Modern high-velocity projectiles produce temporary cavities and can thus cause extensive tissue destruction along the bullet path. It is still unclear whether gelatin blocks, which are used as a well-accepted tissue simulant, allow the effects of projectiles to be adequately investigated and how these effects are influenced by caliber size.Barium titanate particles were distributed throughout a test chamber for an assessment of wound contamination. We fired .22-caliber Magnum bullets first into gelatin blocks and then into porcine hind limbs placed behind the chamber. Two other types of bullets  were then shot into porcine hind limbs. Permanent and temporary wound cavities as well as the spatial distribution of barium titanate particles in relation to the bullet path were evaluated radiologically.p < 0.05) in the mean results for all parameters. There were significant differences between the bullets of different calibers in the depth to which barium titanate particles penetrated the porcine hind limbs. Almost no particles, however, were found at a penetration depth of 10 cm or more. By contrast, gas cavities were detected along the entire bullet path.A comparison of the gelatin blocks and hind limbs showed significant differences (Gelatin is only of limited value for evaluating the path of high-velocity projectiles and the contamination of wounds by exogenous particles. There is a direct relationship between the presence of gas cavities in the tissue along the bullet path and caliber size. These cavities, however, are only mildly contaminated by exogenous particles. Gunshot injuries not only cause direct tissue damage but often also lead to contamination by foreign material along the bullets path ,2. The rClinical radiological examinations of gunshot injuries often show the presence of gas cavities in the tissue along the bullet path which provide first information about the temporary wound cavity. Radiological and clinical examinations alone, however, have thus far not been able to reliably demonstrate the presence or absence of exogenous particles in these gas cavities. Whether or not a wound is contaminated, however, might help to estimate the caliber and ammunition of the used rifle in gunshot wounds.It is difficult to standardize the effects of gunshot injuries on tissue not least because it is virtually impossible to perform direct systematic examinations of gunshot injuries in living tissues. For this reason, there are many well-established models for studying gunshot injuries ,14. OrdnThe objective of this study was therefore to compare bullet paths and wound contamination in gelatin blocks and tissue and to assess the influence of caliber size.The study was approved by the local ethics committee at the Hannover Medical School, Germany (EK/2009).0 945-970 m/s).The firing tests were performed with bullets of different calibers, i.e. .22-caliber Magnum bullets, .222-caliber Winchester bullets and 6.5 × 57 mm bullets . All bullets were full metal jacketed. The projectiles had different weights, but comparable similar initial velocities  with a particle size of 3-6 μm were placed inside the chamber. Three air pressure valves were attached to the chamber (at the bottom and on the left and right sides) and connected to an air compressor. The porcine hind limbs and the gelatin blocks were mounted on a support frame that was positioned directly behind the chamber in such a way that the gelatin blocks and hind limbs could be placed in the line of fire.The gelatin blocks that were used in our experiments consisted of 20% porcine gelatin  and water. The size of the blocks was 120 mm by 120 mm by 180 mm. The hind limbs were obtained from freshly slaughtered pigs with a total weight of approximately 65 kg. They were separated from the body in the median plane in order to facilitate their handling during the tests.The experimental set-up described above was used to fire .22-caliber Magnum bullets into gelatin blocks (n = 6) and hind limbs (n = 6). The other two types of bullets  were fired only into porcine hind limbs (n = 6 each).One shot was fired into each gelatin block and hind limb. At the time of firing, the gelatin blocks had a temperature ranging between +8°C and +10°C. They were mounted on the support frame in such a way that the bullet passed right through the block. Before each shot, 5.0 g of barium titanate were placed inside the test chamber. An air-permeable filter paper was inserted in the chamber to separate the interior of the chamber from the barrel of the rifle. A gelatin block was placed directly against the opposite side of the chamber. Shortly before a shot was fired, the compressor was used to expose the chamber to a blast of compressed air (0.5 bar) and to generate a cloud of barium titanate particles inside the chamber. A shot was then fired from the rifle that was mounted on the rifle support.Barium titanate was again placed inside the chamber and the porcine hind limbs were positioned against the chamber in such a way that they tightly sealed the chamber. The chamber was again exposed to a blast of compressed air and a shot was fired from each of the three rifles into the hind limbs.After each shot, 16-slice computed tomography (CT) scans  of the gelatin blocks and hind limbs were obtained. All scans were performed at 120 kV and 200 mA.The data obtained were made available in a digital format (DICOM) and transferred to a personal computer for further analysis. Voxim software  was used for statistical analysis. For this purpose, the gelatin blocks were virtually cut into 20-mm-thick vertical slices and evaluated. Once the center of the gelatin block was identified, the mean diameter of the permanent cavity was calculated. The distances from eight sites on the wall of the permanent cavity to the center of the block were measured radially and the results were averaged for each vertical slice.Likewise, the distances from eight sites on the wall of the temporary cavity to the center of the block were measured radially and the results were averaged for each vertical slice. In order to determine the depth of infiltration in the gelatin blocks, the distances between barium titanate particles detected inside the temporary wound cavity and the center of the gelatin block at eight sites was measured. The results were averaged for each vertical slice.Similarly, the permanent and temporary cavities and the distribution of barium titanate particles inside the temporary wound cavity were assessed in the hind limbs. For this purpose, the path of the bullet was determined precisely on the basis of radiological parameters such as the presence of gas and the entrance and exit wounds. Two-cm-thick CT slices were obtained perpendicular to the bullet path and analyzed using the aforementioned parameters.Results are expressed as means ± SEM. Differences between groups were evaluated with an analysis of variance (ANOVA) on ranks. Differences within groups were analyzed by one-way repeated measures ANOVA. Student-Newman-Keuls or Dunn's post-hoc tests were used to isolate specific differences. A p-value < 0.05 was considered significant.After firing into the gelatin blocks and hind limbs, no projectiles or projectile fragments were detected in the specimens. Entrance and exit sites were found in all specimens.p < 0.05) between the two groups of specimens in all three parameters investigated. Along the entire bullet path, the mean diameter of the permanent wound cavity in the gelatin blocks was significantly larger than that measured in the hind legs .The results that we obtained for the diameter of the temporary wound cavity along the bullet path were similar to those obtained for the permanent cavity. The diameter increased from the entrance site, reached its maximum at a penetration depth of 120-140 mm and then decreased again Figure . A compaIrrespective of the caliber used, the diameter of the area where barium titanate particles were found continuously decreased from the entrance site to the exit site. The diameter of these areas depended on the caliber used and increased with caliber size. At a penetration depth of 100 mm or more, almost no barium titanate particles in the hind limbs were detectable (2 C).A direct comparison of ordnance gelatin blocks and porcine hind limbs showed significant differences in permanent cavities, temporary cavities and the infiltration depth of barium titanate particles.In porcine hind limbs, the diameter of the bullet path and the depth of infiltration of barium titanate particles along the bullet path increased with caliber. At a penetration depth of 100 mm or more, however, we detected almost no more exogenous particles in the hind limbs.Gunshot injuries can be examined using a wide variety of methods such as high-frequency video analysis, animal testing, and shots into human tissue simulants -18. TissOur study revealed not only significant differences between gelatin blocks and porcine hind limbs in terms of the permanent wound cavity, the temporary wound cavity and the infiltration of exogenous particles but even opposite trends in the parameters investigated. This clearly shows that the results obtained by firing projectiles and especially high-velocity projectiles into gelatin blocks can be transferred only to a limited extent to complex anatomical structures made of different tissues. The porcine hind limbs that we used in our study consisted primarily of skin with subcutaneous fat and muscle tissue. According to Jussila et al., the transferability of the results to other types of tissue is very limited .In our study, we used high-velocity projectiles of different calibers that do not fragment. Although the projectiles exhibited similar velocities, they caused significantly different degrees of tissue destruction and wound contamination in porcine hind limbs. The diameter of the permanent and temporary wound cavities were found to increase with caliber size. The permanent wound cavity is caused by the sound wave preceding the bullet and the displacement of tissue by the projectile. In soft tissues, however, the permanent wound cavity often collapses behind the projectile on account of the elastic behavior and displacement of tissue . High-veA loss of skin integrity in association with a gunshot injury almost always leads to wound contamination by either the projectile itself or by pathogens that are present in the air or on the skin . Our resGas cavities are nevertheless formed as a result of the compression of tissue in the temporary wound cavity and can be regarded as evidence of irreversible tissue compression. In summary, we must critically address the question of whether investigations in gelatin can be transferred to complex tissues in terminal ballistics.This study provides modern treatment strategies for gunshot injuries.Gelatin is only of limited value for evaluating the path of high-velocity projectiles and the contamination of wounds by exogenous particles. There is a direct relationship between the presence of gas cavities in the tissue along the bullet path and caliber size. These cavities, however, are only mildly contaminated by exogenous particles.The authors declare that they have no competing interests.CS, MR, MS, HK, MRU and NCG conceived of the study and participated in its design and coordination. CS and MR made substantial contributions to data acquisation and conception of manuscript and drafted and designed the manuscript. NCG and MRU were involved in revising the manuscript. All authors read and approved the final manuscript.The article processing charges are funded by the Deutsche Forschungsgemeinschaft (DFG), "Open Acess Publizieren".The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2482/12/6/prepub


