Pedigree, demographic, square-root transformed maximum alcohol (SRMAXAPD) and maximum cigarette (MAXCPD) consumption, and genome-wide scan data from the Framingham Heart Study (FHS) were used to investigate genetic factors that may affect alcohol and cigarette consumption in this population-based sample.A significant sister:sister correlation greater than spouse correlation was observed for MAXCPD only. Single-point sib-pair regression analysis provided nominal evidence for linkage of loci to both SRMAXAPD and MAXCPD consumption traits, with more significant evidence of linkage to SRMAXAPD than to MAXCPD. One genomic region, chr9q21.11, exhibits significant multi-point sib-pair regression to SRMAXAPD.SRMAXAPD exhibits greater evidence for genetic linkage than does MAXCPD in the FHS sample. Four regions of the genome exhibiting nominal evidence for linkage to SRMAXAPD in the FHS sample correspond to regions of the genome previously identified as linked to alcoholism or related traits in the family data set ascertained on individuals affected with alcohol dependence known as COGA. Data from the ongoing NHLBI-supported Framingham Heart Study (FHS) on cardiovascular disease (CVD) was made available to Genetic Analysis Workshop 13 (GAW13). Two behaviors of general medical and psychiatric interest collected from this community-based sample were included in the data, i.e., alcohol consumption and cigarette consumption. Increased cigarette consumption in the FHS sample is associated with the development of CVD , but incth century, due to health concerns and restrictions placed on this behavior [The consumption of these two substances varies significantly based on both sex and age and there has been a long-term decline in the consumption of cigarettes in the US in the latter half of the 20behavior . The conbehavior . The genbehavior . MeasureN = 346) were included. Individual outlier trait values > +4 standard deviations were changed to missing for analysis (without outliers); the individuals whose values were converted were predominantly male for all three traits but were mostly (≥80%) from Cohort 1 for APD traits  and from Cohort 2 for MAXCPD (N = 10). Removal of outliers brings SRMAXAPD and MAXCPD traits much closer to normality  measure and 2881 with a maximum cigarette consumption (MAXCPD) measure. Descriptive statistics of the MAXAPD and MAXCPD traits and a square root transformation of MAXAPD, SRMAXAPD, are reported in Table Familial correlations of relative and parental pairs (sex-specific and non-sex-specific) without extreme positive outliers for MAXCPD and SRMAXAPD are reported in Table p-value < 0.01  of marker loci that were nominally linked (p < 0.01) with the maximum alcohol consumption traits, SRMAXAPD. A broad region on chromosome 9 exhibited the most significant evidence for linkage, with the maximum linkage evidence at ~66 cM (Table N = 9) with evidence for nominally significant (p < 0.05) linkage and no loci at p < 0.021. The low number of loci exhibiting nominal evidence for linkage to MAXCPD suggests that the MAXCPD trait, as investigated in this linkage analysis, lacks power to detect the influence of genetic susceptibility factors on maximum cigarette consumption.In the genome-wide search for linkage evidence to maximum alcohol consumption, we found a number N = 7 of markp-values for the significance of linkage analysis results were not substantially different from asymptotic p-values for either trait, suggesting that assumptions of the SIBPAL regression model apply to the phenotypic and genotypic data analyzed in this study. Only the regions of maximum linkage to SRMAXAPD on chromosome 9 and the single-point linkage analysis result to SRMAXAPD on chromosome 17 at 89 cM provided statistical evidence for linkage at p < 0.0007, a level considered "significant" by Lander and Kruglyak [Empirical Kruglyak . MultiplLinkage studies of alcohol- and cigarette-related traits have identified regions of the genome with more than nominal evidence for linkage . RegionsWe observed several marker loci with nominal evidence for linkage to the square-root transformed maximum alcohol consumption traits, SRMAXAPD, in the Framingham Heart Study sample. Some of the regions of the genome have been previously linked to alcoholism or related traits in Caucasian samples based on different ascertainment criteria.The traits of interest were defined as the maximum reported number of grams of alcohol per day (MAXAPD), and the maximum reported number of cigarettes smoked per day (MAXCPD). Exams 1, 2, 4, and 7 from FHS Cohort 1 and exams 1, 2, 3, and 4 from FHS Cohort 2 were chosen to assess MAXAPD and MAXCPD to utilize multiple exams at the earliest age possible to obtain measurements. Covariates of interest included cohort, age of maximum consumption measure, and sex.GAW13 FHS data were imported into a Microsoft Access database and exported in appropriate files for descriptive statistical analysis in SPSS Advanced Statistics or Microsoft Excel and familial correlation and linkage analysis in S.A.G.E. . Familiap-values were obtained for single-point linkage analysis of MAXCPD and SRMAXAPD to evaluate possible deviation from asymptotic p-values. Multi-point linkage analysis was performed using IBD distributions at multiple markers for MAXCPD and SRMAXAPD on those chromosomes showing nominal evidence  for linkage at two or more consecutive loci.SIBPAL from S.A.G.E. 4.2 was used to model the sib-pair covariance of traits reported as a function of marker allele identity-by-descent (IBD) sharing. Our analyses used estimated IBD information from the GENIBD procedure described above to perform single-point linkage analysis in which the mean corrected cross product of the trait was regressed onto the IBD information one trait at a time. The single-point linkage analysis was carried out separately for traits MAXAPD, SRMAXAPD, and MAXCPD, treated as continuous variables. The covariates sex, age of trait report, and cohort were included in the regression models. SRMAXAPD single-point linkage analysis was only performed without outliers. Empirical AB nominated the traits, performed the descriptive and FCOR analyses, and wrote the manuscript, XY performed the linkage analysis of the alcohol consumption traits, YB performed the linkage analysis of the cigarette consumption trait, MB performed the GAW13 FHS databasing, selection, reduction, transformation, and export, and AG and LG provided analysis direction and recommendations at each phase of the analysis.


Preventing Chronic Disease (low-cost MCH), which costs $2.90 and is marketed to maternal child health programs, and a Consumer Reports best buy (CR best buy), which costs $20.00 and was rated by Consumer Reports as a best buy for accuracy, ease of use, and value . Participants were asked to wear two pedometers — one on each hip — every day for 6 days, to record on a preformatted daily log sheet the number of steps measured by each pedometer, and to track any problems with use, such as forgetting, dropping, or losing the pedometers. Participants were told that they did not have to increase their physical activity for the study. The CR best-buy pedometer functioned on all days worn. Counts on both pedometers were accidentally reset on approximately 10% of user-days worn. The low-cost MCH pedometer fell off or was dropped more frequently than the CR best-buy model (7% versus 2% of user-days worn). In all instances of resetting or dropping, the pedometers continued to measure steps throughout the day. Although the difference between the two models in mean daily step counts for the 6-day study period was only 75 steps, on 45% of the 78 days on which both models were worn and functioning, the daily step counts differed between the two models by more than 2000 steps  did not wear the pedometers on at least 1 day; from a total of 168 user-days in the study, pedometers were worn on 133 user-days . The most common reasons for not wearing the pedometers were forgetting (28 user-days), losing the pedometers (4 user-days), and wearing a dress (3 user-days). The low-cost MCH pedometer functioned on only 80 user-days, representing 60% of user-days worn, while the 00 steps . Using mIn this study, the low-cost MCH pedometer model was found to be largely ineffective. Although the CR best-buy model was more reliable in terms of user-days functioning, developers of walking programs using pedometers need to consider cost in addition to reliability. In this study, the benefit of keeping costs low was offset by the poor functioning of the low-cost MCH pedometer model. Programs should carefully evaluate the reliability of any pedometer before use in an intervention.This study had the following limitations: all participants were employees of a large public health organization and may have been more aware of physical activity interventions; the data were collected through self-report; and participants were not asked to change their physical activity, unlike participants in an intervention program. This feasibility study revealed some difficulties in the day-to-day use of pedometers, including the pedometers not being worn and accidentally resetting counts. The high proportion of women who forgot to wear the pedometer on at least 1 day during the short study duration of 6 days (50%) suggests that programs using pedometers to promote physical activity must also incorporate motivational techniques and reminders for participants.


