New Kinase Family 4) in the kinome with unknown substrates and biological functions. Various high throughput studies indicate that STK35 could be involved in various human diseases such as colorectal cancer and malaria.The human kinome containing 478 eukaryotic protein kinases has over 100 uncharacterized kinases with unknown substrates and biological functions. The Ser/Thr kinase 35  is a member of the NKF 4 . Since it is the first of further homologous kinases we termed it as STK35L1. The STK35L1 protein (58 kDa on SDS-PAGE), but not STK35 (44 kDa), was found to be expressed in all human cells studied  and was down-regulated after silencing with specific siRNA. EGFP-STK35L1 was localized in the nucleus and the nucleolus. By combining syntenic and gene structure pattern data and homology searches, two further STK35L1 homologs, STK35L2 (previously known as PDIK1L) and STK35L3, were found. All these protein kinase homologs were conserved throughout the vertebrates. The STK35L3 gene was specifically lost during placental mammalian evolution. Using comparative genomics, we have identified orthologous sets of these three protein kinases genes and their possible ancestor gene in two sea squirt genomes.In this study, we found that the previously published coding sequence of the STK35 gene and termed it as STK35L1. We identified a new third STK35-like gene, STK35L3, in vertebrates and a possible ancestor gene in sea squirt genome. This study will provide a comprehensive platform to explore the role of STK35L kinases in cell functions and human diseases.We found the full-length coding sequence of the  Eukaryotic protein kinases that phosphorylate serine/threonine (Ser/Thr) or tyrosine (Tyr) residues are the largest superfamily of enzymes, comprising ∼1.7% of all human genes www.kinase.com; 1, 7, 8]. In Kinomer database, the best match for the STK35 kinase sequence is within the group TKL (Tyrosine Kinase Like) STK35 and its homolog PDIK1L (PDLIM1 (CLP36) Interacting Kinase 1 Like) shares 69% protein sequence identity in kinase domain. They are members of the NKF4 (New Kinase Family 4) Ser/Thr kinases (STK) family and classified in group “Other” in the human kinome [STK35 has also been named as Clik1 (CLP36 Interacting Kinase 1) based on one study that showed an association of STK35 with CLP36 after overexpression of both proteins in osteosarcoma cells Plasmodium berghei sporozoites The biological function and substrates of STK35 are not known. The STK35 gene was found upregulated specifically in colorectum cancer In the present study, we describe the correct genomic organization and coding sequence of STK35, now renamed STK35L1, which was localized in the nucleus and nucleolus. We identified a new kinase subfamily containing three STK35L genes conserved in vertebrates. The present comprehensive study will provide a platform to further analyze the functional role and regulation of the STK35L kinases.Previously, it has been shown that myc-tagged Clik1 (STK35) translocated from the nucleus to actin stress fibers upon coexpression with EGFP-CLP36 in U2OS osteosarcoma cells http://symatlas.gnf.org) To investigate STK35 mRNA expression in different cell types, total RNA was isolated from endothelial, HEK and HeLa cells and analyzed by RT-PCR. A fragment of the expected size for STK35 (273 bp) was amplified with STK35 specific primers  in all cTo analyze the expression of endogenous STK35 protein in cells, rabbit polyclonal peptide antibodies against a C-terminal STK35 peptide were raised. To test the specificity of the antibody, cell lysates of untransfected and endothelial cells transiently transfected with plasmids encoding for EGFP-tagged STK35 proteins were used as negative and positive controls respectively. The antibody recognized the over-expressed proteins (∼72 kDa for EGFP-STK35) and a prominent band at about 58 kDa specifically whereas the pre-immune serum of the same rabbit did not detect these bands (data not shown). Importantly, we could not observe in transfected and non-transfected cells a band at 44.6 kDa, the theoretical calculated molecular weight of endogenous STK35. During the course of the study a commercial polyclonal goat anti-STK35 peptide antibody against the C-terminal amino acid sequence (ELETRMDQVTCAA) of STK35 became available. This antibody recognized the EGFP-STK35 fusion proteins which contain the C-terminal part of the protein specifically  but not a truncated construct lacking this sequence (EGFP-STK35Δ72-400) . The bloSTK35 gene splice variants from data set of Ensembl and NCBI human build. However, no splice variants co-relation can be established. Therefore, we undertook a careful genome analysis of the STK35 locus.To analyze further, nuclear lysates of endothelial, HEK and HeLa cells were prepared. A prominent band at 58 kDa was recognized by the anti-STK35 antibody in nuclear extracts of HEK, HeLa and endothelial cell  and macrhttp://www.genomatix.de/). We used STK35 cDNA (NM_080836) sequence for complete analysis of the STK35 locus in human genome. The STK35 gene is located on chromosome 20 subregion p13 (www.softberry.com). We identified a start codon surrounded by a Kozak consensus sequence and an open reading frame encoding a protein of 534 amino acids instead of the 401 amino acids predicted earlier for STK35  in an inverse manner . By homology search in Gallus genome, we found that the STK35L1 gene was truncated and the exon 1 and 2 were missing. The expression of the truncated STK35L1 gene in Gallus is supported by an mRNA sequence submitted in the gene bank (BU403355).Gene synteny is a useful tool for predicting functional interaction and relatedness among genes within cluster. Selective processes are essential to preserve the organization of these clusters in closely related species. In the human genome, the s genome , we foune manner . MoreoveSTK35L2 (previously known as PDIK1L) is localized on chromosome 1 in human. In mammals, STK35L2 was conserved differently: the locus is flanked by a marker tetrad EXTL1-TRIM63-PHARAH2-STMN1  genome, STK35L3 is gene flanked by PNOC and ELP3  mammals  Strongylocentrotus purpuratus (sea urchins) Drosophila melanogaster (flies) Caenorhabditis elegans (worms) STK35L1 like gene in two genomes of sea squirts - Ciona intestinalis (JGI Ciona genome accession id -fgenesh3_pg.C_chr_07q001099/Ensembl - ENSCINP00000006268) and Ciona savignyi (Ensembl–ENSCSAV00000013846) with identity of ∼45% with human STK35L1 (STK35 kinase gene (SPU_003710) had been reported. However by careful comparison of the protein sequence of sea urchin kinase with the STK35L1 protein, we found that the sea urchin kinase shared only 10% identity with STK35L1 in the kinase domain. To confirm whether the sea urchin kinase is clustered together with STK35L1 homologs or not in the evolutionary tree, we created a phylogenetic tree of randomly selected representative kinases with STK35 homologs, sea urchin and Ciona STK35L1 like kinases  in comparison to other kinases. An ancestor gene of STK33 is found in sea anemone genome To compare the evolution of  created . This fiSTK35L1 homologs were determined. STK35L1 homologs have variable exon-intron organization patterns . This corroborates that STK35L1 homologs do not share orthology with other serine threonine kinases.Gene structures attribute phylogenetic evidence to genes and aid in assigning origin of given genes. To compare gene structures, exon-introns architectures of different patterns . STK35L1introns’  and ‘twointrons’  patternsintrons’  pattern.om Ciona , suggestSTK35 gene (code for 401 amino acids) is incomplete. The newly identified sequence of STK35 gene codes for a protein of 534 amino acids which was termed as STK35L1. By western blot analysis of endothelial cell lysates using two different specific polyclonal anti-STK35 antibodies, we found that STK35L1 was expressed in all mammalian cells studied. By using the siRNA approach, we could confirm that the 58 kDa protein is endogenously expressed STK35L1 protein. In contrast, no evidence for the expression of STK35 was obtained by western blotting using the antibodies directed against the C-terminal peptide of STK35.In the present study we found that the previously published coding sequence of the Endogenous STK35L1 was found to be localized in the nucleus. Moreover, EGFP-STK35L1 was also localized in the nucleolus suggesting a role in gene regulation. A previous report suggested that STK35 may have role in actin cytoskeleton rearrangement as the interaction of the co-transfected STK35 and CLP36 proteins led to relocalization of Clik1 from the nucleus to actin stress fibers STK35 locus in human genome by using various approaches. Based on CAGE tags, we found three transcription start regions within a conserved region of 1035 bp upstream of the previously defined STK35 start codon. CAGE is a high-throughput method to measure expression levels by counting large amounts of sequenced capped 5′ ends of transcripts, termed CAGE tags Gallus genomic sequence is unable to solve this issue; it can be only clarified with further improvement of genomic assembly of chicken.The identification of the full protein coding gene sequence is obviously crucial for the determination of the biological function of the kinase. Therefore, we analyzed the STK35L2 gene is present in all vertebrates in conserved micro-environment without any exceptions. One of the most exciting outcomes of the present analysis is the identification of a third member in this family as STK35L3. Its orthologs are found from fish to marsupial mammal. However, this gene is lost in placental mammals after divergence of marsupial and placental mammals at ∼173–190 MY ago. Placental mammals have a conserved syntenic organization, but no STK35L3 gene. It gives a hint that this gene was lost due to positive selection and might be involved in physiology related to laying eggs or egg development.STK35L1 is identified in two Ciona species that are the closest living relatives of vertebrates STK35 in sea urchin. Furthermore, no genuine homologs of STK35L1 in worms, flies, echinoderms and lancelets were found even though these groups of invertebrate models possess easily identifiable ser/thr kinases. Thus, it is most likely that the STK35L1 homologs evolved before the divergence of the vertebrate lineage/Ciona lineage about 550 MY ago.The three sets of STK35L1 homologs have independent gene structure patterns, further supporting their orthologous status. Any other known ser/thr kinase family member does not share these patterns of exon-intron organization. An ancestor of the Furthermore, this comprehensive study will provide a platform to further analyze the functional role and regulation of the STK35L1 kinase, and gene transcription in various physiological and patho-physiological conditions.Oligonucleotides and siRNAs were synthesized from MWG Biotech AG . Rabbit polyclonal anti-STK35 antibody was generated against the C-terminal peptide 507–521 (DMLANNPQDRPDAF) were generated by Biotrend Chemikalien GmbH . Polyclonal goat STK35 antibody was from Everest Biotech . Anti-EGFP antibody was kindly gifted by Prof. Schleicher .STK35L1 gene . First-strand cDNAs were synthesized with Omniscript reverse transcriptase kit  using random hexamer primers as per manufacturer's