ARID1A as a novel tumor suppressor gene coding for a chromatin remodeling protein that is mutated in UBC. Here, we assess ARID1A alterations in two series of patients with UBC. In the first tumor series, we analyze exons 2–20 in 52 primary UBC and find that all mutant tumors belong to the aggressive UBC phenotype (high grade non-muscle invasive and muscle invasive tumors) (P = 0.05). In a second series (n = 84), we assess ARID1A expression using immunohistochemistry, a surrogate for mutation analysis, and find that loss of expression increases with higher stage/grade, it is inversely associated with FGFR3 overexpression (P = 0.03) but it is not correlated with p53 overexpression (P = 0.30). We also analyzed the expression of cytokeratins in the same set of tumor and find, using unsupervised clustering, that tumors with ARID1A loss of expression are generally KRT5/6-low. In this patient series, loss of ARID1A expression is also associated with worse prognosis, likely reflecting the higher prevalence of losses found in tumors of higher stage and grade. The independent findings in these two sets of patients strongly support the notion that ARID1A inactivation is a key player in bladder carcinogenesis occurring predominantly in FGFR3 wild type tumors.Urothelial bladder cancer (UBC) is heterogeneous at the clinical, pathological, genetic, and epigenetic levels. Exome sequencing has identified  Urothelial bladder carcinoma (UBC) constitutes a heterogeneous clinical and pathological entity and subjects presenting with these tumors display a highly variable outcome: approximately 20% of patients with non-muscle invasive bladder cancer (NMIBC) are cured after the first resection whereas 60% undergo multiple lifetime recurrences and 15–20% progress and develop muscle-invasive bladder cancer (MIBC). This important clinical event entails cystectomy and poor prognosis: 50% of patients with MI tumors - either at presentation or during the evolution of the disease - die from the cancer. Patients with low grade (LG) NMIBC rarely progress whereas patients with high grade (HG) NMIBC are at high risk FGFR3 activating mutations, mainly in association with low grade; FGFR3 mutant tumors have a good prognosis and a low risk of progression to MIBC PIK3CA mutations occur in approximately 15% of tumors and tend to be associated with FGFR3 alterations RAS and FGFR3 mutations are mutually exclusive, the former occurring in 5–10% of tumors FGFR3 wild type tumors include at least two subgroups: those that are of low grade and stage, display papillary growth pattern, and an overall good prognosis, and those that are of high grade and are generally associated with aneuploidy and an aggressive clinical course. In contrast, Tp53 alterations are associated with HG-NMIBC and with MIBC FGFR3, PIK3CA, and RAS - and tumor suppressors participate in UBC In agreement with the diverse clinical course, genetic analyses of UBC have revealed a wide heterogeneity ARID1A mutation through UBC exome sequencing and a manuscript recently described the occurrence of ARID1A mutations in this tumor ARID1A mutations and loss of expression display an inverse association with FGFR3 mutations, are independent of p53 alterations, and are mainly associated with poor-prognosis UBC.We recently identified a truncating ARID1A sequencing, comprises 52 UBC cases prospectively recruited between 2009–2011 at Hospital del Mar  from which fresh tumor DNA was available. The characteristics of patients included in this series are summarized in We studied two tumor series. The first, used for ARID1A mutational analysis was performed in cases from the first series, essentially as described ARID1A variants identified in the exome sequencing study. Relevant primers can be found in FGFR3 mutational analysis was performed on cases from both series as described elsewhere ARID1A sequence (RefSeq NM_139135) as obtained from UCSC Genome Browser Sequence tags from all samples were independently aligned using Novoalign V2.07.04  versus the genomic http://www.gepas.org/) was used to pre-process the data, obtaining the average values for all probes mapping within a single locus. Three tumor subgroups were defined according to tumor stage/grade and known prevalence of genetic alterations: LG-NMIBC , HG-NMIBC , and MIBC (≥T2). The average expression of genes of interest was calculated for each of these groups and values were normalized with respect to those of the best prognosis group. An Anova limma analysis was performed on the pre-processed data using the POMELO online software (http://pomelo2.bioinfo.cnio.es/); differentially expressed genes were subsequently identified through a t-test, using an FDR adjusted P-value <0.5 as threshold of significance.Normalized bladder cancer gene expression data were obtained from NCBI Gene Expression Omnibus (GEO) database for studies GSE89 Immunohistochemical analyses were performed on tissue microarrays containing cores representative of the corresponding tumor, obtained from formalin-fixed paraffin-embedded tissue blocks from patients included in the second series. The following antibodies were used: ARID1A (2 µg/mL) , FGFR3 (8 µg/mL) , p53 , β-CAT , E-CAD (1∶50) , P-CAD (1∶75) , Ki67 , KRT5/6 , KRT14 (1∶25) , and KRT20 . Antigen retrieval and IHC were carried out as described elsewhere 3 puromycin-selected cells were seeded in triplicate in 6-well plates; 4 days later, cells were washed with PBS, fixed in methanol, and incubated with 0.5% crystal violet in 25% methanol. Because the interfered cells did not form compact colonies, counting did not provide an accurate measurement of growth. Crystal violet was eluted in 10% acetic acid and absorbance was quantified at 590 nm using a biophotometer (Eppendorf).SW800, 253J, 639V, VMCUB-3, SW1710, SCaBER, and HEK293 cells were purchased from the American Type Culture Collection ; RT112 Cells in log-growth phase were collected 48 h after puromycin selection and lysed in RIPA buffer  supplemented with the Complete protease inhibitor (Sigma) and a phosphatase inhibitor (Sigma) cocktails. After sonication, proteins (50 µg) were fractionated by SDS-PAGE using a discontinuous 4% concentrating-6% resolving gel, transferred to a nitrocellulose membrane, and incubated with anti-ARID1A monoclonal antibody  (1∶1000 dilution). After washing, anti-mouse or anti-rabbit horseradish peroxidase-labeled antibodis (Amersham Biosciences) were added. Rabbit anti-Myosin-IIa  served as a loading control. Reactions were developed using enhanced chemiluminiscence (Amersham Biosciences).ARID1A mRNA analysis: CCCCTCAATGACCTCCAGTA (forward) and ATCCCTGATGTGCTCACTCC (reverse). All reactions were performed in triplicate, and expression levels were normalized to individual HPRT values. Statistical analyses. Categorical data were reported by numbers and percentages. Associations between ARID1A mutation/loss of expression and the main characteristics of the patients were assessed using the chi-square test, T test, ANOVA, Mann-Whitney (MW), or Kruskal-Wallis (KW) as appropriate. Associations between markers were evaluated using the chi-square test. Survival data were analysed using Kaplan-Meier curves and the differences between curves were assessed with the log-rank test. Statistical significance was considered at 0.05. R Software  was used for statistical analysis.Total RNA was isolated from cells in log-growth phase using the GenElute Mammalian Total RNA kit (Sigma). Following DNase treatment , RNA was reverse-transcribed  and 20 ng RNA-equivalent were used for RT-qPCR using a 7900H Fast Real Time PCR System (Applied Biosystems). The following primers were used for ARID1A through the initial analysis of 2 bladder cancer exomes. ARID1A mutations have been reported to be frequent in ovarian clear cell carcinomas We identified a truncating mutation in ARID1A were analyzed in 5 UBC cell lines and 52 primary tumors. Exons 2–20 of Six primary tumors harbored 7 mutations predicted to be damaging (6/52 = 11.5%); 5 of them were nonsense and 2 were missense (N2066D and S571L) . One tumSix additional mutations, predicted to be damaging, were detected at a frequency (<10%) that precluded confirmation by Sanger sequencing; 5 of them led to missense substitutions .ARID1A and FGFR3 in the 50 tumors for which this information was available: all 5 tumors that had truncating ARID1A mutations had FGFR3 wild type sequences , suggesting that the two genes are involved in different genetic pathways. To expand this analysis, we took advantage of the data reported by Gui et al. ARID1A and 9/97 tumors had a mutation in FGFR3; 1/97 had a mutation in both genes supporting a lack of association between both genetic alterations in this tumor subgroup.We compared the mutational status of ARID1A pathogenic mutations have been reported to be associated with loss of protein expression −6; KW P = 3×10−5), in agreement with the observation that ARID1A mutations are more common in this tumor subgroup.FGFR3 activating mutations are generally associated with FGFR3 mRNA and protein overexpression Tp53 inactivation is generally associated with p53 nuclear overexpression FGFR3 mutations  and were higher in the low grade NMIBC than in the more aggressive tumor groups of high grade NMIBC and MIBC . In contrast, p53 nuclear overexpression was increasingly higher with increasing tumor stage/grade, reflecting protein accumulation associated with Tp53 mutations . ARID1A scores were significantly correlated with FGFR3 expression scores (Spearman correlation P = 0.03) but not with p53 scores (p = 0.30) (Immunohistochemistry was used as a surrogate to assess the status of two of the main genes involved in UBC:  = 0.30) .ARID1A and FGFR3 are differentially expressed in the three UBC subgroups at the RNA level, we analyzed two independent public UBC expression datasets and confirmed that both ARID1A and FGFR3 mRNA expression levels are significantly lower in MI-UBC, in agreement with the fact that FGFR3 mutations are associated with FGFR3 mRNA overexpression and are less frequent in aggressive tumors  showed a significantly lower rate of tumor recurrence (P = 0.011) but had a higher rate of tumor progression (P = 0.112), further indicating that loss of ARID1A expression is associated with more aggressive tumors .To assess the role of ARID1A in UBC, we knocked it down in cultured UBC lines. Six of 12 UBC lines analyzed lacked ARID1A expression, including VMCUB-3 .ARID1A mutations, are wild type for Tp53Tp53 - had no consistent effects on colony formation Click here for additional data file.Figure S2Mutation occurrence and frequency.Panel A. SNV frequency plotted against sequencing depth. Panel B. SIFT predictions for mutations comparing findings occurring for variants called at high vs. low frequency (threshold at 10%).(TIF)Click here for additional data file.Figure S3Sanger sequencing verification of all mutations detected in the resequencing study. All mutations detected at a frequency >10% were verified in both tumors and VMCUB-3 cells. The wild type sequence in normal leukocyte DNA is also shown for selected cases.(TIF)Click here for additional data file.Figure S4Relationship between ARID1A levels and those of other well-established UBC markers. UBC cases were classified in three categories: low grade NMI (TaG1 and TaG2 tumors), high grade NMI (TaG3 and T1G3 tumors), and MI (>T2 tumors). Nonhierarchical clustering of IHC scores for ARID1A, FGFR3, KRT5/6, KRT14, KRT20, β-CAT, Ki67, ECAD, and P-CAD was performed. IHC scores are shown using a green-red color code. Color bars under the dendogram include information about prognosis (pistachio/bourbon), tumor stage and grade (tones of blue), and FGFR3 mutational status (grey/black) when known. White squares indicate that information for that parameter is not available.(TIF)Click here for additional data file.Figure S5Effects of ARID1A knockdown in the 253J UBC cells.Panel A. ARID1A was knocked-down using three different shRNAs in the 253J cells. The knock-down was efficient at both the protein and mRNA levels. The bars represent the relative quantification of ARID1A mRNA levels taking non-targeting shRNA interfered cells as controls. Panel B. The quantification colony formation is shown, with error intervals of results from triplicate experiments denoting SEM. ARID1A knockdown was associated with reduced colony formation. Representative morphology changes in the cultured cells interfered for shNT (scrambled shRNA) and one of the shRNAs targeting ARID1A are shown.(TIF)Click here for additional data file.Table S1List of primers used for ARID1A resequencing and Sanger sequencing.(TIF)Click here for additional data file.Table S2Summary of reads per exon in the ARID1A resequencing study. Exon starting and ending positions are shown, along with the exon length in base pairs and the average sequencing depth.(TIF)Click here for additional data file.