Manganese superoxide dismutase (MnSOD) plays a critical role in the detoxification of mitochondrial reactive oxygen species, constituting a major cellular defense mechanism against agents that induce oxidative stress. A genetic polymorphism in the mitochondrial targeting sequence of this gene has been associated with increased cancer risk and survival in breast cancer. This base pair transition (-9 T > C) leads to a valine to alanine amino acid change in the mitochondrial targeting sequence. A polymorphism has also been identified in the proximal region of the promoter (-102 C>T) that alters the recognition sequence of the AP-2 transcription factor, leading to a reduction in transcriptional activity. The aim of our study was to investigate possible associations of the -102 C>T polymorphism with overall and relapse-free breast cancer survival in a hospital-based case-only study.MnSOD -102 C>T polymorphism and survival was examined in a cohort of 291 women who received chemotherapy and/or radiotherapy for incident breast cancer. The MnSOD -102 C>T genotype was determined using a TaqMan allele discrimination assay. Patient survival was evaluated according to the MnSOD genotype using Kaplan–Meier survival functions. Hazard ratios were calculated from adjusted Cox proportional hazards modeling. All statistical tests were two-sided.The relationship between the MnSOD -102 genotype . Similarly, when the homozygous and heterozygous variant genotypes were combined, there remained a significant reduction in relapse-free survival in this group .In an evaluation of all women, there was a borderline significant reduction in recurrence-free survival with either one or both variant alleles (CT + TT) when compared with patients with wild-type alleles (CC) . When the analysis was restricted to patients receiving radiation therapy, there was a significant reduction in relapse-free survival in women who were heterozygous for the MnSOD -102 variant allele appears to be associated with an improved recurrence-free survival in all patients, and more dramatically in subjects who received adjuvant radiation therapy.The  Breast cancer represents one of the most common cancers among women residing in the United States and worldwide ,2. In 20BRCA1 and BRCA2, but these genes only account for 5% of all breast cancer cases [Genetic risk factors have also been identified, such as the high-penetrance cancer susceptibility genes, er cases ,5. It haer cases .The development of breast cancer has been linked to the degree of oxidative stress, particularly with perturbations in the delicate balance between reactive oxygen species (ROS) and oxidative defenses . ROS are2-) to hydrogen peroxide (H2O2) and oxygen (O2). Three distinct types of superoxide dismutases have been identified in human cells: a homodimeric cytosolic CuZnSOD [Superoxide dismutase catalyzes the dismutation of the superoxide radical  and the risk of cancer. A significant gene dose-response relationship has been observed in breast cancer ,15; howe2O2) and superoxide anions (O2-), and its efficacy may be related to the ability to neutralize these effects. Because mitochondrial MnSOD is responsible for catalyzing the conversion of superoxide radicals, there has been a growing belief that gene variants could impact the efficacy of radiation therapy for breast cancer. Similarly, the chemotherapeutic agents most commonly utilized in the treatment of breast cancer, adriamycin and cyclophosphamide, generate ROS that may be effected by MnSOD activity. We recently found that the combination of the MnSOD -9 genotype and the myeloperoxidase genotype led to a threefold decrease in the hazard of death among women treated for breast cancer [Since radiation therapy remains an integral part of the multimodality therapy in the treatment of breast cancer, determining maximal efficacy among patients remains of the utmost importance. Radiation therapy exerts its antitumor effects through the increased formation of ROS, including hydroxyl radicals (OH), hydrogen peroxide (Ht cancer . This asMnSOD promoter sequence (MnSOD -102 C>T) has been shown to change the binding pattern of AP-2, leading to a reduction in transcriptional activity [MnSOD promoter and breast cancer treatment outcomes has not, however, been reported. Similarly, there have been no published reports of the effects the MnSOD -102 C>T polymorphism on protein function.The polymorphism in the signal sequence, a mutation in the activity . We receactivity ,22. A reMnSOD -102 C>T polymorphism within a case-only study of breast cancer patients to evaluate possible associations with overall and relapse-free survival in relation to the patients' adjuvant therapy.The aim of our study was therefore to investigate the possible associations of the As previously described ,23, patiArchived normal lymph nodes or skin, stored in paraffin blocks in the Pathology Department, were used as a source of DNA for genotyping as described previously ,23. HospSections (50 μm thick) were cut from archived paraffin normal tissue blocks, the tissue was deparaffinized, and DNA was extracted using a commercially available kit , as previously described ,23.MnSOD -102 C>T polymorphism was determined using a technique developed in our laboratory [MnSOD promoter region. TaqMan Universal PCR Master Mix (Applied Biosystems) was used to prepare the PCR. The two-times mix was optimized for TaqMan reactions and contained AmpliTaq-Gold DNA polymerase, AmpErase, deoxyribonucleotide triphosphate with uracil triphosphate, and a passive reference. Primers, probes, and genomic DNA were added to final concentrations of 300 nM, 100 nM, and 0.5–2.5 ng/μl, respectively.The boratory . Single Controls (no DNA template) were run to ensure there was no amplification of contaminating DNA. The amplification reactions were carried out in an ABI Prism 7700 Sequence Detection System (Applied Biosystems) with two initial hold steps  and 50 cycles of a two-step PCR .MnSOD promoter region. The -102 nucleotide was determined by the fluorescence ratio of the two single nucleotide polymorphism-specific fluorogenic probes. The fluorescence signal increases when the probe with the exact sequence match binds to the single-stranded template DNA and is digested by the 5'-3' exonuclease activity of AmpliTaq-Gold DNA polymerase (Applied Biosystems). Digestion of the probe releases the fluorescent reporter dye (either FAM or VIC) from the quencher dye.The fluorescence intensity of each sample was measured at each temperature change to monitor amplification of the 278-base-pair MnSOD and overall survival were evaluated using the Kaplan–Meier survival function. The survival time was calculated as the time from diagnosis to death or to the last contact date for living subjects. Both heterozygotes and homozygotes were assessed separately in relation to the referent. Cox proportional hazard models were constructed to assess potential confounding effects of other breast cancer prognostic factors, including age, stage with nodal status, and estrogen and progesterone receptor status. The final multivariate-adjusted models shown include those factors that either changed the estimated effect by 10% or more in a best-fitting model, which was developed by starting with a full model and then excluding covariates that did not improve the overall fit. Survival analysis according to MnSOD genotype was conducted separately by treatment group (i.e. chemotherapy or radiation therapy). All analyses were conducted using SAS software . All statistical tests were two sided.Crude associations between genotypes for Acceptable tissue samples and complete baseline information were obtained for 291 cases (Table MnSOD -102 (CT or TT), there was a nonsignificant impact of genotype on overall survival   Table . When thMnSOD genotypes and relapse-free survival   of women receiving radiation therapy for breast cancer, who found no effects of the MnSOD -9 polymorphisms on clinically detectable skin reactions to therapy [MnSOD -102 polymorphism plays in MnSOD function, or could be due to differential effects of MnSOD variability on normal and tumor tissue or, finally, could be due to the small number of subjects (n = 80) in the study. Since the MnSOD -9 polymorphism leads to partial inner mitochondrial membrane transport arrest [MnSOD -102 polymorphism affects the AP-2 binding and thus reduces transcriptional activity, however, there may be a greater effect from this polymorphism than from other polymorphisms that have been reported.Our results are in contrast to a smaller study , we are unable to establish the effects that each individual therapy had on overall survival and on relapse-free survival. Since there is a similarity in the effects that chemotherapy in breast cancer treatment and radiation therapy induce (generation of ROS), however, it is less likely that individual treatment evaluation will lead to different results. Radiation therapy and the chemotherapeutic agents most commonly utilized in the treatment of breast cancer, adriamycin and cyclophosphamide, exert their antitumor effects through the increased formation of ROS, including hydroxyl radicals (OH), hydrogen peroxide (H2O2), and superoxide anions (O2-), and the efficacy may be related to the ability of the patients'/tumors' ability to neutralize these effects. Since MnSOD is not involved in the metabolism of any specific chemotherapeutic agent we believe these results will remain consistent, but they need to be evaluated in a larger study.Even with this report indicating the efficacy effects of ROS producing therapies and the relationship to In summary, this reports demonstrates the first evaluation that decreases in MnSOD by the variant genotype are associated with improved relapse-free survival in women treated for breast cancer.MnSOD = manganese superoxide dismutase; PCR = polymerase chain reaction; ROS = reactive oxygen species.The authors declare that they have no competing interests.RM participated in design of the study and manuscript preparation. JA participated in the statistical analysis. SN contributed to the study design and to the analysis and interpretation of the data. DH participated in design of the study and manuscript preparation. MD and BM participated in the design of the assay methods. CA contributed to the study design, analysis, and interpretation of the data, and to manuscript preparation.


