Sc), which result from the conformational conversion of physiological prion proteins (PrPC). PrPC are variable post-translationally modified glycoproteins, which exist as full length and as aminoterminally truncated glycosylated proteins and which exhibit differential detergent solubility. This implicates the presence of heterogeneous phenotypes, which overlap as protein complexes at the same molecular masses. Although the biological function of PrPC is still enigmatic, evidence reveals that PrPC exhibits metal-binding properties, which result in structural changes and decreased solubility. In this study, we analyzed the yield of PrPC metal binding affiliated with low solubility and changes in protein banding patterns. By implementing a high-speed centrifugation step, the interaction of zinc ions with PrPC was shown to generate large quantities of proteins with low solubility, consisting mainly of full-length glycosylated PrPC; whereas unglycosylated PrPC remained in the supernatants as well as truncated glycosylated proteins which lack of octarepeat sequence necessary for metal binding. This effect was considerably lower when PrPC interacted with copper ions; the presence of other metals tested exhibited no effect under these conditions. The binding of zinc and copper to PrPC demonstrated differentially soluble protein yields within distinct PrPC subtypes. PrPC–Zn2+-interaction may provide a means to differentiate glycosylated and unglycosylated subtypes and offers detailed analysis of metal-bound and metal-free protein conversion assays.Prion diseases are characterized biochemically by protein aggregation of infectious prion isoforms (PrP Sc. PrPSc originates from a host encoded prion protein (PrPC) by conformational conversion. As the mechanism of the folding of PrPSc is not yet clear, the conversion is associated with dramatic changes in biochemical and biophysical properties. PrPC is sensitive to proteolysis, is completely hydrolysed, and has high α-helix content [Sc demonstrates an increase of β-sheet structures [C is post-translationally modified by the formation of a glycophosphatidyl-inositol (GPI) anchor and a disulphide bond. Glycans attach to one or two asparagine residues yielding in di-, mono- and unglycosylated proteins [Prion diseases are fatal neurodegenerative disorders characterized clinically by a long incubation period followed by a rapid course of disease, and biochemically by the accumulation of the infectious prion protein PrP content ; whereasructures  leading proteins .C proteins are characterized by heterogeneous phenotypes in distinct brain regions and display several subtypes, which overlap distinct protein patterns identified by means of differential detergent solubility [C is known to be a metalloprotein having the capability of binding multiple zinc and copper ions that stimulate the endocytosis of PrP. Additionally, the protein is thought to be associated with metal-dependent enzymatic functions and with copper homeostasis [C can be truncated at the aminoterminus under physiological conditions generating a glycosylated C1 fragment which is present in brains in substantial amounts [PrPlubility –5. The dlubility –9. PrPC eostasis –15. A hieostasis . For eaceostasis –18. Botheostasis –20. Addi amounts .C proteins. It is not known whether the modifications, the impact of metal binding or both are essential for the development of prion diseases. In comparison to the incidence of various existing PrPC types expressed in normal tissue and brain, very few PrPSc types have been identified in diseased species. This suggests that different PrPC isoforms may vary in their potential for conformational conversion. To reduce the conversion efficiency to PrPSc, it is important to first identify and target PrPC subtypes with a high-yield conversion. In this study, we performed metal-binding analyses on phenotypes of heterogeneous brain PrPC isoforms derived from uninfected humans, bovine, sheep and mice in order to identify protein subtypes with either high or low solubility. Our results reveal that PrPC markedly exhibited a lower solubility when zinc was bound to the protein, whereas copper binding showed little effect on solubility. Differential solubility as a result of metal binding offers a new tool to examine PrPC isoforms bound to protein complexes.The post-translational modifications themselves and the structural changes due to metal ion interactions increase the variability of PrPMonoclonal anti-PrP antibodies (mabs) SAF34 and SAF70 were prepared by immunizing knock-out mice with formic acid-denatured, hamster scrapie-associated fibrils (263K) . Mab SAFBrain tissue was obtained from TSE-free humans, sheep, bovine and mice. Human brain tissue derived from five individuals was pooled from several unspecified sample regions and comprised mostly of cortex and cerebellum. All experiments on human subjects were conducted in accordance with the Declaration of Helsinki and all procedures were carried out with the adequate understanding and written consent of the subjects. Brain tissue samples were obtained from the Brain Bank, Institute of Neuropathology at the University Hospital Münster . ConsentBrain tissues were weighed and suspended in nine volumes of Tris buffered saline  in order to prepare 10% (w/v) homogenates followed by the application of 2% N-octyl-β-D-glucopyranoside (OGP). Electric homogenizers were used for consistent homogenization followed by intensive ultrasonication as described . Samples2, ZnCl2, MnCl2, MgCl2, NiSO4, CaCl2, and CoCl2 , each at a concentration of 1 mM or otherwise indicated, were added to the samples and incubated at room temperature for 30–60 min. To separate cellular prion protein fractions of high and low solubility, homogenates were centrifuged at 16,000 × g for 10 min resulting in supernatants and pellets. Pellets were washed with TBS to remove residual supernatant proteins and were resuspended in SDS-loading buffer.Divalent cations such as CuClC, samples were incubated with the metal chelator ethylene diamine tetraacetic acid  or with sodium dodecylsulphate (SDS) in indicated concentrations at room temperature for 30 min, followed by centrifugation as described.To demonstrate the metal binding to PrPThe enzyme bromelain was purchased from Sigma-Aldrich  as a chromatographically purified protease from pineapple stem. Protein suspensions from brain were treated enzymatically with bromelain at 37°C with gentle agitation using a concentration of 50 µg/ml for 60 min. Proteolysis was terminated by heating at 99°C for 10 min.C was accomplished by incubating the membranes with the antibodies indicated at room temperature over night. Membranes were washed with TBST prior to incubation with peroxidase-conjugated goat anti-mouse immunoglobulin antibodies  as secondary antibody for 2 h at room temperature. After washing with TBST PrPC bands were visualized using a chemiluminescence enhancement kit .Samples resuspended in SDS-loading buffer were denatured at 99°C for 5 min. Proteins were separated using sodium dodecyl sulphate polyacrylamide (13%) gel electrophoresis (SDS-PAGE) in a mini slab gel apparatus . After electrotransfer onto polyvinylidene difluoride (PVDF) membranes  using a semi-dry blotting system , non- specific binding was blocked on membranes by incubation in TBS containing 0.05% Tween 20 (TBST) and 1% (w/v) non-fat dry milk powder for 1 h at room temperature. Specific detection of PrPC induced a considerable shift of proteins into a PrPC isoform of low solubility retrievable in the pellet after high-speed centrifugation , which is non-ionic, and does not denature proteins. After centrifugation, the highly soluble proteins in the supernatants were exposed to various metal ions prior to incubation. Although we exclusively used proteins with high solubility, the binding of zinc to PrPfugation . The incC was incubated with increasing concentrations of the zinc ion . In contrast, low PrPC signals were observed for the full-length glycosylated isoforms. In the pellets, however, high signals were detected for the full-length diglycosylated PrPC band; these intensified as Zn2+ concentrations were increased. This result indicated that binding of Zn2+ to PrPC is directly correlated to the solubility reduction of the full-length diglycosylated form of PrPC. This differential solubility effect as a result of Zn2+-PrPC interaction was not observed with Cu2+ and Mg2+ under the same conditions and specificity was demonstrated by using the neuron specific enolase (NSE) as a control protein, which remained highly soluble within the range of Zn2+concentration used ..C-Zn2+ wC bound to Zn2+ were detected in the pellet fraction, whereas glycosylated C1 fragment and unglycosylated PrPC was abundant in the supernatant  and sodium dodecylsulphate (SDS) . High solubility of zinc and copper-bound PrPC was also induced by treatment with the protein denaturing detergent sodium dodecylsulphate (SDS).The effect of PrPte (SDS) . Mouse aC-Zn2+ phenotype is the result of metal binding to the octarepeats, we partially truncated the full-length protein by enzymatic treatment with bromelain obtained from Ananas comosus. The amino-terminal region was cleaved off, whereas a stable core protein consisting of glycosylated and nonglycosylated isoforms was generated (C from changing to low solubility. This result indicates an interaction of Zn2+ with the N-terminal region of PrPC.Metal ions bind within the octarepeats at histidine residues with high affinity, whereas zinc binds with lower affinity ;24. To venerated . RemovalC following combined and serial incubations with copper and zinc ions (2+-PrPC-phenotype predominated, even following simultaneous incubation with both copper and zinc ions in identical concentrations (C—were abundant in the pellet fraction when using mab SAF34 as a detection antibody. The full-length diglycosylated PrPC from wild-type mice and the full-length monoglycosylated derived from the mutant T182N mice showed high signal intensity in the pellet, whereas the truncated glycosylated C1 fragment and the unglycosylated wild-type PrPC isoforms dominated the supernatant when using mab SAF70. This Zn2+-PrPC-phenotype persisted as well in samples that had been treated serially with different metal ions (C was observed in the pellets after Zn2+ ions had been independently added to the proteins. Collectively, zinc binding to PrPC had an important influence on decreasing the solubility of PrPC, whereas copper binding failed to neutralize the Zn2+-PrPC-phenotype. This suggests an additional metal-protein interaction and a different biochemical characteristic independent of the copper-PrPC interactions.In the next series of experiments, we analysed the solubility of PrPinc ions . The Zn2trations . The thrtal ions . Using tC remain an enigma, it is known that PrPC interacts with several metal ions such as