Chronic low-grade inflammation has long been recognized as the central link between obesity and type 2 diabetes (T2D). The novel subset of T helper (Th) cells, Th22, plays an emerging role in chronic inflammation. We investigated the potential association between Th22 and the pathogenesis of obesity and T2D.Ninety T2D inpatients (T2D group), 30 healthy participants with BMI ranged from 19 to 23.9 kg/m2 (CTL group) and 30 metabolically healthy obese controls with BMI ≥ 30 kg/m2 (MHO group) were employed in our study. Peripheral frequencies of Th22 and Th1 and Th17 cells were determined by flow cytometry based on their specific cytokine patterns. Cytokine levels in fresh plasma were quantified by ELISA.*P<0.0001] and CTLs . Furthermore, other than Th1/Th17, previously frequently described participants in obesity and T2D, there was a strong correlation between Th22 frequency and the homeostasis model of assessment for insulin resistance index  and HOMA for β-cell function .Compared to that in CTL group , peripheral frequency of Th22 cells was significantly increased in MHO group  and in T2D group . There was a consistent notable increase in plasma interleukin (IL)-22 of T2D patients [47.56 (30.55–76.89) pg/mL] as compared with that of MHO group [36.65 (29.52–55.70) pg/ml; There were increased Th22 frequencies and IL-22 levels in obesity and T2D. Elevated Th22 and IL-22 also aided in the differentiation of MHO from T2D patients. The notable correlation implied that Th22 might play a more determinant role in both insulin resistance and β-cell impairment. Parallel to type 2 diabetes (T2D), obesity has become a worldwide epidemic concern An appropriate balance between pro-inflammatory and anti-inflammatory T-cell subsets is essential to maintain immune homeostasis and avoid inflammatory diseases. Previous studies in a rodent model of T2D identified an elevation of T helper (Th)1 and Th17 subsets accompanied by a significant decrease of regulatory T cells (Tregs), which may directly trigger activation of innate immunity and thus inflammation, and contribute greatly to insulin resistance + T cells characterized by their production of interleukin-22 (IL-22), Th22 cells are emergent constitutor in autoimmune and inflammatory diseases + CCR6+ CCR10+ phenotype and do not express IL-17, CCL20, IL-23R, CD161 (Th17 markers), IL-4 (Th2 marker), or IFN-γ (Th1 marker) + T (Th0) cells, whose activation can be positively regulated by IL-6 and TNF-α + T subsets that include Th1, Th17, and Treg cells is frequently reported, there is no study at present on the frequencies and function status of Th22 cells in obesity and T2D patients. In the present study, we determined the frequencies of peripheral Th22, Th1, and Th17 cells and plasma levels of the cytokines IL-22, IFN-γ, IL-17, IL-6, and TNF-α. The relevance between Th22 cells and clinical patient data was evaluated.To date, substantial progress in understanding the interactions and counterbalance between helper T-cells in inflammatory status suggests the possibility that new helper T subsets may also play a role in either feedback regulation of immune/inflammatory cell polarization or direct instruction to tissue cells. Recently identified as a distinct lineage of CD42) and metabolically healthy obese  control groups were formed with volunteers from the community and hospital staff. Subjects in the MHO group had increased weight but normal glucose tolerance and lipid profiles. A detailed clinical record was registered for each subject, including history of disease and physical and laboratory examinations. Exclusion criteria were any signs of autoimmune disorders, recent infections, tumors, fever of any cause, and significantly elevated erythrocyte sedimentation rate. Participants did not receive immunosuppressive or immunomodulatory drugs for at least three months during sampling. The Medical Ethical Committee of Qilu Hospital approved this study. A signed consent form was obtained from each participant. Baseline clinical parameters are summarized in Enrollment took place between April 2012 and January 2013 in Qilu Hospital, Shandong University, China. Subjects were assigned to three groups. The T2D group comprised clinically definite T2D inpatients from the Qilu Hospital Department of Endocrinology. Healthy  for further incubation or isolation was drawn into BD Vacutainer® Heparin (sodium) Tubes . Heparinized peripheral whole blood (400 µL) diluted with an equal volume of RPMI 1640 medium containing 50 ng/mL phorbol myristate acetate (PMA), 2 µg/mL ionomycin, and 3.4 µg/mL monensin  was incubated for 4.5 h at 37°C in 5% COStimulated whole blood was stained for flow cytometric study to identify cytokine-producing cells. After incubation, 100 µL blood was stained with PerCP-Cy5.5–conjugated anti-human CD4 monoclonal antibodies  at room temperature for 20 min. The sample was then treated with an equal volume of Fix-Perm reagent A for 15 min, and then washed with pre-cooled washing buffer. Subsequently, Fix-Perm reagent B was added for further permeabilization for intracellular staining and erythrocyte lysis. The sample was incubated for 20 min together with PE-conjugated anti–IFN-γ monoclonal antibodies , FITC-conjugated anti–IL-17A monoclonal antibodies , and eFluro680-conjugated anti–IL-22 monoclonal antibodies . Isotope controls were used for each staining procedure as negative controls and for fluorescence compensation. All antibodies were from eBioscience, San Diego, CA, USA. Fix-Perm reagents were from Invitrogen . All samples were washed and collected using BD Accuri C6 Flow Cytometer. Data were analyzed with FlowJo 7.6.The plasma levels of Th1/Th17/Th22-predominantly secreted cytokines IFN-γ, IL-22, IL-17A, and the cytokines previously reported as major drivers of Th22 commitment from Th0 cells, i.e., IL-6 and TNF-α, were determined with a quantitative sandwich ELISA according to the manufacturer's recommendations . The minimum detectable concentration of IFN-γ, IL-22, IL-17A, IL-6 and TNF-α is 0.30, 2.56, 0.55, 0.37, 0.42 pg/ml respectively.P-value of less than 0.05 was considered statistically significant.Results are expressed as mean ± SEM or median (range). Differences in Th22, Th17, and Th1 cell frequencies; plasma cytokine levels; and expression of specific transcription factors in PBMC among T2D, MHO patients, and CTLs were determined by using ANOVA, and differences between two groups were determined by using the Newman–Keuls multiple comparison test (q test). Data not normally distributed were analyzed with the Kruskal–Wallis test (H test) and Dunn test. Odds ratio was calculated in a 2×2 arranged table with Fisher's exact test. The Pearson or Spearman correlation test was used for correlation analysis depending on data distribution. Partial correlation analysis was conducted to role out confounding factors. SPSS 18.0 or GraphPad Prism 5.0 were used to perform all tests and generate values. A + IFNγ−IL-17−IL-22+), Th1 (CD4+ IFNγ+), and Th17 (CD4+ IFNγ−IL-17+) cells are depicted in *P<0.0001) and (1.90 folds) T2D patients . Even higher percentages (1.31 folds) of Th22 cells were observed in T2D compared to MHO patients (*P<0.05)  .*P<0.0001). And in obese people who had an elevated Th22, diabetes might occur 8.636 times as often as in those with normal Th22 frequency(*P = 0.0148), indicating an potential value of Th22 in predicating obese outcomes. However, since Th22 frequency is a continuous variable and there is currently no wide-accepted categorical threshold, we could only make artificial definition according to our data. Moreover, statistical values should be for reference only since the cases of participants in our study did not represent disease prevalence in the natural population.To further evaluate the increased risk associated with elevated Th22 for health people to develp obesity and for obese people to develop diabetes respectively, the odds ratio was calculated in a 2×2 table (with continuity correction of 0.5 to each cell when needed) (see *P<0.0001) and CTLs  (*P<0.0001). However, our findings failed to demonstrate a significant difference in Th17 frequency between T2D and MHO patients  as compared with MHO patients  ( n = 28) . No signpatients .r = 0.2378, *P = 0.0037, n = 147, Spearman analysis; r = 0.3549, *P<0.0001, n = 147, Spearman analysis; r = 0.4387, *P<0.0001, n = 147, Spearman analysis; As preliminary exploration of the interactions between the three pro-inflammatory helper T subsets, inter-subset correlation was evaluated. There was a significantly positive correlation between the frequencies of peripheral Th22 and Th1 cells  and T2D patients (n = 45), plasma levels of the corresponding predominant cytokines IL-22, IFN-γ, and IL-17 were measured by using ELISA. As shown in *P<0.0001) and CTLs . However, there was no significant difference between MHO and CTLs (P>0.05). Similarly, the concentrations of plasma IL-17 from our blood donors were consistent (*P<0.0001) and MHO patients . There was no significant difference between MHO patients and CTLs (P>0.05). There were also significantly higher concentrations of IFN-γ in MHO  and T2D patients  as compared to CTLs . No significant difference was found between MHO and T2D patients (P>0.05) (To test the hyperactivity of the pro-inflammatory CD4nsistent . There w(P>0.05) .r = 0.4904, *P<0.0001, Pearson analysis) as well as between Th17 frequency and IL-17 concentration . However, there was no statistical correlation between peripheral Th1 cells and non-stimulated circulating levels of IFN-γ  . As shown in nalysis) .*P<0.01) and T2D patients  as compared with CTLs . There were also significantly higher concentrations of TNF-α in T2D patients as compared to MHO patients (*P<0.0001) (*P<0.05), we found no significant difference between MHO patients  and CTLs as well as T2D patients (P>0.05)  and IL-6 levels  to Th22 frequency.TNF-α and IL-6 have been reported as major drivers of Th22 polarization<0.0001) . While s(P>0.05) . Moreover = 0.6771, *P<0.0001, Pearson analysis)  index. Further, partial correlation analysis in female (n = 22) and male (n = 28) was conducted with the confounding effects of age and sex removed. Significant correlationship between Th22 frequencies and ln(HOMA-IR) values was still present in both male  and female  subsets of participants.As our study revealed an expanded Th22 population in patients with MHO and T2D, we further studied the potential role of this hyperactive phenotype in the development of impaired insulin sensitivity. The homeostasis model of assessment for insulin resistance index (HOMA-IR) has been widely accepted as a good indicator of insulin resistance, especially in patients with comparatively well preserved islet β-cell functions, e.g., MHO patients, prediabetic individuals (impaired glucose tolerance or impaired fasting glucose), and some early-diagnosed T2D patients nalysis) . Howevernalysis)  nor Th17nalysis) , previour = −0.7264, *P<0.0001, Pearson analysis)  and female  subsets of participants respectively.As an even more significant expansion of the Th22 population and elevated IL-22 secretion was observed in T2D patients, we investigated the idea of whether the hyperactive Th22 phenotype correlated with the decompensation of β-cell function. The HOMA for β-cell function (HOMA-β) was introduced as a parameter of the residual basal insulin-secreting function of β cells in patients with later-stage T2D nalysis) . There wnalysis) . CorrelaAlong with T2D, obesity shares a common pathological process of insulin resistance. It has been proven that insulin resistance is the core initiating and persisting factor in T2D and obesity-associated metabolic syndrome, while the steady loss of β-cell function results in the aggravation of hyperglycemia and development of diabetic complications Compared with the well-known Th1, Th2, Th17, and Treg subsets, Th22 is a newly identified helper T-cell subset with a specific phenotype and distinct function. Th22 cells possess a specific cytokine profile that includes IL-22 and TNF-α, both frequently reported to play important roles in chronic inflammation and tumorigenesis + IFN-γ−IL-17−IL-22+) in both obesity and T2D patients as compared with healthy donors. The increased Th22 population could even aid in distinguishing MHO patients from T2D patients. The increase in Th22 cells was more significant in comparison with Th1 and Th17 cells, the previously described pro-inflammatory subsets in obesity and T2D. There were also consistently increased levels of IL-22 in both MHO and T2D patients, which was in concert with the previously described inflammatory cytokines. These results indicate that Th22 cells might play a greater role in the immunologic disturbance contributing to disease progression.To study whether the Th22 subset is involved in the process of obesity and T2D, the percentages of peripheral Th22 cells in each participant were determined by using flow cytometry. Our study first identified a notably expanded peripheral Th22 population  values with Th22 but not Th1 or Th17 frequencies indicates that Th22 cells may contribute greatly to insulin resistance in both MHO and T2D patients. As we observed an even more hyperactive Th22 phenotype in T2D patients, we further investigated whether Th22 cells exerted a negative effect on islet β-cell function. Again, there was a strong negative correlation of Th22 cells with the clinical indicator of basal β-cell function, ln(HOMA-β). This finding preliminarily demonstrates a causative or even determinant role of Th22 cells in the progressive loss of β-cell function. However, the underlying mechanisms have yet to be discussed.To explore the sources of the expanded Th22 population, we determined the plasma levels of TNF-α and IL-6, which have been identified as major drivers of Th22 lineage commitment The study of how metabolic pressures affect the modulation of immune homeostasis


