Hydration Dynamics of a Peripheral Membrane Protein.
Water dynamics in the hydration shell of the peripheral membrane protein annexin B12 were studied using MD simulations and Overhauser DNP-enhanced NMR. We show that retardation of water motions near phospholipid bilayers is extended by the presence of a membrane-bound protein, up to around 10 Å above that protein. Near the membrane surface, electrostatic interactions with the lipid head groups strongly slow down water dynamics, whereas protein-induced water retardation is weaker and dominates only at distances beyond 10 Å from the membrane surface. The results can be understood from a simple model based on additive contributions from the membrane and the protein to the activation free energy barriers of water diffusion next to the biomolecular surfaces. Furthermore, analysis of the intermolecular vibrations of the water network reveals that retarded water motions near the membrane shift the vibrational modes to higher frequencies, which we used to identify an entropy gradient from the membrane surface toward the bulk water. Our results have implications for processes that take place at lipid membrane surfaces, including molecular recognition, binding, and protein-protein interactions.


Isolation and properties of 600-kDa and 23-kDa haemolymph proteins from the tsetse fly, Glossina morsitans: their possible role as biological insecticides.
The haemolymph of the tsetse fly, Glossina morsitans morsitans, contains a high (lipophorin) and a low molecular weight protein of high densities, 1.11 and 1.29 g/ml, respectively. The purification of the proteins was achieved by a combination of density gradient ultracentrifugation and reported gel permeation chromatography. The lipophorin is of high molecular weight (M(r) integral of 600,000) and consists of two apoproteins, apolipophorin I (M(r) integral of 250,000) and apolipophorin II (M(r) integral of 80,000) both of which are glycosylated. Lipophorin also has a pI of 6.1. However, electrophoresis under non-denaturing and denaturing conditions showed the low molecular weight protein to be a single polypeptide chain (M(r) integral of 23,000). Amino acid analysis revealed a relatively high content of the acidic amino acids as well as serine and glycine. The protein contained lipids as shown by Sudan Black staining but was unglycosylated. Using rabbit antiserum against the isolated protein in immunodiffusion and immunoblotting experiments, no cross-reactivity was detected with haemolymph samples from insects representing six orders. In conclusion, the finding of lipophorin suggests that, although flies primarily utilize proline for their energy needs, there is an active transport mechanism for the supply of lipid requirements. However, the results for the low molecular weight protein indicate that the protein is unique to Glossina, suggesting that it may have an important role in the physiology of this insect and is therefore a significant target for vector management.


Cytokine preconditioning of engineered cartilage provides protection against interleukin-1 insult.
During osteoarthritis and following surgical procedures, the environment of the knee is rich in proinflammatory cytokines such as IL-1. Introduction of tissue-engineered cartilage constructs to a chemically harsh milieu may limit the functionality of the implanted tissue over long periods. A chemical preconditioning scheme (application of low doses of IL-1) was tested as a method to prepare developing engineered tissue to withstand exposure to a higher concentration of the cytokine, known to elicit proteolysis as well as rapid degeneration of cartilage. Using an established juvenile bovine model system, engineered cartilage was preconditioned with low doses of IL-1α (0.1 ng/mL, 0.5 ng/mL, and 1.0 ng/mL) for 7 days before exposure to an insult dose (10 ng/mL). The time frame over which this protection is afforded was investigated by altering the amount of time between preconditioning and insult as well as the time following insult. To explore a potential mechanism for this protection, one set of constructs was preconditioned with CoCl2, a chemical inducer of hypoxia, before exposure to the IL-1α insult. Finally, we examined the translation of this preconditioning method to extend to clinically relevant adult, passaged chondrocytes from a preclinical canine model. Low doses of IL-1α (0.1 ng/mL and 0.5 ng/mL) protected against subsequent catabolic degradation by cytokine insult, preserving mechanical stiffness and biochemical composition. Regardless of amount of time between preconditioning scheme and insult, protection was afforded. In a similar manner, preconditioning with CoCl2 similarly allowed for mediation of catabolic damage by IL-1α. The effects of preconditioning on clinically relevant adult, passaged chondrocytes from a preclinical canine model followed the same trends with low-dose IL-1β offering variable protection against insult. Chemical preconditioning schemes have the ability to protect engineered cartilage constructs from IL-1-induced catabolic degradation, offering potential modalities for therapeutic treatments.


Personal protective equipment for preventing highly infectious diseases due to exposure to contaminated body fluids in healthcare staff.
In epidemics of highly infectious diseases, such as Ebola, severe acute respiratory syndrome (SARS), or coronavirus (COVID-19), healthcare workers (HCW) are at much greater risk of infection than the general population, due to their contact with patients' contaminated body fluids. Personal protective equipment (PPE) can reduce the risk by covering exposed body parts. It is unclear which type of PPE protects best, what is the best way to put PPE on (i.e. donning) or to remove PPE (i.e. doffing), and how to train HCWs to use PPE as instructed. To evaluate which type of full-body PPE and which method of donning or doffing PPE have the least risk of contamination or infection for HCW, and which training methods increase compliance with PPE protocols. We searched CENTRAL, MEDLINE, Embase and CINAHL to 20 March 2020. We included all controlled studies that evaluated the effect of full-body PPE used by HCW exposed to highly infectious diseases, on the risk of infection, contamination, or noncompliance with protocols. We also included studies that compared the effect of various ways of donning or doffing PPE, and the effects of training on the same outcomes. Two review authors independently selected studies, extracted data and assessed the risk of bias in included trials. We conducted random-effects meta-analyses were appropriate. Earlier versions of this review were published in 2016 and 2019. In this update, we included 24 studies with 2278 participants, of which 14 were randomised controlled trials (RCT), one was a quasi-RCT and nine had a non-randomised design. Eight studies compared types of PPE. Six studies evaluated adapted PPE. Eight studies compared donning and doffing processes and three studies evaluated types of training. Eighteen studies used simulated exposure with fluorescent markers or harmless microbes. In simulation studies, median contamination rates were 25% for the intervention and 67% for the control groups. Evidence for all outcomes is of very low certainty unless otherwise stated because it is based on one or two studies, the indirectness of the evidence in simulation studies and because of risk of bias. Types of PPE The use of a powered, air-purifying respirator with coverall may protect against the risk of contamination better than a N95 mask and gown (risk ratio (RR) 0.27, 95% confidence interval (CI) 0.17 to 0.43) but was more difficult to don (non-compliance: RR 7.5, 95% CI 1.81 to 31.1). In one RCT (59 participants) coveralls were more difficult to doff than isolation gowns (very low-certainty evidence). Gowns may protect better against contamination than aprons (small patches: mean difference (MD) -10.28, 95% CI -14.77 to -5.79). PPE made of more breathable material may lead to a similar number of spots on the trunk (MD 1.60, 95% CI -0.15 to 3.35) compared to more water-repellent material but may have greater user satisfaction (MD -0.46, 95% CI -0.84 to -0.08, scale of 1 to 5). According to three studies that tested more recently introduced full-body PPE ensembles, there may be no difference in contamination. Modified PPE versus standard PPE The following modifications to PPE design may lead to less contamination compared to standard PPE: sealed gown and glove combination (RR 0.27, 95% CI 0.09 to 0.78), a better fitting gown around the neck, wrists and hands (RR 0.08, 95% CI 0.01 to 0.55), a better cover of the gown-wrist interface (RR 0.45, 95% CI 0.26 to 0.78, low-certainty evidence), added tabs to grab to facilitate doffing of masks (RR 0.33, 95% CI 0.14 to 0.80) or gloves (RR 0.22, 95% CI 0.15 to 0.31). Donning and doffing Using Centers for Disease Control and Prevention (CDC) recommendations for doffing may lead to less contamination compared to no guidance (small patches: MD -5.44, 95% CI -7.43 to -3.45). One-step removal of gloves and gown may lead to less bacterial contamination (RR 0.20, 95% CI 0.05 to 0.77) but not to less fluorescent contamination (RR 0.98, 95% CI 0.75 to 1.28) than separate removal. Double-gloving may lead to less viral or bacterial contamination compared to single gloving (RR 0.34, 95% CI 0.17 to 0.66) but not to less fluorescent contamination (RR 0.98, 95% CI 0.75 to 1.28). Additional spoken instruction may lead to fewer errors in doffing (MD -0.9, 95% CI -1.4 to -0.4) and to fewer contamination spots (MD -5, 95% CI -8.08 to -1.92). Extra sanitation of gloves before doffing with quaternary ammonium or bleach may decrease contamination, but not alcohol-based hand rub. Training The use of additional computer simulation may lead to fewer errors in doffing (MD -1.2, 95% CI -1.6 to -0.7). A video lecture on donning PPE may lead to better skills scores (MD 30.70, 95% CI 20.14 to 41.26) than a traditional lecture. Face-to-face instruction may reduce noncompliance with doffing guidance more (odds ratio 0.45, 95% CI 0.21 to 0.98) than providing folders or videos only. We found low- to very low-certainty evidence that covering more parts of the body leads to better protection but usually comes at the cost of more difficult donning or doffing and less user comfort. More breathable types of PPE may lead to similar contamination but may have greater user satisfaction. Modifications to PPE design, such as tabs to grab, may decrease the risk of contamination. For donning and doffing procedures, following CDC doffing guidance, a one-step glove and gown removal, double-gloving, spoken instructions during doffing, and using glove disinfection may reduce contamination and increase compliance. Face-to-face training in PPE use may reduce errors more than folder-based training. We still need RCTs of training with long-term follow-up. We need simulation studies with more participants to find out which combinations of PPE and which doffing procedure protects best. Consensus on simulation of exposure and assessment of outcome is urgently needed. We also need more real-life evidence. Therefore, the use of PPE of HCW exposed to highly infectious diseases should be registered and the HCW should be prospectively followed for their risk of infection.


CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor I with chromatin.
Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase CDC7. Here we show that another kinase, CDC28, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of CDC28 but not of CDC7 substantially reduce the in vivo phosphorylation of Cac1p. However, mutations in the putative CDC7 target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a cdc28-1 mutant and to a lesser extent in a cdc7-1 mutant. In addition, mutations in the Cac1p-phosphorylation sites by both CDC28 and CDC7 reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate CDC28 in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly.