Cu2+, Zn2+, Mn2+, Pd2+, Pt2+ and Co2+ when sufficient concentrations are present [2+ and Cu2+ are accompanied by structural changes and decreased solubility [C isoforms using high-speed centrifugation in conjunction with metal binding. The interaction of zinc with PrPC induced an important shift from high to low protein solubility, mainly affecting the full-length glycosylated isoforms. Although both metals bound to the amino-terminal region, copper binding did not reveal the structural changes affiliated with low solubility under these conditions. Copper binds to histidine residues via the octarepeats and to single sites outside this region with high efficiency with a Kd of 6.7 to 14 µM [d was determined as approximately 200 µM [Although the biological functions of PrP present –30; thes present . The binlubility –20. Howeto 14 µM ;24;32–33y 200 µM .2+ acts as a bridge between PrP proteins [2+ having a greater influence on these interactions than Cu2+. This Zn2+ induced PrP-PrP interaction may have decreased protein solubility concurrent with the level of Zn2+ bound PrP that we observed in the pellet fraction. Copper ions seem to be unable to stimulate PrP interactions with the efficiency of Zn2+.The interaction between PrP proteins seems to be mediated by copper and zinc binding within the amino-terminal region of PrP , which cproteins –36. Suchproteins , with ZnC differentiation based on metal binding provides a technique capable of distinguishing PrPC isoforms bound within protein complexes. This may also enable the separation of full-length glycosylated and full-length nonglycosylated as well as truncated glycosylated isoforms, due to the decreased solubility of Zn2+ bound full-length glycosylated PrPC in comparison to truncated PrP. Zn2+ selectively bound to full-length PrPC but not to truncated and nonglycosylated PrPC attributed to lack of the octarepeat sequence in the C1 fragment. As various PrP isoforms coexist in homogenized solutions, the separation of metal-bound and unbound PrPC proteins based on different conformation and aggregation types can easily be accomplished by incorporating a high-speed centrifugation step.In conclusion, PrP


Recombination is a common feature of retroviral biology and one of the most important factors responsible for generating viral diversity at both the intra-host and the population levels. However, relatively little is known about rates and molecular processes of recombination for retroviruses other than HIV, including important model viruses such as feline immunodeficiency virus (FIV).env gene sequences (n = 355) isolated from 43 naturally infected cats. We demonstrated that recombination is abundant in natural FIV infection, with over 41% of the cats being infected with viruses containing recombinant env genes. In addition, we identified shared recombination breakpoints; the most significant hotspot occurred between the leader/signal fragment and the remainder of env.We investigated recombination in complete FIV env as an important site for recombination and highlight potential limitations of the current phylogenetic classification of FIV based on partial env sequences. Furthermore, the presence of abundant recombinant FIV in the USA poses a significant challenge for commercial diagnostic tests and should inform the development of the next generation of FIV vaccines.Our results have identified the leader/signal fragment of The online version of this article (doi:10.1186/s12977-014-0080-1) contains supplementary material, which is available to authorized users. This applies particularly to the open reading frame of the env gene which, in contrast to primate retroviruses, contains an unusually long leader/signal region  as a putative major parent and determined Chicago strains P10 and P2 as putative minor parents for M2, M31, M33, M47, M50, P4, P5, P7, P13, P14, P15, P18 and M20, M48, respectively [Table env sequences from three cats carrying non-monophyletic groups of viruses revealed that sequences from cat P8 at time point C were non-recombinant clade B, while time point A sequences were classified as A/B recombinants. It is intriguing that all time point C sequences in this cat contained premature stop codons [see Additional file env virus that outcompeted the recombinant strain. In cat P21, sequences at time point B appeared to be entirely clade B, while those at time point C showed an incongruent assignment and were identified as clade A/B mosaics [Table Recombination analysis of There was a notable difference in terms of the abundance of clade A recombinants compared to very few recombinant sequences clustering with clade B sequences Figure . Based oenv genes with shared recombination breakpoints and with inconsistent clade assignment circulated widely in the Memphis and Chicago cohorts, with over 41% of tested cats being infected with a recombinant form.The phylogenetic relationship of the sequences from Chicago and Memphis cohorts was in agreement with previously published data demonstrating the presence of clade A and clade B viruses in the USA . Howevergag, was reported in a Canadian study, suggesting the existence of enzootic recombinant forms circulating in Ontario [Recombination is an important event in retroviral evolution, which, in cases of super-infection, can lead to the emergence of novel viral variants. Although there was no evidence of more severe clinical manifestations in cats infected with recombinant strains , newly c Ontario . Given tenv gene predominantly comprised clade A derived sequences. These A/B recombinant strains hypothetically possessed equivalent or greater fitness compared to the parental A strains and were fixed in the population as CRFs of FIV. In contrast, A/B recombinants that were predominantly clade B derived were conspicuously rare, suggesting that these recombinants incurred a fitness cost.It is intriguing that clade A sequences were more likely to have a mosaic composition than those which were originally assigned to clade B. It has been proposed that clade B viruses are evolutionarily older, more host adapted and less pathogenic . It is penv sequences were examined and it is likely that recombination processes within env were accompanied by recombination events elsewhere in the genome, such that additional, as yet unidentified, breakpoints exist [Our analyses revealed the existence of at least one common recombination break point, located at the stem of the V1/V2 loop of the SU of FIV Env. Common recombination breakpoints, or hotspots, have been reported in both FIV  and HIV ts exist ,17.in vitro [env variants identified in our study must therefore have suffered no fitness cost, or might even have possessed some fitness advantage, compared to parental sequences.Although recombination occurs frequently in vitro -36, it hin vitro . The vasin vitro . The recenv gene) underestimated the role of recombination using a simplified phylogenetic classification. Applying the common approach of examining the V3-V5 hypervariable regions of the env gene will likely result in the misidentification of intra- and inter-subtype recombinants and erroneous subtype assignment. For example, the recombinant structure of viruses such as M2, M33 or P7 would have been misclassified if only 700 bp fragments of the env had been examined. Indeed, previous studies focusing solely on the V3-V5 region of env [The results of the present study highlight the risk of examining partial gene sequences. The detection of recombination increases with the nucleotide length screened and therefore the majority of previous studies  was used in place of the error prone Taq polymerase to avoid mutations arising during the PCR amplification of env sequences. Furthermore, in order to minimize template switching during the PCR, three independent amplifications were set up from each blood sample. The presence of similar recombinants, particularly those with shared recombination points, over a period of 18 months provides strong evidence that the amplicons identified were indeed circulating sequences rather than products of PCR-induced errors or polymerase template switching.Potential limitations of this study include PCR sampling and cloning bias ,40. Howeenv sequences sharing a common breakpoint separating the leader/signal region from the remaining part of env were highly abundant in naturally infected cats in the USA. This finding is intriguing, particularly since the feline and ungulate lentiviruses possess unusually long leader/signal sequences compared to primate retroviruses [env could play an important role in virus biology and immune evasion, as has been described for signal sequences in other viruses [Recombinant oviruses . The loc viruses . These f viruses ,43 curre viruses . FurtherFIV were isolated from three serial blood samples collected at 6 monthly intervals from naturally infected domestic cats (n = 43). Cats were enrolled into the study based on a history of FIV diagnosis by ELISA , regardless of breed, age, sex or health status. All cats were FeLV antigen negative . FIV-positive status was confirmed by virus isolation . Within Cats from two cohorts displayed contrasting clinical outcomes of infection. The clinical signs were mild or unapparent in the Chicago cohort, while the outcome of infection in Memphis was dramatically different, with mortality rate reaching 63% and lymphoma being the most common cause of death .The study and its aims were reviewed and approved by the University of Glasgow Ethics Committee and the Purdue Animal Care and Use Committee. Cat owners provided written informed consent for their participation in the study.env genes (~2500 bp) were amplified directly from whole blood using a nested PCR protocol [see Additional file E. coli MAX Efficiency® DH5α™ Competent Cells (Invitrogen). Each VR1012-FIV env construct was sequenced using Big Dye Terminator v1.1 kit (Applied Biosystems) on an Applied Biosystems 3130xl capillary sequencer. Special measures were taken to avoid the possibility of contamination, both in the clinical and the laboratory settings: cats were double identified prior to blood sampling; PCR reactions were prepared in a designated UV treated room; and fresh, unopened reagents were used at each separate time point throughout the 18 month study period.Multiple full length FIV env sequences from 43 cats available for analysis from the two cohorts [see Additional file There were 355 serial Maximum likelihood (ML) trees were constructed in MEGA5  under thenv sequences obtained from GenBank (n = 19), were subjected to rigorous five-fold recombination testing: 1) The initial recombination analysis included the entire data set and was performed with the Datamonkey webserver [Sequences from the study group n = 355), together with reference full-length ebserver , employiebserver  and usinebserver ; 2) The ebserver  were con, togetheebserver  and confebserver ; and 5) ebserver .


