Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India.
A Swedish patient of Indian origin traveled to New Delhi, India, and acquired a urinary tract infection caused by a carbapenem-resistant Klebsiella pneumoniae strain that typed to the sequence type 14 complex. The isolate, Klebsiella pneumoniae 05-506, was shown to possess a metallo-beta-lactamase (MBL) but was negative for previously known MBL genes. Gene libraries and amplification of class 1 integrons revealed three resistance-conferring regions; the first contained bla(CMY-4) flanked by ISEcP1 and blc. The second region of 4.8 kb contained a complex class 1 integron with the gene cassettes arr-2, a new erythromycin esterase gene; ereC; aadA1; and cmlA7. An intact ISCR1 element was shown to be downstream from the qac/sul genes. The third region consisted of a new MBL gene, designated bla(NDM-1), flanked on one side by K. pneumoniae DNA and a truncated IS26 element on its other side. The last two regions lie adjacent to one another, and all three regions are found on a 180-kb region that is easily transferable to recipient strains and that confers resistance to all antibiotics except fluoroquinolones and colistin. NDM-1 shares very little identity with other MBLs, with the most similar MBLs being VIM-1/VIM-2, with which it has only 32.4% identity. As well as possessing unique residues near the active site, NDM-1 also has an additional insert between positions 162 and 166 not present in other MBLs. NDM-1 has a molecular mass of 28 kDa, is monomeric, and can hydrolyze all beta-lactams except aztreonam. Compared to VIM-2, NDM-1 displays tighter binding to most cephalosporins, in particular, cefuroxime, cefotaxime, and cephalothin (cefalotin), and also to the penicillins. NDM-1 does not bind to the carbapenems as tightly as IMP-1 or VIM-2 and turns over the carbapenems at a rate similar to that of VIM-2. In addition to K. pneumoniae 05-506, bla(NDM-1) was found on a 140-kb plasmid in an Escherichia coli strain isolated from the patient's feces, inferring the possibility of in vivo conjugation. The broad resistance carried on these plasmids is a further worrying development for India, which already has high levels of antibiotic resistance.


Prevention of farm injuries in Denmark.
This study examined the effects of a 4-year randomized intervention program that combined a safety audit with safety behavior training in the prevention of farm injuries. From a random sample of farms in the county of Ringkoebing, Denmark, 393 farms with 1597 residents and employees participated in a weekly self-registration of work-related accidents and injuries during 1 year. Worktasks and time at risk were recorded. A questionnaire including items on safety behavior was also mailed to each farm. Thereafter, the farms were randomly assigned to an intervention or control group. Two hundred and one farms with 990 persons at risk participated in the intervention study. The main outcome measures were the number and severity of accidents, safety behavior, and farmsite safety audits. Pre- and postmeasurements showed a substantial reduction in injury rates in the intervention group in comparison with a slight reduction in the control group. In a multivariate regression analysis the intervention effect was estimated to be a 30% injury-rate reduction of all injuries, while there was a 42% reduction for medically treated injuries only. Although none of these effects are statistically significant with the present sample size, their magnitude and direction support an intervention effect. The measures of safety behavior revealed significant improvements, and this finding supports the conclusion that the intervention effect was positive, since they concern some of the mediating factors on the pathway from intervention to improved injury rates. This intervention, which focused on safety behavior and was performed as a randomized controlled trial, was followed by a substantial reduction in the number of farm injuries. The reduction was particularly marked for the more severe injuries demanding medical treatment.


Negotiating explanations: doctor-patient communication with patients with medically unexplained symptoms-a qualitative analysis.
Patients with medically unexplained physical symptoms (MUPS) seek explanations for their symptoms, but often find general practitioners (GPs) unable to deliver these. Different methods of explaining MUPS have been proposed. Little is known about how communication evolves around these explanations. To examine the dialogue between GPs and patients related to explanations in a community-based clinic for MUPS. We categorized dialogue types and dialogue outcomes. Patients were ≥18 years with inclusion criteria for moderate MUPS: ≥2 referrals to specialists, ≥1 functional syndrome/symptoms, ≥10 on the Patient Health Questionnaire-15 and GP's judgement that symptoms were unexplained. We analysed transcripts of 112 audio-recorded consultations (39 patients and 5 GPs) from two studies on the Symptoms Clinic Intervention, a consultation intervention for MUPS in primary care. We used constant comparative analysis to code and classify dialogue types and outcomes. We extracted 115 explanation sequences. We identified four dialogue types, differing in the extent to which the GP or patient controlled the dialogue. We categorized eight outcomes of the sequences, ranging from acceptance to rejection by the patient. The most common outcome was holding (conversation suspended in an unresolved state), followed by acceptance. Few explanations were rejected by the patient. Co-created explanations by patient and GP were most likely to be accepted. We developed a classification of dialogue types and outcomes in relation to explanations offered by GPs for MUPS patients. While it requires further validation, it provides a framework, which can be used for teaching, evaluation of practice and research.


Coexistence of optically active radial and axial CdTe insertions in single ZnTe nanowire.
We report on the growth, cathodoluminescence and micro-photoluminescence of individual radial and axial CdTe insertions in ZnTe nanowires. In particular, the cathodoluminescence technique is used to determine the position of each emitting object inside the nanowire. It is demonstrated that depending on the CdTe deposition temperature, one can obtain an emission either from axial CdTe insertions only, or from both, radial and axial heterostructures, simultaneously. At 350 °C CdTe grows only axially, whereas at 310 °C and 290 °C, there is also significant deposition on the nanowire sidewalls resulting in radial core/shell heterostructures. The presence of Cd atoms on the sidewalls is confirmed by energy dispersive X-ray spectroscopy. Micro-photoluminescence study reveals a strong linear polarization of the emission from both types of heterostructures in the direction along the nanowire axis.


Betahistine co-treatment ameliorates dyslipidemia induced by chronic olanzapine treatment in rats through modulation of hepatic AMPKα-SREBP-1 and PPARα-dependent pathways.
Second-generation antipsychotics including olanzapine are associated with weight gain, dyslipidemia and other metabolic disorders. Both animal and clinical studies have shown that co-treatment with betahistine (a histamine H1 receptor agonist/H3 receptor antagonist) is effective in controlling olanzapine-induced weight gain. In the present study, we investigate whether co-treatment with betahistine is able to prevent dyslipidemia induced by chronic olanzapine treatment and the underlying mechanisms. Female rats were orally administered with olanzapine (1 mg/kg, t.i.d.) for 3.5 consecutive weeks and then a 2.5-week drug withdrawal. Then, rats were divided into 4 groups for 5 weeks treatment: (1) vehicle, (2) olanzapine-only (1 mg/kg, t.i.d.), (3) betahistine-only (9.6 mg/kg, t.i.d.), and (4) olanzapine and betahistine (O+B) co-treatment. After completing treatment, hepatic mRNA expression was measured by qRT-PCR, while the protein levels were detected by western blot. In our study, olanzapine-only treatment significantly increased triglyceride accumulation and non-esterified fatty acids (NEFA), and upregulated mRNA expression of sterol regulatory element binding protein 1 (SREBP-1) and its target genes, while these alterations were ameliorated by O+B co-treatment. Hepatic AMP-activated protein kinase α (AMPKα) was activated in the O+B co-treatment group, with a significant reduction in nuclear SREBP-1 protein expression but an increased expression of peroxisome proliferator-activated receptor-α (PPARα) and its-responsive molecule(CPT1A), compared with olanzapine-only treatment. In addition, olanzapine significantly increased hepatic histamine H1 receptors, while O+B co-treatment significantly reversed them to normal levels. This study provided the first evidence that betahistine could act on hepatic H1 receptors via modulation of AMPKα-SREBP-1 and PPARα-dependent pathways to ameliorate olanzapine-induced dyslipidemia in rats.


Cerebral blood flow and cerebrovascular autoregulation in a swine model of pediatric cardiac arrest and hypothermia.
Knowledge remains limited regarding cerebral blood flow autoregulation after cardiac arrest and during postresuscitation hypothermia. We determined the relationship of cerebral blood flow to cerebral perfusion pressure in a swine model of pediatric hypoxic-asphyxic cardiac arrest during normothermia and hypothermia and tested novel measures of autoregulation derived from near-infrared spectroscopy. Prospective, balanced animal study. Basic physiology laboratory at an academic institution. Eighty-four neonatal swine. Piglets underwent hypoxic-asphyxic cardiac arrest or sham surgery and recovered for 2 hrs with normothermia followed by 4 hrs of either moderate hypothermia or normothermia. In half of the groups, blood pressure was slowly decreased through inflation of a balloon catheter in the inferior vena cava to identify the lower limit of cerebral autoregulation at 6 hrs postresuscitation. In the remaining groups, blood pressure was gradually increased by inflation of a balloon catheter in the aorta to determine the autoregulatory response to hypertension. Measures of autoregulation obtained from standard laser-Doppler flowmetry and indices derived from near-infrared spectroscopy were compared. Laser-Doppler flux was lower in postarrest animals compared to sham-operated controls during the 2-hr normothermic period after resuscitation. During the subsequent 4-hr recovery, hypothermia decreased laser-Doppler flux in both the sham surgery and postarrest groups. Autoregulation was intact during hypertension in all groups. With arterial hypotension, postarrest, hypothermic piglets had a significant decrease in the perfusion pressure lower limit of autoregulation compared to postarrest, normothermic piglets. The near-infrared spectroscopy-derived measures of autoregulation accurately detected loss of autoregulation during hypotension. In a pediatric model of cardiac arrest and resuscitation, delayed induction of hypothermia decreased cerebral perfusion and decreased the lower limit of autoregulation. Metrics derived from noninvasive near-infrared spectroscopy accurately identified the lower limit of autoregulation during normothermia and hypothermia in piglets resuscitated from arrest.


Laser speckle reduction by phase range limited computer generated hologram in laser projection display system.
The speckle phenomenon is an annoyance in laser projection display systems. We propose a novel speckle suppression method that utilizes the interference concept on a pixel point, which reduces the speckle contrast (SC) of the project image by limiting the phase distribution range in the optical field. The SC formula is derived in the uniform interval phase range for partially developed speckle conditions, showing that the SC can be lowered by lessening the phase range limitation. In the ideal simulation model, the SC can be reduced from 98.77% to 0% as the phase range limitation varies from 2π to 0. The phase range limitation model is a novel method using a computer generated hologram to provide beam shaping and phase limitation. In a more realistic simulation model, the SC is reduced from 99.18% to 16.68%.


