Neutrophils induce damage to respiratory epithelial cells infected with respiratory syncytial virus.
The mechanisms by which respiratory syncytial virus (RSV) infection induces bronchiolitis and airway disease are unclear. The presence of large numbers of polymorphonuclear leukocytes (PMN) in the airways of infants with RSV infection suggests a potential role of PMN in airway injury associated with RSV infection. To investigate the potential role of neutrophils in RSV bronchiolitis, human alveolar type II cells (A549 cells) were infected with different doses of RSV for 6-48 h. A 51Cr-releasing assay was used to measure PMN-induced damage and image analysis was used to determine PMN adhesion and detachment of epithelial cells. The results showed that RSV infection of epithelial cells enhanced PMN adherence in a dose- and time-dependent pattern, RSV infection alone could damage and detach epithelial cells to a limited extent and PMN significantly augmented RSV infection-induced damage and detachment of epithelial cells. These data suggest that respiratory syncytial virus infection of respiratory epithelial cells enhances neutrophil adhesion to the epithelium and that activated neutrophils augment the damage and detachment of epithelium infected with the virus. Polymorphonuclear leukocytes may contribute to the pathogenesis of respiratory syncytial virus airway disease by inducing epithelial damage and cell loss.


The ADPKD genes pkd1a/b and pkd2 regulate extracellular matrix formation.
Mutations in polycystin1 (PKD1) account for the majority of autosomal dominant polycystic kidney disease (ADPKD). PKD1 mutations are also associated with vascular aneurysm and abdominal wall hernia, suggesting a role for polycystin1 in extracellular matrix (ECM) integrity. In zebrafish, combined knockdown of the PKD1 paralogs pkd1a and pkd1b resulted in dorsal axis curvature, hydrocephalus, cartilage and craniofacial defects, and pronephric cyst formation at low frequency (10-15%). Dorsal axis curvature was identical to the axis defects observed in pkd2 knockdown embryos. Combined pkd1a/b, pkd2 knockdown demonstrated that these genes interact in axial morphogenesis. Dorsal axis curvature was linked to notochord collagen overexpression and could be reversed by knockdown of col2a1 mRNA or chemical inhibition of collagen crosslinking. pkd1a/b- and pkd2-deficient embryos exhibited ectopic, persistent expression of multiple collagen mRNAs, suggesting a loss of negative feedback signaling that normally limits collagen gene expression. Knockdown of pkd1a/b also dramatically sensitized embryos to low doses of collagen-crosslinking inhibitors, implicating polycystins directly in the modulation of collagen expression or assembly. Embryos treated with wortmannin or LY-29400 also exhibited dysregulation of col2a1 expression, implicating phosphoinositide 3-kinase (PI3K) in the negative feedback signaling pathway controlling matrix gene expression. Our results suggest that pkd1a/b and pkd2 interact to regulate ECM secretion or assembly, and that altered matrix integrity may be a primary defect underlying ADPKD tissue pathologies.


The association between physical activity and a composite measure of sleep health.
Physical activity has been associated with several individual dimensions of sleep. However, the association between physical activity and sleep health, a construct that emphasizes the multidimensional nature of sleep, has not been explored. This analysis examined the relationship between physical activity and a composite measure of sleep health. A total of 114 adults (66% female, 60.3 ± 9.2 years) were included in the analyses. Participants reported daily light-intensity physical activity (LPA) and moderate- and vigorous-intensity physical activity (MVPA) via diary, while wearing a pedometer (Omron HJ-720ITC) to measure daily steps. Sleep health was measured using the RU_SATED questionnaire, which addresses regularity of sleep patterns, satisfaction with sleep, daytime alertness, and sleep timing, efficiency, and duration. Multiple linear regression, binary logistic regression, and analysis of covariance (ANCOVA) were utilized for analyses. Mean sleep health score was 9.6 ± 2.4 (0 [poor]-12 [good]). Participants reported 62.9 ± 66.0 and 51.2 ± 51.2 min/day of LPA and MVPA, respectively, and took 5585.5 ± 2806.7 steps/day. Greater MVPA was associated with better sleep health (β = 0.27, P = 0.005) and sleep health scores differed between those reporting < 30 min/day and ≥ 60 min/day of MVPA (P = 0.004). Greater MVPA was associated with higher odds of having good sleep satisfaction (OR = 1.58 [1.14-2.20], P < 0.01), timing (OR = 2.07 [1.24-3.46], P < 0.01), and duration (OR = 1.48 [1.02-2.18], P = 0.04). Pedometer-based physical activity and LPA were not related to sleep health or its individual dimensions. In middle- to older-aged adults, higher-intensity activity, but not lower-intensity or volume of activity, was associated with greater sleep health. These data suggest that physical activity intensity may be important for sleep health.


Single-Molecule Microscopy Reveals Dynamic FLNA Interactions Governing SSTR2 Clustering and Internalization.
The cytoskeletal protein filamin A (FLNA) has been suggested to play an important role in the responsiveness of GH-secreting pituitary tumors to somatostatin receptor subtype 2 (SSTR2) agonists by regulating SSTR2 expression and signaling. However, the underlying mechanisms are unknown. In this study, we use fast multicolor single-molecule microscopy to image individual SSTR2 and FLNA molecules at the surface of living cells with unprecedented spatiotemporal resolution. We find that SSTR2 and FLNA undergo transient interactions, which occur preferentially along actin fibers and contribute to restraining SSTR2 diffusion. Agonist stimulation increases the localization of SSTR2 along actin fibers and, subsequently, SSTR2 clustering and recruitment to clathrin-coated pits (CCPs). Interfering with FLNA-SSTR2 binding with a dominant-negative FLNA fragment increases SSTR2 mobility, hampers the formation and alignment of SSTR2 clusters along actin fibers, and impairs both SSTR2 recruitment to CCPs and SSTR2 internalization. These findings indicate that dynamic SSTR2-FLNA interactions critically control the nanoscale localization of SSTR2 at the plasma membrane and are required for coupling SSTR2 clustering to internalization. These mechanisms explain the critical role of FLNA in the control of SSTR2 expression and signaling and suggest the possibility of targeting SSTR2-FLNA interactions for the therapy of pharmacologically resistant GH-secreting pituitary tumors.


Ex vivo-expanded human regulatory T cells prevent the rejection of skin allografts in a humanized mouse model.
Composite tissue transplantation effectively reconstructs the most complex defects, but its use is limited because of harmful immunosuppression and the high susceptibility of skin to rejection. Development of tolerance is an ideal solution, and protocols using regulatory T cells (Tregs) to achieve this have been promising in experimental animal models. The aim of this study was to investigate the ability of human Tregs to regulate immune responses to a human skin allograft in vivo. We isolated and expanded naturally occurring CD127loCD25+CD4+ human Tregs from peripheral blood mononuclear cells (PBMCs) and examined their phenotype and suppressive activity in vitro. Using a clinically relevant chimeric humanized mouse system, we transplanted mice with human skin grafts followed by allogeneic populations of PBMCs with or without Tregs derived from the same PBMC donor. Ex vivo-expanded Tregs maintain the appropriate Treg markers and retain suppressive activity against allostimulated and polyclonally stimulated autologous PBMCs in vitro. Mice receiving allogeneic PBMCs alone consistently reject human skin grafts, whereas those also receiving Tregs display stable long-term human skin transplant survival along with a reduction in the CD8+ human cellular graft infiltrate. We show for the first time the unique ability of human Tregs to prevent the rejection of a skin allograft in vivo, highlighting the therapeutic potential of these cells clinically.


Large outbreak of Salmonella Thompson related to smoked salmon in the Netherlands, August to December 2012.
On 15 August 2012, an increase in the number of Salmonella Thompson cases was noticed by the Salmonella surveillance in the Netherlands. A case–control study was performed, followed by a food investigation. In total 1,149 cases were laboratory-confirmed between August and December 2012 of which four elderly (76–91 years) were reported to have died due to the infection. The cause of the outbreak was smoked salmon processed at a single site. The smoked salmon had been continuously contaminated in the processing lines through reusable dishes, which turned out to be porous and had become loaded with bacteria. This is the largest outbreak of salmonellosis ever recorded in the Netherlands. The temporary closure of the processing site and recall of the smoked salmon stopped the outbreak. An estimated four to six million Dutch residents were possibly exposed to the contaminated smoked salmon and an estimated 23,000 persons would have had acute gastroenteritis with S. Thompson during this outbreak. This outbreak showed that close collaboration between diagnostic laboratories, regional public health services, the national institute for public health and the food safety authorities is essential in outbreak investigations.


A Review of Classical Swine Fever Virus and Routes of Introduction into the United States and the Potential for Virus Establishment.
Classical swine fever (CSF) is caused by CSF virus (CSFV) which can be the source of substantial morbidity and mortality events in affected swine. The disease can take one of several forms (acute, chronic, or prenatal) and depending on the virulence of the inoculating strain may result in a lethal infection irrespective of the form acquired. Because of the disease-free status of the United States and the high cost of a viral incursion, a summary of US vulnerabilities for viral introduction and persistence is provided. The legal importation of live animals as well as animal products, byproducts, and animal feed serve as a potential route of viral introduction. Current import regulations are described as are mitigation strategies that are commonly utilized to prevent pathogens, including CSFV, from entering the US. The illegal movement of suids and their products as well as an event of bioterrorism are both feasible routes of viral introduction but are difficult to restrict or regulate. Ultimately, recommendations are made for data that would be useful in the event of a viral incursion. Population and density mapping for feral swine across the United States would be valuable in the event of a viral introduction or spillover; density data could further contribute to understanding the risk of infection in domestic swine. Additionally, ecological and behavioral studies, including those that evaluate the effects of anthropogenic food sources that support feral swine densities far above the carrying capacity would provide invaluable insight to our understanding of how human interventions affect feral swine populations. Further analyses to determine the sampling strategies necessary to detect low levels of antibody prevalence in feral swine would also be valuable.