New progresses into the molecular and cellular mechanisms of autism spectrum disorders (ASDs) have been discussed in 1 day international symposium held in Pavia  on July 4th, 2014 entitled “synapses as therapeutic targets for autism spectrum disorders” . In particular, world experts in the field have highlighted how animal models of ASDs have greatly advanced our understanding of the molecular pathways involved in synaptic dysfunction leading sometimes to “synaptic clinical trials” in children. Autism comprises a wide range of neuro-developmental disorders known as autism spectrum disorders (ASDs), characterized by deficits in verbal and non-verbal communication, impaired social interactions, restricted interest, and stereotyped behavior. ASDs affect 1 over 150/200 children, with onset before 3 years of age. The higher incidence of these disorders in recent years can be attributed to the improvement of the diagnostic criteria in general and to the increased attention of the medical community  entitled “synapses as therapeutic targets for autism spectrum disorders.” We discussed how animal models greatly advanced our understanding of the molecular pathways involved in synaptic dysfunction in ASDs leading sometimes to “synaptic clinical trials” in children.Christian Rosenmund  talked about current work obtained in his laboratory on the cellular mechanisms underlying the Rett syndrome (RTT), a postnatal neurological disorder affecting 1/10000 girls. RTT is caused by loss of function mutations in the X-linked gene encoding the transcriptional repressor, methyl-CpG binding protein 2 (MECP2). Rett patients exhibit impaired motor coordination, learning deficits, irregular breathing, and seizures. Recent findings show that over-expression of wild-type (WT) MeCP2, due to gene duplications, causes also mental retardation and progressive neurological symptoms in males. Neurological phenotypes involve an imbalance of excitation and inhibition in neural circuits caused by too little or too much MeCP2 activity. Using a reduced experimental system consisting in growing single neurons on micro-island cultures, he provided evidence for specific determination of cell intrinsic mechanisms of MeCP2 regulation  briefly synthesized the state of the art research supporting the notion that dysfunctions of group-I metabotropic glutamate receptor 5 (mGlu5) are implicated in the pathophysiology of fragile X syndrome (FXS), the most common form of inherited intellectual disability and an important monogenic cause of autism. In most cases, FXS is caused by mutations in the fragile X mental retardation gene 1 (FMR1), silencing of the gene and ensuing lack of the encoded fragile X mental retardation protein (FMRP), an RNA binding protein involved in RNA metabolism and protein synthesis. FMRP is present at synapses and controls local mRNA translation upon synaptic activity. Although much work has focused on dysregulation of mGlu5 signaling to synaptic protein synthesis, emerging evidence finds abnormal mGlu5 receptor interactions with its scaffolding Homer protein, which results in mGlu5 receptor dysfunction and phenotypes independent of signaling to protein synthesis  mice  results in autistic-like behavior associated with a decreased in PC excitability. These effects can be prevented by rapamycin, suggesting a possible therapeutic role for mTOR inhibition in TSC-related ASDs  described recent data on tuberous sclerosis complex (TSC), caused by haploinsufficiency of the Shank3 and Ib2 genes. The latter encodes for the synaptic protein IB2 that takes part to the NMDA receptor interactome. Electrophysiological recordings combined with voltage-sensitive dye imaging in Ib2 KO mice  discussed modifications of cerebellar circuits in an animal model of the Phelan–McDermid syndrome. This autistic syndrome is caused by a deletion of the terminal portion of chromosome 22 (22q13.3) that involves both Nlgn3 gene detected in a family with children affected by ASDs  basket cells  illustrated the neuroligin (NL) model generated in Südhof’s laboratory by introducing in mice a single mutation (R451C) of the human −/−) mice before and after spatial learning. The homeobox-containing transcription factor has been associated to ASD. En2−/− mice show anatomical and behavioral “ASD-like” features, including loss of forebrain interneurons , associated with a marked down-regulation of neurofibromin expression in the hippocampus. ERK phosphorylation, known to be induced in the presence of neurofibromin deficiency, is increased in the hippocampus of En2−/− mice after spatial learning. Treatment of En2−/− mice with lovastatin, an indirect inhibitor of ERK phosphorylation, markedly reduced ERK phosphorylation in the dentate gyrus, but was unable to rescue learning deficits in MWM-trained mutant mice  reported a recent study on the neurofibromin-extracellular-regulated kinase (ERK) cascade in the hippocampus of WT and engrailed-2 knock-out  discussed how understanding and treating neurological and psychiatric disorders is conditioned by a better knowledge of developmental processes. In the developing brain ionic currents, network activity, and molecular processes have unique features and follow a developmental sequence adapting them to their adult functions. Together with Spitzer  highlighted recent studies showing the involvement of synaptic plasticity mechanisms in the pathophysiology of ASDs (Oberman et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


South Asians are up to four times more likely to develop type 2 diabetes than white Europeans. It is postulated that the higher prevalence results from greater genetic risk. To evaluate this hypothesis, we: (1) systematically reviewed the literature for single nucleotide polymorphisms (SNPs) predisposing to type 2 diabetes in South Asians; (2) compared risk estimates, risk alleles and risk allele frequencies of predisposing SNPs between South Asians and white Europeans; and (3) tested the association of novel SNPs discovered from South Asians in white Europeans.MEDLINE, Embase, the Cumulative Index to Nursing and Allied Health Literature (CINAHL) and the Cochrane registry were searched for studies of genetic variants associated with type 2 diabetes in South Asians. Meta-analysis estimates for common and novel bi-allelic SNPs in South Asians were compared with white Europeans from the DIAbetes Genetics Replication And Meta-analysis (DIAGRAM) consortium. The population burden from predisposing SNPs was assessed using a genotype score.Twenty-four SNPs from 21 loci were associated with type 2 diabetes in South Asians after meta-analysis. The majority of SNPs increase odds of the disorder by 15–35% per risk allele. No substantial differences appear to exist in risk estimates between South Asians and white Europeans from SNPs common to both groups, and the population burden also does not differ. Eight of the 24 are novel SNPs discovered from South Asian genome-wide association studies, some of which show nominal associations with type 2 diabetes in white Europeans.Based on current literature there is no strong evidence to indicate that South Asians possess a greater genetic risk of type 2 diabetes than white Europeans.The online version of this article (doi:10.1007/s00125-014-3354-1) contains peer-reviewed but unedited supplementary material, which is available to authorised users. Type 2 diabetes is a metabolic disorder resulting from the interplay between insulin secretion and action. The prevalence of type 2 diabetes is increasing globally . CurrentThe purpose of this systematic review is to: (1) establish risk estimates for SNPs predisposing South Asians to type 2 diabetes; (2) compare risk estimates, risk alleles and risk allele frequencies (RAFs) of type 2 diabetes predisposing SNPs between South Asians and white Europeans; and (3) explore the association of novel SNPs discovered from South Asians in a large cohort of white Europeans.MEDLINE, Embase, the Cumulative Index to Nursing and Allied Health Literature (CINAHL) and the Cochrane registry  were searched for studies of genetic variants associated with type 2 diabetes in South Asians. South Asians were defined as individuals originating from India, Pakistan, Bangladesh or Sri Lanka. The search strategy was developed in consultation with a research librarian and did not restrict by type of genetic variant, language of study or study design . The full search strategy is presented in electronic supplementary material (ESM) Table p < 10−3 were considered for meta-analysis.Conference abstracts, narrative reviews and other systematic reviews were excluded after full-text review. Primary studies investigating genetic variants other than bi-allelic SNPs  or with fewer than 25 cases were excluded. These restrictions were implemented to ensure that included studies could be combined and were of sound quality. Additionally, to ensure robustness of our results, SNPs from included studies carried forward to meta-analysis were limited to those that previously reached genome-wide significance in any ethnicity. If datasets were published more than once, publication with the largest sample size or the one recommended by the senior author was used for meta-analysis. Two reviewers conducted a full-text assessment and extracted data on participant characteristics , study design  and results . For genome-wide association studies (GWAS) in South Asians, SNPs associated with type 2 diabetes at Using recommendations from the STrengthening the REporting of Genetic Association Studies (STREGA) guidelines , the folκ statistic. Allelic ORs were calculated using RAFs and sample size for cases and controls and assessed for significance using Fisher’s exact test. The ORs were not adjusted for any covariates. ORs for SNPs in linkage disequilibrium (LD) (r2 > 0.8) obtained from independent cohorts were meta-analysed using a random-effects model weighted by inverse variance. LD was estimated using 1000 Genome Centre d'Etude du Polymorphisme (CEU) (Utah residents with northern and western European ancestry) data from SNAP   rs4812829, ST6GAL1 rs16861329, and AP3S2 rs2028299) were independently replicated in South Asians [The systematic review included four GWAS in South Asians, which identified nine SNPs associated with type 2 diabetes at genome-wide significance. With the exception of 05–1.10) , 39, theh Asians .SGCG rs9552911 was monomorphic in white Europeans. The remaining SNPs were directionally consistent with South Asian estimates and three  were nominally (p < 0.05) associated with type 2 diabetes   [KCNQ1 rs2237897 association was reported in only two studies among South Asians and thus could be a product of the winner’s curse [Meta-analysis of risk alleles for most SNPs increased the odds of type 2 diabetes by 15–35% among South Asians. Notable exceptions were the ulations  and beca’s curse .ADAMTS9 rs4607103, CDC123 rs12779790, JAZF1 rs864746, KCNQ1 rs231362, DGKT-TMEM195 rs2191349 , GCK rs1799884, GCKR rs780094, MTNR1B rs10830963, PROX1 rs340874, and TSPAN8 rs7961581  and demonstrated a high degree of heterogeneity .Fifteen GWAS signals for type 2 diabetes in white Europeans, East Asians, and Singaporean Malay –46 were In general when comparing effect sizes of SNPs associated with type 2 diabetes from our meta-analysis with DIAGRAM estimates in white Europeans, the risk from SNPs predisposing to type 2 diabetes did not differ substantially between the groups. However, the point estimates were more precise and CIs tighter among the white Europeans because of the larger sample size. Our observation of no significant difference in risk estimates is not surprising because SNPs evaluated in South Asians are selected for homogeneity as they were first discovered in white Europeans and then replicated in South Asians. If there is a difference in genetic risk between the ethnic groups, it probably does not result from polymorphisms common to both groups. The results of our paper are supported by those recently published by