protocol. cDNAs from these cell types were subjected for gene expression analysis using primers. These primers were designed from different regions of the 5L1 gene .STK35L1 gene was obtained using a multistep cloning strategy. In the first step PCR, we amplified 3′ end of STK35L1 (bp 400–1605) using primers Clik1_long400F and STK35R_HINDIII  as per the manufacturer's instruction. CLP36 gene was amplified as describe previously The full-length coding sequence of STK35 and ST35L1 was amplified by PCR from a cDNA pool of human umbilical vein endothelial cell total RNA. The PCR-amplified product of STK35 was cloned into XhoI and HindIII sites of pEGFP-C1 vector (Clontech) to obtain STK35 fused with EGFP. The full length of _HINDIII . In seco2, and 37°C in complete endothelial growth medium . In all experiments, human umbilical vein endothelial cells up to third passage were used. HeLa and HEK cells were grown in OptiMEM medium (Invitrogen) supplemented with 10% fetal calf serum.Human umbilical vein endothelial cells were obtained and cultured as described previously Endothelial cells were transfected using the nucleofaction transfection technology developed by Amaxa GmbH as per manufacturer's instructions.After 8–10 h of transfection, cells were washed fixed with 3.7% formaldehyde in PBS for 10 minutes at 4°C. For staining of the DNA, cells were incubated with Hoechst 33258 dye solution (1 µg/ml) in PBS for 10 minutes. Cells were observed with a Zeiss LSM510 confocal laser-scanning microscope. The microscope function was controlled by a light manager through the software LSM 510 META.Transfected or untransfected endothelial cells or nuclear lysates (prepared by using nuclei isolation kit from sigma as per manufacturer's protocol) of HEK, HeLa and endothelial cells were dissolved in an equal volume of 2× Laemmli buffer. Equal amounts of proteins in the samples were subjected to SDS-PAGE and then transferred to nitrocellulose membrane at 200 mAmp for 60 min at 4°C using the Mini Trans-Blot electrophoresis cell (Bio-Rad). Membranes were blocked with 5% (w/v) nonfat milk and incubated with the respective primary and secondary antibodies. The dilutions of the primary antibodies were: anti-STK35 (1∶250) and anti-EGFP (1∶250). The dilution of the secondary antibody (horseradish peroxidase-conjugated) was 1∶5000. The membranes were developed with SuperSignal West Pico chemiluminescent substrate (Pierce) and exposed to Hyperfilm (Amersham Biosciences).STK35L1/PDIK1L as query sequence from the different genomic databases  was used for initial analysis of STK35 gene in human genome. To ensure correct gene structures of all putative novel kinases, gene structures were predicted using GENSCAN www.softberry.com). Intron-exon structures were determined with aid of GENEWISE www.softberry.com).ElDorado tool from Genomatix GmbH (http://www.jgi.doe.gov/), Tetraodon genome browser at the Genoscope (http://www.genoscope.cns.fr/externe/tetranew/) and UCSC genome browser To verify the orthology, microsynteny across different genomes were analyzed using NCBI mapviewer, ENSEMBL genome browser Protein alignments of protein kinases were generated with CLUSTALX 1.83 STK35L1 and its exons in two species were carried out by Emboss tool ‘needle’at EBI .Pairwise protein alignments for All phylogeny trees were created with full length sequences of representative serine threonine kinases from different organisms with help of Neighbor Join (NJ) method Figure S1http://symatlas.gnf.org.Messenger RNA expression of STK35 in various human tissues and cells. The expression profile of STK35 in 79 human tissues is shown here. The expression data was obtained from (0.05 MB TIF)Click here for additional data file.Figure S2Multiple protein sequence alignment of human STK35L1 homologs from human (Hsap), opossum (Mdom) and its homolog from Ciona (Cint). Human STK35L1 kinase domain has 45.2% identity and 63.7% similarity with Ciona homolog. The position of kinase domain (based on human kinase)is indicated by blue line.(0.54 MB TIF)Click here for additional data file.Figure S3Expression analysis of STK35. Expression analysis of STK35. To verify the expression of STK35 mRNA, RNA and cDNA were prepared from endothelial cells. 455 bp and 755 bp PCR products (Lane 2 and 3) were amplified with STK35 specific primers from cDNA pools. No PCR product was amplified in negative control (reverse transcriptase polymerase was excluded during RT-PCR).(0.11 MB TIF)Click here for additional data file.Table S1List of the primers and their sequences.(0.04 MB DOC)Click here for additional data file.Table S2List of the genomes of selected species investigated in the present study.(0.04 MB DOC)Click here for additional data file.Table S3Ensembl accession identifiers of STK35L1, STK35L2 (PDIK1L), and STK35L3 genes in different organisms. * NCBI accession id. # Species specific paralogs.(0.12 MB DOC)Click here for additional data file.Table S4List of the Markers flanking different STK35L1, PDIK1L, and STK35L3.(0.04 MB DOC)Click here for additional data file.


Attention Deficit Hyperactivity Disorder (ADHD) is a costly and prevalent disorder in the U.S., especially among youth. However, significant disparities in diagnosis and treatment appear to be predicted by the race and insurance status of patients.This study employed a web-based factorial survey with four ADHD cases derived from an ADHD clinic, two diagnosed with ADHD in actual evaluation, and two not. Randomized measures included race and insurance status of the patients. Participants N = (187) included clinician members of regional and national practice-based research networks and the U.S. clinical membership of the Society of Teachers of Family Medicine. The main outcomes were decisions to 1) diagnose and 2) treat the cases, based upon the information presented, analyzed via binary logistic regression of the randomized factors and case indicators on diagnosis and treatment.ADHD-positive cases were 8 times more likely to be diagnosed and 12 times more likely to be treated, and the male ADHD positive case was more likely to be diagnosed and treated than the female ADHD positive case. Uninsured cases were significantly more likely to be treated overall, but male cases that were uninsured were about half as likely to be diagnosed and treated with ADHD. Additionally, African-American race appears to increase the likelihood of medicinal treatment for ADHD and being both African-American and uninsured appears to cut the odds of medicinal treatment in half, but not significantly.Family physicians were competent at discerning between near-threshold ADHD-negative and ADHD positive cases. However, insurance status and race, as well as gender, appear to affect the likelihood of diagnosis and treatment for ADHD in Family Medicine settings. Attention Deficit Hyperactivity Disorder (ADHD) is a wide-spread and behavioral condition in the U.S. It affects roughly 7% to 9% of the U.S. population aged 4-17 years . By one The data are unclear on the precise amount of ADHD diagnosis and treatment occurring in primary care settings, but it is fair to say that it is substantial. In fact, as much as 65% - 85% of initial ADHD diagnosis and treatment occurs in primary care settings (e.g. Pediatric or Family Medicine practices) ,12. OnceBoth the American Academy of Child and Adolescent Psychiatry and the American Academy of Pediatrics have issued formal guidelines for the diagnosis and treatment of ADHD -19. HoweIn addition to apparent variability in clinical practices, a wide array of social factors appear to cause disparities in diagnosis and treatment for ADHD, especially between races, genders, and socio-economic indicators -32. StudADHD diagnosis and treatment rates clearly fall along several socio-demographic lines. Gender, geography, wealth, and ethnicity all seem to play roles, in addition to race and insurance status ,30,34-38Given the apparent socioeconomic disparities that have been observed in ADHD diagnosis and treatment, as well as the particular issues that appear to be present when cases of suspected ADHD present in primary care offices, a vignette-based factorial web survey experiment designed to test the effects of two social factors on the likelihood of diagnosis and treatment of Attention Deficit Hyperactivity Disorder (ADHD) in children, as applied by primary care providers (PCPs), and focused upon Family Physicians (FPs), was conducted in a convenience sample of PCPs. This study has focused upon race and insurance status in an isolated fashion, as these two items may co-vary . The speA more complete description of the methods employed in this study is available elsewhere . HoweverWhite/African-American), (privately insured/uninsured) . At page bottom, immediately following the vignette, were three questions, asking the respondent to select a) a diagnosis from a set of four  and b) a treatment option, from a set of six . Once finished with the first vignette and set of questions, respondents were taken to randomized versions of Vignettes 2 through 4, with the same set of questions and options after each.The vignettes utilized in this experiment were derived from four cases seen at a local ADHD specialty clinic, with two of the cases nominally meeting criteria for ADHD upon formal evaluation and two cases of suspected ADHD that ultimately did not meet the criteria for diagnosis, upon evaluation. The four cases were intentionally distributed as two sub-threshold cases, 1 male, 1 female, and two "true" ADHD cases, again, 1 male, 1 female. These cases where selected by two psychologists specializing in ADHD diagnosis, who sent case information on anonymous abstracting forms. Information from the abstracting forms was utilized to create a 3-paragraph, narrative vignette describing each case. All four of the cases were derived from actual patients who had been psychometrically tested for the presence of ADHD after referral from an external source. Marginal, as opposed to "clear-cut," cases were used in order to isolate the effects of patient race and insurance status from large variations in presentation of symptoms from case to case.Some confidence in the content validity of the vignettes employed in this study may be taken from the fact that they were drawn from the facts of actual cases. Nevertheless, additional steps were taken to assure that the vignettes performed reliably when employed within the instrument, i.e. that they produced reasonably predictable diagnostic recommendations when read by multiple clinicians from a variety of relevant specialties. Additionally, the vignettes were modified iteratively until they were both reliable in eliciting appropriate responses from a beta panel of diagnosticians, as well as directionally accurate .Following the abstraction and vignette generation, each of the four vignettes were submitted to a panel of 18 primary care physicians and mental health experts blinded to the "true" diagnosis in each case in a draft version of the web-based instrument. Each rater selected a diagnosis and treatment from a set of options for each vignette and was asked to provide summary comments about what affected their decision in a text box following each vignette.The alpha panel test was conducted in May and June of 2008. Results were formally analyzed by