Bartonella in immunocompromised patients are caused by B. henselae and B. quintana. Unlike immunocompetent hosts who usually develop milder diseases such as cat scratch disease and trench fever, immunocompromised patients, including those living with HIV/AIDS and posttransplant patients, are more likely to develop different and severe life-threatening disease. This paper will discuss Bartonella's manifestations in immunosuppressed patients and will examine Bartonella's interaction with the immune system including its mechanisms of establishing infection and immune escape. Gaps in current understanding of the immunology of Bartonella infection in immunocompromised hosts will be highlighted.Most infections by genus  Bartonella species which are important etiologic agents in human disease. The discussion focuses on a review of what is known of the immunology of Bartonella, particularly in immunocompromised hosts. In this paper, we present a summary of the basic characteristics of Bartonella are fastidious hemotropic facultative intracellular Gram-negative bacteria of the alpha-2 subgroup of Proteobacteria [Bartonella, B. bacilliformis, B. henselae, and B. quintana infections have been most commonly implicated in human disease and B. henselae and B. quintana account for the majority of morbidity and mortality among immunocompromised individuals [bacteria , 2. Of tividuals –5. Bartonella species implicated in human disease has unique epidemiology. While the vast majority of Bartonella species have an identified zoonotic reservoir such as domesticated animals and farm animals, the animal reservoir for both B. quintana and B. bacilliformis have not yet been identified. While B. bacilliformis is transmitted by the sand fly and the major vector for B quintana is the human body louse, B quintana has also been found in rodent fleas, including rat fleas, and thus a potential for a rodent vector has been hypothesized [Each of the major thesized , 7. B. henselae occurs throughout the world. It is a zoonosis transmitted from the natural reservoir, cats (Felis catus), usually via cat scratch or bite and less commonly by a vector such as cat fleas  or ticks [or ticks . Cross-sor ticks . Humans B. quintana occurs worldwide, with improved sanitation and living conditions, disease caused by B. quintana has dramatically decreased and now occurs primarily in small epidemics in conditions characterized by crowding and poor sanitation. Although B. quintana which is spread by human body lice (Pediculus humanis corporis) [B. quintana's pathway of invasion [Humans are likely the natural reservoir for orporis) . Infecteinvasion . Wild roinvasion , 7.B. bacilliformis is endemic in mountainous areas of Peru, Columbia, and Ecuador [Lutzomyia sandfly, but the animal reservoir has yet to be identified.  Ecuador . It is tBartonella species in humans, little is known about the humoral and cellular immune mechanisms involved in establishment and persistence of Bartonella infection in humans. Experimental studies using B. tribocorum inoculation onto an immune-competent rat model have shown that the brief bacteremia after inoculation is followed by a 3–5-day bacteremia-free window before resurgence of persistent intraerythrocytic bacteremia that lasts for 8–11 weeks [Despite extensive knowledge about the disease syndromes caused by 11 weeks  or endot11 weeks , and, fi11 weeks .Bartonella lipopolysaccaride (LPS) is composed of a unique combination of Lipid A and long-chain fatty acids and likely contributes to the bacteria's ability to evade the immune system [Bartonella initially evades the innate immune system because its surface molecules are not recognized by TLR-4 on dendritic cells or macrophages, [B. quintana may even antagonize the Toll-like receptor 4 (TLR4) [B. quintana resulted in the downregulation in human monocytes of nearly all cytokines normally produced by TLR4 in response to LPS [B. quintana's antagonism of TLR4 may be responsible for the absence of symptoms normally associated with bacteremia with Gram-negative organisms, such as septic shock.e system . Empiricophages, , therebyophages, , 18. The4 (TLR4) , which ie to LPS , 20 and B. henselae can avoid lysosomal fusion and acidification after the bacteria invades phagocytes such as endothelial cells and macrophages. For instance, B. henselae containing vacuoles (BCVs) had delayed development or complete lack of typical endocytic markers [B. henselae, the endocytic markers were in normal ranges. Such Dendritic cells within cat scratch disease (CSD) granuloma in immune-competent humans that are chronically infected with Bartonella are also able to limit the infection to the same lymph node region via a localized B-cell-mediated humoral immune activation that is regulated by dendritic cells [Bartonella in cardiac valves, other vasoproliferative lesions (bacillary angiomatosis or peliosis). Exactly how local bacterial control is lost in immunocompromised subjects is unclear.Furthermore,  markers . When thic cells . On the Bartonella infection. The old Bartonella skin test involved an intradermal injection of suppurative material derived from lymph nodes from cat scratch disease patients and was interpreted as positive if their skin developed a large erythematous area within 48 hrs. This test is a classic delayed type hypersensitivity reaction, mediated through the Th1 immune response. In vitro studies have also supported the use of a Th1 response with mice splenocytes showing increased production of the Th1 cytokines IFNγ and IL-12 in response to B. henselea when compared with controls [B. henselae [Historically, clinical and in vitro studies implicated a Th1 immune response to controls . The rolhenselae .Bartonella infection. Acute infection in immune-competent subjects with CSD have revealed upregulation of proinflammatory  and anti-inflammatory (IL-10) cytokines [Bartonella, a cytokine milieu that may allow an acute infection to persist especially given the anti-inflammatory nature of IL-10 [Bartonella infection persistence and role in vasoproliferation is better understood from cytokine profiles, and less so from the traditional roles of humoral or cellular immunity.More recent studies have focused on the role of cytokines in acute and chronic ytokines . On the of IL-10 , 25, 26.of IL-10 , 27 and of IL-10 , but notBartonella species in immunocompromised patients has been hampered by the lack of good animal model [Bartonella. Chiaraviglio et al. described a murine model for chronic Bartonella infection using B. taylorii in SCID/Beige mice  [Bartonella growing in extracellular aggregates in the spleen and liver. As the model is still relatively new, they did not present any further characterization of the immune response. However, it is clear that intraerythrocytic persistence is characteristic of infections in immune-competent hosts while endothelial/periendothelial persistence leading to vasoproliferation is characteristic of an infection in immune-compromised hosts [Most of the research in infection involving immunocompromised hosts has focused on the establishment of bacterial stimulated angiogenesis. Understanding of the immune defense of al model . Since tficient) . The groed hosts .B. quintana and B. henselae infections in patients with cell-mediated immunodeficiency such as HIV and posttransplant patients on immunosuppressive therapy [ B. henselae and B. quintana induce their characteristic vasoproliferative lesion, bacillary angiomatosis, or peliosis, in immunosuppressed patients, by direct and indirect effects on endothelial cells.Bacillary angiomatosis is the most common sequelae of  therapy , 23, 30.B. henselae and B. quintana infections persist within periendothelial extracellular matrix resulting in sustained, localized bacterial replication within collagen tissue [B. henselae and B. quintana. A further analysis of the antiapoptotic mechanisms of some B. henselae strains isolated from HIV-infected patients revealed that an anti-apoptotic state may be effected through B. henselae strain-specific upregulation of antiapoptotic or downregulation of proapoptotic factors that work through the mitochondrial intrinsic apoptotic pathway [Bartonella infections may play a role, albeit unclear, in the development of bacillary angiomatosis given that elevated levels have been reported both animal [Whereas most pathogenic bacteria achieve tissue destruction and wider dissemination by inducing apoptosis on host cells, n tissue . This lon tissue . In addi pathway . As noteh animal  and humah animal , but noth animal .κB is also upregulated by Bartonella, resulting in increased leukocyte rolling and adhesion and causes a proinflammatory environment [B. henselae strains that infect HIV patients and vasoproliferation and inflammation. Such work may shed more light into pathogenicity of B. henselae and host susceptibility factors in HIV-positive patients. NF-ironment . AdditioB. bacilliformis itself induced an immunocompromised state. Although B. bacilliformis is not widespread in distribution and has not been described as a cause of bacillary angiomatosis or hepatic peliosis in immunocompromised patients, it is notable that the acute bacteremic phase of B. bacilliformis causes significant immunosuppression by overloading the reticuloendothelial system. These patients are predisposed to infections such as salmonella and tuberculosis [rculosis . Bartonella infections caused by B. henselae or B. quintana in the immunocompromised host may be characterized by fever of unknown origin, culture-negative endocarditis, osteomyelitis, or angioproliferative lesions that may affect virtually any organ system, but have a predilection for the skin, liver, and spleen. B. henselae or B. quintana infection in HIV/AIDS patients [Bacillary angiomatosis lesions and fever are the most common manifestation of patients , 9. The patients –35, genipatients , and othSimilar vascular lesions are seen in both bacillary peliosis and splenitis. Patients present with hepato- and/or splenomegaly as blood filled cysts proliferate in these organs. Bartonella, Coxiella, and Chlamydia species. Results from tissue culture are not always positive and take a long time given the fastidious nature of the bacterium. PCR is favored as a faster, sensitive, and specific diagnostic test [Confirming the diagnosis can be challenging as serology has a 50–95% sensitivity and demonstrates cross-reaction between tic test , 38. B. henselae [B. henselae infection was complicated by acute graft rejection in 2 renal transplant patients [B. henselae on decreasing the donor tolerance of the graft or enhancing graft antigenicity. In liver transplant patients, B. henselae can present with hepatic masses [Persistent fever following close contact with cats is the most common presenting symptom among posttransplant patients who are infected with henselae . B. henspatients . It is uc masses , hepaticc masses , 43, or c masses .B. quintana (less so B. henselae) infection among homeless patients with chronic alcoholism [Bartonella endocarditis include preexisting valvular lesions and exposure to human body lice. Subacute, culture-negative endocarditis is the most common clinical presentation of coholism , 45, 46.B. quintana may be cultured from the blood, but given the prolonged time required for culture, other diagnostic tests are preferred. An indirect immunofluorescent IgG antibody test directed against Bartonella species with titer of 1 : 800 or higher has a 95% positive predictive value for diagnosing Bartonella endocarditis [carditis . Diagnoscarditis .Bartonella species are important zoonotic agents of human disease. Immunocompromised hosts are more susceptible to infection from B. henselae or B. quintana which results in a more severe clinical picture as compared with the normal host. More recent work explores role of cytokines and chemokines in subverting the immune system, and subsequently a persistent infection characterized by vasoproliferation in immunocompromised humans. The mechanisms of establishing infection and immune evasion in immunocompromised hosts are still incompletely understood and are areas where further studies are needed.


