CUB and SUSHI multiple domains 1 (CSMD1) gene as a statistically strong risk factor. CSMD1 is a complement control-related protein suggested to inhibit the classical complement pathway, being expressed in developing neurons. However, expression of CSMD1 is largely uncharacterized and relevance for behavioral phenotypes is not previously demonstrated. Here, we assess neuropsychological behaviors of a Csmd1 knockout (KO) mouse in a selection of standard behavioral tests. Deregulation of neuropsychological responses were observed in both the open field and the elevated plus maze tests, in which KO mice spent 55% and 33% less time than WT littermate mice in open areas, respectively. Altered behaviors were also observed in tail suspension and to higher acoustic stimuli, for which Csmd1 KO mice showed helplessness and moderate increase in startle amplitude, respectively. Furthermore, Csmd1 KO mice also displayed increased weight-gain and glucose tolerance, similar to a major phenotype of the metabolic syndrome that also has been associated to the human CSMD1 locus. Consistent with a role in the control of behaviors, Csmd1 was found highly expressed in the central nervous system (CNS), and with some expression in visceral fat and ovary, under tissue-specific control by a novel promoter-associated lncRNA. In summary, disruption of Csmd1 induces behaviors reminiscent of blunted emotional responses, anxiety and depression. These observations suggest an influence of the CSMD1 schizophrenia susceptibility gene on psychopathology and endophenotypes of the negative symptom spectra.Recent meta-analyses of schizophrenia genome-wide association studies (GWASs) have identified the  Schizophrenia is a severe psychiatric disorder of high heritability with a lifetime risk of approximately 1%. Onset of symptoms typically occurs in young adulthood, with chronic deregulation of behavior and emotions, including psychotic symptoms , emotional flattening and cognitive dysfunctions. Affective symptoms and anxiety are also regarded as key findings in this complex disorder CSMD1 and CSMD2, as well as to the CR1 locus (C3 receptors) PGC) identified CSMD1 as one of only seven verified loci in a large schizophrenia GWAS-based meta-analysis involving about 18.000 patients and 34.000 controls We previously found that the activity of complement-related factors (including complement component C3) was induced during long-term potentiation (LTP) of synaptic strength in the hippocampus of awake rats – a mechanistic model of learning and memory CSMD1 gene encodes a complement control-related protein with multiple copies of CUB and Sushi, a single transmembrane domain and an intracellular signaling peptide. The function of the CSMD1 protein is sparsely described, but inhibition of complement C3 activation is demonstrated in vitroCSMD1 has been reported in autoimmune disease  CSMD1 in immune responses remains to be further described. Distinct from a role in classic immunological pathways, it has also become clear that complement activity is tightly controlled in the brain, regulating C3/CR3-dependent axonal pruning by phagocytic microglia. This mechanism ensures precise wiring of neuronal circuits in the visual system during development The CSMD1 in psychopathology, GWAS studies on bipolar disorder (BIP) and major depressive disorder (MDD) also include nominal significant associations of genetic markers in CSMD1, which can be found in supplemental data in relevant literature CSMD1 may affect shared symptomology in several psychiatric disorders CSMD1 expression on neuropsychological endophenotypes using gene knock out (KO) mice. In the current study, we describe a constitutive Csmd1 KO mice and determine the effect of Csmd1 expression on behaviors using a selection of classical neuropsychological tests.Further suggesting a role of Csmd1 in the regulation of neuropsychological behaviors, we used a constitutive gene deletion to disrupt Csmd1 gene expression in mice. The Reference Sequence collection describes mouse Csmd1 as a 1.65 Mb long and 72 exon-rich gene, which encodes a 14 kb mRNA. To block expression through a minimal transgenic manipulation, a constitutive knockout (KO) of a 1 kb sequence from Csmd1 exon1/intron1 was generated  with highest levels in the cortex, while expression in peripheral tissues was not observed except for low levels in visceral fat and ovary (Csmd1 mRNA and protein expression was documented by QPCR (exon1–2 junction-specific assay) and immunoblotting (custom-made goat anti-Csmd1 antibody) on cortex samples, respectively (Csmd1 expression (Csmd1 KO (n = 4) and WT (n = 4) mice to the mouse genomic reference sequence, respectively  in exon 1, as documented by QPCR (exon 32–33 specific assay)  . We ther)  in the KO mice (Csmd1 promoter-associated (pas) long non-coding (lnc) RNA to be differentially expressed   the expression of pas-lncRNA was constitutively high  . The pasping CNS . In contely high . Thus, tCsmd1 KO mice . However, preliminary examinations of the original repository Csmd1 KO model revealed possible neuropsychological dysfunctions (n = 4–8 mice/genotype)  . AlthougCsmd1 KO and WT littermate mice, using a separate line of mice on a similar mixed genetic background (n = 23–24 mice/genotype). Mice were generated in one breeding scheme using heterozygote female and heterozygote male mice (except for the EPM test). Testing of littermate offspring started at age 10 weeks. Non-stressful tests were conducted on the same set of animals with a one-week interval in the sequence considered most optimal . We also recorded diurnal locomotor activity and metabolism for each animal, to control for underlying phenotypes that could interfere with the behavioral assessment in specific tests.We therefore performed a comprehensive analysis of behaviors and assessed a larger number of homozygous Csmd1 on the free adaption and exploration of a novel environment was first investigated by introducing the mice to the open filed (OF) arena: The time spent in the center of the field (open/exposed area) as opposed to a path along the borders  was recorded. Csmd1 KO mice spent 55% less time in the center , but travelled a similar total distance during the test period, as compared to the WT mice  .The effect of  WT mice . Csmd1 Kime bin) : WT miceCsmd1 expression on the exploration of new and open arenas, we examined the behavior of a separate  set of Csmd1 KO and WT littermate control mice in the elevated plus maze (EPM). In the EPM, mice are free to enter “closed” arms  or “open” arms (exposed platforms). While WT mice explored both corridors and platforms, Csmd1 KO mice spent 33% less time on the open platforms  .To replicate the effect of = 0.001)  and stop= 0.001) . The marCsmd1 KO and WT mice, finding a moderate, but significant elevation of startle amplitude to higher acoustic stimuli in Csmd1 KO mice   of acoustic startle response may further indicate defects in the gating of neuronal signals and is often identified in candidate genes of psychotic disorders = 0.043) . Examinae reflex .Csmd1 on affect-like response was analyzed by the tail suspension test (TST), measuring the total time spent immobile. The test is classically used to investigate the effect of anti-depressant drugs and associates immobility with features of depression (learned helplessness) Csmd1 KO mice spent 25% more time immobile as compared to WT littermates (both genders combined)  (Csmd1 expression to the state of learned helplessness.The effect of  = 0.02) . This fiCsmd1 was also assessed for the cognitive process of recognition memory, as we previously identified a possible role of complement control-related genes in LTP Csmd1 KO mice displayed a marked increase in exploratory activity to both familiar and novel objects, as compared to WT mice  indexes between mice genotypes , rearing, licking (drinking) and metabolic activities were measured in a Comprehensive Cage Monitoring System (CCMS) using non-invasive automated recordings.After completing the assessment of behaviors in OF, NORT, PPI and TST, we examined the behavior of Csmd1 depletion increased the total horizontal activity counts in male mice . In contrast, the total horizontal activity of female Csmd1 KO mice remained unaffected .The CCMS-based analyses revealed some genotype-specific activities in the dark-period only, but not of equal effect in both genders : In the ale mice . The incaffected , but witCsmd1 depletion on neuropsychological behaviors was identified in both genders.Changes in diurnal activity were gender and dark period specific, and therefore not believed to interfere with neuropsychological behaviors observed in the standard tests. The standard tests were performed during defined times of the light periods and the effect of ad libitum). We identified a significant accumulation of body weight over time, especially in female mice   did not differ between Csmd1 KO and WT mice (data not shown).We routinely monitored the weight of all animals during regular handling and when examining behaviors (water and food  = 0.01) . To gain WT mice . CCMS-baCSMD1 schizophrenia susceptibility gene by demonstrating that Csmd1 expression affects the deregulation of neuropsychological behaviors in mice, thereby showing a potential functional relevance of the this gene locus in psychopathology. Using a transgenic mouse line depleted of Csmd1 expression, we demonstrate that Csmd1 is primarily expressed in the brain  in Csmd1 KO mice  mouse genetic background was assessed for changes with respect to behaviors of social interaction, anhedonia, pre-pulse inhibition (PPI) of startle reflex and locomotor response to d-amphetamine Csmd1 KO mice. PPI is the only test overlapping with our report, and we both find no genotypic effects. However, considering the possible association of human CSMD1 with depression and anxiety-linked disorders with suspected genetic overlap to schizophrenia (see introduction), we tested relevant behaviors in Csmd1 KO mice in response to basic startle response, open field, elevated plus maze and tails suspension. Although we assess different behavioral test than Distler et al., positive findings may also be influences by additional factors: The mice used in the two studies are derived and maintained on separate mouse strains. While we used RNA sequencing to confirm genomic depletion and mechanisms of CNS-specific expression, residual expression and KO of the Csmd1 DNA segment is not characterized by genomic analyses by Distler et al. Also, we use a higher number of mice and analyze possible gender-mediated difference in behaviors. Analyzing high number of mice may be essential to compensate for increased behavioral variance that may be linked to heterozygous breeding patterns on mixed genetic backgrounds.Recently, a similar Csmd1 KO mice, may mask the effects of the Csmd1 KO in complex context-dependent tests like e.g. social learning. Also, we control for diurnal activity pattern of each mouse in our study, and therefore applied single housed mice prior to testing. Housing conditions may trigger some of the underlying susceptibility for neuropsychological deficits in Csmd1 KO mice. Social isolation in both young and adult mice has in recent reports been demonstrated to be a relevant stressor generating deficits in the function of neuronal circuits and behaviors Csmd1 KO mice to capture the influence of environment (stressors) x gene interactions is to be examined in future studies.Furthermore, anxiety- and depression-like behaviors, i.e. phenotypes we identified in Csmd1 KO mouse captures several major clinical phenotypes that have been associated to the human CSMD1 locus, both psychiatric disorders and somatic conditions. The presence of weight gain and altered glucose tolerance in Csmd1 KO mice is consistent with the strong association of CSMD1 to the body-mass-index and measures of the metabolic syndrome in the large Framingham Heart Study Csmd1 on metabolism as part of a complex biological system Csmd1 KO mice is to be explored in future studies.The Csmd1 on psychiatry-relevant behaviors. A prominent molecular feature of CSMD1 is the extracellular repeated pattern of CUB and Sushi, domains primarily found in complement- and development-related proteins An unresolved question is which molecular and cellular mechanisms mediate the influence of It has become increasingly clear that immune molecules, in addition to their roles in classical immunological pathways, contribute to brain development and function Csmd1 in adult brain may therefore indicate a relevance of the complement pathway in neuronal processes underlying the risk of schizophrenia, and may contribute to further insight into the suspected link between immunological factors and disease risk in psychiatric disorders.Our identification of predominant expression of These experiments were approved by the Norwegian Committee for Animal Research in accordance with European Community Council Directives.Csmd1 gene knock-out (KO) mouse model  was generated with embryonic stem cells derived from 129SvEvBrd mice, in which a 1,070 bp genomic sequences of the exon 1– intron 1 junction was replaced with a LacZ/Neo selection cassette Csmd1 in exon 1. In the present study, Csmd1 knockout mice were bred on a mixed background of 129SvEvBrd:C57BL/6NTac and genotyped according to the provided protocol . We used a high number of mice in the breeding strategy and tested a high number of littermate offspring, to control for potential effects mediated by variance in the random genetic contribution form the two mouse strains. For this study, Csmd1 KO and WT control mice were generated from the F2 generation using 30 heterozygote (HET) female mice and 15 HET male mice. From this we obtained 130 male  and 141 female  mice. Breeding of the Csmd1 KO genotype followed Mendelian inheritance. For this initial characterization of Csmd1 KO mice, we also controlled for genotypic effects on locomotor activity, which may interfere with the assessment of neuropsychological behaviors: Diurnal activities were measured in the compressive cage monitoring system (CCMS) for three days on single mice  . This preliminary study indicated that mice were in general healthy and fertile with 8–10 pups per litter, but showed signs of deregulation of specific neurological functions See .actb. Endogenous control by actb was compared to arbp mRNA and eukaryotic 18S mRNA and found stable across multiple tissues (data not shown). The following PCR primers  were used for quantitative SYBR green real-time RT–PCR : csmd1∶5′-TGTGCGTGTGGAATATCTGC, 5′- AAGCCAGGACTTTCAATGG; pas-lncRNA: 5′-GCCACCATTGAAAGGAGGTA, 5′-CAGAAAGGCATAGCAAAGGC; 5′-actb: CTTCTCCAGGGAGGAAGAGG, 5′-TACAGCTTCACCACCACAGC.Mice were first sedated with Isoba®vet before being deeply anaesthetized by intraperitonal injection of Pentobarbital. Transcardial perfusion with ice-cold NaCl (9 mg/ml) was performed before dissection of tissues. Isolated tissue samples were homogenized with TissueLyser (QIAGEN) and RNA isolated by RNeasy Mini Kit . The RNA concentration was measured using a NanoDrop ND-1000 spectrophotometer. cDNA was synthesized from 50 ng RNA with Superscript III First-Strand Synthesis System for RT–PCR , as previously described st Century Biochemicals lnc., MA, USA) was generated against the peptide sequence: Nt-QRVTETLAAWNDHR (encoded by Csmd1 exon 9). The antibody detected a protein band corresponding to full-length Csmd1 protein, when using freshly prepared protein homogenates obtained from mouse cortical tissue. Tissue samples were homogenized in 500 µl RIPA  and protein concentrations were determined following the DC Protein Assay . Proteins were separated on 3–8% Tris Acetate gel and transferred onto PVDF membranes . Detection was performed with donkey anti-goat IgG–HRP (1∶10 000) and Enhanced Chemiluminescence .A custom made affinity-purified goat anti-Csmd1 antibody . Ribosomal RNA was depleted with the RiboMinus™ Eukaryote Kit for RNA-Seq . Whole transcriptome cDNA was made from RNaseIII fragmented ribosomal depleted RNA following the protocol of the Library Builder™ Whole Transcriptome Core Kit . Samples were barcoded, pooled and sequenced on the SOLiD 5500xl system using a read length of 75 bp.Adapters from the sequence reads were trimmed using custom perl scripts. We used TopHat 1.3.3 Csmd1 KO mice  and 24 WT mice . Mice were housed individually 2 weeks before start of the first test. Tests were performed in the following sequential order, starting at the age of 10 weeks: 1st week: open field (OF); 2nd week: novel object recognition test (NORT); 3rd week: startle reflex and prepulse inhibition test (PPI); 4th week: tail suspension test (TST); 5–6th week: comprehensive cage monitoring system (CCMS). Testing was performed in the first phase of the light cycle between 6 AM and 12 PM, and standard acclimation procedures were applied for each behavioral test. The experiment was recorded and motion tracked by Viewpoint where applicable. Elevated plus maze test (EPM) was performed on a separate colony of female littermate mice, obtained from a heterozygous breeding strategy (8 Csmd1 KO and 13 WT mice) and was tested for a period of 6 minutes in the EPM arena at 12 weeks of age. A brief description of behavioral tests can be found in Neuropsychological behaviors were examined in a selection of standard tests using 23 p<0.05. Data are shown as mean ± SEM in figures or text. Behavioral data were analyzed with GraphPad Prism , and statistical significance for expression analyses was examined in Excel (Microsoft Corporation).Statistical significance was evaluated with un-paired t-test or 2-way analysis of variance (ANOVA) as applicable. Significance threshold was set to File S1Supporting information.(DOCX)Click here for additional data file.