Direct tumor visual feedback during free breathing in 0.35T MRgRT.
To present a tumor motion control system during free breathing using direct tumor visual feedback to patients in 0.35 T magnetic resonance-guided radiotherapy (MRgRT). We present direct tumor visualization to patients by projecting real-time cine MR images on an MR-compatible display system inside a 0.35 T MRgRT bore. The direct tumor visualization included anatomical images with a target contour and an auto-segmented gating contour. In addition, a beam-status sign was added for patient guidance. The feasibility was investigated with a six-patient clinical evaluation of the system in terms of tumor motion range and beam-on time. Seven patients without visual guidance were used for comparison. Positions of the tumor and the auto-segmented gating contour from the cine MR images were used in probability analysis to evaluate tumor motion control. In addition, beam-on time was recorded to assess the efficacy of the visual feedback system. The direct tumor visualization system was developed and implemented in our clinic. The target contour extended 3 mm outside of the gating contour for 33.6 ± 24.9% of the time without visual guidance, and 37.2 ± 26.4% of the time with visual guidance. The average maximum motion outside of the gating contour was 14.4 ± 11.1 mm without and 13.0 ± 7.9 mm with visual guidance. Beam-on time as a percentage was 43.9 ± 15.3% without visual guidance, and 48.0 ± 21.2% with visual guidance, but was not significantly different (P = 0.34). We demonstrated the clinical feasibility and potential benefits of presenting direct tumor visual feedback to patients in MRgRT. The visual feedback allows patients to visualize and attempt to minimize tumor motion in free breathing. The proposed system and associated clinical workflow can be easily adapted for any type of MRgRT.


Brachial plexus injury.
A 28-year-old man shot himself in the left posterior triangle of the neck with a shotgun. At the initial operation secondary repair of the resultant brachial plexus injury was decided upon in view of the difficulty in assessing lesions in continuity at this point after injury. The patient had total brachial plexus palsy. Nine weeks after the injury sensory and motor function were returning and the only element of the brachial plexus not showing evidence of nerve fibre continuity was the musculocutaneous nerve. Sural nerve autografts were sutured between the trimmed proximal and distal stumps of this nerve. By 4 months after the injury there was further improvement in both sensory and motor function, and by 18 months there was sensation in the autonomous zones of both median and ulnar nerves and good return of muscle power.


Understanding variation for clinical governance: an illustration using the diagnosis and treatment of sore throat.
The aim of clinical governance is to improve clinical care. An understanding of the information contained in variation is central to any improvement effort. We must distinguish between variation intrinsic to a process (common cause variation) and variation caused by extrinsic factors (special cause variation). The control chart is a method of distinguishing between these two kinds of variation: it is used in industry to effect improvement and may be useful in primary care. To illustrate the use of control charts to distinguish between common cause and special cause variation and to guide appropriate action. Analysis of diagnostic and treatment decisions for sore throat. Single practice in the West Midlands. We identified each general practitioner's (GP's) consultations for sore throat over a two-year period. We grouped these into two diagnostic categories (tonsillitis and non-tonsillar throat infection) and two treatment categories (antibiotics and no antibiotics). These data were illustrated graphically as XY control charts. In this practice, a special cause affects one GP's diagnosis--he is less likely to use the term 'tonsillitis'. A special cause also affects his treatment decisions--he is more likely to prescribe antibiotics. Diagnostic and treatment differences between the remaining GPs are consistent with common cause variation. In this practice, action to improve the quality of diagnosis and treatment of sore throat shouldfocus on investigating why one practitioner's diagnosis and treatment differs from that of his colleagues. Control chart analysis is valuable because it enables users to obtain practical guidance for action.


Phosphatidylinositol 3-kinase signaling in proliferating cells maintains an anti-apoptotic transcriptional program mediated by inhibition of FOXO and non-canonical activation of NFkappaB transcription factors.
Phosphatidylinositol (PI) 3-kinase is activated by a variety of growth factor receptors and the PI 3-kinase/Akt signaling pathway is a key regulator of cell proliferation and survival. The downstream targets of PI 3-kinase/Akt signaling include direct regulators of cell cycle progression and apoptosis as well as a number of transcription factors. Growth factor stimulation of quiescent cells leads to robust activation of PI 3-kinase, induction of immediate-early genes, and re-entry into the cell cycle. A lower level of PI 3-kinase signaling is also required for the proliferation and survival of cells maintained in the presence of growth factors, but the gene expression program controlled by PI 3-kinase signaling in proliferating cells has not been elucidated. We used microarray analyses to characterize the changes in gene expression resulting from inhibition of PI 3-kinase in proliferating cells. The genes regulated by inhibition of PI 3-kinase in proliferating cells were distinct from genes induced by growth factor stimulation of quiescent cells and highly enriched in genes that regulate programmed cell death. Computational analyses followed by chromatin immunoprecipitations demonstrated FOXO binding to both previously known and novel sites in promoter regions of approximately one-third of the up-regulated genes, consistent with activation of FOXO1 and FOXO3a in response to inhibition of PI 3-kinase. NFkappaB binding sites were similarly identified in promoter regions of over one-third of the down-regulated genes. RelB was constitutively bound to promoter regions in cells maintained in serum, however binding decreased following PI 3-kinase inhibition, indicating that PI 3-kinase signaling activates NFkappaB via the non-canonical pathway in proliferating cells. Approximately 70% of the genes targeted by FOXO and NFkappaB regulate cell proliferation and apoptosis, including several regulators of apoptosis that were not previously known to be targeted by these transcription factors. PI 3-kinase signaling in proliferating cells regulates a novel transcriptional program that is highly enriched in genes that regulate apoptosis. At least one-third of these genes are regulated either by FOXO transcription factors, which are activated following PI 3-kinase inhibition, or by RelB, which is activated by PI 3-kinase via the non-canonical pathway in proliferating cells.


Reproductive rights and options available to women infected with HIV in Ghana: perspectives of service providers from three Ghanaian health facilities.
Owing to improved management of HIV and its associated opportunistic infections, many HIV-positive persons of reproductive age are choosing to exercise their right of parenthood. This study explored the knowledge of health workers from two Ghanaian districts on the reproductive rights and options available to HIV-positive women who wish to conceive. Facility-based cross-sectional in design, the study involved the entire population of nurse counselors (32) and medical officers (3) who provide counseling and testing services to clients infected with HIV. Both structured and in-depth interviews were conducted after informed consent. Two main perspectives were revealed. There was an overwhelmingly high level of approbation by the providers on HIV-positive women's right to reproduction (94.3%). At the same time, the providers demonstrated a lack of knowledge regarding the various reproductive options available to women infected with HIV. Site of facility, and being younger were associated with practices that violated client's right to contraceptive counseling (p < 0.05) in each case. Some of the providers openly expressed their inability to give qualified guidance to HIV-positive women on the various reproductive options. Taken together, these findings suggest that many HIV-positive clients do not receive comprehensive information about their reproductive options. These findings highlight some of the problems that service providers face as HIV counselors. Both service providers and policy makers need to recognize these realities and incorporate reproductive health issues of HIV-persons into the existing guidelines.


Radiostereometric Analysis Permits In Vivo Measurement of Very Small Levels of Wear in TKA.
Revision of TKA as a result of polyethylene wear is decreasing, but long-term wear performance of polyethylene is still a topic of interest to surgeons and device manufacturers seeking to improve longevity. Measuring wear of modern, wear-resistant implants has been described using radiostereometric analysis (RSA). Performing in vivo measurements would establish whether implant retrieval studies are representative of wear in well-performing knees. For a single knee implant system, we sought to determine (1) the linear wear rate using RSA; (2) the association between demographic factors and wear rate; and (3) the association between limb alignment and wear rate. A total of 49 patients with a minimum followup of 10 years (median, 12 years; range, 10-20 years) were retrospectively selected. During the examined period, 4082 TKAs were performed of which 2085 were the implant examined in this study. There were 71 of these patients who met the criteria including an available full-leg radiograph postoperatively, and 34 of these patients returned for examination along with 15 additional from a separate RSA study that also met the criteria. All patients received a posterior-stabilized, cobalt-chromium-on-conventional polyethylene total knee implant from a single implant system, which was the most commonly used at our institution at the time. Patients underwent standing RSA examinations from 0° to 120° of flexion at a single time point without the use of marker beads. Linear wear rates (including creep) were measured based on intersections between the femoral component and tibial insert models. Associations between wear and patient age at surgery, sex, height, weight, body mass index, tibial insert size, and limb alignment were examined. Using the maximum linear wear rate from any flexion angle, the lateral rate was 0.047 mm/year (interquartile range [IQR], 0.034-0.066 mm/year) and the medial rate was 0.052 mm/year (IQR, 0.040-0.069 mm/year). Using the median of the linear wear rates across all flexion angles, the lateral rate was 0.027 mm/year (IQR, 0.017-0.046 mm/year) and the medial rate was 0.038 mm/year (IQR, 0.022-0.054 mm/year). This rate for males was 0.049 mm/year medially (IQR, 0.042-0.077 mm/year) and 0.032 mm/year laterally (IQR, 0.026-0.059 mm/year), and for females was 0.027 mm/year medially (0.016-0.039 mm/year) and 0.020 mm/year laterally (IQR, 0.013-0.032 mm/year). The wear rate for males was greater medially (difference = 0.022 mm/year, p < 0.001) and laterally (difference = 0.012 mm/year, p = 0.008). There were associations between greater wear and increasing height (ρ = 0.48, p < 0.001 medially and ρ = 0.30, p = 0.04 laterally), decreasing body mass index (ρ = -0.31, p = 0.03 medially), and greater implant size (ρ = 0.34, p = 0.02 medially). Increasingly varus leg alignment was associated with greater medial wear (ρ = 0.33, p = 0.02). Greater wear rates were associated with demographic factors and leg alignment. Further RSA wear studies of other modern implant systems would provide complementary information to retrieval studies and valuable data on wear resistance. Good wear resistance was demonstrated by well-performing implants in patients at long-term followup with wear magnitudes in agreement with reported values from retrieval studies.