Increased serum resistance in Klebsiella pneumoniae strains producing extended-spectrum beta-lactamases.
The aim of this study was to determine whether there is an association between serum resistance, O serotypes, and the production of extended-spectrum beta-lactamases (ESBLs) in Klebsiella pneumoniae. Ninety ESBL-producing and 178 non-ESBL-producing K. pneumoniae isolates gathered in five European countries were O serotyped and tested for sensitivity to the serum's bactericidal effect. The frequency of serum-resistant isolates was higher among ESBL-producing strains (30%; 27/90 isolates) than among non-ESBL-producing strains (17.9%; 32/178 isolates) (P = 0.037; odds ratio [OR] = 1.96; 95% confidence interval [95% CI] = 1.08 to 3.53). Although O1 was the most common O serotype in both Klebsiella groups, its frequency among ESBL-producing strains was significantly higher (59%; 53/90 isolates) than among non-ESBL producers (36%; 64/178 isolates) (P = 0.0006; OR = 2.5; 95% CI = 1.52 to 4.29). Furthermore, the prevalence of the O1 serotype was higher among serum-resistant strains of both ESBL-producing (74%; 20/27isolates) and non-ESBL producers (75%; 24/32 isolates) than among serum-sensitive ESBL producers (52.4%; 33/63 isolates) and non-ESBL producers (27.4%; 40/146 isolates). Serum resistance among ESBL-producing strains (36%; 17/47 isolates) versus non-ESBL-producing strains (16%; 27/166 isolates) was also significantly higher after the exclusion of clonal strains (P = 0.0056; OR = 2.9; 95% CI = 1.41 to 6.01). Sixteen ESBL types were detected, among which the frequency of serum resistance was significantly lower among the SHV-producing strains (9/48 isolates) than among the TEM producers (16/35 isolates) (P = 0.016; OR = 3.65; CI = 1.3 to 9.7). Curing ESBL-coding plasmids did not influence the serum resistance of the bacteria; all six plasmid-cured derivatives maintained serum resistance. The present findings suggest that ESBL-producing strains have a greater pathogenic potential than non-ESBL-producing strains, but the linkage between O serotypes, serum resistance, and ESBL production remains unclear at this stage.


Predicting long-term outcome after traumatic brain injury using repeated measurements of Glasgow Coma Scale and data mining methods.
Previous studies have identified some clinical parameters for predicting long-term functional recovery and mortality after traumatic brain injury (TBI). Here, data mining methods were combined with serial Glasgow Coma Scale (GCS) scores and clinical and laboratory parameters to predict 6-month functional outcome and mortality in patients with TBI. Data of consecutive adult patients presenting at a trauma center with moderate-to-severe head injury were retrospectively analyzed. Clinical parameters including serial GCS measurements at emergency department, 7th day, and 14th day and laboratory data were included for analysis (n = 115). We employed artificial neural network (ANN), naïve Bayes (NB), decision tree, and logistic regression to predict mortality and functional outcomes at 6 months after TBI. Favorable functional outcome was achieved by 34.8% of the patients, and overall 6-month mortality was 25.2%. For 6-month functional outcome prediction, ANN was the best model, with an area under the receiver operating characteristic curve (AUC) of 96.13%, sensitivity of 83.50%, and specificity of 89.73%. The best predictive model for mortality was NB with AUC of 91.14%, sensitivity of 81.17%, and specificity of 90.65%. Sensitivity analysis demonstrated GCS measurements on the 7th and 14th day and difference between emergency room and 14th day GCS score as the most influential attributes both in mortality and functional outcome prediction models. Analysis of serial GCS measurements using data mining methods provided additional predictive information in relation to 6-month mortality and functional outcome in patients with moderate-to-severe TBI.


Multi-pathway control of the proliferation versus meiotic development decision in the Caenorhabditis elegans germline.
An important event in the development of the germline is the initiation of meiotic development. In Caenorhabditis elegans, the conserved GLP-1/Notch signaling pathway regulates the proliferative versus meiotic entry decision, at least in part, by spatially inhibiting genes in the gld-1 and gld-2 parallel pathways, which are proposed to either inhibit proliferation and/or promote meiotic development. Mutations that cause constitutive activation of the GLP-1 pathway, or inactivation of both the gld-1 and gld-2 parallel pathways, result in a tumorous germline in which all cells are thought to be proliferative. Here, to analyze proliferation and meiotic entry in wild-type and mutant tumorous germlines, we use anti-REC-8 and anti-HIM-3 specific antibodies as markers, which under our fixation conditions, stain proliferative and meiotic cells, respectively. Using these makers in wild-type animals, we find that the border of the switch from proliferation to meiotic entry is staggered in late-larval and adult germlines. In wild-type adults, the switch occurs between 19 and 26 cell diameters from the distal end, on average. Our analysis of mutants reveals that tumorous germlines that form when GLP-1 is constitutively active are completely proliferative, while tumors due to inactivation of the gld-1 and gld-2 pathways show evidence of meiotic entry. Genetic and time course studies suggest that a third pathway may exist, parallel to the GLD-1 and GLD-2 pathways, that promotes meiotic development.


Magnitude and pattern of improvement in processes of care for hospitalised children with diarrhoea and dehydration in Kenyan hospitals participating in a clinical network.
WHO recommends optimisation of available interventions to reduce deaths of under-five children with diarrhoea and dehydration (DD). Clinical networks may help improve practice across many hospitals but experience with such networks is scarce. We describe magnitude and patterns of changes in processes of care for children with DD over the first 3 years of a clinical network. Observational study involving children aged 2-59 months with DD admitted to 13 hospitals participating in the clinical network. Processes of individual patient care including agreement of assessment, diagnosis and treatment according to WHO guidelines were combined using the composite Paediatric Admission Quality of Care (PAQC) score (range 0-6). Data from 7657 children were analysed and improvements in PAQC scores were observed. Predicted mean PAQC score for all the hospitals at enrolment was 59.8% (95% CI: 54.7, 64.9) but showed a wide variation (variance 10.7%, 95% CI: 5.8, 19.6). Overall mean PAQC score increased by 13.8% (95% CI: 8.7-18.9, SD between hospitals: ±8.2) in the first 12 months, with an average 0.9% (95% CI: 0.3-1.5, SD ± 1.0) increase per month and plateaued thereafter, and changes were similar in two groups of hospitals joining the network at different times. Adherence to guidelines for children admitted with DD can be improved through participation in a clinical network but improvement is limited, not uniform for all aspects of care and contexts and occurs early. Future research should address these issues.


Efficacy of immunotherapy with TG4040, peg-interferon, and ribavirin in a Phase 2 study of patients with chronic HCV infection.
TG4040 is a modified vaccinia Ankara (MVA) virus that expresses the hepatitis C virus (HCV) proteins NS3, NS4, and NS5B. We performed a phase II open-label study to determine the efficacy, safety, and immunotherapeutic properties of TG4040 in combination with pegylated interferon α-2a and ribavirin (PEG-IFNα/RBV) in patients with chronic HCV infection. Treatment-naive patients with HCV genotype 1 infection were assigned randomly to 1 of the following groups: PEG-IFNα/RBV for 48 weeks (group A, n = 31), PEG-IFNα/RBV for 4 weeks followed by PEG-IFNα/RBV for 44 weeks with 6 injections of TG4040 (group B, n = 63), or TG4040 for 12 weeks (7 injections) followed by PEG-IFNα/RBV for 48 weeks with 6 injections of TG4040 (group C, n = 59). The primary end point was complete early virologic response (cEVR), defined as HCV-RNA level less than 10 IU/mL after 12 weeks of PEG-IFNα/RBV treatment. In group C, 64.2% of evaluable patients achieved cEVR, compared with 30.0% in group A and 45.9% in group B (P = .0003 for group C vs A). A higher percentage of patients achieved a sustained virologic response 24 weeks after therapy ended in group C (58.2%) than in groups A (48.4%) or B (50.8%). HCV- and MVA-specific T-cell responses were observed predominantly in group C. As expected, most patients given injections of TG4040 developed anti-MVA antibodies. The combination of TG4040 and PEG-IFNα/RBV was reasonably well tolerated. However, PEG-IFNα-associated thrombocytopenia developed in 3 patients who carried the class II HLA allele DRB01*04. A higher percentage of patients with chronic HCV infection who received immunotherapy with TG4040 followed by TG4040 and PEG-IFNα/RBV achieved a cEVR compared with patients who received only PEG-IFNα/RBV therapy. These findings show that immunotherapies that activate T cells are effective in patients with chronic HCV infection. ClinicalTrials.gov number, NCT01055821.


Development in vitro of Marsupials: a comparative review of species and a timetable of cleavage and early blastocyst stages of development in Monodelphis domestica.
The development of marsupial oocytes and embryos in vitro is reviewed. Most stages of development have been cultured successfully, usually in a complex medium with added fetal calf serum. Simpler media without added serum have been developed for fertilization and cleavage in vitro. Culture systems have been established for oocyte maturation and fertilization in the grey short-tailed opossum and for cleavage from the zygote to the early expanding unilaminar blastocyst in a number of other marsupials. Survival in vitro of the unilaminar and early bilaminar blastocyst stages is limited in all species examined. In contrast, late bilaminar, trilaminar, embryonic and fetal stages develop at rates approximating those in vivo. More stages have been cultured successfully in Sminthopsis macroura than in any other species. It has been cultured from the late bilaminar blastocyst to within 18 h of birth. Stages of cleavage and unilaminar blastocyst formation of Monodelphis domestica timed by videotaping mating animals, proceeded at similar rates in vivo and in vitro. As in other marsupials, cleavage in this opossum is characterized by a polarized conceptus. This polarity is expressed in the distribution of organelles in the zygote and the localization of secretion of the extracellular matrix material into the cleavage cavity and of the initial cell-zona attachment. Because cell-cell adhesion follows cell-zona adhesion, a unilaminar blastocyst forms without the development of an intervening morula stage.


