CHRNA4  were genotyped and two-step cluster analysis was used for phenotypic cluster characterization. Haplotype estimation was determined by HapStat module of R 2.0 software. Three different phenotypic clusters were identified and the C3 cluster was characterized by the highest ZSDS and MNWS scores compared to others. Furthermore, lifetime prevalence of major depression was significantly higher in the C3 cluster (p = 0.019). In genetic association tests, this cluster was also significantly associated with rs3787138 genotypes (p = 0.004) while haplotype analyses of three SNPs  revealed that the risk for C3 phenotype was almost three times higher in GCC haplotype carriers compared to others (permp = 0.013). This is the first report on a significant association between CHRNA4 variants and a subgroup of smokers characterized by massive withdrawal symptoms and affective vulnerability. Identification of such a phenotypic cluster can be a pivotal step for further pharmacogenetic studies on ligands of the alpha4 nAChR subunit. Our results suggest that performing cluster analysis in genetic association studies can be proposed for complex disorders.Heterogeneous phenotypes of complex disorders pose a great challenge for genetic association studies and for the development of personalized treatment strategies. Cluster analysis of phenotypic data has been recently proposed as a reliable auxiliary method for such studies. A cohort of 236 treatment-seeking smokers was investigated after overnight nicotine abstinence. Alpha4 nicotinic acetylcholine receptor (nAChR) subunit-related phenotypes were assessed by the Fagerström Test for Nicotine Dependence (FTND), exhaled carbon monoxide (CO) measurements, the Minnesota Nicotine Withdrawal Scale (MNWS) and the Zung Self-Rating Depression Scale (ZSDS). Seven tag SNPs (single-nucleotide polymorphisms) across  CHRNA4) because among nicotinic acetylcholine receptors (nAChRs) those containing alpha4 subunit have the highest affinity for nicotine, the primary psychoactive component of tobacco CHRNA4 in multiple smoking-related phenotypes including nicotine dependence, withdrawal and affective symptoms, association studies have provided partly inconsistent results. Positive associations between nicotine dependence and CHRNA4 were reported in case-control and family studies CHRNA4CHRNA4 was not significant on smoking cessation outcome in a nicotine replacement therapy (NRT) treatment study Nicotine dependence is the most prevalent psychiatric disorder in the world and it is responsible for the highest preventable mortality in developed countries CHRNA4 is also implicated in the development of mood disorders. Significant associations between CHRNA4 and depression and loneliness were demonstrated in a study of elderly population CHRNA4CHRNA4 and affective phenotype according to these reports Accumulative data support that depressive phenotypes and smoking have a multifaceted relationship  but other factors did not differ from men. Severe depression symptoms (above 48 points on ZSDS) were detected in 7.5% of the sample which is significantly higher than the Hungarian average of current episode of MDD (major depressive disorder) The average number of cigarettes per day was 21.2±8.4, breath CO level was 19.0±8.7 and FTND mean score was 6.3±1.2. Mean MNWS score was 12.01±6.1. Mean Zung Self-Rating Depression scale score was 37.7±7.4 in the total population. Comparing different variables between the two genders we found that women scored significantly higher on ZSDS . C1 was characterized by the lowest MNWS (8.8±3.8) and ZSDS (34.1±5.1) scores and CO levels (15.0±3.6) (p = 0.019). FTND scores and CPD (cigarettes/day) were also different among the three subgroups identified. Both C2 and C3 had significantly higher FTND score than of C1  but FTND score did not differ between C2 and C3. In line with the highest exhaled CO level in C2, significantly greater amount of CPD was reported in C2 than in C1 . Gender distribution did not differ significantly among the three clusters.Two-step cluster analysis resulted in the strongest model when MNWS, exhaled CO level and ZSDS score were stepped into the model. In this model 3 different clusters were identified with significantly differing variables (5.0±3.6) , Table 25.0±3.6) , Table 25.0±3.6) , Table 2p = 0.013); and effects of rs3787138 and rs3787140 on MNWS score variance were significant but not robust enough for Bonferroni corrections  (p = 0.004) while C allele carriers of rs3787140 had almost double the chance to have C3 phenotype with a nominal significance (OR = 1.67 (95% CI = 0.69–4.04); p = 0.013). ZSDS, exhaled CO and CPD had no significant relation to SNPs in single marker association models.We tested the effects of all SNPs on dependent variables under five models. Single marker association tests provided three nominally significant results: effect of rs6090378 on FTND score (ctively) . Howeverp = 0.013, globalp = 0.054). The highest MNWS score was associated with the GCC (p = 0.04) and the lowest score with the ATT haplotypes  showed that three haplotypes had greater than 5% frequency in the sample . The frequency of the GCC haplotype was found to be the highest in the C3 cluster  and the model was also significant (permp = 0.018). Consequently, GLM  analysis revealed that odds ratio for having the C3 phenotype was almost three times higher in subjects with GCC haplotype compared to others  among smokers which is characterized by significantly more severe withdrawal symptoms with robust affective vulnerability; not only average score on the self-reported depression scale (ZSDS) but also lifetime prevalence of major depression were significantly higher in this subgroup. Of note, the size of this cluster is not negligible: about 20% of the study population belonged to this subgroup. Genotype and haplotype association tests showed that this special phenotype is determined by genetic components: the chance of belonging to the C3 phenotype group was almost three times higher in those carrying the GCC haplotype compared to other haplotype carriers.The risk haplotype identified in our study has a component described earlier as a risk allele associated with different phenotypes in multiple studies. These findings suggest that C allele carriers of rs1044396 (middle SNP of our haplotype) were more prone to develop nicotine dependence CHRNA4 yielded significant increase in acetylcholine sensitivity which can be a reasonable explanation for more serious withdrawal symptoms in carriers of hypersensitive genetic variant of CHRNA4 in our sample CHRNA4 exon 5 in emotional processes have also been confirmed The pivotal role of rs1044396 in receptor function was suggested by several authors. Convergent evidence hints that this missense mutation of exon 5 in From a pharmacogenetic point of view, it was demonstrated previously that rs2236196 and also rs3787138  were associated with treatment response to nicotine supplementation therapy and/or varenicline. These results raise the possibility that members of the C3 cluster have altered response to cessation promoting agents which may suggest that smoking cessation studies should not focus on smokers as a whole but rather on phenotypically different subgroups of them CHRNA4 variants Of note, exhaled CO levels were not the highest in the genetically vulnerable cluster in our cohort. Nevertheless, this finding is partly in line with negative results on the association between serum cotinine levels and CHRNA4 variants.In previous studies it has been demonstrated that smokers with a history of depression report more severe withdrawal symptoms CHRNA4 haplotypes on smoking-related traits using a special phenotypic clustering method. With the help of the phenotypic cluster analysis the possible role of CHRNA4 in smoking behavior and pharmacological actions of cessation agents can be nuanced. Further pharmacological studies are required to extend our knowledge on the role of CHRNA4 in the treatment of nicotine dependence. Based on these findings we propose cluster analysis combined with haplotype association test as an alternative method for identifying genetically predisposed part of complex disorders and for pharmacogenomic trials.In conclusion, we identified a clinically remarkable subgroup within smokers characterized by salient withdrawal symptoms and depressive signs. Here we presented a unique approach and studied the effect of Written informed consent was obtained from all participants and the study was approved by the the Scientific and Research Ethics Committee of the Medical Research Council of Hungary (ad.8-303/2009-1018EKU).A cohort of 350 treatment-seeking smokers from 5 Hungarian Cessation Centers was investigated in the present study. The time between the last smoked cigarette and the clinical interview was not a criterion for study entry, but for the current analysis we selected only those individuals who reported an overnight nicotine abstinence (n = 236). Participants were interviewed by a detailed background questionnaire on smoking-related variables such as quantity and quality of smoking, nicotine content of the cigarette regularly smoked by them and family history of smoking. Nicotine dependence was assessed by the Fagerström Test for Nicotine Dependence (FTND) CHRNA4 were genotyped with Sequenom MassArray technology and the iPLEX Gold chemistry . In this method allele discrimination is based on primer extension with single mass-modified nucleotides followed by MALDI-TOF mass spectrometry. Genotyping was performed by the Technology Centre, Institute for Molecular Medicine Finland (FIMM), University of Helsinki. Genotyping quality was examined by a detailed QC procedure consisting of success rate checks, duplicated samples and positive and negative control samples. Genotyping was done blinded to the phenotypic data. SNPs within CHRNA4 were selected based on literature and the International HapMap project 2>95%) constructing one haploblock  covering the ploblock  accordinTwo-step cluster method was used for phenotypic cluster analysis based on Ward’s aggregation criterion performed in SPSS 20.0 software. Smoking-related phenotypes, such as FTND score, smoked cigarettes/day (CPD), exhaled CO level, MNWS and ZSDS scores for depressive phenotype were stepped into the model. The strongest model was chosen for genetic association tests (higher than 0.5 of silhouette measure of cohesion and segregation). Haplotype analyses were performed with GLM and HapScore tests under additive model using HapStat modul of R 2.0 software. Rare haplotypes with a frequency less than 5% were excluded from analyses. To avoid false positive results Bonferroni’s correction in single marker association tests and permutation procedures with 1000 random permutations were performed by R 2.0 software similarly to our earlier publication Table S1Significant results of single marker association tests.(DOCX)Click here for additional data file.Table S2Basic characteristics of the investigated SNPs.(DOCX)Click here for additional data file.


