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The integrin-adhesome network, which contains >150 proteins, is mechano-transducing and located at discreet positions along the cell-cell and cell-extracellular matrix interface. A small subset of the integrin-adhesome is known to maintain normal muscle morphology. However, the importance of the entire adhesome for muscle structure and function is unknown. We used RNA interference to knock down 113 putative Caenorhabditis elegans homologs constituting most of the mammalian adhesome and 48 proteins known to localize to attachment sites in C. elegans muscle. In both cases, we found >90% of components were required for normal muscle mitochondrial structure and/or proteostasis vs. empty vector controls. Approximately half of these, mainly proteins that physically interact with each other, were also required for normal sarcomere and/or adhesome structure. Next we confirmed that the dystrophy observed in adhesome mutants associates with impaired maximal mitochondrial ATP production (P < 0.01), as well as reduced probability distribution of muscle movement forces compared with wild-type animals. Our results show that the integrin-adhesome network as a whole is required for maintaining both muscle structure and function and extend the current understanding of the full complexities of the functional adhesome in vivo., (© The Author(s).)

Background: In children, community-acquired pneumonia is a frequent cause of emergency department (ED) presentation and hospital admission. Quality primary care may prevent some of these hospital visits., Aims: The aim of this study was to identify primary care factors associated with ED presentation and hospital admission of preschool-aged children with community-acquired pneumonia., Methods: A case-control study was conducted by enrolling three groups: children presenting to the ED with pneumonia and admitted (n = 326), or discharged home (n = 179), and well-neighbourhood controls (n = 351). Interviews with parents and primary care staff were conducted and health record review was performed. The association of primary care factors with ED presentation and hospital admission, controlling for available confounding factors, was determined using logistic regression., Results: Children were more likely to present to the ED with pneumonia if they did not have a usual general practitioner (GP) (odds ratio (OR) = 2.50, 95% confidence interval (CI) = 1.67-3.70), their GP worked ⩽ 20 h/week (OR = 1.86, 95% CI = 1.10-3.13) or their GP practice lacked an immunisation recall system (OR = 5.44, 95% CI = 2.26-13.09). Lower parent ratings for continuity (OR=1.63, 95% CI = 1.01-2.62), communication (OR = 2.01, 95% CI = 1.29-3.14) and overall satisfaction (OR = 2.16, 95% CI = 1.34-3.47) increased the likelihood of ED presentation. Children were more likely to be admitted when antibiotics were prescribed in primary care (OR = 2.50, 95% CI = 1.43-4.55). Hospital admission was less likely if children did not have a usual GP (OR = 0.22, 95% CI = 0.11-0.40) or self-referred to the ED (OR = 0.48, 95% CI = 0.26-0.89)., Conclusions: Accessible and continuous primary care is associated with a decreased likelihood of preschool-aged children with pneumonia presenting to the ED and an increased likelihood of hospital admission, implying more appropriate referral. Lower parental satisfaction is associated with an increased likelihood of ED presentation.

Background and Objective: The role of two recently identified and closely related T-helper cell subsets - regulatory T-cells [Tregs; forkhead box P3-positive (FOXP3(+) )] and Th17 cells [interleukin-17-positive (IL-17(+) )] - in periodontal disease is yet to be determined. Tregs are essential in maintaining peripheral tolerance and regulating the immune response. Th17 cells play a critical role in several autoimmune diseases, inflammation and host defence. The aim of this study was to determine the presence of FOXP3(+) Tregs and IL-17(+) cells, and their possible spatial interaction, in diseased periodontal tissues., Material and Methods: Twenty-nine archival tissues with nonspecific gingival inflammation were grouped based on the intensity (minimally or intensely inflamed) and nature (T-cell predominant or B- and plasma-cell predominant) of the inflammatory infiltrate. Using double-labelling immunohistochemistry, the concomitant presence of FOXP3(+) and IL-17(+) cells was determined and their spatial relationship was established. In addition, the proportions of FOXP3(+) and IL-17(+) cells were compared between the groups., Results: Of the 29 gingival specimens investigated, 17 were intensely inflamed (≥ 1000 inflammatory cells per 0.12 mm(2) ) and 12 were minimally inflamed (≤ 600 cells per 0.12 mm(2) ). Based on the percentage of CD19(+) B-cells and plasma cells collectively and CD3(+) T-cells, gingival tissues were also grouped into B- and plasma-cell-predominant gingival tissues (n = 21; 50.7% total B- and plasma cells vs. 19.1% T cells; p < 0.001) and T-cell-predominant gingival tissues (n = 8; 61.0% T-cells vs. 15.2% B- and plasma cells; p = 0.007). More FOXP3(+) cells than IL-17(+) cells were observed in all archival gingival tissues examined. A trend towards an increased number of FOXP3(+) cells was observed for intensely inflamed gingival tissues (6.7%) and for B- and plasma-cell-predominant tissues (6.4%) compared with minimally inflamed gingival tissues (4.6%) and T-cell-predominant gingival tissues (4.5%). However, no statistically significant difference in the mean percentage of FOXP3(+) cells between the groups was observed. Interestingly, FOXP3(+) cells were significantly correlated with the B- and plasma-cell/T-cell ratio in B- and plasma-cell-predominant tissues (r = 0.713, p < 0.001). Overall, there were very few IL-17(+) cells (< 1%). All IL-17(+) cells identified in this study had an ovoid/plasmacytoid morphology and were larger in size compared with adjacent inflammatory cells. IL-17(+) and FOXP3(+) cells were not adjacent to each other in any of the areas examined, suggesting that FOXP3(+) Tregs do not directly interact with IL-17(+) cells in diseased gingival tissues. IL-17(+) /FOXP3(+) cells were not detected in the tissues examined., Conclusion: These results show that FOXP3(+) cells are more prominent than IL-17(+) cells in periodontal disease processes, which may suggest a predominant role for FOXP3(+) cells in periodontal disease. Further studies are required to characterize these cells more precisely and to understand, in more detail, their roles in the pathophysiology of periodontal disease., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Recurrent and advanced primary pelvic cancers present a complex clinical issue requiring multidisciplinary care and radical extended surgery. Sacral resection is necessary for tumors that invade posteriorly but is associated with increased morbidity and mortality., Objective: This study aimed to analyze the morbidity and survival associated with pelvic exenteration involving sacrectomy for advanced pelvic cancers at a single institution., Design: This study used patient demographics, operative and pathologic reports, and prospective survival data to determine factors affecting patient outcomes., Settings: Data were collected for patients who had operations between July 1998 and April 2012 at Royal Prince Alfred Hospital., Patients: One hundred patients underwent pelvic exenteration with a sacrectomy for advanced pelvic cancers. Sacrectomy was performed for 18 primary and 61 recurrent rectal cancers, 17 anal cancers, and 4 other cancers., Main Outcome Measures: This study looked at postoperative major and minor morbidity rates, as well as disease-free and overall survival rates after sacral resection. It compared the outcomes of high sacrectomy (at or above S2) versus low sacrectomy., Results: Clear margins were achieved in 72 of 100 patients. The overall complication rate was 74% (43% major and 67% minor) with no 30-day or in-hospital mortality. Estimated overall and disease-free survival rates after curative resection were 38% and 30% at 5 years. Involved margins (p = 0.006), lymph node involvement (p = 0.008), and anterior organ invasion (p = 0.008) had a negative impact on patient survival. High sacrectomy increased the incidence of neurologic deficit postoperatively (p = 0.04) but did not alter the rate of R0 resection or patient survival., Limitations: Retrospective data were required to analyze patient morbidity, as well as operative and pathologic factors., Conclusions: This series supports sacral resection for curative surgery in advanced pelvic cancers, achieving excellent R0 and long-term survival rates. Cortical bone invasion and high sacrectomy were not contraindications to surgery and had acceptable outcomes.