Domains containing the β-grasp fold are utilized in a great diversity of physiological functions but their role, if any, in soluble or small molecule ligand recognition is poorly studied.Using sensitive sequence and structure similarity searches we identify a novel superfamily containing the β-grasp fold. They are found in a diverse set of proteins that include the animal vitamin B12 uptake proteins transcobalamin and intrinsic factor, the bacterial polysaccharide export proteins, the competence DNA receptor ComEA, the cob(I)alamin generating enzyme PduS and the Nqo1 subunit of the respiratory electron transport chain. We present evidence that members of this superfamily are likely to bind a range of soluble ligands, including B12. There are two major clades within this superfamily, namely the transcobalamin-like clade and the Nqo1-like clade. The former clade is typified by an insert of a β-hairpin after the helix of the β-grasp fold, whereas the latter clade is characterized by an insert between strands 4 and 5 of the core fold.Members of both clades within this superfamily are predicted to interact with ligands in a similar spatial location, with their specific inserts playing a role in the process. Both clades are widely represented in bacteria suggesting that this superfamily was derived early in bacterial evolution. The animal lineage appears to have acquired the transcobalamin-like proteins from low GC Gram-positive bacteria, and this might be correlated with the emergence of the ability to utilize B12 produced by gut bacteria.This article was reviewed by Andrei Osterman, Igor Zhulin, and Arcady Mushegian. The β-grasp fold (β-GF) was first recognized in ubiquitin and the immunoglobulin-binding (IG-binding) domains of Gram-positive cocci ,2. SinceAs part of our larger effort to understand the evolutionary and structural basis for the functional versatility of this widespread fold (for example see reference ) we wereTo identify potential novel versions of the β-GF that bind soluble ligands, we initiated comprehensive structural comparisons with various previously characterized members of the fold (see β-grasp fold in SCOP database) using the DALI program. Several of these searches retrieved the C-terminal domain of the transcobalamin protein with significant Z-scores. For example, searches initiated with MoaD proteins  retrieved the C-terminal domain of transcobalamin (PDB: 2bbc) with Z-scores of ~7. Transcobalamin is an animal-specific protein that binds cobalamin (vitamin B12), and is involved in its uptake by animal cells . TranscoTetraodon nigroviridus, gi: 47226456, region: 325–425) recovered closely related eukaryotic orthologs and paralogs fused to N-terminal α/α toroid modules (iteration 1), solo transcobalamin C-terminal domains with predicted signal peptides , and several prokaryotic proteins . In order to exhaustively recover all divergent homologs, we conducted transitive searches with all above-detected members and also evaluated all hits below the threshold of PSI-BLAST searches for the presence of potentially homologous domains. We also prepared HMMs and PSSMs from the alignment of this region of all proteins recovered with significant expect-values  and used these to search all completely sequenced genomes. These searches consistently retrieved hits to multiple sequence repeats in a group of bacterial cell-surface/secreted sugar-binding proteins involved in polysaccharide export with significant e-values . Inclusion of polysaccharide export proteins in profiles for further searches additionally recovered the N-terminal region of the ComEA family of DNA uptake receptors of Gram positive bacteria , PduS-like cobalamin reductases , the middle domain of the 51 kD subunit (F chain) of the NADPH-quinone oxidoreductase complex I  and the RnfC subunit of the oxidoreductases encoded by the bacterial Rnf operons [Rhodobacter RnfC, E: 10-6; iteration 14). This latter set of proteins was more similar to the homologous region recovered in the polysaccharide export proteins than to the transcobalamin C-terminal domain  searches for homologs using PSI-BLAST and the HMMER package respectively. In addition to orthologs of transcobalamin, intrinsic factor and solo C-terminal domains from fishes, these searches retrieved numerous prokaryotic proteins, which were either present as stand-alone β-GF domains or in large multidomain proteins. For example, a search initiated with the β-GF domain of puffer fish transcobalamin  as many members of this superfamily are known or predicted to bind soluble ligands (See below for further details).We hereinafter refer to the homologous β-GF domains found in all these proteins as the A comprehensive multiple alignment for the SLBB superfamily Figure  was prepThe alignment also helped us to classify the SLBB superfamily into several distinct families. The Transcobalamin C-terminal domain clade is unified by the presence of the above-described β-hairpin insert within the β-GF that plays an important role in contacting the ligand Fig . This β-The Nqo1-like clade includes at least two distinct families: 1) the first includes the NADPH-quinone oxidoreductase complex I subunit Nqo1 (51 kD/F chain), the RnfC oxidoreductase subunit, and the PduS-like cobalamin reductases. 2) The second family contains polysaccharide export proteins and the DNA receptor ComEA. This clade is unified by the presence of a small, often α-helical insert, in the "connector arm" between the fourth and fifth strands of the domain Figure . In someWhile the interaction between B12 and the transcobalamin-like SLBB domain involves the unique β-hairpin insert, key contacts are also contributed by the core fold  or C-terminal  to the SLBB , the FIVAR (Pfam entry: PF07554) sugar-binding domain , the fasciclin domain  and a β-propeller domain . Given that many of these domains are often involved in interactions with polysaccharides, they might play a role in tethering these proteins to the cell surface by binding peptidoglycan or capsular polysaccharides [Syntrophomonas the SLBB domain is found in putative extracellular enzymes fused to sulfite oxidase-like molybdopterin cofactor binding domain (e.g. gi: 71491441) [Most members of the transcobalamin C-terminal domain clade of the SLBB superfamily contain signal peptides, and several also contain the C-terminal Gram-positive anchor motif , suggestB Figure . As the charides -23. Oftecharides . These o1491441) . It is lMethanosarcina) and AraC-like families   Figure . These pIn the Nqo1-like clade, polysaccharide export proteins are predicted to be secreted or periplasmic proteins and contain an absolutely conserved N-terminal β-strand-rich domain followed by 1–8 repeats of the SLBB domain Figure . They apThe remaining three groups of proteins, namely NqoI, RnfC and PduS, within the Nqo1-like clade of the SLBB superfamily share a common architectural core consisting of a fusion between an N-terminal Rossmannoid domain and an SLBB domain. Unlike classical Rossmann fold domains of oxidoreductases with 5–7 strands, the Rossmannoid domain of these proteins has a 4-stranded core in a 3214 order  coupled The roles of these versions of the SLBB remain enigmatic; however there is evidence that the PduS protein might bind soluble ligands. The PduS gene typically belongs to a large mobile operon coding proteins required for the biogenesis of carboxysome/polyhedral bodies, which contains enzymes involved in propanediol degradation. The PduS has been shown to strongly bind cob(I)alamin and was characterized as a bifunctional cob(II)alamin and hydroxycobalamin (cob(III)alamin) reductase catalyzing the formation of cob(I)alamin. Cob(I)alamin is the immediate precursor of Ado-cobalamin, which serves as an essential coenzyme for the diol dehydratase in degradation of 1,2-propanediol ,31. It iThe phyletic and domain-architecture distributions show that the SLBB superfamily is well-represented and has diversified across the entire bacteria superkingdom. Their sporadic presence in archaea and the stronger affinity of the different eukaryotic versions to their bacterial counterparts suggests that the SLBB superfamily was derived early in evolution of bacteria, most probably from one of the many ancient β-grasp fold domains. The two major families in the NqoI-like clade appear to have a pan-bacterial distribution suggesting that this clade had already differentiated into versions associated with cell wall related functions (ComEA-Polysaccharide export protein family) and intracellular oxido-reductase related functions . Of the latter group the NqoI protein of the respiratory complex-I is seen across bacteria and was transferred to the eukaryotic lineage during the primary endosymbiotic event that generated the eukaryotic cell with mitochondria. RnfC and PduS proteins have more restricted phyletic distributions and are likely to be late derivatives of the more ancient Nqo1 lineage. The transcobalamin C-terminal clade is very divergent in sequence and appears to be a lineage-specific innovation in Gram positive bacteria that was recruited specifically for transporting extracellular B12-like cofactors or their precursors. Subsequently, a specific version that combined the α/α toroid domain with the SLBB domain appears to have been laterally transferred to the animal lineage early in its evolution. This event probably conferred on animals the ability to directly absorb cobalamin synthesized by bacteria in the gut. In parallel, there appear to have been sporadic transfers of large extracellular multidomain versions as well as intracellular versions fused to DNA-binding HTH domains to certain euryarchaeal lineages.In this context, it is of interest to note that the discovery of soluble ligand binding versions of the β-GF points to a noteworthy structure-function analogy with the RNA-recognition motif (RRM)-like fold. In functional terms, the RRM-like fold has long been known to bind a range of soluble ligands such as amino acids, sugars and co-factors. Notable examples of these include the ACT domain superfamily and the amino acid-binding domain of the LRP-like transcription factors ,35,36. BIn conclusion, we show that the β-GF domains found in transcobalamin, polysaccharide export proteins, ComEA, PduS, and RnfC and Nqo1-like oxidoreductases define a novel superfamily, several of which might interact with different soluble ligands. This investigation provides the possible evolutionary scenario for the origin of the vitamin B12 uptake in animals via transcobalamin and intrinsic factor. It also provides leads for new investigations into B12 metabolism in bacteria and other aspects of protein-ligand interaction in competence, cell-surface biochemistry, and respiratory electron transfer.Searches of the PDB database with query structures were conducted using the DALI program . StructuThe manuscript "A novel superfamily with the β-grasp fold involved in binding diverse soluble ligands" by A. M. Burroughs is an excellent and insightful comparative genomics study of a very interesting domain family broadly conserved across all kingdoms of Life. The authors took on a very challenging task of classifying multiple representatives of this superfamily on the edge of detectable homology. Such a task is particularly daunting for non-enzymatic proteins, where the divergence of sequence driven by adaptation to new biological tasks and contexts is much more pronounced than for enzymes. This study tracks down a complex evolution of the family and reveals an apparent functional theme, binding of small molecule ligands, such as B12 and its analogs, that "runs in the family" from bacteria to mammals. In addition to sensitive and sophisticated homology-based methods, the authors broadly used genome context analysis, which was one of the key success factors in pursuing this challenge. Of particular interest is the observation that in course of a fascinating evolutionary reshuffling, not only the ligand binding specificity, but the actual functional context of this domain could have changed on a number of occasions. One of the most remarkable events is a proposed recruitment of this domain into the context of transcriptional