Botrytis cinerea, the subcellular distribution of hydrogen peroxide (H2O2) was monitored over a time frame of 96 h post inoculation (hpi) in Arabidopsis thaliana Col-0 leaves at the inoculation site (IS) and the area around the IS which was defined as area adjacent to the inoculation site (AIS). H2O2 accumulation was correlated with changes in the compartment-specific distribution of ascorbate and glutathione and chloroplast fine structure. This study revealed that the severe breakdown of the antioxidative system, indicated by a drop in ascorbate and glutathione contents at the IS at later stages of infection correlated with an accumulation of H2O2 in chloroplasts, mitochondria, cell walls, nuclei and the cytosol which resulted in the development of chlorosis and cell death, eventually visible as tissue necrosis. A steady increase of glutathione contents in most cell compartments within infected tissues (up to 600% in chloroplasts at 96 hpi) correlated with an accumulation of H2O2 in chloroplasts, mitochondria and cell walls at the AIS indicating that high glutathione levels could not prevent the accumulation of reactive oxygen species (ROS) which resulted in chlorosis. Summing up, this study reveals the intracellular sequence of events during Botrytis cinerea infection and shows that the breakdown of the antioxidative system correlated with the accumulation of H2O2 in the host cells. This resulted in the degeneration of the leaf indicated by severe changes in the number and ultrastructure of chloroplasts , chlorosis and necrosis of the leaves.In order to study the mechanisms behind the infection process of the necrotrophic fungus  Even though the importance of H2O2 and antioxidants for the defense against necrotrophic fungal pathogens are well understood it still remains unclear in which cell compartments H2O2 accumulates during necrotrophic fungal infection and how it correlates to subcellular contents of antioxidants. Such information is crucial to understand the compartment-specific mechanisms behind the infection process of necrotrophic pathogens.The infection of plants by necrotrophic pathogens is characterized by the release of phytotoxins, cell wall degrading enzymes and an oxidative burst which is triggered by the pathogen and finally leads to plant cell death. Early visible signs of these events are the appearance of chlorotic regions followed by necrosis Botrytis cinereaBotrytis cinerea infection ROS accumulation has been observed around the penetrated cell walls, at the plasma membrane and inside cells close at and surrounding the necrotic IS Botrytis cinerea infection is due to the production of phytotoxins, cell wall degrading enzymes and the oxidative burst which eventually leads to the death of the cells One of the best studied necrotrophic fungal pathogens is 2O2 during Botrytis cinerea infection in Arabidopsis Col-0 on a high resolution and to correlate it with subcellular ascorbate and glutathione contents.Thus, the aim of this study was to investigate the compartment-specific accumulation of HArabidopsis thaliana [L.] Heynh. ecotype Columbia (Col-0) were grown on “Naturahum” potting soil  in growth chambers with 8/16 h day/night photoperiod. Day and night temperatures were 22°C and 18°C, respectively, the relative humidity was 60% and the plants were kept at 100% relative soil water content. Light intensity varied between 110 and 140 µmol m−2 s−1.After stratification for 4 d at 4°C seeds of Arabidopsis thaliana Col-0 were placed in petri dishes containing filter paper wetted with sterile water. A 50 µl drop of a Botrytis cinerea (strain: Bo510) spore suspension was delivered onto the abaxial side of each leaf (approximately 1×105 spores ml−1). Spores were preincubated in 3 g L−1 Gamborg's B5 medium  supplemented with 25 mM glucose for 2 h to stimulate germination. Control leaves (CL) were treated likewise but without spores in the medium. Petri dishes were sealed and stored at light conditions described above.Cut leaves of 8 weeks-old Botrytis cinerea. Due to preliminary investigations these time points were chosen as clear changes could not be determined at earlier time points and as cells at the IS showed massive cell death at 96 hpi which made it impossible to obtain adequate ultrastructure for investigations at later time points.Different parameters were investigated for the IS and for the surrounding leaf tissue, which was defined as AIS. Values were then compared to CL, which had been mock-inoculated. As chloroplasts showed the strongest changes in antioxidative capacity the fine structure of chloroplasts was additionally monitored during the infection period. Samples for the different experiments were harvested at the time of the inoculation (0 h), 12, 24, 48, and 96 hpi with the fungal pathogen 2O2 was performed according to Großkinsky et al. Detection of H2) of at least three different leaves for each treatment and time point (see frames in 3) in 50 mM MOPS-buffer (pH 7.2) for subcellular H2O2 visualization. Samples for ultrastructural and cytohistochemical investigations were then transferred into glass vials and fixed for 90 min at RT in the above mentioned solutions. Samples for the visualization of subcellular H2O2 distribution were incubated with CeCl3 solution for 60 min and then fixed in 2.5% glutardialdehyde in 0.06 M Sørensen phosphate buffer at pH 7.2 for 90 min at RT. For ultrastructural analysis and H2O2 localization samples were then rinsed in 0.06 M Sørensen phosphate buffer (4 times for 15 min each) and post-fixed in 1% osmium tetroxide in 0.06 M Sørensen phosphate buffer for 90 min at RT. The samples were then dehydrated in a graded series of increasing concentrations of acetone . Pure acetone was then exchanged for propylene oxide and the specimens were gradually infiltrated with increasing concentrations of Agar 100 epoxy resin  mixed with propylene oxide for a minimum of 3 h per step. Samples were finally embedded in pure, fresh Agar 100 epoxy resin  and polymerized at 60°C for 48 h. Ultrathin sections (80 nm) were cut with a Reichert Ultracut S ultramicrotome , post-stained with lead citrate (1% dissolved in 0.6 M NaOH) and uranyl-acetate (2% dissolved in aqua bidest) for 15 min. Sections were then observed in a Philips CM10 TEM. For cytohistochemical investigations samples were rinsed in 0.06 M Sørensen phosphate buffer (pH 7.2) for 4 times 15 min after fixation. They were then dehydrated in increasing concentrations of acetone  at RT for 20 min at each step. Subsequently, specimens were gradually infiltrated with increasing concentrations of LR-White resin  mixed with acetone (90%) for a minimum of 3 h per step. Samples were finally embedded in pure, fresh LR-White resin and polymerized at 50°C for 48 h in small plastic containers under anaerobic conditions. Ultrathin sections (80 nm) were cut with a Reichert Ultracut S ultramicrotome .Preparation of samples for TEM and immunogold labeling of ascorbate and glutathione was performed as described previously 2O2 was determined with a modified method according to Blokhina et al. 3 were digitized and the percentage of areas covered with CeCl3 precipitates were measured automatically using the software package Cell D with the particle analysis tool . Measurements were performed in different visually identified and manually traced cell structures such as cell walls, mitochondria, plastids, nuclei and the cytosol. A minimum of 40 sectioned cell structures from at least 15 different cells were evaluated for CeCl3-staining. The obtained data were statistically evaluated using Statistica .The accumulation of HChanges in the number of chloroplasts and their inner structures were evaluated according to Zechmann et al. Immunogold labeling of glutathione and ascorbate was done according to Zechmann et al. Micrographs of randomly photographed immunogold labeled sections were digitized and gold particles were counted automatically using the software package Cell D with the particle analysis tool  in different visually identified and manually traced cell structures . For statistical evaluation at least four different samples were examined. A minimum of 20 (peroxisomes and vacuoles) to 60 (other cell structures) sectioned cell structures of at least 15 different cells were analyzed for gold particle density per sample. The obtained data were statistically evaluated using Statistica .Several negative controls were made to support the specificity of the immunogold procedure. Negative controls were treated either with (i) gold conjugated secondary antibody (goat anti-rat IgG for ascorbate and goat anti-rabbit IgG for glutathione) without prior incubation of the section with the primary antibodies, (ii) non-specific secondary antibody (goat anti-rabbit IgG for ascorbate and goat anti-rat IgG for glutathione), (iii) pre-immune serum instead of the primary antibody and (iv) primary antibody against ascorbate and glutathione pre-adsorbed with an excess of reduced and oxidized ascorbate and glutathione, respectively for 2 h prior to labeling of the sections. For the latter a solution containing either 10 mM of ascorbic acid, dehydroascorbic acid, reduced or oxidized glutathione was incubated with or without 0.5% glutardialdehyde for 1 h. Excess glutardialdehyde was saturated by incubation for 30 min in a solution of 1% (w/v) BSA. The resulting solutions were both used in independent experiments to saturate the anti-ascorbate and glutathione antibodies for 2 h prior to its use in the immunogold labeling procedure described above. Labeling on sections treated as negative controls showed no or only very few gold particles bound to ascorbate and glutathione which was similar to previous results obtained by using the same methods in different plant species Botrytis cinerea did not show symptoms before 48 hpi  when compared to CL. In mitochondria glutathione labeling at the AIS was found to be significantly decreased 24 hpi (−33%) but showed no significant changes at other time points when compared to CL. At the IS glutathione labeling in chloroplasts was strongly increased 12 hpi (141%), 24 hpi (335%) and 48 hpi (92%). A similar pattern was found in nuclei, peroxisomes and the cytosol where glutathione labeling density was significantly increased at the IS 12 hpi , and 24 hpi , when compared to CL. At the AIS a strong and significant increase of glutathione specific labeling was observed in chloroplasts, nuclei, and the cytosol 24 hpi , 48 hpi , and 96 hpi  when compared to CL. In peroxisomes of the same leaf area density of glutathione labeling was found to be increased 12 hpi (93%), 24 hpi (214%), 48 hpi (227%) and 96 hpi (328%) when compared to CL . GlutathBotrytis cinerea inoculated plants at 12, 24, 48 and 96 hpi. Mock-inoculated plants contained about 17 and 8 chloroplasts in cell sections of the palisade and spongy parenchyma, respectively. At the IS the number of chloroplasts decreased significantly in cell sections of both tissues 48 hpi  when compared to CL  throughout the infection. At the IS this drop occurred earlier and was more severe than at the AIS showing a good correlation with the accumulation of H2O2. Similar results have been obtained by electron paramagnetic resonance within Arabidopsis leaves where a massive depletion of ascorbic acid correlated with