differentiate phenotypes?
Difficult-to-treat asthma, severe asthma or refractory asthma are all terms defining a fraction of asthmatics (5-10%) whose asthma cannot be controlled with a combination of high-dose inhaled corticoids together with long-acting b2 agonists.One major step before claiming that a patient has difficult-to-treat asthma is to ensure that asthma is the correct diagnosis. This involves taking a history of symptoms suggestive of asthma together with objective evidence of variable airflow limitation and/or airway hyperresponsiveness, calling for an intensive initial investigation taking in associated comorbid conditions and multiple re-evaluations over a period of at least 6 months.The pathophysiology of severe/refractory asthma is likely to be different from that of the mild to moderate form. The immune mechanisms underlying the inflammatory process are not purely Th2. The contribution of allergic mechanisms is usually less prominent than in mild to moderate asthma, and other environmental factors such as air pollution, including tobacco smoke and viruses, may be determinant. Involvement of small airways causing air trapping appears to be crucial in making asthma refractory to classic treatment.Several phenotypes have been identified on the basis of demographic, functional, pathological and clinical characteristics, which may sometimes overlap.A cluster analysis has identified two clusters specific to refractory asthma: an early onset symptom-predominant asthma and a late onset inflammation-predominant form.Teasing out mechanisms underlying different phenotypes is essential in finding a new treatment target to improve asthma control in these patients.


The angiogenic response to PLL-g-PEG-mediated HIF-1α plasmid DNA delivery in healthy and diabetic rats.
Impaired angiogenesis is a major clinical problem and affects wound healing especially in diabetic patients. Improving angiogenesis is a reasonable strategy to increase diabetes-impaired wound healing. Recently, our lab described a system of transient gene expression due to pegylated poly-l-lysine (PLL-g-PEG) polymer-mediated plasmid DNA delivery in vitro. Here we synthesized peptide-modified PLL-g-PEG polymers with two functionalities, characterized them in vitro and utilized them in vivo via a fibrin-based delivery matrix to induce dermal wound angiogenesis in diabetic rats. The two peptides were 1) a TG-peptide to covalently bind these nanocondensates to the fibrin matrix (TG-peptide) for a sustained release and 2) a polyR peptide to improve cellular uptake of these nanocondensates. In order to induce angiogenesis in vivo we condensed modified and non-modified polymers with plasmid DNA encoding a truncated form of the therapeutic candidate gene hypoxia-inducible transcription factor 1α (HIF-1α). HIF-1α is the primarily oxygen-dependent regulated subunit of the heterodimeric transcription factor HIF-1, which controls angiogenesis among other physiological pathways. The truncated form of HIF-1α lacks the oxygen-dependent degradation domain (ODD) and therefore escapes degradation under normoxic conditions. PLL-g-PEG polymer-mediated HIF-1α-ΔODD plasmid DNA delivery was found to lead to a transiently induced gene expression of angiogenesis-related genes Acta2 and Pecam1 as well as the HIF-1α target gene Vegf in vivo. Furthermore, HIF-1α gene delivery was shown to enhance the number endothelial cells and smooth muscle cells - precursors for mature blood vessels - during wound healing. We show that - depending on the selection of the therapeutic target gene - PLL-g-PEG nanocondensates are a promising alternative to viral DNA delivery approaches, which might pose a risk to health.


When experts disagree (and better science won't help much): using structured deliberations to support endangered species recovery planning.
Progress on recovery plans to conserve endangered species is often blocked due to the lack of an effective framework that technical experts and other knowledgeable stakeholders can use to examine areas of agreement or disagreement about the anticipated effects of management actions. Multi-party, multi-interest resource management deliberations, although increasingly common, are difficult in the context of recovery planning due to the range of potentially affected environmental, economic, and social concerns. These deliberations are further complicated by frequent disagreements among technical experts about how to identify and address various sources of biological uncertainty. We describe the development of a decision-aiding framework as part of an inter-agency plan to assist recovery of endangered Gulf of Maine Atlantic salmon (Salmo salar), using a structured decision making approach that encouraged constructive deliberations based on rigorous analysis. Results are summarized in terms of developing an explicit set of management objectives, clarifying and prioritizing hypotheses concerning barriers to recovery, comparing alternative management initiatives in light of biological uncertainty, and incorporating resource constraints to generate preferred sets of actions. Overall, the use of a structured approach to making recovery decisions improved inter-agency cooperation and facilitated dialogue, understanding, and agreement among participating experts. It also helped to create a defensible basis for further internal discussions as well as for communicating with external stakeholders, including resource users and political decision makers.