Inverse probability weighting methods were used to account for countries without AMR data.Escherichia coli, 5.8% (95% CI 1.8–9.7%) for carbapenem-resistant (CR) E. coli, 66.9% (95% CI 47.1–86.8%) for 3GCR Klebsiella pneumoniae, and 23.4% (95% CI 7.4–39.4%) for CR K. pneumoniae. The projected AMR prevalence in 2030 was 77% (95% CI 55–99.1%) for 3GCR E. coli, 11.8% (95% CI 3.7–19.9%) for CR E. coli, 58.2% (95% CI 50.2–66.1%) for 3GCR K. pneumoniae, and 52.8% (95% CI 16.3–89.3%) for CR K. pneumoniae.The estimated prevalence of AMR in 2015 was 64.5%  42–87%) for third-generation cephalosporin-resistant (3GCR) E. coli and K. pneumoniae in most parts of the world by 2030, supporting both the need to enhance stewardship efforts and to prioritize research and development of new antibiotics for resistant Enterobacteriaceae.The models suggest that third-generation cephalosporins and carbapenems could be ineffective against a sizeable proportion of infections by  Enterobacteriaceae, specifically Escherichia coli and Klebsiella pneumoniae, is a critical threat to human health  is now a global problem, and resistance in n health . Infecti strains . Carbape strains . AMR alr strains . ProjectData on population and gross national income per capita (GNIPC) from the World Bank and on AMR from ResistanceMap, a global repository of AMR data from quality-assured and accredited hospitals and laboratory networks, were used . CountriWith few exceptions, low- and middle-income countries are less likely to monitor AMR; therefore, high-income countries are overrepresented in the ResistanceMap database. Not taking this into account may lead to selection bias and an underestimation of the prevalence of AMR because of the strong negative association between GNIPC and the prevalence of AMR . To overE. coli and 43 countries with AMR data for K. pneumoniae. In countries with E. coli AMR data, the median number of AMR point estimates was 14 (interquartile range 1–15), and 31 were high-income countries. In countries with K. pneumoniae AMR data, the median number of AMR point estimates was 10 (interquartile range 2–14), and 28 were high-income countries. No country had AMR data beyond 2015.The study included 45 countries with AMR data for E. coli, 5.8% (95% CI 1.8–9.7%) for carbapenem-resistant (CR) E. coli, 66.9% (95% CI 47.1–86.8%) for 3GCR K. pneumoniae, and 23.4% (95% CI 7.4–39.4%) for CR K. pneumoniae. The projected annual variation (slope) of AMR was 0.83% (95% CI 0.73–0.93%) for 3GCR E. coli, 0.4% (95% CI 0.12–0.68%) for CR E. coli, −0.58% (95% CI −1.46% to 0.3%) for 3GCR K. pneumoniae, and 1.96% (95% CI 0.59–3.33%) for CR K. pneumoniae. The projected AMR prevalence in 2030 was 77% (95% CI 55–99.1%) for 3GCR E. coli, 11.8% (95% CI 3.7–19.9%) for CR E. coli, 58.2% (95% CI 50.2–66.1%) for 3GCR K. pneumoniae, and 52.8% (95% CI 16.3–89.3%) for CR K. pneumoniae. Projections for individual countries with at least four AMR point estimates using simple linear regression are presented in the Supplementary material.Forecast estimates of global AMR are presented in E. coli globally will be 3GCR, and over half of K. pneumoniae invasive isolates will be CR. The consequences of the high prevalence of AMR could be devastating for health systems . CR K. pneumoniae had the highest annual increase of AMR, which could reach 53% by 2030, but the confidence intervals were wide, indicating uncertainty of the projections. The projected increase in the prevalence of CR E. coli was more modest. However, empirical treatment of infections will need to cover 3GCR, leading to an increased use of carbapenems, and this, in turn, may accelerate the pace of CR.The models showed that the annual variation in the prevalence of 3GCR Enterobacteriaceae are part of the human gut microbiota, and the spread of AMR is facilitated by conditions that are more common in resource-poor settings, such as suboptimal sewage systems and a lack of access to clean water (Enterobacteriaceae can emerge anywhere and spread around the globe (an water . Previouhe globe . Isolatehe globe .K. pneumoniae. In addition, it was not possible to distinguish the case mix of community- and hospital-acquired infections among the countries included in the study, and the high prevalence of AMR in some countries could be influenced by a higher proportion of hospital-acquired infections (This study has important limitations. The total population of all countries included in the study was approximately a third of the world population and was biased towards high-income countries. While IPI models were used to attempt to correct for underrepresentation of low- and middle-income countries, more surveillance data are urgently needed to improve current and future estimates of AMR. The projections for future levels of AMR were based on linear models, which assumed no changes in the growth rate of resistance. They also did not account for saturation or stabilization of AMR levels, as was observed with 3GCR fections .E. coli and K. pneumoniae in most parts of the world in the not-too-distant future. Empirical antimicrobial therapy for sepsis or for urinary tract or abdominal infections might shift to non-beta-lactam antibiotics, which, in turn, may lead to an increase in AMR in other antibiotic groups. These results underscore the need to improve the judicious use of antimicrobials and support recent World Health Organization recommendations to prioritize the research, discovery, and development of new and effective antibiotic treatments for beta-lactam-resistant Enterobacteriaceae (These results suggest that if current trends were to continue, third-generation cephalosporins and carbapenems could become ineffective against eriaceae .The funders had no role in the study design, in the collection, analysis, and interpretation of the data, in the writing of the report, or in the decision to submit the article for publication.This study used data available in the public domain and thus did not require ethics approval.There are no conflicts of interest to disclose.