an increase in ROS Botrytis cinerea the decrease of ascorbate contents, insufficient activities of related antioxidative enzymes and a general shift towards the oxidative state at the cellular and organelle level was related to disease progress 2O2 observed here prove that the decreasing capacity of the antioxidative system triggers an oxidative burst leading to chlorosis and eventually cell death during susceptible Botrytis cinerea infection in Arabidopsis. This conclusion is supported by changes in glutathione contents at the IS which showed a strong increase 12 and 24 hpi followed by a strong drop 48 and 96 hpi when H2O2 accumulation reached its maximum. Whereas subcellular glutathione contents 96 hpi are similar to what has been reported previously for tomato Botrytis cinereaROS like H2O2 accumulation could be observed by DAB and CeCl3 staining at the IS at 24 hpi similar effects could be found at the AIS at 48 hpi. Additionally, the situation of H2O2 accumulation at the AIS at 96 hpi mimicked the situation at the IS at 48 hpi. Similar effects of the antioxidative response could be observed at these time points as ascorbate contents dropped earlier at the IS when compared to the AIS in most cell compartments. In addition, both the initial increase and the drop of glutathione contents at the final stages of infection happened earlier at the IS than at the AIS in most cell compartments. The reason for the delayed response of the AIS can be explained by delayed symptom development as the fungal hyphae have to establish the infection at the IS first before they can spread into other areas of the AIS. Thus, we can conclude that H2O2 accumulation and the antioxidative response of the AIS lagged behind but mimicked the situation at the IS.Generally, the response of the AIS lagged behind that of the IS. While HBotrytis cinerea2O2 in chloroplasts at the AIS at this time point. This could be due to a shift from reduced to oxidized glutathione despite higher activity of glutathione reductase which was demonstrated for chloroplasts in Botrytis infected tomato plants at this time point and could also be observed in whole leaves Botrytis cinerea infection 2O2 leading to oxidative damage visible in the form of chlorosis at the AIS and subsequently necrosis caused by massive cell dead at the IS 96 hpi.On the subcellular level it is remarkable that the AIS showed a massive accumulation of glutathione in all cell compartments of up to 600% in chloroplasts at 96 hpi when glutathione contents dropped to background levels at the IS. Again, this increase in glutathione contents can be correlated with the up-regulation of genes encoding enzymes responsible for glutathione synthesis found in Arabidopsis infected with Botrytis cinerea also induced severe changes in chloroplast fine structure. Interestingly, plastoglobuli contents were massively increased 24 hpi and 48 hpi at the IS and 48 hpi and 96 hpi at the AIS. These results correlate well with a strong decrease in thylakoid content 96 hpi at the AIS. Plastoglobuli are considered to be an important storage subcompartment of degenerated thylakoid membranes 2O2 inside the chloroplasts triggered thylakoid degradation which resulted in a strong increase in plastoglobuli size until complete disintegration of chloroplasts.Infection of Arabidopsis with Botrytis cinerea interaction with susceptible Arabidopsis plants induced a severe oxidative bust starting in cell walls at 24 hpi at the IS and spread to mitochondria, chloroplasts, nuclei and the cytosol at later stages. Cell walls, mitochondria and chloroplasts seem to be the main origin of H2O2 as an accumulation occurred in these cell compartments much earlier than in the cytosol and nuclei. Early responses (time points before 24 hpi) observed in other plant species in previous studies seem to be rather local either triggered by oxidative burst in epidermis cells or by ROS produced by the fungal hyphae. The breakdown of the antioxidative system indicated by a strong drop in ascorbate and glutathione levels at later stages of infection correlated with the accumulation of ROS which resulted in the development of chlorosis, necrosis and eventually cell death and was associated with severe changes in chloroplast structure and number.Summing up, this study demonstrates that ROS production during Figure S1Light microscopical images of leaf sections from Arabidopsis thaliana Col-0 inoculated with Botrytis cinerea. Images in A and D show sections of CL at the beginning (0 h) and at the end of the experiment (96 h), respectively, at the mock-inoculation site. Images in B, C and E, F show leaves at the AIS and at the IS, respectively, at 48 hpi  and 96 hpi . Whereas fungal structures remain absent in sections of the AIS  hyphae (arrows) on top (48 hpi) and inside (96 hpi) the leaves could be found at the IS. Bars = 50 µm.(TIF)Click here for additional data file.Figure S2Transmission electron micrographs showing ascorbate specific labeling at the AIS. Gold particles bound to ascorbate were detected at the AIS in leaf sections from Arabidopsis thaliana Col-0 0 h (A), 24 hpi (B), 48 hpi (C) and 96 hpi (D) with Botrytis cinerea. Bars = 1 µm. C = chloroplasts with or without starch (St), CW = cell walls, ICS = intercellular spaces, M = mitochondria, N = nuclei, Px = peroxisomes, V = vacuoles.(TIF)Click here for additional data file.Figure S3Transmission electron micrographs showing ascorbate specific labeling at the IS. Gold particles bound to ascorbate were detected at the IS in leaf sections from Arabidopsis thaliana Col-0 0 h (A), 24 hpi (B), 48 hpi (C) and 96 hpi (D) with Botrytis cinerea. Note that 96 hpi fungal hyphae (H) containing gold particles bound to ascorbate and only remnants of organelles such as plastids (P) could be found in the degenerated leaf cells (D). Bars = 1 µm. C = chloroplasts with or without starch (St), M = mitochondria, N = nuclei, Px = peroxisomes, V = vacuoles.(TIF)Click here for additional data file.Figure S4Transmission electron micrographs showing glutathione specific labeling at the AIS. Gold particles bound to glutathione were detected at the AIS in leaf sections from Arabidopsis thaliana Col-0 0 h (A), 24 hpi (B), 48 hpi (C) and 96 hpi (D) with Botrytis cinerea. Bars = 1 µm. C = chloroplasts with or without starch (St), CW = cell walls, ICS = intercellular spaces, M = mitochondria, N = nuclei, Px = peroxisomes, V = vacuoles.(TIF)Click here for additional data file.Figure S5Transmission electron micrographs showing glutathione specific labeling at the IS. Gold particles bound to glutathione were detected at the IS in leaf sections from Arabidopsis thaliana Col-0 0 h (A), 24 hpi (B), 48 hpi (C) and 96 hpi (D) with Botrytis cinerea. Note that 96 hpi fungal hyphae (H) containing gold particles bound to glutathione and only remnants of organelles such as plastids (P) could be found in the degenerated leaf cells (D). Bars = 1 µm. C = chloroplasts with or without starch (St), CW = cell walls, M = mitochondria, N = nuclei, Px = peroxisomes, V = vacuoles.(TIF)Click here for additional data file.Table S1Arabidopsis thaliana.Total number of gold particles bound to ascorbate and glutathione in mock inoculated  Values are means with standard errors and document the total amount of gold particles bound to ascorbate and glutathione per µm2 in different cell compartments of mock inoculated Arabidopsis thaliana [L.] Heynh. ecotype Columbia (Col-0). n.d. = not detected. n>20 for peroxisomes and vacuoles and n>60 for other cell structures. Different lowercase letters indicate significant differences (P<0.05) analyzed with the Kruskal-Wallis test followed by post-hoc comparison according to Conover.(TIF)Click here for additional data file.Table S2Arabidopsis thaliana Col 0.Total number and size of chloroplast fine structures in mock inoculated  Values are means with standard errors and document the relative area of internal chloroplast structures and the size of chloroplast in µm2 detected by TEM on a longitudinal ultrathin section within the mesophyll of mock inoculated leaves in Arabidopsis thaliana [L.] Heynh. ecotype Columbia (Col-0). Different lowercase letters indicate significant differences (P<0.05) analyzed with the Kruskal-Wallis test followed by post-hoc comparison according to Conover. N>20 from at least four different samples.(TIF)Click here for additional data file.


CDKN2A (p16INK4a) and RB1 tumor suppressor genes in TGCTs.Testicular germ cell tumors (TGCTs) are the most frequent malignances in young adult men. The two main histological forms, seminomas and nonseminomas, differ biologically and clinically. pRB protein and its immediate upstream regulator p16INK4a are involved in the RB pathway which is deregulated in most TGCTs. The objective of this study was to evaluate the occurrence of loss of heterozygosity (LOH) of the Forty TGCTs (18 seminomas and 22 nonseminomas) were analyzed by polymerase chain reaction using the restriction fragment length polymorphism or the nucleotide repeat polymorphism method.CDKN2A was found in two (6%) out of 34 (85%) informative cases of our total TGCT sample. The observed changes were assigned to two (11%) nonseminomas out of 18 (82%) informative samples. Furthermore, LOH of the RB1 was detected in two (6%) out of 34 (85%) informative cases of our total TGCT sample. Once again, the observed changes were assigned to two (10.5%) nonseminomas out of 19 (86%) informative samples. Both LOHs of the CDKN2A were found in nonseminomas with a yolk sac tumor component, and both LOHs of the RB1 were found in nonseminomas with an embryonal carcinoma component.LOH of the The higher incidence of observed LOH in nonseminomas may provide a clue to their invasive behavior. Testicular germ cell tumor (TGCT) is diagnosed mainly after puberty and is the most frequent malignancy in young adult men51 phase-controlling mechanism centered around the pRB protein. Different cancers seem to have altered different key components of that mechanism, which may be connected with gene activity patterns in different target cells.7The cell cycle regulatory pathway deregulated in almost all human tumors appears to be the G0 and the early G1 phase, hypophosphorylated pRB is complexed with the transcription factor E2F.1, a significant hyperphosphorylation of the pRB by CDK4 and CDK6 in complex with D cyclins  occurs.9The pRB is essential in cell cycle regulation and its function is regulated by phosphorylation. In GCDKN2 locus at chromosomal region 9p21 encodes p16INK4a tumor suppressor protein involved in the RB cell cycle control pathway.1/S phase transition by inhibiting the activity of CDK4 and CDK6. Thus, by inhibiting pRB phosphorylation, p16INK4a can promote the formation of a pRB-E2F repressive transcriptional complex, which blocks cell cycle progression past G1/S restriction point.11The RB1 and inactivating mutations or promoter methylation of the CDKN2A (p16INK4a) tumor suppressor gene, as well as the overexpression of the cyclin D2/CDK4 complex.13In diverse types of cancer the RB pathway becomes deregulated through alterations in one or more of its components. The most common defects of the RB pathway are mutations or deletions of CDKN2A p1INK4a tumCDKN2A and RB1 tumor suppressor genes in TGCTs.The objective of this study was to evaluate