TSP-1 secreted by bone marrow stromal cells contributes to retinal ganglion cell neurite outgrowth and survival.
Bone marrow stromal cells (BMSCs) are pluripotent and thereby a potential candidate for cell replacement therapy for central nervous system degenerative disorders and traumatic injury. However, the mechanism of their differentiation and effect on neural tissues has not been fully elucidated. This study evaluates the effect of BMSCs on neural cell growth and survival in a retinal ganglion cell (RGCs) model by assessing the effect of changes in the expression of a BMSC-secreted protein, thrombospondin-1 (TSP-1), as a putative mechanistic agent acting on RGCs. The effect of co-culturing BMSCs and RGCs in vitro was evaluated by measuring the following parameters: neurite outgrowth, RGC survival, BMSC neural-like differentiation, and the effect of TSP-1 on both cell lines under basal secretion conditions and when TSP-1 expression was inhibited. Our data show that BMSCs improved RGC survival and neurite outgrowth. Synaptophysin, MAP-2, and TGF-beta expression are up-regulated in RGCs co-cultured with BMSCs. Interestingly, the BMSCs progressively displayed neural-like morphology over the seven-day study period. Restriction display polymerase chain reaction (RD-PCR) was performed to screen for differentially expressed genes in BMSCs cultured alone or co-cultured with RGCs. TSP-1, a multifactorial extracellular matrix protein, is critically important in the formation of neural connections during development, so its function in our co-culture model was investigated by small interfering RNA (siRNA) transfection. When TSP-1 expression was decreased with siRNA silencing, BMSCs had no impact on RGC survival, but reduced neurite outgrowth and decreased expression of synaptophysin, MAP-2 and TGF-beta in RGCs. Furthermore, the number of BMSCs with neural-like characteristics was significantly decreased by more than two-fold using siRNA silencing. Our data suggest that the TSP-1 signaling pathway might have an important role in neural-like differentiation in BMSCs and neurite outgrowth in RGCs. This study provides new insights into the potential reparative mechanisms of neural cell repair.


Industry-university collaborations in Canada, Japan, the UK and USA--with emphasis on publication freedom and managing the intellectual property lock-up problem.
As industry-university collaborations are promoted to commercialize university research and foster economic growth, it is important to understand how companies benefit from these collaborations, and to ensure that resulting academic discoveries are developed for the benefit of all stakeholders: companies, universities and public. Lock up of inventions, and censoring of academic publications, should be avoided if feasible. This case-study analysis of interviews with 90 companies in Canada, Japan, the UK and USA assesses the scope of this challenge and suggests possible resolutions. The participating companies were asked to describe an important interaction with universities, and most described collaborative research. The most frequently cited tensions concerned intellectual property management and publication freedom. IP disagreements were most frequent in the context of narrowly-focused collaborations with American universities. However, in the case of exploratory research, companies accepted the IP management practices of US universities. It might make sense to let companies have an automatic exclusive license to IP from narrowly defined collaborations, but to encourage universities to manage inventions from exploratory collaborations to ensure development incentives. Although Canada, the UK and US have strong publication freedom guarantees, tensions over this issue arose frequently in focused collaborations, though were rare in exploratory collaborations. The UK Lambert Agreements give sponsors the option to control publications in return for paying the full economic cost of a project. This may offer a model for the other three countries. Uniquely among the four countries, Japan enables companies to control exclusively most collaborative inventions and to censor academic publications. Despite this high degree of control, the interviews suggest many companies do not develop university discoveries to their full potential. The steps suggested above may rebalance the situation in Japan. Overall, the interviews reveal the complexity of these issues and the need for flexibility on the part of universities and companies.


CaspR: a web server for automated molecular replacement using homology modelling.
Molecular replacement (MR) is the method of choice for X-ray crystallography structure determination when structural homologues are available in the Protein Data Bank (PDB). Although the success rate of MR decreases sharply when the sequence similarity between template and target proteins drops below 35% identical residues, it has been found that screening for MR solutions with a large number of different homology models may still produce a suitable solution where the original template failed. Here we present the web tool CaspR, implementing such a strategy in an automated manner. On input of experimental diffraction data, of the corresponding target sequence and of one or several potential templates, CaspR executes an optimized molecular replacement procedure using a combination of well-established stand-alone software tools. The protocol of model building and screening begins with the generation of multiple structure-sequence alignments produced with T-COFFEE, followed by homology model building using MODELLER, molecular replacement with AMoRe and model refinement based on CNS. As a result, CaspR provides a progress report in the form of hierarchically organized summary sheets that describe the different stages of the computation with an increasing level of detail. For the 10 highest-scoring potential solutions, pre-refined structures are made available for download in PDB format. Results already obtained with CaspR and reported on the web server suggest that such a strategy significantly increases the fraction of protein structures which may be solved by MR. Moreover, even in situations where standard MR yields a solution, pre-refined homology models produced by CaspR significantly reduce the time-consuming refinement process. We expect this automated procedure to have a significant impact on the throughput of large-scale structural genomics projects. CaspR is freely available at http://igs-server.cnrs-mrs.fr/Caspr/.


VKORC1 polymorphisms, haplotypes and haplotype groups on warfarin dose among African-Americans and European-Americans.
Although the influence of VKORC1 and CYP2C9 polymorphisms on warfarin response has been studied, variability in dose explained by CYP2C9 and VKORC1 is lower among African-Americans compared with European-Americans. This has lead investigators to hypothesize that assessment of VKORC1 haplotypes may help capture a greater proportion of the variability in dose for this under-represented group. However, the inadequate representation of African-Americans and the assessment of a few VKORC1 polymorphisms have hindered this effort. To determine if VKORC1 haplotypes or haplotype groups explain a higher variability in warfarin dose, we comprehensively assessed VKORC1 polymorphisms in 273 African-Americans and 302 European-Americans. The influence of VKORC1 polymorphisms, race-specific haplotypes and haplotype groups on warfarin dose was evaluated in race-stratified multivariable analyses after accounting for CYP2C9 (*2, *3, *5, *6 and *11) and clinical covariates. VKORC1 explained 18% (30% with CYP2C9) variability in warfarin dose among European-Americans and 5% (8% with CYP2C9) among African-Americans. Four common haplotypes in European-Americans and twelve in African-Americans were identified. In each race VKORC1 haplotypes emerged into two groups: low-dose (Group A) and high-dose (Group B). African-Americans had a lower frequency of Group A haplotype (10.6%) compared with European-Americans (35%, p < 0.0001).The variability in dose explained by VKORC1 haplotype or haplotype groups was similar to that of a single informative polymorphism. Our findings support the use of CYP2C9, VKORC1 polymorphisms (rs9934438 or rs9923231) and clinical covariates to predict warfarin dose in both African- and European-Americans. A uniform set of common polymorphisms in CYP2C9 and VKORC1, and limited clinical covariates can be used to improve warfarin dose prediction for a racially diverse population.


Visual attention is not deployed at the endpoint of averaging saccades.
The premotor theory of attention postulates that spatial attention arises from the activation of saccade areas and that the deployment of attention is the consequence of motor programming. Yet attentional and oculomotor processes have been shown to be dissociable at the neuronal level in covert attention tasks. To investigate a potential dissociation at the behavioral level, we instructed human participants to move their eyes (saccade) towards 1 of 2 nearby, competing saccade targets. The spatial distribution of visual attention was determined using oriented visual stimuli presented either at the target locations, between them, or at several other equidistant locations. Results demonstrate that accurate saccades towards one of the targets were associated with presaccadic enhancement of visual sensitivity at the respective saccade endpoint compared to the nonsaccaded target location. In contrast, averaging saccades, landing between the 2 targets, were not associated with attentional facilitation at the saccade endpoint. Rather, attention before averaging saccades was equally deployed at the 2 target locations. Taken together, our results reveal that visual attention is not obligatorily coupled to the endpoint of a subsequent saccade. Rather, our results suggest that the oculomotor program depends on the state of attentional selection before saccade onset and that saccade averaging arises from unresolved attentional selection.


Causes and circumstances of maternal death: a secondary analysis of the Community-Level Interventions for Pre-eclampsia (CLIP) trials cohort.
Incomplete vital registration systems mean that causes of death during pregnancy and childbirth are poorly understood in low-income and middle-income countries. To inform global efforts to reduce maternal mortality, we compared physician review and computerised analysis of verbal autopsies (interpreting verbal autopsies [InterVA] software), to understand their agreement on maternal cause of death and circumstances of mortality categories (COMCATs) in the Community-Level Interventions for Pre-eclampsia (CLIP) cluster randomised trials. The CLIP trials took place in India, Pakistan, and Mozambique, enrolling pregnant women aged 12-49 years between Nov 1, 2014, and Feb 28, 2017. 69 330 pregnant women were enrolled in 44 clusters (36 008 in the 22 intervention clusters and 33 322 in the 22 control clusters). In this secondary analysis of maternal deaths in CLIP, we included women who died in any of the 22 intervention clusters or 22 control clusters. Trained staff administered the WHO 2012 verbal autopsy after maternal deaths. Two physicians (and a third for consensus, if needed) reviewed trial surveillance data and verbal autopsies, and, in intervention clusters, community health worker-led visit data. They determined cause of death according to the WHO International Classification of Diseases-Maternal Mortality (ICD-MM). Verbal autopsies were also analysed by InterVA computer models (versions 4 and 5) to generate cause of death. COMCAT analysis was provided by InterVA-5 and, in India, by physician review of Maternal Newborn Health Registry data. Causes of death and COMCATs assigned by physician review, Inter-VA-4, and InterVA-5 were compared, with agreement assessed with Cohen's κ coefficient. Of 61 988 pregnancies with successful follow-up in the CLIP trials, 143 maternal deaths were reported (16 deaths in India, 105 in Pakistan, and 22 in Mozambique). The maternal death rate was 231 (95% CI 193-268) per 100 000 identified pregnancies. Most deaths were attributed to direct maternal causes (rather than indirect or undetermined causes as per ICD-MM classification), with fair to good agreement between physician review and InterVA-4 (κ=0·56 [95% CI 0·43-0·66]) or InterVA-5 (κ=0·44 [0·30-0·57]), and InterVA-4 and InterVA-5 (κ=0·72 [0·60-0·84]). The top three causes of death were the same by physician review, InterVA-4, and InterVA-5 (ICD-MM categories obstetric haemorrhage, non-obstetric complications, and hypertensive disorders); however, attribution of individual patient deaths to obstetric haemorrhage varied more between methods (physician review, 38 [27%] deaths; InterVA-4, 69 [48%] deaths; and InterVA-5, 82 [57%] deaths), than did attribution to non-obstetric causes (physician review, 39 [27%] deaths; InterVA-4, 37 [26%] deaths; and InterVA-5, 28 [20%] deaths) or hypertensive disorders (physician review, 23 [16%] deaths; InterVA-4, 25 [17%] deaths; and InterVA-5, 24 [17%] deaths). Agreement for all nine ICD-MM categories was fair for physician review versus InterVA-4 (κ=0·48 [0·38-0·58]), poor for physician review versus InterVA-5 (κ=0·36 [0·27-0·46]), and good for InterVA-4 versus InterVA-5 (κ=0·69 [0·59-0·79]). The most commonly assigned COMCATs by InterVA-5 were emergencies (68 [48%] of 143 deaths) and health systems (62 [43%] deaths), and by physician review (India only) were health systems (seven [44%] of 16 deaths) and inevitability (five [31%] deaths); agreement between InterVA-5 and physician review (India data only) was poor (κ=0·04 [0·00-0·15]). Our findings indicate that InterVA-5 is less accurate than InterVA-4 at ascertaining causes and circumstances of maternal death, when compared with physician review. Our results suggest a need to improve the next iteration of InterVA, and for researchers and clinicians to preferentially use InterVA-4 when recording maternal deaths. University of British Columbia (grantee of the Bill & Melinda Gates Foundation).