Inflammatory responses significantly contribute to neuronal damage and poor functional outcomes following intracerebral hemorrhage (ICH). Soluble epoxide hydrolase (sEH) is known to induce neuroinflammatory responses via degradation of anti-inflammatory epoxyeicosatrienoic acids (EET), and sEH is upregulated in response to brain injury. The present study investigated the involvement of sEH in ICH-induced neuroinflammation, brain damage, and functional deficits using a mouse ICH model and microglial cultures.ICH was induced by injecting collagenase in both wild-type (WT) C57BL/6 mice and sEH knockout (KO) mice. WT mice were injected intracerebroventricularly with 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), a selective sEH inhibitor, 30 min before ICH. Expression of sEH in the hemorrhagic hemisphere was examined by immunofluorescence and Western blot analysis. The effects of genetic deletion or pharmacological inhibition of sEH by AUDA on neuroinflammatory responses, EET degradation, blood-brain barrier (BBB) permeability, histological damage, and functional deficits were evaluated. The anti-inflammatory mechanism of sEH inactivation was investigated in thrombin- or hemin-stimulated cultured microglia.ICH induced an increase in sEH protein levels in the hemorrhagic hemisphere from 3 h to 4 days. sEH was expressed in microglia/macrophages, astrocytes, neurons, and endothelial cells in the perihematomal region. Genetic deletion of sEH significantly attenuated microglia/macrophage activation and expression of inflammatory mediators and reduced EET degradation at 1 and 4 days post-ICH. Deletion of sEH also reduced BBB permeability, matrix metalloproteinase (MMP)-9 activity, neutrophil infiltration, and neuronal damage at 1 and 4 days. Likewise, administration of AUDA attenuated proinflammatory microglia/macrophage activation and EET degradation at 1 day post-ICH. These findings were associated with a reduction in functional deficits and brain damage for up to 28 days. AUDA also ameliorated neuronal death, BBB disruption, MMP-9 activity, and neutrophil infiltration at 1 day. However, neither gene deletion nor pharmacological inhibition of sEH altered the hemorrhage volume following ICH. In primary microglial cultures, genetic deletion or pharmacological inhibition of sEH by AUDA reduced thrombin- and hemin-induced microglial activation. Furthermore, AUDA reduced thrombin- and hemin-induced P38 MAPK and NF-κB activation in BV2 microglia cultures. Ultimately, AUDA attenuated N2A neuronal death that was induced by BV2 microglial conditioned media.Our results suggest that inhibition of sEH may provide a potential therapy for ICH by suppressing microglia/macrophage-mediated neuroinflammation.The online version of this article (10.1186/s12974-017-1005-4) contains supplementary material, which is available to authorized users. Intracerebral hemorrhage (ICH), the spontaneous extravasation of blood into the brain parenchyma, is the second most frequent stroke subtype after ischemic stroke . ICH hasEpoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid derived from the cytochrome P450 (CYP450) enzymes, are potent intracellular lipid signaling molecules that possess important biological activities, including vasodilatation, anti-inflammation, and cellular signaling regulation . Recent All animal protocols were carried out according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health  and were approved by the Animal Research Committee at Cheng Hsin General Hospital . Male wild-type (WT) C57BL/6 mice were obtained from BioLASCO . Male sEH knockout (KO) mice in the C57BL/6 background were purchased from Jackson Laboratory . Animals were housed under conditions of controlled temperature (22–25 °C) and humidity (40–60%) with a 12-h/12-h dark cycle and were allowed free access to water and food.2 at 37 °C as previously described [The mouse BV2 microglial and N2A cell lines were cultured in Dulbecco’s modified Eagle’s media  supplemented with 10% heat-inactivated fetal bovine serum , 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere of 5% COescribed .5 cells per well) in RPMI 1640 Medium with 10% inactivated FBS and used for the experiments 24 h after. The purity of cultured microglia was higher than 99%, as verified by glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule 1 (Iba1) immunohistochemical staining  WT or sEH KO mice as described previously . BrieflyZ)-enoic acid  for 3, 6, or 24 h. For collection of conditioned media, BV2 microglia were plated and incubated with 100 ng/mL LPS, 10 U/mL thrombin, or 10 μM hemin in the absence or presence of 10 μM AUDA for 24 h. Cell-free supernatant