regulation in some bacteria. The latter theme of recruiting former enzymes and, as we see now, other types of proteins with natural affinity to certain ligands, is being recognized as one of the most important strategies of "natural engineering" of effector-binding domains in transcriptional regulators. Although this bioinformatics analysis alone does not allow to precisely identifying specific functions for all SLBB subfamilies, it certainly provides a perfect starting point for many case studies driven by specific research interests of various experimental groups. This constitutes a broad impact of this paper, which goes beyond the innovative bioinformatics methodology and obvious implications in the field of domain classification and evolution. The paper is written very clearly, with sufficient details of methods and key results and with helpful illustrations. The Supplementary material providing the entire list of SLBB domain superfamily including the information about their genome context, is highly valuable. This reviewer strongly supports the publication of this paper "as is" in Biology Direct, a perfect home for this wonderful study.We appreciate the comments, and particularly would like to emphasize that we feel the findings in this paper can be used as starting points for experimental work that will contribute significantly to the study of several important biological pathways.In this study, the authors report a novel domain superfamily within the β-grasp fold. The predicted distinct property of this domain is binding diverse soluble ligands, thus it is termed Soluble-Ligand-Binding β-grasp or SLBB. Motivation for this work came from expectation  that some members of the ubiquitous β-grasp fold are involved in binding small ligands.First, the authors searched the PDB database with known structures to retrieve related structures and then used an array of sensitive sequence-based searches (hidden-Markov-model- or position-specific-scoring-matrix-based) to identify remote homologs. Technically, it is very well done and described in sufficient detail. The authors took advantage of available structures to guide editing of the multiple alignment, which in turn allowed them to associate conserved positions with the structural features.Biological function prediction came primarily from the contextual analysis, which was quite thorough. The most interesting prediction is that the primary role of one of the two SLBB clades is binding vitamin B12 and its precursors.Overall, this is an interesting, well-executed study and defining novel domain families and assigning potential biological functions is very important. I would like to add that it is also important to deposit the newly described domain to leading domain databases, such as Pfam. I have not seen plans for doing so in this study. This reviewer is guilty of not always doing so either, but still it should be a rule rather than exception. Perhaps, two version of SLBB could be produced capturing characteristics of the two clades.We could not agree more regarding the importance of domain databases, and we will be submitting two versions of the SLBB to Pfam.Essentially, there is only one major concern with respect to generalization of biological function prediction. I think the members of the domain clade are divergent enough to worry about B12 and its precursors being the only or even the main ligand. The statement in the abstract seems to address this issue – the authors rightly indicate that "members of this superfamily are likely to bind a range of soluble ligands, including B12"; however in the text it becomes more stringent. For instance, on page 3, when describing SLBB fusions with HTH domains in transcription factors, it is suggested that these proteins "respond to B12 or its precursors". I would suggest toning down the claim that all members of the transcobalamin clade bind exclusively B12 and its precursors. Perhaps, the authors did not intend to make it sounds like that, but it does.We do admit that there might be a greater diversity of bound ligands, rather than B12 alone. Only in the cases where there was contextual evidence for involvement in B12 metabolism do we suggest it as a most likely ligand. Following the suggestion of the reviewer, we have altered the wording in the text to clarify the possibility of greater diversity in terms of bound ligands, especially with the HTH-fused versions.I have no concerns about the main observation of the extended beta-grasp SLBB superfamily, or about functional inferences from the contextual analysis of genes that contain various versions of this domain. I think, however, that the analogy with the RRM fold early in the manuscript is a distraction at that point of the authors' reasoning. Generally, I suppose that this type of domain/fold recognition work may benefit from being more logical and less chronological: that is, even if analogy with RRM played a role in authors' own thinking about the SLBB superfamily, I want to first focus on what has been actually observed. On the other hand, I would like to see a discussion of RRM and other analogous folds at the end of the manuscript, when it could serve a more useful purpose of establishing some trends in evolution .We admit this might provide a better flow for the article. Accordingly, we have now shifted the discussion of the parallels between the β-grasp and RRM folds to the Results and Discussion section.LA and SB made the initial discovery reported here. The complete sequence and structure analysis and preparation of figures and supplementary material was performed by AMB, LMI and SB. The paper was written by AMB and LA.Cartoon representations of SLBB domains. File 1 contains cartoon representations of the transcobalamin and the Nqo1 middle domain showing the structural similarity and innovations associated with the potential binding site for soluble ligands.Click here for fileComprehensive list of proteins of the SLBB superfamily, their gene neighborhoods, and domain architectures. Alignments of the SLBB domain will be submitted to the Pfam domain database [database .Click here for file


While a number of studies report high prevalence of mental health problems among injured people, the temporal relationship between injury and mental health service use has not been established. This study aimed to quantify this relationship using 10 years of follow-up on a population-based cohort of hospitalised injured adults.The Manitoba Injury Outcome Study is a retrospective population-based matched cohort study that utilised linked administrative data from Manitoba, Canada, to identify an inception cohort (1988–1991) of hospitalised injured cases (ICD-9-CM 800–995) aged 18–64 years , which was matched to a non-injured population-based comparison group . Pre-injury comorbidity and post-injury mental health data were obtained from hospital and physician claims records. Negative Binomial regression was used to estimate adjusted rate ratios (RRs) to measure associations between injury and mental health service use.Statistically significant differences in the rates of mental health service use were observed between the injured and non-injured, for the pre-injury year and every year of the follow-up period. The injured cohort had 6.56 times the rate of post-injury mental health hospitalisations  and 2.65 times the rate of post-injury mental health physician claims . Adjusting for comorbidities and pre-existing mental health service use reduced the hospitalisations RR to 3.24  and the physician claims RR to 1.53 .These findings indicate the presence of pre-existing mental health conditions is a potential confounder when investigating injury as a risk factor for subsequent mental health problems. Collaboration with mental health professionals is important for injury prevention and care, with ongoing mental health support being a clearly indicated service need by injured people and their families. Public health policy relating to injury prevention and control needs to consider mental health strategies at the primary, secondary and tertiary level. Previous research suggests that psychosocial and mental health sequelae are some of the most disabling consequences of injury -4. HowevConclusions from previous studies that have described the prevalence of post-injury mental health have been limited by the study methods used. These include small sample sizes ,8, non pThe aim of this paper was to quantify the relationship between injury and mental health service use for 10 years post-injury event, controlling for demographic factors and pre-existing comorbidities, including pre-existing psychiatric conditions. Clarification of mental health conditions as a risk factor, potential confounder and outcome of injury is essential for improving injury prevention strategies and post-injury care.The Manitoba Injury Outcome Study is a population-based retrospective matched cohort study using linked administrative health data from the province of Manitoba, Canada ,14. The International Classification of Diseases, Ninth edition, Clinical Modification (ICD-9-CM) codes 800–995 , in the first or second diagnostic fields of the hospital record. The first injury-related hospital record was designated as the index injury.The injured cohort included all persons aged 18–64 years, resident in the province of Manitoba, who were hospitalised with an injury between 1 January 1988 and 31 December 1991 . Injury related hospitalisations were identified using the A non-injured comparison cohort was randomly selected from the total remaining province population, identified using the Manitoba population registry, matched on Aboriginal status, age, gender and geographic location of residence of the injured case. The health claims databases and population registry can be linked using unique identification numbers. Residents of personal care homes (PCH), patients in extended hospital care, and persons not resident in the province for 12 months prior to the admission date of the index record, were excluded.The injured cohort was analysed by the nature of injury codes (ICD-9-CM 800–995), and subgroup comparisons were made with the matched comparison group. Seven subgroups were created across ICD subchapter headings . Injury Severity Scores (ISS) were generated by ICDMAP-90© software from Johns Hopkins University. An ISS of 16 or greater was considered to be a major injury, an ISS of 9–15 a moderate injury, and a mild injury was defined as an ISS of 1–8 [For both the injured and non-injured cohorts, frequency and types of pre-existing conditions were identified from their health service use records during the 12-months prior to the index injury . In addiPre-existing comorbidity for the two cohorts was also quantified using the Dartmouth-Manitoba version of the Charlson Comorbidity Index (CCI) . The CCITwo outcome variables were used to explore long-term post-injury mental health service use – counts of post-injury mental health related hospital separations and ambulatory physician claims (excluding emergency department presentations). All hospital records and physician claims with a primary diagnosis of ICD-9-CM 'Mental Disorders' codes (290–319) were identified for the 10 year period following the date of the index injury. To identify patterns of post-injury mental health service use, the hospital and physician claims data were further separated into the ICD-9-CM subchapter codes for mental disorders.Using the population registry information, the total time a person was alive, living in Manitoba and eligible for health coverage, was calculated for the 10 years following the index injury date.Statistical significance of differences between injured and non-injured cohorts for pre-injury rates of health service use was assessed by chi-squared test for categorical data and with the Mann-Whitney U test for continuous data because of non-normal distributions. All tests were two sided with a 5% level of significance.Negative Binomial regression was used to estimate crude and adjusted rate ratios (RRs) between exposure (injury) and outcome  ,21. ThosTo identify patterns in post-injury mental health service use, the hospital and physician claims data were categorised into the ICD-9-CM subchapter codes for mental disorders.Analysis was completed using SAS version 8.2.In total, 21,032 injured cases were identified. Most injuries occurred in males (64%) and in the younger age categories 18–34 years (54%). Approximately 40% of cases lived in urban regions, 40% in rural areas and the remaining 20% resided in remote northern areas of the province. 