Lymph node ratio  is reported to be superior to the absolute number of nodes involved (pN stage) in classifying patients at high versus low risk of death following breast cancer. The added prognostic value of LNR over pN in addition to other prognostic factors has never been assessed.All patients diagnosed with lymph node positive, non-metastatic invasive breast cancer at the National University Hospital (Singapore) and University of Malaya Medical Center  between 1990–2007 were included (n = 1589). Overall survival of the patients was estimated by the Kaplan Meier method for LNR  and pN staging . Adjusted overall relative mortality risks associated with LNR and pN were calculated by Cox regression. The added prognostic value of LNR over pN was evaluated by comparing the discriminating capacity (as indicated by the c statistic) of two multivariate models, one including pN and one including LNR.LNR was superior to pN in categorizing mortality risks for women ≥60 years, those with ER negative or grade 3 tumors. In combination with other factors , substituting pN by LNR did not result in better discrimination of women at high versus low risk of death, neither for the entire cohort (c statistic 0.72 [0.70–0.75] and 0.73 [0.71–0.76] respectively for pN versus LNR), nor for the subgroups mentioned above.In combination with other prognosticators, substitution of pN by LNR did not provide any added prognostic value for South East Asian breast cancer patients. Axillary lymph node status is one of the most important prognostic factors for breast cancer et al showed that LNR, categorized as low >0 and <0.2, intermediate 0.2 to 0.65 and high risk >0.65 to 1, was better at predicting breast cancer specific mortality than pN staging Increasing evidence suggests that the Lymph Node Ratio (LNR) , is a superior prognostic indicator compared to the absolute number of nodes involved Prognostication, however, is a multivariable process, as the outcome of a disease is determined by a variety of (sometimes interacting) factors, and breast cancer is no exception. In addition to axillary lymph node status, prognosis is determined by a variety of factors, including, age, tumor size, grade, receptors status and treatment. Despite the large number of studies that have addressed LNR, not one has assessed the added prognostic value of LNR over pN in predicting overall survival after breast cancer. Via this study we aim to assess the added prognostic value of LNR over pN staging in the South East Asian setting by comparing the pN and LNR prediction models in terms of (1) predictive power, (2) discrimination and (3) net reclassification improvement of patient into appropriate risk categories of all cause mortality.Data for this study were obtained from the Singapore Malaysia Hospital-based Breast Cancer Registry The NUH breast cancer registry started in 1995 and contains information on 2,449 consecutive breast cancer patients diagnosed between 1990 and 2007. The UMMC breast cancer registry started in 1993 contains information on 3,320 patients diagnosed between 1993 and 2007. Details on both these registries are described elsewhere in situ breast cancer (N = 317) and stage IV disease (N = 535) were excluded. In total 1589 patients were included for analysis.We selected women diagnosed with non metastatic primary invasive breast cancer, with information on the number of excised and the number of positive axillary lymph nodes. Patients receiving neoadjuvant chemotherapy (N = 312), patients with a node negative (pN0) axilla (N = 2352), patients with missing information on exact number of lymph nodes involved (N = 664), with Information recorded for each patient included age at diagnosis, ethnicity , year of diagnosis, place of diagnosis , date of death or date of last contact. Tumor characteristics included tumor size , estrogen (ER) and progesterone receptor (PR) status , grade . In terms of axillary dissection, we collected information on total number of axillary nodes examined and number of positive axilary nodes. LNR was categorized into three categories including, low (>0 and <0.2), intermediate (0.2 to 0.65) and high category (>0.65 to 1) groups as previously reported Life table analysis was performed to calculate survival probabilities for the three pN categories and the three LNR categories. After testing for proportionality, we performed univariate Cox proportional hazard analysis to identify variables that were significantly associated with all cause mortality. Multivariate Cox proportional hazard analysis was applied 1) to calculate adjusted mortality risks and 2) to identify which combination of factors best predicted overall survival. For this we entered all variables univariately associated with overall survival with a p-value <0.2 into the model and used stepwise backward regression and maximum likelihood method to find the optimal fit. Internal validation of each model was done by bootstrap resampling.Two models (A and B) were constructed. Each model contained the same baseline variables, i.e., age, radiotherapy, surgery type, grade and tumor size (base model). Model A contained pN stage in addition to the base model variables while Model B contained LNR in addition to the base model variables. From the final models, adjusted Hazard Ratio for pN and LNR were derived.Base model : age, radiotherapy, surgery type, grade and tumor size.Model A: age, radiotherapy, surgery type, grade, tumor size and pN stage.Model B: age, radiotherapy, surgery type, grade, tumor size and LNR.In order to ascertain the added prognostic value of LNR over pN, we compared the discriminative capacity of model A with model B. Discrimination indicates how well the model is able to distinguish between patients who will experience the outcome (death) and those who will not. Discrimination was assessed by the Concordance (c) statistic, the interpretation of which is equivalent to the area under the receiver operating characeristic (ROC) curve, that is, a c statistic of 0.5 indicates no discrimination above chance, whereas a c statistic of 1.0 indicates perfect discrimination. Comparison of c statistics between the model including pN Stage (Model A) with the one including LNR (Model B) tells whether one model is better in discriminating between poor and good survivors, and thus superior in predicting survival. Model calibration–the agreement between predicted risks and observed mortality risks–was assessed using the Hosmer Lemeshow test by comparing the predicted survival and the observed survival at 3-year follow-up.Finally, the c statistic has been criticized for being insensitive in comparing models and for having little direct clinical relevance. Therefore, we calculated the Net Reclassification Improvement (NRI), which assesses the ability of a model including a new prognostic marker (LNR - model B) to more accurately reclassify individuals into higher or lower risk(of death) category compared to model A, i.e., to check whether model B was better at correctly reclassifying patients into high risk and low risk groups based on their predicted survival probability as compared to model A. The NRI is the difference in proportions of patients moving up and down risk categories  among patients with the event of interest (death) versus those without (in our case patients who died within 3 years of follow up versus those who survived).The NRI is similar to the percentage reclassified but distinguishes between movements in the correct direction (patients moving up the risk categories for event patients (deaths) and down for nonevent patients (survivors)) The NRI is calculated as follows:up,event  =  number of events moving up/number of events.Pdown,event  =  number of events moving down/number of events.Pup,nonevent  =  number of nonevents moving up/number of non events.Pdown,nonevent  =  number of nonevents moving down/number of non events.Pup,event - Pdown,event) – .NRI  =  .In order to estimate PAfter a recent publication suggested that LNR is particularly informative in subgroups of patients (i.e. patients with unfavorable tumor characteristics and younger patients) we performed subgroup analyses by age (<60 years and ≥60 years), receptor status (ER- vs ER+) and grade  All analyses were performed using STATA version 11.According to the LNR classification, 758 (47.7%) patients were categorized as low category (>0 and <0.2), 574 (36.1%) as intermediate category (0.2 to 0.65) and 257 (16.2%) as high category (>0.65 to 1) LNR corresponding to low, intermediate and high risk of death respectively. For classic pN staging, 879 (55.2%) were pN1, 447 (28.1%) pN2 and 263 (16.7%) pN3 . In all,Five year survival probabilities for the patients categorized by LNR were 79%, 70% and 43% for low, intermediate and high category LNR groups respectively . Five yeIn univariate Cox regression analysis, age at diagnosis, place of diagnosis, year of diagnosis, ethnicity, receptor status (ER and PR), treatment, grade, stage, tumor size, pN staging were independently and significantly associated with all cause mortality . After mBoth models A (base model plus pN) and B (base model plus LNR) were well calibrated . In terms of discriminating ability, both models performed equally well, as shown by the c statistic for model A of 0.72 (95% CI 0.70 to 0.75) and c statistic for the model B of 0.73 (95% CI 0.71 to 0.76). The substantial overlap between the two 95% confidence intervals indicated that LNR did not provide any added prognostic value when compared to pN staging in predicting all cause mortality.Based on individual predicted survival probabilities (from both pN staging and LNR models), when patients were categorized into tertiles of low, intermediate and high risk of death, the LNR model reclassified an additional 8.0% (n = 49) of patients with the event (death) into high risk groups and incorrectly reclassified 4.5% (n = 29) of the patients with the event into low risk groups. Among the patients without the event , an additional 5.6% (n = 52) of patients were reclassified into low risk groups while 5.7% (n = 53) of the patients without the event were reclassified into high risk .Subgroup analysis showed that LNR was superior to pN staging in categorizing patients’ risk of death for patients aged 60 years and above, patients with ER negative tumors and patients with high grade tumors, as in, for these subgroups, 95% confidence intervals (CIs) for intermediate and high risk LNR groups did not overlap while they did for the pN2 and pN3 categories. However, in terms of discriminating ability, models for all subgroup analyses including LNR performed as well as the models including pN respectively, as attested by the c statistics and largely overlapping 95% CIs . There wAlthough a majority of the patients (∼83%) did have at least ten lymph nodes examined, about 17% of the patients had less than 10 nodes removed during axillary dissection. We performed a subgroup analysis to assess the added prognostic value of LNR for patients with less than 10 nodes retrieved but even for this subset of patients, both pN staging and LNR predicted all cause mortality equally well (data not shown). Different cut offs for LNR were tested for the entire dataset but no new cut offs of LNR for South East Asian patients were established.This study shows that pN staging as well as the LNR are comparable in predicting overall survival of women with breast cancer, except for patients aged 60 or more, patients with ER negative tumors and patients with high grade tumors. Here, LNR was superior in categorizing patients into intermediate and high risk strata as compared to pN stage. However, in combination with other prognostic factors, LNR did not provide any additional prognostic information over pN staging, neither for the entire cohort, nor for the subgroups of older women and those with ER negative of grade 3 disease. The fact that LNR was not superior to the pN staging was seen in other Asian studies as well There are several independent but interrelated prognostic factors that predict for recurrence and survival of breast cancer patients. These include amongst others, tumor size, axillary nodal status, histopathology, steroid receptors, HER 2 status, proliferative rate, ploidy, and oncogene amplification The number of lymph nodes retrieved and examined is highly dependent on surgical expertise, the institution’s protocol and the pathologists’ experience Results from our study showed that LNR and pN were equally good at predicting all cause mortality overall but within certain subgroups , LNR was better at categorizing patients into risk categories. The intermediate category LNR was truly intermediate for these subgroups, i.e., the 95% Confidence Interval (CI) of the Hazard Ratio overlapped neither the low nor the high category LNRs, whereas the pN2 and pN3 CIs overlapped . Thus inRecent studies have indicated that full axillary clearance following a positive sentinel node biopsy does not affect survival in certain (low risk) categories of breast cancer patients We acknowledge that our study suffers from several shortcomings, including a relatively short follow up time. In addition, we assessed all cause mortality as our end point as no data on cause of death was available. This could have led to a mixing of effects as this analysis allowed for competing risks of death. Also, additional information on HER2/NEU receptor status, socioecomonic status and comorbidity could have allowed for a deeper understanding of the association.Among South East Asian breast cancer patients, both the Lymph Node Ratio and the pN staging system seem to be equally good at predicting all cause mortality based on the cut offs used for LNR in this study. LNR may be better than pN in dividing tumors into high vs low risk for certain subgroup of patients, but LNR has no added prognostic value over pN staging in addition to other prognosticators.Table S1Multivariate Cox regression analysis for all cause mortality by different subgroups.(DOCX)Click here for additional data file.