Ataxia telangiectasia gene mutations in leukaemia and lymphoma.
Ataxia telangiectasia (AT) is a rare multisystem, autosomal, recessive disease characterised by neuronal degeneration, genome instability, and an increased risk of cancer. Approximately 10% of AT homozygotes develop cancer, mostly of the lymphoid system. Lymphoid malignancies in patients with AT are of both B cell and T cell origin, and include Hodgkin's lymphoma, non-Hodgkin's lymphoma, and several forms of leukaemia. The AT locus was mapped to the chromosomal region 11q22-23 using genetic linkage analysis in the late 1980s and the causative gene was identified by positional cloning several years later. The ATM gene encodes a large protein that belongs to a family of kinases possessing a highly conserved C-terminal kinase domain related to the phosphatidylinositol 3-kinase domain. Members of this kinase family have been shown to function in DNA repair and cell cycle checkpoint control following DNA damage. Recent studies indicate that ATM is activated primarily in response to double strand breaks and may be considered a caretaker of the genome. Most mutations in ATM result in truncation and destabilisation of the protein, but certain missense and splicing errors have been shown to produce a less severe phenotype. AT heterozygotes have a slightly increased risk of breast cancer. Atm deficient mice exhibit many of the symptoms found in patients with AT and have a high frequency of thymic lymphoma. The association between mutation of the ATM gene and a high incidence of lymphoid malignancy in patients with AT, together with the development of lymphoma in Atm deficient mice, supports the proposal that inactivation of the ATM gene may be of importance in the pathogenesis of sporadic lymphoid malignancy. Loss of heterozygosity at 11q22-23 (the location of the ATM gene) is a common event in lymphoid malignancy. Frequent inactivating mutations of the ATM gene have been reported in patients with rare sporadic T cell prolymphocytic leukaemia (T-PLL), B cell chronic lymphocytic leukaemia (B-CLL), and most recently, mantle cell lymphoma (MCL). In contrast to the ATM mutation pattern in AT, the most frequent nucleotide changes in these sporadic lymphoid malignancies were missense mutations. The presence of inactivating mutations, together with the deletion of the normal copy of the ATM gene in some patients with T-PLL, B-CLL, and MCL, establishes somatic inactivation of the ATM gene in the pathogenesis of lymphoid malignancies, and strongly suggests that ATM functions as a tumour suppressor. The presence of missense mutations in the germline of patients with B-CLL has been reported, suggesting that some patients with B-CLL may be constitutional AT heterozygotes. The putative hereditary predisposition of B-CLL, although intriguing, warrants further investigation.


Two Phase 3 Trials of Dupilumab versus Placebo in Atopic Dermatitis.
Dupilumab, a human monoclonal antibody against interleukin-4 receptor alpha, inhibits signaling of interleukin-4 and interleukin-13, type 2 cytokines that may be important drivers of atopic or allergic diseases such as atopic dermatitis. In two randomized, placebo-controlled, phase 3 trials of identical design (SOLO 1 and SOLO 2), we enrolled adults with moderate-to-severe atopic dermatitis whose disease was inadequately controlled by topical treatment. Patients were randomly assigned in a 1:1:1 ratio to receive, for 16 weeks, subcutaneous dupilumab (300 mg) or placebo weekly or the same dose of dupilumab every other week alternating with placebo. The primary outcome was the proportion of patients who had both a score of 0 or 1 (clear or almost clear) on the Investigator's Global Assessment and a reduction of 2 points or more in that score from baseline at week 16. We enrolled 671 patients in SOLO 1 and 708 in SOLO 2. In SOLO 1, the primary outcome occurred in 85 patients (38%) who received dupilumab every other week and in 83 (37%) who received dupilumab weekly, as compared with 23 (10%) who received placebo (P<0.001 for both comparisons with placebo). The results were similar in SOLO 2, with the primary outcome occurring in 84 patients (36%) who received dupilumab every other week and in 87 (36%) who received dupilumab weekly, as compared with 20 (8%) who received placebo (P<0.001 for both comparisons). In addition, in the two trials, an improvement from baseline to week 16 of at least 75% on the Eczema Area and Severity Index was reported in significantly more patients who received each regimen of dupilumab than in patients who received placebo (P<0.001 for all comparisons). Dupilumab was also associated with improvement in other clinical end points, including reduction in pruritus and symptoms of anxiety or depression and improvement in quality of life. Injection-site reactions and conjunctivitis were more frequent in the dupilumab groups than in the placebo groups. In two phase 3 trials of identical design involving patients with atopic dermatitis, dupilumab improved the signs and symptoms of atopic dermatitis, including pruritus, symptoms of anxiety and depression, and quality of life, as compared with placebo. Trials of longer duration are needed to assess the long-term effectiveness and safety of dupilumab. (Funded by Sanofi and Regeneron Pharmaceuticals; SOLO 1 ClinicalTrials.gov number, NCT02277743 ; SOLO 2 ClinicalTrials.gov number, NCT02277769 .).


Standardized Evaluation System for Left Ventricular Segmentation Algorithms in 3D Echocardiography.
Real-time 3D Echocardiography (RT3DE) has been proven to be an accurate tool for left ventricular (LV) volume assessment. However, identification of the LV endocardium remains a challenging task, mainly because of the low tissue/blood contrast of the images combined with typical artifacts. Several semi and fully automatic algorithms have been proposed for segmenting the endocardium in RT3DE data in order to extract relevant clinical indices, but a systematic and fair comparison between such methods has so far been impossible due to the lack of a publicly available common database. Here, we introduce a standardized evaluation framework to reliably evaluate and compare the performance of the algorithms developed to segment the LV border in RT3DE. A database consisting of 45 multivendor cardiac ultrasound recordings acquired at different centers with corresponding reference measurements from three experts are made available. The algorithms from nine research groups were quantitatively evaluated and compared using the proposed online platform. The results showed that the best methods produce promising results with respect to the experts' measurements for the extraction of clinical indices, and that they offer good segmentation precision in terms of mean distance error in the context of the experts' variability range. The platform remains open for new submissions.


The association of 2-year-old training milestones with career length and racing success in a sample of Thoroughbred horses in New Zealand.
There is increasing evidence that exercise early in life has a positive effect on musculoskeletal health. At present, there is little whole population research investigating the effect of racing as 2-year-olds on future racing career. To investigate the association between attaining training milestones as 2-year-olds with length of career and racing success in Thoroughbred horses in New Zealand. Retrospective data were obtained of the 2001/02-born Thoroughbred foal crop. The 3 training milestones were: registered with a trainer, trialled and raced. The association of the training milestones with career length was measured using the outcomes: number of race starts and number of years raced, in a Cox regression model. Logistic regression models analysed the association of the training milestones with the outcomes: won or placed in a race. Linear regression was performed to assess the association of training milestones with total career earnings. Of 4683 horses in the population; 3152 horses were registered with a trainer, 2661 horses trialled and 2109 horses raced. Horses that raced as 2-year-olds had significantly (P<0.001) more race starts than those first raced as 3-year-olds or older, this was also true when the 2-year-old year data were omitted. Horses that raced as 2-year-olds had significantly (P<0.001) more years racing. Horses registered with a trainer, trialled or raced as 2-year-olds were more likely to have won or been placed in a race than those that achieved the milestones as 3-year-olds or older. Horses that first trialled and raced as 2-year-olds had greater total earnings than those that first trialled or raced at a later age. Two-year-old training milestones had a strong association with positive racing career outcomes. Horses in training or racing as 2-year-olds may have better musculoskeletal health throughout life than horses that are first in training or racing at a later age.


Cost-effectiveness of vortioxetine versus venlafaxine (extended release) in the treatment of major depressive disorder in South Korea.
To assess the cost-effectiveness of vortioxetine versus venlafaxine XR (extended-release) in major depressive disorder (MDD) patients in South Korea. A 1-year cost-effectiveness analysis from a limited societal perspective was performed using a combined model consisting of a decision-tree and a Markov model. Patients entered the model when initiating or switching antidepressant treatment following inadequate response to previous treatment. Remission, relapse and recovery were the main health states. Vortioxetine dominated venlafaxine XR, with quality-adjusted life year (QALY) gains of 0.0131 and cost savings of KRW 623,229/year [US$530/year] from a limited societal perspective. Safety contributed more than efficacy to the incremental QALY gains. More patients were in recovery after initial treatment and after 1 year with vortioxetine (31%, 40%) compared to venlafaxine XR (23%, 36%). Vortioxetine remained dominant in 98% of probabilistic simulations. Vortioxetine dominated venlafaxine XR in South Korea and is a relevant treatment option for MDD patients initiating or switching therapy.


Mechanical chest compression for out of hospital cardiac arrest: Systematic review and meta-analysis.
To summarise the evidence from randomised controlled trials of mechanical chest compression devices used during resuscitation after out of hospital cardiac arrest. Systematic review of studies evaluating the effectiveness of mechanical chest compression. We included randomised controlled trials or cluster randomised trials that compared mechanical chest compression (using any device) with manual chest compression for adult patients following out-of-hospital cardiac arrest. Outcome measures were return of spontaneous circulation, survival of event, overall survival, survival with good neurological outcome. Results were combined using random-effects meta-analysis. Studies were identified by searches of electronic databases, reference lists of other studies and review articles. Five trials were included, of which three evaluated the LUCAS or LUCAS-2 device and two evaluated the AutoPulse device. The results did not show an advantage to the use of mechanical chest compression devices for survival to discharge/30 days (average OR 0.89, 95% CI 0.77, 1.02) and survival with good neurological outcome (average OR 0.76, 95% CI 0.53, 1.11). Existing studies do not suggest that mechanical chest compression devices are superior to manual chest compression, when used during resuscitation after out of hospital cardiac arrest.


Cross Validation Through Two-Dimensional Solution Surface for Cost-Sensitive SVM.
Model selection plays an important role in cost-sensitive SVM (CS-SVM). It has been proven that the global minimum cross validation (CV) error can be efficiently computed based on the solution path for one parameter learning problems. However, it is a challenge to obtain the global minimum CV error for CS-SVM based on one-dimensional solution path and traditional grid search, because CS-SVM is with two regularization parameters. In this paper, we propose a solution and error surfaces based CV approach (CV-SES). More specifically, we first compute a two-dimensional solution surface for CS-SVM based on a bi-parameter space partition algorithm, which can fit solutions of CS-SVM for all values of both regularization parameters. Then, we compute a two-dimensional validation error surface for each CV fold, which can fit validation errors of CS-SVM for all values of both regularization parameters. Finally, we obtain the CV error surface by superposing K validation error surfaces, which can find the global minimum CV error of CS-SVM. Experiments are conducted on seven datasets for cost sensitive learning and on four datasets for imbalanced learning. Experimental results not only show that our proposed CV-SES has a better generalization ability than CS-SVM with various hybrids between grid search and solution path methods, and than recent proposed cost-sensitive hinge loss SVM with three-dimensional grid search, but also show that CV-SES uses less running time.