'Other' accidents 28.5%, accidental falls 22.9%, transport related 18.0%, and homicide or injury inflicted by others 10.9% accounted for the majority of the external causes. Further details of these characteristics have been published previously .prior to the injury event than their matched non-injured counterparts. Injured people had higher CCI scores, more hospitalisations, longer average length of stay in hospital, a greater number of physician claims and were more likely to have been admitted to hospital or seen a physician multiple times for a mental health condition, musculoskeletal condition or for a previous injury. Further details of these differences have been published previously [The injured cohort had significantly more all-cause health service use in the 12 months eviously .Mental health conditions were the most frequent cause of hospitalisations for the injured cohort in the pre-injury period. The injured cohort had 9.31 times the rate of separations from hospital for a mental health condition in the 12 months prior to the injury than the non-injured cohort    and post-injury mental health physician claims .Rates of post-injury mental health hospitalisations and physicians claims varied across different injury types Table . After aAdjusted RRs for post-injury mental health hospitalisations during follow-up showed some increase as the severity of the injury increased Table . The minThe injured cohort had 20.3 times the number of post-injury hospitalisations and 4.2 times the number of physician claims for personality disorders than the comparison cohort. Almost eight times more post-injury hospitalisations in the injured cohort were related to alcohol or drugs, compared with the non-injured. Relative differences for each of the other mental health subchapter types were of a fourfold magnitude.While the injured cohort had greater numbers of mental health hospitalisations and physician claims, the proportion of service use for particular mental health conditions was similar in both cohorts. Almost half of all post-injury mental health hospitalisations were for non-psychotic or personality disorders (injured 49.3% and non-injured 46.8%), a third of all post-injury mental health hospitalisations had a primary diagnosis of affective and delusional psychoses (injured 31.5% and non-injured 32.8%) and the remaining 20% were for organic psychoses. The majority of mental health physician claims were for non-psychotic or personality disorders (injured 79.2% and non-injured 86.2%), followed by claims for affective and delusional psychoses (injured 18.0% and non-injured 12.1%). Of all the injury types, poisonings cases were most likely to have subsequent personality disorder diagnoses.The current study aimed to describe and quantify the relationship between injury and subsequent mental health service use, as well as the contribution of pre-existing mental health conditions on post-injury mental health service use. We found an increased risk of mental health service use for at least 10 years following the injury event. While the greatest difference in mental health service use between the injured and non-injured occurred during the year prior to the injury event, statistically significant differences remained for the 10 years following injury, even after adjusting for demographic characteristics and pre-injury health status, including pre-existing psychiatric conditions. While there was a marked increase in health service use for alcohol and drug related conditions and personality disorders in the injured cohort, the proportional distributions of all mental health conditions were similar in both cohorts.The study that was most similar in methods to the current study was the one undertaken by Dryden et al in 2004 , who exaWhile the present study did not specifically aim to identify risk factors for injury, the observed pre-injury rates of mental health service use suggest that the presence of mental health conditions may be associated with an increased risk of injury. This finding is consistent with previous studies that have attempted to measure the prevalence of pre-injury mental health conditions in samples of injured people and compare it to population norms. Patterson et al , found tIn the current study, over and above pre-existing conditions and those of self-harm, injury appeared to be associated with a threefold increase in mental health hospitalisations and 1.4 times the number of mental health physician claims in the decade following the injury. These data support the results of other studies which found pre-existing mental health conditions as both a risk factor for injury and a confounder of the relationship between injury and poor mental health outcomes following injury ,27.While there is a plausible causal pathway linking injury and post-injury mental health episodes of depression, anxiety or alcohol use, it is not obvious why injury would cause a personality disorder. The effect was likely to be due to the injury triggering a pre-existing latent personality disorder or by an increased rate of diagnosis through the increased contact with the health care system .The use of a non-injured comparison group is a key element for attributing effects which have occurred a considerable time after the exposure . While, When considering these data sources for epidemiological research, it is important to recognise the limitations and constraints of using administrative data that were primarily developed for accounting purposes. The Manitoba data have been used extensively in health services and epidemiological research by a number of different groups and there has been a strong emphasis on improving data quality -31. WhilWhile administrative data lack details of individual risk factors, previous studies conducted in this population support the validity of deriving proxy measures of health status from health service use data, though the limitations of this practice have been acknowledged ,33. SomeThis study demonstrated that confounding factors, including pre-existing mental health conditions, accounted for almost half the mental health service use attributable to injury. However, after adjusting for confounding factors, the injured cases continued to have a significantly increased risk of post-injury mental health service use compared to the non-injured. Separating the consequences of injury for those with pre-existing mental health problems from those without pre-existing mental health problems is important because the effects of injury were two-fold: 1) injuries increased mental health service use for those with pre-existing mental health problems and 2) injuries lead to mental health service use among those without pre-injury mental health conditions. These findings indicate the presence of pre-existing mental health conditions is a potential confounder when investigating injury as a risk factor for subsequent mental health problems. Collaboration with mental health professionals is important for injury prevention and care, with ongoing mental health support being a clearly indicated service need by injured people and their families. Public health policy relating to injury prevention and control needs to consider mental health strategies at the primary, secondary and tertiary level.International Classification of Diseases, Ninth edition, Clinical Modification; ISS, Injury Severity Score; LOS, length of stay; PCH, personal care home; PYs, person-years; RR, rate ratio; SCI, spinal cord injury.CI, confidence interval; ICD-9-CM, The author(s) declare that they have no competing interests.CMC conceived the study, participated in its design, conducted the data collection, analysis and manuscript preparation. DVP, EVK and RJM all directly participated in the research conception, design and execution; preparation of the manuscript; and approved the final version submitted.This research was conducted, in part, at the School of Population Health, University of Queensland and was financially supported by the Centre of National Research on Disability and Rehabilitation Medicine, University of Queensland.The pre-publication history for this paper can be accessed here:


Mycobacterium tuberculosis.The whole blood interferon-gamma assay  has not been fully evaluated as a baseline tuberculosis screening test in Japanese healthcare students commencing clinical contact. The aim of this study was to compare the results from the QFT with those from the tuberculin skin test (TST) in a population deemed to be at a low risk for infection with M tuberculosis-specific antigens (early-secreted antigenic target 6-kDa protein [ESAT-6] and culture filtrate protein 10 [CFP-10]) were also measured. Of the 536 healthcare students, most of whom had been vaccinated with bacillus-Calmette-Guérin (BCG), 207 (56%) were enrolled in this study. The agreement between the QFT and the TST results was poor, with positive result rates of 1.4% vs. 27.5%, respectively. A multivariate analysis also revealed that the induration diameter of the TST was not affected by the interferon-gamma concentration after exposure to either of the antigens but was influenced by the number of BCG needle scars (p = 0.046). The whole blood interleukin-10 assay revealed that after antigen exposure, the median increases in interleukin-10 concentration was higher in the subgroup with the small increase in interferon-gamma concentration than in the subgroup with the large increase in interferon-gamma concentration .Healthcare students recruited at Okayama University received both the TST and the QFT to assess the level of agreement between these two tests. The interleukin-10 levels before and after exposure to As a baseline screening test for low-risk Japanese healthcare students at their course entry, QFT yielded quite discordant results, compared with the TST, probably because of the low specificity of the TST results in the BCG-vaccinated population. We also found, for the first time, that the change in the interleukin-10 level after exposure to specific antigens was inversely associated with that in the interferon-gamma level in a low-risk population. Tuberculosis continues to be a heavy burden on human health At Okayama University Hospital, tuberculosis patients are incidentally referred to the hospital before a definite diagnosis has been confirmed, even though the hospital does not have a tuberculosis-specific ward. Thus, not only healthcare workers, but also healthcare students studying at our hospital are at risk of tuberculosis exposure. As an infection control policy, a TST must be taken by all healthcare students and is optional for first-year postgraduate students prior to the start of clinical training, similar to the policies of many other medical universities Mycobacterium tuberculosis but not by BCG strains. Recent studies have shown that this blood assay has a moderate sensitivity for the detection of latent tuberculosis infection in the range of 70–80%, but is highly specific (over 95%) as compared with the TST even in BCG-vaccinated patients with varying risks of infection with M tuberculosis or active tuberculosis M tuberculosis.Unfortunately, the TST has several major limitations; one of them is the possible occurrence of false-positive test results in individuals vaccinated with bacillus-Calmette-Guérin (BCG) Unlike Th1-mediated cytokines such as interferon-gamma that promote the killing of infected cells, regulatory cytokines including interleukin-10 have been implicated in the suppression of in vitro antigen-specific cellular immune responses in tuberculosis. Their down-regulatory mediators may be important players in controlling the excessive synthesis of pro-inflammatory cytokines and subsequent tissue damage M tuberculosis. The