rotating the matrix of responses so that each vignette represented a row and each "response" was the diagnosis assigned by each of the 18 respondents. Cronbach's Alpha split-half correlation was calculated for the matrix. Additionally, the comments entered into the text boxes were analyzed for appropriate content, e.g. that respondents were not openly declaring social biases or blatantly incorrect knowledge while diagnosing each case. Finally, panel respondents confidentially made recommendations on improving each vignette either in direct conversation, by e-mail, or via a secondary web interface. A second goal of the alpha test was to assess the pure functionality of the instrument and web interface, including the speed, data collection and delivery mechanisms, randomization engine, password functioning, etc.The instrument, vignettes, and collection interface were modified based upon initial data analyses, content analysis of remarks included in question #3 after each vignette, direct verbal feedback, and observation of the manner in which the instrument and interface functioned. The second iteration of the survey instrument was successfully beta-tested in August and September, 2008, and produced a reasonable reliability score (Crohnbach's α = .866).A pre-study power analysis, performed using G*3 Power 3.0.5 , determiSeveral strategies were utilized to obtain and surpass the target N. First, distribution through regional and national Practice-Based Research Networks (PBRNs) insured that physicians interested in participating in research studies were invited to participate; such interest is an assumed pre-requisite for members of PBRNs. Additionally, going to both regional and national PBRNs, as well as approaching the non-PBRN general membership of professional societies such as the Society of Teachers of Family Medicine, opened the recruitment pool up to literally thousands of individuals. As an incentive for participation, invitees were offered a $25 stipend plus the opportunity to earn free Continuing Medical Education in the future (currently under development).2 statistics were computed to determine whether there were any significant differences in the distribution of respondent characteristics between vignettes randomized as White/Insured, White/Uninsured, African-American/Insured, or African-American/Uninsured . Absence of significant differences in respondent characteristics across the four factorial combinations was accepted as evidence that the randomization procedure was effective, and hence it was unnecessary to control for any non-significant respondent characteristics.Analyses of the dependent variables were conducted in several steps. Before proceeding to regression analyses, χThe data were then analyzed by creating binary diagnostic and treatment outcome variables. For diagnostic outcomes, cases given "no diagnosis" or Oppositional Defiant Disorder (ODD) were labelled 0 and cases given ADHD or Bipolar (BP) were labelled 1. ODD and BP were so infrequently selected that removing them from the analysis made essentially no statistical difference. Similarly, treatment selections were grouped into those that would result in a medicinal intervention , and those that would not . Binary logistic regressions were calculated for each dependent variable (Diagnosis & Treatment) using the responses from the four cases, modelled against race, insurance status, case gender, ADHD status of the case, and interaction effects.Collection of responses for the full data set (N = 187) occurred primarily from September through December, 2008, with a small number of late respondents entering the survey in January and early February, 2009. All respondents completed the survey. Respondents came from 35 states, with the largest number coming from New York (roughly 30%), followed by Georgia (roughly 9%) and Pennsylvania (roughly 7%). The sample was heavily skewed towards White respondents (87.7% White) and was evenly split between male and female respondents . Physicians educated in the United States were also over-represented at 94.6%, compared to the population of total U.S. physicians (74.5%)  and to F172 of 187 respondents identified themselves as practicing within the Family Medicine specialty, with the rest identifying themselves as Pediatricians. Respondents were mostly MD's (86.6%), followed by DO's (11.2%), with one physician respondent holding a non-U.S. medical credential (M.B.B.S). Three mid-levels (Nurse Practitioners or Physician Assistants) also responded. Average time in practice was approximately 16 years and was fairly evenly distributed across a range of 1 to 36 years (S.D. = 9.71 years). Most respondents saw at least some poor and/or non-white patients in their practices, with 39% indicating their patient panel consisted mostly of poor/non-white or mostly poor/white patients. An additional 17.1% selected an "Other" option to describe their patient population but the majority of these respondents textually indicated a diverse patient population that included poor patients, non-white patients, or both. Almost two thirds of respondents indicated either Urban, Suburban, or mixed Urban/Suburban practice settings (64.1%). The full distribution of sample characteristics is displayed in Tables Several exploratory analyses that incorporated respondent characteristics as covariates, including type of professional credential, level of training, normal patient mix (peds/adult and race/class), and practice setting  did not reveal any of these covariates to be statistically significant. Of possible note, years of training appeared to approach significance, and so number of years in practice was included in the final binary logistic regression models.Neither race nor insurance status appears to be an independent predictor of diagnosis in the clustered, four-case model presented in Table The effects of race and insurance status upon treatment decisions were more direct. Cases randomized as African-American were slightly more likely to be medicated, an effect which approached but did not reach significance , and being uninsured significantly increased the likelihood of being medicated . Cases randomized as both African-American and uninsured were less likely to be medicated, although this effect was not significant. As with diagnostic decisions, treatment decisions were highly predicted by true ADHD status of the cases. Furthermore, a gender contrast once again appears between the ADHD-positive cases, with Case 3  being over 11 times more likely to be medicated or treated with combined medication and psychotherapy  than the male, ADHD-negative reference case, while Case 4  was only about 3 times as likely than the reference case to be medicated .This study was a focused exploration into how primary care physicians, and specifically Family Physicians, approach the diagnosis and treatment of ADHD in children. It was specifically looking at how social factors may influence an ostensibly objective clinical process. Given this central focus, it is important to begin this discussion by clearly stating that the respondents, as a whole, were effective at correctly discriminating between ADHD and non-ADHD cases in their diagnostic decisions. ADHD-positive cases were significantly more likely to be diagnosed and treated in all models constructed with these data. Furthermore, the cases that study participants were reading and responding to were intentionally marginal; all displayed at least some symptoms consistent with an ADHD diagnosis. Also, the participants in the study did not have the opportunity to utilize follow-up questions, formal diagnostic processes  or consults, or otherwise acquire additional case information, as they would in an actual clinical setting. Despite the uncertainty and unfamiliarity with ADHD guidelines identified by other studies in primary care and FP populations ,24,29,43Insurance status appears to play a somewhat complicated role in determining which children are diagnosed with and treated for ADHD in a primary care setting. Being uninsured lowered the odds of diagnosis in male cases, an effect that comports with trends repeatedly observed elsewhere . This diThe picture is somewhat different for treatment decisions. Both African-American race and uninsured status individually increase the odds of medicinal or combined medicinal/psychotherapeutic treatment, with the insurance effect being significant, and the race effect approaching significance. However, when interacted, the two variables together serve to cut the odds of treatment in half, although this interaction effect was not significant in this relatively small study. Also observed was a very significant interaction between male gender and being uninsured, with this combination of factors reducing the odds of treatment by half, in line with the observed effect on diagnosis. It would be speculative to suggest explanations for this effect at this point, but this finding underscores the need for further study of gender and ADHD. The addition of both gender-null and insurance-null cells in a future follow-up study would help resolve this question.Fundamentally, what may be drawn from the current study about insurance status is that patients presenting as uninsured are generally less likely to be diagnosed with and treated for ADHD. This effect is apparently moderated by both race and gender. These latter interactions require further examination, however.Race was a non-factor in predicting diagnostic outcomes in this study. However, it appears to play a more important role in determining physician decisions about treatment, as well as in moderating the effect of insurance status. As noted above, the positive effect observed in the female ADHD positive case when presented as uninsured was completely and significantly reversed when the case was both African-American and uninsured. When grouped by ADHD status, the ADHD-positive cases were more than twice as likely to be diagnosed when identified as African-American, but only about a third as likely to be diagnosed when identified as both African-American and uninsured. This indicates that some but not all of this effect may be due to its presence in Case 4. While neither race alone nor the race × insurance interaction were statistically significant in the four-case model, the trends remained consistent - African-American race alone increased the odds of treatment, but the interaction between race and insurance status decreased these odds. The fundamental role of race appears to moderate the effects of insurance status.There were several central challenges in the design of this study. First was the need to generate an adequate sample size. The study population consisted of busy, over-surveyed primary care physicians. Physician surveys have notoriously low rates of response, and the methods that prove most effective - monetary incentives and mixed web/paper survey distribution - are generally costly and labor-intensive ,45. GiveAn obvious challenge for this study was the fact that the respondents were reacting to purely textual vignettes, as opposed to live, actual or standardized patients. Despite this weakness, vignette-based studies have a long history of effectively demonstrating the effects of discrete factors while holding a body of information