fractions were applied to N2A cells for 48 h to evaluate the changes in cell viability. All group experiments were performed independently four or five times.BV2 microglia and primary mouse microglia were stimulated with either 10 U/mL thrombin or 10 μM hemin in the absence or presence of varying concentrations of 12-(3-adamantan-1-yl-ureido)-dodecanoic acid  or 14,15-epoxyeicosa-5(n = 5–6/group) and double immunofluorescence labeling. The second study investigated the anti-inflammatory and neuroprotective effects of sEH deletion. Experimental methods employed were as follows: (1) histology staining (n = 5–7/group), (2) cytokine enzyme-linked immunosorbent assays (ELISAs) and matrix metalloproteinase (MMP)-9 zymography (n = 6/group), (3) EET and 14,15-DHET ELISAs (n = 4/group), (4) Evans blue dye extravasation (n = 5–6/group), and (5) hemoglobin assay (n = 5/group). Cytokine ELISAs and MMP-9 analyses were performed on the same experimental group whereas other tests were performed on different experimental groups. The third study evaluated the anti-inflammatory and neuroprotective effects of sEH inhibition by AUDA, a selective sEH inhibitor, which has been widely used to evaluate the biological role of sEH [n = 5–6/group), (2) Western blot analysis, cytokine ELISAs, and MMP-9 zymography (n = 6–7/group), (3) EET and 14,15-DHET ELISAs (n = 4/group), (4) Evans blue dye extravasation (n = 6/group), (5) hemoglobin assay (n = 5/group), and (6) behavioral and body weight assessments (n = 14/group). Western blot, cytokine ELISAs, and MMP-9 analyses were performed on the same experimental group.Eight- to 10-week-old mice were randomized into different treatment groups using computer-generated random numbers. All outcome measurements and analyses were performed in a blinded manner. Sample sizes are determined by power analysis based on our pilot data and previous studies. A total of 409 mice (306 WT and 103 sEH KO) were used. Mice that had neurologic deficit scores greater than 15 or less than 3 at 3 h post-ICH were excluded from the study. Fifty-nine WT and 17 sEH KO mice were excluded due to neurologic deficit standard or death after ICH . Sixty-five additional sham-operated control mice were used for biochemical assays and Evans blue quantification (44 WT and 21 sEH KO). Four normal mice were used for Western blot analysis (2 WT and 2 sEH KO). Four studies were conducted. The first study examined the specificity of the anti-sEH antibody and the temporal profile and cellular localization of sEH expression after ICH. These assessments included Western blot analysis  or an equal volume of vehicle (1% DMSO) was given by intracerebroventricular (i.c.v.) injection 30 min before ICH as previously described . BrieflyMice were sacrificed by transcardial perfusion following terminal anesthesia with sodium pentobarbital  for histological examinations. Frozen sections (10 μm) were stained with cresyl violet, Fluoro-Jade B , and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling  as previously described . For immTo assess the cellular source of sEH, double immunofluorescence labeling was performed by simultaneous incubation of sections with rabbit anti-sEH  overnight at 4 °C with mouse anti-CD11b , rat anti-GFAP , mouse anti-neuronal nuclei antigen , and rat anti-CD31 . To assess proinflammatory microglia/macrophages, sections were incubated overnight at 4 °C with rabbit anti-Iba1 , together with rat anti-CD16/32 . Sections were washed, then incubated with Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibodies  for 2 h.2 in 10–12 non-overlapping fields immediately adjacent to the hematoma using a magnification of ×200 as previously described [FJB, TUNEL, MPO, or Iba1 staining was quantified in three consecutive sections from the hemorrhagic core at the level of 0.24 mm from the bregma. The number of positive cells was counted in an area of 920 × 860 μmescribed . Iba1-poescribed . MicroglInjury volumes, striatum atrophy, and striatum enlargement ratios were quantified using cresyl violet-stained sections at 20 rostral-caudal levels that were spaced 200 μm apart as previously described . SectionColocalization of the microglia/macrophage marker (Iba1) and proinflammatory microglia/macrophage activation marker (CD16/32) was analyzed as previously described , 29. BriIL-1β, IL-6, macrophage inflammatory protein-2 (MIP-2), and monocyte chemoattractant protein-1 (MCP-1) , along with EETs , and 14,15-DHET , were measured in brain homogenates or cell lysates using commercially available ELISA kits.Evans blue dye  was injected into the tail vein and allowed to circulate for 1 h. The mice were then transcardially perfused with