following data were collected: history of prior tuberculosis or exposure to a person with tuberculosis, and other tuberculosis risk factors like having an immunosuppressive condition  or having taken immunosuppressive drugs in the 3 months prior to enrollment. Information regarding any clinical symptoms, previous TST results, BCG vaccination status (as reported by the prescribing physician and based on questions about past BCG vaccination and scar inspection), and demographic, clinical, and radiological data was also collected at the time of enrollment. Human immunodeficiency virus testing was not performed because of the low prevalence of positive human immunodeficiency virus expected in this young, educated population.All participants in this trial were prospectively recruited at Okayama University, Japan, between May and July, 2006. The subjects consisted of medical, nursing and dental students who were enrolled at the beginning of their clinical training, all of who were older than 18 years of age. First-year postgraduate students were also recruited. Subjects who received both whole blood assay and TST were considered to be eligible for this study. The protocol was approved by the ethics review committee of Okayama University. After providing written consent, the subjects were asked to complete a questionnaire about possible risk factors for exposure to For the TST, 0.1 mL of tuberculin purified protein derivative (PPD)  was injected intradermally into the volar aspect of the forearm and the transverse induration diameter was measured 48 hours later, as described previously M tuberculosis-specific antigens ESAT-6 or CFP-10, the concentration of interferon-gamma in the four plasma samples from each subject was determined using an ELISA. We interpreted the test results, after subtracting the value of the negative control well from the IU amount of interferon-gamma measured in the wells stimulated by the M tuberculosis-specific antigens as follows: positive if the interferon-gamma concentration in either of the antigen wells was ≥0.35 IU/mL; negative if the concentrations in both antigen wells were <0.35 IU/mL and the concentration in the positive control well was ≥0.5 IU/mL; and indeterminate if the concentration in both antigen wells was <0.35 IU/mL and the concentration in the positive control well was <0.5 IU/mL in the positive-control well. The technicians performing the whole blood interferon-gamma assay had already been trained by the manufacturers and were unaware of the subjects' tuberculosis status in this study.The whole blood interferon-gamma assay was performed using the QuantiFERON-TB-2G  as described elsewhere We measured the protein level of interleukin-10 in whole blood incubated overnight with antigens, using an ELISA kit . This assay also involved the same two steps; after the blood samples were separately incubated with CFP-10 and ESAT-6 and the nil and mitogen (phytohemagglutinin) controls for 18 hours at 37°C, the concentration of interleukin-10 in the four plasma samples from each subject was determined using an ELISA. The detection limit for interleukin-10 was <0.1 pg/mL. The results for ESAT-6 and CFP-10 were expressed as the detected concentration of interleukin-10 minus the concentration of interleukin-10 in the respective nil control plasma. This blood assay was also performed in a blind manner.Agreement between the tests was quantified using the κ statistic. A logistic regression analysis for positive TST results (induration≥15mm) and for positive blood assay results was performed to adjust the potential confounding factors including age, prior tuberculin skin test, number of BCG scars, student category and increase in the interleukin-10 concentrations when exposed to either of the antigens. The Mann-Whitney U test was used for comparisons among groups. Because our goal was to recruit all healthcare students commencing clinical training, we did not determine a formal sample size. All p-values were two-sided, and significance was set at a p-value of ≤0.05.Of the 536 students to whom the outline of this study was announced , 371  had an induration diameter of ≥5 mm, 124 (59.9%) had an induration diameter of ≥10 mm, and 57 (27.5%) had an induration diameter of ≥15 mm. The distribution of induration diameters is displayed in Regarding the whole blood interferon-gamma assay , five suvs. <15 mm) was not affected by the interferon-gamma concentration after exposure to either of the antigens , but was influenced by the number of needle scars from the BCG vaccination . In contrast, the whole blood assay result was not affected by the number of BCG scars .Considering abovementioned two subjects would have previous episodes of tuberculosis infections (the first case with the strong TST reaction and the third case with the positive blood test result), the specificity of blood assay was calculated as 96.6% (198/205) . RegardiThis assay was assessable for all 207 subjects. The median baseline concentration of interleukin-10 (negative control) was 2.9 pg/mL (range: 0.2 to 39.9 pg/mL). This value was not associated with the baseline concentration of interferon-gamma (negative control) (median baseline interleukin-10 levels: 2.8 vs. 3.0 pg/mL for the low (<0.07 IU/mL) and high (≥0.07 IU/mL) baseline interferon-gamma concentrations, respectively; p = 0.263; Historically, infection control policies in many hospitals have recommended pre- and post-employment routine screening of healthcare workers using the TST To overcome the high false-positive TST results, several studies have been conducted to assess the specificity of interferon-gamma assays in low-risk healthcare students without active tuberculosis In contrast, Pai et al. recently showed concordant results between the two tests in Indian medical and nursing students at the start of their clinical studies One of our limitations is that we did not fully assess the sensitivity of the blood assay because of few subjects with active tuberculosis. The recent meta-analysis revealed the sensitivity of this assay did not seem to be significantly higher than that of TST despite its high specificity We should note another obvious limitation in our study-the possibility of bias due to a fairly high non-response rate. Only 207 (39%) of 536 students agreed to undergo both tests, and this may have introduced a selection bias that affected the positive TST and blood assay rates. Thus, our results should be carefully interpreted.Mycoplasma hyopneumoniae surface antigen P71 alone produced higher levels of interleukin-10, while those vaccinated with an ESAT-6:P71 fusion protein secreted higher levels of interferon-gamma but failed to produce interleukin-10 Interleukin-10 was first detected based on its cytokine synthesis inhibitory activity, mainly on macrophages In conclusion, we found in this prospective study that the results of an interferon-gamma assay were quite discordant with those for the TST in a medical university setting, probably because of the influence of BCG vaccination on the TST results. Given this unavoidable influence of BCG vaccination and the relatively low estimated prevalence of tuberculosis infection in Japan, the blood assay seems to be useful as a baseline tuberculosis screening test for Japanese healthcare students, despite the need for further researches. Additionally, we report, for the first time, that the interleukin-10 level after exposure to specific antigens was inversely associated with the interferon-gamma level in the reported population.


This investigation was to evaluate the Patients with known or suspected bronchial carcinoma were randomized to receive 200 mg 5-ALA via inhalation 1, 2, 3, 4 or 6 hours before flexible fluorescence bronchoscopy was performed. Macroscopically suspicious areas as well as areas with visually detected porphyrin fluorescence and normal control sites were measured spectroscopically. Biopsies for histopathology were obtained from suspicious areas as well as from adjacent normal areas.Fluorescence bronchoscopy performed in 19 patients reveals a sensitivity for malignant and premalignant changes (moderate dysplasia) which is almost twice as high as that of white-light bronchoscopy, whereas specificity is reduced. This is due to false-positive inflammatory lesions which also frequently show increased porphyrin fluorescence. Malignant and premalignant alterations produced fluorescence values that are up to 5 times higher than those of normal tissue. According to the pharmacokinetics of porphyrin fluorescence measured by spectroscopy, the optimum time range for 5-ALA application is 80–270 min prior to fluorescence bronchoscopy, with an optimum at 160 min.According to our results we propose inhalation of 5-ALA 160 min prior to fluorescence bronchoscopy, suggesting that this time difference provides the best tumor/normal tissue fluorescence ratio. In-vitro experiments show tumor/normal tissue fluorescence ratios best between 110 and 160 min after exposure to 5-ALA I = Intensity [spectroscopically measured value]Spectroscopy was performed in all macroscopically suspicious areas as well as in areas showing porphyrin fluorescence. Each measurement was repeated three times. In addition, biopsies were obtained from these areas. As a control, adjacent non-suspicious areas were also analyzed spectroscopically and biopsied. The histological results of the biopsies were categorized as "Normal", "Inflammation", "Metaplasia", "Dysplasia Grade I-III " or "Malignant". Up to "Mild Dysplasia" the findings were classified as "Benign". All other findings were classified as "(Pre)Malignant". The application-time dependent spectral PPIX values according to equation 1 were fitted for the benign (≤ mild dysplasia) and the (pre)malignant (≥ moderate dysplasia) histologic findings separately. The fit function used was a normal distribution applied to a logarithmic time scale. The two histological ensembles were determined and further analyzed with the Mann-Whitney rank sum test.Nineteen patients were investigated. Basline characteristics of all patients are displayed in table Figure When tumor tissue is compared to normal tissue, a reduced autofluorescence, but a marked increase in PPIX fluorescence becomes evident. Sensitivity, specificity, negative predictive values, and positive predictive values were calculated from the visual ratings of the findings obtained by white-light and fluorescence bronchoscopy in comparison to histology Figure . FluoresEventually the calculated fluorescence values were plotted against the time between 5-ALA application and bronchoscopy Figure . It is dIn contrast to white-light bronchoscopy, pharmacologically induced fluorescence offers certain advantages. The present data provide evidence that the pharmacologically active process of 5-ALA uptake and metabolism produces a higher sensitivity than white-light bronchoscopy alone. However, this advantage is partly compensated by a reduced specificity, since e.g. some areas of inflammation or metaplasia can generate false-positive results. In this context the issue of "per lesion analysis" has to be taken into consideration since it may represent a potential flaw in the statistical evaluation concerning sensitivity, specificity and predictive values, as impressively demonstrated by Chang et al. . As onlyin-vitro studies with co-cultures, best fluorescence intensities were to be expected between 110 and 160 min after inhalation of 5-ALA [According to of 5-ALA . Our resThe observed heterogeneity of 5-ALA-induced fluorescence intensity in premalignant and malignant changes may be a distinctive feature of 5-ALA metabolism as well as the patterns of tumor invasion. This was also found in experiments with co-cultures, even after correction for tumor cell density . Correla5-ALA-supported fluorescence bronchoscopy enables an increased sensitivity in the bronchoscopic detection of endobronchial malignant and premalignant changes. The clinical implication of this method is the possibility to discover very early-stage lung cancer, in order to markedly improve healing rates and prognosis. With 160 min we propose an optimized time-point in the application of 