Preformed Donor-specific Antibodies Against HLA Class II and Graft Outcomes in Deceased-donor Kidney Transplantation.
Many kidney transplant centers in the United States report both HLA class I and II antibodies detected by sensitive solid-phase assays (SPAs) to United Network for Organ Sharing as unacceptable antigens, significantly reducing the compatible donor organ pool and prolonging waiting time for highly sensitized patients. However, the clinical relevance of all detected donor-specific antibodies (DSAs) by SPA is not unequivocal, because fluorescence intensity does not always accurately reflect antibody pathogenicity. Our center does not exclude patients from transplantation based on DSA class II. We performed a retrospective analysis in 179 deceased-donor kidney transplant recipients with solely DSA class II before transplant and patients without DSA and compared graft survival, rejection, and clinical outcomes. Patient survival was also compared with matched controls on the waiting list. Patients transplanted with DSA class II showed a clear survival benefit compared with matched patients who remained on dialysis or were waitlisted on dialysis/transplanted at 5 years (100%, 34%, and 73%, respectively). After a mean follow-up of 5.5 years, there was no significant difference in death-censored graft survival between transplanted patients without DSA and those with preformed DSA class II (adjusted HR 1.10; 95% confidence interval, 0.41-2.97), although the incidence of rejection was higher in recipients with DSA class II (adjusted HR 5.84; 95% confidence interval, 2.58-13.23; P < 0.001). Serum creatinine levels at 1, 3, and 5 years posttransplant did not differ between groups. No predictors of rejection were found, although patients who received basiliximab induction therapy had higher incidence of rejection (100%) compared with those who received antithymocyte globulin (52%). We conclude that for highly sensitized patients, deceased-donor kidney transplantation with DSA class II yields a survival benefit over prolonged waiting time on dialysis. Instead of listing DSA class II as unacceptable antigens, an individual approach with further immunologic risk assessment is recommended.


Genomic and physiological analyses of the zebrafish atrioventricular canal reveal molecular building blocks of the secondary pacemaker region.
The atrioventricular canal (AVC) is the site where key structures responsible for functional division between heart regions are established, most importantly, the atrioventricular (AV) conduction system and cardiac valves. To elucidate the mechanism underlying AVC development and function, we utilized transgenic zebrafish line sqet31Et expressing EGFP in the AVC to isolate this cell population and profile its transcriptome at 48 and 72 hpf. The zebrafish AVC transcriptome exhibits hallmarks of mammalian AV node, including the expression of genes implicated in its development and those encoding connexins forming low conductance gap junctions. Transcriptome analysis uncovered protein-coding and noncoding transcripts enriched in AVC, which have not been previously associated with this structure, as well as dynamic expression of epithelial-to-mesenchymal transition markers and components of TGF-β, Notch, and Wnt signaling pathways likely reflecting ongoing AVC and valve development. Using transgenic line Tg(myl7:mermaid) encoding voltage-sensitive fluorescent protein, we show that abolishing the pacemaker-containing sinoatrial ring (SAR) through Isl1 loss of function resulted in spontaneous activation in the AVC region, suggesting that it possesses inherent automaticity although insufficient to replace the SAR. The SAR and AVC transcriptomes express partially overlapping species of ion channels and gap junction proteins, reflecting their distinct roles. Besides identifying conserved aspects between zebrafish and mammalian conduction systems, our results established molecular hallmarks of the developing AVC which underlies its role in structural and electrophysiological separation between heart chambers. This data constitutes a valuable resource for studying AVC development and function, and identification of novel candidate genes implicated in these processes.


Novel Phenotypic Fluorescent Three-Dimensional Co-Culture Platforms for Recapitulating Tumor in vivo Progression and for Personalized Therapy.
Because three-dimensional (3D) in vitro models are more accurate than 2D cell culture models and faster and cheaper than animal models, they have become a prospective trend in the biomedical and pharmaceutical fields, especially for personalized and targeted therapies. Because appropriate 3D models can be customized to mimic the in vivo microenvironment wherein various cell populations grow within an intricate but well organized extracellular matrix (ECM), they can accurately recapitulate physiological and pathophysiological progressions. The majority of cancers are carcinomas, which originate from epithelial cells, and dynamically interact with non-malignant cells including stromal cells (fibroblasts), vascular cells (endothelial cells and pericytes), immune cells (macrophages and mast cells), and the ECM. Employing a tumor monoclonal colony, tumor xenograft or patient cancer biopsy into an in vivo-like microenvironment, the native signaling pathways, cell-cell and cell-matrix interactions, and cell phenotypes are preserved and our fluorescent phenotypic 3D co-culture platforms can then accurately recapitulate the tumor in vivo scenario including tumor induced angiogenesis, tumor growth, and metastasis. In this paper, we describe a robust and standardized method to co-culture a tumor colony or biopsy with different cell populations, e.g., endothelial cells, immune cells, pericytes, etc. The procedures for recovering cells from the co-culture for molecular analyses, imaging, and analyzing are also described. We selected ECM solubilized extract derived from Engelbreth-Holm-Swam sarcoma cells. Because the 3D co-culture platforms can provide drug chemosensitivity data within 9 days that is equivalent to the results generated from mouse tumor xenograft models in 50 days, the 3D co-culture platforms are more accurate, efficient, and cost-effective and may replace animal models in the near future to predict drug efficacy, personalize therapies, prevent drug resistance, and improve the quality of life.


Localization of spectrin in mammalian brain.
Spectrin is a major skeletal component of the erythrocyte membrane and is essential in controlling cell shape and structural stability. The brain has also been found to be rich in an immunoreactive and structural analogue of spectrin. In the present study, spectrin was localized in the mouse brain by indirect immunofluorescence using an antibody to erythrocyte spectrin that cross-reacts specifically with the alpha and beta subunits of brain spectrin. Spectrin antigens were concentrated in neuronal perikarya and cell processes. Synaptic structures and axons were observed to have little detectable spectrin antigen by immunofluorescence methodology. The cell bodies of glia had a less intense immunoreactivity in contrast to neurons, and glial processes and myelin were unstained. Cell nuclei of neural cells were not fluorescent. These results show that (a) spectrin is found in all regions of mammalian brain and its intensity corresponds to neural cell density, (b) different neural cell types contain variable spectrin content, and (c) within a single neural cell, the regional disposition of spectrin varies.


Na (+)/H (+)exchange in the tumour microenvironment: does NHE1 drive breast cancer carcinogenesis?
Ionic messengers signal several critical events in carcinogenesis, including metastasis, the leading cause of patient mortality. The aberrant metabolic, proliferative and anti-apoptotic nature of neoplastic cells can be traced to the abnormal expression of their ion transporters and related signalling networks. In this manuscript, we discuss Na(+)/H(+)flux, as mediated by the sodium-hydrogen exchanger isoform 1 (NHE1), a major ion transporter involved in tumourigenesis. Allosteric activation of NHE1 by external stimuli is controlled by phosphorylation of key amino acids on its cytosolic C-terminal tail, which also acts as a signal scaffold for its regulation by intracellular protein and lipid binding partners. In breast cancer cells, pH homeostasis and proton dynamics are disrupted early in transformation. This constitutively activates NHE1, causing a reversal of the plasma membrane pH gradient, resulting in a more alkaline intracellular pH and a more acidic extracellular pH. NHE1-mediated cellular alkalinization potentiates cytoskeletal remodelling, mobilizing cells for directed migration. Concomitant redistribution of NHE1 to invadopodia, where increased proton extrusion promotes proteolytic digestion of the extracellular matrix, primes cells for invasion into the bloodstream. NHE1 hyperactivity therefore heralds an important stage in cancer cell development, critically facilitating the acquisition of the invasive phenotype necessary for metastasis to occur. The potential for targeting NHE1 in the development of novel chemotherapeutic applications is explored.


The effects of background white noise on memory performance in inattentive school children.
Noise is typically conceived of as being detrimental for cognitive performance; however, a recent computational model based on the concepts of stochastic resonance and dopamine related internal noise postulates that a moderate amount of auditive noise benefit individuals in hypodopaminergic states. On the basis of this model we predicted that inattentive children would be enhanced by adding background white noise while attentive children's performance would deteriorate. Fifty-one secondary school pupils carried out an episodic verbal free recall test in two noise conditions. In the high noise condition, verb-noun sentences were presented during auditory background noise (white noise, 78 dB), and in the low noise condition sentences were presented without noise. Exposure to background noise improved performance for inattentive children and worsened performance for attentive children and eliminated episodic memory differences between attentive and inattentive school children. Consistent with the model, our data show that cognitive performance can be moderated by external background white noise stimulation in a non-clinical group of inattentive participants. This finding needs replicating in a larger sample using more noise levels but if replicated has great practical applications by offering a non-invasive way to improve school results in children with attentional problems.


Stimulus-dependent learning and memory in the neotropical ant Ectatomma ruidum.
Learning and memory are major cognitive processes strongly tied to the life histories of animals. In ants, chemotactile information generally plays a central role in social interaction, navigation and resource exploitation. However, in hunters, visual information should take special relevance during foraging, thus leading to differential use of information from different sensory modalities. Here, we aimed to test whether a hunter, the neotropical ant Ectatomma ruidum, differentially learns stimuli acquired through multiple sensory channels. We evaluated the performance of E. ruidum workers when trained using olfactory, mechanical, chemotactile and visual stimuli under a restrained protocol of appetitive learning. Conditioning of the maxilla labium extension response enabled control of the stimuli provided. Our results show that ants learn faster and remember for longer when trained using chemotactile or visual stimuli than when trained using olfactory and mechanical stimuli separately. These results agree with the life history of E. ruidum, characterized by a high relevance of chemotactile information acquired through antennation as well as the role of vision during hunting.