DIAGRAM, the Asian Genetic Epidemiology Network Type 2 Diabetes (AGEN-T2D) Consortium, and the South Asian Type 2 Diabetes (SAT2D) Consortium . Their sFigure PPARG rs1801282, the risk allele is responsible for increased fat storage and may have been advantageous in ancient environments with unpredictable food supply and high level of activity, but now predisposes to type 2 diabetes [We did not find a trend in RAFs; specifically, RAFs were not consistently higher in South Asians. Risk alleles from six SNPs in this analysis were the major/common alleles in both ethnicities. Negative selection usually prevents risk alleles from becoming common unless they are advantageous . In the diabetes . Howeverdiabetes .SGCG rs9552911 is not present in white Europeans. The putative existence of private polymorphisms in South Asians is supported by presence of assortative mating resulting from prolonged geographical and cultural isolation of this region from white Europeans [r2 values with neighbouring SNPs have some differences . Fine mapping analyses will determine whether the same functional variants are responsible for increased risk in both groups and if the effect is comparable, or if the seven SNPs tag unique functional variants in South Asians.Finally, we tested novel SNPs derived from South Asians in white Europeans; only three were associated with type 2 diabetes. Interestingly, one of the novel SNPs, rs1048886, is a functional missense mutation resulting in a change from glutamine to arginine in the UPF0369 protein involved in immune response . Additiouropeans . Non-assNo difference in genotype score between South Asians and white Europeans was observed, which is consistent with our finding that no trend exists for variation in RAFs. It should be noted that our conclusion is based on an assumption of homogeneity in effect sizes between the groups, supported by our primary analysis. Our conclusion is not consistent with some literature, which shows a greater population burden in South Asians based on unweighted genotype scores , 55. HowGCKR rs780094 in the primary report [This is the first meta-analysis that compares genetic risk of type 2 diabetes in South Asians and white Europeans. Potential limitations of our study include use of unadjusted allelic OR, which precluded us from accounting for SNP–type 2 diabetes associations altered by adiposity. Use of unadjusted allelic OR depended largely on the unavailability of appropriately adjusted data. However, we informally compared adjusted estimates reported in the study with our unadjusted allelic OR and found the adjustment to make little difference; for example, the OR for y report  was 0.87Second, publication and time-lag biases may exist whereby significant results are published more often than negative studies. To minimise these biases, we conducted a thorough search of literature and consulted experts to identify as many eligible studies as possible. We were unable to produce funnel plots to formally assess publication bias because fewer than ten studies were used to meta-analyse a majority of SNPs.Third, some SNPs in this meta-analysis were only investigated in two cohorts and their summary estimates should be interpreted with caution.Finally, sample sizes varied with each SNP in South Asians but were consistent for all SNPs in white Europeans, which may create artificial differences between the groups as a result of differences in precision. Our conclusions regarding the genetic heterogeneity between South Asians and white Europeans are applicable only to genetic risk from bi-allelic common SNPs as our investigation was limited to this type of variant.MTNR1B SNP modestly associated with type 2 diabetes (OR between 1.10 and 1.15) identified four rare variants that led to loss of melatonin binding and signalling capacity  [The question of why South Asians have a greater risk for type 2 diabetes remains unanswered from a genetic perspective as risk from common SNPs predisposing to type 2 diabetes does not differ. Our results are particularly important as they emphasise the need for future research to explore heritable epigenetic changes, epistasis and gene–environment interactions to answer this question , 61, rat7–14.82) . Using a7–14.82) , 67, whiSimilar effect sizes for SNPs predisposing to type 2 diabetes are apparent among South Asians and white Europeans, but there is variation in RAFs. Additionally, some novel SNPs are present in South Asians. Given the current literature, there is no strong evidence to indicate that currently known genetic variants explain the higher risk of type 2 diabetes in South Asians compared with white Europeans.ESM Table 1(PDF 43 kb)ESM Table 2(PDF 51 kb)ESM Table 3(PDF 168 kb)ESM Table 4(PDF 113 kb)Below is the link to the electronic supplementary material.


Fmr1 KO) recapitulates several of the neurologic deficits associated with Fragile X syndrome (FXS). As tactile hypersensitivity is a hallmark of FXS, we examined the sensory representation of individual whiskers in somatosensory barrel cortex of Fmr1 KO and wild-type (WT) mice and compared their performance in a whisker-dependent learning paradigm, the gap cross assay. Fmr1 KO mice exhibited elevated responses to stimulation of individual whiskers as measured by optical imaging of intrinsic signals. In the gap cross task, initial performance of Fmr1 KO mice was indistinguishable from WT controls. However, while WT mice improved significantly with experience at all gap distances, Fmr1 KO mice displayed significant and specific deficits in improvement at longer distances which rely solely on tactile information from whiskers. Thus, Fmr1 KO mice possess altered cortical responses to sensory input that correlates with a deficit in tactile learning.The fragile X mental retardation 1 mutant mouse ( Fragile X Syndrome (FXS) is a leading inheritable cause of mental impairment, affecting approximately 1∶4000 males and 1∶8000 females in the United States Fmr1 mutant mouse exhibits several phenotypes similar to Fragile X syndrome including increased seizure susceptibility and hyperactivity, as well as deficits in spatial and motor learning Fmr1 KO mice display abnormal sensory gating during prepulse inhibition as well as cortical hyperexcitability Fmr1 KO mice exhibit differences in somatosensory responsiveness akin to the characteristic tactile hypersensitivity to sensory stimuli in FXS is unknown. In addition, whether Fmr1 KO possess normal tactile-dependent learning is unclear.This The gap crossing (GC) learning paradigm is a prime example of a distance detection/object localization task Fmr1 mutants and wild-type (WT) mice. Fmr1 KO mice displayed greater cortical responses to whisker stimulation, normal initial performance on the gap cross task, but significantly lower improvement with experience at whisker-dependent distances. Thus, we conclude that disruption of Fmr1 gene function alters cortical responses to sensory stimuli and perturbs tactile learning.Here we examined the cortical responses to whisker stimulation and the rate of tactile learning for Pde6b+ Tyrc-ch/AntJ; stock# 4828, Jackson Laboratory) and Fmr1 KO mice  were maintained and all experiments conducted according to protocols approved by the Children’s Hospital Los Angeles Institutional Animal Care and Use Committee. Mice were anesthetized by isoflurane inhalation and euthanized by carbon dioxide asphyxiation in accordance with approved protocols. The Children’s Hospital Los Angeles Institutional Animal Care and Use Committee specifically approved this study. Protocol number 264-12.FVB wild-type  and food and water were available ad libitum except in gap cross groups. Mice trained and tested on the gap cross were individually housed at the start of training through completion of the 6 days of testing and moderately food restricted as normal chow was allocated on a daily basis to maintain 90–95% initial body weight. All mice were 12–14 weeks of age at the time of the study.The gap cross assay was performed with a custom-built robot . In brief, the gap cross assay system is a closed-loop robotic environment with motor controlled units and sensing elements. The mouse behaves upon raised platforms driven by independent linear actuators. The platforms are equipped with servo-motor doors and positional sensors. Data acquisition and control algorithms are both executed online for real-time dynamic control and offline for more advanced analysis. Independent linear actuators move the Plexiglass platforms to generate a range of gap-distances from nose (<4.5 cm) to whisker (5–8 cm) distances in increments of 0.5 cm. To monitor the location of the mouse, IR motion sensors are at the back and edge of each platform. Near the edge of each platform are servo-controlled doors that prevent exploratory behavior during repositioning of the platforms. The linear motors, servos, and motion sensors are USB controlled through microcontroller boards (Arduino Mega 2560 and the Quadstepper Motor Driver) that feed to a quad-core CPU.Motor positions are processed on a quad-core CPU using the Arduino and Matlab programming environments. Platform position, door status (open/closed) and feeders are real-time controlled using the Arduino C-based development environment (ADE). Custom-built feeders delivered a small sugar pellet  following a successful cross. Motion sensor data are continuously acquired and pre-processed within ADE and are visualized and stored in real time. Specifically, sensor activity is encoded as behavioral performance metrics including successful and failed crossing events. Successful trials are defined as trials in which the mouse approaches the gap and crosses. Failures are defined as trials in which the mouse approaches the gap and then retreats back. This information is computed in real-time. Behaviors are segmented into interactive events at the gap and the system is structured as a two state machine: exploration and adjustment. During exploration, the motors are disabled and the system continuously acquires behavioral data through the motion sensors. During adjustment the doors close to halting exploration and motors reposition the platforms for the next exploration phase. Transitions between the two states are triggered by behavioral events (i.e. successful/failed gap-crossing).Mice were handled for 10 minutes a day for one week prior to beginning the task. The day before training began, mice were habituated to the gap cross apparatus. They were placed in the chamber with background white noise (60–65 dB) for 20 minutes in white light, immediately followed by 20 minutes in the dark. A bridge was placed over the gap to prevent exploration of the gap and gap crossing behavior. All training sessions took place in a light-tight enclosure in the presence of background white noise. Food was provided to the mice at least one hour after their final training session.Each training session lasted for 20 successful trials or a maximum of 20 minutes. The training lasted a total of 6 days, with 2 sessions per day for a total of 12 consecutive sessions. Training sessions were separated by at least 6 hours. All sessions began with a trial at 3.0 cm, the shortest distance tested. The position of the mouse was tracked with motion sensors placed at the back and near the edge of each platform. As a mouse traversed the platform, these sensors recorded its progressive position. A successful trial was identified as any trial in which the mouse successfully crossed the gap between the home and target platforms and activated the motion sensor at the back of the target platform. These trials were rewarded with a 5 mg casein pellet delivered from an automated feeder. A failed attempt was defined as an attempt in which the mouse explored the edge of the home platform and returned to the back of the platform. Following each success or failure, the subsequent gap distance was determined using an adaptive learning algorithm designed to decrease the predictability of the next gap distance.The