DISC1 has emerged as one of the most credible and best supported candidate genes for schizophrenia and related neuropsychiatric disorders. Furthermore, increasing evidence – both genetic and functional – indicates that many of its protein interaction partners are also involved in the development of these diseases. In this study, we applied a pooled sample 454 sequencing strategy, to explore the contribution of genetic variation in DISC1 and 10 of its interaction partners  to schizophrenia susceptibility in an isolated northern Swedish population. Mutation burden analysis of the identified variants in a population of 486 SZ patients and 514 control individuals, revealed that non-synonymous rare variants with a MAF<0.01 were significantly more present in patients compared to controls , providing further evidence for the involvement of DISC1 and some of its interaction partners in psychiatric disorders. This increased burden of rare missense variants was even more striking in a subgroup of early onset patients , highlighting the importance of studying subgroups of patients and identifying endophenotypes. Upon investigation of the potential functional effects associated with the identified missense variants, we found that ∼90% of these variants reside in intrinsically disordered protein regions. The observed increase in mutation burden in patients provides further support for the role of the DISC1 pathway in schizophrenia. Furthermore, this study presents the first evidence supporting the involvement of mutations within intrinsically disordered protein regions in the pathogenesis of psychiatric disorders. As many important biological functions depend directly on the disordered state, alteration of this disorder in key pathways may represent an intriguing new disease mechanism for schizophrenia and related neuropsychiatric diseases. Further research into this unexplored domain will be required to elucidate the role of the identified variants in schizophrenia etiology.In recent years,  With a heritability of ∼60–80% Schizophrenia (SZ) is a severe psychiatric disorder affecting ∼1% of the population worldwide. The disease is characterized by Disrupted in Schizophrenia 1 (DISC1), which was originally identified via a balanced t chromosomal translocation segregating with a wide spectrum of psychiatric disorders in a large Scottish pedigree DISC1 genetic variation in conferring susceptibility to psychiatric illness One recent and promising exception is Nevertheless, except for the translocation no specific causal variant has yet been identified, , and most compelling evidence so far has come from functional genomic and cell biological analyses, suggesting an essential role of DISC1 in neuronal development, including adult neurogenesis and signaling in vivo confirmed protein interactors Research into DISC1's multiple interaction partners has proved very valuable in elucidating its biological functions, further implicating it in essential processes of brain development and adult neuronal function At present, however, little is known on the role of genetic variation in the individual DISC1 interactors, or combinations of the different interaction partners, in the development of these disorders. Therefore, detailed studies of the various components of the DISC1 pathway will be essential to fully understand the role of this complex molecular network in conferring susceptibility to psychiatric diseases. Studying a set of candidate genes in convergent molecular risk networks – like the DISC1 pathway – is an attractive strategy, as it allows to investigate the cumulative effects of (moderately deleterious) mutations that, because of their molecular connection, could cause genetic impairment of pathway activity and thereby lead to phenotypic effects. Hereby, it is of particular importance to search for rare mutations (with a low population frequency (<1%), and a relatively high penetrance), as it has been suggested that these variants, or combinations thereof, could explain a substantial fraction of common disorders like SZ Of course, identifying rare variants requires genotyping large populations of individuals, which is costly and time-consuming, especially when one seeks to study large candidate gene sets typically involved in complex diseases. An interesting approach to minimize cost and time, is through sample pooling that in combination with massively parallel sequencing allows the identification and simultaneous quantification of (rare) variants in multiple individuals PAFAH1B1), PDE4B, NDE1, NDEL1, TRAF3IP1, YWHAE, and ZNF365) in schizophrenia patients versus control individuals. The candidate genes were first selected based on their convincingly reported interaction with DISC1. Next, both some more established/studied interactors – having (suggestive) evidence for involvement in psychiatric illness – and some new, potentially interesting candidates were included ), justifying a comparison between the patient and control sequencing data.The number of reads was homogeneously distributed over the different amplicon pools, except for multiplex reaction 12, where a lower number of reads was obtained .2 = 0.98) across a wide range of frequencies, demonstrating the accuracy of our DNA sample pooling  with their actual frequencies for each of the validated variants. The observed and predicted frequencies correlated very well , of which 61 were located in the coding and untranslated regions of the target genes. These 61 variants were further validated by SNP genotyping in the complete association sample,  (57 variants), or Sanger sequencing in the original subject population (80 patient and 80 control samples) (4 variants), resulting in a final set of 50 confirmed coding and UTR variants .Variant frequencies of the 50 confirmed variants are listed in The genotype distribution of variant PAFAH1B1 r.1250C>T (rs6628) was not in HWE, and was omitted from further statistical analyses.At the allelic level, only 1 variant, NDE1 p.Y279Y ( = rs17283846), showed a significant difference between patients and control individuals, exhibiting a higher frequency in the latter  having a MAF below 1%, and 50% (25/50) having a MAF smaller than 2%. Some of these variants were uniquely present in patients  or in controls .i.e. comprising all 49 variants in HWE) did not differ between the 486 patients and 514 control individuals genotyped, neither in the complete set of candidate genes (p = 0.75), nor on an individual gene basis . However, when the variants were stratified based on MAF and variant type, several interesting effects were observed  (N = 163), medium-onset (21 - <35) (N = 266) and late-onset (35 - <60) (N = 57) patients, and the burden of rare missense mutations (MAF <1%) was investigated in these groups. It was found that the early-onset patients had a particularly high burden of these rare missense variants: in this subset of patients, we found 12.9 mutant alleles per 100 individuals, versus 8.6 in the complete SZ sample and 4.7 in controls . This burden decreased to 7.9 mutant alleles per 100 individuals in the medium onset group , and was zero in the late-onset group .The observed increased burden of rare missense mutations in early onset patients also remains significant after Bonferroni correction .DISC1 (one donor and one acceptor site), which were predicted by both of the splice-site prediction algorithms used. Neither of these two alternative splice variants corresponded to the splice isoforms described by Nakata and co-workersFor all variants, the degree of nucleotide conservation was assessed using the GERP (Genome Evolutionary Rate Profiling) score. Missense and silent mutations were further examined for potential splicing defects. Several potential effects on splicing were predicted, with varying consistencies across the different matrices  and 5. TThe variants causing amino acid substitutions were further evaluated by analyzing evolutionary amino acid conservation using SIFT, PolyPhen and Panther. The resulting conservation scores indicated that the majority of identified amino acid substitutions in our data set had either little or no functional effect , or required more homologous sequence data in order to make reliable predictions. Two variants however, were concordantly predicted to be potentially damaging: ATF5 p.R167C  and DISC1 p.S704C ( = rs821616) . In addition, one variant (DISC1 p.L607F) ( = rs6675281) was predicted to be possibly damaging by SIFT and Panther, but scored as benign by PolyPhen .The missense mutations were also examined for putative disruptions of specific structural and functional properties using various predictors in the SNPeffect toolsuite . Single amino acid replacements can affect the protein's structure and dynamics, they can disrupt functional sites or affect the cellular processing of the protein. None of the missense variants detected in this study caused significant changes in any of the properties examined. The lack of three dimensional structures of the proteins in the DISC1 pathway, or even structural models of close homologs, hindered a detailed analysis of the influence of the mutations on protein stability.PAFAH1B1), GRB2 and YWHAE have ≥40% disordered residues  and NDE1 r.1041A>C ( = rs2075511). Interestingly, ATF5 r.871G>A is located in a potential miRNA target site, and may therefore interfere with ATF5 expression and function. However, as none of these effects survived multiple testing correction, further independent replication of these findings will be required.Six variants were found with a statistically significant frequency difference between patients and controls. These include two synonymous mutations, NDE1 Y279Y ( = rs17283846) (p = 0.025) and TRAF3IP1 T23T ( = rs13398676) (p = 0.006). Although the functional consequences of silent mutations may not be obvious, recent studies have shown that these variants might modify protein abundance, structure and/or activity via alterations in mRNA stability DISC1, none of rare variants identified here overlapped with the 5 ultra-rare cohort-specific variants previously described by Song et al. The observed scarcity of statistically significant main effects in our data set does not necessarily rule out the involvement of the DISC1 pathway in the susceptibility for SZ in our population, but may (at least partly) be attributed to the relatively high occurrence of rare variants, having frequencies too low to be able to run adequate statistical comparisons. Indeed, over 35% of all identified coding variants (18/50) have a MAF below 1%, and 50% (25/50) were present at a MAF smaller than 2%. Though the frequencies of these rare variants were not significantly different between patients and controls, they often have odds ratios higher than 1.5 (resp. lower than 0.67), and several are unique in one or the other group , 5, 6. ATo understand the potential role of the identified rare variants in SZ etiology, we evaluated the mutation burden (defined as the average number of mutations per person) in patients versus controls. Under a model in which rare mutations increase risk, we would expect to observe a greater burden in patients compared to control individuals. There was no difference in overall mutation burden between the genotyped patients and control individuals. Yet, when mutation burden was defined not simply as the total number of variants – including neutral polymorphisms – but evaluated as subgroups of variants (based on MAF and variant type), we found that schizophrenia patients were 1.85 times as likely as controls to harbor rare variants (MAF<0.01) causing amino acid substitutions (including frameshifts) , PDE4B (1 mutation), ATF5 (1 mutation), TRAF3IP1 (3 mutations) and ZNF365 (1 mutation)  . Though per base , and mayper base  section.PAFAH1B1, GRB2, nor YWHAE contained a single missense variant.In order to estimate the potential risk associated with the identified missense variants, a range of protein structural and functional properties was investigated. Rather unexpectedly, we found that none of the 22 identified missense variants caused any significant effect on the various properties examined. This absence led us to the observation that 8 of 11 proteins under study showed a remarkably high occurrence of intrinsically disordered regions (IDRs). Indeed, all proteins except LIS1, GRB2 and YWHAE were found to have ≥40% of disordered residues by DisProt analysis , panel AIDRs are segments of proteins that do not definitively fold and remain flexible and unordered. These proteins take up different structures upon binding to different targets, and thereby exhibit functional flexibility ATF5 R167C, DISC1 L607F ( = rs6675281) and S704C ( = rs821616). Interestingly, two of these variants (DISC1 S704C and L607F) were recently reported to have an actual functional effect DISC1 L607F and S704C corresponds to already known biological consequences greatly underlines the value of our in silico predictions, also for other, unknown variants. This is especially interesting as to ATF5 R167C, which was also predicted to be damaging, but not previously reported. This variant corresponds to a novel, rare mutation, having an odds ratio of 2.6 (95% CI: 0.50–13.46). Further studies of this variant are warranted to clarify its relation to disease.In this study, ∼90% of all identified missense variants (including the rare mutations underlying the increased burden in patients versus controls) are located in an IDR. Interestingly, some of the (rare) variants indentified in this study fall into known binding regions on one or more of the interactors . E.g., ADISC1) and, importantly, these genes' functional network.To our knowledge, this study is the first describing a comprehensive resequencing analysis of the DISC1 pathway in schizophrenia. Our results provide support for a model of SZ pathogenesis that includes the effects of multiple rare variants, residing in different vulnerable genes, which may in turn be functionally linked into pathways and networks. This model is consistent with the theory presented by Eyre-Walker in silico prediction programs, as a majority of the proteins encoded by our candidate genes contain large regions of intrinsically disordered residues. Though amino acid conservation analysis may provide a first hint of potential functional effects, it does not tell the whole story, as disorder-based signaling is a complex process, depending on multiple factors including alterations in protein context, alternative splicing and post-translational modifications in se, hopefully encouraging further research into this complex area, and providing new clues to our understanding of the complex etiology of SZ and other (psychiatric) disorders. Indeed, as an increasing amount of evidence is beginning to emerge that many important biological functions depend directly on the disordered state, alteration of this disorder may play a crucial role in the pathogenicity of many complex diseases (including SZ), thereby adding another level of complexity to the study of their molecular mechanism, and providing exciting new perspectives for future research.Assigning potential functional significance to identified variants is a major challenge in genomics research. In this work, a wide array of functional properties was examined to predict possible deleterious effects of the variants. Using these tools, we were able to predict several potential effects on splicing and miRNA target motifs. Yet, alterations of protein structure or function were hard to track down using standard DSM-IV criteria for SZ In a first phase of the study we used DNA of 80 SZ patients  and 80 control individuals  for 454 sequencing based variant discovery. These individuals were selected on the basis of their early age at disease-onset (18.55±3.36), from a larger association sample consisting of 486 unrelated SZ patients  and 514 unrelated control individuals . All originated from a geographically isolated population living in the county of Västerbotten in Northern Sweden. They were all Caucasians and none were of Finnish, Norwegian or Lappish descent. All patients fulfilled the http://www.betula.su.se/en/) The control population had a mean age of 58.0 (±13.0) years at inclusion. They originated from the same geographical area as the patients and were randomly selected from the Betula study, described in detail elsewhere . No population substructure was observed in the association sample (data not shown).The patient-control sample was controlled for population stratification by the genotyping of 37 microsatellite (STR) markers via the use of standard genotyping and scoring methods. Statistical tests for population stratification were performed using the program STRUCTURE , each comprising 40 DNA samples. Hereto, an equal amount of each sample  was combined, and the resulting DNA pool was adjusted to a final concentration of 10 ng/µL.To control the efficiency of DNA pooling, the relative abundance of 3 SNP alleles was measured by pyrosequencing and compared to the allele frequencies of the individual samples constituting the pools (Supporting ATF5) amplified the correct fragment (conversion rate  = 98.7%). The two failed ATF5 amplicons were omitted from further experiments. Multiplex PCR reactions were performed for each DNA pool and 1 individual patient DNA sample, also contained in one of the patient pools . The target sequence was covered by 155 amplicons with an average length of ∼221 bp, resulting in ∼34 kb of sequence. The 155 amplicons were amplified in 12 multiplex PCR reactions. Simplex PCR reactions of the amplicons showed that all except two of the primer pairs , and the concentration of the eluates measured using a Nanodrop spectrophotometer . Finally, for each DNA pool , the 12 purified multiplex PCR products were mixed, taking into account the concentration of each multiplex reaction and its number of constituent amplicons, to obtain an equal representation of every amplicon in the final PCR mixtures.The final mix of 155 amplicons of each sample was sequenced using the standard amplicon sequencing protocol on a 454 GS-FLX genome sequencer (Roche Applied Science) according to the manufacturer's instructions. For each of the 4 pools, 1 lane of a 2-lane Bead Loading gasket on a 70×75 mm PicoTiterPlate was loaded, and sequenced from both directions. The individual DNA sample was sequenced using 1 lane of a 16-lane Bead Loading gasket.The generated standard 454 flow files were analyzed using NovoSNP 4.0 (beta), an in-house developed software program for the identification of variants in resequencing experiments. In short, NovoSNP 4.0 uses the quality and height of the flow at the variation position and the neighboring flows for SNP identification. Further, it takes into account the number and ratio of reads showing the variant – thereby allowing for the analysis of pooled sequencing data – and favors variants seen in both directions. Finally, the program creates a database of all identified variants, for which the flows can be visually inspected .All variants with a frequency ≥0.8% were examined, allowing for a secure cutoff level to detect singleton variants, which theoretically have a frequency of 1.25% in a pool of 40 samples.Finally, all potential variants (except 4) were validated using iPLEX SNP genotyping in the complete association sample . For technical reasons, 4 variants were genotyped by traditional Sanger-based sequencing in the original subject population (80 patient and 80 control samples) (Supporting http://pngu.mgh.harvard.edu/purcell/plink/) was used to calculate genotype deviation from Hardy-Weinberg equilibrium (HWE), by an exact test gPlink version 2.050 only for variant discovery, inclusion of an equally large control sample in the discovery phase, does not lead to an inflation of type I errors Differences in mutation burden  between patients and controls were assessed by two-sided t-tests, using SPSS version 16.0.2 . The data were thereby stratified according to type  and MAF . Empirical p-values were generated using the max(T) permutation approach, based on 100000 replicates. The level of significance for all statistical tests was 0.05. When correcting for multiple testing, Bonferroni corrective measures were taken to control false positive rates. All association analyses were performed on the complete sample (i.e. including the discovery samples). Contrary to the use of patient samples in silico analyses, including assessment of nucleotide and amino acid conservation, effects on potential splice sites and cis-acting elements, potential disruption of miRNA target sites and predicted transcription factor binding sites, and alterations of functional and structural properties of the proteins. Finally, sequences where also examined for intrinsically disordered regions using DisProt (http://www.ist.temple.edu/disprot/Predictors.html). A detailed description of the applied methods is provided in Supporting To investigate potential deleterious effects associated with the identified variants, each variant was subjected to a battery of Figure S1Boxplot showing the distribution of the number of reads per amplicon in each multiplex PCR reaction, for control and patient pools. The observed read count is uniformly distributed across the different amplicon pools (with an average of 1362 reads/amplicon), except for multiplex reaction 12. °: outliers  *: extreme outliers .(PDF)Click here for additional data file.Figure S2Allele frequencies estimated from pooled DNA samples (as determined by GS-FLX sequencing) versus the actual frequencies  in the different pools. °: outlier .(PDF)Click here for additional data file.Figure S3Overview of the known interaction domains between the different proteins investigated, along with the positions of the variants identified in this study. Protein lengths are given between brackets. Binding sites between two proteins are indicated along the line connecting them, with the binding site(s) on a certain protein closest to that protein . The identified missense mutations are shown in a white area within the proteins' oval. Rare missense mutations (MAF ≤1%) are underlined, and mutations located in one of the binding sites are shown in italic. Note that the positions of many of the binding sites were not described/found in literature (indicated with ‘?’).(PDF)Click here for additional data file.Figure S4Schematic representation of the overall domain architecture of each of the proteins investigated, highlighting the regions of predicted disorder (orange), along with regions having known homologous domains (blue). On each protein, the identified missense mutations are indicated. Mutations lying outside a disordered region are marked with an extra line.(PDF)Click here for additional data file.Table S1DISC1 interaction partners included in the study, and evidence for their involvement in psychiatric disease.(PDF)Click here for additional data file.Table S2Allele frequencies of pooled DNA and individual samples, as determined by pyrosequencing.(PDF)Click here for additional data file.Table S3Properties of amplicon subset selected for false negative rate estimation using Sanger sequencing.(PDF)Click here for additional data file.Table S4Mutation burden of identified rare nonsynonymous mutations (MAF<1%), stratified by gene.(PDF)Click here for additional data file.Text S1Supplementary Materials and Methods, Results and Discussion.(PDF)Click here for additional data file.