The union of different genomes in the same nucleus frequently results in hybrid genotypes with improved genome plasticity related to both genome remodeling events and changes in gene expression. Most modern cereal crops are polyploid species. Triticale, synthesized by the cross between wheat and rye, constitutes an excellent model to study polyploidization functional implications. We intend to attain a deeper knowledge of dispersed repetitive sequence involvement in parental genome reshuffle in triticale and in wheat-rye addition lines that have the entire wheat genome plus each rye chromosome pair. Through Random Amplified Polymorphic DNA (RAPD) analysis with OPH20 10-mer primer we unraveled clear alterations corresponding to the loss of specific bands from both parental genomes. Moreover, the sequential nature of those events was revealed by the increased absence of rye-origin bands in wheat-rye addition lines in comparison with triticale. Remodeled band sequencing revealed that both repetitive and coding genome domains are affected in wheat-rye hybrid genotypes. Additionally, the amplification and sequencing of pSc20H internal segments showed that the disappearance of parental bands may result from restricted sequence alterations and unraveled the involvement of wheat/rye related repetitive sequences in genome adjustment needed for hybrid plant stabilization. Triticum aestivum and Secale cereale genomes, respectively [Repetitive DNA sequences have been extensively studied in large plant genomes, corresponding up to 83% and 92% of ectively . These iectively –7. FurthTriticosecale), synthesized through the hybridization of wheat (Triticum spp.) and rye , has been broadly used in the production of wheat-rye addition, substitution, and translocation lines by backcrossing to wheat [Hordeum vulgare L.) [In Situ Hybridization (FISH) experiments that those methodologies target dispersed sequences preferentially clustered in rye sub-telomeric chromosome domains [In polyploids, where more than two basic sets of chromosomes share the same nucleus, an irreversible outbreak of paternal genome reorganization has been described in several plant systems . Polyploto wheat . The invgare L.) ,15. IRAPgare L.) ,15. Moreet al. [The presence of rye chromatin in wheat background has been detected through RAPD analysis ,16. The et al. . We haveOPH20 RAPD marker and pSc20H sequence  were useRAPD marker obtained with the OPH20 10-mer primer  Experim. Minor aThe triticale banding profile was compared with wheat and rye parental lines and with wheat-rye addition lines produced by controlled backcrossing of octoploid triticale to hexaploid wheat followed by self-fertilization. The analysis of triticale banding profile showed two characteristic rye-origin bands and only the ~650 bp wheat-origin band. The wheat-origin ~850 bp band is consistently absent in the triticale banding profile . The anaAll bands observed in wheat and rye parental lines using OPH20, except the rye-origin ~1500 bp band previously sequenced in rye cv. Imperial , were gAegilops tauschii clone BAC RI43D6 hypothetical protein and plastid acetyl-CoA carboxylase (Acc-1) genes  BLAST alignment revealed 87% homology with the following published sequences: 60897.1, ); A. tau97474.1, ) within T. aestivum chromosome 3B-specific BAC library contig ctg0464b  revealed 97% sequence similarity with T. aestivum cv. Chinese Spring clone BAC 400N24 containing a TaCKX2.3 cytokinin oxidase/dehydrogenase gene that was mapped to chromosome 3D  NCBI nucleotide alignment revealed 86% sequence similarity with only one published sequence, a 64430.1, ), coveri92901.1, ). OPH20W64430.1, ) within gypsy-like partial sequences isolated from distinct Secale species  recently published [Triticum sp. loci: 81% with T. aestivum chromosome 3B-specific BAC library, contig ctg0091b  BLAST revealed a sequence similarity between 83% and 97% with several retrotransposon ublished . Rye ori64428.1, ); 80% wi94981.1, ); 80% wi37335.1, ); and 7926565.1, ).Differences between wheat-rye addition lines for amplification products using OPH20 10-mer primer were detected based on the presence or absence of pSc20H band. Thus, we further delineated a deeper analysis of such sequences using three pairs of primers designed to amplify pSc20H internal segments, namely 20H1 (698 bp); 20H2 (699 bp); and 20H3 (700 bp) .Identical bands with the expected size for the three referred pSc20H internal segments were amplified from wheat, rye and triticale. Moreover, using DNA from all wheat-rye addition lines as template, similar bands were obtained for the 20H1 segment  as well DNA dilutions used as templates in the initial OPH20 10-mer PCR experiment and in the PCR reactions using primers to the pSc20H internal segments were virtually the same. Therefore, our results clearly show that although pSc20H rye-origin ~1500 band is not amplified in wheat and in wheat lines with the addition of 2R, 3R, 4R and 7R rye chromosomes, all internal fragments of pSc20H, covering a total extension of 1226 bp are present both in wheat and in the whole set of wheat-rye addition lines.Bands amplified from wheat, rye and 1R, 3R, 4R 5R, 6R, and 7R addition lines using primers for 20H1 internal segment of pSc20H sequence were gel-isolated, purified and cloned for sequence analysis. Sequences amplified from different clones obtained from the same band revealed 100% homology with each other.The 20H1 sequence amplified from rye  shares 94% sequence similarity with the expected segment of pSc20H (pSc20H_20H1) and 20H1 sequences amplified from wheat addition lines of 1R, 5R and 6R rye chromosomes share 97% , 95%  and 96% 20H1 6R, accession number JX120164) homology with rye 20H1 sequence, respectively. 20H1 sequences amplified from the three wheat-rye addition lines referred to above share a level of sequence similarity between 96% and 98% with each other.T. aestivum cv. Chinese Spring clone BAC 400N24 containing TaCKX2.3 cytokinin oxidase/dehydrogenase gene  shares only 81% sequence similarity with the expected pSc20H segment (pSc20H_20H1) and 83% with 20H1 sequence amplified from rye. BLAST nucleotide alignment of the wheat 20H1 sequence revealed 95% homology with 92901.1, ) in a reT. aestivum BAC 400N24. The other one encompasses 20H1 from rye and wheat addition lines 1R, 5R and 6R as well as pSc20H.The alignment of 20H1 sequences is presented in Although it is presently widely established that polyploidization leads to the appearance of new phenotypes and eventually to the creation of new species with higher plasticity , a deepeet al. [et al. [et al. [Regarding wheat-origin bands, in triticale only OPH20Wheat650 is present, with the OPH20Wheat850 wheat-specific band being absent. However both bands are present in all wheat-rye addition lines, which is thought to result from a backcross of triticale to wheat involved in the production of those lines. On the contrary, the two bands amplified in rye are present in triticale, being however polymorphic in wheat-rye addition lines: OPH20Rye1300 sequence is absent in all the wheat-rye addition lines and pSc20H-like 1500 bp sequences are amplified in three of the seven lines. Although not referred to by the authors, in [et al.  and Ko e [et al.  did not The results that we attained confirm parental genome restructuring shown mainly in the disappearance of bands observed in progenitor profiles as previously described through Amplified Fragment Length Polymorphism (AFLP) analysis ,30 as wegypsy-type retrotransposon LTR sequence [Aegilops loci containing d-genome HMW-glutenin [copia-like retrotransposon sequences of a T. aestivum [The molecular tools used not only constitute an original approach to detect sequence rearrangements induced by polyploidization, but yield also novel outcomes concerning the analysis of specific sequences affected by genome merger. In fact, sequence analysis of bands amplified from wheat with OPH20 primer revealed sequence similarity with both repetitive and coding loci. The wheat restructured sequence absent in triticale (OPH20Wheat850) revealed sequence similarities with a sequence . A rye-sglutenin  and acetglutenin  as well aestivum , statingaestivum .T. aestivum a 3D-specific BAC containing a TaCKX2.3 cytokinin oxidase/dehydrogenase gene (OsCKX2). This gene was reported to affect yield in rice and was recently characterized in wheat [To further examine the extension of parental genome remodeling affecting pSc20H, three pairs of primers designed to amplify distinct internal overlapped segments  were used. Unexpectedly, all internal sequences were amplified on all genotypes analyzed including the ones where pSc20H-like sequences were not amplified . Such results demonstrate that differences concerning pSc20H amplification observed in the present study as well as in previous ones ,16 may rin wheat . Thus, wT. aestivum BAC containing the OsCKX2 gene locus [Additionally, the 20H1 sequences phylogenetic tree presented in ne locus . Thus, tT. aestivum L. “Chinese Spring” , diploid rye S. cereale L. “Imperial” , the correspondent synthetic octoploid triticale , and a set of seven wheat-rye addition lines, each composed of the entire hexaploid wheat genome plus a single pair of rye homologous chromosomes. Wheat-rye addition lines were produced through controlled backcrossing of the octoploid triticale to the wheat parent, followed by successive generations of self-fertilization and subsequent rye chromosome selection. The rye cultivar “Imperial” used is highly inbred and the octoploid triticale and their corresponding wheat-rye addition lines are at least 35 generations old [The following plant materials were used: hexaploid wheat ions old . All seeSeeds from all genotypes were germinated and grown in controlled conditions at a 16 h light (20 °C)/8 h dark (20 °C) cycle. Genomic DNA was isolated from fresh young leaves of 8-week-old plants using modified cetyltrimethylammonium-bromide (CTAB) method . The pre2, 0.8 mM dNTP’s, 1.6 μM primer, 1.5 U Taq polymerase, 20 μg BSA, 10 ng DNA template and the following program: 5 min at 94 °C; 45 cycles of 1 min at 94 °C, 1 min at 36 °C, and 2 min at 72 °C; and a final 5-min extension at 72 °C.PCR analysis was performed using OPH20 10-mer as primer , a RAPD 2, 0.25 mM dNTP’s, 1 mM each primer, 0.5 U Taq polymerase, 50 ng DNA template and the following program: 5 min 94 °C, 30 cycles of 45 s 94 °C, 45 s 60 °C and 1 min of 72 °C, and a final extension of 5 min at 72 °C.To further characterize the pSc20H specific sequence, it was amplified using the referred OPH20 10-mer primer , three ® TA Cloning® Kit in pCR2.1 vector and sequenced. The sequences obtained were analyzed through BLAST nucleotide alignment on NCBI and sequence similarity between them was verified using BioEdit version 7.1.3.0 sequence alignment editor . Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 5 [PCR products were run on 1.7% agarose gels using as molecular weight marker 1 Kb Plus DNA Ladder (Invitrogene), detected by ethidium bromide and photographed using a BIO-Rad GEL DOC 2000. OPH20 10-mer PCR and pSc20H internal segments amplification banding profiles presented ,3 were cersion 5 .et al. [RAPD methodology using OPH20 10-mer marker initially developed by Freancis et al.  to achieet al. . The abset al.  and rye et al. ,16 seem