learning algorithm incorporates the progressive history of successful crosses during a session. Beginning with the first trial at 3.0 cm, the next gap distance was chosen randomly from a uniform distribution of distances (in 0.5 cm increments) 1.0 cm less than the maximum distance crossed in the session (to a minimum of 3.0 cm) to 1.5 cm greater than the maximum distance crossed in the session (to a maximum distance of 7.0 cm). This process was then repeated iteratively until 20 successful trials or 20 minutes had elapsed, completing the session .Fmr1 KO mice (12–14 weeks of age) were used. Mice were anaesthetized with isoflurane  throughout surgery. Body temperature was maintained with a biofeedback heatplate (Physitemp). A circular region of the skull over barrel cortex was thinned to allow visualization of blood vessels at the brain surface without perturbing the underlying dura. A 3 mm diameter #1 thickness cover glass (Bellco) was placed on the thinned skull, affixed with cyanoacrylate and sealed with dental acrylic. A small aluminum bar with tapped screw holes was embedded into the acrylic to stabilize the animal for subsequent imaging sessions. Animals received buprenorphine (0.1 µg/g body weight) and baytril in water (0.1 mg/ml) post-surgery. Their water was also supplemented with carprofen (0.025 mg/ml) throughout the imaging series. Animals were given at least 2 days to recover before intrinsic signal optical imaging.Male FVB wild-type (WT) and Imaging was performed adapted from the temporally-encoded optical imaging approach developed to study visual cortex Green light (530 nm±30 nm) was used to visualize cerebral vascularization and red light (620 nm±20 nm) to image intrinsic signals. The imaging plane was focused ∼200–400 µm below the pial surface. Images were acquired at 10 frames per second at 1024×1024 pixels per image at 12-bit depth with a high-speed camera . Custom acquisition and analysis software (C++ and Matlab) spatially binned images and the magnitude of the response (ΔR/R) at the stimulus frequency was extracted from a complete time series for each pixel by Fourier analysis Fmr1 KO and WT mice, we employed optical imaging of intrinsic signals (OIS) to measure cortical responses to whisker stimulation. OIS is a non-invasive measure of tissue reflectance correlated with neural activity Fmr1 mutants and WT mice (Fmr1 KO mice displayed significantly greater size of response (pixels) relative to WT mice across magnitude of response thresholds  and the subsequent six sessions (7–12) at all gap distances tested for WT and  KO mice . Both ge KO mice . At thesstances’ . The perstances’ . Both WTistances .Fmr1 KO mice did not display significant improvement at these whisker-dependent gap distances  = 2.338, p = .14; n = 9, two-way RM-ANOVA)  . Thus, Fmr1 KO mice exhibit a deficit in tactile learning that correlates with abnormal cortical sensory representation of whiskers in barrel cortex.In contrast to shorter distances, at longer distances  mice rely exclusively on information from their whiskers to detect the target platform. Overall, performance declines with increasing gap distance . At thesM-ANOVA) . The imp t-test) . SimilarFXS is characterized by cognitive impairment, anxiety, developmental delay, increased seizure susceptibility, and behavioral hyper-excitability. Altered sensory responses and reduced sensory integration are also symptoms of FXS. In particular, disrupted somatosensory processing, which is often described as tactile defensiveness, is well documented in patients with FXS FMR1 KO mice manifest several phenotypes similar to symptoms of FXS Fmr1 KO mice display alterations in synaptic function and plasticity FMR1 KO mice, it remains unclear whether cortical responses to tactile stimulation are aberrant in the somatosensory cortex of Fmr1 KO mice.Fmr1 KO correlate with a deficit in a whisker-dependent and barrel cortex-dependent tactile learning task. However, as OIS is an indirect measure of neural activity, we cannot exclude the possibility that either differences in coupling of neural activity to factors mediating the change in tissue reflectance or different effects of isoflurane on WT and Fmr1 KO mice could contribute to the differences in cortical responses to whisker stimulation.We examined tactile learning by comparing the performance of Fmr1 KO and WT mice on a whisker-dependent sensory learning paradigm, the gap cross task. Fmr1 KO mice displayed initial performance on this task indistinguishable from WT mice and normal learning at shorter ‘nose’ gap distances where sensory information regarding the position of the target platform was presumably more definitive. However, Fmr1 KO mice exhibited significantly less improvement in performance at whisker-dependent distances. Thus, aberrant cortical responses to whisker stimulation we observed in Fmr1 KO mice or whether this learning impairment results from aberrant neural circuitry elsewhere in brain is unclear at present. For example, this learning deficit could result from a dysfunction in motor cortex. We considered whether the increased anxiety-like behaviors exhibited by Fmr1 KO mice might contribute to their deficits in the gap cross assay Fmr1 KO mice are unlikely to be a major contributor to the learning deficit specific to whisker-dependent gap distances we observe. Future studies will be required both to discriminate between these possibilities as well as ascertain if performance by Fmr1 KO mice on the gap cross assay is improved by administration of mGluR5 antagonists or GABAB agonists reported to rescue some aspects of the phenotype Fmr1 KO mice However, this perceptual learning task is not exclusively reliant on cerebral cortex or the somatosensory system. Distance detection and object localization integrates motor and sensory activity of both subcortical and cortical circuitry


Type II diabetes is an established cause of vascular impairment. Particulate air pollution is known to exacerbate cardiovascular and respiratory conditions, particularly in susceptible populations. This study set out to determine the impact of exposure to traffic pollution, with and without particle filtration, on vascular endothelial function in Type II diabetes. Endothelial production of nitric oxide (NO) has previously been linked to vascular health. Reactive hyperemia induces a significant increase in plasma nitrite, the proximal metabolite of NO, in healthy subjects, while diabetics have a lower and more variable level of response. Twenty type II diabetics and 20 controls (ages 46–70 years) were taken on a 1.5hr roadway traffic air pollution exposure as passengers. We analyzed plasma nitrite, as a measure of vascular function, using forearm ischemia to elicit a reactive hyperemic response before and after exposure to one ride with and one without filtration of the particle components of pollution. Control subjects displayed a significant increase in plasma nitrite levels during reactive hyperemia. This response was no longer present following exposure to traffic air pollution, but did not vary with whether or not the particle phase was filtered out. Diabetics did not display an increase in nitrite levels following reactive hyperemia. This response was not altered following pollution exposure. These data suggest that components of acute traffic pollution exposure diminish vascular reactivity in non-diabetic individuals. It also confirms that type II diabetics have a preexisting diminished ability to appropriately respond to a vascular challenge, and that traffic pollution exposure does not cause a further measureable acute change in plasma nitrite levels in Type II diabetics. Epidemiological and toxicological studies have amply demonstrated associations between acute and chronic exposure to traffic-related air pollutants (TRAPs) and cardiorespiratory health effects . TRAPs aThe relative importance of the various components of the complex TRAPs mixture, as well as the biological pathways by which different TRAPs components may cause adverse health effects, are not well-understood. Exposure to TRAPs, diesel exhaust and particles increases inflammatory responses in animal and human respiratory tracts, in addition to cell systems. Oxidative stress is considered to be a key mechanism in these adverse responses , 17–22.In previous studies, we found that nitrite and nitrate, relatively stable oxidation products of nitric oxide metabolism, were increased in exhaled breath condensate (EBC) among asthmatics after controlled exposure to diesel exhaust  and sub Type 2 diabetes mellitus (T2DM) is associated with increased risk for acute and chronic cardiovascular disease –28. StudThe metabolically active vascular endothelium regulates blood flow through modulation of vascular smooth muscle tone via production of and response to molecules such as nitric oxide (NO) –33. NO m2-) and subsequently into nitrate (NO3-) in the vasculature. Nitrite, although shorter-lived, is considered a more meaningful reflection of NO production, as it reflects NO produced by the endothelial cell lining of blood vessels, while nitrates in circulation may originate from alternate sources such as food constituents and preservatives [An increase in tissue metabolic demands due to physiological or chemical stressors, particularly oxidative stress, normally results in hypoxic vasodilation mediated by NO. NO has a short half-life and is rapidly oxidized into nitrite  when ultrasound measurements of arterial diameter are made, other investigators have directly measured NO metabolites in blood as a relevant endpoint , 37. In Previous studies have shown that adults with T2DM have reduced flow-mediated dilation , 40 on dWe hypothesized that 1.5-hr rides in a passenger vehicle in morning rush-hour traffic on a major highway would cause acute increases in oxidative stress in the peripheral vasculature manifested by impaired generation of NO, observed as its metabolite nitrite, in response to a standard ischemic stimulus, and that the PM component of TRAP is responsible for these effects. We further hypothesized that the impairment in generation of NO would be greater in subjects with T2DM compared to healthy control subjects.Twenty subjects with T2DM aged 46 to 70 years and 20 non-diabetics were recruited from the community and from endocrinology clinics at RWJMS. All participants were medically cleared for the study with the following exclusions: pregnant and breast feeding women, unstable kidney/liver disease, unstable coronary artery disease or arrhythmia, peripheral arterial disease, uncontrolled hypertension, asthma, other pulmonary disease, cigarette smoking, or the inability to ride comfortably in a car for 2 hours. Prior to participation all subjects signed an informed consent approved by the UMDNJ (now Rutgers University) Internal Review Board.Treating physicians adjusted diabetes medications prior to each study visit to account for the lack of breakfast on study mornings (fasting after midnight) and blood glucose was monitored before and after the ride to avoid hypoglycemia. Subjects began pre exposure testing at approximately 7am with all study testing completed within a 4-hr window. Subjects lived within 20 km of the Environmental and Occupational Health Sciences Institute (EOHSI) and were encouraged to avoid highways when driving to reach the testing facility on days of their exposures. Exposure to weekday rush-hour traffic pollution occurred in a Ford Taurus on the New Jersey Turnpike (NJTPK). Each subject rode in the rear seat of the passenger vehicle, which was driven by professional drivers. The car rides were approximately 1.5 hours in length and approximately 79 miles round trip primarily in the diesel-enriched NJTPK truck/bus lanes . Windows were maintained closed but external air intakes were open (non-recirculation mode) with the fan on medium setting. Each subject participated in two randomly ordered exposure sessions at least one week apart. A powered air purifying respirator (PAPR) was