Synthesis of acyl fluorides via photocatalytic fluorination of aldehydic C-H bonds.
Acyl fluorides are versatile acylating agents owing to their unique stability. Their synthesis, however, can present challenges and is typically accomplished through deoxyfluorination of carboxylic acids. Here, we demonstrate that acyl fluorides can be prepared directly from aldehydes via a C(sp2)-H fluorination reaction involving the inexpensive photocatalyst sodium decatungstate and electrophilic fluorinating agent N-fluorobenzenesulfonimide. This convenient fluorination strategy enables direct conversion of aliphatic and aromatic aldehydes into acylating agents.


Scalable variable-index elasto-optic metamaterials for macroscopic optical components and devices.
Optical metamaterials with an artificial subwavelength structure offer new approaches to implement advanced optical devices. However, some of the biggest challenges associated with the development of metamaterials in the visible spectrum are the high costs and slow production speeds of the nanofabrication processes. Here, we demonstrate a macroscale (>35 mm) transformation-optics wave bender (293 mm2) and Luneburg lens (855 mm2) in the broadband white-light visible wavelength range using the concept of elasto-optic metamaterials that combines optics and solid mechanics. Our metamaterials consist of mesoscopically homogeneous chunks of bulk aerogels with superior, broadband optical transparency across the visible spectrum and an adjustable, stress-tuneable refractive index ranging from 1.43 down to nearly the free space index (∼1.074). The experimental results show that broadband light can be controlled and redirected in a volume of >105λ × 105λ × 103λ, which enables natural light to be processed directly by metamaterial-based optical devices without any additional coupling components.


Rethinking Priorities: Cost of Complications After Elective Colectomy.
To compare incremental costs associated with complications of elective colectomy using nationally representative data among patients undergoing laparoscopic/open resections for the 4 most frequent diagnoses. Rising healthcare costs have led to increasing focus on the need to achieve a better understanding of the association between costs and quality. Among elective colectomies, a focus of surgical quality-improvement initiatives, interpretable evidence to support existing approaches is lacking. The 2009 to 2011 Nationwide Inpatient Sample (NIS) data were queried for adult (≥18 years) patients undergoing elective colectomy. Patients with primary diagnoses for colon cancer, diverticular disease, benign colonic neoplasm, and ulcerative colitis/regional enteritis were included. Based on system-based complications considered relevant to long-term treatment of elective colectomy, stratified differences in risk-adjusted incremental hospital costs and complications probabilities were compared. A total of 68,462 patients were included, weighted to represent 337,887 patients nationwide. A total of 16.4% experienced complications. Annual risk-adjusted incremental costs amounted to >$150 million. Magnitudes of complication prevalences/costs varied by primary diagnosis, operative technique, and complication group. Infectious complications contributed the most ($55 million), followed by gastrointestinal ($53 million), pulmonary ($22 million), and cardiovascular ($11 million) complications. Total annual costs for elective colectomies amounted to >$1.7 billion: 11.3% was due to complications [1.9% due to current Centers for Medicare and Medicaid Services (CMS) complications]. The results highlight a need to consider the varied/broad impact of complications, offering a stratified paradigm for priority setting in surgery. As we move forward in the development of novel/adaptation of existing interventions, it will be essential to weigh the cost of complications in an evidence-based way.


Soy isoflavones ameliorate the adverse effects of chemotherapy in children.
Genistein sensitizes cancer cells to chemotherapy and radiation by modulating cell survival pathways. At the same time, genistein's antioxidant and anti-inflammatory effects may protect normal tissues from adverse effects of chemotherapy and radiation, which are largely due to oxygen-free radicals and inflammation. We conducted a small pilot study with a soy isoflavone mixture containing 8 mg of genistein in children receiving chemotherapy and/or radiation to investigate genistein's potential toxicity preventive effect. We monitored clinical and laboratory parameters in children with cancer who received their first cycle of chemotherapy without genistein and the subsequent cycles with genistein. Patients served as their own controls, and the clinical-laboratory data from the first cycle were compared to the data from subsequent cycles. Nine cycles of chemotherapy were administered without genistein and 57 cycles with genistein. Patients experienced less myelosuppression, mucositis, and infection when they received genistein with chemotherapy. During supplementation, serum genistein levels were 2 to 6 times higher compared to presupplementation levels. Patients who received abdominal radiation reported less pain and diarrhea when they took the genistein supplement. Further clinical investigation of soy isoflavones in pediatric cancer patients receiving chemotherapy and/or radiation should be conducted.


Phase retardance of periodic multilayer mirrors.
A periodic multilayer reflector composed of two nonabsorbing materials is found to have a phase retardance that is a function of the refractive indices of the materials, the periodic design, and the incidence angle. The addition of a reflection enhancing periodic multilayer to a mirror causes the phase retardance to change to a unique value that depends on the properties of the added layers and the incidence angle. After reaching this limiting value the retardance is not changed by the further addition of reflection enhancing layers with the same periodic structure. Equations for the limiting phase retardance of a periodic multilayer reflector are provided.


Leptin receptor gene Gln223Arg polymorphism is not associated with obesity and metabolic syndrome in Turkish children.
The aim of the study was to investigate the relationship between leptin receptor gene (LEPR) Gln223Arg polymorphism and obesity in Turkish children. Ninety-two obese and 99 lean children (between 5-15 years) were included in the study. Twenty-three of the obese children were diagnosed with metabolic syndrome. Blood samples were collected for morning fasting blood glucose, insulin, leptin, and lipid level measurements. LEPR Gln223Arg polymorphism was analyzed by restriction fragment length polymorphism. Significant differences were observed in anthropometric measurements, fasting blood glucose, insulin, leptin, and lipid levels between obese and lean children. Serum leptin levels were markedly higher in obese children. No significant association was noted between Gln223Arg polymorphism and serum leptin, insulin and lipid levels. There were no differences in the genotype frequencies or allele distribution for Gln223Arg polymorphism among obese, obese with metabolic syndrome and lean children. Our findings suggest that there is no association between Gln223Arg polymorphism and obesity in Turkish children.


The process of whole person healing: "unstuckness" and beyond.
The purpose of the present study was to fully explore the descriptions of patients' experiences of change after receipt of whole systems of complementary and alternative medicine (CAM) treatment. The aim was to develop an understanding of "unstuckness," including characterization of states, processes, and modifying factors. This was a secondary descriptive qualitative analysis, using techniques borrowed from phenomenology and grounded theory. Three existent datasets collected at two different universities in the United States and Canada were used in the secondary analysis. Patients with chronic illnesses (including cancer and multiple nonmalignant conditions) who were treated with different packages of care were interviewed for the primary three studies (n = 76 with over 150 interview sessions). Complete data sets from these participants were used in this secondary analysis. OUTCOME MEASURES/DATA COLLECTION TECHNIQUES: Original transcripts were coded asking specific research questions about the experience of change subsequent to whole systems treatments. Data clearly indicated experiential differences between stuckness, unsticking, and unstuckness. Descriptors and characteristics of each state were identified, as was an initial grounded theory of change or transformation that occurs as an outcome of whole medical systems of CAM. The results provide preliminary conceptualizations and descriptions of the impact that CAM whole systems interventions may have on the individual' s life courses. This constitutes a first step in the identification, measurement, and evaluation of whole systems outcomes in a clinical setting. The emerging conceptualization of the process from stuckness to transformation may also provide a link between clinical research and systems science theory.


Frequent occult infection with Cytomegalovirus in cardiac transplant recipients despite antiviral prophylaxis.
Despite antiviral prophylaxis, a high percentage (over 90%) of heart transplant patients experience active cytomegalovirus (CMV) infection, diagnosed by detection of viral DNA in peripheral blood polymorphonuclear leukocytes within the first few months posttransplantation. Viral DNA was detected in mononuclear cells prior to detection in granulocytes from CMV-seropositive recipients (R+) receiving a heart from a CMV-seropositive donor (D+). Based on assessment of systemic infection in leukocyte populations, both R+ subgroups (R+/D- and R+/D+) experienced a greater infection burden than the R-/D+ subgroup, which was aggressively treated because of a higher risk of acute CMV disease. Despite widespread systemic infection in all at-risk patient subgroups, CMV DNA was rarely (< 3% of patients) detected in transplanted heart biopsy specimens. The R+ patients more frequently exceeded the 75th percentile of the CMV DNA copy number distribution in leukocytes (110 copies/10(5) polymorphonuclear leukocytes) than the R-/D+ subgroup. Therefore, active systemic CMV infection involving leukocytes is common in heart transplant recipients receiving prophylaxis to reduce acute disease. Infection of the transplanted organ is rare, suggesting that chronic vascular disease attributed to CMV may be driven by the consequences of systemic infection.


Suppressor of cytokine signalling (SOCS) 1 and 3 enhance cell adhesion and inhibit migration towards the chemokine eotaxin/CCL11.
Suppressors of cytokine signalling (SOCS) proteins regulate signal transduction, but their role in responses to chemokines remains poorly understood. We report that cells expressing SOCS1 and 3 exhibit enhanced adhesion and reduced migration towards the chemokine CCL11. Focal adhesion kinase (FAK) and the GTPase RhoA, control cell adhesion and migration and we show the presence of SOCS1 or 3 regulates expression and tyrosine phosphorylation of FAK, while also enhancing activation of RhoA. Our novel findings suggest that SOCS1 and 3 may control chemotaxis and adhesion by significantly enhancing both FAK and RhoA activity, thus localizing immune cells to the site of allergic inflammation.