constant. For example, Lutfey, McKinley and colleagues have recently utilized vignettes to examine both the cognitive pre-dispositions of physicians as they gather patient information, as well as the resulting decisions of such cognitive processes. For example, Lutfey and colleagues recently used a vignette based approach to do precisely this for clinical decision making scenarios involving Coronary Heart Disease and Depression -49. AnotAdditionally, the physician sample obtained for this study was clearly not representative of the U.S. physician population as a whole. Recruitment relied in some cases upon pass-along invitations by network directors, as well as upon snow-ball techniques from colleague to colleague. An accurate response rate is therefore difficult to measure. It is safe to say, however, that the response rate fell below 10%. This study therefore relied upon a sample of convenience. It furthermore relied upon those who were willing and able to open and respond to an e-mail invitation and web-based survey instrument.There were pragmatic decisions made regarding the structure of the data matrix that may be viewed as study weaknesses as well. Given the extreme difficulty in recruiting large samples of physicians to complete surveys, the use of four vignettes per respondent allowed for the potential to quadruple the power of the study, and this proved crucial. However, the use of four cases per respondent introduced the issue of panel-oriented autocorrelation in the variance-covariance matrix. This proved to be of only moderate concern, and was generally addressable by incorporating between-subject and between-case variables into the analytic models, obviating the need for more advanced statistical measures which dilute the efficiency or interpretability of results to some extent. Some issues could not, however, be addressed. For example, the ability to analyze the interaction between clinician and patient race was severely limited by multicollinearity, and clinician/patient gender interactions were severely penalized by the data structure as well. Both had to be eliminated altogether. Additionally, it must be noted that the truly experimental phase of this study is limited to the analyses of the individual cases, which may have been underpowered. Although useful, the two-case and four-case analyses combined multiple vignettes, thus eliminating the ability to control for all variation other than race and insurance status. These analytic phases must therefore be considered quasi-experimental.Presently, reform of the U.S. healthcare system is under robust and serious discussion. At the core of this discussion is a presidential plan to reduce health disparities, drive down healthcare costs, increase usage of electronic medical records, and universalize health insurance, improve coordination of care, and enhance quality of care . In suchSeveral recommendations along these lines may be made based upon the current study. First, further development and evolution of guidelines for the diagnosis and treatment of ADHD ,27,55,56Additionally, as noted by Starfield , the proGiven both the results and the weaknesses described above, the first and foremost recommendation is that additional research is needed. Specifically, an expansion of the vignette-based approach is in order, which should have the following aims:1. Incorporation of gender as a factorial variable, along with race and insurance status2. Increase in study N3. Increase inclusion of Pediatricians4. Increase inclusion of non-Academics.Additionally, follow-up studies incorporating data from actual practice should be conducted, to triangulate and corroborate the findings presented here. Ultimately, a multi-method approach employed across convergent studies will be necessary and is warranted. Finally, utilization of the methods employed in this study to examine the effects of patient characteristics in other medical and mental health specialties, as well as in educational professionals, is warranted.What this study has examined is but one very particular context where disparities in the provision of healthcare, and specifically the diagnosis and treatment of ADHD, may be generated. Clearly, there is much more work to be done in examining other sources of disparity. However, what may be determined from the results presented here is that insurance status clearly impacts the provision of ADHD services in primary care. Ameliorating health insurance disparities, and improving guidelines to enhance their applicability to uninsured, referral-deprived, and non-white/non-male patients are one step toward accomplishing an egalitarian distribution of services for ADHD in children.The author declares that they have no competing interests.CPM designed the project, conducted all procedures and analyses, and wrote the text.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2296/11/11/prepubADHD Vignettes used in this study. Vignettes 1 & 2 were designed to be sub-clinical, non-ADHD, and were derived from true cases where ADHD was ruled out upon a full evaluation of the patient. Vignettes 3 & 4 were derived from cases where a full evaluation led to a diagnosis of ADHD.Click here for file


Children with attention-deficit/hyperactivity disorder (ADHD) frequently display poor judgment and risk taking in their everyday behavior, but there are little empirical data on decision-making cognition in this disorder. The objectives of the study were to assess the effects of stimulant medication on decision making in ADHD and compare performance on the Cambridge Gamble Task between boys with and without ADHD.Twenty-one boys (aged 7–13) diagnosed with ADHD underwent a double-blind, placebo-controlled trial of methylphenidate (.5 mg/kg) during which they performed the Cambridge Gamble Task (CGT). A healthy age-matched control group was tested on two occasions off drug.The ADHD group bet more conservatively on the methylphenidate session than on the placebo session. In comparison with healthy control subjects, the ADHD group made more poor decisions, placed their bets more impulsively, and adjusted their bets less according to the chances of winning. Poor decision making was correlated with parent-reported symptoms and disruptive behavior in the ADHD group.Methylphenidate reduced risk-prone betting behavior on the CGT. Compared with control subjects, children with ADHD display a number of decision-making deficits on the task, and the measure of rational decision making may serve as an ecologically valid neuropsychological marker of impairment. Attention-deficit/hyperactivity disorder (ADHD), a prevalent psychiatric disorder characterized by hyperactivity, impulsivity, and inattention, is diagnosed by pervasive maladaptive behaviors during childhood . ChildrePharmacotherapy with methylphenidate (MPH) improves behavioral symptoms and cognitive function  in ADHD , produciParental written informed consent and ethics committee approval were obtained.n = 21) were males, aged 7 to 13, and stabilized on methylphenidate with no primary learning disabilities or concomitant neurological, psychiatric, or behavioral disorders . A healthy control group (HC)  was recruited with posters from the local community.Psychiatrists referred consecutive attendees to a childhood ADHD outpatient clinic. Diagnoses following DSM-IV guidelines including pervasiveness of symptoms  were estThe ADHD boys underwent a double-blind, placebo-controlled, crossover design of a single .5 mg/kg dose of methylphenidate or placebo. One child received .25 mg/kg (10 mg) due to his high weight and low therapeutic dose. Participants abstained from MPH for 21 to 28 hours  prior to testing sessions. Methylphenidate reaches peak plasma concentration in approximately 2 hours . Questio0), prior to cognitive testing (t1), and after testing (t2).The Mood and Feeling Questionnaire  measuredwww.camcog.com)   assessedcog.com) .1+ t2− t0) , covaried for age, examined ADHD-P versus HC performance on all CGT measures and ADHD-MPH versus HC for measures that significantly responded to drug manipulation. Healthy control group data were assessed for practice effects using repeated measures ANOVA then averaged across visits, since ADHD-P visits were counterbalanced. Effect sizes were calculated as d = (μ1− μ2)/√[(σ12 + σ22)/2]  were determined using Pearson's product-moment correlation coefficient. Other summary questionnaire measures were not analyzed.Repeated-measures analysis of variance (ANOVA), with between-subject factors of methylphenidate/placebo order, compared the ADHD group on placebo (ADHD-P) versus methylphenidate (ADHD-MPH) on CGT measures and change in VAS factors (t t2− t0) . Deliber t2− t0)  to decre σ22)/2] . The relt = −.57, p = .571; meanADHD = 10.00, standard deviationADHD = 2.05; meanHC = 10.32, standard deviationHC = 1.59), test order , days between visits , years of education , and distribution of younger . The ADHD group had higher disruptive behavior ratings  and a trend toward higher depressive symptoms . The HC group showed no significant practice effects on the CGT .Attention-deficit/hyperactivity disorder and HC groups were matched for age . Participants receiving methylphenidate at visit 1 reported feeling calmer than on placebo .On methylphenidate, the ADHD group bet significantly fewer points than on placebo  but did F = 2.96, p = .09, d = .53).The ADHD-P group performed less optimally than HC on rational choices across all box ratios, risk adjustment, and impulsivity index but did not differ on amount bet or deliberation times , Table 1Totalr18 =−.673, p = .002; AchenbachTotalr18 =−.670, p = .002; AchenbachInternalizingr18 =−.506, p = .038; AchenbachExternalizingr18 =−.545, p = .024) . No other behavioral rating scores correlated significantly with CGT performance (p ≥ .195), although low variance on rating scales in control subjects may have limited our sensitivity to detect additional correlations.For ADHD-P, higher behavioral ratings were associated with poor quality of decision making (Conners = .024) . For HC,Methylphenidate significantly reduced CGT betting behavior in children with ADHD without affecting performance on other measures. These methylphenidate effects replicated findings in frontal variant frontotemporal dementia (fvFTD) patients, where MPH reduced betting . The venHigh CGT betting increases potential losses, therefore indicating risk taking. Despite their abnormal pattern of betting, the ADHD group did not bet high amounts for all ratios. However, case-control comparisons indicated significant decision-making impairments in the ADHD group on other CGT variables. On placebo, the ADHD group bet more impulsively than control subjects, selecting high or low bets early in the bet sequence. This shortens the task duration, consistent with ADHD performance profiles on delayed reward tasks, indicating delay aversion  or motorSeveral factors may underpin the deficits in rational choices and risk adjustment. Children with ADHD modulate their behavior according to rates and magnitudes of gains and losses less than their peers , who do Poor quality decision making was associated with ADHD behavioral symptoms. Methylphenidate induced conservative betting behavior in childhood ADHD but did not ameliorate their decision-making deficits.