Community-based obesity treatment programs for children that have a large program reach are a priority. To date, most programs have been small efficacy trials whose findings have yet to be up-scaled and translated into real-world settings. This paper reports on the process evaluation of a government-funded, translated obesity treatment program for children in Australia. It describes the characteristics and reach of children participating in the New South Wales (NSW) Ministry of Health Go4Fun® program.th percentile and no co-morbidities were eligible at no cost. Parents/carers self-refer via a toll-free phone number, text messages, online registration or via secondary referrals. LHDs deliver a 16 to 20-session program based on length of school term, holidays and recruitment challenges. Both parent/carer and child attend bi-weekly after school sessions. Parent-reported socio-demographic and measured child weight characteristics are presented using descriptive statistics. Differences between completers (attended at least 75% of sessions) and non-completers were assessed using chi-square tests, independent sample t-tests and adjusted odds ratios. Analyses were adjusted for clustering of programs.Delivered across the state of NSW  by Local Health Districts (LHDs), Go4Fun® is a community-based, multidisciplinary family obesity treatment program adapted from the United Kingdom Mind Exercise Nutrition Do it (MEND) program that targets weight-related behaviours. Children aged 7-13 years with a BMI ≥85Between 2009 and 2012, a total of 2,499 children  participated in the Go4Fun® program. Children were mainly from low-middle socioeconomic status (76.5%), resided in major cities (63.3%), and 5.7% were Aboriginal. At baseline, 96.5% of children were overweight or obese. Mean BMI-z-score was 2.07 (0.41) and 94.5% had a waist-to-height ratio ≥0.5. More than half (57.9%) completed at least 75% of sessions. Amongst completers , girls , non-Aboriginal children  and children residing in less socially disadvantaged areas  were significantly more likely to complete the program.The Go4Fun® program successfully reached the targeted population of overweight/obese children at socioeconomic disadvantage and is a rare example of an up-scaled translational program. In Australia’s most populous state, New South Wales  program is an example of a community based child obesity program which has demonstrated efficacy in weight outcomes ,9 The utGo4Fun has been widely disseminated and therefore process evaluation is now prudent. Information generated from the process evaluation is especially important for building evidence for real-world, up-scaled programs which have different characteristics to small, controlled efficacy programs. This type of information informs policy makers and program deliverers on program strengths, weaknesses, and areas that need improvement. This paper describes the up-scaling of Go4Fun in NSW and the characteristics of the population it has reached and retained since inception in 2009, including the characteristics of children who completed and did not complete the program.2) of the LHD.In 2008 the National Partnership Agreement on Preventive Health (NPAPH) was announced by the Australian government providing $AUD932.5 million dollars in funding to strengthen Australia’s investment and infrastructure in preventative health. One of the initiatives under the Agreement is the Healthy Children Initiative (HCI) which delivers programmatic activities to 0-18 year olds promoting healthy weight, healthy eating and physical activity. In NSW, one of the HCI programs is Go4Fun, which is supported and funded by the state-wide Office of Preventive Health and locally implemented by Health Promotion Services of Local Health Districts (LHD). Briefly, the NSW health service is divided geographically into 15 LHD . Program funding is allocated according to the prevalence of child overweight/obesity and geographical spread  ≥85A staged process for up-scaling was used with initial funding provided to employ program managers to deliver in two rural and regional LHD in July 2009, then funding to other LHDs to employ program managers was provided in a phased manner to enable the facilitation of the program across the state. The program was managed in each LHD however different models were used with some contracting private organisations such as Private Practice Dieticians, Divisions of General Practice and Aboriginal Medical Services to manage and deliver programs. Other LDH’s employed a program manager and contracted facilitators or allied health professionals to deliver programs.The prevalence of overweight/obesity is significantly higher among NSW children from low socioeconomic backgrounds , so thesA number of strategies were undertaken to promote Go4Fun including advertising in newspapers and publications; attendance at community events with activities and distribution of promotional items ; radio advertising; letterbox drops. Examples of Go4Fun advertising messages included “Free fun program for kids to become fitter, healthier and happier”; “Do you have children 7 to 13 years old? Are you worried about their weight?”; “Healthy, Active, Happy, Kids”; “Healthy lifestyle program for kids”; “Children and their families become fitter, healthier and happier”. Each LHD undertakes local recruitment utilising local media, engaging partnerships  and providing workshops and professional development opportunities to raise program awareness.Families may self-refer via a toll-free phone number, a text message or online registration to the program and secondary referrals were accepted from health professionals, organisations and community members. The top three referral sources accounting for the majority of referrals were (i) Schools ; (ii) Newspapers and local media articles and; (iii) Referrals from health professionals. Two other important referral sources were word of mouth and community groups; and local advertising in the community .Parent must accompany their child to the program so it was developed as a 20-bi-weekly (i.e. 10 weeks) after school program run during school terms however LHDs have adapted the program to 16, 18 or 20 sessions according to the timing of school terms, public holidays and to increase participant numbers in programs. It was left to the discretion of each LHD to decide which sessions to omit or modify. The theoretical basis of Go4Fun is to engage families in the process of weight management by addressing key components for individual-level behavioural change  . ParentaA range of health professionals are trained to deliver Go4Fun including: dieticians, nutritionists, exercise physiologists, physiotherapists, fitness leaders, social workers and health promotion officers and are delivered in a range of settings including community health centres, sport centres, and schools. LHD’s with high social disadvantage and with a high proportion of Aboriginal people, Aboriginal and welfare organisations such as the Aboriginal Medical Services, Aboriginal health workers and Anglicare are contracted to deliver programs often at their venues or the organisations referring participants into programs. Facilitators are required to complete two days of face-to-face training (including taking standard anthropometric measurements) plus online training facilitated by MEND Australia.For process evaluation, we describe the child’s socio-demographic and risk factor profile, commensurate with the intended target group, the dose  and reach . Facilitators enter participant data into the MEND Australia proprietary database.At enrolment, parents completed a questionnaire on behalf of their child providing socio-demographic information including their child’s sex, date of birth, postcode of usual residence and Aboriginal status. Postcode of residence was used as a proxy for socio-economic status (SES), based on the Australian Bureau of Statistics’ Socio-Economic Indexes for Areas (SEIFA) and the Accessibility-Remoteness Index of Australia Plus (ARIA+) which determines geographical remoteness. SEIFA values were divided into quintiles  and ARIA+measures were categorised as major city, inner regional, outer regional, remote/very remote ,13.2), and waist circumference (cm) for waist-to-height ratio (WtHtr). Children’s BMI-for-age z-scores were calculated from the United States Centers for Disease Control and Prevention 2000 reference data [At enrolment, facilitators measured children’s height (m) and weight (kg) for BMI  to adjust standard errors and 95% confidence intervals (95% CI) for clustering of programs. All data collected between 2009 and 2012 were included in the analysis. Percent attendance was calculated, taking into account different program lengths  and program “completers” were defined as having attended at least 75% of sessions. Descriptive statistics describe socio-demographic and weight-related characteristics of participants. Sex differences, as well as differences between “completers” and “non-completers”, were determined by chi-square analyses for categorical variables and independent samples t-tests for continuous variables. Adjusted odds ratios (AOR) of the likelihood of children completing the program based on sex, Aboriginal status, ARIA+and SEIFA were derived from complex samples logistic regression. Statistical significance was accepted at P < 0.05.Between July 2009 and December 2012, 296 Go4Fun® programs have been conducted in NSW. Children’s baseline socio-demographic and weight characteristics are presented in Table More than half of children (57.9%) and their parents/carers attended at least 75% of sessions. Figure Overall, participants’ feedback at the end of the program showed that 95.5% of parents indicated a good or very good level of satisfaction with the program and 87.1% of children indicated that they were either happy or very happy with the program. Some examples of parent statements included “I was sceptical at first but I even lost 4 kg”; “All around a very informative and useful program that we can implement for the whole family” and “It’s changed what we eat and our exercise habits”. Similarly some examples of the children’s statements included “The things we learnt were useful and we had lots of fun playing the games”; “The games and activities were very fun and made you fitter at the same time” and “Meeting up with new friends and getting fit in the process”.In contrast to small-scaled interventions, process evaluation on large, up-scaled programs in the real world are limited. This is a significant evidence gap as the implementation of population-based intervention programs, such as Go4Fun®, in the real world face far greater challenges than the implementation of small efficacy trials that are controllable. More importantly, government decision-makers need information on a program’s strengths, weaknesses and where improvements can be made so that there is a continuous quality improvement process that underpins and