Patient-reported upper extremity outcome measures used in breast cancer survivors: a systematic review.
(1) To identify English-language published patient-reported upper extremity outcome measures used in breast cancer research and (2) to examine construct validity and responsiveness in patient-reported upper extremity outcome measures used in breast cancer research. PubMed, Cumulative Index to Nursing and Allied Health Literature, and ProQuest MEDLINE databases were searched up to February 5, 2013. Studies were included if a patient-reported upper extremity outcome measure was administered, the participants were diagnosed with breast cancer, and the study was published in English. A total of 865 articles were screened. Fifty-nine full text articles were assessed for eligibility. A total of 46 articles met the initial eligibility criteria for aim 1. Eleven of these articles reported means and SDs for the outcome scores and included a comparison group analysis for aim 2. Construct validity was evaluated by calculating effect sizes for known-group differences in 6 studies using the Disabilities of Arm, Shoulder and Hand (DASH), University of Pennsylvania Shoulder Score, Shoulder Disability Questionnaire-Dutch, and 10 Questions by Wingate. Responsiveness was analyzed comparing a treatment and control group by calculating the coefficient of responsiveness in 5 studies for the DASH and 10 Questions by Wingate. Eight different patient-reported upper extremity outcome measures have been reported in the peer-review literature for women with breast cancer; some that were specifically developed for breast cancer survivors (n=3) and others that were not (n=5). Based on the current evidence, we recommend administering the DASH to assess patient-reported upper extremity function in breast cancer survivors because the DASH has the most consistently large effects sizes for construct validity and responsiveness. Future large studies are needed for more definitive recommendations.


Probabilistic methods for verbal autopsy interpretation: InterVA robustness in relation to variations in a priori probabilities.
InterVA is a probabilistic method for interpreting verbal autopsy (VA) data. It uses a priori approximations of probabilities relating to diseases and symptoms to calculate the probability of specific causes of death given reported symptoms recorded in a VA interview. The extent to which InterVA's ability to characterise a population's mortality composition might be sensitive to variations in these a priori probabilities was investigated. A priori InterVA probabilities were changed by 1, 2 or 3 steps on the logarithmic scale on which the original probabilities were based. These changes were made to a random selection of 25% and 50% of the original probabilities, giving six model variants. A random sample of 1,000 VAs from South Africa, were used as a basis for experimentation and were processed using the original InterVA model and 20 random instances of each of the six InterVA model variants. Rank order of cause of death and cause-specific mortality fractions (CSMFs) from the original InterVA model and the mean, maximum and minimum results from the 20 randomly modified InterVA models for each of the six variants were compared. CSMFs were functionally similar between the original InterVA model and the models with modified a priori probabilities such that even the CSMFs based on the InterVA model with the greatest degree of variation in the a priori probabilities would not lead to substantially different public health conclusions. The rank order of causes were also similar between all versions of InterVA. InterVA is a robust model for interpreting VA data and even relatively large variations in a priori probabilities do not affect InterVA-derived results to a great degree. The original physician-derived a priori probabilities are likely to be sufficient for the global application of InterVA in settings without routine death certification.


Open reading frames carried on UL/b' are implicated in shedding and horizontal transmission of rhesus cytomegalovirus in rhesus monkeys.
Implicit with the use of animal models to test human cytomegalovirus (HCMV) vaccines is the assumption that the viral challenge of vaccinated animals reflects the anticipated virus-host interactions following exposure of vaccinated humans to HCMV. Variables of animal vaccine studies include the route of exposure to and the titer of challenge virus, as well as the genomic coding content of the challenge virus. This study was initiated to provide a better context for conducting vaccine trials with nonhuman primates by determining whether the in vivo phenotype of culture-passaged strains of rhesus cytomegalovirus (RhCMV) is comparable to that of wild-type RhCMV (RhCMV-WT), particularly in relation to the shedding of virus into bodily fluids and the potential for horizontal transmission. Results of this study demonstrate that two strains containing a full-length UL/b' region of the RhCMV genome, which encodes proteins involved in epithelial tropism and immune evasion, were persistently shed in large amounts in bodily fluids and horizontally transmitted, whereas a strain lacking a complete UL/b' region was not shed or transmitted to cagemates. Shedding patterns exhibited by strains encoding a complete UL/b' region were consistent with patterns observed in naturally infected monkeys, the majority of whom persistently shed high levels of virus in saliva for extended periods of time after seroconversion. Frequent viral shedding contributed to a high rate of infection, with RhCMV-infected monkeys transmitting virus to one naïve animal every 7 weeks after introduction of RhCMV-WT into an uninfected cohort. These results demonstrate that the RhCMV model can be designed to rigorously reflect the challenges facing HCMV vaccine trials, particularly those related to horizontal transmission.


An abridged version of the Cochrane review of exercise therapy for chronic fatigue syndrome.
Chronic fatigue syndrome (CFS), also known as myalgic encephalomyelitis (ME) is estimated to affect between 2 in 1000 and 2 in 100 adults depending on how diagnostic criteria are applied. Patients with CFS have long-lasting fatigue in addition to symptoms including muscle pain, concentration and sleep problems. These symptoms cause significant disability and distress to the people affected. This review is an update of a previous Cochrane review (2004) that showed that exercise therapy was a promising treatment for adults with CFS. The aim of this systematic review was to determine the effects of exercise therapy for patients with CFS. Systematic review. Health care settings. Participants over 18 years with a primary diagnosis of CFS, able to attend an outpatient clinic for exercise therapy, were included. We searched electronic databases, including SPORTDiscus, up to May 2014 using a comprehensive list of free-text terms for CFS and exercise. Randomized clinical trials from all health care settings with participants over 18 years with a primary diagnosis of CFS, able to attend an outpatient clinic for exercise therapy, were included. We have included 8 randomized clinical studies that reported data from 1518 participants. Seven studies used aerobic exercise such as walking, swimming, or cycling and one study used non-aerobic exercise. The exercise therapies lasted between 12 and 26 weeks. Meta-analysis was done when appropriate. Exercise therapy was more effective at reducing fatigue than "passive" treatments or no treatment at end of treatment. Exercise therapy also had a positive effect on people's daily physical functioning, sleep quality and self-rated overall health. Nearly twice as many patients reported improvement self-rated overall health after exercise therapy (40 per 100) compared to standard treatment (22 per 100). The evidence was too sparse and/or of too low quality to conclude if exercise therapy has an effect on pain, quality of life, anxiety or depression. Exercise therapy was not found to worsen symptoms for people with CFS, while serious side effects were rare in all exercise and comparison groups. Patiens with CFS may generally benefit from and feel less fatigued following exercise therapy, and no evidence suggests that exercise therapy may worsen outcomes. Exercise therapy should be considered.


Use of laboratory-grown bacterial alginate in copper removal.
Industrial production leads to toxic heavy metal pollution in water bodies. Copper is one of the examples that requires removal from effluents before being discharged. It is difficult and sometimes very expensive to remove toxic heavy metals by conventional treatment techniques. This study aims to remove copper by the use of bacterial alginate as a non-conventional technique. Bacterial alginates (natural polymers composed of mannuronic and guluronic acid monomers) were synthesized by Azotobacter vinelandii ATCC(®) 9046 in a laboratory fermentor under controlled environmental conditions. The alginates produced, with a range of different characteristics in terms of monomer distribution and viscosity, were investigated for maximum copper uptake capacities. The average copper uptake capacities of alginates produced were found to be about 1.90 mmol/L Cu(2+)/g alginate. Although the GG-block amount of alginates was varied from 12 to 87% and culture broth viscosities were changed within the range of 1.47 and 14 cP, neither the block distribution nor viscosities of alginate samples considerably affected the copper uptake of alginates.


MRI and neuropsychological differences in early- and late-life-onset geriatric depression.
We sought to determine whether geriatric patients with late-life-onset major depression have more subcortical hyperintensities on MRI and greater cognitive impairment than age-matched geriatric patients with early-life-onset major depression, suggesting that subcortical disease may be etiologic in late-life depression. Most negative studies of the clinical significance of subcortical hyperintensities on MRI in geriatric patients have sampled from a restricted range of subjects, have employed limited batteries of neuropsychological tests, or have not quantified MRI changes; the present study attempted to address these limitations. Thirty subjects from a geriatric psychiatry inpatient service who were over 60 years of age and presented with major depression were divided into groups with onset of first depression after age 60 (mean = 72.4 years, 15 women, 0 men), and onset of first depression before age 60 (mean = 35.8 years, 12 women, 3 men). Quantitative analysis of MRI yielded the volume of: periventricular hyperintensities (PVH) and deep white-matter hyperintensities (DWMH). Subjects were administered a neuropsychological battery and measures of depression by raters blind to age of onset. The late-onset group had significantly more PVH and DWMH. They were also more impaired on executive and verbal and nonverbal memory tasks. Discriminant analysis using the severity or subcortical signal hyperintensities on MRI, cognitive index, and depression scores correctly predicted late versus early onset of depression in 87% of the early-onset group and 80% of the late-onset group. These findings suggest that late-life-onset depression may be associated with an increased severity of subcortical vascular disease and greater impairment of cognitive performance.


Platelet-derived growth factor CC-mediated neuroprotection against HIV Tat involves TRPC-mediated inactivation of GSK 3beta.
Platelet-derived growth factor-CC (PDGF-CC) is the third member of the PDGF family, and has been implicated both in embryogenesis and development of the CNS. The biological function of this isoform however, remains largely unexplored in the context of HIV-associated dementia (HAD). In the present study, we demonstrate that exposure of human neuroblastoma cells SH-SY5Y to HIV transactivator protein Tat resulted in decreased intrinsic expression of PDGF-CC as evidenced by RT-PCR and western blot assays. Reciprocally, pretreatment of SH-SY5Y cells with PDGF-CC abrogated Tat-mediated neurotoxicity by mitigating apoptosis and neurite & MAP-2 loss. Using pharmacological and loss of function approaches we identified the role of phosphatidylinositol 3-kinase (PI3K)/Akt signaling in PDGF-CC-mediated neuroprotection. We report herein a novel role about the involvement of transient receptor potential canonical (TRPC) channel 1 in modulation of calcium transients in PDGF-CC-mediated neuroprotection. Furthermore we also demonstrated PDGF-CC-mediated inactivation of the downstream mediator--glycogen synthase kinase 3β (GSK3β) evidenced by its phosphorylation at Ser-9. This was further validated by gain and loss of function studies using cells transfected with either the wild type or mutant GSK3β constructs. Intriguingly, pretreatment of cells with either the PI3K inhibitor or TRPC blocker resulted in failure of PDGF-CC to inactivate GSK3β, thereby suggesting the intersection of PI3K and TRPC signaling at GSK3β. Taken together our findings lead to the suggestion that PDGF-CC could be developed as a therapeutic target to reverse Tat-mediated neurotoxicity with implications for HAD.