Our ultimate goal is to detect the entire human microbiome, in health and in disease, in a single reaction tube, and employing only commercially available reagents. To that end, we adapted molecular inversion probes to detect bacteria using solely a massively multiplex molecular technology. This molecular probe technology does not require growth of the bacteria in culture. Rather, the molecular probe technology requires only a sequence of forty sequential bases unique to the genome of the bacterium of interest. In this communication, we report the first results of employing our molecular probes to detect bacteria in clinical samples.While the assay on Affymetrix GenFlex Tag16K arrays allows the multiplexing of the detection of the bacteria in each clinical sample, one Affymetrix GenFlex Tag16K array must be used for each clinical sample. To multiplex the clinical samples, we introduce a second, independent assay for the molecular probes employing Sequencing by Oligonucleotide Ligation and Detection. By adding one unique oligonucleotide barcode for each clinical sample, we combine the samples after processing, but before sequencing, and sequence them together.Overall, we have employed 192 molecular probes representing 40 bacteria to detect the bacteria in twenty-one vaginal swabs as assessed by the Affymetrix GenFlex Tag16K assay and fourteen of those by the Sequencing by Oligonucleotide Ligation and Detection assay. The correlations among the assays were excellent. This category included novel bacteria, which were defined as previously . The novThe Tag4 array contains 8-μm features. Each 20-mer barcode is replicated and dispersed five times on the array . We haveL. delbrueckii produced no signals above background (unoccupied 20-mers on the Tag4 array). Therefore, we employed these six probes as the negative controls. We calculated the average fluorescence signal and standard deviation for the six L. delbrueckii probes. To minimize false positives at this stage of the development of the molecular probe technology, we calculated the average plus five standard deviations. We employed that number as the cut-off between negative and positive for each molecular probe on a Tag4 array. Also to minimize false positives at this stage of the development of the molecular probe technology, we required concordance of the data. A majority (> 50%) of the molecular probes for any given bacterium must have been positive for us to call a bacterium present. There is a potential problem with this procedure that is related to possible strain variation in genome sequence: i.e., genome sequence variation within the same species. Any given molecular probe could be authentically positive for one strain and authentically negative for another.On all the Tag4 arrays, the six molecular probes for Acinetobacter baumannii , B. fragilis , Bifidobacterium longum , and two probes for T. pallidum . Therefore, the data from these five molecular probes were excluded from the analyses. Two of three probes for Gardnerella vaginalis (ED116 and ED121B) were also positive for all five simulated clinical samples, when there was no G. vaginalis DNA present in any sample. Since we would not call a bacterium present or absent on the basis of one molecular probe, G. vaginalis was excluded from the analyses. What remained for evaluation of the simulated clinical samples were 183 molecular probes representing 39 bacteria.For the five simulated clinical samples, five molecular probes were positive for all samples whether their corresponding DNA was present or not: one probe each for Lactobacillus probes). We abandoned the data therefrom: two probes for A. baumannii  were positive for twenty and nineteen samples, respectively; two probes for G. vaginalis ; two probes for Streptococcus pneumoniae  were positive for twelve and thirteen samples, respectively; one probe for S. pyogenes  was positive for ten samples; and one probe for Fusobacterium nucleatum  was positive for seventeen samples. The data from all six Enterobacter probes (leaving none) were excluded. G. vaginalis and Pseudomonas aeruginosa were left with only one molecular probe each. Since we would not make a present/absent determination on the basis of one molecular probe, G. vaginalis and P. aeruginosa were removed from consideration within the clinical samples. Only two promiscuous probes were shared between both sets of Tag4 data: ED116 and ED121B . Whereas one A. baumannii probe (ED211) was promiscuous in the simulated clinical sample data, two other A. baumannii probes (ED212 and ED213) were promiscuous in the clinical sample data. What remained for the authentic clinical samples assayed on the Tag4 array were (192 - 17 =) 175 molecular probes representing 37 bacteria.We conducted an analogous process for detecting promiscuous molecular probes within the Tag4 data for the twenty-one clinical samples. Again, to minimize false positives at this stage of the development of the molecular probe technology, we identified molecular probes positive for ten or more  of the clinical samples  files of the microarray data are publicly available on the Stanford Genome Technology Center website The primers used to amplify the product for SOLiD sequencing are presented in Table S1 , was performed at the University of California Santa Cruz Genome Sequencing Center. We have published our procedure for the library preparation of the samples for SOLiD sequencing ,26. We fin silico to create the expected reference sequences. The forward and reverse complements of all molecular tag reference sequences were translated from base space into color space using a custom perl script. We trimmed 20 bases from the 5' end of each read to remove the adapter.Molecular tag sequences were ligated and amplified We aligned the sequence reads to each reference molecular tag sequence using a publically available Smith-Waterman local alignment in colorspace with affine gap penalties . We deteL. delbrueckii as the negative control. The average number of SOLiD reads and standard deviation for the six were calculated. Again, to minimize false positives at this stage of the development of the molecular probe technology, we used the average plus five standard deviations as the cut-off between negative and positive for each molecular probe. Also to minimize the number of false positives at this stage of the development of the molecular probe technology, concordance of the data was required. A majority of the molecular probes for any given microbe must have been positive to score the microbe as present. The same caveats as for the Tag4 data analysis apply.Analogously to the processing of the Tag4 data, we employed the data for the six probes for G. vaginalis) and ED675 (L. jensenii) were positive for all five simulated clinical samples, when neither DNA was present in any. ED611 (B. longum) and ED121B  were positive for four of the five simulated clinical samples. Therefore, the data from these four probes were excluded from the analyses. As only one G. vaginalis probe remained, G. vaginalis was removed from further consideration. That left 187 molecular probes representing 39 bacteria.We identified promiscuous molecular probes for the five simulated clinical samples. ED116 (Lactobacillus). That group included sixteen molecular probes: A. baumannii , B. fragilis , B. longum , G. vaginalis , L. jensenii , Staphylococcus aureus , S. agalactiae , T. pallidum , and Ureaplasma urealyticum . Unfortunately, there were no remaining molecular probes for G. vaginalis, and S. agalactiae was left with only one molecular probe. Since we would not make a present/absent determination on the basis of one molecular probe, S. agalactiae was removed from consideration within the clinical samples.  What remained for the authentic clinical samples were (192 - 17 =) 175 molecular probes representing 38 bacteria.There were SOLiD data for fourteen clinical samples. Since these were sequenced with the simulated clinical samples, the identical negative control was employed. We identified promiscuous molecular probes for the clinical samples. We excluded the data for any probe positive for seven (50%) or more samples , ED611 (B. longum), and ED675 (L. jensenii). Overall, only two probes were promiscuous in all four sets of data: ED116 and ED121B . ED611 (B. longum) was promiscuous in three of the four sets. No other probes were that promiscuous.The four promiscuous probes from the SOLiD data for the simulated clinical samples were also promiscuous within the clinical samples: ED116 and ED121B . Figure S2. Quantitative data for the SOLiD assay for simulated clinical sample C (SCC). Figure S3. Quantitative data for the SOLiD assay for simulated clinical sample D (SCD). Figure S4. Quantitative data for the SOLiD assay for simulated clinical sample E (SCE).are from . Table SClick here for file