The mevalonate pathway (MVP) and the anti-angiogenic effect of bisphosphonates have been shown to play a role in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). This study determined the effect of the bisphosphonate, zoledronic acid and the replenishment of the MVP by geranylgeraniol on human gingival fibroblasts. Cell viability, apoptosis, morphological analysis using transmission electron microscopy, and gene expression for vascular endothelial growth factor A, bone morphogenic protein 2, ras homologue gene family member B, epiregulin and interferon-alpha were conducted. Results showed cellular viability was decreased in the presence of zoledronic acid and the co-addition of zoledronic acid with geranylgeraniol restored cell viability to control levels. Caspase 3/7 was detected in zoledronic-acid-treated cells indicating apoptosis. Transmission electron microscopy revealed dilation of the rough endoplasmic reticulum with zoledronic acid and the appearance of multiple lipid-like vesicles following the addition of geranylgeraniol. Zoledronic acid significantly (P < 0.05, FR > ± 2) up-regulated vascular endothelial growth factor A, bone morphogenic protein 2, ras homologue gene family member B and epiregulin at one or more time points but not interferon-alpha. Addition of geranylgeraniol resulted in a reduction in the expression of all five genes compared with zoledronic-acid-treated human gingival fibroblasts. The study concluded geranylgeraniol partially reversed the effects of zoledronic acid in human gingival fibroblasts both at the cellular and genetic levels, suggesting the regulation of these genes is mediated via the mevalonate pathway., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: Diffusion kurtosis imaging (DKI) is a recent improvement over diffusion tensor imaging that characterizes tissue by quantifying non-gaussian diffusion using a 3D fourth-order kurtosis tensor. DKI needs to consider three constraints to be physically relevant. Further, it can be improved by considering the Rician signal noise model. A DKI estimation method is proposed that considers all three constraints correctly, accounts for the signal noise and incorporates efficient gradient-based optimization to improve over existing methods., Methods: The ternary quartic parameterization is utilized to elegantly impose the positivity of the kurtosis tensor implicitly. Sequential quadratic programming with analytical gradients is employed to solve nonlinear constrained optimization efficiently. Finally, a maximum likelihood estimator based on Rician distribution is considered to account for signal noise., Results: Extensive experiments conducted on synthetic data verify a MATLAB implementation by showing dramatically improved performance in terms of estimation time and quality. Experiments on in vivo cerebral data confirm that in practice the proposed method can obtain improved results., Conclusion: The proposed ternary quartic-based approach with a gradient-based optimization scheme and maximum likelihood estimator for constrained DKI estimation improves considerably on existing DKI methods., (Copyright © 2013 Wiley Periodicals, Inc.)