5-ALA prior to the performance of fluorescence bronchoscopy. In this context, this study can contribute importantly to the efficiency of fluorescence bronchoscopy, particularly with regard to the in-vivo kinetics of 5-ALA. Clinical trials, however, will have to evaluate the significance and the clinical relevance of this method. Of particular interest will be the comparison with autofluorescence bronchoscopy and, especially, whether the addition of inhaled 5-ALA can further improve this technique, since a large multicenter trial has recently shown a benefit for autofluorescence bronchoscopy over white light bronchoscopy .The author(s) declare that they have no competing interests.HH carried out the bronchoscopic examinations, participated in spectroscopy and drafted the manuscript. JP carried out all spectroscopic measurements, took part in writing the manuscript and performed the statistical analysis. HS, RB, FG and RMH conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Self-generated movement is a key aspect of cellular life. From a single-celled amoeba hunting for food or mates to a human neutrophil finding and killing an infectious microbe, cells use many of the same molecular components to move.Directed motility results from asymmetric activities in the cell, as localized signaling networks produce different physical and morphological changes at a cell's leading and trailing edge. This type of polarity occurs when chemical signals—associated with food, pheromones, bacteria, or antigens, for example—trigger and amplify internal signaling cascades that recruit proteins at polar ends of the cell to organize different actin structures at each end.To modify its actin cytoskeleton in response to these chemoattractants, a fast-moving cell like the neutrophil must make one set of actin assemblies in front and another, completely different set in the rear, using different signaling pathways at each site. At the front, actin-rich structures form protrusions that project the cell forward. At the rear, actin–myosin contractile complexes retract the cell's trailing edge, helping it keep pace with the leading front edge. These structures and signaling pathways must be segregated so the assemblies can move in a coordinated manner. Segregation is reinforced by signaling pathway feedback loops, which in turn are likely controlled by specifically targeted regulatory protein complexes. Many questions remain, however, about the identity and function of the proteins that generate and maintain this polarity during chemotaxis. In a new study, Orion Weiner, Henry Bourne, Marc Kirschner, and their colleagues describe a family of protein complexes that regulate polarity circuits at the leading edge of migrating neutrophils.One important regulator of polarity and cytoskeletal remodeling during chemotaxis is a protein called Rac. Neutrophils use Rac to control the positive feedback loops that stabilize its leading edge and Rho to oversee the myosin-driven contractions that bring along the rear. Rac induces actin polymerization under the direction of WAVE proteins, which mediate actin rearrangements and cell migration in processes as diverse as development and tumor metastasis. Rac and WAVE indirectly interact through a multicomponent protein complex called the WAVE regulatory complex.Working with a neutrophil-like cell line, the authors focused on one of these components of the WAVE regulatory complex, Hem-1. The authors tagged Hem-1 with fluorescent protein to monitor its movements. In the absence of a chemical signal, Hem-1 is seen throughout the cytosol. But when a chemoattractant stimulates the cell, the proteins rapidly travel to the periphery, then accumulate at the leading edge as the cells polarize.The authors suspected that Hem-1 complexes might regulate other proteins in addition to WAVE. If so, Hem-1 should exist in higher concentrations than WAVE2 (the WAVE protein most abundant in neutrophils), exist in distinct biochemical milieus, and associate with other proteins. Biochemical assays confirmed these predictions and showed that over 60% of Hem-1 did not associate with WAVE2, making a strong case that Hem-1 targets other proteins.To find out what those targets might be, Weiner et al. isolated complexes containing Hem-1 to identify their components. As expected, they retrieved other members of the WAVE regulatory complex, but also found “excellent candidates” for regulating the positive and negative feedback loops required for neutrophil polarity, including proteins that could keep myosin complexes from acting in the front of the cell. The most abundant candidates likely associate with Hem-1 complexes that do not contain WAVE2, the authors concluded, because the majority of Hem-1-containing complexes do not associate with WAVE2.Reducing Hem-1 concentrations in the neutrophils produced defects in actin polymerization, cell polarity, and morphology in the depleted cells, demonstrating Hem-1's essential role in these processes. Given Rac's importance in actin polymerization and cell polarity orchestration, the authors analyzed its activity in Hem-1-depleted cells. They found that Hem-1 complexes, which act downstream of Rac, are also required for Rac activation. This is consistent with a Rac positive feedback loop in which Rac increases its own activation via Hem-1 complexes. Positive feedback loops are highly conserved elements in cell polarity circuits from yeast to slime molds to neutrophils. Hem-1 supports this feedback loop through mechanisms that operate independently of and in addition to WAVE-regulated actin assembly. The authors go on to show that Hem-1 also protects the leading edge from the contractile influence of myosin.Altogether, these results suggest that Hem-1 protein complexes function to organize the leading edge of migrating neutrophils. Here, these versatile complexes steer multiple inputs, such as Rac, to the appropriate output at the leading edge—whether it be stimulating WAVE-regulated actin polymerization, promoting feedback loops that stabilize the protrusions, or segregating activated myosin to ensure proper chemotaxis. The authors' next round of experiments will investigate the upstream signals that regulate the targets of Hem-1 complexes and help the cell reach its destination.


Accurate Recently, we demonstrated the appealing performance of SVRMHC, an SVR-based quantitative modeling method for peptide-MHC interactions, when applied to three mouse class I MHC molecules. Subsequently, we have greatly extended the construction of SVRMHC models and have established such models for more than 40 class I and class II MHC molecules. Here we present the SVRMHC web server for predicting peptide-MHC binding affinities using these models. Benchmarked percentile scores are provided for all predictions. The larger number of SVRMHC models available allowed for an updated evaluation of the performance of the SVRMHC method compared to other well- known linear modeling methods.SVRMHC is an accurate and easy-to-use prediction server for epitope-MHC binding with significant coverage of MHC molecules. We believe it will prove to be a valuable resource for T cell epitope researchers. Major histocompatibility complex molecules (MHCs) are polymorphic glycoproteins residing on cell membranes. In the cellular immune system, MHC molecules bind small peptide fragments, or epitopes, derived from antigens and host proteins, and present them to T cells, thus inducing downstream immune system responses. Computational prediction and modeling of epitope-MHC binding is of considerable interest because it can greatly facilitate epitope screening, with tremendous concomitant savings in time and experimental effort. Over the past ~15 years, many such computational methods have been proposed (for a comprehensive review see -5) or maWe recently developed SVRMHC, a support vector machine regression (SVR)-based method for modeling peptide-MHC binding. SVRMHC is a sequence-based quantitative method that makes predictions about the exact binding affinity of the peptide. As a kernel-based approach, SVRMHC demonstrates the excellent modeling performance enjoyed by other SVM-based methods such as SVMHC  and HLA-Subsequently, we constructed and validated SVRMHC models for over 40 MHC alleles. In this report, we describe the SVRMHC server, which predicts T-cell epitopes using these models. In addition to the predicted binding affinity, the SVRMHC server calculates a percentile score for each input peptide benchmarked against a pool of ~528,500 peptides. These were derived from 1,000 proteins picked randomly from the Swiss-Prot database. Construction of a large number of SVRMHC models has allowed a better comparison to be made between the SVRMHC and the additive method, which we discuss briefly in this report.SVRMHC model construction was carried out in locally developed C and Matlab programs. LibSVM was used for SVR-related implementation . The webq2 (for class I models) or cross-validated r (for class II models). The configuration that offers the highest prediction performance was chosen for the final model. LOO (leave-one-out) or 7-fold cross-validation was used when assessing the performance of class I models, and 5-fold cross-validation was used when evaluating class II model performance. The final model set included 39 nonamer models, together with 2 octamer models (for H2-Kb and H2-Kk) and 1 decamer model (for A*0207).The data used for constructing the SVRMHC models was obtained from the AntiJen database  March 3 and 2. Tr) no longer improved, or when an iteration threshold was exceeded (this number was set to 4).The class II SVRMHC model construction was more complicated than the class I case because the longer input sequences required alignment to the model's nonameric "core sequence". We took an approach similar to the iterative self-consistent (ISC) strategy described earlier . First, r scores. Therefore, "mean" alignment was implemented in the SVRMHC server.Three different sequence alignment protocols – "mean", "max", and "combi" – were used in  when makIn ROC-based comparisons, previous SVRMHC models out-performed several well-known methods when identifying strong binding peptides . This suAt the SVRMHC prediction server, the user can paste a protein sequence (either as plain text or in FASTA format) into the "Input Sequence" text area, or upload a local sequence file to the server. The user then selects the target MHC allele. Optionally, the user can enter either a pIC50 threshold or a percentile score threshold. The prediction results  will be displayed either in the order in which they occur in the input protein sequence or sorted as a list in descending order of predicted pIC50 values.Of the 42 final SVRMHC models included in the server (see Tables q2 than the "additive method" models before outlier removal [In our previous report , we show removal ,22. Howe removal .SVRMHC server is an accurate and easy-to-use server for predicting epitope-MHC binding. It offers significant coverage in terms of MHC molecules and this study has reconfirmed model performance. SVRMHC will continue to expand as more binding data becomes available. We believe the SVRMHC server will become a valuable resource for researchers interested in predicting T cell epitopes.. Questions and comments are welcomed through the site.SVRMHC server is publicly accessible from the URL JW carried out some of the SVRMHC model construction work, most of the benchmarking and statistic analysis, and produced all compiled models for server construction. WL constructed the majority of SVRMHC models, and performed analysis on model configurations. QX organized the binding data from AntiJen, and executed most of the additive model construction work for performance comparison with SVRMHC models. YR constructed the server web site. DRF provided the data for constructing the SVRMHC models, gave significant assistance and advice on essential issues of the model construction, and helped to write the manuscript. TL conceived of and coordinated the study, performed some of the analysis, and drafted and finalized the manuscript. All authors read and approved the final manuscript.