Serotonin genes have been hypothesized to play a role in the etiology of attention-deficit hyperactivity disorder (ADHD); prior work suggests that serotonin may interact with psychosocial stressors in ADHD, perhaps via mechanisms involved in emotional dysregulation. Because the development of behavioral and emotional regulation depends heavily both on the child's experience within the family context and the child's construals of that experience, children's appraisals of inter-parental conflict are a compelling candidate potentiator of the effects of variation within the serotonin transporter gene promoter polymorphism (5HTTLPR) on liability for ADHD.304 youth from the local community underwent a multi-informant diagnostic assessment procedure to identify ADHD cases and non-ADHD controls. Youth also completed the Children's Perception of Inter-Parental Conflict (CPIC) scale to assess appraisals of self-blame in relation to their parents' marital disputes. The trialleic configuration of 5HTTLPR (long/short polymorphism with A> G substitution) was genotyped and participants were assigned as having high (La/La N = 78), intermediate , or low  serotonin transporter activity genotypes. Teacher reported behavior problems were examined as the target outcome to avoid informant overlap for moderator and outcome measures.Hierarchical linear regression analyses indicated significant 5HTTLPR × self-blame interactions for ADHD symptoms. Examination of the interactions indicated positive relations between reports of self-blame and ADHD symptoms for those with the high and low serotonin activity genotypes. There was no relation between self-blame and ADHD for those with intermediate activity 5HTTLPR genotypes.Both high and low serotonergic activity may exert risk for ADHD when coupled with psychosocial distress such as children's self-blame in relation to inter-parental conflict. Results are discussed in relation to the role of serotonin in the etiology of the ADHD and related externalizing behaviors. Children living with a step-parent did not report significantly higher levels of conflict properties or self-blame than did children living with two biological parents (ps > .33). Despite this, as a precaution we covaried family composition  in the main G × E analyses (with no effect on the results).Children in the ADHD group were less likely to be living with both biological parents than non-ADHD children. This latter difference in regard to family constellation may potentially influence reports of self-blame, as children experiencing parental separation and/or divorce may be more likely to observe frequent and intense marital conflict and therefore, may have higher levels of self-blame. Whereas children who had experienced parental separation and/or divorce reported higher scores on conflict properties . There were also no significant main effects of 5HTTLPR genotype when dimensional measures of ADHD were examined. As seen in Table Table There were no significant differences in reports of self-blame across the three genotype groups (p = .22), suggesting an absence of gene-environment correlation between 5HTTLPR genotype and children's report of self-blame. Lack of correlation between this genetic marker and this environmental measure signals that this particular rGE effect is unlikely to emerge as spurious interaction findings in the present analyses .2 = .11], indicating an increase in total ADHD symptoms with higher reports of self-blame. The linear (low activity as risk) and non-linear (low and high activity as risk) main effects of 5HTTLPR genotype were nonsignificant as was the linear × self-blame interaction  However, the non-linear × self-blame interaction was significant . Examination of the simple slopes revealed that there was no relationship between ADHD symptoms and self-blame for those with the intermediate activity genotypes . In contrast, a significant and positive relationship between self-blame and ADHD emerged for those with the high  and low  activity genotypes . All modeling results reported herein include these covariates. Results revealed a significant main effect of self-blame for the total ADHD Index raw score . There was no main effect of 5HTTLPR genotype group using either the linear (p = .29) or non-linear (p = .54) coding schemes. The linear × self-blame interaction was nonsignificant (p = .54). The non-linear × self-blame interaction showed a trend, but was also not significant  . For Cognitive Problems, there was a significant main effect of self-blame . The linear and non-linear main effects of 5HTTLPR genotype were again nonsignificant as was the linear 5HTTLPR × self-blame interaction . In contrast, the non-linear 5HTTLPR × self-blame interaction was significant . Examination of the simple slopes revealed a similar pattern of results. For youth with the low and high activity serotonin genotypes, there was a significant and positive relationship between self-blame and Hyperactivity . Yet, there was no relationship between self-blame and ADHD for those with the intermediate serotonin activity genotypes  . Further, non-linear 5HTTLPR × self-blame interactions remained significant after adjusting for a number of covariates, including gender, age, ethnicity, family composition, and overall level of conflict frequency/intensity .2 = .08]. The main effect of 5HTTLPR genotype as well as the linear 5HTTLPR × self-blame interactions were again nonsignificant (ps > .63). The non-linear 5HTTLPR genotype × self-blame showed a trend in the expected direction, but did not reach significance .To evaluate degree of internal replication of results, G × E regression models were repeated with parent report on the Conners' Rating Scale ADHD index and the two symptom dimension scales with uncorrected p values. For the ADHD index, results again indicated a significant main effect of self-blame  and Hyperactivity . Similar to results with teacher report, all main effects of 5HTTLPR genotype (linear and non-linear coding schemes) as well as the linear 5HTTLPR genotype × self-blame interactions were not significant for both Cognitive Problems  and hyperactivity . The non-linear 5HTTLPR genotype × self-blame interaction was not significant for Cognitive Problems . It also feel shy of significance for hyperactivity . In sum, non-linear G × E interactions were significant in predicting teacher report of ADHD symptoms , but were shy of significance when parent report was used as the dependent measure.The significant main effect of self-blame was again observed in predicting parent report for both Cognitive Problems . Both the linear and non-linear 5HTTLPR × self-blame interactions were non-significant, although there was a trend toward significance for the linear 5HTTLPR × self-blame interaction (p = .10). Results for parent report of Oppositionality were similar. A main effect of self-blame emerged , whereas all G × E interactions involving self-blame and 5HTTLPR showed no hint of an effect (ps > .41).As secondary analyses, we examined the G × E interaction on the Conners' Oppositionality scale. G × E models predicting teacher report of Oppositionality yielded a main effect of self-blame , however all other main effects and all G × E interactions were nonsignificant (ps > .24). Overall, these results indicate that while the non-linear 5HTTLPR × self-blame interaction did not generalize to other disruptive behaviors, self-blame itself may have exert some main effects for ADHD as well as other disruptive behaviors.2 = .03, total R2 = .13] as well as for hyperactive symptoms . The non-linear 5HTTLP × self-blame interaction predicting teacher inattentive symptoms was marginally significant, in the expected direction . Again, relationships between self-blame and ADHD symptoms were only positive and significant for those with the low and high 5HTTLPR activity genotypes.To enable comparisons with future studies that may rely on ADHD symptom scores, we here report G × E interactions using the lower powered tests with the DSM-IV ADHD Rating Scale. As with the Conners, the non-linear 5HTTLPR × self-blame interaction was significant in predicting teacher report of total ADHD symptoms  but not for parent report of inattention  were included as a covariate, the non-linear interaction terms remained significant in predicting teacher report on the Conners' ADHD Index [b = .19, 95% confidence interval .08-.31, p = .002, ΔRAs a contrast test, we examined appraisals of conflict properties  as a moderator in the G × E analyses. While conflict properties exerted a main effect on both parent and teacher reports of ADHD symptoms (p < .01), the linear and non-linear 5HTTLPR × conflict properties interactions were not significant using either parent or teacher report (ps > .69). This suggested that results for self-blame could not be attributed to measurement artifact.The sample consisted of children and adolescents covering a wide age range (6-18 years). Serotonin systems have been suggested to function differently in pre and post-pubertal children . While w2 = .02, total R2 = .14) but only showed a trend toward significance in pre to-mid pubertal children . While these data are preliminary, they may reflect potential differences in serotonin functioning with age and development. The influence of development on the functioning of the serotonin system, including any potential role for G × E interactions for ADHD involving serotonin system genes, remain critical questions to address in future studies.These exploratory results revealed that the non-linear 5HTTLPR × self-blame interactions predicting teacher report on the Conners' ADHD Index remained significant for post and late-pubertal youth , along with a particularly salient marker of environmental risk .both high and low serotonergic activity genotypes exert risk and that these risk mechanisms are modulated by salient psychosocial stressors.The current study provides evidence of G × E effects for ADHD involving 5HTTLPR and youth appraisals of self-blame. Our analytic methods allowed us to examine a hypothesis previously untested at the genetic level - namely that both high and low serotonergic activity genotypes  exert risk for ADHD symptoms. Findings from both the functional serotonin literature as well as from molecular genetic association studies have yielded seemingly conflicting findings about whether increased or reduced serotonergic activity is related to ADHD. The present results suggest that at the genetic level, The results were generally consistent across informant, although results for parent ratings were shy of the significance levels seen with teacher ratings. For teacher report, the pattern of results indicated significant non-linear 5HTTLPR × self-blame interactions for hyperactivity/impulsivity but not inattention or cognitive problems. The interactions revealed that youth appraisals of self-blame were significantly related to ADHD symptoms for children with the low activity  and high activity (La/La) 5HTTLPR genotypes. Those with the intermediate activity genotypes , on the other hand, appeared to be immune to whatever effects self blame was having on hyperactivity/impulsivity. The interaction was not accounted for by age, gender, or ethnicity, by family composition, by overall levels of conflict frequency/intensity, or by main effects of 5HTTLPR genotype or youth reports of self-blame. It was also not likely to be due to measurement artifact, because another scale (conflict properties) showed no hint of interaction.Analysis of the symptom dimensions appeared to indicate some preliminary evidence for specificity of effects. The non-linear 5HTTLPR × self-blame interactions were non-significant or marginally significant for teacher and parent report of cognitive problems. In contrast, non-linear interactions were significant for teacher report and marginally significant for parent report of hyperactivity. The interaction again revealed that for youth with the high and low serotonin activity genotypes, the relationship between self-blame and hyperactivity was positive and significant. When examined again via the ADHD Rating Scale, a similar pattern emerged, however, the effect appeared attenuated for both parent and teacher report using DSM-IV symptom counts, perhaps due to the loss of power using the less well-distributed scores on that scale. Some potential specificity in terms of G × E effects for the ADHD symptom dimensions may make sense, as hyperactivity-impulsivity and inattention have been described as being partially separable at the genetic, neural, and temperament levels ,73,74 asThe effects appeared to be somewhat specific for ADHD, as the non-linear 5HTTLPR × self-blame interactions showed no hint of an effect for oppositional defiant disorder symptoms (by teacher report) or conduct problems (by parent report). In contrast, the linear 5HTTLPR × self-blame interaction showed marginal significance for oppositional defiant disorder symptoms. If that result were to prove stronger in future work, it could indicate that only individuals with the low activity 5HTTLPR genotypes are vulnerable to development of oppositional and conduct problems .Post-hoc data checks indicated that results were not generalizable to any appraisals of inter-parental conflict, as conflict properties failed to show significant moderation in the G × E analyses. Thus, there appears to be something about self-blame that is important for ADHD specifically. This is line with current work from our own laboratory (which included a portion of this sample) that indicates that among the CPIC scales, self-blame is a significant and unique predictor of ADHD symptomatology .In addition, while serotonin functioning and serotonin genes have also shown association with a number of conditions, including mood disorders, the current findings were not explainable by a history of comorbid depression symptoms. Furthermore, while effects were stronger in late and post-pubertal youth, non-linear 5HTTLPR × self-blame interactions continued to show marginal significance in younger children . Thus, while developmental timing and its relationship with serotonergic functioning may be accounting for some of the effects - and will be an interesting avenue for future study - the non-linear 5HTTLPR × self-blame interactions observed here could not be fully explained developmentally.With regard to the genetic literature for ADHD, our results failed to replicate a main effect of 5HTTLPR genotype with ADHD symptoms that has been previously reported -78. UnliThe overall results are also suggestive of a potential heterozygote advantage, which has been suggested as a potential explanation for some diseases, including mental disorders . HoweverCertain limitations are important to note. First, we did not have parent DNA available for the majority of our sample, thus the use of family-based analyses was not possible. While unlikely, we cannot rule out population stratification effects. Second, because our sample is cross-sectional, we could not examine the longitudinal relationships between appraisals of self-blame and ADHD symptoms. It may be the case that more frequent inter-parental conflict is the result of having a child with more severe ADHD symptoms and that over time, children view themselves (perhaps correctly) as being responsible for their parents' marital problems. As is always the case with single studies of genetic or environmental effects, our findings may be false-positives. Thus, replication of these results in other samples is necessary.The influence of development on the functioning of the serotonin system, including any potential role for G × E interactions for ADHD involving serotonin system genes, remains in need of further investigation in more costly studies, which may be justified by results such as the current one. In addition, the influence of age regarding comprehension of the 48-item CPIC remains in need of further exploration. While we took several steps to assure correct comprehension and completion of the CPIC in younger children, future work extending on these results may be well-served by considering the CPIC-Y designed for younger children .Overall, our study is among the first to examine relationships between 5HTTLPR and ADHD as well as interaction effects using the triallelic genotype configuration. Results suggest that both the low- and high-activity 5HTTLPR genotypes increase risk for ADHD symptoms within the context of higher levels of youth self-blame in relation to their parents' marital conflict.The authors declare that they have no competing interests.MN formulated study hypothesis, participated in data collection, carried out the analyses, and wrote the manuscript draft. KF oversaw genotyping procedures, obtained funding, aided in hypothesis generation, and provided manuscript revision. IW provided statistical consultation, formulated data analytic plan, and provided manuscript revision. KJ completed genotyping assays on the sample. JTN advised on hypothesis formulation, obtained funding, assisted with data analysis and manuscript revision. All authors read and approved the final manuscript.