Objective: To determine the vitamin D dose necessary to achieve serum 25-hydroxyvitamin D (25(OH)D) concentration ≥ 20 ng/mL during infancy., Methods: A randomized, double-blind, placebo-controlled trial in New Zealand. Pregnant mothers, from 27 weeks' gestation to birth, and then their infants, from birth to age 6 months, were randomly assigned to 1 of 3 mother/infant groups: placebo/placebo, vitamin D3 1000/400 IU, or vitamin D3 2000/800 IU. Serum 25(OH)D and calcium concentrations were measured at enrollment, 36 weeks' gestation, in cord blood, and in infants at 2, 4, and 6 months of age., Results: Two-hundred-and-sixty pregnant women were randomized. At enrollment, the proportions with serum 25(OH)D ≥ 20 ng/mL for placebo, lower-dose, and higher-dose groups were 54%, 64%, and 55%, respectively. The proportion with 25(OH)D ≥ 20 ng/mL was larger in both intervention groups at 36 weeks' gestation (50%, 91%, 89%, P < .001). In comparison with placebo, the proportion of infants with 25(OH)D ≥ 20 ng/mL was larger in both intervention groups to age 4 months: cord blood (22%, 72%, 71%, P < .001), 2 months (50%, 82%, 92%, P < .001), and 4 months (66%, 87%, 87%, P = .004), but only in the higher-dose group at age 6 months (74%, 82%, 89%, P = .07; higher dose versus placebo P = .03, lower dose versus placebo P = .21)., Conclusions: Daily vitamin D supplementation during pregnancy and then infancy with 1000/400 IU or 2000/800 IU increases the proportion of infants with 25(OH)D ≥ 20 ng/mL, with the higher dose sustaining this increase for longer.

Objectives: To describe the experience of sacrectomy with extended radical resection in the treatment of locally recurrent rectal cancer., Background: Resections of the bony pelvis, especially the sacrum, are becoming more common as part of extended radical exenterations for patients with recurrent rectal cancer. However, sacrectomy has been shown to carry a significant decrease in survival. Morbidity rates have been associated with the level of the sacrectomy (ie, >S3 junction)., Methods: An analysis was conducted using prospective data from patients with recurrent rectal cancer who underwent pelvic exenteration involving sacrectomy from July 1998 until June 2011. The impact of the proximal level of sacrectomy [low (≤S3) vs high (≥S2-S3 disc)] was compared., Results: Of 240 exenteration patients, 79 underwent sacrectomy, with 49 for recurrent rectal cancer. An R0 margin was achieved in 36 (74%) patients. Achievement of clear operative margins (R0) conferred a large and significant benefit for disease-free survival compared with R1 and R2 resections (median 45 months vs 19 and 8 months, respectively; P = 0.045). Complications were reported in 40 (82%) patients, with major and minor complications in 19 (39%) and 38 (78%) patients, respectively. The proximal level of the sacrectomy (high vs low) did not significantly impair the ability to achieve a clear margin and was not associated with an increase in major or minor complications., Conclusions: This large, single-center series has demonstrated that extended pelvic exenteration involving sacrectomy has excellent R0 margins and survival rates for recurrent rectal cancer. A high sacrectomy has comparable results with a more distal abdominosacral resection.

An international working group within the European LeukemiaNet gathered, aiming to determine the role of flow cytometry (FC) in myelodysplastic syndromes (MDS). It was agreed that FC has a substantial application in disease characterization, diagnosis and prognosis. FC may also be useful in predicting treatment responses and monitoring novel and standard therapeutic regimens. In this article the rationale is discussed that flow cytometry should be integrated as a part of diagnostic and prognostic scoring systems in MDS.

Background and Purpose: Multiple antibiotic resistant strains of plague are emerging, driving a need for the development of novel antibiotics effective against Yersinia pestis. DNA adenine methylation regulates numerous fundamental processes in bacteria and alteration of DNA adenine methlytransferase (Dam) expression is attenuating for several pathogens, including Y. pestis. The lack of a functionally similar enzyme in humans makes Dam a suitable target for development of novel therapeutics for plague., Experimental Approach: Compounds were evaluated for their ability to inhibit Dam activity in a high-throughput screening assay. DNA was isolated from Yersinia grown in the presence of lead compounds and restricted to determine the effect of inhibitors on DNA methylation. Transcriptional analysis was undertaken to determine the effect of an active inhibitor on virulence-associated phenotypes., Key Results: We have identified a series of aryl stibonic acids which inhibit Dam in vitro. The most active, 4-stibonobenzenesulfonic acid, exhibited a competitive mode of inhibition with respect to DNA and a K(i) of 6.46 nM. One compound was found to inhibit DNA methylation in cultured Y. pestis. The effects of this inhibition on the physiology of the cell were widespread, and included altered expression of known virulence traits, including iron acquisition and Type III secretion., Conclusions and Implications: We have identified a novel class of potent Dam inhibitors. Treatment of bacterial cell cultures with these inhibitors resulted in a decrease in DNA methylation. Expression of virulence factors was affected, suggesting these inhibitors may attenuate bacterial infectivity and function as antibiotics., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

To the authors' knowledge, no published studies have examined the use of locking plates on injuries of the anterior pelvic ring. The purpose of this study was to determine whether locked plates provide enhanced stability in the treatment of pubic symphyseal disruptions. Completely unstable pelvic injuries were simulated in pelvic Sawbones (model 1301; Pacific Research Laboratories, Vashon, Washington) and 2 different fixation constructs used for anterior fixation (4-hole, 3.5-mm pubic symphysis plate with all locked or all unlocked screws). Adjunctive sacroiliac screw fixation with a single 7.3-mm screw placed into S1 was used in all specimens. Specimens were analyzed for motion at the pubic symphysis and sacroiliac joints using a Material Testing System (MTS Systems Corporation, Eden Prairie, Minnesota). Each specimen was subjected to compressive loading in a single-limb stance. Side loading was also examined. The main outcome measurement was motion at the pubic symphysis and sacroiliac joints and overall construct stiffness. No significant difference existed in overall construct stiffness between the 2 methods of pubic symphysis fixation. The motions at the pubic symphysis or injured sacroiliac joints were not significantly different. In addition, motion at the pubic symphysis joint with lateral load was not improved with a locking construct.No significant difference existed between 4-hole locked or unlocked constructs used for fixation of the pubic symphysis. No apparent advantage of locking screws exists for disruptions of the pubic symphysis, and recent reports have questioned the possibility of catastrophic failure., (Copyright 2012, SLACK Incorporated.)