utilized for both car rides. Exposures were either filtered (PAPR with HEPA filter in the respirator housing) or unfiltered (PAPR without HEPA filter). Subjects and technicians were blinded to presence or absence of the filter, which was randomized between rides.A five minute arm occlusion consisted of inflation of a blood pressure cuff to 50mmHg above systolic blood pressure. The cuff was placed on the biceps approximately 2–3 cm superior to the olecranon process allowing access to the median cubital vein for phlebotomy.2EDTA and immediately placed on ice. Draws were completed at baseline and 60 seconds following release of the upper arm occlusion. Samples were fractionated by centrifugation (13000 rpm for 20 minutes). The plasma was drawn off and aliquoted into three 1.5mL plastic eppendorf tubes. One plasma aliquot was placed on ice and analyzed for nitrite within 4 hours of sampling. The remaining plasma was frozen and stored at -80°C for future biochemical analyses.Blood draws were taken prior to and immediately following TRAP exposure from the median cubital vein in the test arm. Samples were collected into a BD plastic whole blood tube with spray-coated K2-), was analyzed using an ozone-based chemiluminescent technique with a Sievers Nitric Oxide Analyzer 280. NO2- analysis was completed at room temperature utilizing potassium iodide and acetic acid as reductants and argon as an inert carrier gas. This reaction specifically reduces NO2- into NO, but cannot reduce higher oxides of nitrogen (like nitrate (NO3-)) to NO. Standards for the assays were prepared by serial dilutions of sodium nitrite (Sigma Aldrich) with ultrapure water. Volumes of 10 μL of standards and 40 μL of sample were injected into the reaction vessel to give adequate reaction signal. Integrations of resulting peaks were calculated in the Liquid program to produce areas under the curve (AUCs) from standards and samples. Sample concentrations were calculated utilizing a formulated regression line from known standard concentrations.The NO metabolite, nitrite (NO2.5) at 1-min intervals using TSI SidePak model AM510 aerosol monitors  with the calibration factor set at 0.32 ; number concentrations of particles with aerodynamic diameter from 0.01 to 1.0 μm at 1-min intervals using a condensation particle counter, TSI model 3007 . Pollutants measured in the vehicle cabin only were: back carbon using an AE-51 microaethalometer  at flow rate of 100 ml/min and 1-min time base; nitrogen dioxide (NO2) collected on triethanolamine-coated Sep-Pak cartridges , analyzed using HPLC-UV as previously reported [We made the following measurements with inlets at the subject’s breathing zone inside the respirator (PAPR) face piece and separately near the center of the vehicle cabin: mass concentrations of PM with median cut-point of 2.5 μm -1)x100% where β was the regression coefficient associated with the dummy variable representing exposure (= 1 when unfiltered and = 0 for filtered). Confidence intervals were calculated similarly using the endpoints of the 95% confidence intervals for β.Linear models with random effects to account for repeated measures were used to assess the main effects of diabetic status and exposure as well as the interaction of diabetic status and exposure on the nitrite measures. Log transformations were taken of nitrite values in order to stabilize the variance and ensure that the response variables were normally distributed. When examining exposure, the pre-exposure measure was included as a covariate (accounting for regression to the mean) and a random intercept for subject controlled for correlation within subject due to repeated exposure sessions. Ratios were calculated of changes due to the filtered versus unfiltered conditions, for example, as , while the diabetic cohort consisted of a male majority (75%). No subject had a blood glucose level below 90 mg/dl before or following a ride.Subject characteristics are summarized in 2.5 concentration was 83.9% lower (p< 0.0001) and mean particle number concentration was 99.9% lower (p<0.0001) during HEPA-filtered rides compared to unfiltered rides.The pollutant measurements show no significant differences inside the vehicle cabin between filtered and unfiltered rides as the filter only affected the air directly inhaled by the subject through the PAPR . HoweverWe examined whether car ride pollutant exposures would reduce vascular NO immediately after a ride, either at rest or post-ischemia (hyperemia). The control group’s resting (pre-ischemia) plasma nitrite concentrations were found to be lower following unfiltered rides than filtered rides  . DiabetiPrior to the ride there was greater variability in resting nitrite levels within the diabetic group, as well as in their response to ischemic challenge, when compared to controls . GeometrConsistent with a healthy vascular response, controls showed a statistically significant 15.9% increase in plasma nitrite levels following ischemia (p = 0.017) prior to the car rides . In contFollowing the car rides the significant nitrite increase from ischemia seen in controls prior to rides  is no loThis study set out to determine the impact of both type II diabetes and an acute exposure to TRAP on vascular reactivity through nitric oxide and its proximal metabolite, plasma nitrite. The findings of this study included (i) higher resting nitrite levels in diabetics than controls; (ii) a borderline significant decrease among control subjects in resting nitrite following the unfiltered car ride as compared to resting nitrites after the filtered ride; (iii) increased nitrites in controls but not diabetics during reactive hyperemia prior to exposure and abolition of this increase following acute TRAP exposure.Our findings showed baseline nitrite levels in diabetics to be substantially elevated compared to controls. This is consistent with a previous study that showed subjects with endothelial dysfunction to have increased baseline vascular nitrites . This inSubjects with diabetes showed a small decrease in baseline nitrite levels following the unfiltered rides as compared to filtered rides, while controls displayed a more substantial, borderline significant, decrease. It is possible that TRAP may have a greater impact on the baseline level of NO in healthy vascular systems than on those with disease-related endothelial dysfunction and consequently a pre-existing increase in NO production.Following reactive hyperemia but prior to the car ride, control subjects showed a significant increase in nitrite levels, while this response was blunted in diabetics. Allen showed a similar insufficient response in T2DM subjects following a graded exercise test . It was anticipated that diabetics would have this limited ability to respond effectively to stimuli, as their vascular systems may be compromised by chronically higher levels of oxidative stress and elevated NO scavenging. Additionally, a study by O’Neill showed diabetic subjects to have lower flow-mediated vascular reactivity than those subjects considered at risk for diabetes. This physiological response is consistent with the lack of nitrite response to reactive hyperemic stimulation among diabetics in our study. As nitrite represents a proximal measure of eNOS function these results are consistent with reduced eNOS function as the molecular mechanism of this physiological response.Our diabetic subjects displayed no change from their blunted pre-exposure reactive hyperemia response following the car ride. Their decreased responses during reactive hyperemia prior to exposure may indicate an already compromised vascular system, as discussed above. A recent study by Zanobetti (2014) showed no association between decreases in BAFMD and increases in ambient particulate pollution using a 5 day integrated sample in type II diabetics . These dThe significant increase in plasma nitrite within the control group following reactive hyperemia is no longer present after exposure to TRAP (neither filtered nor unfiltered). This biochemical response is consistent with the observations of a study by Brook who found significant brachial artery vasoconstriction in a healthy population following acute exposure to a mixture of concentrated ambient fine particles and ozone . The absIn contrast to our findings, the diabetic subjects of the O’Neill study had altered vascular reactivity in association with multiple day increases in ambient air pollution, while the at-risk group displayed no change in response to air pollution. Since our diabetic subjects appeared to have severely comprised vascular NO responses prior to exposure, it is plausible that the impact of a relatively brief 2 hour car ride may not have been potent enough to further alter NO production in non-responsive systems. There is also a distinct possibility that physiological changes in brachial artery diameter occur even in the absence of significantly altered NO levels .The nature and length of exposure time may also play an important role in eliciting vascular changes. Our exposures were limited to 2 hours on highly trafficked roadways, while the O’Neill exposure metrics were average ambient urban pollution over 1–6 days in the Boston area. It is possible that a longer traffic exposure, repeated exposures over several days, or a later follow-up time may be necessary for impact on vascular reactivity. In addition, O’Neill also discussed sensitivity of diabetic subjects to pollution effects on vascular function as a possible reason for their findings; however, using the biochemical measure of nitrite, we found that diabetics appeared to be less sensitive than their non-diabetic counterparts to exposure to TRAP.Our control population responded with more modest nitrite increases during reactive hyperemia than those previously reported. This may be due to their more advanced age, elevated BMIs, and co-morbid conditions (other than diabetes) compared with subjects in previous reports . The conThis study demonstrated the adverse vascular effects of a two hour traffic exposure in an older non-diabetic population. The most pronounced change was seen in the decreased vascular reactivity response in non-diabetics following a car ride associated with increased TRAP inhalation. Before exposure, type II diabetic subjects demonstrated a decreased ability to respond appropriately to a vascular stressor and this was not altered by the TRAP exposure. Since the diabetic subjects show pre-existing compromise of their vascular systems, the extent to which a TRAP exposure affected them is unknown. The decrease of resting nitrite levels after TRAP pollution inhalation, and lowered vascular reactivity in non-diabetics, supports the proposition that real-world TRAP acutely increases endothelial dysfunction and alters NO metabolism leading to inadequate vascular responses to hypoxia. These results further validate the previously observed detrimental effects of TRAP exposure on vascular reactivity in controlled diesel exposure studies, and provides a direct mechanism for the epidemiological observations of MI associated with acute traffic exposure , 61–63.S1 Figadenotes (pre ischemia (resting) nitrite level) subtracted from (post ischemia (reactive hyperemia)nitrite level). bdenotes mean values (thick black line).(A)Control subjects’ change in plasma nitrite response to ischemia (resting nitrites subtracted from reactive hyperemia nitrites)before (pre ride) and following (post ride) exposure to filtered ride (n = 16). Mean changes in nitrite values . (B) Control subjects’ change in plasma nitrite response to ischemia before and following exposure to unfiltered ride (n = 17). Mean changes in nitrite values . (C)Diabetic subjects’ change in plasma nitrite response to ischemia before and following filtered ride (n = 14). Mean change in nitrite values  (D)Diabetic subjects’ change in plasma nitrite response to ischemia before and following unfiltered ride (n = 13). Mean change in nitrite values . (TIF)Click here for additional data file.