the occurrence of the loss of heterozygosity (LOH) of the Fourty TGCT samples (18 seminomas and 22 nonseminomas) were collected from Sisters of Mercy University Hospital and University Hospital Center, Zagreb, Croatia. The samples were formalin-fixed and paraffin-embedded. Clinical and pathological data for 40 TGCTs according to the WHO 2004 classification are shown in For each specimen, 20 μm paraffin-embedded section was prepared for DNA extraction. In addition, 4 μm section was stained with hematoxylin-eosin to identify the tumor and normal tissue areas which were removed separately from the microscopic slide, transferred to microtubes and extracted using QIAamp DNA Mini Kit .23 located close to intron 1 of the CDKN2A gene was analyzed in this study.2, 0.2 μM of each primer , 1x Flexi buffer  and 0.5 U of GoTaq® Hot Start Polymerase . One hundred nanograms of DNA were used in each PCR reaction. PCR amplifications were carried out in a Eppendorf Mastercycler Personal , with cycling times of 96ºC for 5 min (one cycle), then 45 cycles of 96ºC for 30 s, 57ºC for 45 s, and 72ºC for 30 + 1 s. The final step was incubation at 72º C for 10 min. Amplified DNA fragments were analyzed on silver-stained 15% polyacrylamide gels. LOH of CDKN2A was considered to had occured if one out of two alleles (heterozygous samples) of a gene marker was missing or significantly reduced in comparison to alleles from adjacent normal tissue.A previously described polymorphic microsatellite marker hMp16α-I1 consisting of a mononucleotide tract of (A)RB1 was detected using polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP). Amplification with RB1 primers 5′-TCCCACCTCAGCCTCCTTAG-3’ and 5′-GTAGGCCAAGAGTGGCAGCT-3′ used in our study produced a 190 bp segment of intron 17.LOH of CDKN2A gene. From 40 TGCTs, 34 (85%) tumors were informative for this polymorphism, 16 (89%) seminomas and 18 (82%) nonseminomas. Our analysis revealed that two (6%) samples showed LOH of hMp16α-I1 marker. The observed changes were assigned to two nonseminomas  of total TGCTs were heterozygous for this polymorphism; 15 (83%) seminomas and 19 (86%) nonseminomas. LOH was observed in two (6%) samples when looking at the total TGCTs analyzed. Once again the observed allelic losses were assigned to nonseminomas: two samples  is reported to be the most commonly deleted region early in the development of nonseminomas, which may be implicated in their ability to differentiate into various types, for various markers located within this region.20Despite of promoter methylation and mutations being the most common ways of inactivating CDKN2A were found in nonseminomas with a yolk sac tumor component, one sample also having an embryonal carcinoma component. Furthermore, one nonseminoma with the LOH of CDKN2A demonstrated LOH of TP53 gene, and the other showed LOH of the CDH1 gene.21In our study only nonseminomas demonstrated LOH . Both LORB1 gene is often deleted or mutated to an inactive form in a variety of human tumors. Cells of embryonal testes and intratubular germ cell neoplasia (ITGCN) show no expression of pRB, whereas it is expressed in healthy testes during spermatogenesis. The lack of pRB in most TGCTs may, therefore, reflect its deregulation by normal mechanisms in testicular germ cells. However, the lack of pRB may facilitate the transition of those cells to tumor cells of ITGCN and thus contribute to molecular pathogenesis of TGCTs.23The RB1 gene are, along with its mutations, also reported as one of the most common alterations of the RB pathway. Various studies revealed deletions of the RB1 gene region in testicular cancer.et al.RB1 introns 16 and 20, and found LOH in 5% of seminomas and 28% of nonseminomas analyzed within 93% of informative TGCT cases. The location of the RB1 gene is reported to be one of the most commonly involved in allelic imbalance within TGCTs.RB1 in various forms of TGCTs needs to be further elucidated in more detail. Studies also revealed a different pattern of LOH in different histological types of nonseminomas for markers located within the genomic region containing the RB1 gene (13q14), varying from 0% in yolk sac tumor component to 50% in choriocarcinoma.25In contrast, deletions of RB1 gene was found in nonseminomas with an embryonal carcinoma component, and both nonseminomas with LOH of RB1 also demonstrated LOH of the TP53 gene.26In our study, LOH of the CDKN2A, RB1, TP53 and CDH1 in TGCTs may increase their tumorigenic potential by the increased proliferation capacity due to RB1 loss and decreased rate of apoptosis due to TP53 alteration.RB1 are often seen together with alterations of TP53 in variety of different cancers.RB1 and TP53 genes in a cell produces a synergistic effect, which imposes a stronger selective pressure for the cellular transformation. This may also help to explain the high proliferation rate and/or invasiveness of TGCTs with embryonal carcinoma and yolk sac tumor component. A higher incidence of LOH in nonseminomas may provide a clue to their invasive behavior, because for some of the nonseminoma types there seem to be a region of preferential loss , and all of the TGCTs show gain of 12p11–12p12 sequences.28LOH of CDKN2A and RB1 inactivation.24However, the low frequency of observed LOHs in this study could be a consequence of genomic instability in above mentioned nonseminomas, rather than the main cause of


Several scoring system have been developed with the aim to identify clinical deterioration among hospitalized patients and allocate resources in accordance with the degree of deterioration - most without validation. The aim of the present study was to describe to which degree the system "Tidlig Opsporing af Kritisk Sygdom" (TOKS) is able to identify patients who either have or develop severe sepsis or septic shock within 24 after arrival to hospital.A retrospective descriptive study of patients hospitalized with community acquired severe sepsis or septic shock. Patients were identified based at discharge diagnosis ((IDC10 code A40.0-A41.9). Based at a manual evaluation of all patient records patients were included if they within the first 24 hours after arrival to the hospital fulfilled predefined criteria for severe sepsis or septic shock. Vital values registered at arrival to the hospital were identified and used for the present analysis. TOKS score is based at scores for respiratory frequency, saturation, systolic blood pressure, pulse rate, consciousness and temperature. The score range from 0 to 21 with an indication of need for a doctoral evaluation if the score is 3 or higher.335 patients were discharged with a diagnosis of sepsis. 212 fulfilled the criteria for severe sepsis or septic shock within the first 24 hours of hospitalization. One hundred and six (50%) were male, mean age 70.6 years , 103 (49%) had septic shock. Median TOKS score at arrival was 4 (range 0-13). 10/212 (5%) had TOKS=0, indicating no need for measurements of vital values the next 24 hours, 20/212 (9%) had TOKS=1, indicating measurements of vital values every 8 hours, 13/212 (6%) had TOKS=2 indicating control of vital values after one hour, 66/212(31%) had TOKS 3-4 indicating need for evaluation by a junior doctor and 103/121 (49%) had a TOKS score≥5 indicating need for urgent specialist evaluation.14% of the patients who develop severe sepsis or septic shock within 24 hours after arrival to the hospital had a TOKS score at arrival indicating a need for control of vital values every 8 hours or less.


The development of methods for the modification of peptides and proteins under benign aqueous conditions has received a great deal of attention from both industrial and academic laboratories.4O-mesitylenesulfonylhydroxylamine and hexamethylphosphorous triamide.Bacillus lentus SBL(S156C) (1) with 2,5-dibromohexanediamide (2) at pH 8, which afforded intermediate dehydroalanine 3, presumably via the transient formation of sulfonium 4  of "superfolder" green fluorescent protein (GFP) bearing a single surface-accessible cysteine. We were able to synthesise unusually stable cyclic sulfonium species and to employ these in a novel bioconjugation that is complementary to current dehydroalanine-based technologies for substrates for which dehydroalanine formation is not facile. Although the presence of sulfonium ions in peptides and proteins derived from the alkylation of methionine is well established,C of "sup6) with 2 under Davis' conditions  MS indicated quantitative conversion to a species with a molecular weight equivalent to that of sulfonium 8 . Indeed, throughout this study, we observed no evidence for the formation of dehydroalanine 7, or products derived therefrom, even upon incubation of sulfonium 8 for prolonged periods (vide infra). The isolation of this sulfonium species as a stable adduct and its reluctance to undergo elimination to give the corresponding dehydroalanine product 7 was noteworthy and we embarked upon further studies to understand and exploit this unexpected mode of reactivity.We treated GFP(S147C)  with 2 u8 was treated with N-methylbromomaleimide (1 equiv).8, whereas 6 reacted cleanly, thus suggesting that 2 had reacted exclusively on cysteine to generate 8 (see the Supporting Information).In order to gain further evidence for the site of reaction between GFP(S147C) and 2,5-dibromohexanediamide, sulfonium 8 was achieved by varying a number of parameters including the number of equivalents of 2, reaction temperature and reaction time  was apparent by LCMS  excess 2 could be readily removed by repeated diafiltration into fresh buffer. Sulfonium 8 was found to be stable in the absence of nucleophiles at 21 °C for 24 h and at 4 °C for 1 month; although, significant decomposition was observed at 37 °C after 4 h (vide supra). Treatment of 8 with β-mercaptoethanol  afforded bisthioether 10 a  in complete conversion, presumably through ring opening of the cyclic sulfonium  afforded the expected GFP–thioglucose conjugate 10 b as the sole product, with complete conversion after 5 h . We also confirmed that these reactions could be carried out effectively at room temperature (21 °C) in 5 h, or more rapidly at 37 °C or with increased concentrations of the nucleophile . Treatment of sulfonium 8 with phenyl selenol (100 equiv) afforded the mixed thio/seleno bisether 10 d in excellent conversion (entry 6). Although the conversion from reaction between 8 and phthalimide to give 10 e was only modest (entry 7), we were delighted to observe clean reaction in high conversion upon treatment of 8 with sodium azide (1000 equiv) to generate azide-labelled GFP 10 f (entry 8).We then evaluated the reactivity of fter 5 h , entry 1In conclusion, we have shown that treatment of superfolder GFP with bis-alkylating agents can be used to prepare highly stable, yet synthetically useful cyclic protein sulfonium adducts. These species undergo clean addition reactions to allow the introduction of a variety of useful chemical motifs. We envisage that the stability of the generated cysteine-derived sulfonium is presumably strongly correlated to the chemical environment of the alkylated cysteine. This new bioconjugation technique could provide a useful entry into multiply functionalised proteins and further work will be directed toward extending these findings to other systems.