Pharmacodynamic Relationships between Duration of Action of JDTic-like Kappa-Opioid Receptor Antagonists and Their Brain and Plasma Pharmacokinetics in Rats.
JDTic is a potent and selective κ-opioid receptor (KOR) antagonist that reverses U50,488-induced diuresis in rats. It partitions into brain with a duration of action lasting for weeks. In a search for KOR antagonists that do not accumulate in the brain, we compared single doses of five methylated JDTic analogs (RTI-97, -194, -212, -240, and -241) for reversal of U50,488 diuresis and pharmacokinetic (PK) properties. All six compounds showed potent and selective KOR antagonism in a [35S]GTPγS binding assay. Plasma half-lives ranged from 24 to 41 h and brain half-lives from 24 to 76 h. JDTic and RTI-194 showed increasing brain to plasma ratios over time, indicating increasing partitioning into brain and a longer duration of action for reversal of diuresis than did RTI-97. RTI-240 did not show significant brain accumulation. RTI-212 showed no substantive difference between brain and plasma levels and was inactive against diuresis. RTI-241, with a lower brain to plasma ratio than JDTic and RTI-194, formed JDTic as a metabolite, which still reduced diuresis after 9 weeks. The fact that the duration of action was correlated with the brain to blood plasma ratios and area under the concentration-time curves suggests that PK properties could help to predict safety and acceptable duration of action for KOR antagonists.


The Canadian Traumatic Brain Injury Research Consortium: Epitomizing Collaborative Research in Canada.
Traumatic brain injury (TBI) is the leading cause of death in the first half of life and a chronic disability for Canadians of all ages. Despite the recognized importance of TBI, there is no integrated national strategy for research and best practices in Canada. We therefore created the Canadian TBI Research Consortium (CTRC) to build an ideal model of collaboration between Canadian TBI researchers. Our objectives were to: (1) Create a collaborative Canadian research network, (2) improve patient survival, functional outcome, and health through sustainable and scalable evidence-based practice implementation, (3) strengthen the healthcare system for patients with TBI, (4) provide international leadership and collaboration in TBI research, (5) build stronger links with patients and their representatives to help set the research agenda, and to participate in analysis of its impacts, and (6) support current researchers and prepare the next generation of leaders in TBI research. Building on the highly successful 30-year history of the Canadian Critical Care Trials Group, we developed a highly collaborative research group that integrates the multi-disciplinary network of TBI researchers in Canada. The CTRC conducts multi-center clinically relevant practice changing research. Our research is developed around investigator-led project-based research using a programmatic approach and multiple methodologies. Through strong and sustainable career development and mentorship programs, we train and develop the next generation of TBI researchers. Our group is composed of more than 100 Canadian researchers from coast to coast, most of them funded by the Canadian Institutes of Health Research and other granting agencies. In conclusion, the CTRC prioritizes investigator-led TBI research and broadens the research agenda by integrating researchers from different disciplines in the field of TBI research to optimize delivery of care and improve the health of Canadians with TBI. Our goals are being accomplished across the whole continuum of care by conducting clinically relevant and practice-changing research.


Synthesis, characterization, and antimicrobial properties of copper nanoparticles.
Copper nanoparticle synthesis has been gaining attention due to its availability. However, factors such as agglomeration and rapid oxidation have made it a difficult research area. In the present work, pure copper nanoparticles were prepared in the presence of a chitosan stabilizer through chemical means. The purity of the nanoparticles was authenticated using different characterization techniques, including ultraviolet visible spectroscopy, transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and field emission scanning electron microscopy. The antibacterial as well as antifungal activity of the nanoparticles were investigated using several microorganisms of interest, including methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Salmonella choleraesuis, and Candida albicans. The effect of a chitosan medium on growth of the microorganism was studied, and this was found to influence growth rate. The size of the copper nanoparticles obtained was in the range of 2-350 nm, depending on the concentration of the chitosan stabilizer.


Visual working memory declines when more features must be remembered for each object.
The article reports three experiments investigating the limits of visual working memory capacity with a single-item probe change detection paradigm. Contrary to previous reports (e.g., Vogel, Woodman, & Luck, Journal of Experimental Psychology. Human Perception and Performance, 27, 92-114, 2001), increasing the number of features to be remembered for each object impaired change detection. The degree of impairment was not modulated by encoding duration, size of change, or the number of different levels on each feature dimension. Therefore, a larger number of features does not merely impair memory precision. The effect is unlikely to be due to encoding limitations, to verbal encoding of features, or to chunk learning of multifeature objects. The robust effect of number of features contradicts the view that the capacity of visual working memory can be described in terms of number of objects regardless of their characteristics. Visual working memory capacity is limited on at least three dimensions: the number of objects, the number of features per object, and the precision of memory for each feature.


How laminar frontal cortex and basal ganglia circuits interact to control planned and reactive saccades.
How does the brain learn to balance between reactive and planned behaviors? The basal ganglia (BG) and frontal cortex together allow animals to learn planned behaviors that acquire rewards when prepotent reactive behaviors are insufficient. This paper proposes a new model, called TELOS, to explain how laminar circuitry of the frontal cortex, exemplified by the frontal eye fields, interacts with the BG, thalamus, superior colliculus, and inferotemporal and parietal cortices to learn and perform reactive and planned eye movements. The model is formulated as fourteen computational hypotheses. These specify how strategy priming and action planning (in cortical layers III, Va and VI) are dissociated from movement execution (in layer Vb), how the BG help to choose among and gate competing plans, and how a visual stimulus may serve either as a movement target or as a discriminative cue to move elsewhere. The direct, indirect and hyperdirect pathways through the BG are shown to enable complex gating functions, including deferred execution of selected plans, and switching among alternative sensory-motor mappings. Notably, the model can learn and gate the use of a What-to-Where transformation that enables spatially invariant object representations to selectively excite spatially coded movement plans. Model simulations show how dopaminergic reward and non-reward signals guide monkeys to learn and perform saccadic eye movements in the fixation, single saccade, overlap, gap, and delay (memory-guided) saccade tasks. Model cell activation dynamics quantitatively simulate seventeen established types of dynamics exhibited by corresponding real cells during performance of these tasks.


Acrobatic courtship display coevolves with brain size in manakins (Pipridae).
Acrobatic display behaviour is sexually selected in manakins (Pipridae) and can place high demands on many neural systems. Manakin displays vary across species in terms of behavioural complexity, differing in number of unique motor elements, production of mechanical sounds, cooperation between displaying males, and construction of the display site. Historically, research emphasis has been placed on neurological specializations for vocal aspects of courtship, and less is known about the control of physical, non-vocal displays. By examining brain evolution in relation to extreme acrobatic feats such as manakin displays, we can vastly expand our knowledge of how sexual selection acts on motor behaviour. We tested the hypothesis that sexual selection for complex motor displays has selected for larger brains across the Pipridae. We found that display complexity positively predicts relative brain weight (adjusted for body size) after controlling for phylogeny in 12 manakin species and a closely related flycatcher. This evidence suggests that brain size has evolved in response to sexual selection to facilitate aspects of display such as motor, sensorimotor, perceptual, and cognitive abilities. We show, for the first time, that sexual selection for acrobatic motor behaviour can drive brain size evolution in avian species and, in particular, a family of suboscine birds.


A Forensic Exploration of the Microsoft Windows 10 Timeline.
The Microsoft Windows operating system continues to dominate the desktop computing market. With such high levels of usage comes an inferred likelihood of digital forensic practitioners encountering this platform during their investigations. As part of any forensic examination of a digital device, operating system artifacts, which support the identification and understanding of how a user has behaved on their system provide a potential source of evidence. Now, following Microsoft's April 2018 build 1803 release with its incorporated "Timeline" feature, the potential for identifying and tracking user activity has increased. This work provides a timely examination of the Windows 10 Timeline feature demonstrating the ability to recover activity-based content from within its stored database log files. Examination results and underpinning experimental methodologies are offered, demonstrating the ability to recover activity tile and process information in conjunction with the Windows Timeline. Further, an SQL query has been provided to support the interpretation of data stored within the ActivitiesCache.db.


Fibrillation of human serum albumin shows nonspecific coordination on stoichiometric increment of Copper(II).
Protein aggregation is related to a series of pathological disorders the main cause of which are the fibrillar species generated during the process. Human serum albumin (HSA) undergoes rapid fibrillation in the presence of Cu(II) at pH 7.4 in 60% ethanol after 6-h incubation (∼65 °C) followed by room temperature incubation. Here, we have investigated the effect of a stoichiometric variation of Cu(II) on the self-assembly of HSA using Congo red and thioflavin T dye-binding studies, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, electron paramagnetic resonance spectroscopy, fluorescence microscopy and transmission electron microscopy. The simulation of EPR spectra suggests that with the increment in Cu(II) ion concentration, there is a change in ligand field coordination. Kinetic parameters indicate reduced cooperativity that may be related to the nonspecific coordination on increment of Cu(II) concentration. Cu(II) is also able to direct the accumulation of a large number of fibers along with a formation of dense fibrillar network which is evident from microscopic images.


A structure-based distance metric for high-dimensional space exploration with multidimensional scaling.
Although the euclidean distance does well in measuring data distances within high-dimensional clusters, it does poorly when it comes to gauging intercluster distances. This significantly impacts the quality of global, low-dimensional space embedding procedures such as the popular multidimensional scaling (MDS) where one can often observe nonintuitive layouts. We were inspired by the perceptual processes evoked in the method of parallel coordinates which enables users to visually aggregate the data by the patterns the polylines exhibit across the dimension axes. We call the path of such a polyline its structure and suggest a metric that captures this structure directly in high-dimensional space. This allows us to better gauge the distances of spatially distant data constellations and so achieve data aggregations in MDS plots that are more cognizant of existing high-dimensional structure similarities. Our biscale framework distinguishes far-distances from near-distances. The coarser scale uses the structural similarity metric to separate data aggregates obtained by prior classification or clustering, while the finer scale employs the appropriate euclidean distance.