Results demonstrate a significant additional value of rsCTP in the assessment of intermediate coronary artery stenosis found with CTA.The prospective study included 54 asymptomatic high-risk patients who underwent coronary CT angiography (CTA) and regadenoson-induced stress CT perfusion (rsCTP). Diagnostic accuracy of significant stenosis (≥50%) determination was evaluated for CTA alone and CTA + rsCTP in 27 patients referred to ICA due to the positive rsCTP findings. Combined evaluation of CTA + rsCTP had higher diagnostic accuracy over CTA alone (per-segment: specificity 96 versus 68%,  Computed tomography is routinely used for the detection of coronary heart disease (CHD) with an excellent prognostic value . CT angiRecently, the stress CT perfusion (CTP) examination has become a fast-developing method for the functional assessment of an occlusive CHD. A number of studies have already shown a high image quality and diagnostic performance of stress CTP in the assessment of myocardial perfusion in patients with known or clinically suspected CHD .The aim of our study was to assess the contribution of a combined protocol including CTA and regadenoson-induced stress CT perfusion (rsCTP) to diagnostic performance of significant coronary artery stenosis detection in comparison to CTA evaluation.μmol/L or glomerular filtration rate <60 mL/min/1.73 m2). Patients were also screened for contraindications of regadenoson administration: atrioventricular block grades II-III and bronchial asthma.A prospectively conducted study included 54 consecutive patients  at high risk of CHD. Concrete inclusion criteria were (1a) peripheral arterial disease (PAD) in severe stage (Fontaine stages IIb–IV) referred to vascular surgery on aorta and/or iliac arteries or (1b) patients with abdominal aortic aneurysm (AAA) referred to resection, (2) no previous history of CHD or recent clinical symptoms, and (3) patients with sinus rhythm. Patients with contraindication to administration of iodinated contrast media were excluded: previous severe allergic adverse reaction and renal dysfunction  of myocardium, and eventually rest CTP. In a subset of enrolled patients results were compared with ICA that was performed in patients with stress-induced hypoperfusion finding or other indications. The study was approved by the local ethics review board and patients signed informed consent.z-axis flying focal spot technique. Premedication of beta-blockers or nitrates was not performed in any patient.All examinations were performed on a second-generation dual-source CT scanner  with CTA was performed using retrospective ECG gating with the following parameters: 2 × 2 × 64 × 0.6 mm detector collimation, 280 ms rotation time, 0.2–0.4 adaptive pitch factor, and automated modulation of tube voltage and tube current using the CARE kV and CARE Dose4D  with 320 reference mAs/rotation. Automated tube current modulation during R-R interval was used during acquisition. The bolus timing test was performed with 10 mL of contrast medium bolus. The time attenuation curve was analyzed  for the adjustment of optimal scanning start . Iodine contrast medium of 50 mL  was injected at a rate of 6 mL/sec with 50 mL saline flush at the same rate for CTA scan. Image series of 0.75 mm section width, reconstruction increment 0.4 mm, and convolution filter for vessels (I20f) were reconstructed using iterative reconstruction algorithm .μg of regadenoson and consists of 15 repeated scans in 30 sec covering left ventricle myocardium . Dynamic acquisition was triggered at 60% of R-R interval with delay  after the bolus of 35 mL iodinated contrast medium injected at 4 mL/sec followed by 40 mL saline flush at the same rate. Patients were instructed to carry out minimal shallow breathing during the examination.In the next phase, dynamic scan using two alternating table positions (“shuttle-mode”) was performed in the maximal coverage (73 mm) of the left ventricle myocardium during stress activity. The dynamic protocol was delayed 60 sec after slow manual intravenous application of 400 Actual blood pressure (BP) was checked immediately after the acquisition and evaluation of rsCTP was performed (one from intended readers). In the case of finding any hypoperfused areas, dynamic acquisition was repeated at least in 15 minutes' interval with the same acquisition and contrast medium administration parameters. Series of 3 mm section width (reconstruction increment 2 mm) and soft tissue convolution filter (B22f) were performed for subsequent evaluation.ICA was performed within maximal three days' interval after CT examinations using a standard technique and coronary artery stenosis were quantified in a consensus of two interventional cardiologists (more than 10 years of experience). Severity of stenosis was quantified and divided into <50% (nonsignificant) and ≥50% (significant) according to routine praxis. The measurement of fraction flow analysis was not performed in all stenosis, so it was not analysed.CTA and CTPs evaluation was performed independently by two radiologists with specialization in cardiac imaging (8 and 12 years of experience) at the multimodality workstation Syngo MMWP . Reconsideration of discordant findings was performed in consensus. Interobserver agreement analysis of stenosis severity assessment was performed.Identified atherosclerotic plaques were localized in segments of coronary arteries using 16-segment model according to the American Heart Association . StenosiDynamic scans were evaluated using a dedicated software application Syngo Volume Perfusion Body with preset for myocardium  that included automatic motion correction algorithm with possibility of manual adjustment. Colour-coded perfusion maps  and time-invariant reconstructions  were saved for further analysis. In addition, 4D-CT display of first-pass perfusion  with narrow window width and center adjusted by readers was performed to detect hypoperfused areas (in comparison to surrounding tissue). The presence of a myocardial perfusion defect was considered when hypoperfusion persisted for more than 3 cycles (heartbeats). In this way, we tried to reduce the number of artifacts. The 17-segment model for left ventricle myocardium (according to the American Heart Association) was used and perfusion defects were described as stress induced  and fixed.Using fused CTA images and perfusion maps, stenosis and perfusion defects were assessed with maximum emphasis on the correlation of anatomical relationships and areas of the vascular supply to the myocardium. In the presence of stress-induced defect in the territory of the branch with nonsignificant (<50%) stenosis according to CTA, this was reclassified as a significant stenosis. The stenosis determined as significant (≥50%) was reclassified when normal perfusion was found in corresponding territory. Separate evaluation of CTP was not performed.p value of <0.05 is considered a statistically significant difference. Statistical analysis was performed using MedCalc software .Continuous data were presented as mean, standard deviation (SD), or range and categorical variables as percentages. The diagnostic accuracy of CTA and CTA + rsCTP in detection of significant stenosis (50% cut-off) comparing ICA as reference standard was expressed by sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) on a per-segment and per-vessel basis. The improvement of sensitivity and specificity after rsCTP reclassification was assessed using McNemar test and net reclassification improvement index (NRI). The Cohen kappa value was used for interobserver agreement assessment. A All enrolled males were without symptoms of CHD; only two females (4%) stated previous unique episode of atypical chest pain, which was not further investigated. Baseline cohort characteristic is summarized in In average there was no significant change of BP after regadenoson application (systolic and diastolic BP decreased in 18 patients) and any serious adverse effects were not observed. Mean effective dose for CTA was 3.6 mSv ± 0.9, for rsCTP 8.9 mSv ± 2.4, and for rest CTP 8.4 ± 2.1 . Complete results are summarized in Sufficient quality for analysis of CTP was achieved in all patients; in 25 cases (31%) the manual adjustment of motion correction was necessary. Overall 17 (1.6%) segments were not covered within limited range of rsCTP acquisition. In 15 cases it was only basal anterior segment; basal anterior and midanterior segment were missed in only 2 cases. These segments ineligible for evaluation did not mean serious complication for assessment.ICA was performed in a total of 27 patients in whom relevant stress-induced perfusion defects were observed ; no completely fixed perfusion defects were proved . Overall 324 nonstenotic and 122 stenotic segments were recognized on CTA: 50 with mild stenosis (10–39%), 46 with intermediate stenosis (40–70%), and 26 with severe stenosis or complete occlusion (>70%). Stress-induced hypoperfusion in corresponding supply area was observed in 25 (96%) of >70% stenotic segments. On the other hand, no stress-induced perfusion defects were observed in segments corresponding to mild stenosis (10–39%). Altogether 14 mild stenotic segments were excluded from evaluation because of conflict of supply area with severe stenosis; altogether 108 stenotic segments were analyzed.p = 0.002; per-vessel: specificity 95 versus 75%, p = 0.012) and high overruling rate of rsCTP was proved in intermediate stenosis (40–70%). During the CTP evaluation, 15 (37%) of 50–70% stenoses were correctly reclassified due to the normal rsCTP as nonsignificant; one stenosis was reclassified falsely (p = 0.002 and p = 0.012). The additional value of CTP in severity stenosis reclassification was proved using NRI index, 0.32  and 0.21 , in complete number of stenoses. In a subset of intermediate stenoses (40–69%) the benefit of CTP was higher: 0.66  and 0.68 . Complete results are presented in Tables Combined evaluation of CTA + rsCTP had higher diagnostic accuracy over CTA alone  in comparison to combined CTA + CTP assessment .Interobserver agreement regarding stenosis significance assessment was lower in CTA alone assessment . Low specificity and positive predictive value are consequences due to the overestimation of the severity of stenosis caused by atherosclerotic plaques. This fact is the most relevant limitation of CTA, especially with regard to the corresponding risk and exposure of the patient during further examination  in the case of false-positive findings.The results of our study show the addition of the regadenoson-induced stress perfusion examination to CTA is significantly beneficial for diagnostic performance. An increase of specificity and positive predictive value in the identification of ≥50% stenosis was achieved with the combined evaluation of CTA and rsCTP in comparison to the CTA alone. We did not perform a per-patient analysis (limited number of subjects), but only 2 8%) patients with a positive CTA + CTP finding were considered as false positive on the basis of the ICA finding. The additional value of CTP assessment is in accordance with most of the previously published papers % patient. MoreoveKim et al. also demonstrated, in addition to the benefit of stress CTP for diagnostic accuracy, a superior contribution to the detection of ≥50% stenosis, compared with the 70% cut-off value . Althougz-axis has recently been a serious handicap of routine CTP performed on the second-generation dual-source CT scanners. In our cohort, 2% of segments were missed during rsCTP acquisition. Further technical development will certainly eliminate this factor.A limitation of a combined CTA + stress CTP + rest CTP protocol is the higher amount of the administered contrast medium and the radiation dose . The posA selective a2a blocker (regadenoson) represents a very simple and relatively safe option of pharmacological myocardial stress on a CT workplace, without the necessity of two venous ports, as in adenosine administration. We did not register any further serious undesirable reactions in our patients. In a multicentre study (ADVANCE), a similar effect was demonstrated using a regadenoson bolus injection and an adenosine infusion, which is often used for pharmacological stress in CT or MR .Practical usage of CTP and position in the diagnostic algorithm has not been settled. We chose patients with severe PAD or AAA who are limited for physical exercise. These patients undergo thorough preoperative investigation in our institution considering the high risk of latent CHD including cardiac stress test. The importance of stress test in asymptomatic patients prior to major vascular surgery is a widely discussed topic , 19. SevTo the best of our knowledge, there is no published study dealing with stress CTP used in a routine diagnostic algorithm replacing another established method. Also, enrolment of high-risk but asymptomatic patients is uncommon. The comparison of stress CTP with established stress tests in cardiology has been performed. Most recent multicentre studies have demonstrated comparable results in detecting the perfusion defects during stress myocardial perfusion imaging with the application of 99mTc-MIBI and stress CTP . Also, iOur study has several limitations that have to be mentioned. While detecting perfusion defects, we did not assess the extent within the width of the myocardial wall and no quantification procedure was used . There iOur results demonstrate a significant additional value of rsCTP in the assessment of intermediate stenosis (40–70%). A combined CTA + rsCTP protocol was also feasible as an alternative stress test with high diagnostic performance in asymptomatic patients at high risk of CHD, referred for major vascular surgery.