Molecular and functional characterization of the Salmonella typhimurium invasion genes invB and invC: homology of InvC to the F0F1 ATPase family of proteins.
Entry into intestinal epithelial cells is an essential step in the pathogenesis of Salmonella infections. Our laboratory has previously identified a genetic locus, inv, that is necessary for efficient entry of Salmonella typhimurium into cultured epithelial cells. We have carried out a molecular and functional analysis of invB and invC, two members of this locus. The nucleotide sequence of these genes indicated that invB and invC encode polypeptides with molecular masses of 15 and 47 kDa, respectively. Polypeptides with the predicted sizes were observed when these genes were expressed under the control of a T7 promoter. Strains carrying nonpolar mutations in these genes were constructed, and their phenotypes were examined in a variety of assays. A mutation in invC rendered S. typhimurium defective in their ability to enter cultured epithelial cells, while mutations in invB did not. Comparison of the predicted sequences of InvB and InvC with translated sequences in GenBank revealed that these polypeptides are similar to the Shigella spp. proteins Spa15 and Spa47, which are involved in the surface presentation of the invasion protein antigens (Ipa) of these organisms. In addition, InvC showed significant similarity to a protein family which shares sequence homology with the catalytic beta subunit of the F0F1 ATPase from a number of microorganisms. Consistent with this finding, purified preparations of InvC showed significant ATPase activity. Site-directed mutagenesis of a residue essential for the catalytical function of this family of proteins resulted in a protein devoid of ATPase activity and unable to complement an invC mutant of S. typhimurium. These results suggest that InvC may energize the protein export apparatus encoded in the inv locus which is required for the surface presentation of determinants needed for the entry of Salmonella species into mammalian cells. The role of InvB in this process remains uncertain.


THE ETIOLOGY OF BARTONELLA MURIS ANEMIA OF THE ALBINO RAT : THE ISOLATION OF BARTONELLA MURIS.
1. Bartonella muris has been isolated in pure culture on Noguchi's leptospira medium from the blood of the splenectomized adult rat suffering with Bartonella muris anemia. 2. Bartonella muris is a small, actively motile, Gram-negative bacillus. It grows best on media containing blood and on Noguchi's leptospira medium. The optimal temperature for growth is 20-25 degrees C. It produces neither gas nor acid on media containing sugars. It does not hemolyze blood in artificial media. Viability of the cultures in leptospira media was maintained for 36 days. 3. With this culture a severe anemia was produced in rats weighing 30 gm., with the occasional appearance of Bartonella muris bodies on the red cells. The anemia occurred within 24 hours, and lasted for 3 to 5 days with recovery of the rat. Bartonella muris was recovered in pure culture from the blood of these animals. The blood of these rats was infectious for other 30 gm. rats. 4. 3 week old rabbits were infected with cultures of Bartonella muris, a severe anemia resulting after an incubation period of 48 hours. The organism was recovered from the blood on the 5th day after injection. Bartonella muris is non-pathogenic for adult rabbits. 5. A severe anemia was produced in young guinea pigs with cultures of Bartonella muris within 48 hours. The organism was recovered on the 2nd and 5th days after injection. Postmortem examination revealed changes in the organs similar to those found in splenectomized rats suffering with spontaneous Bartonella muris anemia. 6. The infection was reproduced in white mice. In one instance a severe anemia developed on the 5th and 6th days. The organism was recovered on the 6th day. 7. The anemia was produced in splenectomized adult rats of non-carrier stock. The organism was recovered from the blood stream of these rats. A marked leukocytosis was noted (65,000) at the peak of the anemia as is found in the spontaneous disease in infected splenectomized adult rats. 8. Serological tests have thus far failed to demonstrate the production of agglutinins, though complement-fixing antibodies are present in homologous sera in low dilutions.


Concerted activity of IgG1 antibodies and IL-4/IL-25-dependent effector cells trap helminth larvae in the tissues following vaccination with defined secreted antigens, providing sterile immunity to challenge infection.
Over 25% of the world's population are infected with helminth parasites, the majority of which colonise the gastrointestinal tract. However, no vaccine is yet available for human use, and mechanisms of protective immunity remain unclear. In the mouse model of Heligmosomoides polygyrus infection, vaccination with excretory-secretory (HES) antigens from adult parasites elicits sterilising immunity. Notably, three purified HES antigens (VAL-1, -2 and -3) are sufficient for effective vaccination. Protection is fully dependent upon specific IgG1 antibodies, but passive transfer confers only partial immunity to infection, indicating that cellular components are also required. Moreover, immune mice show greater cellular infiltration associated with trapping of larvae in the gut wall prior to their maturation. Intra-vital imaging of infected intestinal tissue revealed a four-fold increase in extravasation by LysM+GFP+ myeloid cells in vaccinated mice, and the massing of these cells around immature larvae. Mice deficient in FcRγ chain or C3 complement component remain fully immune, suggesting that in the presence of antibodies that directly neutralise parasite molecules, the myeloid compartment may attack larvae more quickly and effectively. Immunity to challenge infection was compromised in IL-4Rα- and IL-25-deficient mice, despite levels of specific antibody comparable to immune wild-type controls, while deficiencies in basophils, eosinophils or mast cells or CCR2-dependent inflammatory monocytes did not diminish immunity. Finally, we identify a suite of previously uncharacterised heat-labile vaccine antigens with homologs in human and veterinary parasites that together promote full immunity. Taken together, these data indicate that vaccine-induced immunity to intestinal helminths involves IgG1 antibodies directed against secreted proteins acting in concert with IL-25-dependent Type 2 myeloid effector populations.


Cyclophosphamide versus placebo in scleroderma lung disease.
We conducted a double-blind, randomized, placebo-controlled trial to determine the effects of oral cyclophosphamide on lung function and health-related symptoms in patients with evidence of active alveolitis and scleroderma-related interstitial lung disease. At 13 clinical centers throughout the United States, we enrolled 158 patients with scleroderma, restrictive lung physiology, dyspnea, and evidence of inflammatory interstitial lung disease on examination of bronchoalveolar-lavage fluid, thoracic high-resolution computed tomography, or both. Patients received oral cyclophosphamide (< or =2 mg per kilogram of body weight per day) or matching placebo for one year and were followed for an additional year. Pulmonary function was assessed every three months during the first year, and the primary end point was the forced vital capacity (FVC, expressed as a percentage of the predicted value) at 12 months, after adjustment for the baseline FVC. Of 158 patients, 145 completed at least six months of treatment and were included in the analysis. The mean absolute difference in adjusted 12-month FVC percent predicted between the cyclophosphamide and placebo groups was 2.53 percent (95 percent confidence interval, 0.28 to 4.79 percent), favoring cyclophosphamide (P<0.03). There were also treatment-related differences in physiological and symptom outcomes, and the difference in FVC was maintained at 24 months. There was a greater frequency of adverse events in the cyclophosphamide group, but the difference between the two groups in the number of serious adverse events was not significant. One year of oral cyclophosphamide in patients with symptomatic scleroderma-related interstitial lung disease had a significant but modest beneficial effect on lung function, dyspnea, thickening of the skin, and the health-related quality of life. The effects on lung function were maintained through the 24 months of the study.


A new anoplocephalid (Cestoda) from Tarentola parvicarinata (Lacertilia: Gekkonidae) in Senegal (West Africa).
The present study presents the description of Oochoristica beveridgei n. sp. (Linstowiinae Fuhrmann, 1907). The species was found in the small intestine of Tarentola parvicarinata Joger (Gekkonidae) in East Senegal (West Africa). Among known species of Oochoristica Lühe, 1898, O. beveridgei is most similar to Oochoristica junkea (Johri, 1950) Schmidt, 1986, found in Gekko gecko (Linnaeus) from India. A common feature of both species is the location of the cirrus sac, which lies anterior to the poral part of the ovary. The new species differs from O. junkea in having a higher number of testes, different testes arrangement, wider cirrus sac, and a different shape of ovary. The position of the cirrus sac in O. beveridgei is also similar to that found in Oochoristica gallica Dollfus, 1954, whose host is Psammodromus hispanicus Fitzinger (Lacertidae) from Morocco. The new species differs from O. gallica in that it possesses fewer testes and that these testes have a different arrangement. The location of the cirrus sac in O. beveridgei is also similar to that seen in Oochoristica bivitellolobata Loewen, 1940, whose host is Cnemidophorus sexlineatus (Linnaeus) (Teiidae) from the United States. The latter species has a higher number of testes, with only half of the cirrus sac located anterior to the poral part of the ovary, and possesses a divided vitellarium.


The Endosperm-Derived Embryo Sheath Is an Anti-adhesive Structure that Facilitates Cotyledon Emergence during Germination in Arabidopsis.
Germination sensu stricto in Arabidopsis involves seed-coat and endosperm rupture by the emerging seedling root. Subsequently, the cotyledons emerge rapidly from the extra-embryonic tissues of the seed, allowing autotrophic seedling establishment [1, 2]. Seedling survival depends upon the presence of an intact seedling cuticle that prevents dehydration, which has hitherto been assumed to form the interface between the newly germinated seedling and its environment [3-5]. Here, we show that in Arabidopsis, this is not the case. The primary interface between the emerging seedling and its environment is formed by an extra-cuticular endosperm-derived glycoprotein-rich structure called the sheath, which is maintained as a continuous layer at seedling surfaces during germination and becomes fragmented as cotyledons expand. Mutants lacking an endosperm-specific cysteine-rich peptide (KERBEROS [KRS]) show a complete loss of sheath production [6]. Although krs mutants have no defects in germination sensu stricto, they show delayed cotyledon emergence, a defect not observed in seedlings with defects in cuticle biosynthesis. Biophysical analyses reveal that the surfaces of wild-type cotyledons show minimal adhesion to silica beads in an aqueous environment at cotyledon emergence but that adhesion increases as cotyledons expand. In contrast, krs mutant cotyledons show enhanced adhesion at germination. Mutants with defects in cuticle biosynthesis, but no sheath defects, show a similar adhesion profile to wild-type seedlings at germination. We propose that the sheath reduces the adhesiveness of the cotyledon surface under the humid conditions necessary for seed germination and thus promotes seed-coat shedding and rapid seedling establishment.