CLOCK) gene encodes protein regulation circadian rhythm and also plays some roles in neural transmitter systems including the dopamine system. Several lines of evidence implicate a relationship between attention-deficit hyperactivity disorder (ADHD), circadian rythmicity and sleeping disturbances. A recent study has reported that a polymorphism (rs1801260) at the 3'-untranslated region of the CLOCK gene is associated with adult ADHD.The circadian locomotor output cycles kaput (To investigate the association between the polymorphism (rs1801260) in ADHD, two samples of ADHD probands from the United Kingdom (n = 180) and Taiwan (n = 212) were genotyped and analysed using within-family transmission disequilibrium test (TDT). Bonferroni correction procedures were used to just for multiple comparisons.P = 0.010). There was also evidence of preferential transmission of the T allele of the rs1801260 polymorphism in combined samples from the Taiwan and UK (P = 0.008).We found evidence of increased transmission of the T allele of the rs1801260 polymorphism in Taiwanese samples (CLOCK in susceptibility to ADHD.This study provides evidence for the possible involvement of  Attention-deficit hyperactivity disorder (ADHD) is a common and heritable childhood behavioural disorder characterised by developmentally inappropriate levels of hyperactivity, impulsivity and inattentiveness. ADHD is a very complex disease. Polymorphic variants in several genes involved in regulation of dopamine and related neurotransmitter pathways are reported to be associated with ADHD . Some liVarious types of sleep disorders have been associated with ADHD -14. In aCLOCK gene was reported to be associated with evening preference and delayed sleep timing in a Japanese population and it suggests that this polymorphism may influence a human behavioural pattern and possibly lead to altered daytime brain performance [A polymorphism (rs1801260) of formance . Howeverformance .CLOCK was shown to regulate dopamine neural transmission and the CLOCK mutant mouse showed a pronounced elevation of locomotor activity [In mice study, activity . Abnormaactivity -26.CLOCK gene is located on the long arm of chromosome 4q12 . CLOCK protein is involved in the transcriptional regulation of many circadian output genes and in the core circadian clock [CLOCK locus significantly affects the regulation of transcription [The an clock ,28. Up tcription ,28.CLOCK gene and psychiatric disorders [CLOCK gene and adult ADHD with German background [Recently several groups did genetic association study between a polymorphism (rs1801260) at the 3'-untranslated region (3'-UTR) of the isorders -33. Howeckground .To provide further clarification of the reported association, in this study we examine the role of this polymorphism in two clinical ADHD samples from the UK and Taiwan.For the UK sample, DNA was collected from 180 DSM-IV ADHD combined subtype probands, from both parents for 116 of the ADHD probands and from the mother alone for 64 of the probands. Ninety-six percent of the sample was male subjects. Cases were recruited from child behaviour clinics in South-East England and referred for assessment if they were thought by experienced clinicians to have a diagnosis of the combined subtype of ADHD under DSM-IV criteria, with no significant Axis I co-morbidity apart from oppositional defiant disorder (ODD) (5.5%) and conduct disorder (CD) (15.5%). Intelligence quotient (IQ) was assessed in the majority of the subjects using the Wechsler Intelligence Scale for Children-Third Edition (WISC-III), but for those under 6 years, the Wechsler Preschool and Primary Scale of Intelligence (WPPSI) was applied.Exclusion criteria included IQ less than 70, neurological disorders or brain damage and autism. Only those individuals fulfilling the recruitment criteria after completion of research assessments were included in the study. The age range was 5-15 years at the time of assessment . Parents were interviewed with a modified version of the Child and Adolescent Psychiatric Assessment (CAPA) . InformaThe Taiwanese sample consisted of 212 children with ADHD diagnosed between the ages of 5-15 years . Both parents were available for 114 families, only the mother for 59 families and only the father for 39 families. ADHD cases were ascertained from the Child Psychiatric Clinics in the Chang Gung Memorial Hospital in Taipei area, Taiwan. A diagnosis of ADHD was made according to DSM-IV criteria following completion of a standard maternal interview  and compThe rs1801260 polymorphism was genotyped using the method of PCR and enzyme restriction. Genomic DNA was amplified by using the following primers: 5'-TCCAGCAGTTTCATGAGATGC-3'and 5'-GAGGTCATTTCATAGCTGAGC-3'. The reaction was formed according to the protocol described in the study . The PCRhttp://www.mrc-bsu.cam.ac.uk/personal/frank/software/unphased/. The Bonferroni correction was applied for multiple tests and P < 0.025 was considered to show a statistically significant difference.Family genotype data were analysed using the transmission disequilibrium test (TDT) implemented in UNPHASED program (TDTPHASE) . http://Population frequencies for the SNP (rs1801260) were estimated from parental genotypes. For the UK and the Taiwanese sample the T-allele frequency of the polymorphism are 73% and 87%, respectively; the C-allele frequencies are 27% and 13%, respectively. Allele frequencies of the SNP did not show any significant deviation from those expected according to Hardy-Weinberg equilibrium in either population.2 = 6.701, P = 0.010, OR = 2.06). No significant association was found between this polymorphism and ADHD in the UK sample , while combining the two datasets together the T allele of rs1801260 SNP was significantly over-transmitted to affected probands . Even after correcting P-values using the Bonferroni method for multiple tests a significant association was still found between the T allele and ADHD.TDT analysis Table  showed tCLOCK gene (rs1801260) in two independent samples of ADHD probands from UK and Taiwan. We found a significant over-transmission of the T allele of rs1801260 SNP to ADHD cases in Taiwanese population. No association was observed between this polymorphism and ADHD in the UK sample. We did however find evidence for increased transmission of the T allele of rs1801260 in the Taiwanese and UK samples pooled together (P = 0.008).In this study, we set out to investigate a previously reported finding of association between a single nucleotide polymorphism in the 3'-UTR region of the CLOCK mRNA 3'-UTR region. As for function, this polymorphism was speculated to affect mRNA [CLOCK and schizophrenia and mood disorders in the Japanese population. There was also no association found between rs1801260 SNP and schizophrenia and mood disorders [P = 0.001) between each of the adult ADHD assessment and the rs1801260 polymorphism with at least one T-mutation being the risk allele in a Caucasians of western European origin and German background. Our data suggests that the T-allele of rs1801260 polymorphism may be a risk allele in the development of ADHD and linked or contributing to a causative polymorphism for ADHD in Taiwanese population.In summary, the rs1801260 polymorphism has been detected at position 3111 in the ect mRNA . The rs1ect mRNA . This fiect mRNA ,40. The ect mRNA ,31,32. Hect mRNA . Recent isorders . To our isorders . Our stuisorders , who fouCLOCK gene and susceptibility to the disorder. Our findings support the notion that genetic variation in the 3'-UTR region of CLOCK gene might be a risk factor in the development of ADHD, particularly in the Taiwanese sample studied. Due to the small sample sizes in our study and only one published data on rs1801260 polymorphism investigated in ADHD, further association studies are needed to confirm or refute the finding. Since the 3'-UTR of CLOCK gene may contain sequences that regulate translation efficiency, mRNA stability, and polyadenylation signals, the rs1801260 polymorphism may be associated with other functional polymorphisms in 3'-UTR regions, which act as regulatory domains of the gene, and may influence the circadian rhythms [In conclusion, in this study we used family-based ADHD data in the UK and Taiwanese population to test for an association between rs1801260 SNP at the 3'-untranslated region of  rhythms ,40. TherThe authors declare that they have no competing interests.XX selected the SNP, performed genotyping, genetic analysis and drafted the manuscript. GB revised the manuscript. CKC, YSH and YYW provided the Taiwanese DNA samples and clinical data. CKC also revised the manuscript. PA supervised the study and revised the paper. All authors contributed to the final critical revision of the manuscript.