Flow cytometry (FC) is increasingly recognized as an important tool in the diagnosis and prognosis of myelodysplastic syndromes (MDS). However, validation of current assays and agreement upon the techniques are prerequisites for its widespread acceptance and application in clinical practice. Therefore, a working group was initiated (Amsterdam, 2008) to discuss and propose standards for FC in MDS. In 2009 and 2010, representatives from 23, mainly European, institutes participated in the second and third European LeukemiaNet (ELN) MDS workshops. In the present report, minimal requirements to analyze dysplasia are refined. The proposed core markers should enable a categorization of FC results in cytopenic patients as 'normal', 'suggestive of', or 'diagnostic of' MDS. An FC report should include a description of validated FC abnormalities such as aberrant marker expression on myeloid progenitors and, furthermore, dysgranulopoiesis and/or dysmonocytopoiesis, if at least two abnormalities are evidenced. The working group is dedicated to initiate further studies to establish robust diagnostic and prognostic FC panels in MDS. An ultimate goal is to refine and improve diagnosis and prognostic scoring systems. Finally, the working group stresses that FC should be part of an integrated diagnosis rather than a separate technique.

Aim: To identify risk factors for children developing and being hospitalised with community-acquired pneumonia., Methods: Children <5 years old residing in urban Auckland, New Zealand were enrolled from 2002 to 2004. To assess the risk of developing pneumonia, children hospitalised with pneumonia (n= 289) plus children with pneumonia discharged from the Emergency Department (n= 139) were compared with a random community sample of children without pneumonia (n= 351). To assess risk of hospitalisation, children hospitalised with pneumonia were compared with the children discharged from the Emergency Department. Adjusted odds ratio (OR) with 95% confidence intervals (CIs) were used to estimate the risk of pneumonia and hospitalisation with pneumonia., Results: After adjustment for season, age and ethnicity there was an increased risk of pneumonia associated with lower weight for height (OR 1.28, 95% CI 1.10-1.51), spending less time outside (1.96, 1.11-3.47), previous chest infections (2.31, 1.55-3.43) and mould in the child's bedroom (1.93, 1.24-3.02). There was an increased risk of pneumonia hospitalisation associated with maternal history of pneumonia (4.03, 1.25-16.18), living in a more crowded household (2.87, 1.33-6.41) and one with cigarette smokers (1.99, 1.05-3.81), and mould in the child's bedroom (2.39, 1.25-4.72)., Conclusions: Lower quality living environments increase the risk of pneumonia and hospitalisation with pneumonia in New Zealand. Poorer nutritional status may also increase the risk of pneumonia. Improving housing quality, decreased cigarette smoke exposure and early childhood nutrition may reduce pneumonia disease burden in New Zealand., (© 2011 The Authors. Journal of Paediatrics and Child Health © 2011 Paediatrics and Child Health Division (Royal Australasian College of Physicians).)

Background and Objective: Cell adhesion plays important roles in maintaining the structural integrity of connective tissues and sensing changes in the biomechanical environment of cells. The objective of the present investigation was to extend our understanding of the effect of cyclic mechanical strain on the expression of adhesion-related genes by human periodontal ligament cells., Material and Methods: Cultured periodontal ligament cells were subjected to a cyclic in-plane tensile deformation of 12% for 5 s (0.2 Hz) every 90 s for 6-24 h in a Flexercell FX-4000 Strain Unit. The following parameters were measured: (i) cell viability by the MTT assay; (ii) caspase-3 and -7 activity; and (iii) the expression of 84 genes encoding adhesion-related molecules using real-time RT-PCR microarrays., Results: Mechanical stress reduced the metabolic activity of deformed cells at 6 h, and caspase-3 and -7 activity at 6 and 12 h. Seventy-three genes were detected at critical threshold values < 35. Fifteen showed a significant change in relative expression: five cell adhesion molecules (ICAM1, ITGA3, ITGA6, ITGA8 and NCAM1), three collagen α-chains (COL6A1, COL8A1 and COL11A1), four MMPs (ADAMTS1, MMP8, MMP11 and MMP15), plus CTGF, SPP1 and VTN. Four genes were upregulated (ADAMTS1, CTGF, ICAM1 and SPP1) and 11 downregulated, with the range extending from a 1.76-fold induction of SPP1 at 12 h to a 2.49-fold downregulation of COL11A1 at 24 h., Conclusion: The study has identified several mechanoresponsive adhesion-related genes, and shown that onset of mechanical stress was followed by a transient reduction in overall cellular activity, including the expression of two apoptosis 'executioner' caspases., (© 2011 John Wiley & Sons A/S.)