Sampling and processing blood samples within the South American Youth/Child cARdiovascular and Environmental (SAYCARE) Study.
Obesity and overweight in children and adolescents is increasing rapidly worldwide; however, scarce data have been reported from South America countries. With the purpose of assessing hyperlipidemia, insulin resistance and chronic inflammation, the evaluation of blood biomarkers such as glucose, lipoproteins and chronic inflammation proteins is required. In the context of the SAYCARE study, in children and adolescents (3 to 18 years) from seven South American cities, our aim was to assess the impact of pre analytical conditions on different biomarkers evaluated in 474 fresh serum samples, in different country centers. We also evaluated the stability according to time and frozen storage within this study across the concordance of the results obtained from the 49 blood samples measured in three different centers. Significant correlations as well as concordance were observed in TG, Total-C, HDL-C and glucose between Buenos Aires and São Paulo. The samples evaluated in Teresina and São Paulo presented similar results, with exception of total cholesterol. We observed acceptable concordance between Buenos Aires vs São Paulo and Teresina vs São Paulo, suggesting that samples could be processed in each of these centers. This concordance is a consequence of the strict pre analytical conditions previously established in the SAYCARE study.


Gender dimorphism of the cardiac dysfunction in murine sepsis: signalling mechanisms and age-dependency.
Development of cardiac dysfunction is associated with increased morbidity and mortality in patients with sepsis. Increasing evidence shows that gender determines the degree of inflammatory response of the host and that females tolerate sepsis better than males. It is unknown whether gender affects the cardiac dysfunction in animals or patients with sepsis. To investigate this, male or female C57BL/6 mice were subjected to either lipopolysaccharide (LPS)/peptidoglycan (PepG) co-administration or cecal ligation and puncture (CLP). At 18 hours after LPS/PepG injection or 24 hours after CLP, cardiac function was evaluated by echocardiography. The septic insult caused a significant cardiac dysfunction in both genders. However, the cardiac dysfunction was significantly less pronounced in females in comparison with males subjected to LPS (3 mg/kg)/PepG (0.1 mg/kg) or CLP. Compared with males injected with LPS (3 mg/kg)/PepG (0.1 mg/kg), western blotting analysis of the myocardium from females injected with LPS/PepG revealed i) profound increases in phosphorylation of Akt and eNOS; ii) significant decreases in phosphorylation of IκBα, nuclear translocation of the NF-κB subunit p65, decreased expression of iNOS and decreased synthesis of TNF-α and IL-6 in the heart. However, the gender dimorphism of the cardiac dysfunction secondary to LPS/PepG was not observed when higher doses of LPS (9 mg/kg)/PepG (1 mg/kg) were used. In conclusion, the cardiac dysfunction caused by sepsis was less pronounced in female than in male mice. The protection of female hearts against the dysfunction associated with sepsis is (at least in part) attributable to cardiac activation of the Akt/eNOS survival pathway, decreased activation of NF-κB, and decreased expression of iNOS, TNF-α and IL-6. It should be noted that the observed gender dimorphism of the cardiac dysfunction in sepsis was not seen when a very severe stimulus (high dose of LPS/PepG co-administration) was used to cause cardiac dysfunction.


Quantitative analysis of peripheral tissue perfusion using spatiotemporal molecular dynamics.
Accurate measurement of peripheral tissue perfusion is challenging but necessary to diagnose peripheral vascular insufficiency. Because near infrared (NIR) radiation can penetrate relatively deep into tissue, significant attention has been given to intravital NIR fluorescence imaging. We developed a new optical imaging-based strategy for quantitative measurement of peripheral tissue perfusion by time-series analysis of local pharmacokinetics of the NIR fluorophore, indocyanine green (ICG). Time-series NIR fluorescence images were obtained after injecting ICG intravenously in a murine hindlimb ischemia model. Mathematical modeling and computational simulations were used for translating time-series ICG images into quantitative pixel perfusion rates and a perfusion map. We could successfully predict the prognosis of ischemic hindlimbs based on the perfusion profiles obtained immediately after surgery, which were dependent on the preexisting collaterals. This method also reflected increases in perfusion and improvements in prognosis of ischemic hindlimbs induced by treatment with vascular endothelial growth factor and COMP-angiopoietin-1. We propose that this novel NIR-imaging-based strategy is a powerful tool for biomedical studies related to the evaluation of therapeutic interventions directed at stimulating angiogenesis.


Inhibition of STAT3, FAK and Src mediated signaling reduces cancer stem cell load, tumorigenic potential and metastasis in breast cancer.
Cancer stem cells (CSCs) are responsible for aggressive tumor growth, metastasis and therapy resistance. In this study, we evaluated the effects of Shikonin (Shk) on breast cancer and found its anti-CSC potential. Shk treatment decreased the expression of various epithelial to mesenchymal transition (EMT) and CSC associated markers. Kinase profiling array and western blot analysis indicated that Shk inhibits STAT3, FAK and Src activation. Inhibition of these signaling proteins using standard inhibitors revealed that STAT3 inhibition affected CSCs properties more significantly than FAK or Src inhibition. We observed a significant decrease in cell migration upon FAK and Src inhibition and decrease in invasion upon inhibition of STAT3, FAK and Src. Combined inhibition of STAT3 with Src or FAK reduced the mammosphere formation, migration and invasion more significantly than the individual inhibitions. These observations indicated that the anti-breast cancer properties of Shk are due to its potential to inhibit multiple signaling proteins. Shk also reduced the activation and expression of STAT3, FAK and Src in vivo and reduced tumorigenicity, growth and metastasis of 4T1 cells. Collectively, this study underscores the translational relevance of using a single inhibitor (Shk) for compromising multiple tumor-associated signaling pathways to check cancer metastasis and stem cell load.


Atropisomeric meroterpenoids with rare triketone-phloroglucinol-terpene hybrids from Baeckea frutescens.
Baefrutones A-F (1-6), six new meroterpenoids with rare triketone-phloroglucinol-monoterpene/sesquiterpene frameworks, together with their biosynthetically related intermediate (±)-baeckenon B (7), were isolated from the aerial part of Baeckea frutescens under the guidance of HPLC-Q/TOF-MS2 investigation. Compounds 1-4 represent the first examples of natural meroterpenoids existing as four pairs of inseparable diastereomeric atropisomers (2 : 1, 1H NMR integration) caused by the restricted rotation around the C-6-C-7-C-1' bonds arising from the intramolecular hydrogen bond between C-1 carbonyl and 2'-OH. The discovery of these architectures not only largely enriched the chemodiversity of the meroterpenoid and atropisomer library, but also might be exciting and challenging for asymmetric organic synthesis. Their structures and absolute configurations were established by extensive spectroscopic analysis, X-ray diffraction, and ECD calculations. Compounds 5 and 6 were biomimetically synthesized from 7 and β-caryophyllene via a regioselective oxidative hetero-Diels-Alder reaction, thus providing access to the construction of the 6/6/9/4 tetracyclic ring system. The anti-inflammatory activities of these meroterpenoids were also discussed.


Finite-element model of interaction between fungal polysaccharide and monoclonal antibody in the capsule of Cryptococcus neoformans.
Many microorganisms such as bacteria and fungi possess so-called capsules made of polysaccharides which protect these microorganisms from environmental insults and host immune defenses. The polysaccharide capsule of Cryptococcus neoformans, a human pathogenic yeast, is capable of self-assembly, composed mostly of glucuronoxylomannan (GXM), a polysaccharide with a molecular weight of approximately 2,000,000, and has several layers with different densities. The objective of this study was to model pore-hindered diffusion and binding of the GXM-specific antibody within the C. neoformans capsule. Using the finite-element method (FEM), we created a model which represents the in vivo binding of a GXM-specific antibody to a C. neoformans cell taking into account the intravenous infusion time of antibody, antibody diffusion through capsular pores, and Michaelis-Menten kinetics of antibody binding to capsular GXM. The model predicted rapid diffusion of antibody to all regions of the capsule where the pore size was greater than the Stokes diameter of the antibody. Binding occurred primarily at intermediate regions of the capsule. The GXM concentration in each capsular region was the principal determinant of the steady-state antibody-GXM complex concentration, while the forward binding rate constant influenced the rate of complex formation in each region. The concentration profiles predicted by the model closely matched experimental immunofluorescence data. Inclusion of different antibody isotypes (IgG, IgA, and IgM) into the modeling algorithm resulted in similar complex formation in the outer capsular regions, but different depths of binding at the inner regions. These results have implications for the development of new antibody-based therapies.


CATR: a new generation of autologous blood transfusion.
As the field of surgery advances and the conservative use of banked blood transfusion increases the need for postoperative autotransfusion has increased. By using the CATR, the patient's demands for blood can be more adequately met without causing further complications for the patient. This article presents one method for postoperative cardiothoracic autotransfusion and describes its advantages and risks. A Nursing Plan of Care is included.


Characterization of Monilinia fructicola Strains Resistant to Both Propiconazole and Boscalid.
In 2011 and 2012, significant brown rot disease caused by Monilinia fructicola was observed in a peach orchard in Spartanburg County, SC, despite preharvest fungicide applications of demethylation inhibitor (DMI), quinone outside inhibitor (QoI), and succinate dehydrogenase inhibitor (SDHI) fungicides. All 22 isolates obtained in 2011 from this orchard were sensitive to the QoI fungicide, azoxystrobin, and the methyl benzimidazole carbamate (MBC) fungicide, thiophanate-methyl. Five were resistant to the DMI fungicide, propiconazole, and were selected, together with five propiconazole-sensitive isolates, for further investigations. One of the 10 isolates was resistant to propiconazole but sensitive to the SDHI fungicide, boscalid (EC50 = 0.42 μg/ml), 3 were resistant to propiconazole with intermediate sensitivity to boscalid (EC50 0.72 to 2.1 μg/ml); 2 were sensitive to propiconazole with intermediate sensitivity to boscalid; 3 were sensitive to propiconazole but resistant to boscalid (EC50 ≥ 2.1 μg/ml); and 1 (isolate MD22) was resistant to both propiconazole and boscalid. Disease incidence on detached fruit treated with formulated propiconazole or boscalid was significantly higher for MD22 compared to a sensitive control isolate. Continued monitoring of fungicide resistance in the same orchard in 2012 revealed an increase of isolates resistant to propiconazole from 22.7% in 2011 to 34.7%, and an increase of isolates resistant to both propiconazole and boscalid from 4.5% in 2011 to 18.4%. Propiconazole resistance was always associated with the presence of the 'Mona' mobile element located upstream of the sterol 14α-demethylase (MfCYP51) gene. To investigate whether mutations in the subunits of the succinate dehydrogenase enzyme were involved in boscalid resistance, significant portions of the M. fructicola SdhA, SdhB, SdhC, and SdhD genes were cloned and analyzed for 2 sensitive, 2 boscalid-resistant, and 6 dual-resistant isolates. Although sequence variation was found among the isolates, no single change correlated with resistance. Interestingly, analysis of isolates collected from orchards in 2001 and 2002, prior to the registration of boscalid, revealed a range of sensitivities to boscalid (EC50 0.03 to 3.46 μg/ml) including boscalid-resistant isolates. The presence of boscalid-resistant isolates in the baseline population was unexpected and requires further investigation.