phosphate-buffered saline (PBS), and the ipsilateral hemispheric samples were homogenized in 1 mL of 60% trichloroacetic acid by sonication. After centrifugation at 4500 rpm for 15 min at 4 °C, the supernatants were diluted with ethanol (1:4). The absorbance of Evans blue in the supernatant was measured at 620 nm. Evans blue concentrations were calculated and expressed as μg/g brain tissue using a standard curve.Zymography was performed as previously described . BrieflyThe hemoglobin contents of ICH brains were quantified using a spectrophotometric assay according to previously described methods . DistillWestern blot analyses were performed as previously described . ProteinAll the behavioral tests were performed before ICH and at 1, 4, 7, 14, 21, and 28 days after ICH. Mice were pretrained for both rotarod and beam walking tests for 3 days.The mNSS provides an index of motor, sensory, reflex, and balance tests . The neuThe rotarod test was used to measure motor function and balance . BrieflyMotor function and coordination were also assessed by measuring the ability of mice to pass across an elevated beam . The latThe body swing test for evaluating asymmetrical motor behavior was conducted as previously described with slight modifications . Mice weH-tetrazolium bromide (MTT) assay (Sigma-Aldrich). Data are presented as the percentage of control group. The experiments were repeated four to five times using different batches of primary cultures.NO production was assessed by measuring the nitrite levels of the culture supernatants using the Griess reagent (Sigma-Aldrich). The nitrite content in the samples was calculated based on a standard curve prepared with known concentrations of sodium nitrite. Cell viability was assessed using 3--2,5-diphenyl-2t test was used to test the difference between two groups. Statistical significance was set at P < 0.05.Data are presented as the mean and standard error of the mean (mean ± S.E.M.). One-way or two-way analysis of variance (ANOVA) followed by a post hoc Bonferroni test was used for multiple groups to determine significant differences. Student’s P < 0.001) following ICH and remained elevated at 6 h (P < 0.001), 12 h (P < 0.001), 1 day (P = 0.003), and 4 days  of WT mice, but absent in sEH KO brains Fig. . The sEHP = 0.0038, Fig. P = 0.0049, Fig. P < 0.001; MCP-1: P = 0.001; Fig. P = 0.008; IL-6: P = 0.002; MIP-2: P < 0.001; MCP-1: P = 0.038; Fig. P < 0.05; Fig. P < 0.01; Fig. Since we demonstrated that sEH was upregulated in the hemorrhagic hemisphere following ICH and was colocalized with microglia/macrophages, we hypothesized that sEH inhibition reduces neuroinflammatory responses after ICH. To test this hypothesis, we first used a loss-of-function strategy to evaluate the effect of sEH deletion on microglia/macrophage activation at 1 and 4 days post-ICH. These two time points were chosen because the inflammatory-related signals peak within 4 days after collagenase-induced ICH and decline thereafter , 6, 34. P < 0.001; Fig. P < 0.05; Fig. P = 0.009; Fig. P < 0.001; Fig. P = 0.0298; Fig. P = 0.0167). These data indicate that sEH deficiency sealed the barrier and reduced MMP-9 activity and neutrophil accumulation.We next evaluated the effect of sEH deletion on BBB integrity, as BBB disruption is a major consequence of post-ICH inflammation and contributes to brain edema and subsequent neuronal damage . The EvaP = 0.0024; Fig. P = 0.0012; Fig. P = 0.0359; Fig. P = 0.0214; Fig. To determine whether the observed changes in inflammatory responses and BBB breakdown were reflected in a reduction of neuronal death and apoptosis, histological outcomes were evaluated. The number of FJB-positive degenerative neurons around the hematoma were significantly reduced in sEH KO brains compared to WT brains at both 1 day , decreased EET/14,15-DHET ratio (P = 0.009), and reduced 14,15-DHET level  30 min before collagenase-induced ICH Fig. . We chosP < 0.001; Fig. P = 0.0030; Fig. P < 0.001; Fig. P = 0.02; Fig. We next evaluate whether sEH inhibition by AUDA attenuated neurodegeneration and apoptosis at the acute stage. Treatment with 10 μM AUDA significantly reduced the number of FJB-positive degenerative neurons around the hematoma and levels of cCP-3, a critical effector in apoptosis, compared with vehicle-treated mice at 1 day post-ICH  and 24 h  and in hemin-stimulated BV2 cells at 6 h  and 24 h , respectively  and 24 h  and in hemin-stimulated BV2 cells at 6 h  and 24 h , respectively  and hemin  and hemin  without affecting the level of total P38 , a family of serine/threonine protein kinases that mediates microglial activation . Co-treaP < 0.001, Additional file P = 0.002, Additional