justifies public funding of programs.In terms of dissemination and implementation, the overall process evaluation findings reported here support the benefits of a significant government investment in a community-based child obesity treatment program to reach into socially disadvantaged communities, including Aboriginal and rural children. We found that the up-scaling of Go4Fun® into a real world setting was successful in its reach into Aboriginal families and families from lower socio-economic groups and more geographically remote locations where communities not only face significant greater health disadvantage, but limited access to health care services . Almost There were some important lessons that we learnt during the up-scaling of Go4Fun across an entire jursidiction which assisted with the programs reach into socially disadvantaged families. These include working with local health promotion services to utilise their expertise in engaging with the local communities; supporting the program with comprehensive participant materials; ensuring that programs were delivered at a consistent location which enabled the program to become established in the community; and developing partnerships with key community members. A potential barrier of the program expressed by parents is the time commitment to attend twice weekly for 20 weeks.Further, this process evaluation has provided important information which is necessary to program planning. For example, knowing that the children who did not complete Go4Fun were more likely to be boys, to be Aboriginal, and to come from lower quintiles of social advantage feeds into continued program improvement and development. That families from socially disadvantaged backgrounds were less likely to complete the program is congruent with the literature which shows that there is a clear social gradient in health and health literacy , but this information will inform program managers of the need to adapt Go4Fun® so that it resonates better with boys and socially disadvantaged families. Next steps are to conduct qualitative research through focus groups and individual interviews to understand why these population groups are less likely to complete the program.Although the overall feedback from parents and children about the program was very positive, the twice a week time commitment was a consistent issue across all programs. In response, the Office of Preventative Health is currently piloting a randomised control trial examining the efficacy of using a once a week delivery mode for Go4Fun.The importance of translational research for progressing public health and translating evidence from efficacy trials into practice has been emphasized . IncumbeThe potential of community-based population-wide programs such as the Go4Fun® can only be realised when it is being used by a substantial proportion of the target audience. While a substantial number of overweight/obese children have participated in the Go4Fun® program, this represents an uptake of about 1.6% of overweight/obese children in NSW. There are opportunities to reach more families with overweight/obese children. The delivery of the program continues as part of the NSW Ministry of Health’s Healthy Children Initiative program of work. The Go4Fun® program could benefit from greater dissemination of program outcomes to health professionals and strengthening of clinical referral pathways to encourage secondary referrals; programs to increase parental awareness of unhealthy weight in childhood and; increased investment in state-wide advertising and marketing to promote awareness of the program and encourage self-referral to increase program reach. Next steps are to undertake the outcome evaluation of Go4Fun® and to assess the impact of different doses of attendance on program outcomes.The up-scaled and broadly delivered NSW Go4Fun® program achieved success in terms of reaching almost 2,500 overweight/obese children from socially disadvantaged backgrounds who potentially would not have accessed primary care or tertiary obesity treatment services. To date the program only reached a small proportion of overweight/obese children in the NSW population, highlighting the opportunity to increase efforts toward recruitment to the program and investigate modified program delivery to increase the program to retain socially disadvantaged families. Evaluation of the NSW Go4Fun® program both in terms of reach and effectiveness has national and international relevance, but more importantly has policy implications for the future roll out of the program, which in turn will impact on the effective expenditure of public funds.ARIA+: Accessibility-remoteness index of Australia plus; BMI: Body mass index; CI: Confidence intervals; LHDs: Local health districts; MEND: Mind exercise nutrition do it; NSW: New South Wales; SE: Standard error; SEIFA: Australian bureau of statistics’ socio-economic indexes for areas (SEIFA); SES: Socioeconomic status; UK: United Kingdom.The authors declare that they have no competing interests.DW and CIH designed the study. BN, BOH, LLH and AB conceptualized and designed the study evaluation. BN performed the statistical analyses. All authors participated in drafting the manuscript and revising it for important intellectual content. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/14/140/prepub


Encouraging family forest owners to create early successional habitat is a high priority for wildlife conservation agencies in the northeastern USA, where most forest land is privately owned. Many studies have linked regional declines in wildlife populations to the loss of early successional habitat. The government provides financial incentives to create early successional habitat, but the number of family forest owners who actively manage their forests remains low. Several studies have analyzed participation of family forest owners in federal forestry programs, but no study to date has focused specifically on creation of wildlife habitat. The objective of our study was to analyze the experience of a group of wildlife-oriented family forest owners who were trained to create early successional habitat. This type of family forest owners represents a small portion of the total population of family forest owners, but we believe they can play an important role in creating wildlife habitat, so it is important to understand how outreach programs can best reach them. The respondents shared some characteristics but differed in terms of forest holdings, forestry experience and interest in earning forestry income. Despite their strong interest in wildlife, awareness about the importance of early successional habitat was low. Financial support from the federal government appeared to be important in motivating respondents to follow up after the training with activities on their own properties: 84% of respondents who had implemented activities received federal financial support and 47% would not have implemented the activities without financial assistance. In order to mobilize greater numbers of wildlife-oriented family forest owners to create early successional habitat we recommend focusing outreach efforts on increasing awareness about the importance of early successional habitat and the availability of technical and financial assistance. Encouraging family forest owners (FFO) to manage their forests to create early successional habitat (ESH) is a high wildlife conservation priority in the northeastern United States Several studies have analyzed participation of FFOs in NRCS forestry programs. None of these studies focused specifically on ESH, but some of the findings are relevant. For example, most participants in federal forestry programs were more concerned about “doing the right thing” than maximizing profits, but the financial incentives provided by the programs increased the number of acres treated The objective of our study was to analyze the experience of a group of FFOs who had a strong interest in wildlife and who had been trained to create ESH on their own properties. We conducted the study in RI, one of three states in the Southern New England region of the United States. RI is experiencing ongoing loss of ESH In the current study we surveyed FFOs who participated in the RI Coverts Program, which has trained FFOs to create wildlife habitat on their own properties since 2008. These FFOs represent a small portion of the total population of FFOs, but we believe they can play an important role in creating wildlife habitat, and that it is important to understand how outreach programs can best reach them. Our study addressed three research questions. (a) What characteristics were shared by this group of FFOs ? (b) How important was technical and financial assistance in motivating these FFOs to create ESH? (c) What other factors made some of these FFOs more likely than others to follow up after the training with activities on their own land? To our knowledge, this was the first study that specifically examined the experience of FFOs in creating ESH. Even though our study had a small sample size and was limited to a subset of FFOs with a strong interest in wildlife conservation, our hope is that the results of the study can be used to strengthen forestry outreach programs in the region and encourage greater numbers of wildlife-oriented FFOs to create ESH on their own properties.The University of Rhode Island (URI) Institutional Review Board (IRB) reviews research projects conducted at URI that involve human subjects to ensure that two broad standards are upheld: first, that subjects are not placed at undue risk; second, that they give uncoerced, informed consent to their participation. The application for the current study (Project Title: (239239-2) Study of forestry activities by private landowners in Rhode Island), was approved by the IRB on 6 September 2011. As per the approved IRB proposal, we requested the participants to formally consent to participating in the study. This was done by either checking a box on an on-line survey, or by consenting in writing if they decided to submit a paper version of the survey rather than the on-line version. We retained the records for all participants.We surveyed all FFOs who attended the three day RI Coverts Program from 2008 to 2012. A total of 79 persons representing 54 households attended from 2008 to 2012. We sent one survey per household, and did not include program participants who were not FFOs, such as representatives of conservation agencies. Before distributing the 54 surveys, we announced the study in a newsletter to FFOs distributed by NRCS. We then contacted the program participants by email or regular mail, using the contact information they provided when they registered for the program. We gave the respondents the option of filling out paper or online versions of the survey. The survey included 41 questions that were a mix of multiple-choice questions to generate quantitative data as well as open