The classical form of diagnostic imaging includes X-ray-based imaging systems, magnetic resonance imaging (MRI), and sonographic imaging. Such imaging systems in a given clinical situation document the findings which reflect the end effect of molecular/biochemical changes associated with a particular disease condition. The lack of information at molecular or cellular level as a precursor to a disease condition resulted into emergence of “Molecular Imaging.” This approach is expected to provide an imaging tool that enables visualization, characterization, and measurement of biological events at molecular and cellular level both in human being and other living species. Broadly speaking, molecular imaging investigates molecular signature of the disease through measurement and characterization of biological processes in a molecular or cellular compartment and thus enables opportunities to replace population-based treatment methods by personalized genotype/phenotype-adapted concepts. If this In other words, cell growth or destruction responsible for biochemical changes can be visualized and quantified by using molecular imaging techniques so as to delineate disease pattern specific to an individual(s) in its early stages. Such findings would score in terms of personalized medicine based on physiological and/or biochemical changes particularly when it is well known that anatomical/structural changes in a given disease condition appear much later than the biochemical changes.A wide range of fast emerging techniques rely on visualization of biological events or biochemical changes in a biological system. These include molecular nuclear medicine, magnetic resonance spectroscopy (to identify proteins produced during the progression of the disease), and bioluminescence imaging. These techniques can also be successfully deployed to differentiate between rapidly proliferating tumor cells and normal cells. In addition, these techniques can be used to depict angiogenesis of formation of new blood vessels. Thus the quantification of cellular changes with time enables characterization of disease process.Nuclear medicine with latest developments in terms of positron emission tomography (PET) radiopharmaceuticals and fusiThus, the hybrid imaging owing to its capability to provide physiostructural information shall continue to be the cornerstone in molecular imaging. It wouldIn the era of personalized medicine molecular imaging with appropriate biomarkers/probe would initiate a concept of individualized imaging and hence individualized diagnosis as well as treatment. Such tre


Meles meles) culling as a control strategy. Our study complements previous analyses of the RBCT data (focusing on treatment effects) by presenting analyses of herd-level risks factors associated with the probability of a confirmed bTB breakdown in herds within each treatment: repeated widespread proactive culling, localized reactive culling and no culling (survey-only).The control of bovine tuberculosis (bTB) remains a priority on the public health agenda in Great Britain, after launching in 1998 the Randomised Badger Culling Trial (RBCT) to evaluate the effectiveness of badger (M. bovis positive badgers initially culled within 1500 m of a farm was the strongest predictor of the risk of a confirmed bTB breakdown.New cases of bTB breakdowns were monitored inside the RBCT areas from the end of the first proactive badger cull to one year after the last proactive cull. The risk of a herd bTB breakdown was modeled using logistic regression and proportional hazard models adjusting for local farm-level risk factors. Inside survey-only and reactive areas, increased numbers of active badger setts and cattle herds within 1500 m of a farm were associated with an increased bTB risk. Inside proactive areas, the number of The use of herd-based models provide insights into how local cattle and badger populations affect the bTB breakdown risks of individual cattle herds in the absence of and in the presence of badger culling. These measures of local bTB risks could be integrated into a risk-based herd testing programme to improve the targeting of interventions aimed at reducing the risks of bTB transmission. Mycobacterium bovisM. bovis to human health is low in most developed countries, the main causes of concern related to M. bovis in industrialized countries are epizootics in domesticated and wild mammal populations M. bovis remains a significant livestock zoonosis in the European Union where some member states experience a reemergence of the disease despite significant historical efforts to implement eradication plans. In Great Britain, the disease was eliminated from most cattle herds by 1960, with the exception of infection hotspots in southwest England, after the implementation of a herd testing and slaughter policy M. bovis in wildlife host populations, acting as reservoirs of infection, in particular badgers (Meles meles) Bovine tuberculosis (bTB) remains an important public health concern worldwide as a result of deficiencies in preventing and/or controlling measures targeting the spread of its causative agent M. bovisM. bovis than younger ones Risk factors associated with bTB have been investigated in case-control studies in Europe and the USA M. bovis can infect a wide range of wild animals Trichosurus vulpecula) are the primary wildlife reservoir of bovine bTB in New Zealand Odocoileus virginianus) in Michigan Bison bison athabascae) Syncerus caffer) in Southern Africa Sus scrofa) in Southern Europe M. bovis. The Randomised Badger Culling Trial (RBCT) was launched in 1998 to evaluate the effectiveness of badger culling as a control strategy for bTB in Britain 2) trial areas recruited as matched sets of three, known as “triplets”. Detailed field surveys in all trial areas for which consent was obtained  to identify the most important bTB breakdown risk factors for herds within the RBCT areas.M. bovis) from “unconfirmed breakdowns” (incidents in which one or more cattle reacted to the tuberculin test but infection was not confirmed at postmortem or by culture). Herds with the same County Parish Holding Herd numbers (CPHH: unique herd identifier) which were registered in different treatment groups (n = 14); herds which were archived before the start of the RBCT (n = 22) and herds which showed no evidence of having had a bTB disclosing test during the RBCT (n = 745) were removed from the VETNET records; leaving us with 1306 unique herds recorded in RBCT proactive areas, 1380 unique herds recorded in RBCT survey-only areas and 1320 unique herds recorded in RBCT reactive areas.The Defra animal health information system (VETNET) provided data on cattle bTB tests and herd breakdowns, distinguishing between “confirmed breakdowns” .On the basis of details recorded in the RBCT database, farms were categorized into one of three enterprise types: beef, dairy and other . The median herd size was 72 animals  . The hisM. bovis positive (+) badgers culled, the number/density of active badger setts and the number/density of neighbouring cattle herds within 500, 1000 and 1500 m of all the land parcels belonging to a farm were extracted from the RBCT geodatabase   (to be) in a better position to tackle the disease” Supporting Information S1Univariable models and alternative multivariable models of herd-level bTB risk.(DOC)Click here for additional data file.


With the speeding tendency of aging society, human neurological disorders have posed an ever increasing threat to public health care. Human neurological diseases include ischemic brain injury, Alzheimer's disease, Parkinson's disease, and spinal cord injury, which are induced by impairment or specific degeneration of different types of neurons in central nervous system. Currently, there are no more effective treatments against these diseases. Traditional Chinese medicine (TCM) is focused on, which can provide new strategies for the therapy in neurological disorders. TCM, including Chinese herb medicine, acupuncture, and other nonmedication therapies, has its unique therapies in treating neurological diseases. In order to improve the treatment of these disorders by optimizing strategies using TCM and evaluate the therapeutic effects, we have summarized molecular imaging, a new promising technology, to assess noninvasively disease specific in cellular and molecular levels of living models in vivo, that was applied in TCM therapy for neurological diseases. In this review, we mainly focus on applying diverse molecular imaging methodologies in different TCM therapies and monitoring neurological disease, and unveiling the mysteries of TCM. With the dramatic improvement of average life expectancy and increasing trend of the aged population in recent years, neurological diseases have become a major problem of public health. Human neurological diseases such as ischemic brain injury, Alzheimer's disease (AD), Parkinson's disease (PD), depression, and spinal cord injury are caused by loss and impairment of different types of neurons in central nervous system. Current therapies, mainly focusing on western therapy such as interventional procedures, surgery, and synthetic drug, are limited in their ability to improve neural function because they fail to repair damaged neurons or improve neural regeneration. With intense exchanges between the eastern and western of the world, traditional Chinese medicine (TCM) becomes more popular and is progressively recognized all over the word. Here we mainly focus on using TCM treatment against neurological disorders.TCM is a unique system to diagnose and cure illness, and TCM has been widely investigated, especially in China . TCM is With developing of molecular imaging technology in the early 21st century, the unique theoretical system of medicine and therapy gradually was elaborated. Molecular imaging generally was defined as the visual representation, characterization, and quantification of biological processes at the cellular and molecular levels within intact living organism  IBZM-SPECT imaging. The SPECT imaging showed that the intensity of radionuclide or radiopharmaceutical uptake in RA group is higher than that in sham acupuncture (SA) group  group . By usinTCM including Chinese herbal medicine, acupuncture, and other nonmedication therapies may offer a unique strategy in combating the devastating neurological diseases. A variety of molecular imaging methodologies, such as PET, SPECT, and MRI, have been applied in TCM therapy for neurological disorders, which shift away from classical morphological measures towards the assessment of functional, cellular, metabolic, and molecular information in vivo . In addi However, more effective treatment is critically required not only for ameliorating the clinical symptoms but also slowing down or reversing of the progress of the disease. Thus, it is still far from thoroughly verifying the effectiveness of TCM in neurological diseases treatment, and it shows that we can probe the consequences of TCM quantitatively with a target specific imaging technique. As TCM in general has the reputation of being mysterious, our review offers an example of how TCM therapy in neurological disorders can be explained with modern scientific language.11C, 18F, and 15O -salvianic acid A as a tracer to study its dynamic biodistribution of in the brain and the correlation of its concentration and the changes in brain function.In the future, we also could take advantage of molecular imaging, especially PET imaging, to investigate the pharmacokinetics study of TCM. We choose some biological active components of Chinese herb medicine, which are similar to the tracers of PET in molecular structure. Later, the biological active component, as a tracer, is labelled with a positron emitting isotope, such as  and 15O . For exadiseases . The molIn conclusion, we believe that a combination of TCM and modern molecular imaging techniques will initiate new approaches for neurological diseases treatment.