NLGN3, NLGN4 and scaffolding proteins SHANK2 and SHANK3. Neuroligin proteins play an important role in synaptic function and trans-synaptic signaling by interacting with presynaptic neurexins. Shank proteins are scaffolding molecules of excitatory synapses, which function as central organizers of the postsynaptic density. Sequence level mutations and structural variations in these genes have been identified in ASD cases, while few studies were performed in Chinese population. In this study, we examined the copy numbers of four genes NLGN4, NLGN3, SHANK2, and SHANK3 in 285 ASD cases using multiplex fluorescence competitive polymerase chain reaction (PCR). We also screened the regulatory region including the promoter region and 5′/3′ untranslated regions (UTR) and the entire coding region of NLGN4 in a cohort of 285 ASD patients and 384 controls by direct sequencing of genomic DNA using the Sanger method. DNA copy number calculation in four genes showed no deletion or duplication in our cases. No missense mutations in NLGN4 were identified in our cohort. Association analysis of 6 common SNPs in NLGN4 did not find significant difference between ASD cases and controls. These findings showed that these genes may not be major disease genes in Chinese ASD cases.Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social communication, absence or delay in language development, and stereotyped or repetitive behaviors. Genetic studies show that neurexin-neuroligin (NRXN-NLGN) pathway genes contribute susceptibility to ASD, which include cell adhesion molecules  NLGN3, NLGN4 , NRXN1 and scaffolding proteins SHANK2 and SHANK3Autism spectrum disorder (ASD) is a neurodevelopmental disease with complex genetic and clinical heterogeneity. The core symptoms of ASD are impaired social interactions, deficient communication, restricted interests, and stereotyped activity patterns NLGN3 and NLGN4 are the first discovered ASD-associated genes in this pathway NLGN4 gene was identified in two brothers, one with autism and the other one with Asperger syndrome NLGN4 in ASD NLGN3 was detected in two male ASD siblings until now NLGN3 gene, NLGN4 was more frequently discovered to have ASD-related rare functional mutations in coding regions or promoter regions NLGN4 happen in only a small fraction of ASD cases, their impacts on synaptic function provide an important perspective for understanding the pathogenesis of ASD. Besides the coding region, the regulatory region and copy number of NLGN4 are deserved to be studied in ASD since they may affect the expression level of NLGN4.NLGN proteins, expressed highly in brain, are postsynaptic adhesion molecules interacting with presynaptic NRXNs in a calcium-dependent manner SHANK3 in ASD cases SHANK2 in ASD subjects, suggesting the implication of SHANK2 in ASD SHANK proteins contain multiple interaction sites and assemble many molecules, including guanylate kinase-associated proteins (GKAPs), which links to PSD-95 NLGN4 in a cohort of 285 ASD patients and 384 controls by direct sequencing of genomic DNA using the Sanger method. We also examined the CNVs of NLGN4, NLGN3, SHANK2 and SHANK3 in 285 ASD cases using multiplex fluorescence competitive polymerase chain reaction (PCR). Moreover, six common single nucleotide polymorphisms (SNPs) within NLGN4 were identified and analyzed for the association with ASD.In this study, we sequenced the regulatory region including the promoter region and 5′/3′ untranslated regions (UTR) and the entire coding region of The study was approved by the Ethics Committee of Shanghai Mental Health Center and the Ethics Committee of the School of Life Sciences, Fudan University. Informed, written consent was obtained from the controls, the parents or guardians of the children.A total of 285 patients with ASD were recruited from Department of Child and Adolescent Psychiatry, Shanghai Mental Health Center, China. All probands met DSM-IV diagnostic criteria for autism, Asperger syndrome or pervasive developmental disorder not otherwise specified (PDD-NOS). Cases were excluded if they had known medical disorders or chromosomal abnormalities. The patients were 246 males and 39 females with a mean age of 7.05 (SD = 3.36 years). All individuals were Han Chinese.384 unrelated ethnically-matched healthy controls were recruited from Fudan University. There were 311 males and 73 females.NLGN4 gene was acquired from the UCSC Genome Browser (NM_181332). The promoter region (2000 bp upstream of the transcription start site), six exons including the 5′/3′ UTRs and the open reading frame (ORF), and ten exon-intron junctions were sequenced in 285 ASD patients and 384 controls. Totally, 9110 basepair (bp) of untranslated region and 2451 bp of coding region in NLGN4 were sequenced. Genomic DNA was isolated from whole blood using Puregene Blood Core Kit C . PCR primers were designed by PRIMER 3 (http://frodo.wi.mit.edu/primer3/). The information of primers is shown in NLGN4, while those for exons 1, 2, and 4 can amplify the sequences of NLGN4 and NLGN4Y simultaneously due to sequence homology. PCR was performed in a 10 µl reaction mixture containing 1 µl 10×buffer (Mg2+ Plus), 1 µl dNTP mixture (2.5 mM), 0.2 µl primers (10 µM), 0.05 µl Takara Hotstar Taq (5 U/µl), and 1 µl genomic DNA (20 ng/µl). Thermo cycling conditions consisted of 35 cycles with an initial denaturation at 95°C for 5 min, followed by denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 5 min. We carried out all sequencing reaction by BigDye Terminator version 3.1 in ABI 3100 sequencers .The reference sequence of NLGN4, NLGN3, SHANK2, and SHANK3) were measured by a custom-by-design Multiplex AccuCopy™ Kit  based on a multiplex fluorescence competitive PCR principle as described by Du et al. http://genome.ucsc.edu). Three reference genes  were utilized for normalization. Nine target genomic segments within the four genes (two or three segments for each gene) were chosen for the AccuCopy assay. The forward and reverse primers of target segments were provided in POP1, RPP14, and TBX15) and the S/C ratio for each target fragment was first normalized to three reference genes respectively. The three normalized S/C ratios were further normalized to the median value in all samples for each reference gene respectively and then averaged. If one of the three normalized S/C ratios deviated more than 25% from the average of the other two, it was excluded for further analysis.The copy numbers of four genes  analysis of SNPs and the haplotype association were analyzed using Haploview 4.2 software. LD between the SNPs was measured by a pairwise D′ statistic. The structure of the LD block was examined using the method of Gabriel et al. NLGN4 was detected in our cohort. Direct sequencing of the coding region and regulatory region of NLGN4 identified six common SNPs . They were rs5916355 in the promoter region, rs3810688, rs3810686, rs5916269, rs1882260, and rs140700235 in 3′UTR. Five percent of samples were selected randomly for validation by sequencing in the opposite direction. No genotyping error was detected. Association analysis of these six SNPs with ASD did not show significant difference of allele frequencies between ASD patients and controls (Only one synonymous mutation rs7049300 (Thr311Thr) was found in one ASD individual. No non-synonymous mutation in controls . The LD controls . The polNLGN4 ranged between 0.76 and 1.22 in 246 male samples, and ranged between 1.69 and 2.38 in 39 female samples, which indicated unchanged copy number of NLGN4 for all samples  was identified in two brothers with ASD Since the first report of NLGN3 and NLGN4 to ASD in Chinese population so far NLGN3 and NLGN4 genes were screened and twelve intronic SNPs were genotyped for case-control association analysis in 229 ASDs cases and 184 controls in a Chinese cohort NLGN3 gene was associated with ASD . Six SNPs in the introns of NLGN4, different with the ones examined in our study, were analyzed and showed no frequency difference between ASD cases and controls NLGN4 in 285 Chinese ASD cases by comprehensively testing genetic variants including rare mutations in the coding region and regulatory region, copy number variations and common SNPs. No positive results were identified, suggesting NLGN4 gene is not a major susceptibility gene in our ASD cohort.Only one study investigated the susceptibility of NLGN3, SHANK2, and SHANK3 in this cohort. Shank proteins are scaffolding molecules of excitatory synapses, which function as central organizers of the postsynaptic density and are believed to play an important role in the formation and maturation of dendritic spines. Rare de novo CNVs and nonsense/frameshift mutations in SHANK2 and SHANK3 have been identified in ASD and mental retardation cases SHANK2 in a German cohort of 184 mental retardation cases and a Canadian cohort of 396 ASD probands de novo deletions and a de novo nonsense mutation of SHANK2 were identified in unrelated mental retardation and ASD individuals. Haploinsufficiency of SHANK3 is associated with ASD and mental retardation in human patients and results in altered synaptic plasticity and reduced social behaviors in mice NLGN3, SHANK2 and SHANK3, indicating the limited role of these genes in Chinese ASD patients. Considering that we did not screen these genes for sequence level variants and did not investigate other genes in this pathway like NRXN3 and SHANK1, recently reported to be associated with ASD To investigate the possibility of other genes in neurexin-neuroligin pathway involved in the etiology of ASD, we examined the copy number of NLGN4 or NLGN3 have been reported by different studies involving about 1500 ASD cases, indicating a low frequency (no more than 1%) of rare variants within NLGN3 and NLGN4 in ASD NLGN4 as well as examine the copy numbers of four genes NLGN4, NLGN3, SHANK2, and SHANK3 in Chinese population. Though the results were negative, this study would extend our understanding about the role of these genes in ASD in different populations.One limitation of this study should be considered. Nine missense/nonsense mutations and 3 deletions within In this study, we used the multiplex fluorescence competitive PCR for copy number measurement. This newly developed method has the advantages of multiplex measurement and low measurement variation NLGN4 were identified and studied for the relationship with ASD in our cohort. These SNPs might affect the expression of NLGN4 by binding with transcription factors or microRNA. The SNP rs5916355 in the promoter region of NLGN4 was predicted to be a binding site for transcription factor HFH-2 using the online tool TFSEARCH. Two websites (http://www.microrna.org/microrna/getGeneForm.do and http://www.mirbase.org/search.shtml) were used to predict miRNAs which bind to the 3′UTR sequence of NLGN4 containing five tested SNPs. The variants rs3810688, rs5916269 and rs140700235 were found located within the potential binding sites of has-miR-1293, has-miR-1324 and has-miR-5011-5p, respectively. These SNPs may affect NLGN4 expression by altering the interaction of miRNAs and mRNA. For example, according to the algorithm RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/), the allele A of rs3810688 in the NLGN4 3′UTR has a mismatch with miR-1293 seed region and an altered minimum free energy value , compared with the allele G . Though we did not find any SNP which had significant frequency difference between ASD cases and controls, the validation in more samples and functional study would help us to understand the potential role of these polymorphisms in ASD.Most previously reported studies focused on the mutations in the coding region. In our study, we also pay attention to the genetic variants which may influence the expression level of NLGN4. Six common SNPs in the promoter region and 3′ UTR of NLGN4 in ASD by comprehensive screening of the coding region and regulatory region of NLGN4 for mutations and examining the copy number variations of NLGN4. 285 ASD patients were studied and no missense mutations or deletion/duplication were identified. There was no association between 6 common SNPs in NLGN4 and ASD. These findings showed that NLGN4 may not play an important role in our ASD cohort.In this study, we investigated the role of Table S1The primer and PCR condition of NLGN4 gene.(DOC)Click here for additional data file.Table S2The primers of four genes for multiplex competitive PCR.(DOC)Click here for additional data file.