BAIAP2, which is asymmetrically expressed in the two cerebral hemispheres, with ADHD in European population. The present study aimed to investigate the association between BAIAP2 and ADHD in Chinese Han subjects.Attention-deficit/hyperactivity disorder (ADHD) is a common chronic neurodevelopmental disorder with a high heritability. Much evidence of hemisphere asymmetry has been found for ADHD probands from behavioral level, electrophysiological level and brain morphology. One previous research has reported possible association between BAIAP2 were chosen and performed genotyping for both family-based and case-control association studies.A total of 1,397 ADHD trios comprised of one ADHD proband and their parents were included for family-based association tests. Independent 569 ADHD cases and 957 normal controls were included for case-control studies. Diagnosis was performed according to the DSM-IV criteria. Nine single nucleotide polymorphisms (SNPs) of Transmission disequilibrium tests (TDTs) for family-based association studies showed significant association between the CA haplotype comprised by rs3934492 and rs9901648 with predominantly inattentive type (ADHD-I). For case-control study, chi-square tests provided evidence for the contribution of SNP rs4969239, rs3934492 and rs4969385 to ADHD and its two clinical subtypes, ADHD-I and ADHD-C. However, only the associations for ADHD and ADHD-I retained significant after corrections for multiplicity or logistic regression analyses adjusting the potential confounding effect of gender and age.BAIAP2 in the etiology of ADHD, especially ADHD-I.These above results indicated the possible involvement of  Attention-deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder. The high heritability of approximately 0.76  has suggThe typical development of human cerebral asymmetry has profound effect on the normal lateralization of cognitive and motor functions, such as language and handedness. Several studies have suggested the possible link of the disturbance of cerebral asymmetry with the pathogenesis of neurodevelopmental disorders , such asThe non-right-handedness, especially mixed-handedness, might be a risk indicator for the symptom severity of inattention in ADHD . One of BAIAP2, which is located on 17q25 and encodes brain-specific angiogenesis inhibitor 1-associated protein 2 (BAIAP2), has been suggested to be involved in cerebral asymmetry [BAIAP2 with ADHD, supplying genetic evidence of abnormal left-right brain asymmetries with this disorder [BAIAP2 and its isoform BAIAP2L1 with ADHD [BAIAP2 in rat cerebral cortices [BAIAP2 has also been found to confer risk for autism spectrum disorders (ASD), which shared some genetic risk factors with ADHD [The gene symmetry . Recentldisorder . In addicortices . BAIAP2 ith ADHD .BAIAP2 has been suggested to be one of novel candidate genes for ADHD, but need more work for replication [BAIAP2 and ADHD in Chinese Han subjects.For the potentially important role of cerebral asymmetry in the pathophysiology of ADHD, lication . Our preAll ADHD cases were recruited from child psychiatric clinics of Peking University Institute of Mental Health. Diagnosis was performed according to the DSM-IV criteria by experienced child psychiatrists, using the Clinical Diagnostic Interview Scale (CDIS) , which wThe control sample consisted of 957 subjects from local elementary schools, healthy blood donors from the blood center of the First Hospital of Peking University, and healthy volunteers at our institute. ADHD, other major psychiatric disorders, family history of psychosis, severe physical diseases and substance abuse were excluded (more details have been described in our previously published article ). DemogrAll subjects were treated according to the Declaration of Helsinki and this work was approved by the Ethics Committee of Peking University Health Science Center. Written informed consent was obtained from each subjects or parents of children.Peripheral blood was collected for all subjects included in this study. Then genomic DNA was extracted following the standard protocols using E.Z.N.A.™ Blood DNA Kits .http://www.hapmap.org). Threshold limit was set of r2 > 0.80 for all SNPs with minor allele frequency (MAF) > 0.25. Thirteen tag SNPs were chosen with these criteria. However, only eight tag SNPs were included for genotyping and further association analyses. The other five SNPs were not selected for the location at the same haplotype block or strong to moderate linkage disequilibrium (LD) with those selected SNPs (Table For selection of single nucleotide polymorphisms (SNPs), Haploview version 4.2 was used to pick up tag SNPs based on the CHB database from Hapmap , using Taqman allelic genotyping assays  and follP-value from 0.178 to 0.921). The MAFs for all SNPs were from 0.162 to 0.499. Haploview software was also used to estimate the linkage disequilibrium (LD) of all SNPs and generate haplotype blocks which will be used for multi-marker haplotype-based association tests [Calculation of MAF and analysis of Hardy-Weinberg equilibrium (HWE) were performed using Haploview 4.2 software for cases and controls separately, showing no departure from HWE for any SNP (on tests . As showP < 0.05), further analyses for genotypes based on dominant and recessive models were conducted [For family-based association study, TDT tests were conducted using Haploview to investigate whether there was biased-transmission of alleles or haplotypes in ADHD trios. For case-control study, chi-square tests were conducted using Haploview for both single-marker and multi-marker haplotype-based analyses, to compare the frequencies of alleles and haplotypes between ADHD cases and controls. In addition, comparison of the genotype frequencies was also performed for case-control statistics using SPSS software. Once there was nominally significant association appeared either for alleles or genotypes under additive model , three clinical subtype, and the comparison of genotype and allele frequencies, the adjusted significance was set at P < 0.0012. For multi-marker association tests, haplotype analysis was only conducted when nominal association existed in the single-marker analyses and significance was corrected by 5,000 permutations using Haploview software.For multiple testing corrections of single-marker analyses, Bonferroni corrections were conducted. We firstly used SNPSpD software  to consihttp://pngu.mgh.harvard.edu/~purcell/gpc/), taking the lowest MAF of 0.162 and assuming the prevalence of 0.05, significance level of 0.05 and odd ratio (OR) of 1.5. Under above settings, our sample showed the minimum statistically genetic power of 80.9% for TDT statistics with about 500 efficient trios, and 74.4% for 569 cases vs. 957 controls under additive model.The minimum statistically genetic power for TDT tests and case-control studies was calculated using the Genetic Power Calculator software  and the GA haplotype was potentially under-transmitted    and rs3934492 . One SNP rs4969385 showed association with ADHD-C   and ADHD-I  retained significant after corrections for multiplicity, but not for ADHD-C  (Table BAIAP2 with ADHD (P < 0.05) and ADHD-I (P < 0.05) after adjusting the potential effect of gender and age, but not for ADHD-C (P = 0.086)  a2=5.44, nominal P = 0.019, empirical P = 0.097). For ADHD-I, the GA haplotype of Block2 showed lower frequency  in cases than controls, while the CA haplotype showed higher frequency . For ADHD-C, the CT heplotype of Block3 showed hither frequency in cases . However, none of above association remained significant after permutations.Based on the association from single-marker analyses, we performed haplotype analyses for ADHD, ADHD-I and ADHD-C. As indicated in Table BAIAP2 was associated with childhood ADHD of Chinese Han descent, especially for the predominantly inattentive type (ADHD-I). Analyses for case-control studies indicated different alleles and genotypes distribution between ADHD and controls, and the association was only retained for ADHD and ADHD-I after multiple corrections or adjusting the confounding effect of gender and age, but not for ADHD-C. From TDT tests for trios, the significant association was only indicated for ADHD-I from haplotype analyses.Our results showed that BAIAP2 with ADHD. However, in their study, the association of BAIAP2 with ADHD was only observed in adults, which has also been replicated in an independent population, but not children. The possible explanation for this discrepancy may be that the genetic load for ADHD children may be lower than ADHD adults, while the sample size in the study by Ribasés et al. [BAIAP2 on ADHD.In some extent, our current findings are consistant with previous reports. Ribasés et al.  have anas et al.  may be nD’ = 0.231, r2 = 0.04) and rs4969385 . So we can conclude that the SNP rs8079626 was not associated with ADHD in Chinese Han subjects. This discrepancy may be greatly due to the ethnic difference, that its allele distribution in our current analyses of Chinese Han subjects  was adverse to that of German samples  from the study by Ribasés et al. [In our present study, the SNP rs4969385 was the only associated one with ADHD and ADHD-I after corrections for multiple testing. This SNP has also been reported in adult samples by Ribasés et al. . This res et al. . This adBAIAP2 with ADHD-I subtype. Three subtypes of ADHD were defined in DSM-IV including predominantly inattentive, predominantly hyperactive/impulsive and the combined subtypes [COMT, 5-HT1B, MAOA, CHRNA4, SYP and DDC exhibited associations primarily with ADHD-I, whereas HTR2C, 5-HT1D, ADRA2C, DRD3 and NET1 were associated mainly with ADHD-C [NET1 and ADHD strongly suggested that the haplotype blocks within different regions of NET1 show divergent association based on sex and subtype [Another interesting finding is the specific association of subtypes . A growisubtypes ,33. Attesubtypes . Our preh ADHD-C ,35-38. T subtype . Taken tBAIAP2 in this study and whether other genes played the role for the pathogenesis of ADHD remained unclear. Secondly, we have set MAF > 0.25 for tag SNPs selection and included an additional reported SNP rs4969385 with MAF of 0.162. We could not preclude the rare genetic contribution of uncommon SNPs (MAF <0.162) to the occurrence of ADHD. Thirdly, we have not screened those parents for ADHD diagnosis that we could not explain the genetic mechanisms of BAIAP2 in the etiology of ADHD clearly. Fourthly, ADHD was associated with high rates of comorbidity including dyslexia (reading disorder). In the existing literature, evidence has support the common risk neurobiological phenotype of atypical cerebral asymmetry (ACA) and shared ACA genes for ADHD and dyslexia [Our findings need to be considered in light of some limitations. Firstly, twenty-seven genes have been identified involving in the left-right asymmetric cortical development in humans . We onlydyslexia . The diaBAIAP2 in the predisposition to ADHD, especially ADHD-I. Further studies should investigate the involvement of hemispheric asymmetry genes modulating the difference of left-right hemisphere in ADHD and related cognition.The present family-based and case-control association studies in our Chinese Han populations provide further evidence for the role of ADHD: Attention-deficit/hyperactivity disorder; ADHD-I: ADHD predominantly inattentive type; ADHD-HI: ADHD predominantly hyperactive/impulsive type; ADHD-C: ADHD combined type; SNP: Single nucleotide polymorphisms; CDIS: Clinical Diagnostic Interview Scale.The author’s declare that they have no competing interests.LZH and QQJ designed the study. LL, SL, LZH and LHM participated in data collection and experiments. LL, LZH and WLP participated in the statistical analyses and interpreted the results. LL, SL and QQJ drafted the manuscript. WYF supervised the whole study and revised the manuscript. All authors read and approved the final manuscript.