Comparative Evaluation of Clinical and Radiographic Success of Formocresol, Propolis, Turmeric Gel, and Calcium Hydroxide on Pulpotomized Primary Molars: A Preliminary Study.
Despite various advents in technology, the present era marks a shift to phytotherapeutics and alternative modalities to conventional endodontic treatments. Newer endodontic modalities have been developed inculcating the ancient system of medicine. The present study was done to compare and evaluate the clinical pulp response and radiographic signs after pulpotomy in four groups of primary molar teeth treated with formocresol (control), propolis extract, turmeric gel, and calcium hydroxide respectively. Following ethical clearance, 90 primary molar teeth in 45 pediatric patients, aged between 4 and 9 years, were selected for pulpotomy. These were then randomly divided by split-mouth technique into two groups as experimental (propolis extract/turmeric gel/calcium hydroxide) and control (formocresol) groups. The patients were followed up for 6 months for clinical and radiographic signs and symptoms to evaluate the success of treatment. A comparable clinical and radiographic success rate was seen with all experimental groups as compared to the control (formocresol) group. With concerns about the safety of formocresol appearing in the dental and medical literature for more than 20 years, the materials used in this study can be considered as promising alternatives for formocresol in pediatric endodontic treatment. Hugar SM, Kukreja P, Hugar SS, Gokhale N, Assudani H. Comparative Evaluation of Clinical and Radiographic Success of Formocresol, Propolis, Turmeric Gel, and Calcium Hydroxide on Pulpotomized Primary Molars: A Preliminary Study. Int J Clin Pediatr Dent 2017;10(1):18-23.


Comparison of training and game demands of national level cricketers.
Although the physiological demands of cricket match play are emerging, the demands of contemporary training practices have not been reported. The aim of this study was to quantify the physiological demands of selected cricket training activities and compare these to known match demands. Twenty-eight different training activities were monitored in national academy level cricketers (n = 42) using global positioning system units during a 14-week residential training program. The training activities were classified into 3 categories: conditioning sessions (n = 8), skill sessions (n = 9), and game simulations (n = 11). Conditioning sessions were further classified into high- (n = 4) and low- (n = 4) intensity drills. Time-motion measures included movement patterns (walk, jog, run, stride, and sprint distances), total distance covered, number of sprints, number of high-intensity efforts, maximum speed, and recovery time between high-intensity efforts. Inferential statistics were used to quantify magnitudes of difference between various training drills. Movement patterns were then compared to recently published game data (Twenty20, One-Day, and Multiday games) from the same sample group of cricketers. Conditioning drills were twice as long in duration as skill drills and twice as intense as both the skill and game simulation drills. Exercising heart rates were 9-26% and lactate levels up to 3.5 times higher in conditioning compared to other training drills. Conditioning drills matched or exceeded (up to 10 b·min⁻¹; 5%) peak game heart rates. Conversely, skill and simulation drills replicated mean game heart rates for some, but not all positions. In conclusion, training replicates or exceeds cricket match demands in conditioning-type drills but not in simulation or skill-based drills. Modification of skill and simulation training drill practices will ensure closer replication of match and training demands.


Short term memory for tactile stimuli.
Research has shown that unreported information stored in rapidly decaying visual representations may be accessed more accurately using partial report than using full report procedures (e.g., [Sperling, G., 1960. The information available in brief visual presentations. Psychological Monographs, 74, 1-29.]). In the 3 experiments reported here, we investigated whether unreported information regarding the actual number of tactile stimuli presented in parallel across the body surface can be accessed using a partial report procedure. In Experiment 1, participants had to report the total number of stimuli in a tactile display composed of up to 6 stimuli presented across their body (numerosity task), or else to detect whether or not a tactile stimulus had previously been presented in a position indicated by a visual probe given at a variable delay after offset of a tactile display (i.e., partial report). The results showed that participants correctly reported up to 3 stimuli in the numerosity judgment task, but their performance was significantly better than chance when up to 5 stimuli were presented in the partial report task. This result shows that short-lasting tactile representations can be accessed using partial report procedures similar to those used previously in visual studies. Experiment 2 showed that the duration of these representations (or the time available to consciously access them) depends on the number of stimuli presented in the display (the greater the number of stimuli that are presented, the faster their representation decays). Finally, the results of a third experiment showed that the differences in performance between the numerosity judgment and partial report tasks could not be explained solely in terms of any difference in task difficulty.


Interplay between polyadenylate-binding protein 1 and Kaposi's sarcoma-associated herpesvirus ORF57 in accumulation of polyadenylated nuclear RNA, a viral long noncoding RNA.
Polyadenylate-binding protein cytoplasmic 1 (PABPC1) is a cytoplasmic-nuclear shuttling protein important for protein translation initiation and both RNA processing and stability. We report that PABPC1 forms a complex with the Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein, which allows ORF57 to interact with a 9-nucleotide (nt) core element of KSHV polyadenylated nuclear (PAN) RNA, a viral long noncoding RNA (lncRNA), and increase PAN stability. The N-terminal RNA recognition motifs (RRMs) of PABPC1 are necessary for the direct interaction with ORF57. During KSHV lytic infection, the expression of viral ORF57 leads to a substantial decrease in overall PABPC1 expression, along with a shift in the cellular distribution of the remaining PABPC1 to the nucleus. Interestingly, PABPC1 and ORF57 have opposing functions in modulating PAN steady-state accumulation. The suppressive effect of PABPC1 specific to PAN expression is alleviated by small interfering RNA knockdown of PABPC1 or by overexpression of ORF57. Conversely, ectopic PABPC1 reduces ORF57 steady-state protein levels and induces aberrant polyadenylation of PAN and thereby indirectly inhibits ORF57-mediated PAN accumulation. However, E1B-AP5 (heterogeneous nuclear ribonucleoprotein U-like 1), which interacts with a region outside the 9-nt core to stimulate PAN expression, does not interact or even colocalize with ORF57. Unlike PABPC1, the nuclear distribution of E1B-AP5 remains unchanged by viral lytic infection or overexpression of ORF57. Together, these data indicate that PABPC1 is an important cellular target of viral ORF57 to directly upregulate PAN accumulation during viral lytic infection, and the ability of host PABPC1 to disrupt ORF57 expression is a strategic host counterbalancing mechanism.


Bax accelerates staurosporine-induced but suppresses nigericin-induced neuronal cell death.
Bax, a member of the Bcl-2 multigene family, is known to promote apoptosis. To investigate the role of Bax in an experimentally induced cell death of the murine dopaminergic neuronal cell line (MN9D), we established MN9D cells stably over-expressing murine Bax (MN9D/ Bax) or vector alone (MN9D/Neo). In MN9D/Neo cells treated with either 1 microM staurosporine or 0.1 microM nigericin, a ladder pattern of DNA fragmentation was induced. As expected, over-expression of Bax in MN9D cells accelerated staurosporine-induced cell death as measured by the MTT reduction assay (62.3% survival in MN9D/Neo vs 27.0% survival in MN9D/Bax). Surprizingly, both nigericin-induced cell death and its accompanying DNA fragmentation were largely attenuated in MN9D/Bax cells (22.0% survival in MN9D/Neo vs 86.7% survival in MN9D/Bax). Similar patterns were observed in two other MN9D/Bax cell lines. Cleavage of poly(ADP-ribose)polymerase caused by nigericin was greatly attenuated in MN9D/Bax cells suggesting that, like Bcl-2, Bax suppresses nigericin-induced cell death by inhibiting the activation of cysteine proteases. Thus, our data imply that Bax acts as a negative or positive regulator of cell death depending on the type of death stimulus applied to the cell.


Optical performance monitoring technique using asynchronous amplitude and phase histograms.
We report a new optical performance monitoring technique based on the amplitude and phase histograms of the optical signal. The amplitude and phase histograms are obtained by using an intradyne receiver and the asynchronous delay-tap sampling method. From these histograms, we can evaluate the amplitude and phase Q-factors of the optical signal regardless of its modulation format. In addition, by using these Q-factors, we can estimate the bit-error rate (BER) of the monitoring signal.


Assessment of Leishmaniasis notification system in Santiago del Estero, Argentina, 1990-1993.
Using a capture-recapture method, this study evaluates the completeness of the cutaneous leishmaniasis (CL) surveillance system in four districts of Santiago del Estero province, Argentina, for the period 1990-1993. Four reporting sources were evaluated: medical records kept by health facilities, interviews conducted during a case-control study, and the national and provincial levels of the leishmaniasis surveillance system (LSS). Using the capture-recapture method it was estimated that 210 cases (95% confidence interval [CI]: 202-218) of CL occurred in the four districts during the study period. Completeness of reporting to the leishmaniasis surveillance system at the national level was estimated to be 44.8% (95% CI: 43.2-46.4). The study results indicate that there is substantial underreporting within the LSS, although it did show the appropriate secular trends. The reasons for under-reporting and methods for addressing this problem are discussed.


Structural variants of yeast prions show conformer-specific requirements for chaperone activity.
Molecular chaperones monitor protein homeostasis and defend against the misfolding and aggregation of proteins that is associated with protein conformational disorders. In these diseases, a variety of different aggregate structures can form. These are called prion strains, or variants, in prion diseases, and cause variation in disease pathogenesis. Here, we use variants of the yeast prions [RNQ+] and [PSI+] to explore the interactions of chaperones with distinct aggregate structures. We found that prion variants show striking variation in their relationship with Hsp40s. Specifically, the yeast Hsp40 Sis1 and its human orthologue Hdj1 had differential capacities to process prion variants, suggesting that Hsp40 selectivity has likely changed through evolution. We further show that such selectivity involves different domains of Sis1, with some prion conformers having a greater dependence on particular Hsp40 domains. Moreover, [PSI+] variants were more sensitive to certain alterations in Hsp70 activity as compared to [RNQ+] variants. Collectively, our data indicate that distinct chaperone machinery is required, or has differential capacity, to process different aggregate structures. Elucidating the intricacies of chaperone-client interactions, and how these are altered by particular client structures, will be crucial to understanding how this system can go awry in disease and contribute to pathological variation.