In 2006 the International Organization for Standardization (ISO) published Standard 15883 for Washer-disinfectors (WD), with Part 3 for human waste containers: what states that manual procedures must be avoided as much as possible. I conducted research to get insight in the awareness of ISO15883 and into bedpan management.Awareness rising for safe bedpan management.Conducting a Global survey (2010) of bedpan management, the awareness of ISO15883 and bedpans causative for HAI. I performed Planned and ‘secret shopper‘ visits in hospital sluice rooms (31) on all continents and had Experience exchange with colleagues (>250).Survey- Response:13% in 54 countriesClostridium difficile, Norovirus, MRSA,- HAI (4-21%) reported with: Pseudomonas aeruginosa, Salmonella species and Acinetobacter baumannii- - Majority never searched for bedpans as a potential source- Manual handling is done (65%)- ISO15883 is known in northwest Europe (76%) in the rest of the world (14-37%)Visits- Diapers and urine catheter as replacement for bedpans is regular done- Nurses, nurse aids, relatives and others do the job without training ‘how to’- Personal Protective Equipment is seldom used- Bedpans without covers are transported to a toilet or slobhopper- Manual empty of the content with (bloody) faeces causing splashes and splatters- Regular only ‘cleaning’ by rinsing or spraying with cold water- Disinfection without attention for the right product, duration and prior cleaning- Not well equipped sluice rooms- Macerators or WD are not maintained or validatedExperience exchangeNurses and physicians seldom look after the process and are glad that it is brought to their attention. Most WD or macerators are often malfunctioning or unused. No available guidelines for bedpan management.There is a risk for HAI caused by poorly performed bedpan management. The risk of handling bedpans can be easily ignored because the chosen practice depends on habits, rituals and budget. Manual handling contributes to the risk of environmental contamination and transmission. In case of using machines maintenance and validation must be regular checked. Hospitals have to recognize their weakness in bedpan management and must set improvements and investments.G. Van Knippenberg-Gordebeke Consultant for For MEIKO Germany


Minimally invasive aortic valve replacement (MIAVR) was first described in 1996 as an alternative to median sternotomy for AVR. Its uptake has been sporadic in cardiac surgery units in the UK. It remains unclear and somewhat controversial whether it has superior patient outcomes over conventional sternotomy. This survey aims to explore the reason for the low uptake by consultant surgeons in the UK and assess current opinions regarding the benefits, evidence base and barriers to MIAVR.An online survey was created with 20 questions designed to explore the reasons that the current consultant population uses to base its practise on MIAVR. The link was distributed by the UK Society of Cardiothoracic Surgeons to the consultant members. Opinions on benefits of MIAVR compared with conventional sternotomy were evaluated along with potential barriers and areas for future research. Information regarding use of MIAVR in their clinical practice was obtained.Forty-nine consultants responded (response rate approximately 30%).67% of the consultants have performed MIAVR. 84% of the consultants identified that MIAVR was performed in their unit. 45% who identified themselves as performing MIAVR have carried out less than 15 procedures. 22% have carried out more than 26 procedures.Only 48% of consultants felt there was an overall benefit of MIAVR when compared with conventional. Consultants with less than 10 years experience had a significantly more positive opinion of MIAVR than their more experienced colleagues.These results suggest that consultants’ opinions on the benefits and drawbacks of MIAVR remain divided. It has highlighted some perceived barriers and the desire for further evidence on which to base practice. Cost-benefit analysis would enable units to make an informed decision on the most effective approach to patients requiring AVR.


Background. Inspite of the fact that accidental caustic ingestion is an entirely easily preventable problem, it has however persisted in rural Nigerian communities because the commonly implicated agent which is caustic soda  is sold in open markets without restrictive legislations. This study aims to identify the perpetuating factors of paediatric caustic ingestion and recommend preventive measures. Method. Retrospective analysis of clinical records of our paediatric patients who presented following caustic ingestion between November 2006 and November 2010 was made for demography, socioeconomic status of parent(s), caustic substance ingested with amount (where known), circumstance of ingestion, means of oesophageal evaluation, treatment and outcome. Results. There were 16 paediatric cases of caustic ingestion during the study period with age ranging from 1 to 18 years with mode in the 1–3 years group and male : female ratio 4.3 : 1. In 100% of the cases, the caustic ingestion was accidental, while caustic soda was the agent in 93.7%, and 87.5% of the parents were into local soap and detergent production. In all patients, the oesophagus was evaluated with late barium swallow/meal and oesophagoscopy before treatment. Conclusion. Caustic ingestion among rural children in Nigeria can be prevented. The ingestion of caustic substances induces a wide range of injuries to the gastrointestinal tract, which can be mild or fatal, or leads to chronic disease and is a worldwide problem , 2. CausCaustic agents with a pH level <2 or >12 rapidly penetrate layers of the esophagus resulting in necrosis-induced eschar formation in the mucosa that limits deep tissue penetration. The extent of tissue destruction depends on the physical form, type, and concentration of corrosive agent, premorbid state of the tissue, contact duration, and amount of substance ingested. Esophageal mucosa is thought to be more resistant to acidic than alkaline substances, as alkaline liquids are often highly viscous and thus persist for a longer duration in the esophageal mucosa causing liquefactive necrosis, and serious esophageal injury becomes inevitable once alkaline liquids penetrate deep muscle layers [Caustic ingestion by children has persisted in most developing countries including Nigeria –6. InspiAlarmed by the frequent presentation of rural children to our new cardiothoracic surgery unit with complaints of caustic ingestion, we set out to analyse our experience in the first four years with a view to discover the risk factors to exposure to caustic agents in the rural Nigerian communities and clinicopathologic characteristics of caustic ingestion in our paediatric patients and recommend preventive measures against accidental caustic ingestion.It was analyse the paediatric cases of caustic ingestion in our new cardiothoracic surgery unit with a view to identify the perpetuating factors and recommend preventive measures.This is a retrospective study of paediatric patients who presented to our unit following caustic ingestion between November 2006 and November 2010 (four years). Sources of data included admission records, patients' records, cardiothoracic surgery unit records, and theatre registers. Data extracted for analysis included patients' demographic data, parental socioeconomic data, diagnosis, caustic agent ingested including quantity (where possible), and reason for ingestion, complication, and treatment with outcome. All patients were evaluated in regard to the physical examination, routine laboratory investigations, and radiological assessment performed. Our unit though new and first in the state with a population of 3,902,051 people  is still creating awareness amongst the doctors on its existence and the range of pathologies that should be referred to it.The total number of children who presented following caustic ingestion during the study period was 16 with age ranging from one year and two months to 18 years (mean age = 5 years and 4 months). The modal age group was 1–3 years where 10 patients belonged. There were 13 boys and three girls (M : F = 4.3 : 1). The reason for caustic ingestion was accidental in all the 16 patients (100%), while 15 (93.7%) out of the 16 patients ingested caustic soda. The only ingestion of acid was in the 18-year-old daughter of a civil servant who mistakenly ingested concentrated acid stored in wine bottle in her mother's wardrobe. The highest educational attainment of the mothers of the patients was secondary education in 9 (56%) and primary education in the remaining 44%. Also 87.5% (14) of the mothers of the patients were engaged in local soap making, while one each was a civil servant and farmer, respectively. All the patients (100%) were rural dwellers.Twelve (75%) out of the 16 patients presented to our unit on referral after having presented to and received initial treatment from other hospitals, while only four 25%) presented to our unit early following caustic ingestion. All patients (100%) had barium oesophagogram and late oesophagoscopy during the assessment of the oesophagus. Long-segment oesophageal stricture was present in three (18.7%) of the patients, one of which has had successful oesophageal replacement operation using colonic conduit (Figures 5% presenStamm's gastrostomy was done for seven patients due to severe dysphagia, while feeding jejunostomy was done for one patient because of extensive oesophago-gastro-duodenal strictures. Eight patients were able to swallow well after three to four days of acute-phase treatment.Caustic ingestion in children though still encountered globally is certainly most frequently encountered by thoracic surgeons working in the developing countries where poverty level is high, and there are no restrictions to sale and handling of caustic chemicals , 4–7. ThThis study has collaborated the fact that alkali damages oesophagus more than stomach, , 7 as alPublic enlightenment of present and prospective rural dwellers who are engaged in local soap making on the dangers of caustic ingestion, storage of caustic agent in safe places that cannot be reached by children, separation of soap making from food processing and cooking area of the household, creation of local/community centres for local soap making, incorporation of colour and perfume to caustic soda granules at the point of production to make its solution to possess colour and odour and therefore distinguishable from drinking water,restriction on sale and handling of caustic substances.Caustic ingestion among rural children in Nigeria appears to be increasing in the present research. The stricture rate of 50% contributes to the workload of cardiothoracic surgeons practicing in Nigeria. It is hoped that with widespread adoption of the preventive measures advocated by the present study, the menace can be drastically reduced if not eradicated among rural children.