Attention deficit hyperactivity disorder (ADHD) is a common comorbidity of childhood epilepsy, but the neuroanatomical correlates of ADHD in epilepsy have yet to be comprehensively characterized.Children with new and recent-onset epilepsy with (n = 18) and without (n = 36) ADHD, and healthy controls (n = 46) underwent high resolution MRI. Measures of cortical morphology  and subcortical and cerebellar volumes were compared between the groups using the program FreeSurfer 5.1.Compared to the control group, children with epilepsy and ADHD exhibited diffuse bilateral thinning in the frontal, parietal and temporal lobes, with volume reductions in the brainstem and subcortical structures . There were very few group differences across measures of cortical volume, area or curvature.Children with epilepsy and comorbid ADHD exhibited a pattern of bilateral and widespread decreased cortical thickness as well as decreased volume of subcortical structures and brainstem. These anatomic abnormalities were evident early in the course of epilepsy suggesting the presence of antecedent neurodevelopmental changes, the course of which remains to be determined. Attention deficit hyperactivity disorder (ADHD) is a common neuropsychiatric disorder in children and adolescents. The prevalence rate varies across individual studies, but is similar among pooled results regardless of the diagnostic procedure used and the countries or regions of the populations examined, ranging from 5.9 to 7.1% Children with epilepsy have an increased prevalence of mental health disorders that include ADHD, mood and anxiety disorders, autistic spectrum disorder and conduct problems. These issues have been demonstrated repeatedly in epidemiological and clinical studies which have reported rates 4–5 times higher than the general population and 2.5 times higher than in individuals with other non-neurological chronic conditions Despite the elevated rate of ADHD in youth with epilepsy and its potential relevance for children's well-being In the present study, we conducted a broad and comprehensive characterization of the neuroanatomical correlates of ADHD in childhood epilepsy. We included children with epilepsy with and without ADHD, as well as healthy controls, for the purpose of determining features specifically associated with ADHD that are not associated with epilepsy more generally. Additionally, in order to avoid confounding factors such as chronic seizure activity or long term anti-epileptic drug (AED) effects, as well as the cumulative social and psychological consequences of the disorder, we restricted our examination to children with new- and recent-onset epilepsy.Assessment of ADHD below). Exclusion criteria for controls were: (i) any initial precipitating event (e.g. simple or complex febrile seizures); (ii) any seizure or seizure-like episode; (iii) diagnosed neurological disease; (iv) loss of consciousness greater than 5 min; or (v) other family history of a first-degree relative with epilepsy or febrile convulsions; (vi) ADHD. Further demographic and clinical features of the three groups are provided in A total of 100 research participants comprised three groups of children: new and recent-onset epilepsy with ADHD  and without ADHD , and typically-developing first-degree cousin controls (n = 46). All participants were aged 8–18 years and attended regular schools. Children with epilepsy were recruited from the pediatric neurology clinics at three Midwestern medical centers  and met the following inclusion criteria: (i) newly diagnosed epilepsy within the past 12 months; (ii) no other developmental disabilities (e.g. autism); (iii) no other neurological disorder, and (iv) normal clinical MRI. Children with epilepsy underwent routine MRI as part of their diagnostic workup and all scans were interpreted as unremarkable by the clinical neuroradiologists. This point was later confirmed by the research pediatric neurologist when reviewing the cases for study participation. Inclusion in the Epilepsy ADHD+ group required a diagnosis of ADHD according to DSM-IV First-degree cousins were used as controls rather than siblings or other potential controls groups because: (i) first-degree cousins are more genetically distant from the participants with epilepsy and thus less pre-disposed than siblings to shared genetic factors that may contribute to anomalies in brain structure and cognition; (ii) a greater number of first-degree cousins were available than siblings in the target age range and (iii) the family link was anticipated to facilitate participant recruitment and especially retention over time (which is our intent) compared to more general control populations (e.g. unrelated school mates).This project represents an extension of our previous report To determine ADHD status the Kiddie–Schedule for Affective Disorders and Schizophrenia–Present and Lifetime Version (K-SADS-PL) was used Images were obtained on a 1.5T GE Signa MRI scanner . Sequences acquired for each participant were T1-weighted, three-dimensional (3D) spoiled gradients recall (SPGR) using the following parameters: TE = 5 ms, TR = 24 ms, flip angle  = 40 degrees, NEX = 1, slice thickness  = 1.5 mm, slices  = 124, plane  =  coronal, field of view (FOV)  = 200 mm, matrix = 256×256. All MR images were inspected before image processing. Image quality was rated on a five-point scale (0 = poor 4 = excellent) and we required a minimum quality rating of 3 or better for the scan to be included in this analysis. Further details regarding MRI processing are provided in the supporting information file This study was reviewed and approved by the Institutional Review Boards of all participating institutions . On the day of study participation families and children gave informed consent and assent, respectively. All procedures were consistent with the Declaration of Helsinki . A general linear model was then applied at each vertex to test for group differences in cortical thickness, area, volume and curvature. Age and gender were modeled as covariates for all measures while intracranial volume (ICV) was added as a covariate for measures of cortical area, volume and curvature. To correct for multiple comparisons, a Monte-Carlo simulation was implemented with an initial cluster-forming threshold set to p<0.05. Clusters were then tested against an empirical null distribution of maximum cluster size built using synthesized Z-distributed data across 10,000 permutations, producing cluster-wise p-values (CWP) fully corrected for multiple comparisons. A False Discovery Rate (FDR) of p<0.05 was also independently applied to correct for multiple comparisons. Finally, we examined subcortical structure volumes previously reported to be abnormal in epilepsy and/or ADHD using multivariate analysis of covariance (MANCOVA), with age, gender and ICV as covariates.Analyses focused on differences between the Epilepsy ADHD+, Epilepsy ADHD- and control groups in regard to cortical thickness, area, volume and curvature, as well as volumes of subcortical and brain stem structures. For cortical measurements, a surface-based group analysis using Freesurfer's statistical tool Qdec was performed. The participants' surface data were smoothed using 10, 15 or 20 mm FWHM kernels to optimize significant regionsCortical surface curvature assesses differences in small areas (peak of gyri and sulci), making FDR a more appropriate correction. The curvature index is zero for a locally flat surface and the magnitude increases as the surface becomes more spiked. Sulci and gyri are distinguished by positive and negative curvatures, respectively. Minor dissimilarities surviving FDR are found in two group comparisons  differing significantly from the controls, with the epilepsy groups not differing from each other. Regions of interest showing this pattern included volumes of the right thalamus, and total cerebellum (due to left cerebellum).In this study a comprehensive set of analyses were performed to characterize alterations in cortical morphology, subcortical structures, and cerebellum that may be associated with ADHD in children with epilepsy. By incorporating children with epilepsy with and without ADHD, we were able to identify anatomical abnormalities associated with epilepsy more generally versus abnormalities associated more specifically with ADHD. Three core findings emerged from this investigation: 1) ADHD in children with epilepsy is associated with a distributed pattern of anatomic abnormality involving the cortex and subcortical structures, the cortical morphological abnormalities characterized overwhelmingly by reduced cortical thickness in bilateral areas of the frontal, parietal, and temporal lobes, with minor or no group differences in metrics of cortical surface area, volume or curvature; 2) Children with epilepsy and ADHD exhibited significantly smaller subcortical volumes of the caudate, thalamus, hippocampus, and brainstem; 3) A more general association with epilepsy, unrelated to the presence or absence of ADHD, was seen in reduced volumes of the cerebellum and thalamus. These findings will be reviewed below.First, the main finding is a diffusely distributed pattern of neuroanatomical abnormality in children with epilepsy and ADHD compared to children with epilepsy but without ADHD and typically-developing controls. These results involve broad and widespread cortical and subcortical areas suggesting associations between altered brain networks in ADHD that participate in executive function, attention, and sensorimotor systems We found minimal differences in cortical area or volume when examining the Epilepsy ADHD+ group. Nevertheless, two studies have reported decreased cortical area in children with ADHD compared to controls Second, at the subcortical level, children with epilepsy and ADHD differed significantly from the Epilepsy ADHD- and control groups in the volume of the right basal ganglia, as well as from either the Epilepsy ADHD- or the control group, with the latter two groups comparable to each other across volumes of the left caudate, left thalamus, right hippocampus, and brainstem, with additional reduced volume of total subcortical grey matter. Several of these results are consistent with meta-analyses and reviews of ADHD in the general population Brainstem volume, likewise, is understudied in the ADHD literature, but our result is physiologically reasonable, given that the brainstem hosts the reticular activating system and is the production site for neurotransmitters with a key role in ADHD In the epilepsy ADHD literature several clinical seizure features, including seizure frequency In summary, this study indicates that ADHD, a common comorbidity in pediatric epilepsy, is associated with thinning of cortical regions and diminution of subcortical structures related to attention, executive function and sensorimotor networks.There are differences in the neuroimaging results of this investigation compared to our previous report Our study has both limitations and strengths. Regarding limitations, first noted is that the number of children with epilepsy and ADHD is modest. Second, we were not able to include an ADHD group without epilepsy as too few controls met criteria for ADHD, thereby precluding a true 2×2 analysis. Third, because of the modest sample size, it was not possible to perform analyses examining ADHD as a function of the known subtypes including inattentive, hyperactive, and combined—a very important task for the future. Fourth, the mechanisms that underlie the ADHD comorbidity in epilepsy remain an open question. This investigation does not inform that point. One avenue we are currently investigating is whether family history/family aggregation of ADHD is a pertinent consideration. Despite these limitations, the strengths of the paper include that fact that ADHD is an important complication of childhood epilepsy, the diagnosis of which was defined rigorously based on DSM-IV diagnosis from the K-SADS-PL rather than a parent report questionnaire which thereby improved diagnostic reliability. We included measures of cortical morphology that went beyond cortical thickness, measures that have been understudied in epilepsy in general and epilepsy and ADHD in particular. Because of that, we felt that there was utility in examining these measures comprehensively and indeed it appears that alterations in cortical thickness are a primary correlate of ADHD in epilepsy. Moving forward, we are tracking these children prospectively (2 years and 5-6 years later) and plan to focus on their longitudinal trajectories in the future.ADHD is a common co-occurring condition in childhood epilepsy. This comorbidity of epilepsy has anatomical correlates that are detectable early in the course of the epilepsy suggesting that the altered neuroanatomy is not due to epilepsy chronicity or treatment. The etiology of this anatomical signature and its prospective course remain to be characterized, as does the life course of children with epilepsy with ADHD compared to children with epilepsy without ADHD.Figure S1Cortical curvature comparisons. A) FDR corrected significant differences in curvature. Blue colors indicate regions in which the Epilepsy ADHD+ group has decreased curvature relative to the Epilepsy ADHD- group (fusiform region). Red colors indicate regions in which the Epilepsy ADHD+ group has increased curvature relative to the Epilepsy ADHD- group . B) FDR corrected significant differences in curvature in the right hemisphere. Blue colors indicate regions in which the Epilepsy ADHD - group has decreased curvature compared to the control group. Red colors indicate regions in which the Epilepsy ADHD - group has increased curvature compared to the control group.(TIF)Click here for additional data file.Methods S1MRI processing details.(DOCX)Click here for additional data file.