Identification of cross-species shared transcriptional networks of diabetic nephropathy in human and mouse glomeruli.
Murine models are valuable instruments in defining the pathogenesis of diabetic nephropathy (DN), but they only partially recapitulate disease manifestations of human DN, limiting their utility. To define the molecular similarities and differences between human and murine DN, we performed a cross-species comparison of glomerular transcriptional networks. Glomerular gene expression was profiled in patients with early type 2 DN and in three mouse models (streptozotocin DBA/2, C57BLKS db/db, and eNOS-deficient C57BLKS db/db mice). Species-specific transcriptional networks were generated and compared with a novel network-matching algorithm. Three shared human-mouse cross-species glomerular transcriptional networks containing 143 (Human-DBA STZ), 97 (Human-BKS db/db), and 162 (Human-BKS eNOS(-/-) db/db) gene nodes were generated. Shared nodes across all networks reflected established pathogenic mechanisms of diabetes complications, such as elements of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and vascular endothelial growth factor receptor (VEGFR) signaling pathways. In addition, novel pathways not previously associated with DN and cross-species gene nodes and pathways unique to each of the human-mouse networks were discovered. The human-mouse shared glomerular transcriptional networks will assist DN researchers in selecting mouse models most relevant to the human disease process of interest. Moreover, they will allow identification of new pathways shared between mice and humans.


High-throughput sequencing enhanced phage display identifies peptides that bind mycobacteria.
Bacterial cell wall components have been previously used as infection biomarkers detectable by antibodies. However, it is possible that the surface of the Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), also possesses molecules which might be non-antigenic. This makes the probing of biomarkers on the surface of M. tb cell wall difficult using antibodies. Here we demonstrate the use of phage display technology to identify peptides that bind to mycobacteria. We identified these clones using both random clone picking and high throughput sequencing. We demonstrate that random clone picking does not necessarily identify highly enriched clones. We further showed that the clone displaying the CPLHARLPC peptide which was identified by Illumina sequencing as the most enriched, binds better to mycobacteria than three clones selected by random picking. Using surface plasmon resonance, we showed that chemically synthesised CPLHARLPC peptide binds to a 15 KDa peptide from M.tb H37Rv whole cell lysates. These observations demonstrate that phage display technology combined with high-throughput sequencing is a powerful tool to identify peptides that can be used for investigating potential non-antigenic biomarkers for TB and other bacterial infections.


Homoeobox gene expression in mouse embryos varies with position by the primitive streak stage.
Pattern formation in animal development requires that genes be expressed differentially according to position in the sheets of cells that make up the early embryo. The homoeobox-containing genes of Drosophila are control genes active both in the establishment of a segmentation pattern and in the specification of segment identity. In situ hybridization experiments confirm that these genes are expressed in a segmentally-restricted manner and that their expression presages morphological differentiation of segmental structures. Homoeobox genes have recently been isolated from the mouse and have been shown to be expressed during mouse development. Using in situ hybridization, we show here that expression of the mouse homoeobox gene Mo-10 (ref. 7) is spatially restricted in the developing embryo and that localization of expression is already evident within the germ layers before their morphological differentiation. These findings support the suggestion that the homoeobox genes of mammals, like those of Drosophila, may be important in pattern formation.


Plastic and moldable metals by self-assembly of sticky nanoparticle aggregates.
Deformable, spherical aggregates of metal nanoparticles connected by long-chain dithiol ligands self-assemble into nanostructured materials of macroscopic dimensions. These materials are plastic and moldable against arbitrarily shaped masters and can be thermally hardened into polycrystalline metal structures of controllable porosity. In addition, in both plastic and hardened states, the assemblies are electrically conductive and exhibit Ohmic characteristics down to approximately 20 volts per meter. The self-assembly method leading to such materials is applicable both to pure metals and to bimetallic structures of various elemental compositions.


Chromosome abnormalities in ovarian adenocarcinoma: I. Nonrandom chromosome abnormalities from 244 cases.
Cytogenetics provides important insights into the molecular pathogenesis of human cancers. Although extensive data exist on recurring cytogenetic abnormalities in hematologic cancers, data on individual solid tumor types remain limited. Previous studies of ovarian carcinoma indicated the presence of multiple, complex clonal chromosome abnormalities. Cytogenetics remains one of a few techniques capable of detecting these multiple, simultaneously occurring genetic abnormalities. We describe cytogenetic abnormalities from a series of 244 primary ovarian cancer specimens referred to a single institution. A total of 201/244 cases had fully characterized clonal chromosome abnormalities, of which 134 showed clonal chromosome breakpoints. We used a novel statistical technique to detect nonrandom chromosome breakpoints at the level of chromosome regions. Nonrandom occurrence of chromosome breakpoints was detected at regions 1p1*, 1q1*, 1p2*, 1q2*, 1p3*, 1q3, 3p1*, 1q4*, 6q1*, 6p2, 6q2, 7p1*, 7q1, 7p2*, 11p1*, 11q1, 11q2*, 12p1, 12q2*, 13p1, and 19q1. Simultaneous occurrence of multiple abnormalities was common. However, 120/134 cases had breakpoints at one or more of 13 commonly involved regions (*), suggesting a hierarchy of genetic abnormalities. Among clinical and tumor variables that predict patient survival, tumor grade was significantly associated with the presence of chromosome breakpoints. In additional studies, we show that nonrandom chromosome abnormalities are associated with impaired survival in ovarian cancer and that specific, nonrandomly involved chromosome regions retain significant effects on survival when analyses are controlled for important clinical variables. Additional specific chromosome abnormalities in this series are described, including chromosome gains and losses in near-diploid cases and homogeneously staining regions. These results suggest that recurring, nonrandom chromosome abnormalities are important in the pathogenesis and/or progression of ovarian cancers, and target areas of the genome for molecular genetic studies.


Role of kinins and nitric oxide in the effects of angiotensin converting enzyme inhibitors on neointima formation.
Marked neointima formation occurs after balloon injury to the intima of rat arteries. Angiotensin II has been implicated as a growth factor in this process, since angiotensin converting enzyme (ACE) inhibitors block neointima formation after injury. However, ACE is an important kininase, and its inhibitors may act in part by a kinin-mediated mechanism. Kinins are also known to stimulate synthesis of endothelium-derived relaxing factor/nitric oxide (EDRF/NO) and prostacyclin, both of which have antigrowth effects. To determine whether the effect of ACE inhibitors on neointima formation is due to blockade of angiotensin II synthesis alone and/or inhibition of kinin inactivation, we followed two approaches. First, we compared the inhibition of neointima formation induced by the AT1-type angiotensin II receptor antagonist losartan with that caused by the ACE inhibitor ramipril. We also studied whether a kinin receptor antagonist, Hoe 140, blocks the effect of two different ACE inhibitors, ramipril and enalapril, on neointima formation. In addition, we studied whether the effect of ramipril is blocked by an NO synthesis inhibitor, N omega-nitro-L-arginine-methyl ester (L-NAME). Although both ramipril and losartan significantly reduced neointima formation, ramipril had a more marked effect (p < 0.05 for ramipril versus losartan). The kinin antagonist Hoe 140 reduced the inhibitory effect of ramipril and enalapril by 73% and 62%, respectively. The remaining effect of the ACE inhibitors was now similar to that of losartan. Inhibition of neointima formation by ramipril was also blocked by the NO synthesis inhibitor L-NAME.(ABSTRACT TRUNCATED AT 250 WORDS)


Perception of breathlessness during bronchoconstriction induced by antigen, exercise, and histamine challenges.
Perception of breathlessness was studied in eight patients with mild, stable asthma after a histamine and exercise challenge performed before and 24 and 48 hours respectively after an antigen challenge. FEV1 and perception of breathlessness, evaluated by Borg's 10 point category scale, were measured after each administration of doubling antigen or histamine concentrations to achieve a greater than 20% fall in FEV1, and after six minutes of steady state exercise at 80% of maximal oxygen consumption (VO2max). The geometric mean provocative concentration of histamine causing a 20% fall in FEV1 (PC20) fell from 1.67 mg/ml before antigen challenge to 0.52 mg/ml 24 hours after the challenge. The median maximal % fall in FEV1 with exercise was 24.9% (range 10.5-40.5%) before and 30.6% (range 13.8-52.3%) 48 hours after antigen challenge. The median maximum % fall in FEV1 after antigen inhalation was 20.1% (range 13.3-35.2%) within the first hour; only two subjects had a late fall in FEV1 (23% and 58%). The median (range) of Borg scores obtained when FEV1 was reduced by 20% did not differ significantly for the three types of acute challenges: 1.25 (0.5-2.5) and 1.0 (0.5-3.0) after histamine tests, 1.0 (0.5-4.1) and 1.55 (0.5-2.0) after exercise, and 1.5 (0-3.0) after antigen challenge. In the two subjects who had a late response to antigen the Borg score was reduced for the same % fall in FEV1 as with the early response. It is concluded that the perception of breathlessness does not differ appreciably during the early response to histamine, antigen exposure, or exercise, but that it is reduced during the late asthmatic response. It was not influenced by previous antigen exposure, despite an increase in airway responsiveness.


Characterization of the feline thyroglobulin promoter.
The feline thyroglobulin promoter was identified by a combination of standard polymerase chain reaction (PCR) techniques, using primers designed according to regions of homology in published sequences from other species, then adaptor ligated PCR. A 310 bp fragment of the feline thyroglobulin promoter was generated, including 8 nucleotides of adaptor sequence at the 5' end and, based on the putative transcription start site, 36 nucleotides of the thyroglobulin mRNA (untranslated portion). The homology between the feline promoter sequence (from 193 bp upstream to the putative cap site) and canine, bovine and human sequences was 89%, 81% and 78%, respectively. Transient transfection studies, using reporter constructs in which the feline promoter controlled expression of chloramphenicol acetyl transferase, demonstrated promoter activity in thyroid cells, but no activity in non-thyroid cells. The data presented here demonstrate that the feline thyroglobulin promoter may provide a targeting mechanism for somatic gene therapy of feline thyroid disease.