Cerebellar function is critical for coordinating movement and motor learning. However, events occurring in the cerebellum following spinal cord injury (SCI) have not been investigated in detail. We provide evidence of SCI-induced cerebellar synaptic changes involving a loss of granule cell parallel fiber input to distal regions of the Purkinje cell dendritic tree. This is accompanied by an apparent increase in synaptic contacts to Purkinje cell proximal dendrites, presumably from climbing fibers originating in the inferior olive. We also observed an early stage injury-induced decrease in the levels of cerebellin-1, a synaptic organizing molecule that is critical for establishing and maintaining parallel fiber-Purkinje cell synaptic integrity. Interestingly, this transsynaptic reorganizational pattern is consistent with that reported during development and in certain transgenic mouse models. To our knowledge, such a reorganizational event has not been described in response to SCI in adult rats. Regardless, the novel results of this study are important for understanding SCI-induced synaptic changes in the cerebellum, which may prove critical for strategies focusing on promoting functional recovery. Traumatic spinal cord injury (SCI) damages intrinsic local circuits, descending supraspinal pathways involved in motor function, autonomic systems, and ascending fiber systems conveying sensory information and nonconscious proprioception. To date, there are no effective treatments available that reduce these deficits or promote recovery of function. The lack of successful interventions is based, in part, on an incomplete understanding of the biochemical, physiological, and neuroanatomical changes that occur at different time points following injury. The majority of current research in the field of experimental SCI focuses on preservation, restoration, and activation of residual descending motor systems as well as intact intraspinal local circuits to promote recovery of motor function . SeveralIn rats, Clarke's nucleus relays nonconscious proprioception from spinal levels T1-L2 via the dorsal spinocerebellar tract (dSCT) to cerebellar lobules I, II, III, and VIII , 12. AxoA total of 20 adult female Long-Evans rats  weighing 225–250 gm at the time of surgery were used in this study. Animals were maintained under environmentally controlled conditions and subjected to a 12 h/12 h light/dark cycle with food and water provided ad libitum and acclimatized to the facility for seven days prior to starting the experiments. All procedure and handling techniques were in strict accordance with the National Institutes of Health guidelines for the care and use of laboratory animals and approved by the Institutional Animal Care and Use Committee. Animals were randomized into one of two groups (sham or SCI), with the SCI group subjected to a spinal cord contusion injury using the Infinite Horizon (IH) impactor device  by surgeons blinded to the treatment conditions. Briefly, rats were anesthetized with sodium pentobarbital , and the fur overlying the vertebral column was shaved and a dorsal laminectomy was performed at the tenth thoracic vertebra level (T10) to expose the spinal cord. The vertebral column was then stabilized by clamping the rostral T9 and caudal T11 vertebral bodies, so that the spinal cord remained in a horizontal plane. All SCI animals received a 200 kdyn (impact force) spinal cord contusion using the IH impactor device at the T10 level. This injury force results in a moderate to severe contusion injury in female rats in this weight range . Immedian = 7 SCI, n = 4 sham) was deeply anesthetized with 100 mg/kg (ip) sodium pentobarbital  and transcardially perfused with 100 mL of cold 0.1 M phosphate buffered saline  followed by 400 mL of 4% paraformaldehyde in PBS. Animals were then decapitated and the cerebellum was removed and snap-frozen in cold acetone (kept on dry ice) and stored at −80°C until being sectioned on a cryostat for histological analysis. In addition, a different set of injured animals and two sham controls were anesthetized at 24 hr (n = 3) or 7 days (n = 4) after SCI and decapitated immediately, and the cerebellum was rapidly removed and homogenized in RIPA buffer for western blot analysis of cerebellin-1 (Cbln-1).At 42 days following SCI, one set of rats  were then separated by SDS-PAGE using Criterion 4–20% Tris-HCl gels . The gels were transferred onto PVDF membranes (Millipore) and the membranes were incubated overnight at 4°C with a polyclonal antibody to Cbln-1 . The following day, blots were rinsed for 6 × 10 min in TBS buffer containing 0.1% Tween 20 and incubated with rat-absorbed, species appropriate antibodies conjugated to HRP . Finally, bots were rinsed for 6 × 10 min followed by incubation in Enhanced Chemiluminescence Plus solution  and exposure to chemiluminescent-sensitive film (Hybond CL). The blots were then stripped and reprobed with an antibody to β-tubulin , which served as a loading control as well as a normalizing signal. Quantitative densitometric analysis of Cbln-1 bands normalized to β-tubulin was conducted using ImageJ software (http://rsb.info.nih.gov/ij/).Western blot assays were conducted using procedures routinely conducted in the lab , 18. Brip < 0.05.Densitometric data from western blot were analyzed by ANOVA and Student-Newman-Keuls post hoc analyses using GraphPad Prism 6 . Differences were considered significant if The SMI-31 antibody recognizes high molecular weight phosphorylated neurofilaments in intact axons. Cerebellar sections from sham injured animals contained robust SMI-31 staining of axonal process in the granule cell (GC) layer. SMI-31 positive fibers were also observed coursing perpendicular to the lobule surface in the molecular layer (ML), which is consistent with the known projection pattern of the cerebellar parallel fibers . HoweverA loss of parallel fiber input to Purkinje cell (PC) dendrites has been shown to alter the innervation pattern of cerebellar climbing fibers, which originate in the inferior olive and synapse on the PC cell body and proximal dendritic tree. Therefore, additional cerebellar sections were double-labeled with antibodies to SMI-31 and synapsin-1 to visualize SCI-induced changes in Purkinje cell synaptic patterns. As shown in p < 0.01) and also compared to homogenates obtained from 24 hr post-SCI animals .The loss of parallel fiber staining suggests a disruption of contact with Purkinje cell dendrites. Therefore, we examined cerebellar homogenates from relatively early post-SCI time points for levels of cerebellin-1 (Cbln-1), a cerebellum specific glycoprotein thought to be important for maintaining synaptic integrity of parallel fibers with Purkinje cell dendrites , 20. As The results of this study suggest that a loss of spinocerebellar input due to a contusive injury to the rat thoracic spinal cord alters cerebellar circuitry. This is reflected by a loss of SMI-31 immunoreactive parallel fibers innervating distal segments of Purkinje cell dendrites and an increase in synapsin-1 immunoreactive profiles on cell bodies in the Purkinje cell layer. These structural changes are preceded by a loss of Cbln-1 levels as early as 7 days after injury. We hypothesize that a contusive injury at lower thoracic segments results in a loss of Clarke's nucleus neurons and their axons, which convey ascending nonconscious proprioception feedback to the cerebellum via spinocerebellar input to cerebellar granule cells. As a consequence, there is disruption of granule cell activation and an apparent loss of excitatory parallel fiber influence on the distal Purkinje cell dendrites.The major output out of cerebellar information to higher motor centers begins with the Purkinje cell, which integrates the activity of two excitatory afferents consisting of the parallel fibers from cerebellar granule cells and climbing fibers originating in the contralateral inferior olive. Parallel fiber axons ascend into the outer regions of the molecular layer where they bifurcate and synapse on distal segments of the Purkinje cell dendritic tree. Climbing fibers ascend into the more superficial territory of the molecular layer and terminate on the Purkinje cell bodies and proximal dendrites. During development, Purkinje cells receive inputs from multiple climbing fibers followed by a pruning phase characterized by a strict innervation pattern where a single climbing fiber innervates a specific Purkinje cell. Several studies have demonstrated that the developmental pattern of multiple climbing fiber inputs to a single Purkinje cell is maintained into adulthood under conditions that disrupt excitatory parallel fiber input to Purkinje cell dendrites.Glurδ2) [δ2 is expressed in Purkinje cell dendrites in opposition to Cbln-1-containing parallel fibers, and it has been suggested that Glurδ2 and Cbln-1 interact to stabilize parallel fiber-Purkinje cell synaptic integrity [δ2 gene expression. In support of this, the relatively early decrease in Cbln-1 levels we observed in the present study is consistent with the developmental literature and Cbln-1 gene deletion studies discussed above.For example, a loss of granule cells during development results in a loss of parallel fiber input and maintenance of multiple climbing fiber inputs to Purkinje cells , 22. In Glurδ2) . Glurδ2 ntegrity , 20. We Overall, the results of this study suggest that cerebellar circuitry undergoes significant alterations in response to injuries of the spinal cord that affect ascending spinocerebellar pathways. This novel observation extends our understanding of the pathophysiological consequences of SCI, which may be critical for developing strategies targeting the recovery of coordinated movement and motor learning.