Genotypic and phenotypic characterization of Noonan syndrome: new data and review of the literature.
Noonan syndrome (NS) is an autosomal dominant disorder, characterized by short stature, minor facial anomalies, and congenital heart defects. In approximately 50% of cases the condition is caused by missense mutations in the PTPN11 gene on chromosome 12, resulting in a gain of function of the protein SHP-2. In this study, PTPN11 mutation analysis was performed in 170 NS patients. In 76 (45%) of them a mutation was identified. We report on the distribution of these mutations, as well as on genotype-phenotype relationships. The benefit of the NS scoring system developed by van der Burgt et al. [(1994); Am J Med Genet 53:187-191] is shown, among physicians who consequently based their diagnosis on the NS scoring system the percentage mutation positive subjects was 54%, whereas this percentage was only 39% among physicians who made less use of the scoring system. In two patients with some uncommon manifestations mutations were found in the C-SH2 domain, a region in which defects are not often identified in NS. A trend was observed in patients carrying the 922A --> G change (Asn308Asp) receiving normal education. In one patient with NS and mild juvenile myelomonocytic leukemia (JMML) the mutation 218C --> T (Thr73Ile) was found. This confirms previous findings indicating that individuals with NS with specific mutations in PTPN11 are at risk of developing JMML.


GADD45A and CDKN1A are involved in apoptosis and cell cycle modulatory effects of viscumTT with further inactivation of the STAT3 pathway.
ViscumTT, a whole mistletoe preparation, has shown synergistic induction of apoptosis in several pediatric tumor entities. High therapeutic potential has previously been observed in Ewing's sarcoma, rhabdomyosarcoma, ALL and AML. In this study, we analyzed modulatory effects on the cell cycle by viscumTT in three osteosarcoma cell lines with various TP53 statuses. ViscumTT treatment induced G1 arrest in TP53 wild-type and null-mutant cells, but S arrest in TP53 mutant cells. Blockage of G1/S transition was accompanied by down-regulation of the key regulators CDK4, CCND1, CDK2, CCNE, CCNA. However, investigations on the transcriptional level revealed secondary TP53 participation. Cell cycle arrest was predominantly mediated by transcriptionally increased expression of GADD45A and CDKN1A and decreased SKP2 levels. Enhanced CDKN1A and GADD45A expression further played a role in viscumTT-induced apoptosis with involvement of stress-induced MAPK8 and inactivation of MAPK1/3. Furthermore, viscumTT inhibited the pro-survival pathway STAT3 by dephosphorylation of the two sites, Tyr705 and Ser727, by down-regulation of total STAT3 and its direct downstream targets BIRC5 and C-MYC. Moreover, tests of the efficacy of viscumTT in vivo showing reduction of tumor volume confirmed the high therapeutic potential as an anti-tumoral agent for osteosarcoma.


Variability of inter-team distances associated with match events in elite-standard soccer.
In soccer, critical match events like goal attempts can be preceded by periods of instability in the balance between the two teams' behaviours. Therefore, we determined periods of high variability in the distance between the teams' centroid positions longitudinally and laterally in an international-standard soccer match and evaluated corresponding match events. Position data were collected with AMISCO Pro®. Inter-team distance variability was calculated over a 3-s moving window. Out of the 242 match periods that exceeded the variability criterion, 51 were dead-ball situations. Match events identified through longitudinal inter-team distance primarily related to defending players moving forward-backward after a longitudinal pass. Match events identified through lateral inter-team distance mainly corresponded with defending players moving laterally following sideways passing. One of two goals and two of fourteen goal-attempts were preceded by a period of high variability. Together, periods of highly variable inter-team distance were associated with collective defensive actions and team reorganisation in dead-ball moments rather than goals or goal attempts. Inter-team dynamics quantified (mutual) reorganisation of the teams and marked teams' collective defensive ability to respond to attacking explorations.


Characterization of a sialate-O-acetylesterase (NanS) from the oral pathogen Tannerella forsythia that enhances sialic acid release by NanH, its cognate sialidase.
Tannerella forsythia, a Gram-negative member of the Bacteroidetes has evolved to harvest and utilize sialic acid. The most common sialic acid in humans is a mono-N-acetylated version termed Neu5Ac (5-N-acetyl-neuraminic acid). Many bacteria are known to access sialic acid using sialidase enzymes. However, in humans a high proportion of sialic acid contains a second acetyl group attached via an O-group, i.e. chiefly O-acetylated Neu5,9Ac2 or Neu5,4Ac2. This diacetylated sialic acid is not cleaved efficiently by many sialidases and in order to access diacetylated sialic acid, some organisms produce sialate-O-acetylesterases that catalyse the removal of the second acetyl group. In the present study, we performed bioinformatic and biochemical characterization of a putative sialate-O-acetylesterase from T. forsythia (NanS), which contains two putative SGNH-hydrolase domains related to sialate-O-acetylesterases from a range of organisms. Purification of recombinant NanS revealed an esterase that has activity against Neu5,9Ac2 and its glycolyl form Neu5Gc,9Ac. Importantly, the enzyme did not remove acetyl groups positioned at the 4-O position (Neu5,4Ac2). In addition NanS can act upon complex N-glycans released from a glycoprotein [erythropoietin (EPO)], bovine submaxillary mucin and oral epithelial cell-bound glycans. When incubated with its cognate sialidase, NanS increased sialic acid release from mucin and oral epithelial cell surfaces, implying that this esterase improves sialic acid harvesting for this pathogen and potentially other members of the oral microbiome. In summary, we have characterized a novel sialate-O-acetylesterase that contributes to the sialobiology of this important human pathogen and has potential applications in the analysis of sialic acid diacetylation of biologics in the pharmaceutical industry.


Genetic structuring and migration patterns of Atlantic bigeye tuna, Thunnus obesus (Lowe, 1839).
Large pelagic fishes are generally thought to have little population genetic structuring based on their cosmopolitan distribution, large population sizes and high dispersal capacities. However, gene flow can be influenced by ecological (e.g. homing behaviour) and physical (e.g. present-day ocean currents, past changes in sea temperature and levels) factors. In this regard, Atlantic bigeye tuna shows an interesting genetic structuring pattern with two highly divergent mitochondrial clades (Clades I and II), which are assumed to have been originated during the last Pleistocene glacial maxima. We assess genetic structure patterns of Atlantic bigeye tuna at the nuclear level, and compare them with mitochondrial evidence. We examined allele size variation of nine microsatellite loci in 380 individuals from the Gulf of Guinea, Canary, Azores, Canada, Indian Ocean, and Pacific Ocean. To investigate temporal stability of genetic structure, three Atlantic Ocean sites were re-sampled a second year. Hierarchical AMOVA tests, RST pairwise comparisons, isolation by distance (Mantel) tests, Bayesian clustering analyses, and coalescence-based migration rate inferences supported unrestricted gene flow within the Atlantic Ocean at the nuclear level, and therefore interbreeding between individuals belonging to both mitochondrial clades. Moreover, departures from HWE in several loci were inferred for the samples of Guinea, and attributed to a Wahlund effect supporting the role of this region as a spawning and nursery area. Our microsatellite data supported a single worldwide panmictic unit for bigeye tunas. Despite the strong Agulhas Current, immigration rates seem to be higher from the Atlantic Ocean into the Indo-Pacific Ocean, but the actual number of individuals moving per generation is relatively low compared to the large population sizes inhabiting each ocean basin. Lack of congruence between mt and nuclear evidences, which is also found in other species, most likely reflects past events of isolation and secondary contact. Given the inferred relatively low number of immigrants per generation around the Cape of Good Hope, the proportions of the mitochondrial clades in the different oceans may keep stable, and it seems plausible that the presence of individuals belonging to the mt Clade I in the Atlantic Ocean may be due to extensive migrations that predated the last glaciation.


Costs of Food Safety Investments in the Meat and Poultry Slaughter Industries.
To develop regulations efficiently, federal agencies need to know the costs of implementing various regulatory alternatives. As the regulatory agency responsible for the safety of meat and poultry products, the U.S. Dept. of Agriculture's Food Safety and Inspection Service is interested in the costs borne by meat and poultry establishments. This study estimated the costs of developing, validating, and reassessing hazard analysis and critical control points (HACCP), sanitary standard operating procedures (SSOP), and sampling plans; food safety training for new employees; antimicrobial equipment and solutions; sanitizing equipment; third-party audits; and microbial tests. Using results from an in-person expert consultation, web searches, and contacts with vendors, we estimated capital equipment, labor, materials, and other costs associated with these investments. Results are presented by establishment size (small and large) and species (beef, pork, chicken, and turkey), when applicable. For example, the cost of developing food safety plans, such as HACCP, SSOP, and sampling plans, can range from approximately $6000 to $87000, depending on the type of plan and establishment size. Food safety training costs from approximately $120 to $2500 per employee, depending on the course and type of employee. The costs of third-party audits range from approximately $13000 to $24000 per audit, and establishments are often subject to multiple audits per year. Knowing the cost of these investments will allow researchers and regulators to better assess the effects of food safety regulations and evaluate cost-effective alternatives.


Unusually Facile Thermal Homodienyl-[1,5]-Hydrogen Shift Reactions in Photochemically Generated Vinyl Aziridines.
A range of photochemically generated tri- and tetracyclic vinyl aziridines have been found to undergo a general and surprisingly low temperature ring opening through a [1,5]-hydrogen shift reaction. The rate of the process was found to be highly dependent on the structure and substitution around the azirdine ring and the alkene terminus, with some substrates being observed to undergo ring opening at temperatures as low as 25 °C. The rigid nature of these polycyclic systems precludes a conformational explanation of these rate differences, and an Eyring study confirmed a negligible entropic barrier to the reaction. However, the Eyring plots for two different aziridines systems showed a significant difference in their enthalpies of activation. It is therefore believed that the levels of aziridine ring strain, as well as electronic effects, are the dominant factors in this sequence.