Isolation and characterization of cytotoxic granules from human lymphokine (interleukin 2) activated killer cells.
Cytotoxic granules were isolated from human lymphokine-activated killer (LAK) cells and analyzed for their biochemical properties. Isolated granules of approximately 85-95% purity were obtained by differential centrifugation followed by discontinuous Percoll gradient centrifugation. The murine lymphocyte granule marker N-alpha-carbamazepine-L-lysine thiobenzyl ester-esterase as well as cytotoxic activity toward the human tumor cell lines K562, Raji, Daudi, and CEM were associated with LAK granule fractions. Granule-associated N-alpha-carbamazepine-L-lysine thiobenzyl ester-esterase activity increased in recombinant interleukin 2 expanded human LAK cells in parallel with cytotoxic activity for Raji tumor cell targets. Cytotoxic LAK cell granules mediated calcium-dependent killing of the tumor cell lines K562, Raji, Daudi, and CEM. However, no calcium-dependent hemolytic activity was found. Preincubation of human granules with calcium, a treatment which totally inactivates the hemolytic and cytotoxic activity of murine lymphocyte granules [perforin 1 (P1)] had no effect on human LAK granule cytotoxicity for nucleated cells. Human LAK granules appear to contain P1 detected as cross-reactive antigen detected by mouse anti-P1 and human anti-C9 in Western blot analysis. In addition, Northern blot analysis of polyadenylated RNA isolated from human LAK cells using a murine P1 complementary DNA probe showed a cross-hybridizing 2.8- to 3.0-kilobase mRNA species identical in size to murine P1 mRNA. These results demonstrate that despite similar biochemical composition, functional differences exist between human and murine cytotoxic granules. Human LAK granules were synthesized in response to recombinant interleukin 2 activation and appeared in parallel with cytotoxicity for tumor targets, suggesting an important role for LAK granules in tumor cell cytotoxicity by human LAK cells.


Measuring macroscopic brain connections in vivo.
Decades of detailed anatomical tracer studies in non-human animals point to a rich and complex organization of long-range white matter connections in the brain. State-of-the art in vivo imaging techniques are striving to achieve a similar level of detail in humans, but multiple technical factors can limit their sensitivity and fidelity. In this review, we mostly focus on magnetic resonance imaging of the brain. We highlight some of the key challenges in analyzing and interpreting in vivo connectomics data, particularly in relation to what is known from classical neuroanatomy in laboratory animals. We further illustrate that, despite the challenges, in vivo imaging methods can be very powerful and provide information on connections that is not available by any other means.


Phenotyping for abiotic stress tolerance in maize.
The ability to quickly develop germplasm having tolerance to several complex polygenic inherited abiotic and biotic stresses combined is critical to the resilience of cropping systems in the face of climate change. Molecular breeding offers the tools to accelerate cereal breeding; however, suitable phenotyping protocols are essential to ensure that the much-anticipated benefits of molecular breeding can be realized. To facilitate the full potential of molecular tools, greater emphasis needs to be given to reducing the within-experimental site variability, application of stress and characterization of the environment and appropriate phenotyping tools. Yield is a function of many processes throughout the plant cycle, and thus integrative traits that encompass crop performance over time or organization level (i.e. canopy level) will provide a better alternative to instantaneous measurements which provide only a snapshot of a given plant process. Many new phenotyping tools based on remote sensing are now available including non-destructive measurements of growth-related parameters based on spectral reflectance and infrared thermometry to estimate plant water status. Here we describe key field phenotyping protocols for maize with emphasis on tolerance to drought and low nitrogen.


Generation and Production of Modified Vaccinia Virus Ankara (MVA) as a Vaccine Vector.
The smallpox vaccine based on the vaccinia virus was successfully used to eradicate smallpox, but although very effective, it was a very reactogenic vaccine and responsible for the deaths of one to two people per million vaccinated. Modified Vaccinia virus Ankara (MVA) is an attenuated derivative, also used in the smallpox eradication campaign and now being developed as a recombinant viral vector to produce vaccines against infectious diseases and cancer. MVA can encode one or more foreign antigens and thus can function as a multivalent vaccine. The vector can be used at biosafety level 1, has intrinsic adjuvant properties, and induces humoral and cellular immune responses. Many clinical trials of these new vaccines have been conducted, and the safety of MVA is now well documented. Immunogenicity is influenced by the dose and vaccination regimen, and information on the efficacy of MVA-vectored vaccines is now beginning to accumulate. In this chapter, we provide protocols for generation, isolation, amplification, and purification of recombinant MVA for preclinical and clinical evaluation.


Administrative compensation for medical injuries: lessons from three foreign systems.
The United States requires patients injured by medical negligence to seek compensation through lawsuits, an approach that has drawbacks related to fairness, cost, and impact on medical care. Several countries, including New Zealand, Sweden, and Denmark, have replaced litigation with administrative compensation systems for patients who experience an avoidable medical injury. Sometimes called "no-fault" systems, such schemes enable patients to file claims for compensation without using an attorney. A governmental or private adjudicating organization uses neutral medical experts to evaluate claims of injury and does not require patients to prove that health care providers were negligent in order to receive compensation. Information from claims is used to analyze opportunities for patient safety improvement. The systems have successfully limited liability costs while improving injured patients' access to compensation. American policymakers may find many of the elements of these countries' systems to be transferable to demonstration projects in the U.S.


Yeast Ste23p shares functional similarities with mammalian insulin-degrading enzymes.
The S. cerevisiae genome encodes two M16A enzymes: Axl1p and Ste23p. Of the two, Ste23p shares significantly higher sequence identity with M16A enzymes from other species, including mammalian insulin-degrading enzymes (IDEs). In this study, recombinant Ste23p and R. norvegicus IDE (RnIDE) were isolated from E. coli, and their enzymatic properties compared. Ste23p was found to cleave established RnIDE substrates, including the amyloid-beta peptide (Abeta1-40) and insulin B-chain. A novel internally quenched fluorogenic substrate (Abz-SEKKDNYIIKGV-nitroY-OH) based on the polypeptide sequence of the yeast P2 a-factor mating propheromone was determined to be a suitable substrate for both Ste23p and RnIDE, and was used to conduct comparative enzymological studies. Both enzymes were most active at 37 degrees C, in alkaline buffers and in high salt environments. In addition, the proteolytic activities of both enzymes towards the fluorogenic substrate were inhibited by metal chelators, thiol modifiers, inhibitors of cysteine protease activity and insulin. Characteristics of STE23 expression were also evaluated. Our analysis indicates that the 5' terminus of the STE23 gene has been mischaracterized, with the physiologically relevant initiator corresponding to residue M53 of the publicly annotated protein sequence. Finally, we demonstrate that, unlike haploid-specific Axl1p, Ste23p is expressed in both haploid and diploid cell types. Our study presents the first comprehensive biochemical analysis of a yeast M16A enzyme, and provides evidence that S. cerevisiae Ste23p has enzymatic properties that are highly consistent with mammalian IDEs and other M16A enzymes.


Sulforaphane protects immature hippocampal neurons against death caused by exposure to hemin or to oxygen and glucose deprivation.
Oxidative stress is a mediator of cell death following cerebral ischemia/reperfusion and heme toxicity, which can be an important pathogenic factor in acute brain injury. Induced expression of phase II detoxification enzymes through activation of the antioxidant response element (ARE)/Nrf2 pathway has emerged as a promising approach for neuroprotection. Little is known, however, about the neuroprotective potential of this strategy against injury in immature brain cells. In this study, we tested the hypothesis that sulforaphane (SFP), a naturally occurring isothiocyanate that is also a known activator of the ARE/Nrf2 antioxidant pathway, can protect immature neurons from oxidative stress-induced death. The hypothesis was tested with primary mouse hippocampal neurons exposed to either O(2) and glucose deprivation (OGD) or hemin. Treatment of immature neurons with SFP immediately after the OGD during reoxygenation was effective in protecting immature neurons from delayed cell death. Exposure of immature hippocampal neurons to hemin induced significant cell death, and both pre- and cotreatment with SFP were remarkably effective in blocking cytotoxicity. RT-PCR analysis indicated that several Nrf2-dependent cytoprotective genes, including NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO1), and glutamate-cysteine ligase modifier subunit (GCLM), which is involved in glutathione biosynthesis, were up-regulated following SFP treatment both in control neurons and following exposure to OGD and hemin. These results indicate that SFP activates the ARE/Nrf2 pathway of antioxidant defense and protects immature neurons from death caused by stress paradigms relevant to those associated with ischemic and traumatic injury to the immature brain.


Prognostic value of miR-96 in patients with acute myeloid leukemia.
Aberrant expression of miRNA (miR)-96 is associated with tumorigenesis and tumor progression in several solid cancers. However, little is known about the expression and prognostic value of miR-96 in acute myeloid leukemia (AML). Therefore, the aim of this study was to investigate the correlation of miR-96 expression with clinicopathological features and prognosis of AML. Real-time quantitative RT-PCR assay was performed to evaluate the expression levels of miR-96 in mononuclear cells from bone marrow or peripheral blood specimens in 86 patients with newly diagnosed AML. Compared with normal controls, miR-96 expression was significantly downregulated in patients with newly diagnosed AML (P < 0.001). In analysis of 14 diagnosis/CR-paired samples, the expression level of miR-96 was found markedly elevated in patients after treatment than before (P < 0.001). Moreover, lower levels of miR-96 were associated with a higher white blood cell count, bone marrow blast count (P < 0.001 and 0.022, respectively), and lower hemoglobin and platelet count (P = 0.036 and 0.033, respectively). Although the low-expression group seemed to have a lower CR rate (53.85% vs 70.0%), there was no significant difference between the two groups (P = 0.213). The low-expression group had a lower relapse-free survival (RFS) (P = 0.038) and overall survival (OS) (P = 0.022) compared with the high-expression group during a median follow-up of 20 months. Our data demonstrated that the expression of miR-96 was downregulated in newly diagnosed AML patients and associated with leukemic burden, as well as RFS and OS. This suggests that miR-96 detection might become a potential biomarker of prognosis and monitoring in AML. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1434808553949498.


Molecular characterization of plasmid pMoma1of Moraxella macacae, a newly described bacterial pathogen of macaques.
We report the complete nucleotide sequence and characterization of a small cryptic plasmid of Moraxella macacae 0408225, a newly described bacterial species within the family Moraxellaceae and a causative agent of epistaxis in macaques. The complete nucleotide sequence of the plasmid pMoma1 was determined and found to be 5,375 bp in size with a GC content of 37.4 %. Computer analysis of the sequence data revealed five open reading frames encoding putative proteins of 54.4 kDa (ORF1), 17.6 kDa (ORF2), 13.3 kDa (ORF3), 51.6 kDa (ORF4), and 25.0 kDa (ORF5). ORF1, ORF2, and ORF3 encode putative proteins with high identity (72, 42, and 55 %, respectively) to mobilization proteins of plasmids found in other Moraxella species. ORF3 encodes a putative protein with similarity (about 40 %) to several plasmid replicase (RepA) proteins. The fifth open reading frames (ORF) was most similar to hypothetical proteins with unknown functions, although domain analysis of this sequence suggests it belongs to the Abi-like protein family. Upstream of the repA gene, a 470-bp intergenic region, was identified that contained an AT-rich section and two sets of tandem direct and indirect repeats, consistent with a putative origin of replication site. In contrast to other plasmids of Moraxella, the occurrence of pMoma1 in M. macacae isolates appears to be common as PCR testing of 14 clinical isolates from two different research institutions all contained the plasmid.