During the last year we have developed Open3DQSAR, an open-source tool aimed at high-throughput 3D-QSAR model building and evaluation . During Molecular interaction fields of different kinds  can be seamlessly computed and combined in a 3D-QSAR model from within Open3DQSAR thanks to its interoperability with OpenBabel, Molecular Discovery GRID and most QM packages.In the poster, we describe the recent integration in Open3DQSAR of existing open-source tools to perform conformational analysis and molecular alignment, in order to automatically generate an exhaustive pool of pharmacophore hypotheses for a given dataset. Subsequently, such hypotheses are scored according to their ability to yield a predictive and robust 3D-QSAR model Figure . We appl


Thermotoga petrophila (TpMan) is a hyperthermostable enzyme that catalyzes the hydrolysis of β-1,4-mannoside linkages in various mannan-containing polysaccharides. A recent study reported that TpMan is composed of a GH5 catalytic domain joined by a linker to a carbohydrate-binding domain. However, at this moment, there is no three-dimensional structure determined for TpMan. Little is known about the conformation of the TpMan as well as the role of the length and flexibility of the linker on the spatial arrangement of the constitutive domains. In this study, we report the first structural characterization of the entire TpMan by small-angle X-ray scattering combined with the three-dimensional structures of the individual domains in order to shed light on the low-resolution model, overall dimensions, and flexibility of this modular enzyme at different temperatures. The results are consistent with a linker with a compact structure and that occupies a small volume with respect to its large number of amino acids. Furthermore, at 20°C the results are consistent with a model where TpMan is a molecule composed of three distinct domains and that presents some level of molecular flexibility in solution. Even though the full enzyme has some degree of molecular flexibility, there might be a preferable conformation, which could be described by the rigid-body modeling procedure. Finally, the results indicate that TpMan undergoes a temperature-driven transition between conformational states without a significant disruption of its secondary structure. Our results suggest that the linker can optimize the geometry between the other two domains with respect to the substrate at high temperatures. These studies should provide a useful basis for future biophysical studies of entire TpMan.Endo-β-1,4-mannanase from  Lignocellulosic biomass is the major renewable carbon source in nature with several applications and consists of cellulose, hemicellulose and lignin with an approximate representation of 2∶1:1 The biodegradation of cellulose and hemicellulose polymers involves the concerted action of a variety of hydrolytic enzymes. Cellulases and xylanases have been extensively studied due to their several industrial applications principally relating to biorefineries, however, mannan-degrading enzymes have enjoyed less attention. The major enzymes involved in the biodegradation of mannan polysaccharides are β-mannanase (EC 3.2.178), β-mannosidase (EC 3.2.1.25), and β-glucosidase (EC 3.2.1.21) Thermotoga petrophila (T. petrophila) strain RKU-1 (T) is a hyperthermophilic bacterium isolated from the Kubiki oil reservoir in Niigata (Japan) T. petrophila (TpMan) is an enzyme composed of 667 amino acid residues (Gene Bank: ABQ47550.1). The first 20 amino acid residues are a signal peptide, which are removed after protein exportation. TpMan enzyme presents optimal activity at the temperature range of 81–93°C The hyperthermostable bacterial endo-β-1,4-mannanase (EC 3.2.1.78) from T. petrophila by small-angle X-ray scattering in order to shed light on the low-resolution molecular envelope, overall dimensions, and flexibility of this modular enzyme at different temperatures.In the present study, the bacterial TpMan was analyzed using biophysical techniques as well as with bioinformatics tools. Here, we report the first structural characterization of hyperthermostable endo-β-1,4-mannanase from the bacterium The activity of an enzyme is dependent on pH and temperature, conditions that invoke changes in enzyme folding, conformation, and protonation. As a first step of our studies, the mannan endo-1,4-β-mannosidase activity of TpMan and TpManGH5 were studied as a function of temperature using locust bean gum  as substrate . Our resSR) of TpMan were practically independent of protein concentration over the range 0.5 to 8 mg/mL at 20°C and pH 6  provides a fast and convenient way to monitor the size of proteins in solution and also investigate whether the enzyme aggregates at high temperatures and/or at high protein concentrations and pH 6 . Further.5 mg/mL . After igregates  [16]. AfsR were practically independent of temperature over the range 20 to 75°C at pH 6 and 0.5 mg/mL  evaluated with the Guinier approximation at 20, 50 and 65°C were 32±1 Å, 34±1 Å and 36±1 Å, respectively. The pair distance distribution functions, P(r), evaluated by the indirect Fourier transform with GNOM package maxD) of 105±5 Å, 110±5 Å and 115±5 Å, respectively. The gR values determined for TpMan at 20, 50 and 65°C using the GNOM package were 32.50±0.14 Å, 34.87±0.15 Å and 35.98±0.19 Å, respectively.To obtain more detailed information about the tertiary structure of TpMan and its molecular shape, we submitted TpMan to small-angle X-ray scattering (SAXS) analysis as a function of temperature. The X-ray scattering curves obtained at 8 mg/mL and three different temperatures  for TpMan at pH 6 are shown in ab initio simulations were performed without imposing any symmetry restrictions, and the models described very well the experimental curves. The results, in each case, were compared with each other by the use of the DAMAVER procedure maxD of approximately 105 Å. However, the reconstructed low-resolution model of TpMan at 65°C and pH 6, restored at 18 Å resolution, showed a more elongated shape roughly twice as long as they are wide, with a maxD of approximately 115 Å. The atomic coordinates of the low-resolution models of TpMan are available by request to the corresponding author of this article. A summary of the main SAXS results described in this study for TpMan is given in The low-resolution molecular shape of TpMan was determined from X-ray scattering data with DAMMIN package q2.I vs. q) q <0.15 Å−1), however, it does not decay close to zero at higher q values, presenting a slight elevated baseline. This result suggests that TpMan may exhibit some degree of flexibility at 20°C and pH 6, probably owing to the inherent flexibility between domains. At 50°C and pH 6, the curve shows also a well-defined maximum and presents a subtle decrease of the baseline when compared to the same enzyme at 20°C  (Clostridium thermocellum cellobiohydrolase A (CbhA) A recent study presents the crystal structure of GH5 catalytic domain  (TmMan) . Therefo (TmMan)  from itsFar-UV CD spectroscopy was used to probe the secondary structure of the expressed TpMan and TpManGH5 products to assess their folding in terms of secondary structure and to draw comparisons . The CD The percentages of α-helix, β-strand, and coils (turns and irregular structures) were obtained by deconvolution of the experimental spectra using four independent methods and expressed as a mean and standard deviation . In the case of TpManGH5, the deconvolution of the spectrum yielded the following average values for the secondary structure : 42±4% αThe results of the secondary-structure prediction for the complete TpMan sequence (Gene Bank: ABQ47550.1) and its individual domains are shown in gR and maxD distributions of the structures generated for the initial pool and the distributions of the structures selected during the ensemble optimization are shown for 20, 50 and 65°C in gR and maxD of the structures selected during the ensemble optimization are centered around 31 Å and 103 Å, respectively  was applied ectively . At 50°CmaxD of 104.70 Å and gR = 32.30 Å. The RBM obtained at 50°C shows a particle with a maxD of 111.20 Å and gR = 33.99 Å. Finally, the RBM obtained at 65°C shows a particle with a maxD of 118.20 Å and gR = 34.95 Å. Superposition of the low-resolution models obtained at different temperatures by the dummy atom modeling approach (DAM) and the respective TpMan rigid-body models (RBM) show a very good agreement  is very close to the value obtained from the integral analysis of the scattering curve (gR = 32.50±0.14 Å) using the method implemented in the GNOM package (maxD of 103.90 Å and gR = 32.97 Å (SR = 36±2 Å) is consistent with the SR determined for the TpMan low-resolution model (SR = 38.3 Å) employed the program HYDROPRO maxD of TpMan both indicate that the dimension of the linker is not very large with respect to its mass. Therefore, our results are consistent with a linker with a compact structure and that occupies a small volume with respect to its relative large number of amino acids. Although the resolution of the molecular envelope is limited, it represents the first visualization of a full-length TpMan structure reported to date.The value of the radius of gyration of TpMan (at 20°C and pH 6) calculated from the Guinier plot  of TpMan is a compact domain of unknown biological function Visual inspection of the CD spectrum of TpManGH5 indicates the presence of α-helical secondary structure as evidenced by negative ellipticities around 210 and 220 nm . The speAs can be seen from For a system with a compact shape, the Kratky plot shows a curve with a bell shape, where an initial rising portion is followed by a marked descent thereby forming a well-defined maximum q <0.15 Å−1), however, presents a slight elevated baseline at high q values (q >0.15Å−1). This behavior at q >0.15Å−1 suggests that TpMan presents some level of intrinsic molecular flexibility. Due to their architecture, modular proteins can often adopt several conformations in solution. The ensemble optimization method (EOM) represents an excellent strategy to identify interdomain motions unambiguously gR and maxD of the structures selected during the ensemble optimization are centered around 31 Å and 103 Å, respectively  the thermostabilization of the whole enzyme, ii) adequate degree of molecular flexibility between the structural domains and iii) optimize the geometry between the others domains with respect to the substrate at high temperatures. These studies should provide a useful basis for future biophysical studies of entire TpMan and others multimodular carbohydrases.Based on these observations we advocate that the linker is very important to guarantee the optimal conditions for TpMan enzymatic activity due to at least three factors: Nickel-nitrilotriacetic acid resin (Ni-NTA), imidazole, kanamycin, LB medium, isopropyl-β-D-thiogalactopyranoside (IPTG) and locust bean gum were purchased from Sigma-Aldrich. All chemicals and reagents used in this study were of the highest purity analytical grade.280 nm = 156,900 M−1cm−1 for TpMan and ε280 nm = 108,875 M−1cm−1 for TpManGH5. The final products TpMan and TpManGH5 were then frozen and stored at −80°C and were melted on ice before use.The expression and purification of endo-β-1,4-mannanase (TpMan) and its isolated catalytic core domain (TpManGH5) were carried out as described previously with minor modification Mannan endo-1,4-β-mannosidase activity was determined using as substrate 1% against locust bean gum  dissolved in a buffer. Activity of TpMan and TpManGH5 were measured in the temperature range between 20 and 85°C (20 mM acetate-borate-phosphate buffer adjusted at pH 6). The reaction in each experiment was performed by mixing 20 μL of the diluted enzyme (1.5 μM) with 80 μL of galactomannan for 10 min. The reaction was stopped by the addition of DNS reagent followed by boiling the samples at 100°C water bath for 5 min The size characteristic of the purified TpMan and TpManGH5 samples were examined by means of the Nano-ZS dynamic light scattering system . This system employs a 633 nm laser and a fixed scattering angle (173°). Protein solutions (between 0.5 and 8 mg/mL), in 20 mM acetate-borate-phosphate buffer adjusted at pH 6, were first passed through a 0.22 μm filter , centrifuged at 16,000×g for 10 min at room temperature, and subsequently loaded into a cuvette prior to measurement. The temperature was raised from 20 to 85°C and the samples were allowed to equilibrate for 2 min in each temperature prior to DLS measurements, after which multiple records of the DLS profile were collected. In each case the hydrodynamic radius was obtained from a second order cumulant fit to the intensity auto-correlation function (size distribution by volume).kα) and the sample to detector distance was 0.67 m, providing an q (scattering vector) interval from 0.008 to 0.35 Å−1, where q = 4 π sin(θ)/λ and θ is half the scattering angle. The 2D scattering data was collected on a Vantec2000 detector and the integration of the SAXS patterns were performed by the use of the Bruker SAXS software. The data treatment, normalization to absolute scale and averaging procedures were performed by the use of the SUPERSAXS package .For SAXS measurements, TpMan was measured at different protein concentrations  and temperatures . The samples were passed through a 0.22 μm filter  and centrifuged at 16,000×g for 10 min at room temperature prior to measurement. No concentration effects were detected for the samples. The measurements were carried out on a laboratory SAXS instrument Bruker-Nanostar, placed at the Institute of Physics of University of São Paulo. This equipment is improved by the use of microfocus source Genix3D coupled with Fox3D multilayer optics and two sets of scatter less slits for beam definition, all provided by Xenocs. The samples were kept on a quartz capillary glued to a stainless steel case, which enabled the proper rinse and reuse of the sample holder, permitting an accurate background subtraction. The scattering of water measured on the same sample holders was used to normalize the data to absolute scale. The sample temperature was controlled by a Peltier system. Several 900 s frames were recorded for each sample to monitor radiation damage and beam stability. The wavelength of the incoming monochromatic X-ray beam was λ = 1.54 Å (CugR) of the molecules were determined by two independent procedures: i) by the Guinier equation I(q) = I(0).exp[(−q2.gR2)/3], q<1.3/gR, and ii) by the indirect Fourier transform method using the GNOM package (www.embl-hamburg.de/biosaxs) P(r) was also evaluated with GNOM software and the maximum diameter (maxD) was obtained.The radius of gyration (ab initio procedures implemented in DAMMIN package (www.embl-hamburg.de/biosaxs) maxq. The CRYSOL package (www.embl-hamburg.de/biosaxs) was used to generate theoretical scattering curves from DAMs gR and maxD were determined with the same package.Dummy atom models (DAMs) were calculated from the experimental SAXS by ab initio DAMs were superimposed with de SUPCOMB package et al.www.pymol.org).Rigid-body modeling of the TpMan was performed with the CORAL package Thermotoga maritima endo-β-1,4-mannanase was initially used as input to the Modeller program, together with the atomic coordinates of the latter (PDB 1OH4). The alignment of the TpManIg-like against the sequence of X1 domain from Clostridium thermocellum cellobiohydrolase A was initially used as input to the Modeller program, together with the atomic coordinates of the latter (PDB 3PE9).The remote homology detection servers HHPRED www.expasy.org). The methods used for general secondary-structure prediction were PHD The theoretical extinction coefficients, based on the amino acid composition (Gene Bank: ABQ47550.1), for TpMan and TpManGH5 were obtained from the ProtParam utility available on the ExPaSy server . Eight accumulations were averaged to form the CD spectra, taken using a scanning speed of 100 nm minFigure S1DLS temperature experiments with TpMan. (A) The size distribution by intensity for purified TpMan (0.5 mg/mL) where DLS runs were conducted between 20–85°C. (B) The size distribution by volume for purified TpMan where DLS runs were conducted at 20, 30, 40, 50 and 85°C.(TIF)Click here for additional data file.Figure S2DLS temperature experiments with TpManGH5. (A) The size distribution by intensity for purified TpManGH5 (0.5 mg/mL) where DLS runs were conducted between 20–70°C. (B) The size distribution by volume for purified TpManGH5 where DLS runs were conducted at 20, 30, 40, 50 and 70°C.(TIF)Click here for additional data file.