Streptococcus equi ssp. zooepidemicus  is one of the common pathogens that can cause septicemia, meningitis, and mammitis in domesticated species. M-like protein (SzP) is an important virulence factor of S. zooepidemicus and contributes to bacterial infection and antiphagocytosis. The interaction between SzP of S. zooepidemicus and porcine thioredoxin (TRX) was identified by the yeast two-hybrid and further confirmed by co-immunoprecipitation. SzP interacted with both reduced and the oxidized forms of TRX without inhibiting TRX activity. Membrane anchored SzP was able to recruit TRX to the surface, which would facilitate the antiphagocytosis of the bacteria. Further experiments revealed that TRX regulated the alternative complement pathway by inhibiting C3 convertase activity and associating with factor H (FH). TRX alone inhibited C3 cleavage and C3a production, and the inhibitory effect was additive when FH was also present. TRX inhibited C3 deposition on the bacterial surface when it was recruited by SzP. These new findings indicated that S. zooepidemicus used SzP to recruit TRX and regulated the alternative complement pathways to evade the host immune phagocytosis. Streptococcus equi ssp. zooepidemicus , a member of the Lancifield's group C, is an opportunistic pathogen that could infect a wide variety of non-human species, including important domesticated cattle such as horses, cows, swine, sheep, and dogs. In China, S. zooepidemicus is the major cause of diseases in swine. Occasionally, it can infect humans via zoonotic transmission from the infected animals and cause invasive infections in humans such as septicemia and meningitis. streptococci: this fibrillar, surface-exposed protein deters opsonization of the organism using the alternate complement pathway S. zooepidemicus carry antigens with antiphagocytic characteristics similar to the M proteins expressed from Lancefield group A and G streptococci; hence it was named M-like protein (SzP) S. zooepidemicus from phagocytosis is poorly understood.Thioredoxin (TRX) is a small multifunctional protein with a redox-active dithiol/disulfide in the conserved active site. The functions of TRX are to reduce protein disulfide bonds and to scavenge hydrogen peroxide together with peroxiredoxins S. zooepidemicus from phagocytosis. We identified the SzP/TRX interaction and found that the activity of TRX was not inhibited by this interaction. We also found that TRX could facilitate the antiphagocytic process when it was recruited by SzP anchored on the surface of S. zooepidemicus. Further experiments showed that TRX regulated the alternative complement pathway via C3 convertase inhibition and FH association. TRX alone could inhibit C3 cleavage and C3a production, and the inhibitory effect was additive with FH. TRX was able to inhibit C3 deposition on the bacterial surface when it was recruited by SzP of S. zooepidemicus. To our knowledge, this was the first reported identification of SzP/TRX interaction. It mediated resistance to the phagocytic activity of macrophages. Our findings could contribute to the general understanding on how SzP confers phagocytosis resistance.Our studies were focused on the molecular mechanism(s) by which SzP protects S. zooepidemicus was screened against a porcine pulmonary alveolar macrophage (PAM) cDNA library using the Split-ubiquitin yeast two-hybrid (Y2H) technique. In the Y2H screen, 28 proteins were identified to have a potential interaction with the SzP protein in the yeast cells growing on Trp− Ade− His− Leu− 80 mM aminotriazole media (Sigma). Thioredoxin  was repetitively identified 13 times  was genetically integrated at the N-terminal of each of these 12 candidate proteins using the vector pProLabel-C. pProLabel-C or pProLabel-C fused with the candidate protein were then transfected into HEK 293 cells with plasmid pAcGFP1-C, expressing GFP alone, or with plasmid pAcGFP1-SzP, a derivative of pAcGFP1-C coding for a GFP-SzP fusion protein. For the chemiluminescent detection approach, protein G plus/A agarose beads and anti-GFP monoclonal antibodies were incubated with the transfected HEK293 lysate and the luminescence level of the substrate-ProLabel reaction was measured. As seen in 2O2 before the in vitro binding assay. We found a clear difference of migration in polyacrylamide gels between the H2O2-treated and the DTT-treated TRX  showed that TRX and FH both prevented C3b deposition on the d strain . We detereasing. S. zooepidemicus has developed a multitude of strategies to evade host defenses by interfering many aspects of the immune system. Such interference can normally lead to the down-regulation or inhibition of the inflammatory responses. Previous studies have indicated that SzP in S. zooepidemicus shares similar antiphagocytic characteristics with the M proteins in the Lancefield groups A and G streptococci S. zooepidemicus is a 40.1-kD surface-anchored protein that elicits serum opsonic and protective responses in mice and horses S. zooepidemicus ATCC35246 and investigated the mechanism by which they conferred antiphagocytic effects against the host immune responses.There are generally cross-talks between bacteria and the host immune system. Initial attempt to identify SzP interaction partners using the classical yeast two-hybrid system was not successful. Classical Y2H requires both proteins to interact in the nucleus and the nuclear environment may cause certain proteins to fold improperly and thus abolish their interactions with other proteins The current study identified the interaction between SzP and TRX. We further provided three lines of evidence to verify that SzP was associated with TRX; (i) An interaction between SzP and TRX was screened and confirmed in the split-ubiquitin yeast two-hybrid system. (ii) SzP and TRX were co-immunoprecipitated from pAcGFP1-SzP and pProLabel-TRX transfected HEK293 cells, and the result was detected with ProLabel-tag enzyme activity and confirmed with western-blot. (iii) FH was able to bind with SzP and TRX, as well as the SzP/TRX complex. This suggested that the binding of SzP to TRX was biologically relevant.E. coli to humans. It is a small, ubiquitous, multifunctional protein that contains a redox-active disulfide/dithiol within its active site, -Cys-Gly-Pro-Cys- in vitro. These results could explain why SzP/TRX interaction did not inhibit TRX activity.TRX plays a variety of redox-related roles that are conserved from S. zooepidemicus, we investigated the interaction of S. zooepidemicus with Raw264.7 cells, using the wild strain ATCC35246 (which is virulent to mice) and SzP-knockout strain attenuated in virulence. We employed flow cytometry to detect the recruitment of TRX to the surface of viable S. zooepidemicus. This recruitment was mediated by SzP, since SzP-knockout strain was unable to recruit TRX to the surface. We also found that both the S. zooepidemicus wild type and the SzP-knockout strain were efficiently phagocytized by macrophages when TRX was absent, though the wild strain was harder to be ingested than the SzP-knockout strain. We believed that there should be another mechanism of SzP to elicit antiphagocytosis responses in S. zooepidemicus. The current work provided evidence that the S. zooepidemicus wild strain could avoid being phagocytized much more effectively, whereas the mutant strains were rapidly ingested by Raw264.7 cells even in the presence of TRX. All these results were obtained in the presence of the serum. However, TRX could not facilitate antiphagocytosis responses in the absence of the serum, it was only related to SzP. These results indicated the expression of SzP allowed the bacteria to recruit TRX, which had effects on the complement pathway. Therefore, the SzP/TRX interaction facilitated the antiphgocytic response of the S. zooepidemicusTo better understand how surface protein SzP could contribute to the pathogenecity of S. pyogenes expressing M protein and/or M-like proteins on the cell surface could survive inside the neutrophils S. pyogenes mutant strains that lacked either M protein and/or M-like proteins were rapidly killed. M and M-like proteins display affinity for several human plasma proteins such as IgG S. zooepidemicus elicit antiphagocytosis through its interaction with TRX.Previous report found that the wild-type S. zooepidemicus could evade phagocytosis by a similar method via SzP/TRX interaction. Our results showed that SzP, TRX and SzP/TRX complex were able to bind with FH. This suggested that the SzP/TRX interaction did not prohibit S. zooepidemicus to recruit FH on its surface. We reasoned that although SzP itself could recruit FH to the cell surface, SzP/TRX interaction was still important because TRX could act as a regulator of the alternative complement pathway not only in association with FH but also on its own. TRX acted additively to FH in the alternative complement pathway. The increased level of the capsules may enhance phagocytosis resistance, but could also reduce adhesion to the mucosal surface S. equi. Therefore, S. zooepidemicus has stronger adhesiveness but lower level of capsules, which rendered it more susceptible to phagocytosis S. zooepidemicus. Recruiting FH via SzP and TRX to its cell surface would certainly contribute to phagocytosis evasion. When FH was not abundant, S. zooepidemicus could use TRX as succedaneum of FH. C3 deposition experiments showed that TRX and FH both prevented deposition of C3b on S. zooepidemicus, which was pretreated with both TRX and FH limited deposition of opsonic C3b on the bacterial surface. This indicated that the SzP/TRX interaction contributed to the antiphagocytosis response in S. zooepidemicus via the inhibition of the C3b deposition.FH can inhibit the conversion of C3 to C3a and C3b and inactivate C3b. It is recognized as the main regulator of C3 convertase. Many pathogenic organisms evade phagocytosis by coating their surface with the host FH S. zooepidemicus. The SzP/TRX interaction did not influence the TRX activity and function. It also contributed to the FH recruitment and reduced the C3 deposition on the bacterial surface, allowing S. zooepidemicus to be more evasive to the alternative complement pathways. These mechanisms were very beneficial for S. zooepidemicus to evade phagocytosis of the host immune system. Additional studies of this interaction will undoubtedly help us to understand better how SzP is involved in the antiphagocytosis mechanisms.In conclusion, the SzP/TRX interaction was a novel