Aims: This study provides a systematic review of existing research that has empirically evaluated interventions designed to reduce stigma related to substance use disorders., Methods: A comprehensive review of electronic databases was conducted to identify evaluations of substance use disorder related stigma interventions. Studies that met inclusion criteria were synthesized and assessed using systematic review methods., Results: Thirteen studies met the inclusion criteria. The methodological quality of the studies was moderately strong. Interventions of three studies (23%) focused on people with substance use disorders (self-stigma), three studies (23%) targeted the general public (social stigma) and seven studies (54%) focused on medical students and other professional groups (structural stigma). Nine interventions (69%) used approaches that included education and/or direct contact with people who have substance use disorders. All but one study indicated their interventions produced positive effects on at least one stigma outcome measure. None of the interventions have been evaluated across different settings or populations., Conclusions: A range of interventions demonstrate promise for achieving meaningful improvements in stigma related to substance use disorders. The limited evidence indicates that self-stigma can be reduced through therapeutic interventions such as group-based acceptance and commitment therapy. Effective strategies for addressing social stigma include motivational interviewing and communicating positive stories of people with substance use disorders. For changing stigma at a structural level, contact-based training and education programs targeting medical students and professionals (e.g. police, counsellors) are effective., (© 2011 The Authors, Addiction © 2011 Society for the Study of Addiction.)

The forces that orthodontic appliances apply to the teeth are transmitted through the periodontal ligament (PDL) to the supporting alveolar bone, leading to the deposition or resorption of bone, depending upon whether the tissues are exposed to a tensile or compressive mechanical strain. To evaluate the osteogenic potential of PDL cells, we applied a 12% uni-axial cyclic tensile strain to cultured human PDL cells and analyzed the differential expression of 78 genes implicated in osteoblast differentiation and bone metabolism by real-time RT-PCR array technology. Sixteen genes showed statistically significant changes in expression in response to alterations in their mechanical environment, including cell adhesion molecules and collagen fiber types. Genes linked to the osteoblast phenotype that were up-regulated included BMP2, BMP6, ALP, SOX9, MSX1, and VEGFA; those down-regulated included BMP4 and EGF. This study has expanded our knowledge of the transcriptional profile of PDL cells and identified several new mechanoresponsive genes.

At present, air traffic controllers (ATCOs) exercise strict control over routing authority for aircraft movement in airspace. The onset of a free flight environment, however, may well result in a dramatic change to airspace jurisdictions, with aircraft movements for the large part being governed by aircrew, not ATCOs. The present study examined the impact of such changes on spatial memory for recent and non-recent locations of aircraft represented on a visual display. The experiment contrasted present conditions, in which permission for manoeuvres is granted by ATCOs, with potential free flight conditions, in which aircrew undertake deviations without explicit approval from ATCOs. Results indicated that the ATCO role adopted by participants impacted differently on short-term and long-term spatial representations of aircraft manoeuvres. Although informing participants of impending deviations has beneficial effects on spatial representations in the short term, long-term representations of spatial events are affected deleteriously by the presentation of subsequent information pertaining to other aircraft. This study suggests strongly that recognition of the perceptual and cognitive consequences of changing to a free flight environment is crucial if air safety is not to be jeopardized.

Stimulation of protective immune responses against intracellular pathogens is difficult to achieve using non-replicating vaccines. BALB/c mice immunized by intramuscular injection with killed Francisella tularensis (live vaccine strain) adjuvanted with preformed immune stimulating complexes admixed with CpG, were protected when systemically challenged with a highly virulent strain of F. tularensis (Schu S4). Serum from immunized mice was used to probe a whole proteome microarray in order to identify immunodominant antigens. Eleven out of the top 12 immunodominant antigens have been previously described as immunoreactive in F. tularensis. However, 31 previously unreported immunoreactive antigens were revealed using this approach. Twenty four (50%) of the ORFs on the immunodominant hit list belonged to the category of surface or membrane associated proteins compared to only 22% of the entire proteome. There were eight hypothetical protein hits and eight hits from proteins associated with different aspects of metabolism. The chip also allowed us to readily determine the IgG subclass bias, towards individual or multiple antigens, in protected and unprotected animals. These data give insight into the protective immune response and have potentially important implications for the rational design of non-living vaccines for tularemia and other intracellular pathogens., (Crown Copyright 2007 Dstl.)

MLL fusion proteins are oncogenic transcription factors that are associated with aggressive lymphoid and myeloid leukemias. We constructed an inducible MLL fusion, MLL-ENL-ERtm, that rendered the transcriptional and transforming properties of MLL-ENL strictly dependent on the presence of 4-hydroxy-tamoxifen. MLL-ENL-ERtm-immortalized hematopoietic cells required 4-hydroxy-tamoxifen for continuous growth and differentiated terminally upon tamoxifen withdrawal. Microarray analysis performed on these conditionally transformed cells revealed Hoxa9 and Hoxa7 as well as the Hox coregulators Meis1 and Pbx3 among the targets upregulated by MLL-ENL-ERtm. Overexpression of the Hox repressor Bmi-1 inhibited the growth-transforming activity of MLL-ENL. Moreover, the enforced expression of Hoxa9 in combination with Meis1 was sufficient to substitute for MLL-ENL-ERtm function and to maintain a state of continuous proliferation and differentiation arrest. These results suggest that MLL fusion proteins impose a reversible block on myeloid differentiation through aberrant activation of a limited set of homeobox genes and Hox coregulators that are consistently expressed in MLL-associated leukemias.