file P < 0.001) or 58.6 ± 0.6% (P < 0.001) of the control level, respectively  and 72.1 ± 0.9% (P < 0.001), respectively. These results show that the neurotoxic effects of condition media from the thrombin- or hemin-stimulated BV2 microglia were reduced by AUDA treatment, indicating that the protective effect of AUDA on neuronal viability is at least partly conferred through the inhibition of microglial-induced injury.Since our in vivo results showed that deletion and inhibition of sEH reduced microglial/macrophage activation and attenuated neuronal damage, we further hypothesized that inhibition of sEH reduced microglia-derived neurotoxic factors and thus ameliorated neuronal injury. We first assessed the direct effect of thrombin or hemin on cell viability in N2A cultures. While treatment with 10 U/mL thrombin had no direct effect on neuronal viability assessed by the MTT assay, 10 μM hemin significantly reduced viability of N2A cells to 69.2 ± 1.0% of the control level  and the inflammatory responses they induce contribute to BBB disruption . We demoWe observed that sEH was expressed in astrocytes and neurons, in addition to microglia/macrophages. Thus, apart from these anti-inflammatory actions, inhibition of the sEH activity may provide neuroprotection via other mechanisms. For example, a recent study reported that addition of 14,15-EET to astrocytes after oxygen-glucose deprivation (OGD) increased brain-derived neurotrophic factor (BDNF) expression and astrocyte survival. Additionally, administration of sEH inhibitors to cultured astrocytes after OGD increased vascular endothelial growth factor (VEGF) secretion, which then enhanced Akt pro-survival signaling in neurons leading to less neuronal cell death. Thus, inhibition of sEH may promote production of BDNF and VEGF from astrocytes to improve neuronal survival. Furthermore, inhibition of sEH exhibits direct neuroprotective properties, as evidenced by a report showing that pharmacological inhibition of sEH reduced cell death in cortical neurons exposed to hypoxic injury . CloselyIn conclusion, we show that both genetic deletion and pharmacological inhibition of sEH significantly reduced inflammatory responses, BBB permeability, and limited neurodegeneration after ICH Fig. . InhibitAdditional file 1: Figure S1. Isolation of high purity microglia as verified by immunohistochemical analysis using microglia  and astrocyte  specific markers. Staining for nuclei of cells in culture by DAPI (blue) is also shown. The scale bar is 100 μm. (TIFF 1214 kb)Additional file 2: Figure S2.Effects of saline and heat-inactivated collagenase on apoptosis and inflammatory responses in ipsilateral hemispheres 1 day after injection. Representative immunoblots and bar graphs show that there were no significant differences in (A) body weight change and protein levels of (B) cCP-3, (C) Iba1, and (D) IL-1β between the saline- and heat-inactivated collagenase-injected (HIC) mice. (TIFF 529 kb) (TIFF 601 kb)Additional file 3: Figure S3.##P < 0.01 and ###P < 0.001 vs. sham group; *P < 0.05 and **P < 0.01 vs. vehicle group; †P < 0.05 vs. 1 μM AUDA group . (TIFF 529 kb)Bar graphs of (A) IL-1β, IL-6, and MIP-2 protein levels and (B) EET protein level, EET/14,15-DHET ratio, and 14,15-DHET protein level at 1 day post-ICH. Values are mean ± S.E.M.; Additional file 4: Figure S4.###P < 0.001 vs. control group; **P < 0.01 vs. hemin-stimulation group . (TIFF 521 kb) Effects of 10 μM AUDA on N2A cell viability as assessed by the MTT assay after exposure to thrombin or hemin for 24 h. Values are mean ± S.E.M.;


Electrochemotherapy (ECT) has shown to be an effective treatment for cutaneous and subcutaneous Kaposi sarcoma (KS) lesions. However, no study has investigated the impact of ECT treatment on the kinetics of human herpesvirus type 8 (HHV8), which is considered the necessary causal agent of KS. We aimed to evaluate HHV8 viral load and expression levels in patients affected by classic KS who received one or more ECT treatments and have been followed semi annually for up to four years.A total of 27 classic KS patients were enrolled in this study. Tumour biopsies and blood samples were obtained before ECT treatment. Additional blood samples were collected at six month intervals for 12–48 months. HHV8 viral load and expression profiles of latent (ORF72 and ORF73) and lytic  genes were assessed in all samples by real-time PCR. HHV8 ORF26 and K1 regions were amplified and subjected to direct nucleotide sequencing followed by phylogenetic analysis for variant identification.