Trajectories of heroin use: 10-11-year findings from the Australian Treatment Outcome Study.
To identify trajectories of heroin use in Australia, predictors of trajectory group membership and subsequent outcomes among people with heroin dependence over 10-11 years. Longitudinal cohort study. Sydney, Australia. A total of 615 participants were recruited between 2001 and 2002 as part of the Australian Treatment Outcome Study (66.2% male; mean age 29 years). The predominance of the cohort (87.0%) was recruited upon entry to treatment (maintenance therapies, detoxification and residential rehabilitation), and the remainder from non-treatment settings (e.g. needle and syringe programmes). This analysis focused upon 428 participants for whom data on heroin use were available over 10-11 years following study entry. Structured interviews assessed demographics, treatment history, heroin and other drug use, overdose, criminal involvement, physical health and psychopathology. Group-based trajectory modelling was used to: (i) identify trajectory groups based on use of heroin in each year, (ii) examine predictors of group membership and (iii) examine associations between trajectory group membership and 10-11-year outcomes. Six trajectory groups were identified [Bayesian Information Criterion (BIC) = -1927.44 (n = 4708); -1901.07 (n = 428)]. One in five (22.1%) were classified as having 'no decrease' in heroin use, with the probability of using remaining high during the 10-11 years (> 0.98 probability of use in each year). One in six (16.1%) were classified as demonstrating a 'rapid decrease to maintained abstinence'. The probability of heroin use among this group declined steeply in the first 2-3 years and continued to be low (< 0.01). The remaining trajectories represented other fluctuating patterns of use. Few baseline variables were found to predict trajectory group membership, but group membership was predictive of demographic, substance use and physical and mental health outcomes at 10-11 years. Long-term trajectories of heroin use in Australia appear to show considerable heterogeneity during a decade of follow-up, with few risk factors predicting group membership. Just more than a fifth continued to use at high levels, while fewer than a fifth become abstinent early on and remained abstinent. The remainder showed fluctuating patterns.


Excavations at Schöningen and paradigm shifts in human evolution.
The exceptional preservation at Schöningen together with a mixture of perseverance, hard work, and sheer luck led to the recovery of unique finds in an exceptional context. The 1995 discovery of numerous wooden artifacts, most notably at least 10 carefully made spears together with the skeletons of at least 20 to 25 butchered horses, brought the debate about hunting versus scavenging among late archaic hominins and analogous arguments about the purportedly primitive behavior of Homo heidelbergensis and Neanderthals to an end. Work under H. Thieme's lead from 1992 to 2008 and results from the current team since 2008 demonstrate that late H. heidelbergensis or early Neanderthals used sophisticated artifacts made from floral and faunal materials, in addition to lithic artifacts more typically recovered at Lower Paleolithic sites. The finds from the famous Horse Butchery Site and two dozen other archaeological horizons from the edges of the open-cast mine at Schöningen provide many new insights into the technology and behavioral patterns of hominins about 300 ka BP during MIS 9 on the Northern European Plain. An analysis of the finds from Schöningen and their contexts shows that the inhabitants of the site were skilled hunters at the top of the food chain and exhibited a high level of planning depth. These hominins had command of effective means of communication about the here and now, and the past and the future, that allowed them to repeatedly execute well-coordinated and successful group activities that likely culminated in a division of labor and social and economic patterns radically different from those of all non-human primates. The unique preservation and high quality excavations have led to a major paradigm shift or "Schöningen Effect" that changed our views of human evolution during the late Lower Paleolithic. In this respect, we can view the behaviors documented at Schöningen as a plausible baseline for the behavioral sophistication of archaic hominins of the late Middle Pleistocene and subsequent periods.


Energy level and band alignment for GaAs-alkylthiol monolayer-Hg junctions from electrical transport and photoemission experiments.
A series of p- and n-GaAs-S-C(n)H(2n+1) || Hg junctions are prepared, and the electronic transport through them is measured. From current-voltage measurements, we find that, for n-GaAs, transport occurs by both thermionic emission and tunneling, with the former dominating at low forward bias and the latter dominating at higher forward bias. For p-GaAs, tunneling dominates at all bias voltages. By combining the analysis of the transport data with results from direct and inverse photoemission spectroscopy, we deduce an energy band diagram of the system, including the tunnel barrier and, with this barrier and within the Simmons tunneling model, extract an effective mass value of 1.5-1.6m(e) for the electronic carriers that cross the junctions. We find that transport is well-described by lowest unoccupied and highest occupied states at 1.3-1.4 eV above and 2.0-2.2 eV below the Fermi level. At the same time, the photoemission data indicate that there are continua of states from the conduction band minimum and the valence band maximum, the density of which varies with energy. On the basis of our results, it appears likely that, for both types of junctions, electrons are the main carrier type, although holes may contribute significantly to the transport in the p-GaAs system.


Comparative Analysis of the Flexural Stiffness of Pinniped Vibrissae.
Vibrissae are important components of the mammalian tactile sensory system and are used to detect vibrotactile stimuli in the environment. Pinnipeds have the largest and most highly innervated vibrissae among mammals, and the hair shafts function as a biomechanical filter spanning the environmental stimuli and the neural mechanoreceptors deep in the follicle-sinus complex. Therefore, the material properties of these structures are critical in transferring vibrotactile information to the peripheral nervous system. Vibrissae were tested as cantilever beams and their flexural stiffness (EI) was measured to test the hypotheses that the shape of beaded vibrissae reduces EI and that vibrissae are anisotropic. EI was measured at two locations on each vibrissa, 25% and 50% of the overall length, and at two orientations to the point force. EI differed in orientations that were normal to each other, indicating a functional anisotropy. Since vibrissae taper from base to tip, the second moment of area (I) was lower at 50% than 25% of total length. The anterior orientation exhibited greater EI values at both locations compared to the dorsal orientation for all species. Smooth vibrissae were generally stiffer than beaded vibrissae. The profiles of beaded vibrissae are known to decrease the amplitude of vibrations when protruded into a flow field. The lower EI values of beaded vibrissae, along with the reduced vibrations, may function to enhance the sensitivity of mechanoreceptors to detection of small changes in flow from swimming prey by increasing the signal to noise ratio. This study builds upon previous morphological and hydrodynamic analyses of vibrissae and is the first comparative study of the mechanical properties of pinniped vibrissae.


Characterization of chromosomal architecture in Arabidopsis by chromosome conformation capture.
The packaging of long chromatin fibers in the nucleus poses a major challenge, as it must fulfill both physical and functional requirements. Until recently, insights into the chromosomal architecture of plants were mainly provided by cytogenetic studies. Complementary to these analyses, chromosome conformation capture technologies promise to refine and improve our view on chromosomal architecture and to provide a more generalized description of nuclear organization. Employing circular chromosome conformation capture, this study describes chromosomal architecture in Arabidopsis nuclei from a genome-wide perspective. Surprisingly, the linear organization of chromosomes is reflected in the genome-wide interactome. In addition, we study the interplay of the interactome and epigenetic marks and report that the heterochromatic knob on the short arm of chromosome 4 maintains a pericentromere-like interaction profile and interactome despite its euchromatic surrounding. Despite the extreme condensation that is necessary to pack the chromosomes into the nucleus, the Arabidopsis genome appears to be packed in a predictive manner, according to the following criteria: heterochromatin and euchromatin represent two distinct interactomes; interactions between chromosomes correlate with the linear position on the chromosome arm; and distal chromosome regions have a higher potential to interact with other chromosomes.


Unraveling the rat blood genome-wide transcriptome after oral administration of lavender oil by a two-color dye-swap DNA microarray approach.
Lavender oil (LO) is a commonly used essential oil in aromatherapy as non-traditional medicine. With an aim to demonstrate LO effects on the body, we have recently established an animal model investigating the influence of orally administered LO in rat tissues, genome-wide. In this brief, we investigate the effect of LO ingestion in the blood of rat. Rats were administered LO at usual therapeutic dose (5 mg/kg) in humans, and following collection of the venous blood from the heart and extraction of total RNA, the differentially expressed genes were screened using a 4 × 44-K whole-genome rat chip (Agilent microarray platform; Agilent Technologies, Palo Alto, CA, USA) in conjunction with a two-color dye-swap approach. A total of 834 differentially expressed genes in the blood were identified: 362 up-regulated and 472 down-regulated. These genes were functionally categorized using bioinformatics tools. The gene expression inventory of rat blood transcriptome under LO, a first report, has been deposited into the Gene Expression Omnibus (GEO): GSE67499. The data will be a valuable resource in examining the effects of natural products, and which could also serve as a human model for further functional analysis and investigation.


Orbital volume and shape in Treacher Collins syndrome.
Orbito-palpebral reconstruction is a challenge in Treacher Collins syndrome (TCS). This study investigates orbital phenotypes in TCS using cephalometry and orbital shape analysis. Eighteen TCS and 52 control patients were included in this study, using the Dr Warehouse database. Orbital cephalometry was based on 20 landmarks, 10 planes, 16 angles, and 22 distances. Orbits were segmented. Registration-based, age-specific mean models were generated using semi-automatic segmentation, and aligned and compared using color-coded distance maps - mean absolute distance (MAD), Hausdorff distance (HD), and Dice similarity coefficient (DSC). Symmetry was assessed by mirroring and DSC computing. Central orbital depth (COD) and medial orbital depth (MOD) allowed 100% of orbits to be classified. COD and lateral orbital depth (LOD) were different from the controls. Average MAD between TCS and controls was ≤1.5 mm, while for HD it was >1.5 mm, and for DSC <1. TCS orbits were more asymmetrical than controls, and orbital volumes were smaller when age was considered as a confounding factor, and had a trend for normalization with age. This report emphasizes the importance of combining different morphometric approaches in the phenotype characterization of non-trivial structures such as the orbit, and supports composite skeletal and soft-tissue strategies for the management of the peri-orbital region.


Genome editing and stem cell therapy pave the path for new treatment of sickle-cell disease.
Sickle-cell disease (SCD), also known as sickle-cell anemia, is a hereditary blood disorder characterized by the presence of abnormal hemoglobin, the oxygen-carrying protein found in red blood cells. This devastating hematologic disease affects millions of children worldwide. Currently the only available cure is an allogenic hematopoietic stem cell transplant (HSCT) which is limited by the scarcity of fully-matched donors. SCD is caused by a single nucleotide mutation in the beta-globin gene. Correction of this genetic defect would provide a cure for the disease. Two recent murine studies have provided proof of principle for such a strategy by correcting the mutation in hematopoietic stem cells (HSC) using genome editing techniques. With transformative advances being made in the genome editing field, effective and precise manipulation of cellular genomes is becoming highly feasible. Genome editing techniques in combination with stem cell therapy should provide a safe and curative treatment of various genetic diseases such as SCD.