In Situ Activation and Heterologous Production of a Cryptic Lantibiotic from an African Plant Ant-Derived Saccharopolyspora Species.
Most clinical antibiotics are derived from actinomycete natural products discovered at least 60 years ago. However, the repeated rediscovery of known compounds led the pharmaceutical industry to largely discard microbial natural products (NPs) as a source of new chemical diversity. Recent advances in genome sequencing have revealed that these organisms have the potential to make many more NPs than previously thought. Approaches to unlock NP biosynthesis by genetic manipulation of strains, by the application of chemical genetics, or by microbial cocultivation have resulted in the identification of new antibacterial compounds. Concomitantly, intensive exploration of coevolved ecological niches, such as insect-microbe defensive symbioses, has revealed these to be a rich source of chemical novelty. Here, we report the new lanthipeptide antibiotic kyamicin, which was generated through the activation of a cryptic biosynthetic gene cluster identified by genome mining Saccharopolyspora species found in the obligate domatium-dwelling ant Tetraponera penzigi of the ant plant Vachellia drepanolobium Transcriptional activation of this silent gene cluster was achieved by ectopic expression of a pathway-specific activator under the control of a constitutive promoter. Subsequently, a heterologous production platform was developed which enabled the purification of kyamicin for structural characterization and bioactivity determination. This strategy was also successful for the production of lantibiotics from other genera, paving the way for a synthetic heterologous expression platform for the discovery of lanthipeptides that are not detected under laboratory conditions or that are new to nature.IMPORTANCE The discovery of novel antibiotics to tackle the growing threat of antimicrobial resistance is impeded by difficulties in accessing the full biosynthetic potential of microorganisms. The development of new tools to unlock the biosynthesis of cryptic bacterial natural products will greatly increase the repertoire of natural product scaffolds. Here, we report a strategy for the ectopic expression of pathway-specific positive regulators that can be rapidly applied to activate the biosynthesis of cryptic lanthipeptide biosynthetic gene clusters. This allowed the discovery of a new lanthipeptide antibiotic directly from the native host and via heterologous expression.


Organizing pneumonia. Features and prognosis of cryptogenic, secondary, and focal variants.
Organizing pneumonia (OP) is a non-specific response to many types of lung injury. Clinicians frequently encounter pathology reports of OP in patients with no underlying condition (cryptogenic OP, also known as BOOP or bronchiolitis obliterans OP) or in association with drugs or nonpulmonary disease. The goals of this study are to describe the clinical course and outcomes in patients with 3 clinical variants of OP. A retrospective study of patients with OP seen at the Mayo Clinic, Rochester, Minn, from January 1, 1984, through June 30, 1994, was conducted. Initial features were obtained from medical records. Chest radiographs and pathology specimens were reviewed for this study. Resolution, relapse, and survival were obtained from medical records and a follow-up patient questionnaire. Seventy-four patients had pathologically confirmed OP. Organizing pneumonia was classified into 3 clinical groups: symptomatic cryptogenic OP; symptomatic OP related to underlying hematologic malignant neoplasm, collagen vascular disease, or drugs (secondary OP); and asymptomatic OP presenting as a focal nodule (focal OP). Thirty-seven patients (50%) had cryptogenic OP and 27 patients (36%) had secondary OP. No difference was found between cryptogenic and secondary OP in type or severity of symptoms, signs, laboratory and pulmonary function tests, or radiologic or pathologic findings. Corticosteroids were given at a similar initial dose (prednisone, about 50 mg/d). Resolution of symptoms was more frequent in patients with cryptogenic OP than those with secondary OP. Relapse was infrequent in both of these groups. Five-year survival was higher in patients with cryptogenic OP (73%) than in secondary OP (44%), and respiratory-related deaths were more frequent in patients with secondary OP. Organizing pneumonia was an asymptomatic focal rounded opacity in 10 patients (14%), most often detected on chest radiograph and diagnosed on lung biopsy done for suspicion of lung cancer. Patients with focal OP required no treatment and had no relapse or respiratory-related deaths. Clinical classification of OP is useful to predict clinical course and outcome. Cryptogenic OP most often was a symptomatic bilateral lung process that had an overall favorable prognosis with prolonged corticosteroid therapy. Patients with secondary OP had a high mortality rate when the disease was associated with predisposing conditions or drugs. Patients with asymptomatic focal OP had an excellent prognosis.


Prevalence of sleep deficiency and use of hypnotic drugs in astronauts before, during, and after spaceflight: an observational study.
Sleep deprivation and fatigue are common subjective complaints among astronauts. Previous studies of sleep and hypnotic drug use in space have been limited to post-flight subjective survey data or in-flight objective data collection from a small number of crew members. We aimed to characterise representative sleep patterns of astronauts on both short-duration and long-duration spaceflight missions. For this observational study, we recruited crew members assigned to Space Transportation System shuttle flights with in-flight experiments between July 12, 2001, and July 21, 2011, or assigned to International Space Station (ISS) expeditions between Sept 18, 2006, and March 16, 2011. We assessed sleep-wake timing objectively via wrist actigraphy, and subjective sleep characteristics and hypnotic drug use via daily logs, in-flight and during Earth-based data-collection intervals: for 2 weeks scheduled about 3 months before launch, 11 days before launch until launch day, and for 7 days upon return to Earth. We collected data from 64 astronauts on 80 space shuttle missions (26 flights, 1063 in-flight days) and 21 astronauts on 13 ISS missions (3248 in-flight days), with ground-based data from all astronauts (4014 days). Crew members attempted and obtained significantly less sleep per night as estimated by actigraphy during space shuttle missions (7·35 h [SD 0·47] attempted, 5·96 h [0·56] obtained), in the 11 days before spaceflight (7·35 h [0·51], 6·04 h [0·72]), and about 3 months before spaceflight (7·40 h [0·59], 6·29 h [0·67]) compared with the first week post-mission (8·01 h [0·78], 6·74 h [0·91]; p<0·0001 for both measures). Crew members on ISS missions obtained significantly less sleep during spaceflight (6·09 h [0·67]), in the 11 days before spaceflight (5·86 h [0·94]), and during the 2-week interval scheduled about 3 months before spaceflight (6·41 h [SD 0·65]) compared with in the first week post-mission (6·95 h [1·04]; p<0·0001). 61 (78%) of 78 shuttle-mission crew members reported taking a dose of sleep-promoting drug on 500 (52%) of 963 nights; 12 (75%) of 16 ISS crew members reported using sleep-promoting drugs. Sleep deficiency in astronauts was prevalent not only during space shuttle and ISS missions, but also throughout a 3 month preflight training interval. Despite chronic sleep curtailment, use of sleep-promoting drugs was pervasive during spaceflight. Because chronic sleep loss leads to performance decrements, our findings emphasise the need for development of effective countermeasures to promote sleep. The National Aeronautics and Space Administration.


Dynamics of pectoral fin rowing in a fish with an extreme rowing stroke: the threespine stickleback (Gasterosteus aculeatus).
The dynamics of pectoral fin rowing in the threespine stickleback are investigated by measuring the instantaneous force balance on freely swimming fish throughout the stroke cycle and comparing the measured forces with fin motions and an unsteady, blade-element model of pectoral fin propulsion. Both measured and modeled forces suggest that attached vortex and circulatory forces and not inertial (added mass) forces dominate the force balance. Peak forces occur at midstrokes. There is no evidence for large force peaks at the stroke transitions due to either rapid fin rotation (supination) or rapid fin closure against the body. The energetics of pectoral fin rowing are estimated using the unsteady blade-element model and an indirect method based on the center of mass dynamics. The results indicate that the mechanical efficiency of pectoral fin rowing is low (0.1-0.3) relative to a flapping mechanism and possibly relative to axial undulation at comparable speeds.


Characterization of prostatic epithelial cell lines derived from transgenic adenocarcinoma of the mouse prostate (TRAMP) model.
To develop a syngeneic transplantable system to study immunotherapeutic approaches for the treatment of prostate cancer, three cell lines were established from a heterogeneous 32 week tumor of the transgenic adenocarcinoma mouse prostate (TRAMP) model. TRAMP is a transgenic line of C57BL/6 mice harboring a construct comprised of the minimal -426/+28 rat probasin promoter driving prostate-specific epithelial expression of the SV40 large T antigen. TRAMP males develop histological prostatic intraepithelial neoplasia by 8-12 weeks of age that progress to adenocarcinoma with distant metastases by 24-30 weeks of age. The three cell lines (TRAMP-C1, TRAMP-C2, and TRAMP-C3) express cytokeratin, E-cadherin, and androgen receptor by immunohistochemical analysis and do not appear to have a mutated p53. Although TRAMP-C1 and TRAMP-C2 are tumorigenic when grafted into syngeneic C57BL/6 hosts, TRAMP-C3 grows readily in vitro but does not form tumors. The T antigen oncoprotein is not expressed by the cell lines in vitro or in vivo. The rationale for establishing multiple cell lines was to isolate cells representing various stages of cellular transformation and progression to androgen-independent metastatic disease that could be manipulated in vitro and, in combination with the TRAMP model, provide a system to investigate therapeutic interventions, such as immunotherapy prior to clinical trials.