−7 to 6.9 × 10−4 copies per cell in PBMCs. Overall, latent ORF72 and ORF73 as well as lytic K2, K8 and K10/K10.1 were expressed in all KS biopsies. ORF16 mRNA was detected in 71.4% and both K8.1 and K10.5/K10.6 mRNAs in 57.1% of KS samples. The ORF72, ORF73 and K2 transcripts were amplified in 37.5%, 25% and 25% of PBMCs collected before ECT, respectively. After the first ECT session, complete response was achieved in 20 out of 27 (74.1%) patients and HHV8 DNA was detected in four out of 27 (14.8%) PBMC samples at six month follow up. Phylogenetic analysis of ORF26 amplimers showed that most viral variants belonged to A/C (82.3%), and few to C2 (5.9%) or C3 (11.8%) subtype. The K1/VR1 variants fell into A (33.3%) and C (66.7%) HHV8 clade. No correlation was found between HHV8 subtypes and ECT complete response.All KS biopsies and 46.4% of peripheral blood mononuclear cells (PBMCs) collected before ECT treatment were positive for HHV8 DNA. Viral load ranged from 0.02 to 2.3 copies per cell in KS lesions and 3.0 × 10ECT therapy has a significant effect on HHV8 kinetics in patients with classic KS. The complete remission of patients was accompanied by clearance of circulating virus. Kaposi sarcoma (KS) is a locally aggressive vascular tumour generally presenting with cutaneous multiple patches, plaques or nodules . EpidemiThe frequency of KS has dramatically increased in Europe and United States during AIDS epidemic and remains one of the most frequent tumours among HIV positive patients . The incClassic KS is characterized by cutaneous lesions distributed mainly on the lower extremities and trunk and often associated with venous stasis, lymphedema or hyperkeratosis , 7. DiffHuman herpesvirus type 8 (HHV8) is recognized as the etiological agent of all forms of KS and two lymph proliferative disorders, multicentric Castleman disease and primary effusion lymphoma (PEL) –19. The  HHV8 infects “spindle” shaped cells, endothelial and epithelial cells as well as B lymphocytes, monocytes and CD34+ stem cells –23. HHV8Several studies have evaluated the association between HHV8 viral load in peripheral blood and KS progression in HIV-infected patients , 29. TheThis prospective, single-centre study included 27 consecutive patients diagnosed with classic KS lesions on the inferior limbs  that were treated by ECT at the Istituto Nazionale Tumori “Fondazione G. Pascale” of Naples from February 2009 to November 2013. All KS patients were classified as stage I or II based on objective criteria according to Brambilla staging system . Each paThe ECT treatment regimen was previously described . The trePBMCs were isolated from fresh blood by Ficoll-Hypaque™ density gradient. The mononuclear cell layer was resuspended in 500 μl of RNAlater and stored at −80 °C. Genomic DNA was extracted from the PBMCs and KS biopsies according to previously described protocol . In partTotal RNA was isolated from frozen biopsies and PBMCs using Trizol™ Reagent (Invitrogen™) following the manufacturer’s instructions. Briefly, tissues and PBMCs were resuspended in Trizol reagent and tissues were homogenized by TissueLyser (Qiagen GmbH). After 15 min centrifugation at 13000 rpm, the top phase was collected and total RNA was precipitated with 0.5 volume of isopropyl alcohol 10 min at room temperature. RNA pellet was washed with 75% ethanol, resuspended in sterile water and stored at −80 °C. RNA quality and concentration was analysed with RNA Nano chipassay on Agilent 2100 Bioanalyzer (Agilent Technologies).2, 20 nM SYBR Green I ), 5 pmol of each primer, and 100–500 ng of genomic DNA. All experiments were performed in the CFX96 Real Time System . Dilution series (10 to 104 copies) of genomic DNA extracted from HHV8-infected BCBL-1 cell line (containing 70 copies per cell of HHV8 DNA) were used to construct the standard curve.All DNA samples were subjected to HHV8 quantification by real time PCR using primer pairs amplifying a region within the HHV8 ORF26 as previously described . The PCRTotal RNA (100 ng) was reverse transcribed into cDNA using iScript™ cDNA Synthesis kit  according to manufacturer’s instructions. The reaction was performed in 20 μl reaction mixture containing 4 μl of 5× iScript Reaction Mix, 1 μl of iScript Reverse Transcriptase and nuclease-free water in a Perkin Elmer thermo cycler  with the following steps: 5 min at 25 °C, 30 min at 42 °C and 5 min at 85 °C.A SYBR Green real time PCR method was used to evaluate latent (ORF72 and ORF73) and lytic  gene expression in biopsies and PBMCs. Specially, oligoprimers were designed with Beacon Designer software (Premier Biosoft) and used for real time PCR  while the inner PCR amplifications were performed in 50 μl reaction mixture containing 5 μl of outer reaction. All samples were amplified in the