A Unidirectional Transition from Migratory to Perivascular Macrophage Is Required for Tumor Cell Intravasation.
Tumor-associated macrophages (TAMs) are critical for tumor metastasis. Two TAM subsets support cancer cell intravasation: migratory macrophages guide cancer cells toward blood vessels, where sessile perivascular macrophages assist their entry into the blood. However, little is known about the inter-relationship between these functionally distinct TAMs or their possible inter-conversion. We show that motile, streaming TAMs are newly arrived monocytes, recruited via CCR2 signaling, that then differentiate into the sessile perivascular macrophages. This unidirectional process is regulated by CXCL12 and CXCR4. Cancer cells induce TGF-β-dependent upregulation of CXCR4 in monocytes, while CXCL12 expressed by perivascular fibroblasts attracts these motile TAMs toward the blood vessels, bringing motile cancer cells with them. Once on the blood vessel, the migratory TAMs differentiate into perivascular macrophages, promoting vascular leakiness and intravasation.


Kinase suppressor of Ras signals through Thr269 of c-Raf-1.
We recently established a two-stage in vitro assay for KSR kinase activity in which KSR never comes in contact with any recombinant kinase other than c-Raf-1 and defined the epidermal growth factor (EGF) as a potent activator of KSR kinase activity (Xing, H. R., Lozano, J., and Kolesnick, R. (2000) J. Biol. Chem. 275, 17276-17280). That study, however, did not address the mechanism of c-Raf-1 stimulation by activated KSR. Here we show that phosphorylation of c-Raf-1 on Thr(269) by KSR is necessary for optimal activation in response to EGF stimulation. In vitro, KSR specifically phosphorylated c-Raf-1 on threonine residues during the first stage of the two-stage kinase assay. Using purified wild-type and mutant c-Raf-1 proteins, we demonstrate that Thr(269) is the major c-Raf-1 site phosphorylated by KSR in vitro and that phosphorylation of this site is essential for c-Raf-1 activation by KSR. KSR acts via transphosphorylation, not by increasing c-Raf-1 autophosphorylation, as kinase-inactive c-Raf-1(K375M) served as an equally effective KSR substrate. In vivo, low physiologic doses of EGF (0.001-0.1 ng/ml) stimulated KSR activation and induced Thr(269) phosphorylation and activation of c-Raf-1. Low dose EGF did not induce serine or tyrosine phosphorylation of c-Raf-1. High dose EGF (10-100 ng/ml) induced no additional Thr(269) phosphorylation, but rather increased c-Raf-1 phosphorylation on serine residues and Tyr(340)/Tyr(341). A Raf-1 mutant with valine substituted for Thr(269) was unresponsive to low dose EGF, but was serine- and Tyr(340)/Tyr(341)-phosphorylated and partially activated at high dose EGF. This study shows that Thr(269) is the major c-Raf-1 site phosphorylated by KSR. Furthermore, phosphorylation of this site is essential for c-Raf-1 activation by KSR in vitro and for optimal c-Raf-1 activation in response to physiologic EGF stimulation in vivo.


The H4b minor histocompatibility antigen is caused by a combination of genetically determined and posttranslational modifications.
Minor histocompatibility (H) Ag disparities result in graft-vs-host disease and chronic solid allograft rejection in MHC-identical donor-recipient combinations. Minor H Ags are self protein-derived peptides presented by MHC class I molecules. Most arise as a consequence of allelic variation in the bound peptide (p) that results in TCR recognizing the p/MHC as foreign. We used a combinational peptide screening approach to identify the immune dominant H2K(b)-restricted epitope defining the mouse H4(b) minor H Ag. H4(b) is a consequence of a P3 threonine to isoleucine change in the MHC-bound peptide derived from epithelial membrane protein-3. This allelic variation also leads to phosphorylation of the H4(b) but not the H4(a) epitope. Further, ex vivo CD8(+) T lymphocytes bind phosphorylated Ag tetramers with high efficiency. Although we document the above process in the minor H Ag system, posttranslational modifications made possible by subtle amino acid changes could also contribute to immunogenicity and immune dominance in tumor immunotherapeutic settings.


US Patient-Centered Research Priorities and Roadmap for Bronchiectasis.
Noncystic fibrosis bronchiectasis (bronchiectasis) is an increasingly common chronic lung disease that is difficult to manage because of a lack of evidence on which to base treatment decision-making. We sought to develop a practical list of US-based patient-centered research priorities and an associated roadmap to guide bronchiectasis research. We designed and administered a web-based patient needs assessment survey to establish broad research priorities, convened three stakeholder webinars to confirm the top priorities, obtained written stakeholder feedback, and completed a final consensus survey of objectives. The stakeholder panel consisted of clinical research experts in bronchiectasis, a seven-member patient advisory panel, and representatives from the two key patient advocacy organizations: COPD Foundation and NTM Info and Research Inc. Based on survey results from 459 patients with bronchiectasis, the stakeholder panel identified 27 patient-centered research priorities for bronchiectasis in the areas of bronchiectasis treatment and prevention of exacerbations, improving treatment of exacerbations and infections, improving health-related quality of life, predictors of poor prognosis, understanding the impact of underlying conditions, and conducting patient-centered clinical trials. These priorities should further inform the development and evaluation of both new and previously unproven therapies, with particular attention to the inclusion of patient-reported outcomes. We anticipate a great deal of progress will be made in the field of bronchiectasis in the next decade.


Abnormal social behavior, hyperactivity, impaired remote spatial memory, and increased D1-mediated dopaminergic signaling in neuronal nitric oxide synthase knockout mice.
Neuronal nitric oxide synthase (nNOS) is involved in the regulation of a diverse population of intracellular messenger systems in the brain. In humans, abnormal NOS/nitric oxide metabolism is suggested to contribute to the pathogenesis and pathophysiology of some neuropsychiatric disorders, such as schizophrenia and bipolar disorder. Mice with targeted disruption of the nNOS gene exhibit abnormal behaviors. Here, we subjected nNOS knockout (KO) mice to a battery of behavioral tests to further investigate the role of nNOS in neuropsychiatric functions. We also examined the role of nNOS in dopamine/DARPP-32 signaling in striatal slices from nNOS KO mice and the effects of the administration of a dopamine D1 receptor agonist on behavior in nNOS KO mice. nNOS KO mice showed hyperlocomotor activity in a novel environment, increased social interaction in their home cage, decreased depression-related behavior, and impaired spatial memory retention. In striatal slices from nNOS KO mice, the effects of a dopamine D1 receptor agonist, SKF81297, on the phosphorylation of DARPP-32 and AMPA receptor subunit GluR1 at protein kinase A sites were enhanced. Consistent with the biochemical results, intraperitoneal injection of a low dose of SKF81297 significantly decreased prepulse inhibition in nNOS KO mice, but not in wild-type mice. These findings indicate that nNOS KO upregulates dopamine D1 receptor signaling, and induces abnormal social behavior, hyperactivity and impaired remote spatial memory. nNOS KO mice may serve as a unique animal model of psychiatric disorders.


Recombinant attenuated Salmonella enterica serovar typhimurium expressing the carboxy-terminal domain of alpha toxin from Clostridium perfringens induces protective responses against necrotic enteritis in chickens.
Clostridium perfringens-induced necrotic enteritis (NE) is a widespread disease in chickens that causes high mortality and reduced growth performance. Traditionally, NE was controlled by the routine application of antimicrobials in the feed, a practice that currently is unpopular. Consequently, there has been an increase in the occurrence of NE, and it has become a threat to the current objective of antimicrobial-free farming. The pathogenesis of NE is associated with the proliferation of C. perfringens in the small intestine and the secretion of large amounts of alpha toxin, the major virulence factor. Since there is no vaccine for NE, we have developed a candidate live oral recombinant attenuated Salmonella enterica serovar Typhimurium vaccine (RASV) that delivers a nontoxic fragment of alpha toxin. The 3' end of the plc gene, encoding the C-terminal domain of alpha toxin (PlcC), was cloned into plasmids that enable the expression and secretion of PlcC fused to a signal peptide. Plasmids were inserted into Salmonella enterica serovar Typhimurium host strain chi8914, which has attenuating pabA and pabB deletion mutations. Three-day-old broiler chicks were orally immunized with 10(9) CFU of the vaccine strain and developed alpha toxin-neutralizing serum antibodies. When serum from these chickens was added into C. perfringens broth cultures, bacterial growth was suppressed. In addition, immunofluorescent microscopy showed that serum antibodies bind to the bacterial surface. The immunoglobulin G (IgG) and IgA titers in RASV-immunized chickens were low; however, when the chickens were given a parenteral boost injection with a purified recombinant PlcC protein (rPlcC), the RASV-immunized chickens mounted rapid high serum IgG and bile IgA titers exceeding those primed by rPlcC injection. RASV-immunized chickens had reduced intestinal mucosal pathology after challenge with virulent C. perfringens. These results indicate that oral RASV expressing an alpha toxin C-terminal peptide induces protective immunity against NE.


Concurrence of Frailty and Parkinson's Disease.
Females with Parkinson's disease (PD) are at greater risk of frailty than males. Little is known about how age and disease-related characteristics influence frailty in females with PD because frailty studies often exclude persons with underlying neurological pathologies. To determine age and disease-related characteristics that best explain physical frailty in community-dwelling females with and without PD. Correlation coefficients described relationships between PD-related characteristics and physical frailty phenotype criteria (Cardiovascular Health Study). Regression analysis identified associations between disease-related characteristics and frailty in non-PD and PD females. Community-dwelling. Females with mild to moderate PD (n = 17, mean age = 66 ± 8.5 years) and non-PD (n = 18, mean age = 72 ± 13.2 years) participated. Daily carbidopa-levodopa dose best explained frailty in PD females (β = 0.5), whereas in non-PD females, age (β = 0.7) and comorbidity (β = 0.5) were most associated with frailty. Dopaminergic medication explained frailty in PD and not measures of disease progression (i.e. severity, duration). In females without PD age-related accumulation of comorbidities resulted in greater risk of frailty. This indicates dopaminergic management of PD symptoms may better reflect frailty in females with PD than disease severity or duration. These data suggest the influence of underlying frailty should be considered when managing neurological conditions. Understanding how frailty concurrently exists with PD and how these conditions progress within the aging female will facilitate future care management.