ended questions to generate qualitative data. The survey included three components: (a) general demographics and interest in forestry and wildlife, (b) experience with forestry activities before attending the program, and (c) experience with forestry activities after the training. In the summer of 2011 we surveyed all FFOs who participated in the program between 2008 and 2011. In the summer of 2012 we distributed a shorter version of the survey to the participants of the 2012 program - this version included the first two components of the original survey, but not the section on experience with activities after the training.2LX) when our data met the requirements for expected frequencies.Our small sample size (N = 34) limited the options for statistical analysis, but we used SPSS v. 20 to calculate the Likelihood Ratio Statistic  = 7.197, n = 34, p<0.01) and to have sold timber or firewood before attending the Coverts Workshop (2LX(1) = 6.983, n = 34, p<0.01). However, there were no significant differences in their likelihood of having engaged in forest management activities before attending the Coverts Program  or having followed up after attending the Coverts Program with forest management activities on their own land (2LX(1) = 0.125, 2LX(1) = 0.125, (2LX(1) = 0.118, 2LX (1) = 0.000 respectively; p>0.5 in all cases).Fifty percent of our respondents owned more than 20 ha of forest . The resBefore attending the Coverts Program, 71% of the respondents had already received some forestry advice from friends or relatives and 62% had hired a professional forester. Sixty nine percent were involved in forest certification programs such as the Rhode Island Tree Farm Program, 31% were involved in forest easement programs, and 62% had started to prepare a forest management plan. In addition to managing their own forests, 60% were involved in planning forest management activities on land that did not belong to them, such as land owned by friends, relatives or conservation organizations.All of our respondents expressed a strong interest in wildlife, whether observing wildlife, hunting, or identifying species of wild plants and animals. Sixty eight percent of the respondents who had implemented activities since attending the program could describe some positive impact of their forest management activities, including increased abundance of birds, mammals, amphibians and insects. Awareness about the importance of ESH for wildlife was low: several respondents commented that before attending the program they did not realize that many wildlife species depend on ESH or that this habitat type was declining in New England. Several commented that they viewed clearcutting negatively before visiting other FFOs who had already created ESH. Thirty three percent of the respondents who implemented activities after attending the program said they probably would not have implemented the forestry activities if they had not attended the program.Eighty three percent of our respondents who had attended training at least six months before the survey had already followed up with forest management activities on their own land, and all of the other respondents were planning to implement activities. The respondents implemented a range of forest management activities after attending the Coverts Program . The mosOur respondents varied in terms of interest in earning income from their forests: 12% were not at all interested in earning forest income, while 24% were slightly interested and 65% were at least moderately interested. However, the financial and technical assistance offered by NRCS programs appeared to be an important motivating factor for our respondents. Eighty four per cent of our respondents who had implemented activities after attending the RI Coverts Program had received support from NRCS, and 47% said they would probably not have implemented the activities without the financial assistance. Several respondents mentioned that the process of obtaining NRCS support was complex and time consuming, and/or that they were not aware about these financial assistance programs before attending the Coverts Program.Our findings demonstrate that this subset of FFOs shared some common attributes such as education level and interest in wildlife, but differed in terms of land holdings, prior experience with implementing forestry activities, and interest in earning income from their forests.The FFOs in our study tended to have large forest holdings: half owned more than 20 ha of forest, whereas the National Woodland Owner Survey (NWOS) conducted by the US Forest Service reported that only 3% of FFOs in Southern New England and 0% in RI owned more than 20 ha of forest The strong interest in wildlife expressed by the respondents was not surprising since they had volunteered to participate in a three day training on wildlife issues. More unexpected was their limited understanding about the importance of ESH prior to the training. Several respondents commented that before attending the program they were unaware that many wildlife species depend on ESH and that this habitat type is declining in New England. This applied to several respondents who had already prepared forest management plans before attending the training, which suggests that the consulting forester did not stress the importance of creating ESH during the process of plan preparation. Several respondents commented that they felt negatively about forest clearcutting before attending the Coverts Program, an attitude that is common in other Southern New England states Our respondents appeared to be comfortable networking with other FFOs and receiving advice about forest management. Even before attending the Coverts program, most had received forestry advice from friends, relatives or professional foresters, and many had participated in forest certification programs. Other studies have reported that land owners who receive advice about their forests from professionals or friends are more likely to participate in forestry support programs The main factor affecting how quickly the respondents implemented forest management activities after attending the training appeared to be whether or not the respondent had prepared a forest management plan, a process which can take up to a year. Eighty per cent of the respondents with management plans had implemented additional forest management activities after the training, whereas none without a forest management plan had implemented any activity after the training except for preparing a management plan. As such, we endorse the current NRCS program of providing technical and financial support to encourage FFOs to prepare forest management plans, an important first step in creating wildlife habitat.Our respondents varied in terms of interest in earning income from their forests, but the financial and technical assistance offered by NRCS programs appeared to be an important motivating factor for them. Most respondents who had implemented activities after attending the RI Coverts Program received financial assistance from NRCS and would probably not have implemented the activities without this assistance. We did not see any indications that landowners who are less interested in earning income from their forests were less interested in the financial incentives, as was reported in North Carolina However, several respondents commented that they did not know about the NRCS financial assistance programs before attending the Coverts Training. Other respondents mentioned that the process of obtaining NRCS support was complex and time consuming, which is a common complaint of landowners about federal financial assistance programs Our study examined the characteristics of FFOs who have a strong interest in wildlife. We found that this subset of FFOs share some characteristics such as education level, but differ in terms of land holdings and interest in earning income from their forests. Most had prior experience with implementing forestry activities, but 26% were totally unengaged in forest management before attending the program. A key finding of our study was that despite their strong interest in wildlife, many respondents had not been aware before attending the Coverts Program of either the importance of ESH or the availability of financial support currently available from NRCS. However, these FFOs were willing to implement forest management activities on their own land once they were provided with adequate training and financial incentives. These FFOs represent a small portion of the total population of FFOs, but we believe they can play an important role in creating wildlife habitat, and we recommend the expansion of outreach programs such as the Coverts Program. The sample size of our study was smaller than we would have liked, and we recommend conducting a comparable study with a larger sample size to confirm our findings. Meanwhile we offer these preliminary recommendations for an outreach strategy to motivate greater numbers of wildlife-oriented FFOs to engage in forest management: (a) use existing networks of wildlife-oriented FFOs such as conservation organizations and land trusts to identify FFOs who are not yet engaged in forest management but who can potentially be motivated to create ESH on their own properties; (b) focus the content of outreach efforts on the importance of creating ESH and the availability of NRCS technical and financial assistance; (c) encourage peer-to-peer learning by providing more opportunities for FFOs to visit FFOs who have already created ESH on their own land; (d) develop simpler descriptions of NRCS forestry programs and application procedures to make them more easily understood by FFOs; (e) encourage FFOs with smaller forest holdings (4 ha or less) to manage their forests: these FFOs can make a valuable contribution by creating small patches of ESH which provide critical habitat for many species of shrubland birds