Ethylenediaminetetraacetic acid (EDTA) is the anticoagulant recommended for full blood counts, citrate is recommended for coagulation and platelet studies, and citrate-theophylline-adenosine-dipyridamole (CTAD) inhibits platelet activation. Because the combination of EDTA and CTAD (E/C) is better than EDTA or CTAD alone for measuring platelet parameters on the ADVIA 120 Haematology System, we investigated whether it also offers advantages for the flow cytometric assessment of platelet and/or neutrophil activation and platelet-leucocyte aggregate formation ex vivo. Blood from healthy subjects was collected into E/C or citrate, kept at room temperature or at 4 degrees C, and analysed 0 to 360 min later in the ADVIA 120 and by immunofluorescent flow cytometry. Platelet count, mean platelet volume, number of platelet clumps, mean platelet component, numbers of CD62P(+) platelets and platelet-leucocyte aggregates, and expression of CD11b on neutrophils changed little over 360 min in blood with E/C kept at 4 degrees C. In contrast, one or more parameter changed when blood was kept with E/C at ambient temperature or with citrate at either temperature. The use of E/C in in vitro and in vivo studies is illustrated. Platelet and neutrophil activation status ex vivo can be reliably assessed if blood is collected into E/C, held at 4 degrees C, and analysed within 6 h., (Copyright 2002 Wiley-Liss, Inc.)

Objective: To test the hypothesis that longer plates with the minimum number of screws provide equivalent or superior strength of fixation to standard compression plating using the maximum number of screws., Design: Prospective analysis of biomechanical data was performed., Setting: In vitro experimentation., Interventions: A reproducible osteotomy was made in formalin-fixed ulnae. The osteotomies were stabilized employing six-, eight-, or ten-hole plates with two screws in the outermost holes and two screws in the innermost holes, and compared to stabilization with a six-hole plate with six screws., Main Outcome Measure: Four-point mechanical testing to failure was performed in both apex-dorsal (tension-band) or medial-lateral bending modes. Load-displacement curves were obtained., Results: All of the longer plates with the minimum number of screws were stronger than the six-hole plate with six screws when tested in the medial-lateral and the tension-band mode. The eight-hole plate with four screws, however, was statistically inseparable from the fully loaded six-hole plate. There was no statistical difference between any of the configurations in regard to the stiffness of the fixation., Conclusions: In a cadaveric ulnar osteotomy model stripped of soft tissue, the number of screws is less important than the length of the plate in providing bending strength to the construct.

The Drosophila trithorax- and Polycomb-group (trxG and PcG) proteins maintain activated and repressed transcriptional states at specific target gene loci. The Additional sex combs (Asx) gene is of particular interest as it appears to function in both protein complexes and yet its effects on target genes are more restricted. A novel protein, Tantalus (TAN), was identified in a yeast two-hybrid screen for ASX-interacting proteins that might confer tissue-specific ASX functions. TAN contains consensus nuclear localization sites and binds DNA in vitro. However, its subcellular localization varies in a tissue-specific fashion. In salivary glands, TAN is predominantly nuclear and associates with 66 euchromatic sites on polytene chromosomes, more than half of which overlap with ASX. These loci do not include the homeotic genes of the ANT and BX complexes bound by other PcG and trxG proteins. Rather, tan mutant defects are restricted to sensory organs. We show that one of these defects, shared by Asx, is genetically enhanced by Asx. Taken together, the data suggest that TAN is a tissue-specific cofactor for ASX, and that its activity may be partially controlled by subcellular trafficking., (Copyright 2001 Academic Press.)

Background: A transient expansion of the CD8+ T cell pool normally occurs in the early phase of HIV infection. Persistent expansion of this pool is observed in two related settings: diffuse infiltrative lymphocytosis syndrome (DILS) and HIV associated CD8+ lymphocytosis syndrome., Aim: To investigate a group of HIV infected patients with CD8+ lymphocytosis syndrome with particular emphasis on whether monoclonality was present., Methods: A group of 18 patients with HIV-1 infection and persistent circulating CD8+ lymphocytosis was compared with 21 HIV positive controls. Serum samples were tested for antinuclear antibodies, antibodies to extractable nuclear antigens, immunoglobulin levels, paraproteins, human T lymphotropic virus type 1 (HTLV-1), Epstein-Barr virus, and cytomegalovirus serology. Lymphocyte phenotyping and HLA-DR typing was performed, and T cell receptor (TCR) gene rearrangement studies used to identify monoclonal populations of T cells. CD4+ and CD8+ subsets of peripheral blood lymphocytes were purified to determine whether CD8+ populations inhibited HIV replication in autologous CD4+ cells., Results: A subgroup of patients with HIV-1 infection was found to have expanded populations of CD8+ T cell large granular lymphocytes persisting for 6 to 30 months. The consensus immunophenotype was CD4- CD8+ DRhigh CD11a+ CD11c+ CD16- CD28+/- CD56- CD57+, consistent with typical T cell large granular lymphocytes expressing cellular activation markers. Despite the finding of monoclonal TCR gene usage in five of 18 patients, there is evidence that the CD8+ expansions are reactive populations capable of mediating non-cytotoxic inhibition of HIV replication., Conclusions: A subgroup of HIV positive patients has CD8+ lymphocytosis, but despite the frequent occurrence of monoclonal TCR gene usage there is evidence that this represents an immune response to viral infection rather than a malignant disorder.

From a national perspective, local public health practice continues to face many of the challenges identified in the 1988 Institute of Medicine study. Unless they are addressed broadly, current trends may exacerbate further the capacity of local public health agencies. Efforts to strengthen local practice should focus on workforce development, scale of operations, and resources. New and innovative models of collaboration between academia and local practice are proposed.