Cocaine addiction is accompanied by a decrease in striatal dopamine signaling, measured as a decrease in dopamine D2 receptor binding as well as blunted dopamine release in the striatum. These alterations in dopamine transmission have clinical relevance, and have been shown to correlate with cocaine-seeking behavior and response to treatment for cocaine dependence. However, the mechanisms contributing to the hypodopaminergic state in cocaine addiction remain unknown. Here we review the positron emission tomography (PET) imaging studies showing alterations in D2 receptor binding potential and dopamine transmission in cocaine abusers and their significance in cocaine-seeking behavior. Based on animal and human studies, we propose that the kappa receptor/dynorphin system, because of its impact on dopamine transmission and upregulation following cocaine exposure, could contribute to the hypodopaminergic state reported in cocaine addiction, and could thus be a relevant target for treatment development. Studies imaging the neurochemistry associated with cocaine addiction in humans have largely focused on dopamine signaling in the striatum. These studies show that pre-synaptic dopamine release, in response to the administration of a stimulant, is reduced in cocaine abusers compared to healthy controls. This has important implications for this disorder, since the reduction in dopamine release has been shown to correlate with increased cocaine-seeking behavior. Importantly, the imaging studies were performed at about 14 days abstinence, which has clinical relevance, since previous studies have shown that cocaine abusers who achieve 2 weeks of abstinence have a better treatment response compared to those who do not  allows imaging of the neurochemistry associated with drug and alcohol addiction in the human brain. This imaging modality uses radionuclide-labeled ligands that bind to a specific receptor, and the radioligands used most frequently in addiction research label the dopamine receptors. Radiotracers that label the dopamine type 2 family of receptors (referred to as D2) can also be used to measure changes in extracellular dopamine. This is performed by imaging with radiotracers that are sensitive to changes in extracellular dopamine, and obtaining scans before and after the administration of a psychostimulant (such as amphetamine or methylphenidate). These stimulants increase extracellular dopamine levels, which results in a reduction of dopamine receptors that are available to bind to the radiotracer, shown in Figure The main outcome measure in radioligand imaging studies is receptor binding to the radiotracer, referred to as BPND, defined as the ratio of specific to non-specific binding  of 15–20%  are reduced in cocaine dependence. This is performed by imaging the D2 receptors before and after acute depletion of endogenous dopamine using alpha-methyl-para-tyrosine (AMPT). Thus, imaging after AMPT administration results in an increase in [11C]raclopride binding, as opposed to the decrease seen after stimulant administration , although dopamine neurons fire in response to the receipt of a reward, and during the expectation of a reward. However, dopamine signaling more likely mediates the reinforcing effects of natural rewards and abused drugs, and makes the behavior required to obtain the reward more likely to be repeated Schultz, . HoweverThe imaging studies in human cocaine abusers show that blunted dopamine release correlates with an increase in cocaine self-administration , which can serve as a marker for the integrity of the dopamine neurons  is the class of peptides cleaved from prodynorphin, which include dynorphin A and B (and others) which have a high affinity for the kappa receptor (KOR)  depending on experimental conditions  can be categorized into at least two subgroups according to their projections sites and the proteins they express Gerfen, . The “dis protein and consequently stimulate the production of adenosine 3′, 5′-monophosphate (cAMP) which leads to the activation of protein kinase A (PKA)-dependent signaling pathways. In contrast, D2 receptor inhibits adenylyl cyclase and cAMP/PKA pathways by recruiting inhibitory Gαi. Accordingly, cocaine activates PKA signaling pathway mainly through activation of D1 receptor and manipulation of this pathway alters behavioral responses to cocaine  function together to increase the negative reinforcing effects of cocaine (Koob et al., Studies in human cocaine abusers have also shown that stress increases the risk of drug abuse and relapse (De La Garza et al., Thus, DYN/KOR signaling appears to play a crucial role in reinstating drug-seeking behavior by mediating the negative effects associated with drug cessation and stress-induced drug taking (Koob and Le Moal, The data presented here suggest that blunted striatal dopamine release measured with imaging in cocaine dependence may be associated with an upregulation of DYN. Acting at the KOR of the dopamine terminals, KOR activation would be expected to produce a decrease in striatal dopamine release. Post-mortem studies in cocaine abusers and animal studies show that both KOR and DYN are upregulated following chronic cocaine exposure, and that this effect is long lasting (Spangler et al., Chronic cocaine exposure induces CREB phosphorylation and changes in gene expression, which increase expression of prodynorphin mRNA in the nucleus accumbens in addition to other factors. As described above, excessive DYN signaling results in a decrease in extracellular dopamine release, which has been shown in the imaging studies of human cocaine abusers. These findings suggest that increasing signaling at the dopamine receptors may be an appropriate treatment approach, but clinical studies using dopamine agonists have not shown efficacy (Amato 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.


This review outlines the effects of 1,2,3,4-tetrahydroisoquinoline (TIQ) and its derivative, 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ), endogenous substances imbued with high pharmacological potential and broad spectrum of action in brain. 1MeTIQ has gained special interest as a neuroprotectant, and its ability to antagonize the behavioral syndrome produced by well-known neurotoxins . This review is thus focused on mechanisms of action of 1MeTIQ in behavioral, neurochemical, and molecular studies in rodents; also, effects of TIQ and 1MeTIQ on dopamine metabolism; and neuroprotective properties of TIQ and 1MeTIQ in vitro and in vivo. Finally, antiaddictive properties of 1MeTIQ will be described in cocaine self-administered rats. Findings implicate TIQ and especially its methyl derivative 1MeTIQ in unique and complex mechanisms of neuroprotection in various neurodegenerative illnesses of the central nervous system. We believe that MAO inhibition, free radicals scavenging properties, and antagonism to the glutamatergic system may play an essential role in neuroprotection. In addition, the results strongly support the view that 1MeTIQ has a considerable potential as a drug for combating substance abuse, through the attenuation of craving. Simultaneously, THP was identified in the urine of parkinsonian patients on l-DOPA  medication  receptor antagonists , is a neuroprotective compound. Among several endogenous TIQs 1MeTIQ has a special position, as very early it was described in the brain —chemical species known to damage cellular macromolecules , and this damage can lead to equally damaging secondary products -enantiomers of TIQ and 1MeTIQ were most potent in this respect. These findings suggest that tetrahydroisoquinoline analogs profoundly stimulate dopamine release, resulting in the competitive inhibition of [11C]raclopride binding to dopamine D2 receptors, but not loss of receptor number - and (S)- in respect of their effects on dopamine catabolism. While both enantiomers increased the concentrations of dopamine and its extraneuronal metabolite, 3-methoxytyramine (3-MT) in rat striatum, they differently affected dopamine catabolism. Thus, (R)-1MeTIQ increased both the level of the final dopamine metabolite homovanillic acid (HVA) (by about 70 %) and the rate of dopamine metabolism (by 50 %), while (S)-1MeTIQ depressed the DOPAC  and HVA levels , and attenuated the rate of dopamine metabolism -1,2-dimethyl-5,6-dihydroxy-TIQ, (R)-N-methyl-salsolinol, and TIQ  induced by glutamate in mouse embryonic primary cell cultures . Dependence is defined as a need for continual drug exposure to avoid a withdrawal syndrome which is characterized by physical or motivational disturbance when the drug is withdrawn.Addiction is a complex disease process of the brain which results from recurring drug intoxication and is modulated by genetic, experiential, and environmental factors. Drug addiction is one of the most difficult medical and social problems, as no effective pharmacotherapy has been available so far. Until recently, it was believed that addiction was associated with neuroplasticity in the cortico-striatal brain circuitry, which is important for adaptive behavior and predominantly involved reward processes mediated by limbic circuits, whereas results from recent neuroimaging studies have implicated additional brain areas, especially the frontal cortex , and opiates increases the locomotor activity of animals. Repeated administration of the drug of abuse induces neurobiological changes, such that after 10 days of withdrawal, acute administration of the drug produces an even greater increase in locomotor activity, a process called sensitization. Both behavioral sensitization, self-administration, and drug-reinstatement of seeking behavior are the major models of drug addiction  and addiction evoked by drugs of abuse.