Several indices have been proposed to assess disease activity in patients with Systemic Lupus Erythematosus (SLE). Recent studies have showed a prevalence of flare between 28–35.3%, persistently active disease (PAD) between 46%–52% and serologically active clinically quiescent (SACQ) disease ranging from 6 to 15%. Our goal was to evaluate the flare, PAD and SACQ rate incidence in a cohort of SLE patients over a 2-year follow-up.We evaluated 394 SLE patients. Flare was defined as an increase in SLEDAI-2K score of ≥4 from the previous visit; PAD was defined as a SLEDAI-2K score of ≥4, on >2 consecutive visits; SACQ was defined as at least a 2-year period without clinical activity and with persistent serologic activity.Among the 95 patients eligible for the analysis in 2009, 7 (7.3%) had ≥1 flare episode, whereas 9 (9.4%) had PAD. Similarly, among the 118 patients selected for the analysis in 2010, 6 (5%) had ≥1 flare episode, whereas 16 (13.5%) had PAD. Only 1/45 patient (2.2%) showed SACQ during the follow-up.We showed a low incidence of flare, PAD and SACQ in Italian SLE patients compared with previous studies which could be partly explained by ethnic differences. Monitoring of disease activity is an important aspect in the management of patients affected by Systemic Lupus Erythematosus (SLE) as was recently pointed out in a core-set of recommendations proposed by the European League Against Rheumatism (EULAR) intention to treat the flare Flare is one of the most commonly used outcome measures in the core-set of indices evaluated in clinical trials on SLE. By using the existing disease activity indices, several definitions of flare have been proposed. Thus, a critical question is how to best define SLE flare. One of the most used was proposed by Gladman and colleagues in 2000 More recently, Nikpour and colleagues underlined that such definition of flare does not capture patients who have a disease course characterized by periods of persistently active disease (PAD), defined as a SLEDAI-2K score ≥4, excluding serology alone, on ≥2 consecutive visits “Serologically active clinically quiescent” (SACQ) disease was proposed as another outcome measure. This index identifies patients clinically quiescent despite persistent serologic activity, and appears to have a prevalence of 6–15% in SLE patients Thus, our goal was to evaluate the incidence of flare, PAD, and SACQ in a cohort of Italian SLE patients over a two-year follow-up.Policlinico Umberto I”, Rome, approved the study. At each visit, the patients underwent a complete physical examination, the clinical and laboratory data were collected in a standardized, computerized, and electronically-filled form, which included demographics, past medical history with date of diagnosis, co-morbidities, previous and concomitant treatments.SLE patients referred to the Lupus Clinic of the Rheumatology Unit, Sapienza University of Rome (Sapienza Lupus Cohort) were enrolled in a prospective study. SLE diagnosis was performed according to the revised 1997 American College of Rheumatology (ACR) criteria Disease activity was assessed with the SLEDAI-2K, while chronic damage was measured with the Systemic Lupus International Collaborating Clinics/ACR Damage Index score (SLICC) Prospectively collected data were used to determine the incidence of flares and PAD in 2009 and 2010. According to Nikpour and colleagues, flare was considered as an increase in SLEDAI-2K score of ≥4 from the previous visit with a minimum interval of 2 months between visits; PAD as a SLEDAI-2K score ≥4, excluding serology alone, on ≥2 consecutive visits, with a minimum interval of 2 months between visits SACQ was defined as a period of at least two years without clinical activity but with persistent serologic activity .The patients evaluated for flare and PAD did not have any restriction concerning medications given. The patients evaluated for SACQ were taking only anti-malarials, while those receiving corticosteroids or immunosuppressive medications were excluded We used version 13.0 of the SPSS statistical package. Normally distributed variables were summarized using the mean ± SD, and non-normally distributed variables by the median and range. Wilcoxon’s matched pairs test and paired t-test were performed accordingly. Univariate comparisons between nominal variables were calculated using chi-square test or Fisher-test where appropriate. Two-tailed P values were reported, P values less than 0.05 were considered significant.Ninety-five and one-hundred eighteen SLE patients were eligible for the study in 2009 and 2010, respectively, as they underwent at least two visits per year. The main clinical and laboratory parameters are reported in The mean ± SD time interval between visits used to diagnose ﬂare and PAD in 2009 and 2010 was 3.6±0.6 months and 3.5±0.6 months, respectively.Among the 95 patients selected for the analysis in 2009, 7 (7.3%) had 1 flare episode (only 1/7 showed 2 flare episodes), whereas 9 (9.4%) had PAD. Similarly, among the 118 patients selected for the analysis in 2010, 6 (5%) had 1 flare episode (none more than 1 episode), whereas 16 (13.5%) had PAD.In 2009, 1 patient showed both flare and PAD. One patient had flare in 2009 and in 2010, while 5 patients showed PAD in both years.versus 128.4±84.6 months, P = 0.02 in 2009; 188.4±100.08 versus 135.8±89.5 months, P = 0.03 in 2010).Notably, 5/7 patients (71.4%) in 2009 and 5/6 (83.4%) in 2010 of the patients who experienced flares were not on immunosuppressive drugs. The patients who experienced flares showed a significantly longer disease duration compared with those who did not experience flares in both years of observation .The clinical characteristics of the patients with PAD and the involved organ/systems are reported in The occurrence of flare was associated with a history of nervous system involvement . The logistic regression analysis confirmed such association (P = 0.008). The occurrence of PAD was associated with a history of arthritis, renal and nervous system involvement . The logistic regression analysis confirmed the association only with nervous system involvement (P = 0.01).Forty-five patients were eligible for the evaluation of SACQ. Only 1 patient (2.2%) had SACQ during the two-year follow-up. The patient (D.G.) was a 37-year-old female, with a disease duration of 144 months, who was taking hydroxychloroquine and, in the absence of clinical manifestations, showed persistent complement reduction and elevated anti-dsDNA antibodies (titer ≥1∶40).In this prospective study, we showed that, in a large cohort of Italian patients affected by SLE, flares and PAD are relatively infrequent conditions. In fact, in 2009 flares were observed in 7% and PAD in 9.4% of our patients, while in 2010 flares were observed in 5% and PAD in 13.5% of our patients, respectively.SLE is a prototype of systemic autoimmune diseases, characterized by heterogeneity of clinical features and presence of a wide autoantibodies profile. The great heterogeneity of SLE determined a still open debate, concerning the possibility that SLE is a single disease with varied phenotypes or a similar phenotype shared by a variety of different diseases with diverse pathogenic mechanisms SLE is characterized from heterogeneous degrees of severity as well as unpredictable disease flares and remissions. Several experimental trials are in progress to evaluate new biologic drugs to treat patients affected by SLE. However, the remitting and relapsing course of the disease and the heterogeneity of SLE features make the design and the interpretation of clinical trials difficult. Quantification of disease activity is mandatory to identify patients eligible to participate in clinical trials and, thereafter, to establish the efficacy of the drug examined.Data published in the literature suggested that the disease activity in SLE patients could be evaluated by using laboratory markers and/or global indices. Conventional biomarkers for the assessment of disease activity include anti-dsDNA antibodies and serum complement levels. However, the “classical” markers of activity are not specific and accurate in differentiating between disease flares and other concomitant conditions, such as infections. Recent research has provided data about new potential biomarkers to guide clinical decision-making in the management of SLE patients Disease activity indices are helpful in the routine assessment of SLE patients, as suggested by recent EULAR recommendations for monitoring patients with SLE Several indices have been applied to evaluate disease activity, such as SLEDAI, European Consensus Lupus Activity Measurement (ECLAM) and British Isles Lupus Assessment Group index (BILAG) Recent randomized controlled trials for new biologic treatment in patients affected by SLE used a new composite assessment, called SLE Responder Index (SRI), that combines the SELENA-SLEDAI, BILAG and Physician Global Assessment (PGA). A responder according to the SRI is defined as having ≥4 point reduction from baseline in SELENA-SLEDAI score and no new BILAG A score and no more than one new BILAG B organ domain score compared with baseline and no worsening in PGA. When all three criteria are met, the patient is a responder at that time point according to the SRI Several definitions of flare were proposed and used in clinical trials and observational studies. Nevertheless, how to define the best SLE flare is still an open and critical question. Global scoring systems, such as the SLEDAI, are easy to perform and widely standardized. They are used to define flare; nonetheless they only take a snapshot of the patient rather than indicating the trend of the disease.Thus, Nikpour et al. proposed a new outcome measure, the so-called “persistently active disease” (PAD), to define those patients not in remission but without flare. They addressed that these patients require the most frequent monitoring We have already reported a preliminary study on 63 SLE patients who were referred to the Sapienza Lupus Clinic followed during 1 year of follow-up (September 2008–September 2009) in which we observed a lower incidence of flare and PAD compared with the Canadian cohort  Occurrence of flares and PAD in our SLE cohort was associated with longer disease duration. Moreover, most patients who experienced flares were not taking an immunosuppressive drug. Thus, longer disease duration and the absence of an immunosuppressive treatment should be considered risk factors for the worsening of disease activity.In the logistic regression analysis NPSLE involvement was associated with both flare and PAD. This is in agreement with the definition of flare, as well as of PAD, probably due to the fact that in the SLEDAI, used in the formulation of both indices, neuropsychiatric manifestations account for the highest scores.SACQ is another important outcome measure that was first suggested by Gladman et al. in 1979. They described a subset of patients who had persistent serologic activity (elevated anti-dsDNA antibody levels and/or hypocomplementemia) despite clinical quiescence Our study showed a low incidence of flares, PAD and SACQ in Italian SLE patients compared with previous studies where the results could be only partly explained by ethnic differences. This may suggest that definition of disease activity is critical for SLE management, and that timing for immunosuppressive treatment suspension should be carefully evaluated. In this view, it is very important to improve, select and use indices of outcome in SLE in order to better assess and treat patients. Flares, PAD and SACQ could be considered useful parameters of clinical evaluation in SLE patients in monitoring disease progression and response to treatment.