Fidelity of the humoral immune response requires that quiescent B lymphocytes display membrane bound immunoglobulin M (IgM) on B lymphocytes surface as part of the B cell receptor, whose function is to recognize an antigen. At the same time B lymphocytes should not secrete IgM until recognition of the antigen has occurred. The heavy chains of the secretory IgM have a C-terminal tail with a cysteine instead of a membrane anchor, which serves to covalently link the IgM subunits by disulfide bonds to form “pentamers” or “hexamers.” By virtue of the same cysteine, unassembled secretory IgM subunits are recognized and retained (via mixed disulfide bonds) by members of the protein disulfide isomerase family, in particular ERp44. This so-called “thiol-mediated retention” bars assembly intermediates from prematurely leaving the cell and thereby exerts quality control on the humoral immune response. In this essay we discuss recent findings on how ERp44 governs such assembly control in a pH-dependent manner, shuttling between the cisGolgi and endoplasmic reticulum, and finally on how pERp1/MZB1, possibly as a co-chaperone of GRP94, may help to overrule the thiol-mediated retention in the activated B cell to give way to antibody secretion. In the arms race between pathogens and hosts, vertebrates developed a long-range weapon of high precision: the antibody. Antibodies are produced by activated B lymphocytes. Once they are secreted, the antibodies spread throughout the organism to recognize and bind to specific epitopes on pathogens. As such, the pathogens are stigmatized as a target for complement or phagocyte mediated attack and  elimination. All weaponry is hazardous, because of the risk that it may “go off” at the wrong moment and aim at the wrong target. Thus, it comes as no surprise that the immune system is reined in by a wealth of safety measures . Safety dictates that B lymphocytes should not secrete antibodies, unless there is a confirmed sighting of an antigen, that is, when there is a good match between the antigen and the particular antibody which the B lymphocyte happens to express on its surface. At that moment, the B lymphocyte commits to become a plasma cell, which arguably is one of the most prolific secretory cells in the metazoan kingdom as it releases in bulk the antibody that specifically targets that very antigen. How can it be that the same cell that first obstinately prevents antibodies from leaking out makes a volte-face in a matter of a few days and starts to spit out antibodies by the millions? Here we discuss recent findings that shed new light on this fascinating question.μ heavy chains (HC) that are covalently linked by a disulfide bond. Each HC is also covalently linked by a disulfide bond to a light chain (LC). Both the HC and the LC are composed of domains that all adopt a so-called Ig-fold, and each of these domains is stabilized by an intradomain disulfide bond. The HCs have one variable domain (VH) at the N terminus and four constant domains (referred to as CH1–CH4 from N to C terminus), while the LCs have next to their N terminal variable domain (VL) a single constant domain (CL). The CL is juxtaposed to the CH1 and the VL to the VH at the tips of the BCR  of Igs, with each B lymphocyte displaying only one particular variant that matches a single particular antigenic epitope. The tetrameric (H2L2) IgM that forms the core of the BCR is anchored to the plasma membrane via transmembrane domains at the C-terminal part of the Ig-μm HCs downstream of CH4 [To ensure that the antibodies that are secreted recognize the exact same antigen as was originally sighted by or presented to the B lymphocyte, the B cell receptor (BCR) and the antibody are in fact two manifestations of the same molecular device. The core of the BCR is formed by two Ig- the BCR . Togethe2L2 units  with one cysteine that is key both for preventing premature secretion and for the assembly of mature secretory IgM (see below). Once it commits to the plasma cell stage, the TP cysteine is used, in fact, to covalently link H2L2 “monomeric” units in polymeric secretory IgM by disulfide bonds [2L2)6 or as “pentamers” (H2L2)5J, in which a third antibody component, the J-chain, joins the “monomeric” units, again by disulfide bonds [Secretory IgM consists of nearly identical HL2 units . The LCsde bonds . Altogetde bonds   and the Igs are no exception. The nascent polypeptides obtain glycans and disulfide bonds in the ER as they fold with assistance of the array of ER resident chaperones and oxidoreductases . In factH1 domain of the HC and facilitates its retention in the early secretory pathway, until LCs manage to displace BiP, and the heteromeric complex is “approved of” by the proximal ER quality control systems [Except for the lectins calnexin and ERGIC-53, the chaperones listed above are all soluble proteins that display a tetrapeptide KDEL (or KDEL-like) sequence at their C terminus. In case any of these chaperones either alone or in complex with a folding intermediate leaves from the folding promoting environment of the ER to stray along the secretory pathway, they will be captured by the KDEL receptors in the ERGIC or cisGolgi. From there the KDEL receptors with their cargo are shuttled back to the ER for further folding attempts , 17. Thi systems , 19.2L2 “monomeric” units oligomerize into mature secretory IgM. The task at hand is twofold; on the one hand polymerization must be promoted and polymers must be let go of by the ER quality control mechanisms; on the other hand unassembled “monomers” must be barred from traveling further along the secretory pathway and retrieved for further polymerization attempts  cysteines. In that case, the clients may displace one of the ERp44 molecules forming a mixed disulfide with the other. The free ERp44 then can team up with another free ERp44, whereupon formation of the disulfide that links the ERp44 homodimer may be catalyzed by PDI, Ero1α, or the like. Indeed, a substantial fraction of ERp44 is present in the cell as a disulfide linked dimer [The pH regulation of ERp44 may explain how the assembly process of IgM is patrolled by virtue of missing (disulfide) links. Yet, despite the elucidation of this elegant assembly control, there are several missing links in our understanding of how B cells finally commit to IgM secretion. For one, little is known about the mechanisms that promote the incorporation of J chains into polymers that as a result become “pentamers” and how the plasma cell balances its “hexamer” and “pentamer” output . Then, s partner , provide partner , 37. Anoμs HC but secretes little if any antibody . Herpesviridae are notorious for their misleading of the immune system. Upholding herpes family values, an estimated one-third of the KSHV genome indeed is dedicated to immune evasion , and as 6 bp deletion in chromosome 22 at the q11.2 location. This 22q11.2 deletion syndrome is a relatively prevalent (1 in 2000–4000 live births) genetic disorder that can lead to a wide range of symptoms including congenital heart disease, renal and/or skeletal abnormalities, and learning difficulties, owing to the haploidy of the ~30–40 genes in the affected region [As opposed to KSHV driven shipwrecking of pERp1/MZB1, another pathologic condition involves uncontrolled pERp1/MZB1 expression. MicroRNAs (miRs) contribute to the regulation of gene expression through annealing with target mRNAs, causing their degradation or translational inhibition. A recent report now implicates pERp1/MZB1 as the most prominent target of miR-185 , while id region . Importad region  that mayAll these findings indeed point at pERp1/MZB1 as key for IgM assembly. Yet, how pERp1/MZB1 would assist IgM polymerization remains unclear. With the idea in mind that thiol-mediated retention should be counteracted, it was explored whether pERp1/MZB1 could fulfill a role as thiolreductase, but evidence for such activity was meager  to nonexAfter evaluating sequence homologies anew, we now report that pERp1 is part of the saposin-like protein family with, as its closest relatives the canopy homolog proteins CNPY1-4; an alignment is shown in Mammalian CNPY1 has not been studied but in zebrafish it associates with the fibroblast growth factor (FGF) receptor. This activity is essential for proper FGF signaling in the developing brain of the fish . CNPY2 wSeveral cochaperones of the cytosolic chaperone HSP90 have been identified , but forIn all Ig secreting vertebrates (from cartilaginous fish onwards) the first wave of the antibody response appears to involve polymeric IgM, whether “tetrameric” (in teleost fish), “pentameric”, or “hexameric” . Polymer


Immunosuppression in breast cancer has been reported in women and in the highly metastatic mouse mammary tumor model 4 T1. The immunosuppressive environment complicates the use of the humoral response against the tumor as an immunodiagnostic tool. IgM has not been used in immunodiagnostic in part because its antitumor responses, both innate and adaptive, have not been studied in function of time in breast cancer.We show a new approach to analyzing the mouse humoral immune response, and compare the evolution with time of IgG and IgM responses against the antigens of 4 T1 cells.The study is based on 2-dimensional immunoblotting detection of antigens from 4 T1 cells by the IgG and IgM antibodies in the serum of female mice injected with 4 T1 cells.There was a high variability in the intra-and inter-mouse response. Variability in the IgM response was manifested as a pattern of spots that could become a multibinomial variable of 0 and 1, which could represent a signature of the immune response. Different numbers of spots was found in the IgG and IgM responses from week 1 to 5. On average, the IgM had more but the IgG response decrease with the time. The natural IgM at t = 0 responds stronger than w1; the adaptive response of both IgM and IgG were elicited where, with the former being stronger better than the latter. Antigens that are recognized by some female mice in the first week are also recognized by other female mice at time 0. Contamination of the natural IgM makes difficult use the adaptive IgM as a tool for immunodiagnostic.IgM and IgG response varied with the time and individuals. Spot variation in 2D pattern for the natural IgM could be expressed as a binomial signature, which opens up the way to correlate a particular pattern with resistance or susceptibility. This uncovers a battery of IgMs for each individual to confront cancer or infections. The possibility to differentiate between adaptive IgM antibodies from the natural IgM will allow investigation of the adaptive IgM for early immunodiagnosis. During a lifetime, human beings are permanently exposed to the development of transformed cells, which arise spontaneously or by contact with trigger factors. Immune surveillance of cancer leads to the generation of anti-tumor antibodies from the early stages of the disease. Once a The images in Figure + cells in the apparent absence of antigen stimulation, which constitutes most of the circulating IgM-1-propanesulfonate; SDS: Sodium dodecyl sulfate; HCl: Hydrochloric acid; KCl: Potassium chloride; NaOH: Sodium hydroxide; ∑: Summation; t = 0: Time 0.The authors declare that they have no competing interests.MDZ carried out aspects of the mouse experiments and 2D Immunoblots, reviewed the manuscript its scientific veracity. RH participated in the execution of the mouse studies and participated in all experiments. POS conceived, designed and analyzed the collective results of all the experiments, and wrote the manuscript. All the authors read and approved the final manuscript.MDZ has a Doctoral fellowship from the Consejo Nacional de Ciencia yTecnología, México. RH is a Master of Science and has an academic position at the Instituto de Investigaciones Biomédicas. POS is a PhD in Biochemistry and is a Researcher at the Instituto de Investigaciones Biomédicas. All work at the Universidad Nacional Autónoma de México.