Talimogene Laherparepvec in Combination With Ipilimumab in Previously Untreated, Unresectable Stage IIIB-IV Melanoma.
Combining immunotherapeutic agents with different mechanisms of action may enhance efficacy. We describe the safety and efficacy of talimogene laherparepvec (T-VEC; an oncolytic virus) in combination with ipilimumab (a cytotoxic T-lymphocyte-associated antigen 4 checkpoint inhibitor) in patients with advanced melanoma. In this open-label, multicenter, phase Ib trial of T-VEC in combination with ipilimumab, T-VEC was administered intratumorally in week 1 (10(6) plaque-forming units/mL), then in week 4 and every 2 weeks thereafter (10(8) plaque-forming units/mL). Ipilimumab (3 mg/kg) was administered intravenously every 3 weeks for four infusions, beginning in week 6. The primary end point was incidence of dose-limiting toxicities. Secondary end points were objective response rate by immune-related response criteria and safety. Median duration of treatment with T-VEC was 13.3 weeks (range, 2.0 to 95.4 weeks). Median follow-up time for survival analysis was 20.0 months (1.0 to 25.4 months). Nineteen patients were included in the safety analysis. No dose-limiting toxicities occurred, and no new safety signals were detected. Grade 3/4 treatment-related adverse events (AEs) were seen in 26.3% of patients; 15.8% had AEs attributed to T-VEC, and 21.1% had AEs attributed to ipilimumab. The objective response rate was 50%, and 44% of patients had a durable response lasting ≥ 6 months. Eighteen-month progression-free survival was 50%; 18-month overall survival was 67%. T-VEC with ipilimumab had a tolerable safety profile, and the combination appeared to have greater efficacy than either T-VEC or ipilimumab monotherapy.


Improving epistemological beliefs and moral judgment through an STS-based science ethics education program.
This study develops a Science-Technology-Society (STS)-based science ethics education program for high school students majoring in or planning to major in science and engineering. Our education program includes the fields of philosophy, history, sociology and ethics of science and technology, and other STS-related theories. We expected our STS-based science ethics education program to promote students' epistemological beliefs and moral judgment development. These psychological constructs are needed to properly solve complicated moral and social dilemmas in the fields of science and engineering. We applied this program to a group of Korean high school science students gifted in science and engineering. To measure the effects of this program, we used an essay-based qualitative measurement. The results indicate that there was significant development in both epistemological beliefs and moral judgment. In closing, we briefly discuss the need to develop epistemological beliefs and moral judgment using an STS-based science ethics education program.


Mouthing activity data for children age 3 to <6 years old and fraction of hand area mouthed for children age <6 years old in Taiwan.
Non-dietary ingestion is an important exposure pathway for children owing to their frequent hand-to-mouth and object-to-mouth activities involving soil and dust contacts. We used videotaping and the computer-based translating methods to quantify the mouthing activity information for 24 children ages 3 to <6 years old living in Taiwan. We also reviewed the entire mouthing activity data collected during the project to determine the lesser studied information on hand surface areas mouthed by children ages <6 years old. The median indoor hand-to-mouth and object-to-mouth frequencies were found to be 10 and 4.3 contacts/h, respectively. Hand-to-mouth and object-to-mouth contact frequencies used in exposure assessments for children ages 3 to <6 years old in this study were similar to the recommended values reported in United States. Exposure Factors Handbook for comparable age US children. The average fractions of the hand area mouthed for children 6 to <12 months, 1 to <2 years, 2 to <3 years, and 3 to <6 years old were 0.12, 0.12, 0.13, and 0.09, respectively. The fraction of hand area mouthed by children was found to be significantly and negatively correlated with their age. About half of the total hand-to-mouth contact events involved immersion of part of a hand or a finger into the mouth. The findings from this study extend the available mouthing activity information for 3 to <6 years old children and also provide new data for an Asian country, allowing comparison of results with western values collected mostly in the United States.


Long-term body-weight-supported treadmill training and subsequent follow-up in persons with chronic SCI: effects on functional walking ability and measures of subjective well-being.
Longitudinal, prospective within-subject design. (1) To determine the effects of long-term body-weight-supported treadmill training (BWSTT) on functional walking ability and perceived quality of life in persons with chronic incomplete spinal cord injury (SCI), and (2) to investigate whether training adaptations are maintained following cessation of the BWSTT programme. Hamilton, Ontario, Canada. A group of 14 individuals with chronic (mean 7.4 years postinjury) incomplete SCI (ASIA B & C) participated in thrice-weekly sessions of BWSTT for a period of approximately 12 months (144 sessions). Functional walking ability and indices of subjective well-being were evaluated during the training programme and over an 8-month follow-up. In total, 13 subjects successfully completed the 144 training sessions in the required study period (max. 15 months). Adherence to the thrice-weekly training frequency was 78.8%. All subjects improved in treadmill walking ability (54% reduction in required external body-weight support (BWS), 180% increase in treadmill walking speed, 335% increase in distance walked/session), and six subjects improved their capacity to walk over ground. There were accompanying increases in satisfaction with life and satisfaction with physical function, both of which were significantly correlated with improvements in treadmill walking ability. All but one subject returned for follow-up assessment 8 months post-training; while there was a slight decline in treadmill walking performance, over ground walking scores remained relatively stable. The only change in subjective well-being in the follow-up was a slight decrease in satisfaction with physical function. Thrice-weekly BWSTT for 12 months was an effective stimulus to improve treadmill walking ability and indices of subjective well-being in persons with chronic incomplete SCI, and most of these improvements were maintained for up to 8 months following the cessation of training.


Biosensor zebrafish provide new insights into potential health effects of environmental estrogens.
Environmental estrogens alter hormone signaling in the body that can induce reproductive abnormalities in both humans and wildlife. Available testing systems for estrogens are focused on specific systems such as reproduction. Crucially, however, the potential for significant health impacts of environmental estrogen exposures on a variety of body systems may have been overlooked. Our aim was to develop and apply a sensitive transgenic zebrafish model to assess real-time effects of environmental estrogens on signaling mechanisms in a whole body system for use in integrated health assessments. We created a novel transgenic biosensor zebrafish containing an estrogen-inducible promoter derived with multiple tandem estrogen responsive elements (EREs) and a Gal4ff-UAS system for enhanced response sensitivity. Using our novel estrogen-responsive transgenic (TG) zebrafish, we identified target tissues for environmental estrogens; these tissues have very high sensitivity even at environmentally relevant concentrations. Exposure of the TG fish to estrogenic endocrine-disrupting chemicals (EDCs) induced specific expression of green fluorescent protein (GFP) in a wide variety of tissues including the liver, heart, skeletal muscle, otic vesicle, forebrain, lateral line, and ganglions, most of which have not been established previously as targets for estrogens in fish. Furthermore, we found that different EDCs induced GFP expression with different tissue response patterns and time trajectories, suggesting different potential health effects. We have developed a powerful new model for understanding toxicological effects, mechanisms, and health impacts of environmental estrogens in vertebrates.


Overexpression of Psoriasin (S100A7) Contributes to Dysregulated Differentiation in Psoriasis.
Psoriasin, which is highly expressed in psoriasis, is encoded by a gene located within the epidermal differentiation complex. The aim of this study was to investigate the effect of endogenous psoriasin on disturbed keratinocyte differentiation in psoriasis. Immunohistochemical staining revealed a gradient of psoriasin expression in the psoriatic epidermis with highest expression in the suprabasal, differentiated layers. Induction of keratinocyte differentiation caused concurrent expression of psoriasin and the differentiation marker involucrin. The differentiation-induced psoriasin expression was found to be mediated by the protein kinase C pathway. The downregulation of psoriasin expression by small interfering RNA revealed that psoriasin mediates the expression of involucrin, desmoglein 1, transglutaminase 1 and CD24 in normal differentiation. The lentivirus-mediated overexpression of psoriasin, mimicking the psoriatic milieu, gave rise to an altered regulation of differentiation genes and an expression pattern reminiscent of that in psoriatic epidermis. These findings suggest that psoriasin contributes to the dysregulated differentiation process in the psoriasis epidermis.


Sky-Blue-Emissive Perovskite Light-Emitting Diodes: Crystal Growth and Interfacial Control Using Conjugated Polyelectrolytes as a Hole-Transporting Layer.
A series of poly(fluorene-co-phenylene)-based anionic conjugated polyelectrolytes (CPEs) are prepared with varying sizes of counterions (tetramethylammonium, tetraethylammonium, and tetrabutylammonium (TBA+)) and studied as a hole-transporting layer (HTL) for sky-blue-emissive perovskite light-emitting diodes (PeLEDs). Ionic CPE HTLs improve the wettability, compatibility, and nucleation of perovskite crystals at interfaces, enabling highly crystalline perovskite crystal growth with enhanced light-emitting properties. By incorporating the CPE HTLs containing bulky TBA+ counterions (MPS2-TBA) in place of PEDOT:PSS, the decreased phonon-electron coupling and increased exciton binding energy in perovskites are measured by temperature-dependent photoluminescence (PL) measurements. By increasing the size of counterions in CPE interlayers, the PL intensities and lifetimes of perovskite films increase. Through space-charge-limited current measurements, the lowest trap density is measured in the perovskite film on MPS2-TBA, emphasizing a critical role of larger counterions. Using density functional theory, MPS2-TBA is calculated to show the strongest adsorption affinity toward the interstitial defect of lead ions, explaining its pronounced interfacial defect passivation. The counterion size in CPE interlayers is interpreted as a main factor to determine the adsorption affinity onto perovskite, which determines the interacted area as noncovalent adsorption occurs. Finally, the sky-blue-emissive quasi-2D PeLED with MPS2-TBA shows the highest luminance efficiency (a peak EQE of 2.6% at 489 nm) and significantly improved spectral stability.