Drosophila, several non-catalytic PGRPs function as selective peptidoglycan receptors upstream of the Toll and Imd pathways, the two major signalling cascades regulating the systemic production of antimicrobial peptides. Recognition PGRPs specifically activate the Toll pathway in response to Lys-type peptidoglycan found in most Gram-positive bacteria and the Imd pathway in response to DAP-type peptidoglycan encountered in Gram-positive bacilli-type bacteria and in Gram-negative bacteria. Catalytic PGRPs on the other hand can potentially reduce the level of immune activation by scavenging peptidoglycan. In accordance with this, PGRP-LB and PGRP-SC1A/B/2 have been shown to act as negative regulators of the Imd pathway. In this study, we report a biochemical and genetic analysis of PGRP-SB1, a catalytic PGRP. Our data show that PGRP-SB1 is abundantly secreted into the hemolymph following Imd pathway activation in the fat body, and exhibits an enzymatic activity towards DAP-type polymeric peptidoglycan. We have generated a PGRP-SB1/2 null mutant by homologous recombination, but its thorough phenotypic analysis did not reveal any immune function, suggesting a subtle role or redundancy of PGRP-SB1/2 with other molecules. Possible immune functions of PGRP-SB1 are discussed.Peptidoglycan is an essential and specific component of the bacterial cell wall and therefore is an ideal recognition signature for the immune system. Peptidoglycan recognition proteins (PGRPs) are conserved from insects to mammals and able to bind PGN  and, in some cases, to efficiently degrade it . In  N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues, which are cross-linked together via short peptide bridges. Because PGN is essential to bacteria but absent from eukaryotic cells, it makes an ideal indicator for metazoan immune systems of the presence of bacteria within the organism. In addition, the PGN composition and structure can be markedly different between bacterial species, allowing the immune system to further distinguish between different types of intruders. For example, the PGN of Gram-negative and Gram-positive bacilli-type bacteria differs from the PGN of most Gram-positive bacteria by the replacement of lysine (Lys) with meso-diaminopimelic acid (DAP) at the third position in the peptide chain In the last decades, peptidoglycan (PGN), an essential component of virtually all bacteria, has appeared as a key player in host-microorganism interactions N-acetylmuramoyl-L-alanine amidase Bombyx moriOn the host side, some protein families have evolved the capacity to interact with PGN, e.g. lysozymes, Nods (for Nucleotide-binding Oligomerisation Domain), and peptidoglycan recognition proteins . PGRPs form a conserved family of proteins sharing a 160 amino acid domain (the PGRP domain) with similarities to bacteriophage T7 lysozyme, a zinc-dependent PGRPs have been classified in two groups according to their enzymatic activity. Indeed, some PGRPs have lost their ancestral amidase activity , while others can still efficiently cleave PGN . Non-catalytic PGRPs have been implicated in functions as diverse as immune receptors, regulators and effectors Drosophila genome encodes 13 PGRPs and many of them function both as activators and regulators of the Toll and Imd pathways meso-DAP-D-Ala; The Drosophila non-catalytic PGRPs function as pattern recognition receptors for PGN, the catalytic PGRPs have demonstrated  or predicted  amidase activity that removes peptides from PGN glycan chains, thereby reducing or eliminating its immunological activity in vivo by scavenging PGN, and also TCT in the case of PGRP-LB Staphylococcus aureusDrosophila PGRPs, only the function of PGRP-SB1 and PGRP-SB2  analysis revealed a basal level of PGRP-SB1 expression throughout Drosophila development; however, its expression level was higher at pupal stage, consistent with a previous Northern blot analysis  has been previously identified as the minimum PGN motif capable of efficiently inducing the Imd pathway E. coli DAP-type PGN. Both the monomer (GlcNAc-MurNAc tetrapeptide unit) and dimer of muropeptide were hydrolyzed by PGRP-SB1 but PGRP-SB1 was much more active on the dimer (243U compared to 4U for the monomer). Kinetic studies of the hydrolysis of GlcNAc-MurNAc tetrapeptide dimer showed a two step-reaction, the faster one leading to disaccharide-octapeptide and disaccharide and the slower one leading to octapeptide and disaccharide  than monomeric ones (TCT (GlcNAc-MurNAc(anhydro)-L-Ala-γ-D-Glu-ccharide . This prric ones .Collectively, our results demonstrate that PGRP-SB1 degrades DAP-type PGN into non-immunostimulatory fragments, but does not degrade TCT, a key activator of the Imd pathway. We also observed that the efficacy of PGRP-SB1 is dependent on two factors, the size of the PGN fragments  and the MurNAc configuration (anhydro or not).PGRP-SB1 in vivo, we aimed to generate a null mutant by homologous recombination. The PGRP-SB1 locus (720 bp) is located on the 3L chromosome at less than 150 bp from PGRP-SB2  and down-regulation of the Imd pathway  Drosomycin (Drs) and Diptericin (Dpt) respectively. PGRP-SBΔ5 mutants expressed Drs and Dpt at a wild-type level after injection of E. carotovora .Our data indicate that, in contrast to all rotovora  and L. i innocua  did not Listeria. Indeed, in these bacteria the PGN participates to the outer cell wall layer and could thus be directly accessible for PGRP-SB1, while in Gram-negative bacteria the PGN is “hidden” by the outer lipopolysaccharide layer. Therefore, we injected L. innocua in flies over-expressing PGRP-SB1 and assessed their viability as well as the bacteria persistence in these flies as compared to wild-type flies. Our data show that PGRP-SB1 over-expression is not sufficient to protect from the morbidity associated with this infection, or to decrease the number of persisting bacteria in the fly at 24 hours  did not reveal a striking immune function for PGRP family has been thoroughly studied in the last decade, both in Drosophila and in vertebrates. We present here an extensive analysis of the expression, enzymatic activity and immune phenotype of PGRP-SB1, one of the last Drosophila PGRPs  whose functions remain uncharacterized in vivo. This is also the first analysis of a complete genetic knockout of two catalytic PGRPs in Drosophila, as previously published in vivo studies of PGRP-LB and PGRP-SC1/2 have relied either on RNA interference or on uncharacterized mutations The PGRP-SB1 expression is highly induced after infection, far more than for any other PGRP and to an extent similar to that of effectors such as antimicrobial peptide genes. PGRP-SB1 is abundantly produced in the fat body, and then released into the hemolymph. It has an amidase activity against DAP-type PGN. Taken together with the in vitro bactericidal activity of PGRP-SB1 against a DAP-type containing bacteria PGRP-SBΔ5 flies, it is expected that the removal of one out of many effectors would not induce a major immune deficiency, due to redundancy or synergy. This could also explain the non-protective effect of PGRP-SB1 over-expression after L. innocua infection. For example, PGRP-SB1 could need the activity of another protein to have sufficient access to its PGN substrate. On the other hand, PGN cleavage by PGRP-SB1 could increase with its over-expression but not be sufficient to eliminate the bacteria in absence of expression of a co-factor. Therefore a function of PGRP-SB1 as an effector remains a possibility.In this study, we have demonstrated that PGRP-SBΔ5 mutants. Firstly, PGRP-SB1/2 could have a function at a specific stage or tissue that would not be revealed by our analysis, based as it was on whole organism infections/responses and restricted to the adult stage. Indeed, PGRP-SB2 is solely expressed at the pupal stage, suggesting that it could have a role during metamorphosis, although we did not notice any developmental defect of PGRP-SBΔ5 mutants. Secondly, PGRP-SB1 could have a more subtle immune function against a specific natural pathogen of the fly, untested here, or in other immune reactions, e.g. phagocytosis  were replaced by the white gene.A deletion of the /2 locus  were cloUAS-PGRP-SB1 transgenic flies, a full-length cDNA of PGRP-SB1 (using the CG9681_cDNA full length IP02762 from DGRC) was inserted in the pDONR221 Gateway entry clone (Invitrogen) and finally subcloned in pTW transgenesis vector. The transgenic flies were obtained by P-element transgenesis. Over-expression of PGRP-SB1 was achieved by crossing the resulting UAS-PGRP-SB1 transgenic flies with flies carrying the ubiquitous daughterless-Gal4 driver. F1 progeny was transferred to 29°C at late pupal stage for optimal efficiency of the UAS/Gal4 system.To generate Erwinia carotovoraE. carotovora, O.D. 200), the DAP-type PGN containing Gram-positive bacteria Listeria monocytogenes , Listeria innocua  or Bacillus megaterium , and the Lys-type PGN containing Gram-positive bacteria Micrococcus luteus  and Enterococcus faecalis . Strains were cultured in Brain-Heart Infusion Broth (BHI - Listeria) or Luria Broth  at 29°C (E. carotovora) or 37°C . E. coli PGN was used at a monomer equivalent concentration of 5 mM and injected into the thorax of adult females at 18.4 nl per fly using a Nanoject apparatus (Drummond™).Systemic bacterial infections were performed by pricking adults in the thorax with a thin needle previously dipped into a concentrated pellet of a bacterial culture in vivo persistence, 9.4 nl of bacterial culture at O.D. 10 were injected into the thorax of female adults (3–4 days old) using a Nanoject apparatus (Drummond™). Persistence of the bacteria was evaluated at 24 and 48 h post-infection by crushing 20 flies in either BHI (L. innocua) or LB  culture medium. Serial dilutions of these extracts were spotted in triplicate on appropriate medium and incubated overnight at 29°C (E. carotovora) or 37°C . Colonies were counted from spots containing more than 10 colonies.For the measurement of E. carotovora supplemented with sucrose, as described previously Oral infections were performed by feeding adults with an O.D. 200 pellet of E. carotovora injection as well as in unchallenged flies over-expressing PGRP-SB1 , but not in extracts from PGRP-SBΔ5 mutants  that also recognizes PGRP-SB1. Indeed, a band of about 18 kDa was detected in wild-type fly extracts 6 h after  mutants . A monoc mutants , were lyDpt, Drs, PGRP-SB1 and PGRP-SB2 mRNA quantification from whole animals or guts, RNA was extracted using RNA TRIzol™. For whole animals, 10 flies were used for each sample; for guts, 20 dissected guts from the crop to just above the malpighian tubules were used. cDNAs were synthesized using SuperScript II (Invitrogen) and PCR was performed using dsDNA dye SYBR Green I (Roche Diagnostics). Primer pairs for Dpt , Drs , PGRP-SB1 , PGRP-SB2  and RpL32  were used. SYBR Green analysis was performed on a Lightcycler (Roche). The amount of mRNA detected was normalized to control RpL32 mRNA values. We used normalized data to quantify the relative level of a given mRNA according to cycling threshold analysis (ΔCt), hence data