R2 > 0.75), and suggests that compensatory plant responses to grazing are an important means of how herbivores might maintain or increase SOC in tropical grasslands.The effects of grazing on soil organic carbon (SOC) dynamics, particularly in the tropics, are still poorly understood. Plant compensation to grazing, whereby plants maintain leaf area (C input capacity) despite consumption  by grazers, has been demonstrated in tropical grasslands but its influence on SOC is largely unexplored. Here, the effect of grazing on plant leaf area index (LAI) was measured in a field experiment in Serengeti National Park, Tanzania. LAI changed little for grazing intensities up to 70%. The response curve of LAI versus grazing intensity was used in a mass balance model, called SNAP, of SOC dynamics based on previous data from the Serengeti. The model predicted SOC to increase at intermediate grazing intensity, but then to decline rapidly at the highest grazing intensities. The SNAP model predictions were compared with observed SOC stocks in the 24 grazed plots of a 10-year grazing exclosure experiment at eight sites across the park that varied in mean annual rainfall, soil texture, grazing intensity and plant lignin and cellulose. The model predicted current SOC stocks very well ( Soil organic carbon (SOC) in grasslands and savannas represents one of the largest reservoirs of carbon on earth , both positive and negative, depending on soil type, precipitation, plant species composition and grazing intensity . These rOf these three important features, the consequence of plant compensation for soil carbon is probably the least well understood. Leaf area index (leaf area/area sampled), or LAI, has been shown to decrease in response to grazing in some contexts but not others . StudiesThe response of LAI to herbivory may be a key driver of soil carbon response to grazing. Leaf area is highly correlated with gross photosynthesis and net carbon (C) assimilation . MaintenThe goal of this paper is to show how leaf area index (LAI), or leaf area/area sampled, and plant allocation to stem and leaf changes with grazing intensity in a tropical savanna and then to explore how such possible compensatory responses might affect soil carbon dynamics. Hence, this paper (1) presents the results of a 2-year grazing exclosure experiment in Serengeti National Park to measure effects of varying grazing intensity on plant LAI and proportion of biomass allocated to stems vs. leaves, (2) develops a model of soil carbon dynamics based on extensive past research in the Serengeti as a framework for evaluating the impact of LAI response to grazing, and (3) compares predictions of this model with observed soil carbon stocks across the eight sites of a 9-year grazing exclosure experiment in which key model input parameters were independently measured.The model of carbon dynamics, called SNAP  accounts explicitly for fixed carbon, as determined by LAI, soil texture and rainfall, and its allocation to roots versus shoots, and its fate: decomposition, consumption by herbivores, combustion in fire or residence in a long-term recalcitrant SOC pool. The model has five input variables: mean annual rainfall, grazing intensity, fire frequency, aboveground proportion of cellulose + lignin, and soil texture (percent sand), based on the suggested most important fates of stable carbon in the Serengeti ecosystem (R2) and tests for differences in slope from a value of 1 and an intercept value of zero. This analysis provides an explicit and quantitative test of the hypothesis that plant compensatory responses are important in the influence of herbivores on soil carbon.To evaluate the role of plant compensation, a sensitivity analysis of model parameters, including grazing intensity, in the SNAP model is conducted. The model also is used to make deductive predictions of current soil carbon stocks under the assumption of a mass balance steady-state resulting from the persistence of mean rainfall, plant lignin and cellulose, soil texture, fire history, and grazing intensity conditions over an extended period of prior years. These predictions are then compared with observed soil carbon measured in grazed plots of the 9-year exclosure experiment using linear regression, with tests for overall model fit (This approach applies a deductive  rather tConnochaetes taurinus), zebra (Equus burchelli), and Thomson’s gazelles (Gazella thomsoni), plus multiple species of resident herbivores >10 kg in size, all distributed heterogeneously (2 (a mean density of 120/km2) , Tanzania. SNP contains nearly 3 million migratory wildebeest (120/km2) . VegetatAcacia tortilis savanna woodland, with dominant grasses Themeda triandra and Digitaria macroblephara, and hosts resident herds of impala (Aepyceros melampus), buffalo (Syncerus caffer), and Thomson’s gazelles (Gazella thomsoni), among others, and is routinely visited by the annual wildebeest and zebra migration. Plant responses to grazing were measured inside and outside of grazing exclosures established 2 years previously in a 10 ha open grassland with less than 5% tree cover near the Serengeti Wildlife Research Centre (2-year exclosure experiment). The proportion of biomass as stem versus leaf was measured for all plants clipped from six randomly selected 15 × 15 cm quadrats, three inside and three outside each of twelve exclosures of various size (10–200 m2) erected between 2009 and 2010 to protect experimental gardens or nutrient addition experiments. LAI, proportion of stem, and aboveground biomass were measured in the unused “buffer zone” just inside each fence and then in quadrats 4 m perpendicular to each fence paired with each quadrat inside the fence. The objective was to encounter different localized grazing intensities that resulted from sampling beneath thorny shrubs or other plant protections versus patches with highly preferred forage species.Plant compensation to grazing was assessed near the Serengeti Wildlife Research Centre near Seronera (34° 50′ E and 2° 25′ S) inside the Park. The area is open Indigofera volkensii, were ubiquitous and reasonably abundant (>2% cover) at all our study sites. Our eight sites included two on the treeless shortgrass plains of southeastern SNP, and the other six sites were representative of woody savannas broadly distributed across SNP  grazing exclosure experiment established by Sam McNaughton in 1999  was usedross SNP .2 leaf∕cm2 ground area) was assessed. Clipped plants for all plant species in a quadrat were taken back to the lab and, while fresh, laid out and traced on graph paper, and then leaves were clipped from stems and stored, dried at 45°C, and weighed separately (as with field biomass). The proportion of leaf for each quadrat was calculated, along with LAI  divided by quadrat area (cm2)).The effect of grazing on plant (grass) allocation to leaf versus stem and on leaf area index, LAI  varies locally and was estimated in both the 2-year and 9-year experiments as the fractional difference in standing aboveground biomass (AGB): GI = 1 − AGBg∕ABGug . GI is ag∕ABGug . AGB wasg∕ABGug , yieldin2SO4), and then in 72% sulfuric acid, followed finally by ashing in a muffle furnace at 500°C for 24 h. Lignin and cellulose fractions were inferred by the mass lost between the remainder following neutral detergent digestion and the remainder after ashing, expressed as a proportion of the original sample mass.To measure lignin pluscellulose fraction (LIGCELL), dried plant material from the entire clipped quadrat was ground through a 0.9 mm (40 mesh) screen in a Wiley mill and then subjected to a sulfuric acid hydrolysis method  were determined from field measurements of soil moisture in the long-term experiment (unpublished data generously provided by Sam McNaughton). Five cm diameter soil cores were taken to a depth of 10 cm each month from December 1999 to June 2002  at each of three grazed plots at the eight sites of the long-term Serengeti grazing experiment. Moisture content was determined gravimetrically and monthly rainfall was measured from rain gauges mounted on 1 m tall fence posts at each site. A recent review  suggeststi soils  (Cosby e3). Both SOC and nitrogen contents were determined with the Walkley-Black method at the Soils Analysis Laboratory at Sokoine University of Agriculture in Morogoro, Tanzania. Reported error was ±0.004 g/gC. Soil composition  was determined by a micropipette method. Soil carbon density was measured by multiplying SOC content by bulk density (g/m2) to 40 cm depth of the pooled cores in each plot. A 40 cm depth was used, as this typically corresponds to the depth of >90% of roots , aboveground production, belowground production, and soil microbial respiration of carbon. For sources and units of literature data, see The flow of fixed carbon through hypothetical tropical grassland was described according to four criteria. First, mass balance was assumed, so that all fixed carbon resided in pools, such as biomass or SOC, or suffered the fate of being combusted, consumed, or respired. Second, this flow was described in the minimum number of fluxes and pools necessary to account for the fate of carbon . Third, The model was based on four major assumptions. First, tropical grasses potentially exhibit compensatory responses to defoliation that maintain similar leaf area, and thus photosynthetic capacity, across a broad range of grazing intensities . Second,2 to 40 cm depth) measured in 2009 in the grazed plots in the long-term grazing experiment represented SOCeq under the reasonable assumption that conditions in these plots had been approximately similar in terms of wildlife use, plant species composition, and climate since 1979 (The SOC (g/m0 − B1 exp(−B2 GI), where Bi are fitted coefficients. Linear regression in SPSS was used to build regression relationships from literature data  was tested with linear regression in SPSS version 19 . Non-linear relationships were also explored, and based on the appearance of the data, the R script “nls” was used with a function Bure data  and to ceq for each site and each paired grazed and ungrazed plot in the 9-year exclosure experiment.The SNAP soil carbon dynamic model was constructed from a number of empirical relationships that contain parameters  estimated with error. A Monte Carlo analysis (P < 0.001) with increasing grazing intensity . However a non-linear function, LAI = B0 − B1 exp(B2 GI) fit the data much better  and visibly matched better the consistently high LAI found at low to intermediate grazing intensity and the dramatic decline in LAI above a grazing intensity of 70%  negative association with increasing sand content (P = 0.0015) decline in association with higher rainfall (P = 0.006) with mean annual rainfall across the eight long-term grazing experimental sites (P < 0.001) associated with existing SOC stocks across the Serengeti landscape  in the long-term grazing exclosures against the logarithms of soil N (ln(SoilN) and mean annual rainfall (ln(RAIN)) revealed that ln(RAIN) was a highly significant factor, but ln(Soil N) was not (P = 0.45). This outcome suggests that N is a far less important factor influencing productivity in the Serengeti than is rainfall. In addition, soil N or soil C:N was not a significant variable explaining residual variation in microbial respiration in Serengeti soils following regression