Immune dysfunction can provoke (multiple) organ failure in severely injured patients. This dysfunction manifests in two forms, which follow a biphasic pattern. During the first phase, in addition to the injury by trauma, organ damage is caused by the immune system during a systemic inflammatory response. During the second phase the patient is more susceptible for sepsis due to host defence failure . The pathophysiological model outlined in this review encompasses etiological factors and the contribution of the innate immune system in the end organ damage. The etiological factors can be divided into intrinsic  and extrinsic components . Of all the factors, the intervention load is the only one which, can be altered by the attending emergency physician. Adjustment of the therapeutic approach and choice of the most appropriate treatment strategy can minimize the damage caused by the immune response and prevent the development of immunological paralysis. This review provides a pathophysiological basis for the damage control concept, in which a staged approach of surgery and post-traumatic immunomonitoring have become important aspects of the treatment protocol. The innate immune system is the main objective of immunomonitoring as it has the most prominent role in organ failure after trauma. Polymorphonuclear phagocytes and monocytes are the main effector-cells of the innate immune system in the processes that lead to organ failure. These cells are controlled by cytokines, chemokines, complement factors and specific tissue signals. The contribution of tissue barrier integrity and its interaction with the innate immune system is further evaluated. Trauma is one of the major causes of mortality in people under the age of 50 in the Western world. Patients die as a direct consequence of their sustained injuries, or by the additional damage caused by subsequent immune reactions . About 5Multiple organ failure after trauma has a multifactorial etiology, which can be divided in endogenous and exogenous factors. Endogenous factors, such as genetic predisposition and physical condition form the basis of the patient s susceptibility for the development of organ failure. Recent studies have shown that genetic variations (e.g. TNF-α polymorphisms) are strongly associated with the development of organ failure . ExogenoTo restore the equilibrium of the excessive pro-inflammatory reaction, an anti-inflammatory response is evoked. In a propitious case, homeostasis is achieved. However, an overreaction of the anti-inflammatory response can lead to either a compensatory anti-inflammatory response (CARS), or a mixed antagonist response (MARS) . In the Tissue damage leads to the activation of neutrophils and macrophages . HemorrhPriming is the result of pre-exposure to priming agents, like granulocyte macrophage colony stimulating factor (GM-CSF) or tumor necrosis factor (TNF-α) ,16. Thesin vitro stimulation) was found between 3 and 24 hours after trauma [The increased oxidative burst (a cytotoxicity associated response) is necessary to prepare the neutrophils for invading micro-organisms. This increased functional response in the form of oxidative radical production correlates with the incidence of SIRS and MOF . It is tr trauma . An elevr trauma . This inr trauma . The harRolling is regulated and controlled by selectins. These proteins undergo interactions with ligands on endothelial cells, which slow down the leukocytes at this surface . E-selecIntegrins are involved in the adhesion of leukocytes to the endothelium. The integrin amβ2, or MAC-1 (CD11b/CD18) and the ligand ICAM-1 (intercellular adhesion molecule 1) form a high affinity stationary connection between leukocyte and endothelium. This is in contrast to the low affinity, reversible binding of selectins. Functional integrins are only expressed upon activation of the neutrophil and are necessary for an adequate transmigration process . An incrICAM-1, normally expressed by activated entothelium, also exists as a soluble factor in serum (sICAM-1) and increased concentrations in septic patients correlate with the incidence of organ failure and mortality ,29. ExprBillions of neutrophils are produced by the bone marrow on a daily basis . NeutropNeutrophils are essential in the pathophysiology of trauma-related organ failure . BlockinNeutrophils are important in the first response to injury, as they form the first natural immunological defense against micro-organisms and occur within 10 minutes after injury is sustained. Subsequent to the initial responders, monocytes/macrophages are recruited. These cells orchestrate the mechanisms involved in wound healing . They fuDown-regulation of MHC-II expression leads to decreased antigen presentation capacity and therefore higher susceptibility for infectious complications. Several authors have shown MHC-II suppression after trauma, which correlated with the incidence of infectious complications. MHC-II suppression on monocytes and macrophages is considered to be one of the most important features of immune suppression after injury. Some authors have suggested CARS to be defined as less than 30% expression of MHC-II on monocytes .In past years many studies focused on the relation between pro- and anti-inflammatory cytokines and the development of SIRS and CARS. Tissue damage causes the endothelial cells, fibroblasts, lymphocytes and tissue-macrophages to produce these cytokines . At firsTNF-α and IL-1β are situated at the beginning of the pro-inflammatory cascade Fig. . IL-1β aBoth IL-1β and TNF-α stimulate the production of IL-6 and IL-8. IL-8 is an important chemokine in the cascade that leads to leukocyte recruitment and activation in the tissues . Productin vitro rapidly released to microbial products (both lipopolysaccharide and Gram-positive exotoxins) [in vivo as high circulating levels of MIF were found in septic and septic shock patients, in contrast to normal levels in non-septic traumapatients [Macrophage migration inhibitory factor (MIF) is a pleiotropic molecule exerting its functions as an anterior pituitary hormone, a pro-inflammatory cytokine and high activity enzyme. It is produced abundantly by monocytes/macrophages and acts in an autocrine/paracrine manner to up-regulate and sustain the activation responses of diverse cell types . MIF is otoxins) . This ispatients . In addipatients . MIF indMIF overrides the anti-inflammatory actions of glucocorticoid and acts via the stimulation of pro-inflammatory cytokines like TNF-α, IL-1β and IL-8 via the NF-κB pathway. MIF prevents apoptosis by reduction of the p53 tumor suppressor gene. Therefore, high concentrations of MIF lead to a sustained pro-inflammatory response and delayed apoptosis of cells of the innate immune system. High concentrations of MIF have been found in the alveolar spaces of patients with ARDS . Those ain vitro 20 hours after stimulation. Increased levels of HMGB-1 result in the disruption of endothelial barrier functions, leading to vascular leakage and tissue hypoperfusion, similar to that observed in sepsis. In vivo increased levels of HMGB-1 are shown in patients with severe sepsis [High-mobility group box (HMGB)-1 was originally identified as a nuclear DNA-binding protein that functions as a structural cofactor for proper DNA-transcriptional regulation and gene expression . Recent e sepsis . In expee sepsis . In an ee sepsis . Neutrale sepsis .IL-10 plays an important role in the anti-inflammatory response. This protein is produced simultaneously with the pro-inflammatory cytokines, but peaks hours later. One of the functions of IL-10 is the negative feedback on the production of TNF-α, IL-6 and IL-8. The cytokine IL-10 plays a pivotal role in the suppression of monocyte function as it directly decreases MHC-II expression . IL-10 cIn vitro is shown that C5a regulates two important aspects of neutrophil function; i) adhesion associated processes and ii) cytotoxic associated processes [Complement is a collection of proteins, which are involved in the protection against micro-organisms. It is one of the most preserved defense mechanisms during the evolution of the immune system. Next to activation by immune complexes complement can bind conserved bacteriological compounds  and altered self-products (e.g. free DNA) via mannose binding lectin, ficolins or complement factor C1q . Complemrocesses . Complemrocesses . The accTrauma not only activates the innate immune response, but also alters the barrier integrity of several organs. Intramedullary osteosynthesis of femur fractures is thought to stimulate the innate immune response on a systemic level and is associated with an increased incidence of ARDS . On the A cut-off point of >800 pg/ml IL-6 has been proposed as a prognostic marker and has been suggested for immunomonitoring in the damage control strategy. Unfortunately, at present no scoring system or prognostic tool is conclusive enough to adequately predict an adverse outcome on an individual level. The complexity of organ failure and the often ambiguous role of the different factors prevents a clear cut target for therapy. Many studies investigated individual mediators or effectors, which limits the interpretation of effector function in the tissues. Furthermore, cytokines often have crosstalk or cumulative effect and insight in the group effect of cytokines and chemokines would provide more accurate information about the net effect.The scoring systems ought to be used to define the appropriate therapy. Damage control surgery and damage control orthopedics are currently used strategies to limit the incidence of organ failure after trauma ,77. TimiSeveral studies have shown a relationship between the severity of trauma and the resulting immune response . The injARDS: Acute respiratory distress syndromeCARS: Compensatory anti-inflammatory response syndromeCRP: C-Reactive proteinGM-CSF: Granulocyte macrophage colony stimulating factorHMGB-1: High mobility group box 1ICAM-1: Intercellular adhesion molecule 1IL-n: Interleukin-nISS: Injury Severity ScoreMAC-1: Macrophage 1MARS: Mixed antagonist response syndromeMHC-II: Major histocompatibility complex IIMIF: Macrophage migration inhibitory factorMODS: Multiple organ dysfunction syndromeMOF: Multiple organ failureROS: Radical oxygen speciessICAM: Soluble ICAMSIRS: Systemic inflammatory response syndromeTGF: Tumor growth factorTNF-α: Tumor necrosis factor αThe author(s) declare that they have no competing interests.FH participated in the design of the review and drafted the manuscript.LK revised the manuscript critically on the content of effector processes till the final version was reached.GR revised the manuscript critically on the mediator processes till the final version was reached.LL participated in the design of the review and revised the manuscript till the final version was reached.The authors have read and approved the final manuscript.