Clearance of influenza virus from the lung depends on migratory langerin+CD11b- but not plasmacytoid dendritic cells.
Although dendritic cells (DCs) play an important role in mediating protection against influenza virus, the precise role of lung DC subsets, such as CD11b- and CD11b+ conventional DCs or plasmacytoid DCs (pDCs), in different lung compartments is currently unknown. Early after intranasal infection, tracheal CD11b-CD11chi DCs migrated to the mediastinal lymph nodes (MLNs), acquiring co-stimulatory molecules in the process. This emigration from the lung was followed by an accumulation of CD11b+CD11chi DCs in the trachea and lung interstitium. In the MLNs, the CD11b+ DCs contained abundant viral nucleoprotein (NP), but these cells failed to present antigen to CD4 or CD8 T cells, whereas resident CD11b-CD8+ DCs presented to CD8 cells, and migratory CD11b-CD8- DCs presented to CD4 and CD8 T cells. When lung CD11chi DCs and macrophages or langerin+CD11b-CD11chi DCs were depleted using either CD11c-diphtheria toxin receptor (DTR) or langerin-DTR mice, the development of virus-specific CD8+ T cells was severely delayed, which correlated with increased clinical severity and a delayed viral clearance. 120G8+ CD11cint pDCs also accumulated in the lung and LNs carrying viral NP, but in their absence, there was no effect on viral clearance or clinical severity. Rather, in pDC-depleted mice, there was a reduction in antiviral antibody production after lung clearance of the virus. This suggests that multiple DCs are endowed with different tasks in mediating protection against influenza virus.


Salmonella infection and carriage in reptiles in a zoological collection.
OBJECTIVE To identify important subspecies and serovars of Salmonella enterica in a captive reptile population and clinically relevant risk factors for and signs of illness in Salmonella-positive reptiles. DESIGN Retrospective cross-sectional study. ANIMALS 11 crocodilians (4 samples), 78 snakes (91 samples), 59 lizards (57 samples), and 34 chelonians (23 samples) at the Bronx Zoo from 2000 through 2012. PROCEDURES Data pertaining to various types of biological samples obtained from reptiles with positive Salmonella culture results and the reptiles themselves were analyzed to determine period prevalence of and risk factors for various Salmonella-related outcomes. RESULTS Serovar distribution differences were identified for sample type, reptile phylogenetic family, and reptile origin and health. Salmonella enterica subsp enterica was the most common subspecies in Salmonella cultures (78/175 [45%]), identified across all reptilian taxa. Salmonella enterica subsp diarizonae was also common (42/175 [24%]) and was recovered almost exclusively from snakes (n = 33), many of which had been clinically ill (17). Clinically ill reptiles provided 37% (64) of Salmonella cultures. Factors associated with an increased risk of illness in reptiles with a positive culture result were carnivorous diet and prior confiscation. Snakes had a higher risk of illness than other reptile groups, whereas lizards had a lower risk. Bony changes, dermatitis, and anorexia were the most common clinical signs. CONCLUSIONS AND CLINICAL RELEVANCE This study provided new information on Salmonella infection or carriage and associated clinical disease in reptiles. Associations identified between serovars or subspecies and reptile groups or clinical disease can guide management of Salmonella-positive captive reptiles.


High-Dose Opioid Prescribing and Opioid-Related Hospitalization: A Population-Based Study.
To examine the impact of national clinical practice guidelines and provincial drug policy interventions on prevalence of high-dose opioid prescribing and rates of hospitalization for opioid toxicity. Interventional time-series analysis. Ontario, Canada, from 2003 to 2014. Ontario Drug Benefit (ODB) beneficiaries aged 15 to 64 years from 2003 to 2014. Publication of Canadian clinical practice guidelines for use of opioids in chronic non-cancer pain (May 2010) and implementation of Ontario's Narcotics Safety and Awareness Act (NSAA; November 2011). Three outcomes were explored: the rate of opioid use among ODB beneficiaries, the prevalence of opioid prescriptions exceeding 200 mg and 400 mg morphine equivalents per day, and rates of opioid-related emergency department visits and hospital admissions. Over the 12 year study period, the rate of opioid use declined 15.2%, from 2764 to 2342 users per 10,000 ODB eligible persons. The rate of opioid use was significantly impacted by the Canadian clinical practice guidelines (p-value = .03) which led to a decline in use, but no impact was observed by the enactment of the NSAA (p-value = .43). Among opioid users, the prevalence of high-dose prescribing doubled (from 4.2% to 8.7%) over the study period. By 2014, 40.9% of recipients of long-acting opioids exceeded daily doses of 200 mg morphine or equivalent, including 55.8% of long-acting oxycodone users and 76.3% of transdermal fentanyl users. Moreover, in the last period, 18.7% of long-acting opioid users exceeded daily doses of 400 mg morphine or equivalent. Rates of opioid-related emergency department visits and hospital admissions increased 55.0% over the study period from 9.0 to 14.0 per 10,000 ODB beneficiaries from 2003 to 2013. This rate was not significantly impacted by the Canadian clinical practice guidelines (p-value = .68) or enactment of the NSAA (p-value = .59). Although the Canadian clinical practice guidelines for use of opioids in chronic non-cancer pain led to a decline in opioid prescribing rates among ODB beneficiaries these guidelines and subsequent Ontario legislation did not result in a significant change in rates of opioid-related hospitalizations. Given the prevalence of high dose opioid prescribing in this population, this suggests that improved strategies and programs for the safe prescribing of long-acting opioids are needed.


Persistence versus escape: Aspergillus terreus and Aspergillus fumigatus employ different strategies during interactions with macrophages.
Invasive bronchopulmonary aspergillosis (IBPA) is a life-threatening disease in immunocompromised patients. Although Aspergillus terreus is frequently found in the environment, A. fumigatus is by far the main cause of IBPA. However, once A. terreus establishes infection in the host, disease is as fatal as A. fumigatus infections. Thus, we hypothesized that the initial steps of disease establishment might be fundamentally different between these two species. Since alveolar macrophages represent one of the first phagocytes facing inhaled conidia, we compared the interaction of A. terreus and A. fumigatus conidia with alveolar macrophages. A. terreus conidia were phagocytosed more rapidly than A. fumigatus conidia, possibly due to higher exposure of β-1,3-glucan and galactomannan on the surface. In agreement, blocking of dectin-1 and mannose receptors significantly reduced phagocytosis of A. terreus, but had only a moderate effect on phagocytosis of A. fumigatus. Once phagocytosed, and in contrast to A. fumigatus, A. terreus did not inhibit acidification of phagolysosomes, but remained viable without signs of germination both in vitro and in immunocompetent mice. The inability of A. terreus to germinate and pierce macrophages resulted in significantly lower cytotoxicity compared to A. fumigatus. Blocking phagolysosome acidification by the v-ATPase inhibitor bafilomycin increased A. terreus germination rates and cytotoxicity. Recombinant expression of the A. nidulans wA naphthopyrone synthase, a homologue of A. fumigatus PksP, inhibited phagolysosome acidification and resulted in increased germination, macrophage damage and virulence in corticosteroid-treated mice. In summary, we show that A. terreus and A. fumigatus have evolved significantly different strategies to survive the attack of host immune cells. While A. fumigatus prevents phagocytosis and phagolysosome acidification and escapes from macrophages by germination, A. terreus is rapidly phagocytosed, but conidia show long-term persistence in macrophages even in immunocompetent hosts.


Design, Isolation, and Spectroscopic Analysis of a Tetravalent Terbium Complex.
Synthetic strategies to yield molecular complexes of high-valent lanthanides, other than the ubiquitous Ce4+ ion, are exceptionally rare, and thorough, detailed characterization in these systems is limited by complex lifetime and reaction and isolation conditions. The synthesis of high-symmetry complexes in high purity with significant lifetimes in solution and the solid state is essential for determining the role of ligand-field splitting, multiconfigurational behavior, and covalency in governing the reactivity and physical properties of these potentially technologically transformative tetravalent ions. We report the synthesis and physical characterization of an S4 symmetric, four-coordinate tetravalent terbium complex, [Tb(NP(1,2-bis-tBu-diamidoethane)(NEt2))4] (where Et is ethyl and tBu is tert-butyl). The ligand field in this complex is weak and the metal-ligand bonds sufficiently covalent so that the tetravalent terbium ion is stable and accessible via a mild oxidant from the anionic, trivalent, terbium precursor, [(Et2O)K][Tb(NP(1,2-bis-tBu-diamidoethane)(NEt2))4]. The significant stability of the tetravalent complex enables its thorough characterization. The stepwise development of the supporting ligand points to key ligand control elements for further extending the known tetravalent lanthanide ions in molecular complexes. Magnetic susceptibility, electron paramagnetic resonance (EPR) spectroscopy, X-ray absorption near-edge spectroscopy (XANES), and density functional theory studies indicate a 4f7 ground state for [Tb(NP(1,2-bis-tBu-diamidoethane)(NEt2))4] with considerable zero-field splitting, demonstrating that magnetic, tetravalent lanthanide ions engage in covalent metal-ligand bonds. This result has significant implications for the use of tetravalent lanthanide ions in magnetic applications since the observed zero-field splitting is intermediate between that observed for the trivalent lanthanides and for the transition metals. The similarity of the multiconfigurational behavior in the ground state of [Tb(NP(1,2-bis-tBu-diamidoethane)(NEt2))4] (measured by Tb L3-edge XAS) to that observed in TbO2 implicates ligand control of multiconfigurational behavior as a key component of the stability of the complex.


Activating Transcription Factor 3 (ATF3) Protects Retinal Ganglion Cells and Promotes Functional Preservation After Optic Nerve Crush.
To investigate the possible role of activating transcription factor 3 (ATF3) in retinal ganglion cell (RGC) neuroprotection and optic nerve regeneration after optic nerve crush (ONC). Overexpression of proteins of interest (ATF3, phosphatase and tensin homolog [PTEN], placental alkaline phosphatase, green fluorescent protein) in the retina was achieved by intravitreal injections of recombinant adenovirus-associated viruses (rAAVs) expressing corresponding proteins. The number of RGCs and αRGCs was evaluated by immunostaining retinal sections and whole-mount retinas with antibodies against RNA binding protein with multiple splicing (RBPMS) and osteopontin, respectively. Axonal regeneration was assessed via fluorophore-coupled cholera toxin subunit B labeling. RGC function was evaluated by recording positive scotopic threshold response. The level of ATF3 is preferentially elevated in osteopontin+/RBPMS+ αRGCs following ONC. Overexpression of ATF3 by intravitreal injection of rAAV 2 weeks before ONC promoted RBPMS+ RGC survival and preserved RGC function as assessed by positive scotopic threshold response recordings 2 weeks after ONC. However, overexpression of ATF3 and simultaneous downregulation of PTEN, a negative regulator of the mTOR pathway, combined with ONC, only moderately promoted short distance RGC axon regeneration (200 μm from the lesion site) but did not provide additional RGC neuroprotection compared with PTEN downregulation alone. These results reveal a neuroprotective effect of ATF3 in the retina following injury and identify ATF3 as a promising agent for potential treatments of optic neuropathies.