Agilent Technologies Sure Cycler 8800 thermal cycler with the following steps: an initial 2 min denaturation at 94 °C, followed by 45 amplification cycles for ORF-26 and 38 cycles for ORF-K1 of 55 °C and 52 °C respectively for 45 s, 68 °C for 1 min, 94 °C for 15 s and a 5 min final elongation at 68 °C. Reaction mixture containing genomic DNA, extracted from NIH 3 T3 murine cell line, was used as negative control and included in every set of 5 clinical specimens.The HHV8 ORF26 region was amplified by seminested PCR using in the outer reaction the oligonucleotide ORF26LR1F1 (5′-GCAGTATCTATCCAAGTA-3′) with ORF26LR1R1 (5′-GGAACCAAGGCTGAT-3′) and in the inner reaction the ORF26LR1F1 with ORF26LR2R2 (5′-ACAGATCGTCAAGCA-3′). The HHV8 K1 region was amplified by seminested PCR using K1LR1F1 (5′-ATCAAGATGTTCCTGTAT-3′) and K1LR1R1 (5′-TATAGTATTTAGTTTGTGACA-3′) in the outer reaction and K1LR1F1 and K1LR2R2 (5′-CATTATTTCCAGAGGTAG-3′) in the inner reaction yielding an amplimer of 250 bp encompassing the hypervariable region VR1 (aa 54–93). The outer PCR amplification reactions were performed in 50 μl reaction mixture containing 300 ng of target DNA, 5 pmol of each primer, 2.5 mM MgClhttp://www.technelysium.com.au/chromas.html) and converted to FASTA format.All HHV8 amplimers were subjected to bidirectional direct sequencing analysis by Eurofins Genomics Srl. Nucleotide sequences were edited with Chromas Lite 2.01 (https://www.ncbi.nlm.nih.gov/nuccore/?term=HHV-8). Reference sequences for each HHV8 ORF26 subtype were DQ984689.1 , DQ984768.1 , DQ984785.1 , DQ984789.1 , and DQ984759.1 ; whereas reference sequences for each HHV8 K1 subtype were AF133038 , AF133039 , JN800486.1 , AF151688.1 , JN800487.1 , AF130284.1 , KF781665.1 , GU097427.1 ,; AF133041 , AF133042 , FJ866517.1 , DQ394064.1 , DQ394068.1 , DQ394038.1  and DQ394054.1 .Multiple alignments of HHV8 nucleotide sequences from the present study and reference strains reported in the GenBank were performed with clustal W tool of MegAlign program of the Lasergene software . All reference sequences were downloaded from the National Centre for Biotechnology Information , followed by the neighbor-joining bootstrap analysis , which wP values were less than 0.05.The statistical analysis was performed using Graph Pad Prism Software version 6.00. Two-tailed Fisher’s exact test were used for comparison of categorical data. U Mann–Whitney test were used to evaluate differences in viral load. Differences were considered to be statistically significant when This study included a total of 27 patients  affected by classic KS, with a median age at the diagnosis of 74 years (range 43–88 years of age) (Table 0), after approximately 6 months (T1) and 12 months (T2) after ECT therapy were available for 27, 20 and 9 patients, respectively. HHV8 sequences have been identified in 100% of KS biopsies, in 13 out of 27 (48.1%) PBMC samples at T0, in 4 out of 20 (20%) at T1 and in 2 out of 9 (22%) at T2. (Table −5 to 1.6 × 10−1 copies per cell in PBMCs at the time of enrolment (T0). The differences in viral load between KS lesions and PBMCs were statistically significant (p = 0.006), Fig. 0, Fig. KS biopsies were available from 16 patients. PBMC samples collected at the time of enrolment , C2  and C3  subtypes, following the nomenclature proposed by Zong et al. .The HHV8 K1 amplimers obtained in 12 tumour samples were subjected to phylogenetic analysis showing a nucleotide divergence among them of 0.7–12.3% corresponding to a 4.9–43.3% amino acid divergence in the VR1 (aa 52–92) sequence. Phylogenetic analysis based on the comparison of newly identified K1 sequences with K1 reference strains available in GenBank showed that four out of 12 (33.3%) HHV8 K1 variants belonged to subtype A and eight (66.7%) to subtype C  differentially expressed in different cell types. In particular, the K9 (vIRF-1) gene is generally co-expressed with ORF73 (LANA-1) in KS cells, while K10.5/K10.6  is mainly transcribed in HHV8-associated lymphomas. Such data suggest that different vIRFs encoding genes are important for HHV8 life cycle and that their expression is regulated by cell specific factors . HosseinIn recent years, much attention has been paid to the association between different subtypes of HHV8 and KS aggressiveness , 44. In This study has shown that ECT treatment of patients with classic KS causes a decline of HHV8 virus load in peripheral blood. The complete remission of KS patients treated with ECT is accompanied in the majority of cases by clearance of the virus in the PBMCs. ECT is very effective in the treatment of classic KS independently from HHV8 viral load, viral gene expression and HHV8 variants.