In 2001 CAD was developed and implemented in Stockholm and was disseminated to 20 other municipalities in Sweden up until 2010. This study investigates the factors related to the implementation and compares Stockholm to the rest of the municipalities.A sequential exploratory method was used which consisted of three steps including a questionnaire and two rounds of interviews. Questionnaires included all communities and the interviews included twelve respondents from three example municipalities in the first phase and four respondents from Stockholm in the second phase. The interviews were analyzed using content analysis.In Stockholm the program was described as having been implemented and sustained over time. The implementation in the example municipalities was perceived as difficult with many obstacles and achieving sustainability was also described as difficult. Two of three municipalities were not active at the time of this study, illustrating that the program only lasted a few years. Factors identified as being related to implementation outcomes were need assessments, participation, support, collaboration and local enthusiasts.The capacity to implement and sustain CAD differed substantially between Stockholm and the other municipalities. If the prerequisites and capacity are not sufficient the implementation is not likely to be successful, even when the activities are promoted on a national level like CAD. The needs of the interventions and the capacity to implement the program should be examined before adopting the program. This was not done, probably because the drive to spread the activity was not sufficiently questioned. This study investigated implementation of the “Club Against Drugs” (CAD) program  in 20 muMany issues and problems arise with the implementation of community-based drug prevention programs because they are complex and do not fall within the framework of one single organization’s boundaries. Instead they require the efforts of many different organisations. Collaboration has thus become an essential element in the success of community-based interventions as well as the reliance on community coalitions to solve complex problems . CollaboThe CAD-program was developed in 2001 by researchers in Stockholm and focused on reducing the availability and use of illegal drugs in the nightclub environment. The program was developed, implemented and managed by STAD (Stockholm prevents Alcohol and Drug problems), a research and development unit within the Stockholm County Council and the Karolinska Institutet. Initially, a study was performed to assess the need for a community-based drug intervention and to gain ideas from community stakeholders on how such a program should be designed. The results indicated that staff at licensed premises had a higher prevalence of last year drug use than the general Swedish population and that observed drug use among guests was high . The procollaboration, education and inspection.In 2002 the Swedish authorities responsible for drug prevention decided to disseminate CAD to other municipalities in Sweden. As a result Gothenburg and Malmö, the second and third largest cities in Sweden, adopted the program and by 2010 a total of 20 communities besides Stockholm had initiated the program. The program was to some extent changed from how it had originally been developed and implemented in Stockholm; e.g. the required length of the education for bar staff was reduced from 2 days to half a day. The core components of the program were For the municipalities and clubs that initiated the program there were a number of demands on their performance and level of activity. Mandatory components for the municipalities included political anchoring, formation of a steering group, conducting a survey examining the situation in clubs and offering education in collaboration with the police. Recommended components included assigning a local coordinator, performing a study using actors (like the one in Stockholm) and offering policy education to club managers. Mandatory components for the clubs were making environmental rounds with a representative from the municipalities’ steering group, working actively with policy, cooperating with the police and educating the staff or at least the key persons. For more details see the CAD website krogarmotknark.se. There were no formal demands for the police. Some funding was available from the Swedish National Institute of Public Health and the additional funding was taken from the general operating budgets of the stakeholders. A national steering group was set up and was responsible for managing the contact with the communities, updating the website and arranging national conferences. More municipalities wished to join but had to wait for the results from an evaluation. As this was taking longer than planned more municipalities were allowed to start working with the program from 2011 and forward.This study investigates the implementation of CAD in the municipalities to which it has been disseminated in contrast to the findings from Stockholm. We wanted to identify what central factors facilitate and hinder implementation and sustainability of a comprehensive prevention program involving multiple stakeholders. We relate these factors to the concept of community capacity and argue that without sufficient capacity one should question the initiation of an implementation.The “Clubs against drugs” program was developed in Stockholm and had in 2010 been disseminated to 20 other municipalities in Sweden. A sequential explanatory mixed method  was usedInterviews were conducted with key informants that included public officials, politicians, police, club owners and doormen from each of the three selected municipalities. Unfortunately, doormen were not interviewed as none were willing to participate. The informants were selected with help from the local CAD coordinators. Twelve people, four from each municipality, were interviewed during the summer and autumn of 2012.Based on the content analysis of these twelve interviews, we wanted to compare the results to those of Stockholm. To do this we interviewed participants from the Stockholm CAD program and asked questions particularly related to implementation factors. The four key informants were the researcher who had been a part of the team at STAD that developed and implemented the initial work, the current coordinator, a police officer who had been involved in the initiation of CAD and a club owner who had also been involved from the start of the program. These interviews were conducted spring 2013. The interviews were semi-structured and conducted by a researcher with a public health background. The first interviews were performed face-to-face and the location was chosen by the respondent and in all cases but one it was held at the respondent’s work place. The Stockholm interviews were performed over the phone. The key questions included how the CAD program was initiated and how the implementation process had progressed, what factors had enabled the process and had there been any barriers, how the collaboration had functioned and what they thought about the future and the sustainability of CAD. The questions asked in the interviews were open ended and gave the respondents space to formulate their answers quite freely. The interview respondents were informed that they would be anonymous and that they could refuse to answer questions or terminate the interview at any point. They were further informed that the interviews would be recorded and were given the opportunity to decline to this.The interviews were transcribed and analyzed using content analysis techniques . The matWe did not require ethical approval for this study because it did not handle sensitive information about individuals  about ethical approval of research that pertains to humans).A total of 55 surveys were returned, out of 101 distributed, giving a response rate of 54%. Fourteen surveys were excluded because the respondents were not involved in CAD during 2008-2010, thus a total of 41 surveys were included in the analysis. Survey respondents were directly involved in the implementation of CAD and included public officials, politicians, club owners, bar staff, doormen and police officers. Response rate was highest among coordinators and roughly equal for politicians, police and club owners. Response rate was lowest among bar staff and doormen.The majority of respondents stated to have strengthened relations and knowledge and increased their collaboration through CAD participants; a smaller part also obtained a better knowledge of methods and increased understanding of other’s work role Table .Perceived support differed between the police officers and the rest of the respondents Table . RoughlyOf the police officers, bar staff and club owners 87% had participated in a CAD educational training and 86% felt that this training had been sufficient for them to feel secure in their roles. All the club owners answering the questionnaire (n = 6) stated that they had developed a drug policy for their club during CAD and four of these perceived the support for this had been adequate. Out of all stakeholders excluding the police 81% answered that the collaboration with the police had been improved. Of the public officials 58% stated that they had completed a survey measuring the negative effects of narcotics. 83% of the respondents answered yes to the question of whether they would participate in CAD if asked today.The categories and themes from the interviews conducted in the three example municipalities are shown in Table The respondents were very interested in working with drug prevention and believed that the “Clubs against drugs” program was an appropriate way to deal with the problem of drug use in the nightclub environment.We were asked by the municipality if we were interested in working with this. Of course we wanted to. It says in the law that this is something that you shouldn’t do, drugs and so on. For the club there was not any doubt, it’s a highly current issue. Club ownerThe support from local politicians and employers was emphasized as an important factor for implementing CAD. The perceived support differed between individuals and municipalities and many perceived this as lacking.Interest exists sporadically, but when the employers do not have this interest and it comes as a directive that “This is a problem and we shall do something about it”, it does not get done. Sorry. Police officerThe respondents, in both interviews and questionnaires, expressed that sufficient resources were necessary to complete program implementation. Resources coming from external sources were perceived as being sufficient but many respondents described the police resources as insufficient.The activities completed in the municipalities varied. In the three example municipalities the local coordinator for CAD initiated the use of the program. In all municipalities a steering group comprised of key personnel was created to support and lead the local work. At the time of the interviews club environment rounds had partly been done in two of the example municipalities and in the third this work had not yet been initiated. In two of the example municipalities the work with CAD had stopped almost completely and they did not, at the time of study, have any activities running.Collaboration was, for all of the interviewees, viewed as a key aspect of local and national work with CAD. Failed collaboration was perceived as a leading cause in the communities where the work had not gone as planned and successful collaboration was described as an essential factor in the community where the work was continued.The club owners and the police officers thought it was important to have a closer relationship with each other. Police officers described the relationship as positive due to the large number of criminal persons that frequently visited these kinds of venues and cited the advantage of becoming more familiar with the club area during the daytime hours.You work with honesty and it is not my fault if someone comes into the club with drugs but rather something we should stop together. So the collaboration was absolutely the best and working with this was best if you viewed it locally, for us. Club ownerThe other stakeholders considered the police to be the weakest link.If we look at this collaboration as resting on three legs – police, club owners and municipalities, the weakest leg in this triad is the police. Public officialThe reasons mentioned for the difficulties collaborating with the police were related to opinions of organizational weaknesses within the police resulting from many reorganizations and also transfers and lack of interest from the police authorities regarding preventive work. This was also the opinion expressed by the interviewed police officers. One of them said that their work in large was determined by acute actions rather than a long term prevention perspective.The police were described as an important part of the CAD work and the other stakeholders did not think it was possible to implement the program without their active participation. If one or a few of the club owners were not interested the work could still continue. According to the respondents the municipalities were important but their role was not as critical compared to that of the police.It doesn’t help if we identify that someone is under influence of drugs if there are no police that can handle it. It’s not the job of the doormen; they have some authority but cannot act as a police. That is what the police are supposed to do, but the police don’t exist. Club ownerAccording to the respondents the work in many municipalities depended on a few dedicated individuals who were pushing the work forward.As long as there is not a developed organization dedicated individuals are needed to keep it going.Police officerThe CAD program was described as a long-term commitment and in need of a plan for sustainability. The respondents talked about lessons they had learned from CAD. One police officer thought that the police had been too unrealistic concerning what they would be able to contribute with in the program and not been able to live up to their promise.One can be wise in hindsight and say that we should have obtained the resources that were needed. Police officerFormal agreements were suggested as a possible solution to this.The categories and themes from the interviews conducted in Stockholm are presented in Table A needs assessment was conducted in Stockholm to determine whether there was a problem with drugs in the nightclub environment and the severity of drug use. This was emphasized as an essential prerequisite for CAD. The study was used to formulate mutual goals and to gain support from managers and politicians. This support was perceived as substantial.Mutual analysis or