are plotted for example as ΔCt Dpt/ΔCt RpL32.For E. coli PGN was purified from the BW25113 Δlpp::CmR strain that does not express the Braun lipoprotein. Digestion of this material by pure SltY lytic transglycosylase was used to produce mg quantities of TCT and dimer of TCT purified as described previously meso-[3H]DAP in the FB8 lysA::kan strain grown in M63 minimal medium supplemented with 0.2% glucose and 100 µg.ml−1 of lysine, threonine and methionine, as previously described. Muropeptide fragments of PGN were generated by digestion with mutanolysin as described in Gram-negative and Gram-positive PGN preparations were described in Amino acid and amino sugar compositions were determined and quantified with a Hitachi L8800 amino acid analyzer (ScienceTec) after hydrolysis of samples with 6 M HCl for 16 h at 95°C. The structure and purity of isolated PGN fragments and synthesized compounds were further confirmed by MALDI-TOF mass spectrometry.His-tagged PGRP-SB1 was expressed in S2 cells and purified as described previously 4, pure PGN (100-20 µg) or PGN fragment (2-20 nmoles), and PGRP-SB1 (1-2 µg). After incubation at 37°C for 26 h or 16 h, the reaction was stopped by addition of 2 µl phosphoric or acetic acid and 500 µl HPLC elution buffer: 50 mM sodium phosphate, pH 4.5. To analyze the effects of PGRP-SB1 on the PGN structure in more detail, samples of PGN were first incubated with PGRP-SB1, as described above, for different periods of time, from 20 min to 24 h. Then, reactions were stopped by boiling for 5 min and mixtures were centrifuged. Supernatants were stored for subsequent HPLC analysis of the released soluble peptides . Pellets of remaining polymeric PGN were treated for 24 h by pure SltY enzyme and the pattern of muropeptides thus generated  was analyzed by HPLC 18 (4.6×150 or 250 mm) (Alltech France) and products were eluted at a flow rate of 0.6 ml.min−1 using a gradient of methanol or acetonitrile from 0 to 20% in 40 or 100 min. Peaks were detected either by measurement of the absorbance at 215 nm  or by detection of radioactivity .The amidase activity of PGRP-SB1 was assayed in a reaction mixture (50-100 µl) containing 50 mM Tris buffer (pH 8), 2 mM ZnSO


Blechnum orientale (Linn) was separately collected from polluted and unpolluted sites to determine whether it could accumulate hazardous pollutants or not. Metal concentrations  both in the fronds and roots and polycyclic aromatic hydrocarbons (PAHs) in the fronds of this fern were quantified. At both sites, roots of B. orientale had significantly higher heavy metals than the fronds. Concentrations of Pb, As, Hg, Cd, and Cu in the fronds at the polluted site were more than 2, 6, 7, 14, 5, and 13 times of those at the unpolluted site. Translocation factor and bioaccumulation factor implied that B. orientale did not have a good ability to transport heavy metals from the roots to the fronds. Total PAHs in the fronds at the polluted site were significantly higher than those at the unpolluted site, indicating the physiological PAHs absorption by B. orientale growing at polluted sites. Uptake of pollutants via stomata might be the main reason causing the significant accumulation of hazardous pollutants in the fronds of B. orientale. Large-scale systematical survey and intensive monitoring on pollutants in this medicinal fern should be necessarily strengthened.A Chinese medicinal fern  Various plants have been identified as traditional medicines for pharmaceutical and dietary therapy by folks for several millennia. Eighty percent of the world's inhabitants were estimated to rely mainly on their traditional medicines for health care . RecentlBlechnum orientale L.  in the fronds at sites severely polluted by heavy metals [−1 in most terrestrial plants [tericide , antioxitericide , antimictericide , and cantericide . Presenty metals . Usuallyl plants .B. orientale accumulated toxic metals and polycyclic aromatic hydrocarbons (PAHs) in a polluted environment and (2) investigate the transportation of heavy metals from roots to fronds of B. orientale. The higher levels of toxic heavy metals  than their limited concentrations in “China Green Trade Standards of Importing and Exporting Medicinal Plants and Preparation (GTSIEMPP)” in some Chinese traditional medicines might press potential risk to human health. Historically, many ferns have been consumed as edible plant and utilized as pharmaceutical species in China. The accumulation and/or the adsorption of toxic pollutants by medicinal ferns have not been intensively concerned up to date. The main objectives of this study were to (1) detect the degree to which B. orientale were collected from two woodlands with different levels of pollution. The first woodland located about 5 km away from a Zn-Pb smelting plant. The second woodland located in a natural reserve. Previous investigation showed that the woodland nearby the smeltery was a polluted site (PS), and the woodland in the reserve was a relatively unpolluted site (UPS) [B. orientale. The clusters were at least 100 m apart and were at least 200 m from the nearest road. About 500 g of fronds and 500 g of roots (fresh weight) were collected from each cluster. Surface soils (0–10 cm depth) beneath each cluster were sampled at the same time. The fronds, roots, and soils from each cluster were treated as a replicate. Thus, ten replicates of fronds, roots, and soils were collected at each site. All samples were kept in paper envelopes and then sealed in polythene bags before being transported to the laboratory.Specimens of te (UPS) . At eachIn the laboratory, the fronds were quickly cleaned with water and then rinsed with deionized water. The roots were first cleaned with water and then rinsed with an ultrasonic cleaning instrument to remove remaining soil particles. All samples were then dried at 65°C to constant weight, ground, and stored in plastic vials for heavy metals analyses. A portion of each frond sample (fresh) was gently washed with tap water, rinsed with deionized water, freeze-dried for 36 h, and ground for PAHs detection.Soil samples were air-dried at room temperature, lightly crushed, and passed through 2 mm acid-washed plastic sieve to remove stones, roots, leaves, and other debris. All samples were ground in a mortar, passed through a 100-mesh polyethylene sieve, and were kept in desiccators.3 in a microwave digestion system. Metal concentrations were quantified with an inductively coupled plasma-mass spectrometer . Precision and accuracy of the determinations were tested through blank and repeated analyses of National Standard Reference material (GBW 07604). Concentrations of heavy metals  and PAHs were determined separately. Samples for metal analyses were digested with concentrated HNOPAHs in fronds were extracted and quantified according to the method detailed by Sun et al. . The 16 t-tests were used to compare the differences of pollutant concentrations in the fronds, roots, and soils between the sites at the confidence level of 95% using software SPSS 10.0 .The results for the concentrations of heavy metals and PAHs were presented as mean and standard deviation (SD). Paired sample Concentrations of the selected heavy metals beneath the ferns at both sites were compared in B. orientale at the two sites were shown in Concentrations of the selected heavy metals in the fronds and roots of B. orientale always had significantly higher metal concentrations than the fronds. All the studied heavy metals except for Mn both in fronds and roots from the polluted site were significantly higher than those from the unpolluted site. For instance, fronds Cu, Zn, Pb, Cd, As, and Hg at the polluted site were more than 2, 6, 7, 14, 5, and 13 times of those at the unpolluted site, respectively. Concentrations of Mn both in fronds and roots were similar between the sites.At both sites, the roots of B. orientale were presented in Concentrations of total PAHs (∑PAHs) and individual PAHs in the fronds of For further analysis of the PAH composition in the fronds, we grouped the PAH compounds into classes according to the number of benzene rings in their structures . Low molThe selected heavy metals in the soils at the polluted site were extremely above their background values in Guangdong forest soils . The res−1, respectively, in raw materials of medicinal plants. In this study, the average values for Pb, As, Hg, Cd, and Cu in the fronds of B. orientale nearby the smelting plant were 66.0, 5.6, 0.4, 7.5, and 21.7 mg·kg−1, respectively, which indicated that Pb, As, Cd, and Cu were above the standard of GTSIEMPP. In contrast, the concentrations of Pb, As, Hg, Cd, and Cu in the fronds at the natural reserve were within the limitation of GTSIEMPP. The results implied potential risk on human health when this fern was collected for medicinal and edible utilization from polluted environments.Heavy metals accumulated in medicinal plants could pose potential toxicity for human being due to the biomagnified effect along food chains . Many feB. orientale had both TF and BF values less than 1.0 at both sites  and bioaccumulation factor (BF), which was the ratio of metal concentration in aboveground tissues to that in roots  and the th sites . A TF vaEspinosa ; namely,B. orientale could accumulate PAHs as well as heavy metals in fronds. The accumulation of PAHs suggested that consumption and utilization of B. orientale fronds growing at polluted sites could be detrimental to human health. Polycyclic aromatic hydrocarbons were another common pollutant with carcinogenic, mutagenic, and teratogenic effects on organisms. Environmental PAHs were mainly from multiply anthropogenic emissions. After being emitted into the atmosphere, PAHs could commonly be absorbed in a vaporous or particulate state by the aboveground tissues of plants. The concentrations of most PAHs in plant tissues were determined by the atmospheric deposition of PAHs from the atmosphere. In the present study, the significantly higher concentrations of both ∑PAHs and several individual PAHs in the fronds from the polluted site indicated that B. orientale might mainly stem from low- or moderate-temperature combustion processes. The higher fraction of HMW-PAHs in the fronds implied that there were additional industrial sources at the polluted site. Clarifying the contributions of these different sources required further quantification of PAHs in the fern, soil, and atmosphere at the both sites. The ratios of LMW-PAHs to HMW-PAHs were often used to discriminate the origins (petrogenic versus pyrogenic) of airborne PAHs . In this B. orientale growing at polluted site and confirmed the nontransportation of heavy metals from roots to fronds. The substantial higher concentrations of the studied heavy metals and PAHs implied potential risk of toxicity on human health when this medicinal fern was harvested from polluted sites. Large-scale systematical survey and intensive monitoring on pollutants in medicinal ferns should be necessarily strengthened. The collection of B. orientale for medicinal utilization from polluted areas should better be abandoned to reduce the intake of hazardous pollutants by human being.The results of this case study revealed the significant adsorption and accumulation of toxic metals and hazardous PAHs by