Molecular machines embedded in a Langmuir monolayer at the air-water interface can be operated by application of lateral pressure. As part of the challenge associated with versatile sensing of biologically important substances, we here demonstrate discrimination of nucleotides by applying a cholesterol-armed-triazacyclononane host molecule. This molecular machine can discriminate ribonucleotides based on a twofold to tenfold difference in binding constants under optimized conditions including accompanying ions in the subphase and lateral surface pressures of its Langmuir monolayer. The concept of mechanical tuning of the host structure for optimization of molecular recognition should become a novel methodology in bio-related nanotechnology as an alternative to traditional strategies based on increasingly complex and inconvenient molecular design strategies. The synthesis of the molecular machine, cholesterol-armed-triazacyclononane  in the subphase are shown in Figure 1 under each condition exhibit monotonic increases without phase transitions. Increase in the nucleotide concentration in the subphase shifted the isotherms to larger molecular areas, suggesting that the molecular packing of 1 was disturbed by interaction between the nucleotides and 1 at the air-water interface. According to a reported method  = 10 mM and π = 5 mN · m-1); K(CMP/GMP) = 3.11 ([Li+] = 10 mM and π = 20 mN · m-1); K(CMP/UMP) = 8.77 ([Li+] = 0 mM and π = 35 mN · m-1); K(AMP/GMP) = 2.22 ([Li+] = 0 mM and π = 20 mN · m-1); K(AMP/UMP) = 9.89 ([Li+] = 0 mM and π = 35 mN · m-1); K(GMP/UMP) = 5.52 ([Li+] = 0 mM and π = 35 mN · m-1). On the other hand, the maximum binding constants for individual nucleotides are: K(CMP) = 1080 M-1 ([Li+] = 10 mM and π = 5 mN · m-1); K(AMP) = 550 M-1 ([Li+] = 0 mM and π = 5 mN · m-1); K(GMP) = 480 M-1 ([Li+] = 0 mM and π = 5 mN · m-1); K(UMP) = 710 M-1 ([Li+] = 0 mM and π = 5 mN · m-1). Therefore, conditions suitable for discrimination of the nucleotides and for most efficient binding of a single nucleotide component can be selected. The molecular recognition system presented here is therefore distinct different from more conventional ones where the structure of recognition components primarily defines binding efficiency of guest molecules.These results clearly indicate that the recognition of aqueous nucleotides can be tuned both by the surface pressure and the presence of LiPrior to this and our other preliminary reports, discrimination of nucleotides has not been easy to achieve because of their structural similarity, and despite its importance in biological and pharmaceutical fields. This research strikingly demonstrates a method for molecular discrimination amongst structurally similar nucleotides by mechanical tuning of a simple host at a dynamic interfacial medium. Recognition and discrimination of ribonucleotides can also be optimized. The concept of mechanical tuning for optimization of molecular recognition should become a novel methodology in bio-related nanotechnology as an alternative to traditional strategies based on increasingly complex and inconvenient molecular design strategies.AMP: adenosine 5'-monophosphate disodium salt; CMP: cytidine 5'-monophosphate disodium salt; GMP: guanosine 5'-monophosphate disodium salt; UMP: uridine 5'-monophosphate disodium salt.The authors declare that they have no competing interests.SS, MM, and HT carried out syntheses of molecular machines. TM, KO, HE, KS, JPH, YS, YK, and KA evaluate molecular recognition at the air-water interface. All authors read and approved the final manuscript.


Most of the presently used percutaneous arterial closure devices (PACD) for hemostasis after interventional vascular procedures are effective, but carry risk of complications by deposition of a foreign body. A new promising externally applied PACD – chitosan-based HemCon Bandage (HCB) was explored in sheep. The HCB hemostatic efficacy and complications occurring with its use were compared to those with the standard manual compression (SMC).Both superficial femoral arteries (SFA) of 9 heparinized sheep were catheterized with an 8F sheath for 5 minutes. After the sheath withdrawal, hemostasis with the HCB was compared with hemostasis achieved with SMC in the contralateral SFA. Iliac angiograms performed by carotid artery approach determined the hemostasis time.The HCB use shortened time to hemostasis with a mean time of 6.9 ± 3.9 minutes versus 10.8 ± 2.8 minutes for the SMC . Seven SFAs in the HCB group and only 1 SFA in the SMC group exhibited hemostasis in 5 minutes. All nine SFAs using the HCB showed femoral artery patency and demonstrated less hematoma (2/9) than in the SMC group (8/9). No complications developed in the HCB group, one SFA occlusion was seen in the SMC group.The externally applied HCB in heparinized sheep was safe and effective. It significantly shortened time to hemostasis at the SFA access sites following 8F sheath removal. Proper application of the HCB was necessary to shorten hemostasis and prevent hematoma formation. The HCB should be tested in a clinically controlled study to evaluate its efficacy in humans. Since the introduction of percutaneous catheterization in 19535Since the early nineties, several types of percutaneous arterial closure devices (PACD) have been introduced to enhance hemostasis after interventional procedures and to decrease the rate of complications. These devices either replace or shorten the time of manual compression at the puncture site.20The study protocol was approved by the Institutional Animal Care and Use Committee. Nine female sheep weighing from 56 to 70 kg were used in this study. A cardiac mobile system  with digital imaging was used for fluoroscopy and angiography. Digital subtraction angiographies were performed with an injector .Preparation of animals and their anesthesia were described in previous paper.The access sites in each animal were the superficial femoral arteries (SFA). One SFA received treatment with the HCB applied with manual compression and the contralateral SFA served as a control with the use of SMC. The sequences of the puncture sites and treatment modes were randomized. With selective iliac angiography, a road map image was created and SFA diameter was measured. Single wall access of the SFA was done under road map guidance with the 21 gauge needle of the micropuncture set . An 8F sheath was then placed into the artery and left there for 5 minutes. During the sheath removal and prior to the use of the HCB 2″ X 2″ in size, mild nonocclusive pressure was first applied above the skin puncture site. After the sheath was completely removed, a small amount of blood was first allowed to seep on the skin access site to contact the bandage and initiate hemostasis. The bandage was then applied with digital nonocclusive pressure. In the control SFA, significant digital pressure was applied during the sheath removal to prevent blood penetration through the puncture tract, as used in clinical practice. Manual pressure was held in both the treatment and control groups for 5 minutes. Angiography was done immediately after pressure relief to confirm hemostasis. If angiography showed extravasation, compression was continued for a further 2.5 minutes. Angiography was then repeated every 2-1/2 minutes until no evidence of bleeding was seen. Compression was reapplied in the interval between repeat angiographies. Lack of extravasation was the endpoint. The study then proceeded on the contralateral SFA. Finally, angiography of each side was performed at about 30 minutes after cessation of bleeding to check the patency and status of the SFA. The access sites were then checked for hematomas defined as loss of definition of the fossa subinguinalis and raised appearance of the skin. The degree of hematoma at the groin was graded: 0 = no hematoma, 1 = slight and 2 = significant. Groin area fullness with prominence <1 cm was considered slight hematoma. At the end of the study, the animals were euthanized.Data were recorded into a worksheet  and summary statistics (mean and standard deviation) were calculated. Student’s t-test was used to determine if there was a statistically significant difference between the control and treatment with regard to achieving hemostasis. A value of P<0.05 was considered significant.SFA diameters ranged from 5.4 to 6.1 mm with all pairs being matched in size. The mean ACT prior to the sheath removal in the HCB group was 404.4 ± 262 seconds and in the control group 371.87 ± 73.1 (p=0.737). The mean time to achieve hemostasis in the HCB group was 6.9 ± 3.9 minutes, while the mean time of the control group was 10.8 ± 2.8 minutes (p=0.019). The results are summarized in et al. in 2007 reviewed 14 PACDs.®Bandage). Clinical studies of D-Stat-Dry, Syvek Patch, Clor-Sur Pad and Chito-Seal showed that these PACDs applied with compression reduce time to hemostasis after femoral artery catheterization compared with SMC and do not increase the complication rate when using 4–6 F sheaths.14Numerous PACDs are now available for achieving rapid hemostasis at percutaneous arterial access sites after diagnostic or interventional procedures. Madigan Literature is available on the hemostatic efficacy of the HCB in traumatic animal models, and on HCB use in trauma patients in the military, in emergency departments and during surgery.The study limitations include the small sample size and the impossibility of performing a blind study due to the distinctive appearance of the HCB. Another limitation is the absence of data on time to ambulation and long-term efficacy of the closure. Important information about time to ambulation, thus, could not be evaluated. Another study should address these limitations and should also include histopathologic evaluation of the SFAs access site.The chitosan-based HCB was effective and shortened the time to hemostasis at SFA access sites following removal of an 8F sheath in heparinized sheep. Proper application of the bandage, however, was necessary to shorten hemostasis and decrease hematoma formation. A controlled clinical study needs to be done for evaluation of hemostatic efficacy of HCB following endovascular interventions in humans.