if SOC was included in a regression model . Finally, the influence of other variables that are often correlated with higher N, such as finer-textured soils \docurements  (4)\docuPL∕0.6 in PL = 0.6, the y-intercept value in t generally declines with increasing rainfall  were accounted for as a function of the number of days in which soil moisture exceeds a threshold for microbial activity, which was determined to be 10% (see Methods). The proportion of days during the typical 240 day wet season in which microbes would be active (WETDAYS) was estimated from the average annual rain fall from the regression in Dung-derived carbon was assumed to be the lignin and cellulose fraction of biomass consumed by grazers. All stable C in dung was assumed to be incorporated in soil because of the activities of termites and dung beetles, which often incorporate dung into soil within 2–5 days , allowinpiration , as detechange in sequestered carbon (ΔSOCt) can be estimated by adding the plant derived SOC (PDSOCt) to the dung derived SOC (DDSOCt) and then subtracting the carbon lost through microbial maintenance respiration (MRESPt) as follows: ΔSOCt = 0, the above equation can be solved for the SOCt term in MRESPt to yield an equilibrium SOCeq. These equations account for all the major inputs and losses of stable C into soil. The eq allows for a relatively straightforward analysis of how different factors influence soil carbon dynamics. It can predict ΔSOCt from knowledge of some initial soil carbon stock The equation for SOCeq increased with higher plant lignin and cellulose content, but decreased with increasing rainfall and soil sand content  to 10% changes in parameter values  shows theq of around 250–400 g∕m2 (around 4%) and an overall uncertainty of around 14% (2) observed across the range of conditions in the Serengeti.The Monte Carlo analysis yielded consistently small standard errors in estimated SOCound 14% . These found 14%  and suggeq was compared to observed SOC (SOCobs) in two ways. First, the average predicted SOCeq and SOCobs were compared at the site level (N = 8), i.e., averaged over all three plots at a site, which reflects that RAIN, FIRE, and SAND% were the same for all three plots. Secondly, to test whether accounting for local variation in grazing intensity and lignin and cellulose (eq and SOCobs were compared at the plot level (N = 24). At the site level, average SOCeq at a site strongly predicted the mean SOC among grazed plots at that site (obs = 0.817 (± 0.12 s.e.m.) SOCeq + 858.1 (± 1178.3 s.e.m.). The slope was not significantly different from 1 and the intercept was not different from zero . Likewise, at the plot level, SOCeq for a plot strongly predicted the mean SOC for that plot (obs = 0.77 (± 0.11 s.e.m.) SOCeq + 1122.1 (± 1101.3 s.e.m.). However, even though the intercept was not different from zero the slope was significantly different from 1 and . Both slopes were less than 1, which suggests that the model may over-predict SOC under conditions that sustain the highest SOC . Very few of the microbial respiration rates measured by Predicted SOCellulose  affectedhat site : SOCobs hat plot : SOCobs The strong correlation with observed carbon stocks  suggestseq eq to decrease with increasing rainfall appears counterintuitive but results from the decline in root biomass and productivity with increasing rainfall that was observed in the one available tropical grassland study  dynamics plays a key role in driving grazer effects on soil carbon dynamics  may haveAnother key factor that is important in most other soil carbon models is temperature, and it perhaps surprising that the model performed well without a response to temperature. In tropical systems like Serengeti, soil temperatures vary from 23°C in the wet season when soils are cooled by evaporation to 26°C in the dry season. This low temperature range may be typical for tropical grasslands . It is pThe mass-balance (SNAP) model developed here contrasts in several ways from current standard models of grazing impacts on SOC, such as CENTURY . First, The simplified model and tests presented here suggests that tracking just the stable carbon pool, in this case lignin and cellulose, successfully predicted variation in SOC stocks across the Serengeti landscape . This isWork derived from the Serengeti, which is a unique ecosystem with the majority of herbivore biomass in migratory large mammal grazers (wildebeest and zebra), is sometimes viewed as inapplicable to other more common grazing systems, or even other ecosystems in Africa. While the Serengeti certainly does not exactly mimic most livestock-dominated grazing systems, it would be unfair to discount the applicability of Serengeti data and functional relationships to other ecosystems. First, the Serengeti and its history of grazing research offer the only such data for tropical grasslands for some measures . Secondly, from Compensation to grazing by plants in Serengeti National Park occurred across a wide range of grazing intensities, maintaining LAI at the expense of stem biomass up to grazing intensities near 70%. Incorporation of this LAI response into a simplified model of soil carbon dynamics, developed with data from the extensive research history in Serengeti, suggests that plant compensation can allow grazers to increase SOC stocks at intermediate grazing intensities and that this influence is stronger at higher fire frequencies. The model performed very well in predicting current SOC stocks at both a plot and site level. The success of the model indicates the potential utility of tracking the fate of stable carbon  rather than focusing on fluxes in understanding SOC dynamics.The predictions of the model, which would likely apply mainly to tropical grasslands, suggest that two factors commonly under management control, grazing and fire, can have large impacts on soil carbon stocks. In particular, there may be potential for land users to (1) reduce fire in lightly grazed, fire-prone areas of Africa and Australia , and (2)


The ubiquitin/proteasome pathway plays a vital role in plant development. But the effects of proteasome malfunction on root growth, and the mechanism underlying this involvement remains unclear. In the present study, the effects of proteasome inhibitors on Arabidopsis root growth were studied through the analysis of the root length, and meristem size and cell length in maturation zone using FM4–64, and cell-division potential using GFP fusion cyclin B, and accumulation of ubiquitinated proteins using immunofluorescence labeling, and autophagy activity using LysoTracker and MDC. The results indicated that lower concentration of proteasome inhibitors promoted root growth, whereas higher concentration of inhibitors had the opposite effects. The accumulation of cyclin B was linked to MG132-induced decline in meristem size, indicating that proteasome malfunction prevented cell division. Besides, MG132-induced accumulation of the ubiquitinated proteins was associated with the increasing fluorescence signal of LysoTracker and MDC in the elongation zone, revealing a link between the activation of autophagy and proteasome malfunction. These results suggest that weak proteasome malfunction activates moderate autophagy and promotes cell elongation, which compensates the inhibitor-induced reduction of cell division, resulting in long roots. Whereas strong proteasome malfunction induces severe autophagy and disturbs cell elongation, resulting in short roots. Root is a very important organ growing downward into the soil to anchor the plant as well as take up water and mineral ions. Root growth is determined by the meristem cell division and subsequently cell elongation-differentiation shr root elongation However, the data available at present appears insufficient to provide complete knowledge of the functions of the UPP during root development. For example, analysis of several proteasome mutations with different degree in proteasome malfunction indicated that the majority of the UPP mutants have shorter root To extend our knowledge of the involvement of the UPP in root growth, we provide here several lines of evidence about effects of the peptide aldehyde proteasome inhibitor on Arabidopsis root growth, providing further insights into the mechanism by which the UPP controls plant growth.−1) and LysoTracker Green (1 mM) were prepared using dimethylsulfoxide (DMSO) as solvent. E-64 (20 mM) and MDC (25 mM) were dissolved in water.MG132, MG115, E-64, and Dansylcadaverine (MDC) were purchased from Sigma. LysoTracker Green and FM4–64 were purchased from Invitrogen. The stock solutions of MG132 (40 mM), MG115 (40 mM), FM4–64  were cold-pretreated at 4°C for 48 h, and cultured in 1/2 MS with 1% Suc for 24 h, and then uniform seedlings of similar size and primary root length were transferred to the same fresh medium containing different proteasome inhibitors MG132, MG115, and Cys-protease inhibitor E-64, for additional 48 h. Each proteasome inhibitors were used at concentrations of 0, 20, 40, and 80 µM. DMSO controls were set up by adding the similar amount of DMSO solvent.Surface sterilized seeds of Images of seedlings were acquired with a Zeiss Stemi SV11 stereoscopic microscope (Germany), equipped with an AxioCam HRc digital camera, and the lengths of the primary roots were measured using AxioVision Rel 4.8. The experiment was repeated thrice with consistent results. The values were expressed as the means ± SD for 15 seedlings, of each treatment.−1) for 2 min, and then visualized using 40× objective of a Zeiss 5 live confocal microscope equipped with an automated microscope stage and a 561 nm laser (Germany). Images were recorded and analyzed with the Zeiss Zen2007 software.Roots of both control and treated were stained with FM4–64 , and then examined under the ZEISS 5 live laser scanning confocal microscope (Germany), with a 488 nm laser for GFP, and a 561 nm laser for FM 4–64. To make the results comparable, laser power and channel setting for GFP were kept identical for all samples.Arabidopsis with GFP-fusion cyclin B (kindly provided by associated Prof. Bai from CNU) was treated with various concentrations of MG 132. All seedlings were stained with FM 4–64 (2.5 µg ml−1) for 2 min. All samples were visualized under the confocal microscope, with a 405 nm laser for MDC, with a 488 nm laser for LysoTracker –Green, and with a 561 nm laser for FM 4–64. To make the results comparable, laser power and channel setting for MDC and LysoTracker-Green were kept identical for all samples.LysoTracker or MDC staining were performed according to earlier studies One-way ANOVA was used to compare the difference between the treated and control pollen tubes. Values of P<0.05 were taken as statistically significant.To assess the roles of the UPP during root growth, seeds of Arabidopsis were cultured under standard conditions for 24 h, and then seedlings with homologous root length were treated with 0, 20, 40 or 80 µM MG132 for additional 48 h, respectively. Microscopic analysis revealed that MG132 treatment has various effects on Arabidopsis root growth in a dose-dependent manner. The seedlings cultured under control conditions for 72 h showed normal morphology with an average length of 4011±785 µm . In contGiven that tripeptide aldehydes can also inhibit calpains as well as proteasome Root growth is sustained