Dissecting the organ specificity of insecticide resistance candidate genes in Anopheles gambiae: known and novel candidate genes.
The elevated expression of enzymes with insecticide metabolism activity can lead to high levels of insecticide resistance in the malaria vector, Anopheles gambiae. In this study, adult female mosquitoes from an insecticide susceptible and resistant strain were dissected into four different body parts. RNA from each of these samples was used in microarray analysis to determine the enrichment patterns of the key detoxification gene families within the mosquito and to identify additional candidate insecticide resistance genes that may have been overlooked in previous experiments on whole organisms. A general enrichment in the transcription of genes from the four major detoxification gene families (carboxylesterases, glutathione transferases, UDP glucornyltransferases and cytochrome P450s) was observed in the midgut and malpighian tubules. Yet the subset of P450 genes that have previously been implicated in insecticide resistance in An gambiae, show a surprisingly varied profile of tissue enrichment, confirmed by qPCR and, for three candidates, by immunostaining. A stringent selection process was used to define a list of 105 genes that are significantly (p ≤0.001) over expressed in body parts from the resistant versus susceptible strain. Over half of these, including all the cytochrome P450s on this list, were identified in previous whole organism comparisons between the strains, but several new candidates were detected, notably from comparisons of the transcriptomes from dissected abdomen integuments. The use of RNA extracted from the whole organism to identify candidate insecticide resistance genes has a risk of missing candidates if key genes responsible for the phenotype have restricted expression within the body and/or are over expression only in certain tissues. However, as transcription of genes implicated in metabolic resistance to insecticides is not enriched in any one single organ, comparison of the transcriptome of individual dissected body parts cannot be recommended as a preferred means to identify new candidate insecticide resistant genes. Instead the rich data set on in vivo sites of transcription should be consulted when designing follow up qPCR validation steps, or for screening known candidates in field populations.


Physics-based simulation of the modulation transfer function in HgCdTe infrared detector arrays.
We have developed a numerical technique for performing physics-based simulations of the modulation transfer function (MTF) of infrared detector focal plane arrays. The finite-difference time-domain and finite element methods are employed to determine the electromagnetic and electrical response, respectively. We show how the total MTF can be decomposed to analyze the effect of lateral diffusion of charge carriers and present several methods for mitigation of such effects. We employ our numerical technique to analyze the MTF of a HgCdTe two-color bias-selectable infrared detector array.


Forward problem of electrocardiography: is it solved?
The relationship between epicardial and body surface potentials defines the forward problem of electrocardiography. A robust formulation of the forward problem is instrumental to solving the inverse problem, in which epicardial potentials are computed from known body surface potentials. Here, the accuracy of different forward models has been evaluated experimentally. Body surface and epicardial potentials were recorded simultaneously in anesthetized closed-chest pigs (n=5) during sinus rhythm, and epicardial and endocardial ventricular pacing (65 records in total). Body surface potentials were simulated from epicardial recordings using experiment-specific volume conductor models constructed from magnetic resonance imaging. Results for homogeneous (isotropic electric properties) and inhomogeneous (incorporating lungs, anisotropic skeletal muscle, and subcutaneous fat) forward models were compared with measured body surface potentials. Correlation coefficients were 0.85±0.08 across all animals and activation sequences with no significant difference between homogeneous and inhomogeneous solutions (P=0.85). Despite this, there was considerable variance between simulated and measured body surface potential distributions. Differences between the body surface potential extrema predicted with homogeneous forward models were 55% to 78% greater than observed (P<0.05) and attenuation of potentials adjacent to extrema were 10% to 171% greater (P<0.03). The length and orientation of the vector between potential extrema were also significantly different. Inclusion of inhomogeneous electric properties in the forward model reduced, but did not eliminate these differences. These results demonstrate that homogeneous volume conductor models introduce substantial spatial inaccuracies in forward problem solutions. This probably affects the precision of inverse reconstructions of cardiac potentials, in which this assumption is made.


Propolis as lipid bioactive nano-carrier for topical nasal drug delivery.
Propolis shows therapeutic properties ascribed to the presence of some flavonoids, phenolic acids, and their esters; it is a natural multifunctional material, solid at room temperature, and composed mainly of resin and waxes. We therefore used propolis as a lipid material to prepare solid lipid nanoparticles (SLNs); SLNs are proposed bioactive medications for topical intranasal therapy. Suitable formulation parameters were studied and the SLNs obtained by the high shear homogenization method were characterized; a selected formulation was viscosized to increase the residence time. Dimensional, morphological, and solid-state characterizations of the formulated SLNs were performed. In vitro and ex vivo permeation tests of diclofenac sodium, the model drug, and polyphenols were carried out. The propolis amount and surfactant concentration represent the key parameters that affect nanoparticle properties in terms of size, drug and polyphenol content, and physical stability. Size dispersions of about 600 nm and 0.4 PI were obtained, which do not change by increasing the viscosity. Drug is encapsulated in SLNs, as demonstrated by FTIR and DSC analyses. In vitro and ex vivo studies prove that drug and polyphenols do not cross the membranes; therefore, propolis-based SLNs could be used as delivery systems of diclofenac and flavonoids for the local treatment of nasal cavity diseases. Due to propolis composition, the proposed formulation could be used as a bioactive medication in which the carrier can exert a complementary effect with the loaded drug.


The additional costs of catheter-related bloodstream infections in intensive care units.
The additional costs of health care-associated infections vary depending on the reimbursement systems of different countries. We estimated the additional costs of central venous catheter-related bloodstream infections (CVC-CRBSI) in Japan, which has a universal health insurance system covering all citizens. We conducted a retrospective matched case-control study. Twenty-two patients with CVC-CRBSI were identified among 2,148 patients treated between October 2011 and May 2014 in the intensive care unit of Tokyo Medical University Hospital (1,015 beds). Twenty-two matched controls were selected on the basis of 5 criteria. The drug and medical material costs and technical fees incurred from the date of catheter insertion until hospital discharge were examined using a fee-for-service system. The additional costs of CVC-CRBSI were calculated as the difference between the costs of cases and controls. The contribution of antimicrobial drugs and the causative microorganism to the additional drug costs were also assessed. The additional costs of CVC-CRBSI were estimated at $57,090 per case. Antimicrobial agents comprised only about 10% of the additional drug costs. The additional costs of Candida infection were almost twice those of CVC-CRBSI caused by other microorganisms. The additional costs of CVC-CRBSI in Japan were estimated at $57,090 per case.


Stability of Cellular Immune Parameters over 12 Weeks in Patients with Major Depression or Somatoform Disorder and in Healthy Controls.
Cellular immune status in major depression (MD) patients differs from that in somatoform disorder (SFD) patients and healthy controls (HC). It is still questionable whether the patterns of immune parameters remain stable over time. Therefore, we studied lymphocyte and monocyte cell counts and neopterin levels in peripheral blood of MD and SFD patients and HC over 12 weeks and tested for correlations between biochemical and psychometric parameters. Thirty-nine patients with MD, 27 with SFD, and 51 HC were recruited. Peripheral blood was drawn at four visits, at 4-week intervals. We assessed the total cell count of B lymphocytes, natural killer (NK) cells, T lymphocyte subpopu-lations, and monocytes by flow cytometry, and neopterin serum levels by ELISA. Psychometric parameters were measured with questionnaires. Counts of lymphocytes, monocytes, and neopterin were stable in the SFD and HC groups. In the MD group, total CD3+, CD3+CD8+, NK cells, and CD3+CD25+ T cells showed inhomogeneous variances in Friedman tests, particularly in females. Neopterin correlated with depressed mood in MD patients, and with body mass index in HC. Cellular immune parameters are stable in HC and SFD. Our results may indicate influences of MD and gender on some cellular immune parameters. This may need to be considered in future immunological studies.


Red blood cell transfusion in patients with traumatic brain injury: a systematic review protocol.
Anemia is a prevalent condition in critically ill patients and red blood cell transfusions are frequent. Although transfusions at low hemoglobin levels have been shown to be associated with equivalent or better outcomes than higher hemoglobin thresholds, clinical equipoise persists in patients with traumatic brain injury considering their susceptibility to secondary cerebral insults such as those from hypoxemia. Our objectives are to estimate the frequency of red blood cell transfusion in patients with traumatic brain injury and to evaluate transfusion thresholds, determinants and outcomes associated with transfusion strategies.We will conduct a systematic review of cohort studies and randomized controlled trials of patients with traumatic brain injury. We will search MEDLINE, Embase, BIOSIS and the Cochrane Library for eligible studies. Two independent reviewers will screen all identified references. Studies including adult patients with traumatic brain injury reporting data on red blood cell transfusions will be eligible. We will collect data on baseline demographics, trauma characteristics, hemoglobin thresholds, blood transfusions and clinical outcomes (mortality, length of stay, complications, and so on). Two independent reviewers will extract data using a standardized form. We will pool cumulative incidences using DerSimonian and Lair random-effect models after a Freeman-Tukey transformation to stabilize variances. We will pool risk ratios or mean differences with random-effect models and Mantel-Haenszel or inverse variance methods in order to evaluate the association between red blood cell transfusion and potential determinants or outcomes. Sensitivity and subgroup analysis according to timing of red blood cell transfusion, traumatic brain injury severity, year of conduction of the study, risk of bias, notably, are planned. We expect to observe high heterogeneity in the proportion of transfused patients across studies and that the global proportion will be similar to the frequency observed in the general medical critically ill population. Our systematic review will allow us to better describe and understand current transfusion practices in patients with traumatic brain injury, a clinical population in which liberal transfusions are still advocated in the absence of evidence-based data. CRD42014007402.


Immunocytochemical distribution of the cannabinoid CB1 receptor in the primate neocortex: a regional and laminar analysis.
Delta-9-tetrahydrocannabinol (Delta9-THC) has profound effects on higher cognitive functions, and exposure to Delta9-THC has been associated with the appearance or exacerbation of the clinical features of schizophrenia. These actions appear to be mediated via the CB1 receptor, the principal cannabinoid receptor expressed in the brain. However, the distribution of the CB1 receptor in neocortical regions of the primate brain that mediate cognitive functions is not known. We therefore investigated the immunocytochemical localization of the CB1 receptor in the brains of macaque monkeys and humans using antibodies that specifically recognize the N- or C-terminus of the CB1 receptor. In monkeys, intense CB1 immunoreactivity was observed primarily in axons and boutons. Across neocortical regions of the monkey brain, CB1-immunoreactive (IR) axons exhibited considerable heterogeneity in density and laminar distribution. Neocortical association regions, such as the prefrontal and cingulate cortices, demonstrated a higher density, and exhibited a unique laminar pattern of CB1-IR axons, compared with primary sensory and motor cortices. Similar regional and laminar distributions of CB1-IR axons were also present in the human neocortex. CB1-IR axons had more prominent varicosities in human tissue, but this difference appeared to represent a postmortem effect as similar morphological features increased in unperfused monkey tissue as a function of postmortem interval. In electron microscopy studies of perfused monkey prefrontal cortex, CB1 immunoreactivity was predominantly found in axon terminals that exclusively formed symmetric synapses. The high density, distinctive laminar distribution, and localization to inhibitory terminals of CB1 receptors in primate higher-order association regions suggests that the CB1 receptor may play a critical role in the circuitry that subserves cognitive functions such as those that are disturbed in schizophrenia.