Salmonella, a ubiquitous Gram-negative intracellular bacterium, is a food borne pathogen that infects a broad range of hosts. Infection with Salmonella Typhimurium in mice is a broadly recognized experimental model resembling typhoid fever in humans. Using a N-ethyl-N-nitrosurea (ENU) mutagenesis recessive screen, we report the identification of Ity16 (Immunity to Typhimurium locus 16), a locus responsible for increased susceptibility to infection. The position of Ity16 was refined on chromosome 8 and a nonsense mutation was identified in the ankyrin 1 (Ank1) gene. ANK1 plays an important role in the formation and stabilization of the red cell cytoskeleton. The Ity16/Ity16Ank1 mutation causes severe hemolytic anemia in uninfected mice resulting in splenomegaly, hyperbilirubinemia, jaundice, extramedullary erythropoiesis and iron overload in liver and kidneys. Ity16/Ity16Ank1 mutant mice demonstrated low levels of hepcidin (Hamp) expression and significant increases in the expression of the growth differentiation factor 15 (Gdf15), erythropoietin (Epo) and heme oxygenase 1 (Hmox1) exacerbating extramedullary erythropoiesis, tissue iron deposition and splenomegaly. As the infection progresses in Ity16/Ity16Ank1, the anemia worsens and bacterial load were high in liver and kidneys compared to wild type mice. Heterozygous +/Ity16Ank1 mice were also more susceptible to Salmonella infection although to a lesser extent than Ity16/Ity16Ank1 and they did not inherently present anemia and splenomegaly. During infection, iron accumulated in the kidneys of +/Ity16Ank1 mice where bacterial loads were high compared to littermate controls. The critical role of HAMP in the host response to Salmonella infection was validated by showing increased susceptibility to infection in Hamp-deficient mice and significant survival benefits in Ank1+/Ity16 heterozygous mice treated with HAMP peptide. This study illustrates that the regulation of Hamp and iron balance are crucial in the host response to Salmonella infection in Ank1 mutants. Salmonella infections in humans are responsible for two major diseases, typhoid fever caused by Salmonella Typhi and Salmonella Paratyphi and a diarrheal disease known as salmonellosis caused by several non host specific serotypes including Salmonella Typhimurium and Salmonella Enteritidis. Typhoid is transmitted by a fecal-oral route through contaminated food and water and is endemic in areas of poor water sanitation. It is estimated by the World Health Organization that there are 21 million new cases of typhoid fever each year resulting in approximately 200,000 deaths. In addition, 1 to 5% of patients become asymptomatic chronic carriers serving as reservoirs from which new Salmonella Typhi infections can be transmitted Salmonella infection Salmonella infection Salmonella Typhimurium infection results in a systemic disease with clinical manifestations resembling those found in humans with typhoid fever. The severity of infection depends on a variety of factors including the dose of the inoculum, as well as the interaction of host and bacterial genetic determinants. Several strains of mice show varying degrees of susceptibility to Salmonella Typhimurium infection with the mouse strain 129 substrains showing the most resistance. The study of the natural variation of the host response to infection with Salmonella Typhimurium in spontaneous mouse mutants have identified important innate immune genes having Mendelian contribution to disease susceptibility and contributing to different mechanisms including pathogen recognition (Tlr4P712H in C3H/HeJ), phagosome transport of divalent cations including iron (Nramp1G169D in C57BL/6J and BALB/cJ) or erythropoiesis and iron metabolism (PklrI90N in AcB61 mice) N-ethyl-N-nitrosurea (ENU) mutagenesis to generate novel mutations responsible for increased susceptibility to Salmonella infection. Recessive ENU induced mutations are bred to homozygosity with the use of a three-generation breeding scheme and challenged with Salmonella Typhimurium. In the current screen we have evaluated 216 pedigrees for their susceptibility to infection and identified 5 deviant pedigrees. In this paper, we report the identification of one of these ENU mutants named Ity16 that carries a nonsense mutation in the gene Ank1. Ank1 encodes a red blood cell (RBC) adaptor protein consisting of three major functional domains: an N-terminal membrane binding, a spectrin binding and a C-terminal regulatory domain Ank1, both spontaneous and ENU-induced, have been described to cause hemolytic anemia Ity16/Ity16Ank1 and +/Ity16Ank1 mice have increased susceptibility to Salmonella Typhimurium infection, although in the latter group the increased susceptibility is delayed and milder. The suppression of hepcidin (Hamp) expression and iron overload contribute to their increased susceptibility to Salmonella infection.Due to the limited amount of natural variation found in classical inbred mice, we have used N-ethyl-N-nitrosurea (ENU) given intraperitoneally. G0 males were outcrossed to 129X1 females to create G1 progeny that were subsequently crossed to DBA/2J. Resulting G2 animals were intercrossed to produce the G3 mice that were phenotyped for their susceptibility to Salmonella infection. Hamptm1Svl knock out mice Slc11a1 (129S6.B6*129S2-Hamptm1Svl).All animal experiments were performed under conditions specified by the Canadian Council on Animal Care and the animal use protocol was approved by McGill University Facility Animal Care Committee. Generation 0 (G0) 129S1 males were mutagenized with a single injection of 150 mg per kg of body weight of ty16/Ity16Ank1 and +/+Ank1 mice using 3730xl DNA Analyzer (Applied Biosystems) from the McGill University and Genome Quebec Innovation Centre.The genome scan was performed using a 1441 SNP panel  on 22 mice. Additional genotyping was performed by microsatellite analysis or restriction enzyme digests of SNP markers. Sequencing of the Ank1 gene was performed on cDNA isolated from spleens of Salmonella Typhimurium strain Keller as described by us previously 2 at day 2 and day 6 post infection and spleen, liver and kidney were removed aseptically, weighed and homogenized. Homogenates were diluted in saline and plated on trypticase soy agar (TSA) overnight. Other groups of mice were coinfected with 2500 CFUs of Salmonella Typhimurium strain Keller and 2500 CFUs of ΔtonB constructed by allelic exchange in serovar Keller Mice were infected with 5000 CFUs of Ank1 transcript (ENSMUST00000121802) and recognized the full length ANK1. The ACTIN antibody and the secondary antibodies for Western detection (anti-rabbit IgG) were purchased from Cell Signaling Technology.Erythrocyte ghost membranes were prepared by osmotic lysis as previously described . PrimarySalmonella Typhimurium and monitored for survival over a period of 14 days. Littermate controls were given 100 µl of PBS intraperitoneally.Mice were treated with 50 µg of HAMP  resuspended in 100 µl of PBS intraperitoneally. Four hours later, mice were infected with 5000 CFUs of Ity16/Ity16Ank1, +/Ity16Ank1 and +/+Ank1 mice and fixed in 10% neutral buffered formalin for 24 hours at 20°C, then placed in 70% ethanol at 4°C before processing and embedding . Embedded tissues were sectioned and stained with hematoxylin and eosin or prussian blue.Tissues were collected from Ity16/Ity16Ank1, +/Ity16Ank1 and +/+Ank1 mice were collected by cardiac puncture and analyzed for CBC & differential and reticulocyte counts . Serum was isolated with a serum separator tube (Sarstedt) and analyzed for bilirubin, blood urea nitrogen (BUN), alanine transaminase (ALT) and aspartate transaminase (AST) .Blood samples from Hamp), ferroportin (Slc40a1), Il6, Il1, Ifng, erythropoietin (Epo), heme oxygenase 1 (Hmox1) and growth differentiation factor 15 (Gdf15) expression. Two housekeeping genes: Tbp (TATA box binding protein) and Hprt (hypoxanthine guanine phosphoribosyl transferase) were used. The relative expression of the genes was normalized to the amount of Tbp and Hprt (endogenous reference) and relative to a calibrator (untreated wild type genotype) for each tissue by using the comparative 2(−Delta Delta Ct) method. The primer sequences are provided in Total RNA was extracted from spleen, liver and kidneys with Trizol Reagent  according to manufacturer instructions. cDNAs were synthesized using SuperScript®II Reverse Transcriptase (InVitrogen). Quantitative PCR was performed on a Chromo4  or StepOnePlus  using SYBR Green (Applied Biosystems) for hepcidin (Data (unless otherwise specified) was analyzed by two-tailed Mann Whitney test and two-way ANOVA using GraphPad Prism 5. Data for qPCR was analyzed in R by ANOVA and Welch two sample t-test Ity16 pedigree is shown in Salmonella infection with 5,000 CFUs and monitored for a period of 14 days. Mortality in the Ity16 pedigree was observed between day 3 post infection and continued until day 11 with 25% of mice succumbing to infection by day 6 (Ity16 G3 mice were plotted according to their genotype at the peak marker (rs32874474) on chromosome 8 (The breeding scheme used to identify the by day 6 . An initby day 6 . DNA samby day 6  that incmosome 8 . All micmosome 8 .Ity16 mice showed they have severe constitutive anemia as demonstrated by low hematocrit levels (25% in mutants compared to 50% in wildtype and heterozygous mice) which decreased to as low as 15% 2 days after infection . Total number of WBC, neutrophils and lymphocytes were significantly higher in Ity16 mutant mice compared to littermate controls before and after infection  met our criteria. ANK1 is primarily known for its structural role in erythrocytes. Ank1 is part of a small gene family which members are adaptor structural components linking lipid membranes to the cytoskeleton showing an essential role in the stability of plasma membranes of many cell types Ank1 gene comprises a total of 44 exons. Several Ank1 splice variants are observed and the full-length isoform encodes for a protein of 1907 amino acids (200−210 kDa). Erythroid Ank1 consists of three major conserved domains including an N-terminal membrane-binding domain, a spectrin-binding domain and a C-terminal regulatory domain containing a death domain motif Ity16 mutant showed a C to T transition at cDNA position 4069 (c.4069C>T) resulting in a nonsense mutation in exon 33 at amino acid position 1357 (p.Gln1357Ter ) (Ity16 mutant mice (Identification of the causative gene for 357Ter ) . This pr357Ter ) . Immunobant mice . A truncIty16/Ity16Ank1 mutant livers and kidneys (Prussian blue positive pigment) compared with wild