antiphagocytic mechanism of Porcine PAM was obtained with consent from one healthy pig under the ethical approval granted by the Nanjing Agricultural University Veterinary College. The protocol was approved by the Science and Technology Agency of Jiangsu Province. The approval ID is SYXK (SU) 2010-0005. All efforts were made to minimize animal's suffering.Raw264.7 cells (ATCC) and HEK 293 cells (ATCC) were maintained in the Dulbecco's modified Eagle medium (DMEM) high glucose  supplemented with 10% fetal bovine serum (FBS). Cells were used from passages 5–20.S. Zooepidemicus wild type strain ATCC35246 and the S. zooepidemicus SzP-knockout strain was constructed by our lab Escherichia coli strains DH5α, DH10B and BL21, Saccharomyces cerevisiae strain NMY51 4-HIS3 ura3::(lexAop)8-lacZ ade2::(lexAop)8-ADE2 GAL4) were used in this study. S. zooepidemicus was cultured with fresh Todd-Hewitt broth (THB) medium. E.coli was cultured with fresh Luria–Bertani (LB) medium. S. cerevisiae was cultured with fresh Yeast Extract Peptone Dextrose Adenine hemisulfate (YPDA) medium.The S. cerevisiae was transformed using the LiAc method Raw264.7 cells and HEK 293 cells were transiently transfected using Lipofectamine 2000 and opti-MEM  according to the manufacturer's instruction. Media were changed 6 h post transfection and the cells were treated or assayed 24 h post transfection. S. zooepidemicus ATCC35246 SzP (GenBank No. EU624402.1), excluding the signal sequence, was amplified by PCR using primers 5′- GCGGCACGGCCATTACGGCCGTTGAGTCAGCTAAGCCTGTA -3′(SfiI) and 5′- GCAGCGCGGCCGAGGCGGCCTTTTCTTTGCGTCTTGTTGAC -3′(SfiI). PCR product was then inserted into the Split-ubiquitin yeast two-hybrid Cub domain vector pDHB1 to generate the ‘bait’ plasmid pDHB1-SzP, which was verified by DNA sequencing. The expression of the bait protein was confirmed. pDHB1-SzP was used to screen a porcine PAM cDNA library (constructed by our lab) for identifying SzP interacting proteins. Positive yeast clones containing the library plasmid encoding Szp interacting proteins were purified and retested for their growth phenotypes. Plasmid DNA preparations for these yeast clones were generated by the Yeast Plasmid Extraction Kit (Biomega). The insert fragment of these prey plasmids was detected by PCR amplification using primers pPR3N-F (5′- GTCGAAAATTCAAGACAAGG -3′) and pPR3N -R (5′- AGCGTGACATAACTAATTAC - 3′). The chosen prey plasmids were amplified in DH5α, recovered through ampicillin selection and identified by DNA sequencing with pPR3N-F primer. The cDNA sequences were used to search GenBank and NCBI BLAST against the porcine genome. After banishing duplication, the remaining plasmids were re-transformed into the yeast cells containing pDHB1-SzP. Liquid β-galactosidase assay was used to retest for their interaction with SzP in the yeast again.We used the Split-ubiquitin yeast two-hybrid DUALhunter system  to identify SzP interaction partners from the porcine macrophages S. zooepidemicus strain ATCC35246 using the vector pAcGFP1-C . ProLabel tag was fuse at the N-terminal of 12 porcine candidate proteins using the vector pProLabel-C (Clontech). These 12 plasmids were each co-transfected with pAcGFP1-SzP into HEK 293 cells and cultured on 100 mm plates. Cells were harvested after 36 h incubation and lysed with the lysis buffer containing 20 mM Tris, 200 mM NaCl, 1 mM EDTA, 0.5 NP-40 and Protease inhibitor Cocktail  and 1× PMSF . Cell lysates were incubated 2 h with anti-GFP monoclonal antibody (Beyotime) at 4°C. Next, 30 µl of protein G plus/A agarose beads (Clontech) was added and incubated at 4°C overnight. Next day, the beads were washed five times with the lysis buffer and the activity of ProLabel was measured using the ProLabel Detection Kit II (Clontech). pAcGFP1-p53 and pProLabel-T were used as the positive control, while pAcGFP1-Lam and pProLabel-T (Clontech) were used as the negative control.GFP was fused at the N-terminal of SzP in the After the initial identification of the Szp/TRX interaction, it was again confirmed with Co-IP and western-blot. Protein G plus/A agarose beads together with anti-mouse TRX polyclonal antibodies  were incubated with HEK293 cell lysates. The samples were washed extensively in the lysis buffer. The beads were boiled in the sample buffer, and the supernatants were loaded onto SDS-PAGE gels. To confirm the SzP/TRX interaction, western-blot against GFP-SzP was performed using anti-GFP monoclonal antibodies.2O2 in Nonidet P-40 buffer for 15 min and then washed 6 times with the buffer prior to SzP incubation. As a control to revert oxidation, a part of the H2O2-treated fusion protein was incubated with buffer containing 100 mM DTT for 15 min prior to SzP incubation. After 2 h incubation, samples were centrifuged and washed five times with Nonidet P-40 buffer. The precipitated proteins were subjected to SDS-polyacrylamide gel electrophoresis and detected by western-blot using anti-SzP monoclonal antibodies Oxidization treatment for TRX was performed following a previously publish protocol 32 and Cys35 to Ser (C32S/C35S) double mutations in TRX using the TRX-pPR3-N vector as the template (Stratagene). Primers were 5′ CTCAGCCACGTGGTCTGGGCCTTCCAAAATGATCAAGCC 3′ and 5′ GGCTTGATCATTTTGGAAGGCCCAGACCACGTGGCTGAG 3′. The mutant plasmid was named mut-TRX-pPR3-N, and transformed into the yeast strain containing SzP-pDHB1 plasmid to detect the interaction between SzP and mutant TRX.We used “QuikChange II Site-directed Mutagenesis Kit” to construct CysTRX activity assay was performed in 96-well plates with an end point insulin assay S. zooepidemicus ATCC 35246 cultured in vitro was performed as described previously 7 CFU/mL wild strain or the SzP knockout was incubated for 30 min at 37°C in 2 mg/mL TRX. The bacteria cells were washed with phosphate-buffered saline , collected by centrifugation, and incubated with rabbit anti-mouse TRX polyclonal antibodies  for 30 min at 37°C. This was followed by incubation with FITC-conjugated goat anti-rabbit IgG secondary antibodies . Surface-bound TRX was fluorescently stained and analyzed by a FACS Calibur (Becton Dickinson). Data were compared by the analysis of variance with Tukey's correction for multiple comparisons and Student's t-test. P values<0.05 were considered significant.The flow cytometry analysis of 5′- GATCCGGCTTGAAGCCTCTATTACTTTCAAGAGAAGTAATAGAGGCTTCAAGC TTTTTTACGCGTG -3′ and iTRX-antisense 5′-TCGACACGCGTAAAAAAGCTTGAAGCCT CTATTACTTCTCTTGAAAGTAATAGAGGCTTCAAGCCG -3′. The oligonucleotides were annealed and cloned into pBAsi-mU6 . The expression of TRX siRNA was regulated by the mU6 promoter. Cells were harvested 24 h and 36 h after pBAsi-mU6-TRX transfection. The lysates were collected for western-blot analysis and total RNA was extracted to confirm TRX knockdown using RT-PCR analysis.siRNA templates were designed using online siRNA Target Finder  to match 20 nonconserved nucleotide sequences within the mouse TRX mRNA. The oligo nucleotide sequences used to create pBAsi-mU6-TRX were iTRX-sense S. zooepidemicus wild type strain was cultured to mid-exponential phase of growth , washed, and resuspended in Dulbecco's phosphate-buffered saline at 1.25×108 CFU/ml. Bacteria cells were labeled with 5.0 µg/ml FITC (Sigma) for 20 min at 37°C and the unbound labels were removed by 2 washes in DPBS. Labeled S. zooepidemicus were resuspended in DMEM and chilled on ice. The Raw264.7 cells (106) or TRX knock-down cells (106) were combined with 107S. zooepidemicus wild type or the SzP knock-out strain in a 96-well microtiter plate on ice. Samples were mixed gently with a pipette and rotated at 37°C for 60 min. At the desired times, samples were placed on ice and analyzed by flow cytometry. Samples were analyzed to determine the total number of Raw264.7 cells with bound/ingested bacteria and then re-analyzed immediately in the presence of an equal volume of Trypan Blue  to measure the number of Raw264.7 cells with ingested bacteria (phagocytosis). A total of 10,000 events were collected per sample and a single gate was used to exclude debris and free bacteria . Phagocytosis percentage was determined by the percentage of FITC positive Raw264.7 cells after quenching with Trypan Blue.The phagocytosis assay was performed using a previously described method with some modifications Binding of SzP and TRX to FH was determined using the method previously described with some modifications 2. C3i, also known as C3(H2O), was generated by five freeze/thaw cycles of the purified C3. Varying concentrations of TRX, SzP, and the SzP/TRX complex were added with or without 10 µg FH, followed by 200 ng factor D . The reaction mixture was incubated at 37°C for 30 minutes. The generation of C3a was measured by ELISA . All experiments included positive controls  and negative controls .C3 convertase activity was measured as previously described with some modifications S. zooepidemicus wild type and SzP-knockout strains (1.5×108 cells in 150 µl of THB medium) were incubated with 10 µg TRX, 10 µg FH or TRX+FH for 30 min. Fresh porcine plasma (180 µl) incubated at 37°C was then added and mixed with shaking. Complement deposition was stopped immediately after 30 min by the addition of 12 µl 0.5 M EDTA (pH 8.0).The C3 deposition assay was performed using a previously reported protocol with some modifications Flow cytometry analysis: The cells were spun down by centrifugation at 13,000 g for 2 min. Cell pellets were washed twice and resuspended in PBS. Surface-bound C3b was detected with anti-C3 antibodies (Sigma) and FITC-labeled goat anti-rabbit IgG (Beyotime). The binding of the antibodies was measured by flow cytometry. A total of 10,000 events were collected per sample and a single gate was used to exclude debris .Western-blot analysis: the cells were spun down and resuspended in 80 µl 1 M hydroxylamine in carbonate buffer (pH 9.0) after washing with PBS-1% SDS twice. The mixture was incubated at 37°C for 1 h with gentle shaking. After centrifugation, aliquots (25 µl) of the supernatant were subjected to SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated with a 1∶100 dilution of anti-C3 antibodies (Sigma), and developed using HRP conjugated goat anti- rabbit IgG antibodies . The bands were visualized by Chemiluminescence with SuperSignal® West Pico Substrate .t-test with a 95% confidence interval. In all experiments, error bars were denoted using the standard deviation (±SD) of the samples in triplicates or more.All statistics were performed using an unpaired two-tailed