It has been proposed that phasic dopamine activity is responsible for learning whereas tonic dopamine is rather responsible for the response vigour . In contHere, we show how tonic dopamine is controlled by the sub-cortical nuclei of the limbic system, especially by the nucleus accumbens (NAc) shell and the ventral pallidum . By embeTo demonstrate our model, we have set up a simple food seeking task  where an


In mice fed SFA for 3 months, provision thereafter of a DHA-enriched diet exacerbated parenchymal apo B retention, concomitant with a significant increase in plasma cholesterol. In contrast, provision of a low-fat diet following chronic SFA feeding had no effect on SFA-induced parenchymal apo B. The findings suggest that in a heightened state of cerebrovascular inflammation, the provision of unsaturated fatty acids may be detrimental, possibly as a consequence of a greater susceptibility for oxidation.Chronic ingestion of saturated fatty acids (SFAs) was previously shown to compromise blood-brain barrier integrity, leading to brain parenchymal extravasation of apolipoprotein B (apo B) lipoproteins enriched in amyloid beta. In contrast, diets enriched in mono- or polyunsaturated (PUFA) oils had no detrimental effect. Rather, n3 and n6 oils generally confer protection via suppression of inflammation. This study investigated in wild-type mice if a PUFA diet enriched in docosahexanoic acid (DHA) restored blood-brain barrier integrity and attenuated parenchymal apo B abundance induced by chronic ingestion of SFA. Cerebrovascular leakage of apo B was quantitated utilising immunofluorescent staining. The plasma concentration of brain-derived S100 Accumulating evidence supports the hypothesis that dietary behaviour and in particular ingestion of fats contribute to Alzheimer's disease (AD) onset and progression. The work in  reportedβ) [β is the predominant component of  “amyloid” (or senile) plaques [β and deposition upon extracellular matrices may also reflect decreased degradation and efflux by epithelial cells of the choroid plexus [β load may be exacerbated if cerebrovascular integrity is compromised and blood-to-brain delivery of peripheral Aβ is increased [In AD, chronic inflammation leading to neuronal loss appears to be primarily associated with cerebrovascular and brain parenchymal deposits of amyloid beta (Aβ) . Derived plaques , 10. Key plaques –13, a phd plexus , 15. Altncreased , 17. Morncreased , 19. β is found associated with triglyceride-rich lipoproteins (TRLs) and cell culture and immunohistochemical studies confirm secretion of Aβ as a TRL from hepatocytes and absorptive epithelial cells of the small intestine [β concentration, following the consumption of a mixed lipid meal and kinetic studies in vivo showing that Aβ serves as a regulating apolipoprotein of TRLs [β pathway may contribute to AD risk. In three strains of amyloid transgenic mice, secretion into plasma of TRL-Aβ was strongly associated with onset and progression of amyloidosis [β concomitant with evidence postprandial dyslipidaemia [Significant plasma Antestine –22. In h of TRLs . Howeverloidosis . Moreoveloidosis . Consistpidaemia . Moreovepidaemia , 28.β and cerebrovascular integrity axis, wild-type (WT) mice were fed diets enriched in either SFA, MUFA, or PUFAs [β. The endothelial tight junction protein occludin was substantially attenuated in SFA-fed mice concomitant with substantially increased enterocytic abundance of Aβ [To explore directly the hypothesis of a dietary fat modulation of TRL-Aor PUFAs . Within ce of Aβ , 29. In The potential cytotoxic properties of SFA are established and the principal mechanisms include mitochondrial respiratory burst resulting in oxidative stress and endoplasmic reticulum dysfunction . Polyunsn = 6 mice per group). All mice were maintained on a 12 h light and dark cycle room, at 22°C and with free access to water and food. The animal housing, handling, and experimental procedures described for this study were approved by the Curtin University Animal Experimentation and Ethics Committee. Six-week-old female WT mice (C57BL/6J) were housed in groups and randomized into their respective diets  for 3 months and then randomised to receive either an LF , PUFA-enriched , or maintained on SFA for a further 2 months. Fatty acid composition for each diet is shown in Blood and brain samples were collected as previously described by Takechi et al. . Mice weμm were cut from the right cerebral hemispheres for each mouse and mounted on Polysine slides . Brain tissues were carefully isolated, washed with chilled phosphate buffer saline , right hemispheres were separated, and fixed in 4% paraformaldehyde for 24 h. The tissues were then cryoprotected with 20% sucrose solution at 4°C for 72 h, frozen in isopentane with dry ice and stored at −80°C. For histology and fluorescence microscopy, serial cryosections of 18 μm) were air-dried for 30 min, rehydrated with PBS and incubated in blocking serum (10% goat serum) for 30 min prior to the application of the antibodies. Cerebral apo B was detected by overnight incubation with the primary antibody polyclonal rabbit anti-apo B  at 1 : 500 dilution, at 4°C. Postovernight incubation and washing with PBS, primary antibody was labelled at room temperature with the secondary goat anti-rabbit IgG-Alexa 488 fluorochrome conjugate  for 2 h. The sections were then washed with PBS and the nuclei were counterstained with DAPI (1 : 1000) for 5 min at room temperature. Thereafter, the stained sections were mounted with antifade mounting medium. Primary antibody was replaced with buffer or an irrelevant serum for negative control tissues.Cerebrovascular leakage of apo B was detected as previously described . Brain cμm optimised by Nyquist. A minimum of nine 3D images were randomly captured per mouse, which include 5 images within the cortex (CTX) and 2 images each from brainstem (BS) and hippocampal formation (HPF). Digital images for photomicroscopy were acquired through AxioCam HRm camera  with an AxioVert 200 M inverted microscope by Zeiss (Germany) at ×200 magnification . Three-dimensional (3D) images were captured through ApoTome optical sectioning methodology (Carl Zeiss). Each 3D image consisted of 6–10 two-dimensional (2D) images and the distance between Z-stack slices was 1.225 Cerebrovascular leakage of plasma protein apo B was quantified within the CTX excluding the hippocampus, BS, and HPF. The pixel intensity of the protein of interest for each 3D image was calculated utilising the automated optical intensity measurement tool in Volocity  and expressed as per unit volume. The investigator was blinded during imaging and quantitation.Plasma cholesterol and triglycerides were determined in duplicate by enzymatic assays . Nonesterified fatty acids (NEFAs) were determined with NEFA-C . β is used as a marker of cerebrovascular inflammation and was measured using ELISA kits according to manufacturers' instructions . Plasma S100β was measured with 30 μL of plasma sample or standard and incubated in precoated microtitre well plates at 4°C overnight. Plates were then incubated with biotinylated secondary antibody and Streptavidin-HRP for 2 h, each. Colour generated with substrate and optical density determined at 492 nm. After adjusting for sample dilution, final concentrations of plasma S100β were extrapolated from standard curve.Plasma S100n = 6 mice per group and minimum of nine 3D images were captured per mouse for detection of apo B leakage. Each 3D image was constructed by stacking of sequential 2D images, therefore generating 324–540 two-dimensional images per group. All data was analysed by either parametric or nonparametric one-way Analysis of Variance to assess the main effects of LF and DHA on dietary SFA-induced blood-brain barrier (BBB) dysfunction and their two-way interactions. Post-hoc comparison of means was done if the associated main effect or interaction was statistically significant within the Analysis of Variance procedure. P values < 0.05 were considered statistically significant.This study utilised β in the SFA→DHA mice compared to SFA alone (β is commonly used as a surrogate marker of brain-to-blood leakage. The S100β is a cytokine produced exclusively by the astrocytes of the central nervous system. Following randomization, differences in food consumption were identified. Mice maintained on SFA or randomized to the LF consumed on average 3 g/day, whereas consumption of the DHA-enriched diet was reduced to 2 g/day. The lower caloric intake of mice on the DHA enriched diet resulted in a slower rate of growth following randomization (We confirm previous studies showing significant CTX > BS > HPF extravasation of apo B lipoproteins in WT mice maintained on an SFA diet for a total of 5 months (3 months plus randomization to SFA for an additional 2 months: Figures FA alone . The promization .β. Cerebral capillary vessels normally have tightly apposed endothelial cells that ordinarily prevent transport of plasma proteins and macromolecules [This study was designed to explore if provision of a diet enriched in DHA attenuated cerebrovascular dysfunction induced by chronic ingestion of an SFA diet. The primary outcome measure was to determine the abundance of brain parenchymal apo B lipoproteins that transport significant endogenous Aolecules . β as mice that were maintained on SFA alone. The primary finding of this study showed that provision of a PUFA diet principally enriched in DHA exacerbated brain parenchymal extravasation of apo B lipoproteins that had been initially induced by chronic ingestion of SFA. Previous studies exploring the effect on cerebrovascular integrity and function by the SFA and PUFA diets described here, as well as an MUFA-enriched diet, showed in C57BL/6J mice that only the SFA diet induced parenchymal accumulation of apo B lipoproteins . Therefoβ, with pathological manifestations prior to Aβ deposition. Exogenous administration of Aβ is vasoconstrictive and vessels treated with Aβ show significant endothelial cell damage with changes in the cell membrane, cytoplasm, nucleus, and other organelles [β secretion and that repeated postprandial excursions may eventually disrupt BBB function. Consistent with this possibility, SFAs were shown to stimulate enterocytic Aβ abundance and released into the circulation associated with postprandial TRL and a similar phenomenon may also occur in liver [β [Several studies have provided evidence of a vasoactive role of Aganelles . Takechiganelles  suggestein liver , 35. Morliver [β , 37. In this study, parenchymal apo B abundance did not correlate with plasma triglyceride concentration. Mice fed the DHA-enriched diet had comparable triglycerides to the SFA group and plasma triglycerides were greatest in SFA mice randomized to LF. Saturated fats often increase plasma NEFA concentration compared to low-fat diets, whereas DHA generally accelerates TRL clearance by facilitating LPL-mediated lipolysis , 39. HowThe most significant lipid effect of randomization to DHA following chronic ingestion of SFA in this study was a twofold increase in plasma cholesterol compared to the mice maintained on the SFA diet, or mice randomied to the LF diet. A number of animal and clinical studies have shown that DHA-enriched diets can increase plasma cholesterol associated with low- and high-density lipoproteins –43. HypeThe anti-inflammatory properties of particularly the n3 and n6 fatty acids have been unequivocally demonstrated in many studies. However, the propensity for PUFA to oxidize may under some circumstances amplify oxidative stress sequelae. Diets enriched in SFA enhance oxidation as a consequence of stimulated mitochondrial function in activated macrophages . DietaryChronic ingestion of diets enriched in SFA commonly causes vascular dysfunction, including in capillary vessels of the brain. The effects of SFA could be described as a response-to-injury phenomenon induced by exaggerated exposure to plasma triglyceride, cholesterol, NEFA, or harmful inflammatory products of lipid metabolism, such as lipid peroxides. Many studies support a role of n3 and n6 fatty acids in the prevention of vascular, based disorders primarily via suppression of inflammatory cascades. Less clear however are the benefits of polyunsaturated oils in the presence of profound inflammation, because of the propensity to generate lipid peroxidation products. In an established model of cerebrovascular dysfunction induced by chronic ingestion of an SFA-enriched diet, provision of DHA amplified the harmful effects. Probable mechanisms include hypercholesterolemia and perhaps fatty acid-induced cytotoxicity. The data suggests that introduction of n3/n6 fatty acids in metabolic conditions that are characterized by heightened systemic inflammation needs to be carefully considered in the context of paradoxical detrimental effects that could occur.