type and heterozygous littermates in non-infected conditions and in mice at 2 days after infection (Ity16/Ity16Ank1 mutant mice suggest that the rapid turnover of RBC in these mice leads to accumulation of iron in the liver and kidneys (secondary hemochromatosis). In contrast, splenic iron content was markedly decreased in Ity16/Ity16Ank1 mutant mice compared to wild type littermates  and during infection (liver and kidney). In fact, we observed glomeruli with enlarged mesangium in Ity16/Ity16Ank1mice, a pathological change that may be compatible with membranoproliferative glomerulonephritis  in the liver of mutant mice where there is iron overload , ferroportin (Slc40a1) and heme oxygenase 1 (Hmox1) and regulators of Hamp expression  were investigated  (Gdf15)  known to be upregulated during Salmonella infection and to impact on Hamp transcription were measured (Ity16/ity16Ank1 mutant mice appeared to be underresponsive to infection-induced cytokines as measured by very little or no induction of Il1 and Il6 (Ity16/ity16Ank1 mice showed marked increases in Il1 (liver and kidneys) and Il6 (liver) mRNA expression during infection  were significantly more susceptible to infection than mice carrying one (+/−Hamp) or two (+/+Hamp) (Mantel-Cox test P = 0.0079) wild type allele at Hamp. These results confirm the importance of Hamp during acute systemic model of Salmonella Typhimurium infection.Because of the severe iron overload present in the liver and kidney of stigated  and 7. Ttermates . On the  Ank1+/+  and 7C. termates  and 7C. the mRNA  and protin (Epo)  and grow (Gdf15) . These atermates . In addimeasured . The spl and Il6 . In the nfection . In theshimurium . We showAnk1+/Ity16 heterozygous mice present an intermediate phenotype with respect to susceptibility to infection  when compared to +/+Ank1 and Ity16/Ity16Ank1 littermates (Ank1+/Ity16 mice did not present any sign of anemia (+/Ity16Ank1 mice presented moderate extramedullary hematopoiesis in the spleen (Hamp expression in the liver (Gdf15 (data not shown).termates . Clinicaf anemia  or splenf anemia . Howeverf anemia . In addie spleen  and lowehe liver  and kidnhe liver  that wasAnk1+/Ity16 mice behaved as wild type littermates for most subphenotypes we have measured including different blood parameters  test p = 0.008) compared to heterozygous mice treated with PBS confirming the importance of HAMP in the host response to Salmonella infection (Early during infection (day 2), rameters  and orgarameters . To undee kidney . At day termates . The inctes  in the gene Ank1 identified in an ENU recessive screen for susceptibility to typhoid-like disease in mice. The mutation consists in a nonsense mutation (p.Gln1357Ter) located in the spectrin binding domain of Ank1. ANK1 protein was not detected in RBC ghosts suggesting that Ity16Ank1 is a null allele although we could not exclude the possibility that low levels of other ANK1 isoforms lacking the N-terminal region may remain undetected. Mice carrying the homozygous allele for Ity16Ank1 present clinicopathological features of human hereditary spherocytosis (HS) which is the most common cause of inherited chronic hemolysis in Europe and North America with a prevalence in population of 1 in 2000 ANK1 gene accounts for about 50% of genetically defined cases of HS and most cases of HS associated with ANK1 mutations We report here the identification of a novel mutation , two additional one were identified in dominant ENU screens for blood cell phenotypes (RBC2Ank1 and E924XAnk1) and the last one, in a dominant screen for resistance to malaria in SJL/J mice (MRI23420Ank1) Ity16, nb, RBC2, E924X or MRI23420 allele at Ank1 present characteristic clinicopathological features of HS including severe anemia, reticulocytosis, splenomegaly with complete effacement of the normal splenic architecture, multiorgan iron overload and low body weight. Embryonic and neonatal lethality is observed and may vary according to the genetic background and the position of the mutation  and survive to at least 6 months of age. In mutant Ank1 mice where there is 100% neonatal mortality, the death was associated with severe clinical signs of jaundice due to massive hemolysis In addition to the mutation described in the current paper, five mutations within the reported . Two of Ity16/Ity16Ank1 mutant mice and other ENU-induced Ank1 mutants (RBC2Ank1 and E924XAnk1) was similar with hematocrit levels varying between 22–27%. Reticulocytosis was present in all mutants and significantly different from control mice. Ity16/Ity16Ank1 mutant mice exhibit extensive iron accumulation in several organs including the kidneys, a pathological observation that was not reported in other Ank1 mutants Salmonella infection because of impaired NADPH-dependent respiratory burst activity Salmonella strain deficient in iron uptake grows better in the iron rich environment of Ity16/Ity16Ank1 liver. We do also show that in heterozygous +/Ity16Ank1 mice, higher bacterial load is detected only in tissues where iron accumulation is detected. These results clearly show the importance of iron overload in susceptibility to Salmonella infection in Ity16/Ity16Ank1 and +/Ity16Ank1 mice.The severity of anemia in Ank1 mutant mice and HS patients, the normal life span of erythrocytes in the peripheral blood is substantially shortened and retention of abnormal erythrocytes by the spleen was shown to be the dominant mechanism for their reduced life-span Hmox1. HMOX1 catalyzes the degradation of heme to carbon monoxide, biliverdin that is converted to bilirubin and ferrous iron Ity16/Ity16Ank1 mutant mice, we observed high serum bilirubin levels and iron overload in the liver and the kidneys but not in the spleen. High Hmox1 mRNA levels was observed in the liver and the kidneys but not in the spleen of mutant mice suggesting that the iron overload present in the liver and kidneys resulted from degradation of heme by Hmox1. Low levels of Hmox1 in the spleen of mutant mice are most likely a consequence of splenic macrophage depletion (data not shown) and could explain the observation that there is no iron deposition in the spleen of mutant mice and less bacterial growth considering that hemophagocytic macrophages may provide a survival niche for SalmonellaHmox1 has been recently shown to impair resistance to Salmonella infection in a context of hemolysis through the suppression of the oxidative burst capacity of neutrophils Hmox1 levels in Ity16/Ity16Ank1 mutant mice may contribute to their susceptibility.In Ity16/Ity16Ank1 mutant mice, the excessive iron load in the kidneys most likely results from the high rate of hemoglobin filtration and reabsorption by renal tubular cells. Iron deposition in glomeruli, and proximal and distal tubules of the kidney has been observed in chronic experimental hemosiderosis Ity16/Ity16Ank1 mutants develop a more severe nephropathy compared to heterozygous and wild type animals and are more susceptible to infection as measured by increased in bacterial proliferation upon infection. The higher degree of iron accumulation in liver and kidneys in older animals is most likely responsible for these observations.It has been shown that the kidney plays an important role in iron metabolism. It has also been reported that a significant amount of serum iron is filtered by the glomeruli and is reabsorbed Ity16/Ity16Ank1 mutant mice is an increase in Epo and Gdf15 levels associated with suppression of Hamp expression. Hamp expression is known to be regulated by intestinal iron absorption, iron recycling by macrophages and iron mobilization from hepatic stores by inhibiting iron export through its binding to Slc40a1 causing its internalization Ity16/Ity16Ank1 mice, low Hamp expression had a modest impact on the expression of Slc40a1 in the liver. The synthesis of Hamp is also upregulated in hepatocytes by inflammatory cytokines and inhibited by anemia, hypoxia and erythropoietic activity Epo is detected in the kidneys. Despite these changes to compensate for erythrocyte demand, erythropoisesis is not efficient and led to massive expansion of the erythroid compartment. GDF15 is a member of the TGFβ superfamily that was shown to negatively regulate the expression of Hamp in vitroHAMP have been observed in β-Thalassemia and it is thought that erythroid expansion influences the regulation of HAMP expression through systemic release of GDF15 from erythroblasts Hamp in response to ineffective erythropoiesis, as observed in Ity16/Ity16Ank1 mutant mice, clearly exacerbate extramedullary erythropoiesis, tissue iron deposition and splenomegaly. During infection, liver Hamp expression levels remain suppressed and Gdf15, Il1 and Il6 expression increased significantly in Ity16/Ity16Ank1 mutant mice compared to wild type littermates. Gdf15 has been shown to be highly expressed in macrophages stimulated with LPS and to be modulated by several cytokines including IL-1 Gdf15 expression observed during infection in Ity16/Ity16Ank1 mutant mice could be explained by the progression of anemia and expansion of the erythroid compartment, by the high expression of cytokines induced by infection with Salmonella or as a consequence of intracellular iron deprivation due to low levels of Hamp expression.Another hallmark of ineffective erythropoiesis in Salmonella susceptibility in Ity16/Ity16Ank1 differs from that observed in the iron overload disorder, hereditary hemochromoatosis (HFE). Hfe-deficient mice do not lack Hamp, present excessive accumulation of iron in the liver and improve resistance to Salmonella infection Hfe-deficient mice, high lipocalin-2 (Lcn2) levels were observed in the liver and the spleen prior infection and increased resistance to infection in these mice was associated with higher induction of Lcn2 expression that was shown to reduce the availability of iron for Salmonella within macrophages Ity16/Ity16Ank1 mutant mice, the spleen and liver expression of Lcn2 did not differ from that observed in littermate controls prior infection however we did observe a significant increased in the spleen and liver expression of Lcn2 during infection (data not shown). There were no significant differences between genotypes in the spleen in contrast levels of Lcn2 were significantly higher (by a factor of 1.5 LOG) in the liver of Ity16/Ity16Ank1 mutant mice compared to littermate controls and parallel the high bacterial loads detected in this organ. Altogether, these data suggest that high levels of Lcn2 expression in a context of low Hamp expression do not protect Ity16/Ity16Ank1 mutant mice from systemic Salmonella infection.