Structure of a DNA glycosylase searching for lesions.
DNA glycosylases must interrogate millions of base pairs of undamaged DNA in order to locate and then excise one damaged nucleobase. The nature of this search process remains poorly understood. Here we report the use of disulfide cross-linking (DXL) technology to obtain structures of a bacterial DNA glycosylase, MutM, interrogating undamaged DNA. These structures, solved to 2.0 angstrom resolution, reveal the nature of the search process: The protein inserts a probe residue into the helical stack and severely buckles the target base pair, which remains intrahelical. MutM therefore actively interrogates the intact DNA helix while searching for damage.


neXtA5: accelerating annotation of articles via automated approaches in neXtProt.
The rapid increase in the number of published articles poses a challenge for curated databases to remain up-to-date. To help the scientific community and database curators deal with this issue, we have developed an application, neXtA5, which prioritizes the literature for specific curation requirements. Our system, neXtA5, is a curation service composed of three main elements. The first component is a named-entity recognition module, which annotates MEDLINE over some predefined axes. This report focuses on three axes: Diseases, the Molecular Function and Biological Process sub-ontologies of the Gene Ontology (GO). The automatic annotations are then stored in a local database, BioMed, for each annotation axis. Additional entities such as species and chemical compounds are also identified. The second component is an existing search engine, which retrieves the most relevant MEDLINE records for any given query. The third component uses the content of BioMed to generate an axis-specific ranking, which takes into account the density of named-entities as stored in the Biomed database. The two ranked lists are ultimately merged using a linear combination, which has been specifically tuned to support the annotation of each axis. The fine-tuning of the coefficients is formally reported for each axis-driven search. Compared with PubMed, which is the system used by most curators, the improvement is the following: +231% for Diseases, +236% for Molecular Functions and +3153% for Biological Process when measuring the precision of the top-returned PMID (P0 or mean reciprocal rank). The current search methods significantly improve the search effectiveness of curators for three important curation axes. Further experiments are being performed to extend the curation types, in particular protein-protein interactions, which require specific relationship extraction capabilities. In parallel, user-friendly interfaces powered with a set of JSON web services are currently being implemented into the neXtProt annotation pipeline.Available on: http://babar.unige.ch:8082/neXtA5Database URL: http://babar.unige.ch:8082/neXtA5/fetcher.jsp.


Program evaluation for accreditation.
Program evaluation in preparation for accreditation provides opportunities for the identification of program strengths, weaknesses, and improvement potential. In the present study, naturalistic inquiry, an evaluation methodology responsive to the concerns of persons with a stake in the program, was successfully used in preparation for the accreditation of an occupational therapy program. The experiences of undergraduate students who implemented the program evaluation as part of an honors elective course describe the process, outcomes, benefits, and problems associated with the use of naturalistic inquiry as a responsive program evaluation.


Feline hyperthyroidism reported in primary-care veterinary practices in England: prevalence, associated factors and spatial distribution.
Feline hyperthyroidism is a commonly diagnosed endocrinopathy that can have a substantial deleterious impact on the welfare of affected cats. This study aimed to estimate the prevalence, associated factors and geographical distribution for feline hyperthyroidism in England, using primary-care veterinary practice clinical data from the VetCompass Animal Surveillance Project. Prevalence was estimated from the overall cat cohort. Associated factor analysis used an age-matched, nested, case-control design with multivariable logistic regression. There were 2,276 cases of feline hyperthyroidism identified from 95,629 cats attending 84 practices from September 2009 to December 2011. Cases were aged 6-25 years. 3.7 per cent of cases and 9.9 per cent of controls were purebred, 56.4 per cent of cases and 56.5 per cent of controls were female, and 88.1 per cent of cases and 86.0 per cent of controls were neutered. The apparent prevalence was 2.4 per cent (95% CI 2.3 to 2.5 per cent) overall, and 8.7 per cent (95% CI 8.3 to 9.0 per cent) in cats aged 10 years or above. Burmese (OR 0.15, 95% CI 0.07 to 0.32, P<0.0001), Persian (OR 0.17, 95% CI 0.08 to 0.33, P<0.0001), Siamese (OR 0.4, 95% CI 0.21 to 0.75, P=0.004) and purebred cats overall (OR 0.33, 95% CI 0.25 to 0.42, P< 0.0001) had lower odds of feline hyperthyroidism than non-purebred cats. Insured cats had increased odds (OR 1.78, 95% CI 1.56 to 2.03, P< 0.001). There was little evidence of spatial variation. This study highlights feline hyperthyroidism as a high-prevalence disease in England, and reports reduced odds of diagnosis in certain breeds and purebred cats overall.


Novel computational methods for increasing PCR primer design effectiveness in directed sequencing.
Polymerase chain reaction (PCR) is used in directed sequencing for the discovery of novel polymorphisms. As the first step in PCR directed sequencing, effective PCR primer design is crucial for obtaining high-quality sequence data for target regions. Since current computational primer design tools are not fully tuned with stable underlying laboratory protocols, researchers may still be forced to iteratively optimize protocols for failed amplifications after the primers have been ordered. Furthermore, potentially identifiable factors which contribute to PCR failures have yet to be elucidated. This inefficient approach to primer design is further intensified in a high-throughput laboratory, where hundreds of genes may be targeted in one experiment. We have developed a fully integrated computational PCR primer design pipeline that plays a key role in our high-throughput directed sequencing pipeline. Investigators may specify target regions defined through a rich set of descriptors, such as Ensembl accessions and arbitrary genomic coordinates. Primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the specified target regions. As part of the tiling process, primer pairs are computationally screened to meet the criteria for success with one of two PCR amplification protocols. In the process of improving our sequencing success rate, which currently exceeds 95% for exons, we have discovered novel and accurate computational methods capable of identifying primers that may lead to PCR failures. We reveal the laboratory protocols and their associated, empirically determined computational parameters, as well as describe the novel computational methods which may benefit others in future primer design research. The high-throughput PCR primer design pipeline has been very successful in providing the basis for high-quality directed sequencing results and for minimizing costs associated with labor and reprocessing. The modular architecture of the primer design software has made it possible to readily integrate additional primer critique tests based on iterative feedback from the laboratory. As a result, the primer design software, coupled with the laboratory protocols, serves as a powerful tool for low and high-throughput primer design to enable successful directed sequencing.


A TinyOS-based wireless neural interface.
The overlay of a neural interface upon a TinyOS-based sensing and communication platform is described. The system amplifies, digitally encodes, and transmits two EEG channels of neural signals from an un-tethered subject to a remote gateway, which routes the signals to a client PC. This work demonstrates the viability of the TinyOS-based sensor technology as a foundation for chronic remote biological monitoring applications, and thus provides an opportunity to create a system that can leverage from the frequent networking and communications advancements being made by the global TinyOS-development community.


Diffusion-limited phase separation in eukaryotic chemotaxis.
The ability of cells to sense spatial gradients of chemoattractant factors governs the development of complex eukaryotic organisms. Cells exposed to shallow chemoattractant gradients respond with strong accumulation of the enzyme phosphatidylinositol 3-kinase (PI3K) and its D3-phosphoinositide product (PIP(3)) on the plasma membrane side exposed to the highest chemoattractant concentration, whereas PIP(3)-degrading enzyme PTEN and its product PIP(2) localize in a complementary pattern. Such an early symmetry-breaking event is a mandatory step for directed cell movement elicited by chemoattractants, but its physical origin is still mysterious. Here, we propose that directional sensing is the consequence of a phase-ordering process mediated by phosphoinositide diffusion and driven by the distribution of chemotactic signal. By studying a realistic reaction-diffusion lattice model that describes PI3K and PTEN enzymatic activity, recruitment to the plasma membrane, and diffusion of their phosphoinositide products, we show that the effective enzyme-enzyme interaction induced by catalysis and diffusion introduces an instability of the system toward phase separation for realistic values of physical parameters. In this framework, large reversible amplification of shallow chemotactic gradients, selective localization of chemical factors, macroscopic response timescales, and spontaneous polarization arise naturally. The model is robust with respect to order-of-magnitude variations of the parameters.


The Lyn kinase C-lobe mediates Golgi export of Lyn through conformation-dependent ACSL3 association.
The Src-family tyrosine kinase Lyn has a role in signal transduction at the cytoplasmic face of the plasma membrane upon extracellular ligand stimulation. After synthesis in the cytoplasm, Lyn accumulates on the Golgi and is subsequently transported to the plasma membrane. However, the mechanism of Lyn trafficking remains elusive. We show here that the C-lobe of the Lyn kinase domain is associated with long-chain acyl-CoA synthetase 3 (ACSL3) on the Golgi in a manner that is dependent on Lyn conformation but is independent of its kinase activity. Formation of a closed conformation by CSK prevents Lyn from associating with ACSL3, resulting in blockade of Lyn export from the Golgi. Overexpression and knockdown of ACSL3 accelerates and blocks Golgi export of Lyn, respectively. The post-Golgi route of Lyn, triggered by ACSL3, is distinct from that of vesicular stomatitis virus glycoprotein (VSV-G) and of caveolin. Moreover, an ACSL3 mutant lacking the LR2 domain, which is required for the catalytic activity, retains the ability to associate with Lyn and accelerate Golgi export of Lyn. These results suggest that initiation of Golgi export of Lyn involves association of ACSL3 with the Lyn C-lobe, which is exposed to the molecular surface in an open conformation.


Allocating scarce medical resources during armed conflict: ethical issues.
We describe ethical issues arising in the allocation of civilian medical resources during armed conflict. Three features are significant in the context of allocating scarce resources in armed conflicts: the distinction between continuous and binary medical resources; the risks of armed conflict itself, and the impact of cultural differences on cases of armed conflict. We use these factors to elicit a modified principle for allocating medical resources during armed conflict, using hemodialysis for patients with end-stage renal disease as a case study.