Polyfluoroalkyl chemicals (PFCs) have been widely used in consumer products. Exposures in the United States and in world populations are widespread. PFC exposures have been linked to various health impacts, and data in animals suggest that PFCs may be potential developmental neurotoxicants.We evaluated the associations between exposures to four PFCs and parental report of diagnosis of attention deficit/hyperactivity disorder (ADHD).Data were obtained from the National Health and Nutrition Examination Survey (NHANES) 1999–2000 and 2003–2004 for children 12–15 years of age. Parental report of a previous diagnosis by a doctor or health care professional of ADHD in the child was the primary outcome measure. Perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorohexane sulfonic acid (PFHxS) levels were measured in serum samples from each child.Parents reported that 48 of 571 children included in the analysis had been diagnosed with ADHD. The adjusted odds ratio (OR) for parentally reported ADHD in association with a 1-μg/L increase in serum PFOS (modeled as a continuous predictor) was 1.03 . Adjusted ORs for 1-μg/L increases in PFOA and PFHxS were also statistically significant , and we observed a nonsignificant positive association with PFNA .Our results, using cross-sectional data, are consistent with increased odds of ADHD in children with higher serum PFC levels. Given the extremely prevalent exposure to PFCs, follow-up of these data with cohort studies is needed. Polyfluoroalkyl chemicals (PFCs) are a class of highly stable man-made compounds. Composed of a variable-length fluorinated carbon backbone and a carboxylate or sulfonate functional group, PFCs have both hydrophobic and oleophobic portions that enable products to repel both oil and water and resist staining. PFCs are widely used in industrial applications as surfactants and emulsifiers and in consumer products such as food packaging, nonstick pan coatings, fire-fighting foams, paper and textile coatings, and personal care products .PFCs are extremely resistant to environmental and metabolic degradation and have been detected globally in the environment and wildlife . PFCs haPFCs are absorbed through ingestion and to a lesser extent through inhalation. Once absorbed, PFCs are eliminated from the human body very slowly. Serum half-life estimates in an occupationally exposed cohort ranged from 5.4 years for perfluorooctane sulfonic acid (PFOS) to 8.5 years for perfluorohexane sulfonic acid (PFHxS) . In a coThe ubiquitous presence and persistence of PFCs in the environment and the human body have led to efforts to understand the toxicologic hazards that may be associated with exposure. Early animal studies focused almost exclusively on exposure to PFOS and PFOA and found several potential effects, primarily related to hepatotoxicity, immunotoxicity, and reproductive and developmental toxicity , 2007. Ain vitro models, PFCs were shown to affect neuronal cell development in a variety of ways, including changes in cell differentiation (in utero exposure to PFOS was linked to reduction in thyroid hormone (circulating thyroxin and triiodothyronine), which is known to regulate brain development  is one of the most common neurodevelopmental disorders in children, with an estimated prevalence between 7% and 16% in the United States . Data suIn the present analyses, we explored the association between ADHD and PFOS, PFOA, PFHxS, and PFNA using cross-sectional data from the National Health and Nutrition Examination Survey (NHANES) 1999–2000 and 2003–2004 cycles. To our knowledge, this is the first study examining the association between PFCs and ADHD.NHANES is a nationally representative, cross-sectional sample of the noninstitutionalized U.S. civilian population. The survey combines in-home interviews and physical examinations in a mobile exam unit to collect data on demographics, socioeconomic status (SES), health conditions, and behavioral and environmental risk factors . DetailsWe used parental report of previous ADHD diagnosis as the primary dependent variable. Questionnaires were administered by trained personnel , 2009b. To improve specificity, we also considered a second definition of ADHD used previously in an assessment of the effects of other environmental exposures on ADHD risk in the NHANES population . The secThe National Center for Environmental Health analyzed serum PFC levels in a one-third sample of all individuals ≥ 12 years of age. Data were available during two nonconsecutive survey cycles, 1999–2000 and 2003–2004. Detailed analytic methods were described previously , 2007b. We investigated a number of covariates and potential confounders in the association between PFCs and ADHD. The demographic variables age, sex, and race/ethnicity were included as covariates based on their role in the NHANES selection procedure and previous research on their association with ADHD . AdditioWe examined the shape of the relationship between the continuous measures of each individual PFC and parent-reported ADHD using a locally weighted regression smoother (LOESS) in S-Plus . Smoothing allowed us to summarize ADHD odds as a function of PFC exposures without imposing a rigid form of dependence. We selected the optimal span size—the window from which data are drawn to estimate the odds of ADHD—by minimizing Akaike’s information criterion . We visup < 0.10) or if they appreciably altered the association between PFC exposure and ADHD [odds ratio (OR) change > 10%]. We included continuous covariates in models as continuous predictors and also explored the use of categorization. A p-value of 0.05 was chosen to indicate the statistical significance of the association between each PFC and ADHD.Logistic regression analyses were performed in SAS  using the Proc SURVEYLOGISTIC procedure, which accounts for stratification and clustering within primary sampling units used to select the NHANES sample. Rather than using NHANES sample weights, we adjusted all models for relevant covariates . This method is considered to be a good trade-off between efficiency and bias . Other pn = 0; PFOA, n = 2; PFNA, n = 12; PFHxS, n = 26). Additionally, because the range of serum values varied considerably for the PFCs we assessed, we conducted additional analyses to provide ORs standardized to an increase in units equal to the interquartile range (IQR) for each PFC to provide comparable effect estimates.Our preliminary investigation of PFC data revealed several children with very high serum PFC levels, particularly PFHxS, PFOA, and PFNA. To ensure that individuals with extreme exposure values were not disproportionately influencing results, we conducted sensitivity analyses excluding observations greater than three times the 75th percentile for each compound , we also performed principal component analysis, a systematic method of reducing the number of correlated observed variables into a smaller number of principal components that account for most of the variance in the observed PFC measures .Of the 571 study participants 12–15 years of age with complete data, parents of 48 (8.4%) reported that their child had ADHD. Of those, 21 (3.6% of the study population) also reported using prescription medications approved for the treatment of ADHD within the last month. We included one child reported to be taking prescription medication for ADHD but not reported to have been diagnosed with ADHD in analyses as a noncase. Details regarding the total sample size are displayed in the Supplemental Material, Figure 1 (doi:10.1289/ehp.1001898).Sex and maternal smoking during pregnancy were significantly associated with parental-report of ADHD in bivariate analyses . Comparep-value < 0.05) dose–response relationship between PFOS exposure and parent-reported ADHD; the OR for each 1-μg/L increase in serum PFOS was 1.03  ]. We observed a significant (01–1.05)  after ad01–1.05) . The samBecause the range of serum values varied considerably for the PFCs we assessed, we also calculated ORs standardized to an increase in units equal to the IQR for each PFC. An increase in serum PFOS levels equal to the IQR was associated with 1.60 times the odds of ADHD . For PFOA, the odds of ADHD increased 1.35 times for an increase equal to the IQR . A 2.9-μg/L increase in serum PFHxS increased the odds of ADHD 1.19 times . The IQR effect for PFNA increased ADHD odds 1.15 times .Several children had very high serum PFC levels, particularly PFHxS, PFOA, and PFNA. Estimated ORs were slightly higher than those reported in p-value = 0.02; PFNA p-value = 0.72).Principal component analyses indicated that PFOS, PFOA, and PFHxS all loaded onto a single component that accounted for 58% of the total variability in all four PFC measures. A second component, which primarily represented PFNA exposure, accounted for an additional 22% of the total variability. We repeated multiple logistic regression analyses including both PFNA and the principal component that represented the weighted combination of PFOS, PFOA, and PFHxS. A positive association with ADHD remained for the combined PFC variable and for PFNA , and consequently reduced the precision, the point estimates were similar regardless of which case definition was used. Similarly, although we included a large number of terms in models for a relatively small number of outcomes, crude estimates of the association between PFCs and ADHD were similar to adjusted estimates. Analyses of the NHANES population conducted previously have shown that parental report of ADHD was related to other environmental exposures   .Detailed questionnaire and laboratory measurement data were available for a number of potential covariates in the NHANES data set, and we able to evaluate the potential for confounding by conditions during the prenatal and early childhood periods and other environmental exposures, such as lead and SES, related to ADHD. Data for potential confounders were generally very complete. However, there may still be residual confounding in the association between PFCs and ADHD. We were unable to assess several potential confounders because they were either not publically available  or not collected in the NHANES data set .Despite these limitations, our analyses have a number of strengths including the use of a representative sample of the U.S. population. Additionally, we were able to consider the association between PFC levels and ADHD odds over a wide range of exposures. We were also able to estimate associations of PFNA and PFHxS with ADHD; to our knowledge, this is the first assessment of the potential developmental neurotoxicity of these PFCs in children.To our knowledge, these analyses are the first to assess the association between PFC levels and ADHD. As a whole, our results are consistent with increased ADHD in children with higher serum levels of PFCs. Given the extremely prevalent exposure to PFCs, further investigation into the impact of PFC exposure on ADHD and other neurodevelopmental endpoints is warranted.