The flow cytometric analysis of leucocytes in whole blood is usually performed on samples in which the erythrocytes have been lysed and the leucocytes fixed. Because lysis and fixation reagents have the potential to introduce artefacts, several commercially available reagents were used to prepare normal and leukaemic lymphocytes for immunophenotypic analysis by flow cytometry, and the results were compared with those obtained from live whole blood. The reagents tested were the ImmunoPrep system and OptiLyse C (Coulter), LF-1000-Lyse and Flow (Harlan), Uti-Lyse (Dako) and FACS Lysing Solution (Becton Dickinson). The effect of each reagent on the apparent expression of CD3, CD5, CD11b, CD45, FMC7, kappa and lambda antigens was determined on lymphocytes from six normal controls and from six patients with chronic lymphocytic leukaemia (CLL). The following observations were made: (i) the time in minutes for each procedure varied markedly and was 1.5, 15, 20, 30 and 30 for the ImmunoPrep system, OptiLyse C, Uti-Lyse, FACS Lysing Solution, and LF-1000, respectively, but only 0.5 min for live whole blood. (ii) The forward and side scatter characteristics were affected by all of the lysis and fixation procedures, and this was most marked for LF-1000-Lyse and Flow. (iii) OptiLyse C gave preparations with poor forward and side scatter resolution due to the presence of residual red cell fragments. (iv) Lysis and fixation procedures did not affect the apparent expression of the CD3, CD45, or FMC7 antigens on normal or CLL samples, but gave highly variable results for the expression of the CD5, CD11b, kappa, and lambda antigens on the CLL samples. We conclude that lysis and fixation procedures can introduce different artefacts in the analysis of normal and leukaemic samples that are best avoided by analysing live whole blood., (Copyright 1999 Wiley-Liss, Inc.)

Background: One major problem with the use of small-diameter nails in the treatment of tibial fractures is the high rate of fatigue fractures of the locking screws. The objective of this study was to correlate such parameters as diameter of locking screws, influence of notch effect, and material with fatigue strength and fatigue limit., Methods: In a biomechanical study, the mechanical properties of 11 different locking screws and screw designs to be used in solid tibial nails were tested under typical fatigue conditions., Results: The results showed that an increase in the screw's diameter significantly increased the fatigue strength of the tested locking screws and prototypes (p < 0.0005). A 20% increase of diameter improved fatigue strength by 25 to 70%. Absence of thread at the location where the screw contacts the nail's aperture (where the main load is transmitted to the screw and where the screw, therefore, usually tends to break) may double the fatigue strength and fatigue limit by avoiding the negative notch effect of the screw's thread. It was further significant that titanium alloys had a higher fatigue strength compared with stainless steel alloys (p < 0.001). However, the fatigue strength of titanium implants is massively reduced by minimal damage to the surface., Conclusion: Optimizing the core diameter of the locking screws at the aperture of the solid tibial nail by using screws without a continuous thread generates a fatigue strength two to three times higher compared with screws with a continuous thread. Increasing fatigue strength of locking screws might decrease malunion in fractures stabilized by small-diameter nails.

The protein products of Polycomb group (PcG) and trithorax group (trxG) genes are required for the maintenance of the transcriptionally repressed and active states, respectively, of the homeotic genes. Mutations in PcG genes produce gain-of-function (posterior) homeotic transformations, while mutations in trxG genes produce loss-of-function (anterior) homeotic transformations. Double mutant combinations between a PcG gene and a trxG gene suppress the homeotic transformations seen with either mutation alone, suggesting that PcG and trxG genes act antagonistically. The PcG gene Additional sex combs (Asx) is interesting because one mutant allele, AsxP1, causes both anterior and posterior homeotic transformations. AsxP1 and other Asx mutations were crossed to mutations in the PcG gene Polycomb (Pc) and the trxG gene trithorax (trx). Asx alleles enhance both PcG and trxG homeotic transformations, showing that Asx is required for both the activation and the repression of homeotic loci. Asx also shows strong allele-specific interactions with the PcG genes Pc and super sex combs (sxc). Together, these data indicate that there are functional interactions between Asx, Pc and sxc in vivo. ASX may interact with a PcG complex containing PC and SXC and mediate activation versus repression at target loci, perhaps by interacting directly with the TRX protein.

The platelet-type thrombin receptor was the first member to be identified in a family of protease activated receptors (PARs) and has been designated PAR-1. We recently reported that the large granular lymphocytes (LGLs) in patients with proliferations of CD8+ cells co-expressed PAR-1 and the expression of PAR-1 correlated with the expression of CD57. Here we show, by three-colour immunofluorescence, that the LGLs from a patient with a rare CD4+ CD57+ monoclonal expansion also expressed PAR-1. Northern blot analysis confirmed the presence of high levels of mRNA for PAR-1 in these LGLs.

The Additional sex combs (Asx) gene of Drosophila is a member of the Polycomb group of genes, which are required for maintenance of stable repression of homeotic and other loci. Asx is unusual among the Polycomb group because: (1) one Asx allele exhibits both anterior and posterior transformations; (2) Asx mutations enhance anterior transformations of trx mutations; (3) Asx mutations exhibit segmentation phenotypes in addition to homeotic phenotypes; (4) Asx is an Enhancer of position-effect variegation and (5) Asx displays tissue-specific derepression of target genes. Asx was cloned by transposon tagging and encodes a protein of 1668 amino acids containing an unusual cysteine cluster at the carboxy terminus. The protein is ubiquitously expressed during development. We show that Asx is required in the central nervous system to regulate Ultrabithorax. ASX binds to multiple sites on polytene chromosomes, 70% of which overlap those of Polycomb, polyhomeotic and Polycomblike, and 30% of which are unique. The differences in target site recognition may account for some of the differences in Asx phenotypes relative to other members of the Polycomb group.