by the root meristem Besides, FM staining also revealed that MG132 treatment is concomitant with a more rapid cell differentiation in the elongation zone . Given tThe final size of an organ depends on the number and volume of its cells In the present study, we also found that MG132 treatment is concomitant with strongly cytoplasmic vacuolization in the elongation zone (Data not shown). Given that most substrate proteins are usually ubiquitinated before being degraded by the proteasome Previous reports on animal system have indicated a crosstalk between the UPP and lysosome/vacuole-dependent autophagy Though the ability of LysoTracker to label autophagy in plants has been demonstrated in various organisms including Arabidopsis Application of proteasome inhibitors is one of the main strategies to study the role of the UPP during plant development. In the present study, we utilized MG132 and MG115, two widely used proteasome inhibitors in both plant and animal systems, to determine the effects of proteasome malfunction on primary root development. The obtained results indicated that different concentration of MG132 or MG115 had diverse effects on root growth . Neverthshr roots Previous reports indicated that weak loss of proteasome function displayed enlarged plant organs, including cotyledons, leaves, petals, shoots and matured seeds, The rate of root growth is determined by the rate of the cell division in the meristematic zone and cell differentiation in the elongation zone rpt 2a and smaller in rpn10 and rpn12aThe final sizes of determinate organs are believed to be controlled not only by cell number and size, but also by compensation, a still largely unclear mechanism that leads to the enlargement in cell size in an organ with reduced cell number Cell enlargement is often associated with endoreplication and/or vacuolization expansion The lysosomal/vacuole dependent autophagy is another main protein degradation system characterized by the formation of autophagic vacuoles and fusing with lysosomes/vacuole to form autophagolysosomes, where proteins are degraded More interestingly, our data indicated that lower concentration of MG132 induced moderate activation of autophagy, associated with promoted cell elongation; while higher concentration of MG132 induced vigorous activation of autophagy, concomitant with disrupted cell elongation. Yet, we still cannot explain the precise mechanism leading to this phenomenon. The most probable explanation is that slight activation of autophagy was capable of protecting the cells through degrading damaged organelles and misfolded proteins, while vigorous activation of autophagy was harmful to cells due to an excessive elimination In summary, our investigation revealed that inhibition of proteasome not only reduced the cell division, but also induced autophagic vacuolization in a dose-dependent manner, resulting in promoted or prevented root elongation. Our data provides providing a further insight into the mechanism by which proteolysis controls root growth.


However, we favor the view that the chronic systemic and brain dysregulated inflammation and the diffuse vascular damage contribute to the establishment of neurodegenerative processes with childhood clinical manifestations. Friend turns Foe early; therefore, implementation of neuroprotective measures to ameliorate or stop the inflammatory and neurodegenerative processes is warranted in exposed children. Epidemiological, cognitive, structural, and functional neuroimaging and mechanistic studies into the association between air pollution exposures and the development of neuroinflammation and neurodegeneration in children are of pressing importance for public health.Chronic exposure to particulate matter air pollution is known to cause inflammation leading to respiratory- and cardiovascular-related sickness and death. Mexico City Metropolitan Area children exhibit an early brain imbalance in genes involved in oxidative stress, inflammation, and innate and adaptive immune responses. Early dysregulated neuroinflammation, brain microvascular damage, production of potent vasoconstrictors, and perturbations in the integrity of the neurovascular unit likely contribute to progressive neurodegenerative processes. The accumulation of misfolded proteins coincides with the anatomical distribution observed in the early stages of both Alzheimer's and Parkinson's diseases. We contend misfolding of hyperphosphorylated tau (HP Air pollution is a significant health problem in megacities around the world –3. In a clinically healthy children and young adults and establish the links with the current mainstream concepts of neurodegeneration. This principled problem thus addresses the relation between neuroinflammation, neurodegeneration, and air pollution exposures with an emphasis on compensatory responses. Dealing with this problem invites the development of linking hypotheses between the domains and the need for intervention, issues addressed in the third part of the paper. Recent works have shed new light on the etiology of Alzheimer's and Parkinson's diseases (AD and PD), with a growing body of evidence that oxidative stress and neuroinflammation are at the core of their etiopathogenesis and that there is a close interplay between environmental factors and neurodegeneration –8. We alμm; PM10), fine particles , and ultrafine particles . Fine and ultrafine PM are of particular interest given their capability to reach the brain [Although there is significant air pollution associated with ozone in MCMA, in this work, we will focus on particulate matter (PM) broadly defined by the diameter of the aerodynamic particles and classified into coarse particles  achieved during the past fifteen years, MCMA residents remain exposed to concentrations of airborne pollutants exceeding current ambient air quality standards for PM and ozone [2.5 as well as significant levels of PM10 associated with lipopolysaccharides (PM-LPS) have been registered historically in Mexico City's air, and marked regional differences in the air pollutants concentrations and composition have been reported within MCMA [Even with the substantial reductions in the concentrations of some criteria pollutants . PM10 concentrations had shown a clear reduction up to 2007; however, concentrations have been slowly back on the rise in the last 5 years. PM2.5 data from the monitoring network [μg/m3. MCMA residents are also exposed to UFPM from ambient air and workplaces. These nano sized PM include combustion sources  and manufactured or engineered nanoparticles (NPs). It is not widely appreciated that nano-sized materials are also present in many consumer products to which large segments of the population are exposed  [CMA 1995–11. PM10 issions) , 22.In this massive exposure chamber, 8 million children and teens <18 y are receiving the impact of the involuntary exposure to the polluted air. credible pathological mechanisms have been elucidated that lend biological plausibility to  findings” [It is important to emphasize that PM exposure has been epidemiologically associated to a wide spectrum of cardiovascular, pulmonary, and CNS effects , 23–25. indings” . Two mecindings” . These pindings” . 2.5 and outdoor exposure hours [α, interleukin-6 (IL-6), and IL-1β, which in turn recruit and increase the activity of other immune cells [Systemic inflammation with increased concentrations of proinflammatory cytokines, chemokines, and potent vasoconstrictors . The concentrations of inflammatory mediators and ET-1 correlate positively with cumulative exposures to PMre hours . Chronicre hours –33. Contre hours . Activatre hours , 34, 35.ne cells , 34–36.γ) [Altered immune responses include significant decreases in the numbers of natural killer cells and increased numbers of mCD14+ monocytes and CD8+ cells. The reduction in the number of NK cells goes along with the low concentrations of interferon gamma (IFN-γ) . MCMA chγ) . MCMA chγ) , 32, 38.γ) . Moreoveγ) –41. Evenγ) . The priγ) , 35. 2.5 [Pulmonary changes in MCMA children living in tobacco free homes include bilateral hyperinflation and increased linear markings observed in chest radiographs and mild bronchial wall thickening, prominent central airways, air trapping and pulmonary nodules identified by computed tomography scans. Abnormal lung function tests based on predicted values are seen in 7.8% of MCMA children. Higher concentrations of endothelin-1 correlate with elevations of mean pulmonary artery pressure, average hours per day spent outdoors, and 7 day cumulative concentrations of fine PM2.5 , 27. β, TNF-α, IL-10, and CD14, and a left ventricle difference in TNF-α, and IL-10 in South versus North Mexico City residents, a key point in relation to the marked difference in pollutant profiles determined by the residence MCMA location [Cardiovascular effects include a significant right ventricle upregulation of IL-1location . The pediatric studies from our laboratory cited in this work were performed in Mexico City clinically healthy children with no known risk factors for pulmonary, cardiovascular, and CNS pathology or cognitive deficits. MCMA children are selected from nonsmoking families and their results compared to age, gender, and socioeconomic status (SES) matched children residing in low polluted places. The detrimental nonCNS effects associated to residency in MC include the following.MCMA children with no known risk factors for neurological or cognitive disorders exhibit significant deficits in a combination of fluid and crystallized cognition tasks versus control children . Fifty-sWhen we analyzed the WMH lesions in relation to the profile of cytokines and chemokines , MCMA WMThese findings suggest a complex modulation of cytokines and chemokines influencing children's white matter hyperintensities, volumetric white matter responses and cognitive outcomes as a result of environmental pollution exposures. In 2002, we published a dog study pointing to the nasal cavity as a major portal of entry of xenobiotics to the brain . The stuβ, and CD14 in supra, and infratentorial regions and cranial nerves including: olfactory bulb, frontal cortex, substantia nigrae, and the vagus nerve [A very critical component of air pollution exposure is neuroinflammation , 50–52. us nerve . The entry of activated lymphocytes, mast cells, and macrophages into the brain parenchyma is a hallmark of chronic inflammatory processes , 53–56. There was extensive vascular damage in the olfactory bulb and in the frontal cortex. In the prefrontal cortex, the vascular damage affects predominantly white matter . The maiUltrafine particles are likely players in the endothelial cell activation and are found in various CNS regions, including the Olfactory bulb . UFPM arA growing body of epidemiologic and experimental data point to particulate matter components of air pollution as well as nanoparticles in the environment as risk factors for neurodegenerative diseases , 58–63. β42) frontal  versus a low polluted city (n = 17) [α-synuclein, activated microglia, extracellular neuromelanin, and pigment-laden macrophages are seen from the dorsal motor nucleus of the vagus level  .  