evaluation, that’s where it starts. Police officerThe participation of the stakeholders was described as necessary and important; the coordinator who started CAD wanted the stakeholders to feel that it was their project.It has been an important part all the time, participation. Former CoordinatorThe coordinator described their work as extensive and exceeding their full-time employment during the first years of CAD. All respondents stressed the importance of coordination, especially when working with multiple stakeholders.Collaboration was described as necessary and well-functioning but the hard work in forming the groundwork to initiate the program was also highlighted. The former coordinator emphasized the importance of their previously positive collaboration with the municipality and the police through STAD and their efforts to reduce alcohol-related problems in the nightclub setting. The difficulties concerning collaboration were described as related to the nature of the stakeholder’s occupations. The club owners were described primarily as businessmen for whom drug use prevention was not the highest priority. Furthermore they were viewed to be in a dependant relationship to the authorities in order to keep their licence to sell alcohol. To develop trust between the authorities and the nightclub industry was described as hard work but also successful overall.There are very competent police officers, very competent public officials and also competent club owners, but it is about keeping this gang together. It’s a quite motley crew. Club ownerCAD had been sustained in Stockholm and the current coordinator described the program as being a natural part of community drug preventive work. To achieve sustainability the importance of formal agreements was highlighted which also assured that CAD remained a high priority. Keeping up interest was emphasised as a challenge for sustaining the work.I believe in agreements and on being clear about what the police commit to doing. Police officerInterest and enthusiasm for CAD was substantial in the communities to which it had been disseminated but at the same time there had been considerable difficulties during the implementation process. There was a discrepancy between the results of the questionnaire and the first interviews. The enthusiasm and contentment expressed in the questionnaires did not, when put in the light of the interviews, correspond with an overall success in implementation. It rather reflected contentment with smaller parts of the program or with the thought of what could be rather than what actually was. Compliance within the program was lacking, the police had had difficulties with their commitments and the work with CAD had ceased almost entirely in two of the three example municipalities. The results differed from the experiences in Stockholm where the work was perceived overall as being successful and sustained. Factors highlighted as important were support, participation and the assessment of needs, among other things. The results are consistent with prior research demonstrating that implementation is complex and that successful implementation requires considerable effort . The capDuring the 1990s Stockholm experienced a significant rise in problems associated with drugs and criminal activity in the nightclub environment and an investigation was launched to determine the magnitude of illicit drug use in the nightlife setting ,7.The development of CAD in Stockholm engaged a broad range of stakeholders in the overall process and participants took part in creating the program based on their specific needs and expertise. This stakeholder community empowerment was lacking in the other municipalities to which CAD was disseminated, as they received a fixed program to implement with small possibility for local variations or adaptation.When the program was developed in Stockholm groundwork had been done for years before, this through collaboration with the clubs on Responsible beverage service (RBS). The organizational prerequisites were in place to facilitate the development and implementation of CAD, this mainly through STAD but also within the police organization. In Stockholm the program had a full-time coordinator who also served as the lead investigator that researched the development, implementation and outcomes of CAD. In the other municipalities the coordinator was a public official that assumed extra duties associated with the implementation of CAD. The coordinator in Stockholm highlighted their full-time focus on CAD as being necessary for success of the project. Considerable time and effort was required by the coordinator to raise interest among various stakeholders, to facilitate stakeholder collaboration and to manage media messages and policy work. In Stockholm there were also additional researchers involved that had a great interest in seeing the success of this project. The external knowledge supporting the other municipalities consisted of the national coordinator who worked 50% with this assignment. Despite the knowledge gained from CAD in Stockholm and the success of the program, technical assistance (TA) was not used in the dissemination of CAD to the rest of the country. The program was designed for use in Stockholm and the developers had no intentions of spreading the program, neither did they partake in the dissemination. Future implementation of similar programs could benefit from the use of TA as this enhances the chances of successful implementation. Currently the CAD program has an annual meeting for participants and this provides the opportunity to make new contacts and share experiences in working with drug problems at the nightclubs. This can, in hindsight, provide a type of technical assistance but more specific efforts to help the dissemination were needed early on.The requirements and investments of the stakeholders who joined CAD varied. The police was the stakeholder that had to make the biggest commitment and organizational change to manage their part, which included more and closer contact with the clubs and an overall shift in priority with more personnel working towards the clubs. The inability by the police to meet the requirements was, in the municipalities to which CAD had been disseminated, the main reason for why the implementation did not go as desired. Reasons for this inability were said to be mainly scarce resources, reorganizations and lack of interest. In Stockholm the police were engaged in the project from the beginning, they already had efforts towards clubs and saw the potential gains of extending those efforts. The respondents in Stockholm expressed that police involvement was a major reason for positive outcomes of CAD and that they were an indispensable part in the program. Respondents also spoke about the importance of prioritizing drug preventive efforts within the community and the need to have resources specifically allocated to the police for their participation in community-based drug prevention efforts; this may not be possible in smaller communities with limited resources. Overall the stakeholders regarded the police as playing a pivotal role in the CAD program and without their presence found little meaning or incentive in following through with their commitments. In municipalities where the police state that they do not have the capacity to allocate resources for their active participation in CAD or where the support and legitimacy within their organization is lacking, initiation of the program should be questioned. As Stockholm is the largest city in Sweden the possibilities to allocate resources are much greater compared to smaller municipalities. Communities with small populations can have special challenges when it comes to implementing, for example with resource allocation . Broad pOne central aspect that differed between Stockholm and the majority of the other municipalities was the support from politicians and persons in leading positions. In Stockholm this was a major supporting factor for the implementation. Support was lacking or at least not as extensive in other municipalities. The police had significant problems due to lack of support. Lack of support was described as the main reason for implementation failure. Support from leaders is an essential part in the implementation process and for sustainability and institutionalization of interventions ,8,20. ItIt was evident in the study that collaboration could affect the implementation in both a positive and a negative direction and this was stressed as an important factor by the interviewees. The stakeholders are mutually dependent on each other and have to rely on the others to do their part. Problems with this lead many to think about the need for formal agreements; something also highlighted in research as important and a recommendation for future implementation of CAD. If program adaptation will be done, something virtually impossible to avoid , it has We found that in many municipalities not all components of CAD had been implemented and that the emphasis was placed on drug-training and on media campaigns. This finding is consistent with previous research illustrating that fidelity is often low when programs are applied in real-world settings . The capOne of the reasons for the less successful implementation of CAD in the smaller communities compared to Stockholm is a lower program readiness. Monetary support from the government was made available for the dissemination of CAD throughout Sweden. When it became possible to apply, too little time between the analysis of the Stockholm results and implementation had passed. The applicability of this for other communities had not been checked and not enough time was available to analyze the needs and prerequisites of the respective communities. As governmental support is only available for a short time span, the necessary analyses to estimate program readiness will often be inadequate.The respondents in the study expressed views that were highly consistent across stakeholders and communities about what factors had affected the implementation. In the communities where the police participation was perceived as weak the same view was shared by the interviewed police officers. A strength of this study was the broad inclusion of key informants which facilitated a verification and comprehension of the expressed perceptions. Another strength was the inclusion of a variety of municipalities from different areas of Sweden and the fact that the results from these were highly consistent.There was a low response rate on the questionnaire, but this is consistent with the overall trend in declining response rates in this type of study . This alThis study was originally done as an evaluation of the implementation between 2008 and 2010, it should be noted that some of the included municipalities had initiated the implementation before 2008. However, we assumed that the implementation factors were the same across all municipalities regardless of the date of program initiation and hence the early start date was not a factor in the selection of example municipalities or in the analysis of the questionnaire or interviews.The factors influencing the implementation and the difference in implementation outcome between Stockholm and the rest of the country can be summarized by the concept of community capacity. In Stockholm the community had a need, the individual organizations had the resources required as well as the ability to cooperate. This was made possible by the coordinator and external knowledge. In all, this indicated a high capacity within the Stockholm community. In the other municipalities some parts of this may have been high but overall the capacity was lacking. By the concept of capacity one can highlight the complexity of implementation prerequisites and the importance of resources within all participating organizations as well as the ability to perceive needs, formulate goals and to collaborate. All of this is needed to implement and sustain complex programs and solve the problems of substance use.There has to be capacity within the implementing organizations including assessment of needs, support, sufficient and long-term funding and formal agreements between stakeholders to facilitate the implementation and sustainability of CAD. Implementation of comprehensive programs is complex and if the capacity to implement is not adequately established beforehand the initiation of implementation should be questioned.It was difficult to find a municipality, besides Stockholm, where CAD had functioned well and was being sustained. In the example municipality that was functioning well according to the questionnaire and where the stakeholders seemed satisfied with the work, there had been no environmental rounds executed and the collaboration with the police was perceived as lacking as they did not have, or did not prioritize, the required resources to attend the clubs as much as wanted. In this instance the level of satisfaction did not relate to proper implementation of program activities. Expressed satisfaction may be based on the shared enthusiasm but this on its own is not sufficient for accomplishing the main goals and objectives of the CAD program.Just as programs need to be adapted to local context, so does the implementation process. The process may vary between different communities and factors may further have different influence depending on the context and the other factors involved . Hence iThe reason for the higher capacity in Stockholm may also be associated with the size of the municipality as well as the ability to mobilize resources and competence. But in this particular case what the respondents highlighted was the perceived need and benefits. There is a need for evaluation and for research programs in locations other than Stockholm and the studied community must have similarities with the communities where the program will be used in the future. The program has to be relevant in all settings and also possible to implement well in all settings.More studies of the efficacy of CAD are needed and it should not be promoted as an effective method without a sufficient amount of evaluation. The Swedish authorities should take more responsibility for which intervention programs they are spreading and should demand evaluation and closer collaboration with researchers.The authors declare that they have no competing interests.EN is the main author and has done most of the data collection. She has been active in writing, as have all the other authors. FS has also been project leader, EW has had main responsibility for theories of collaboration and organization, JG has been the foremost collaborator in Stockholm with personal experience of the start in that city. CP, finally, has made the most valuable comments in finalizing the paper. All authors read and approved the final manuscript.