Polycomb group (PcG) genes of Drosophila are negative regulators of homeotic gene expression required for maintenance of determination. Sequence similarity between Polycomb and Su(var)205 led to the suggestion that PcG genes and modifiers of position-effect variegation (PEV) might function analogously in the establishment of chromatin structure. If PcG proteins participate directly in the same process that leads to PEV, PcG mutations should suppress PEV. We show that mutations in E(Pc), an unusual member of the PcG, suppress PEV of four variegating rearrangements: In(l)wm4, B(SV), T(2;3)Sb(V) and In(2R)bw(VDe2). Using reversion of a Pelement insertion, deficiency mapping, and recombination mapping as criteria, homeotic effects and suppression of PEV associated with E(Pc) co-map. Asx is an enhancer of PEV, whereas nine other PcG loci do not affect PEV. These results support the conclusion that there are fewer similarities between PcG genes and modifiers of PEV than previously supposed. However, E(Pc) appears to be an important link between the two groups. We discuss why Asx might act as an enhancer of PEV.

We studied 45 patients aged 14-66 years who had undergone stem cell transplantation for a variety of malignant conditions at least 12 months previously. Compared to normal controls, they had significantly reduced absolute numbers of CD4+, CD4+ CD45RA+ and CD4+ CD45RO+ T cells and a reduced CD4+ CD45RA+:CD4+ CD45RO+ ratio. In all subsets T-cell numbers were significantly greater 24 months, compared to 12-24 months, after transplantation and there was a nonsignificant trend towards lower T-cell numbers with increasing age. We conclude that the thymus, or putative thymic-equivalent tissue, remains functional in older adults.

Gramicidin A analogs, labeled with 13C in the backbone carbonyl groups and the C-2 indole carbons of the tryptophan-11 and tryptophan-13 residues, were synthesized using t-Boc-protected amino acids. The purified analogs were incorporated into phosphatidylcholine bilayers at a 1:15 molar ratio and macroscopically aligned between glass coverslips. The orientations of the labeled groups within the channel were investigated using solid-state NMR and the effect of a monovalent ion (Na+) on the orientation of these groups determined. The presence of sodium ions did not perturb the 13C spectra of the tryptophan carbonyl groups. These results contrast with earlier results in which the Leu-10, Leu-12, and Leu-14 carbonyl groups were found to be significantly affected by the presence of sodium ions and imply that the tryptophan carbonyl groups are not directly involved in ion binding. The channel form of gramicidin A has been demonstrated to be the right-handed form of the beta 6.3 helix: consequently, the tryptophan carbonyls would be directed away from the entrance to the channel and take part in internal hydrogen bonding, so that the presence of cations in the channel would have less effect than on the outer leucine residues. Sodium ions also had no effect on the C-2 indole resonance of the tryptophan side chains. However, a small change was observed in Trp-11 when the ether lipid, ditetradecylphosphatidylcholine, was substituted for the ester lipid, dimyristoylphosphatidylcholine, indicating some sensitivity of the gramicidin side chains to the surrounding lipid.

Ten analogues of the 26-residue, bee venom peptide, melittin (H3N(+)-GIGAVLKVLTTGLPALISWIKRKRQQ-CONH2), were synthesized, each with 13C enrichment of a single peptide carbonyl carbon. These peptides were incorporated into bilayers of the diether lipid, ditetradecylphosphatidylcholine, aligned between stacked glass plates. Solid-state 13C nuclear magnetic resonance spectra were obtained as a function of the angle between the bilayer planes and the magnetic field of the spectrometers, and at temperatures above and below the lipid gel-to-liquid crystalline transition temperature, Tc. For bilayers aligned with the normal along the applied magnetic field there was no shift in the carbonyl resonances of residues Ile2, Ala4, Leu9, Leu13, or Ala15, with minor changes for residues Val8 and Ile20, and small changes at Val5, Leu6 and Ile17 on immobilization of the peptide below Tc. In contrast, the spectra for bilayers aligned at right angles to the field showed greatly increased anisotropy below Tc for all analogues. From these experiments it was evident that the peptide was well-aligned in the bilayers and reoriented about the bilayer normal. The observed reduced chemical shift anisotropies and the chemical shifts were consistent with melittin adopting a helical conformation with a transbilayer orientation in the lipid membranes. With the exception of Ile17, there was no apparent difference between the behaviour of residues in the two segments that form separate helices in the water-soluble form of the peptide, suggesting that in membranes the angle between the helices is greater than the 120 degrees observed in the crystal form.

This paper reviews the recent experience in Washington State which resulted in creation of a separate state Department of Health. Until the 1989 legislation was passed, Washington was one of 19 states in which public health was organizationally housed in a superagency. The Institute of Medicine, in its 1988 report on The Future of Public Health, recommended that states organize core functions under a separate department of health. The effort in Washington State was led by local public health officials. Success followed four years of hard work during which mistakes were made, coalitions were built, and legislative process was learned.

Myelin basic protein (MBP) from the Whaler shark (Carcharhinus obscurus) has been purified from acid extracts of a chloroform/methanol pellet from whole brains. The amino acid sequence of the majority of the protein has been determined and compared with the sequences of other MBPs. The shark protein has only 44% homology with the bovine protein, but, in common with other MBPs, it has basic residues distributed throughout the sequence and no extensive segments that are predicted to have an ordered secondary structure in solution. Shark MBP lacks the triproline sequence previously postulated to form a hairpin bend in the molecule. The region containing the putative consensus sequence for encephalitogenicity in the guinea pig contains several substitutions, thus accounting for the lack of activity of the shark protein. Studies of the secondary structure and self-association have shown that shark MBP possesses solution properties similar to those of the bovine protein, despite the extensive differences in primary structure.