+/Ity16Ank1 mice to infection with Salmonella Typhimurium. In heterozygous mice, there is only one functional copy of Ank1 which may be limiting to confer a normal structure to the RBC membrane and cause a partial loss of membrane surface. Although the half-life of RBC appears not to be affected in +/Ity16Ank1 mice, small increase in osmotic fragility has been reported which may lead to more RBC destruction by the spleen +/Ity16Ank1 mice although the mice were not anemic. The phenotype observed in +/Ity16Ank1 mice may correspond to the human mild form of HS where patients have compensated hemolysis without anemia. As observed in uninfected Ity16/Ity16Ank1 homozygous mice, liver and kidneys Hamp levels were low compared to controls and levels of Gdf15 were increased. Hamp is mainly produced by the liver but it has been found also to be highly expressed in the apical pole of epithelial cells of distal tubules and collecting ducts of the kidney +/Ity16Ank1 mice, the Salmonella susceptible phenotype appears to be expressed predominantly in the kidney where we found significant accumulation of iron and bacteria. Low levels of Hamp expression in +/Ity16Ank1 heterozygous mice at the time of infection appears to contribute to iron accumulation in the kidney and liver and consequently favor bacterial growth. The central role of Hamp in the host response to Salmonella infection was validated using mice deficient for Hamp. These mice are not anemic and present an important iron overload secondary to low level of Hamp a phenotype similar to the one observed in heterozygous mice Salmonella infection (current paper). Finally, direct evidence of the role of Hamp in the host response to Salmonella infection was obtained by treating +/Ity16Ank1 mice with hepcidin. Hepcidin treatment did not improve resistance of Ity6/Ity16Ank1 mutant mice where iron accumulation in tissues is massive however it was clearly beneficial in +/Ity16Ank1 heterozygous mice. We do not know at the moment the exact mechanism of how hepcidin is improving resistance to infection in +/Ity16Ank1 heterozygous mice. Induction of hepcidin through transgenesis in mouse models of hemochromatosis and β-Thalassemia was shown to alter the pattern of cellular iron accumulation and limit iron overload −/− mice caused a partial redistribution of iron from the liver to the spleen Of particular interest was the intermediate susceptibility of Hamp expression and iron overload contribute to the susceptibility of Ity16/Ity16Ank1 mutant and +/Ity16Ank1 heterozygous mice to Salmonella infection. This emphasizes the importance of iron metabolism and a role for Hamp in susceptibility to systemic Salmonella infection.Overall, the current study shows that the suppression of Figure S1Histopathologic examination of the spleen, liver and kidney of 7 week old +/+ANK1 wild type, +/Ity16ANK1 heterozygous, and Ity16/Ity16ANK1 mutant mice before infection (day 0). H&E stain of uninfected spleen of wild type (A), heterozygous (E), and Ity16 mutants (I). Prussian blue stain of uninfected spleen, liver and kidney, respectively, of wild type , heterozygous , and Ity16 mutants . All pictures are taken at 200×magnification. RP = red pulp, WP = white pulp.(PDF)Click here for additional data file.Figure S2Progression of lesions in kidney and liver 2 days after Salmonella infection of +/+ANK1 wild type, +/Ity16ANK1 heterozygous, and Ity16/Ity16ANK1 mutant mice aged 7 and 24 weeks using hematoxylin & eosin staining. H&E stain of uninfected kidney of wild type , heterozygous , and Ity16 mutants  at 7 and 24 weeks of age respectively. H&E stain of day 2 post infection liver of wild type , heterozygous , and Ity16 mutants  at 7 and 24 weeks of age respectively. All pictures taken at 200×magnification.(PDF)Click here for additional data file.Figure S3Prussian blue staining of liver and kidney of 7 week old +/+ANK1 wild type and +/Ity16ANK1 heterozygous mice at day 2 and day 6 post infection with Salmonella Typhimurium. Prussian blue stain of day 2 post infection liver in wild type (A) and heterozygous (D) mice at 200×magnification. Prussian blue stain of day 6 post infection liver of wild type (B) and heterozygous (E) mice at 200×magnification. Prussian blue stain of day 6 post infection kidney of wild type (C) and heterozygous (F) mice at 1000×magnification.(PDF)Click here for additional data file.Table S1Primer sequences used for quantitative PCR. Two housekeeping genes: Tbp (TATA box binding protein) and Hprt (hypoxanthine guanine phosphoribosyl transferase) were used. Hepcidin (Hamp), ferroportin (Slc40a1), interleukin 6 (Il6), inteleukin 1 (Il1), interferon gamma (Ifng), erythropoietin (Epo), heme oxygenase 1 (Hmox1) and growth differentiation factor 15 (Gdf15).(XLSX)Click here for additional data file.


ANTIS revealed the presence of an appreciable number of sense and antisense peptide pairs within any protein molecule and those portions were designated as antisense homology boxes (AHB).Each peptide should have specific structure determined by its amino acid sequence which may react with its antisense peptide. To generate candidates of complementary peptide (C-pep) reactive to a target amino acid sequence based upon the sense-antisense amino acid relationship. We invented an evolutionary computer program (MIMETIC) that generates C-pep sequences that have a potential to interact with a target peptide .N-terminal alanine generating acetylated PepA (AcPepA).C5a anaphylatoxin is considered to be an effective target for treatment of hyperinflammation since C5a stimulates generation of tumor necrosis factor alpha (TNFα and other inflammatory cytokines. Amino acids 37 to 53 of C5a (RAARISLGPRCIKAFTE) is an antisense peptide to AHBpeptides of the C5a receptor (C5aR), and this has been designated PL37. This region of C5a is presumed to be a potential site for C5aR stimulation. Using the computer program MIMETIC, we generated 19 C-peps to PL37. One of the 7 inhibitory C-peps to PL37 which interfered with C5a function was termed PepA(ASGAPAPGPAGP- LRPMF). To improve stability, we modified PepA by acetylation of its AcPepA rescued Cynomolgus monkeys at lethal shock induced by bacterial LPS (4 mg/kg).The excellent therapeutic effect of AcPepA is due to restriction of high mobility group box 1 (HMGB1) surge induced by the effect of C5a on C5L2, which is the second C5a receptor, since the released HMGB1 has the capacity to stimulate TLR4 as an endogeneous ligand resulting in further activation of inflammatory cells to release inflammatory cytokines forming positive feedback circuit of inflammation.


Soon after introducing recombinant hepatitis B virus (HBV) vaccine, universal neonatal vaccination became the corner stone of the preventive measures for this potentially life threatening infection2]3]. B[3]. B2][. B[3]. BA level of 10 IU/L of anti hepatitis B surface antibody (Anti HBsAb) is usually considered as a protective level against future infections. Although this level was initially determined in studies about passive prophylaxis of HBV infection, the same level was arbitrary applied to active immunization though this was debated by some researchers 9][10]. [10]. 9]12]13][[13][12][[[13][12]Tosun et al.  in a pubThe coverage of complete vaccination of infants against HBV was estimated 69% in 2008 worldwide5] while while5] Some problems such as maintaining cold chain in transportation and handling of vaccine, improper injection and other technical problems in this context are still real challenges in many countries making effectiveness of vaccination, like HBV which need a stable cold chain, lower than expected in real practice 5]8]9][8][9]5][[9][8][9][8][9]5] which prThe HBV infection rate in children received vaccination after determining by Anti HBcAb positivity might be as high as 0.5% . The impAfter all we still confront with this major question: How long the protective effect of neonatal HBV vaccination persists? Is there a need for booster after complete series of vaccination? If so when this booster should be administered and to whom? Should it be universal like the neonatal vaccination itself or only be given after monitoring or given only to selected groups?Answer to these questions is not easy and there are contradictory evidences in the literature. The debate is whether the reduction in antibody levels is indicating lower immunity or not. An anamnestic response may occur when people who were previously immunized against an antigen are re-exposed to the same antigen 10]26].[26].10][.[26].10]The same group reported the rate of breakthrough infection among vaccines was less than 0.1% . Even ch


Since 2003, H5N1 avian influenza strains have become endemic in many countries in Southeast Asia, including Cambodia. In Cambodia alone there have been 26 outbreaks in poultry flocks and 10 human cases with 9 deaths. We have analyzed the genome of a large panel of H5N1 strains isolated from poultry and human between 2004-2010. Several strains have molecular alterations which are predicted to affect sensitivity to neuraminidase inhibitors (NAI), the primary drugs of choice in the treatment of H5N1 infections. In June 2009 the first H1N1 pandemic (H1N1pdm) viruses were detected in Cambodia and have since been the main influenza strains circulating during the epidemic season. The aim of this study was primarily the surveillance of oseltamivir and zanamivir drug resistance in Cambodian H5N1 and H1N1pdm isolates.in vitro assay of neuraminidase (NA) activity, which utilizes the artificial NA substrate 1,2-dioxetane derivative of sialic acid , was used to determine the concentration of drug required to inhibit 50% of NA enzyme activity (IC50).A chemiluminescence-based We have identified a small number of H5N1 outliers with reduced susceptibility to NAIs and have further characterized mutations predicted to affect drug resistance using computer modeling, and recombinant viruses containing these mutations generated by reverse genetics. We have also investigated several previously described resistance mutations in the context of the Cambodian H5N1 virus using reverse genetics. We found no evidence of NAI drug resistance in H1N1pdm viruses in Cambodia.We have monitored NAI sensitivity of H5N1 and H1N1pdm viruses in Cambodia and in general have not found NAI resistant viruses with classical resistance mutations. However, we have identified naturally occurring mutations in H5N1 viruses which reduce the sensitivity to NAIs. The ongoing surveillance and understanding of novel drug resistance mutations is of great importance in the global efforts against influenza disease and pandemics.