Figure(n = 17) . The patus level  to the sus level . There i∼90% of early onset cases [The olfactory bulb pathology deserves special attention because large segments of the world population are exposed to a myriad of toxic substances on a daily basis that have the potential for harming the olfactory system and penetrating the brain via the olfactory epithelium (OE) –71. Extret cases –76. β42 was present in young children regardless of APOE genotype  . In one genotype . The basgenotype . In contgenotype . The chagenotype . The earβ aggregation and clearance in the brain, effects on synaptic plasticity, cell signaling, lipid transport and metabolism, and neuroinflammation [β metabolism and toxicity. The APOE 4 genotype (in contrast to APOE 3) is associated with oxidative stress and chronic inflammation [β metabolism and toxicity and increased oxidative stress and chronic inflammation, we have shown MCMA APOE4 carriers have greater hyperphosphorylated tau and diffuse Aβ plaques versus E3 carriers  [The Apolipoprotein E (APOE) 4 polymorphism influences aging and age-related diseases including the risk for Alzheimer's disease –80. The ammation . APOE reammation . In trauammation . APOE4 a= 0.005) . This ob= 0.005) . This inκB, TNF, IFN, and TLRs are likely players in the significant cognitive deficits observed in children with no risk factors for neurological or cognitive deficits, other than their residency in a highly polluted megacity [In our pediatric studies, the early clinical olfactory deficits appear to be associated with the presence of misfolded proteins, reactive gliosis and vascular damage in the olfactory bulb and the frontal cortex . There imegacity , 33, 77.megacity , 35, 67.What is the role of PM in the neuroinflammatory process described in highly exposed children? What is the relationship between clinical and electrophysiological changes and the described neuropathology? How to interpret the neuropathology hallmarks of AD and PD in a 10 year old child with no family history of neurological diseases? In view of the cognitive, olfactory, auditory, vestibular, and volumetric white matter changes described in exposed children, a series of critical questions arise: 2.5 consists of organic compounds including biologic components from bacteria and fungi, and transition metals with neurotoxic properties [2.5.Let us begin with the issue of particulate matter: Mexico City residents have been chronically exposed to concentrations of particulate matter above the USA standards for the last 26 years , 13, 16.operties –19. Envimicrobial invaders” could translate into signaling pathways that culminate in the transcriptional regulation of immune responsive genes and how the activation of inflammasomes [β to engage innate immune defenses [β into an active form. We have repeatedly shown IL-1β in frontal cortex, olfactory bulb, hippocampus, and the dorsal vagal complex is upregulated in highly exposed children, dogs and mice compared to low pollution controls [pathogens that do not really exist , and neuroinflammation. This is of particular importance as neuroprotective strategies are being explored. Why is PM important for MCMA children? Because fine and ultrafine particles reach their brain by uptake through olfactory neurons and cranial nerves, trafficking of macrophage-like cells loaded with PM from the lung capillary bed to the systemic circulation, and by a direct transfer of ultrafine particles from the systemic circulation and/or red blood cells to brain endothelial cells , 35. Ourmmasomes  could bedefenses . Indeed,controls , 35, 87.P = 0.01). The olfactory bulb neuropathology associated with urban exposures is very similar to the one described in early stages of AD and PD [The relationship between clinical and electrophysiological changes and the described neuropathology is of deep interest to pediatricians working in polluted urban centers. We mentioned olfaction deficits and abnormal UPSIT (University of Pennsylvania Smell Identification Test) scores present in 35.5% of the MCMA teens versus 12% of age matched controls . MoreoveD and PD –96. β amyloid and alpha synuclein in key olfactory nuclei [The central delayed brainstem auditory evoked potentials (BAEPs), auditory impairment and vestibular dysfunction could relate to the extensive brainstem inflammation with accumulation of y nuclei . Neurodey nuclei  are simiy nuclei , 91, 96.β diffuse plaques in the absence of cognitive and brain MRI data in the demised children. However, we have shown a strong relationship between residency, brain structural changes and cognitive deficits [It is difficult to establish the association between cognitive deficits, frontal tau hyperphosphorylation, and amyloid-deficits . MCMA chdeficits . WMH in deficits –106. WMHdeficits . Disruptdeficits . The chadeficits .A difficult question to answer is how to interpret the early neuropathology hallmarks of Alzheimer's and Parkinson's diseases in children with no family history of neurological diseases. In the Alzheimer's brain, tau is abnormally hyperphosphorylated and it is aggregated into paired helical filaments forming neurofibrillary tangles, a histopathological hallmark of the disease . Tau phoin vivo multiphoton imaging in living tau transgenic mice. Caspase activation precedes tangle formation by hours to days, tangles form quickly but persist apparently indefinitely, thus cleavage of tau is enough to cause misfolding of tau followed by nucleation and recruitment of additional tau molecules to the neuronal cell body. Is our description of HPτ in MCMA children's brains an isolated observation in the literature? The answer is no, Braak and Del Tredeci [τ using AT8 in 38/42 cases with no extracellular amyloid β protein deposition or neuritic plaques with the 4G8 antibody. Although these subjects were not healthy, there was no APOE genotyping or a recorded history of environmental exposures, we fully agree with Braak and Del Tredeci [Is tau phosphorylation in children detrimental or protective in the setting of severe air pollution? –114. The Tredeci  examined Tredeci  that theτ and if indeed represents a compensatory neuronal response against oxidative stress. At this time, however, we are of the opinion that given the factors  potentially accounting for the aggregation of tau, tau phosphorylation could represent an early sensor of oxidative stress with all the subsequent detrimental effects if the exposure persist.There are very few arguments about the role of abnormal tau hyperphosphorylation in AD, related tauopathies and under experimental conditions , 114–118β42 is capable of aggregation and misfolding leading to progressive neurodegeneration that develops insidiously over a lifetime. A key issue has to be addressed in this scenario: APOE4 carriers not only have HPτ, but also exhibit significant numbers of Aβ 6E10 diffuse plaques (P = 0.005) in comparison to APOE 3 carriers. Recent work by Cerf et al. [β oligomers, the pathological species responsible for AD; thus we suggest APOE4 carriers are potentially at a higher risk of developing AD if residing in a highly polluted environment. This information is critical given that ∼18% of the MCMA population carries an APOE 4 allele [Likewise, Af et al.  suggests4 allele . α-synuclein confer resistance to oxidative stress downstream of free radical production and scavenging [α-synuclein directly activates microglia inducing the production and release of the proinflammatory cytokine, TNF-α, and increasing antioxidant enzyme expression [Alpha-synuclein aggregation is associated to the pathogenesis of Parkinson's disease and exposure to a myriad of environmental agents, including agrochemicals increases the PD risk , 121. Miavenging . Recent pression . Béraud pression .α-synuclein in MCMA children is present in key regions associated with PD pathology including olfactory bulb, the midbrain, and the lower sections of the brainstem, for example, the medulla oblongata [α-synuclein- and HPπ-related olfactory bulb pathology in young highly exposed children is an ominous sign possibly associated with a number of other nonmotor symptoms related to PD, such as dysautonomia and sleep disturbances. Epidemiological studies addressing nonmotor PD symptoms in highly exposed young urbanites are warranted.It is important to note that blongata , 77. MCMblongata , 74 giveblongata . There iblongata , 74. Damblongata , 74. TheDespite controversy regarding the mechanistic pathways involved in the CNS damage associated with exposure to air pollutants, specifically fine and ultrafine particles of diverse origin, animal models and tissue culture studies have greatly improved our understanding of the mechanistic processes , 69–71. MCMA children experience a chronic, intense state of oxidative stress resulting from lifelong exposures to a severely polluted environment. Children exhibit an early brain imbalance in genes involved in oxidative stress, inflammation, innate and adaptive immune responses, cell proliferation and apoptosis. Neuroinflammation, endothelial activation, endothelial cell hyperplasia, the attachment of white blood cells to the endothelial damaged walls with the reduction of the lumen vessel, high blood concentrations of endothelin-1, and the breakdown of the BBB clearly contribute to cognitive impairment and pathogenesis and pathophysiology of neurodegenerative states , 126. EnThe neuronal accumulation of misfolded proteins in exposed children coincides with the anatomical distribution observed in the early stages of both AD and PD with early clinical evidence of olfactory and cognitive deficits, brain volumetric changes, white matter hyperintense lesions, altered brainstem evoked auditory potentials and autonomic disbalance. There is a complex modulation of cytokines and chemokines influencing structural and volumetric brain responses and cognitive deficits.We contend that misfolding of critical proteins could be a defensive early response to the sustained systemic and CNS inflammation. However, the sustained oxidative stress associated with dysregulated inflammation, both systemic and in the CNS contribute to the establishment of neurodegenerative processes with clinical early counterparts. We strongly support the contention that the nasal , cardiorespiratory and gastrointestinal (vagus) pathways-along with the systemic direct transport of particles to the brain and the dysregulated systemic inflammation are critical in explaining the brain pathology in highly exposed MCMA children. Moreover, these children are at risk of developing Alzheimer's and Parkinson's diseases as adults. We have a 50-year window of opportunity between the early brain changes observed in children and the time when the patient with mild cognitive impairment or dementia will show up at the neurologist's door. Facing the current pediatric clinical and pathology evidence is imperative if we are aiming our efforts to identify and mitigate environmental factors that influence AD and PD pathogenesis.One thing is clear: early implementation of neuroprotective measures to ameliorate or stop the inflammatory and neurodegenerative processes in children is warranted , 87. Ide2.5 above the 2006 standards  [It is important to remember there is a severe and woeful deficit of progress in the development of both AD and PD-modifying therapy , 128. Si5 μg/m3) . An appeEpidemiological, cognitive, and mechanistic studies into the association between air pollution exposures and the development of CNS damage in children are of pressing importance for public health and quality of life.