For the first time, p-type nanofibers have been electrospun onto a conductive fluorine doped tin oxide (FTO) surface. The properties of the NiO nanofibers have been directly compared to that of bulk NiO nanopowder. We have observed a p-type photocurrent for a NiO photocathode fabricated on an FTO substrate. One-dimensional (1D) TiO2 metal oxide nanofibers have been previously reported and applied for solar cell applications [Nickel oxide (NiO) is an attractive semiconductor, because its properties are very useful for various photocatalytic , batteryensional D TiO2 meications ,8. Nanofications . Nanofibications .2 to NiO occurs at 250 °C, Boshloo and Hagfeldt illustrated that at this temperature, cubic NiO (bunsenite) is formed [2 species closer to 500 °C to achieve nanostructures, because the decomposition of most polymers or salt precursors, occurs around 400–500 °C [et al. [p-type photoelectrodes for dye sensitized solar cells (DSSCs) [3)2 this occurs around 170 °C. Fu et al. [Electro-spun nanofibres are usually prepared using a metal containing sol-gel polymer solution to support and shape the fibers. Electrospinning uses an electrical charge to draw very fine fibers from a liquid. When a high voltage is applied to a liquid droplet, the body of the liquid becomes charged and electrostatic repulsion counteracts the surface tension causing the droplet to be stretched. When the liquid is stretched, it forms a stream of liquid that erupts from the needle’s tip. The liquid dries in flight and is elongated by electrostatic repulsion initiated at small bends of the fiber until it is finally deposited on the ground collector. Polymers are then burnt-off along with any other unwanted volatile precursors and the metal oxides are sintered. For this to occur an understanding of the thermal properties of the system is essential. While the conversion of Ni(OH)s formed . While t0–500 °C ,10,11. Fp/n heterojunction. By going from a flat surface to a 3-D surface increases the area for light harvesting while simultaneously gaining a large degree of porosity from the layers of nanofibers. As a result, these types of electrodes enhance the excitation (hole/electron formation) as well as the mobility of charge carriers, hence increases the efficiency of devices [In photovoltaic devices, it is especially important to harvest as much light as possible by producing a large  devices . It stil2 is the most studied sensitized metal oxide material for use in photovoltaic devices, particularly for highly efficient dye-sensitized solar cells [2 films have been under extensive investigation since the discovery of the Grätzel cell [2 based DSSC had an efficiency of 12% [2, remains a challenging process. While tandem dye-sensitized solar cells can provide a route to highly efficient energy conversion, the biggest challenge is finding a p-type photocatalyst comparable to that of TiO2. This is because it is difficult to match the current generated at the photocathode to that of the anode. Lindquist et al. [p-type photocathode should greatly improve the device efficiency by collecting high energy photons at the top of the electrode and low energy photons at the bottom [TiOar cells ,13 and war cells ,15. Whilzel cell , the mosy of 12% ,17. Altht et al.  first pre bottom ,19. While bottom , perylene bottom , and derp-type NiO photocathodes in tandem solar cells include that by Hammarstrom et al. [VOC) of 0.91 V, and an efficiency of 0.55%. More recently, Powar et al. [VOC of 0.79 V but an efficiency of 1.3%, which can be mainly attributed to an impressive short circuit photocurrent (JSC) of 4.44 mA/cm2.Some recent breakthroughs with m et al. , where tr et al.  achievedWhile numerous studies for the sensitization of NiO with organic dyes  have pro2 nanofibers with a 10 μm resolution are shown in The scanning electron microscopy (SEM) images of the polyactonitrile (PAN) Ni(acac)2 nanofibers at 500 °C. The X-ray diffraction (XRD) pattern is shown in NiO nanofibers were obtained by sintering PAN structured Ni(OH)The transmission electron microscope (TEM) images of the NiO nanofibers and selected area electron diffraction (SAED) patterns are shown in et al. in 2012 [et al. in 2001 [versus particles. Despite these subtleties, both particles and fibers exhibit bulk NiO characteristics.X-ray photoelectron spectroscopy (XPS) measurements were undertaken to investigate the chemical state of the elements in the as-synthesized products. In doing, so we looked at the Ni 2p and the Ni valence band spectra.  in 2012 . There a in 2001 . Here, nvs. Ag/AgCl we can clearly observe a photocurrent response in the cathodic direction. Typically, the photocurrent response for p-type semiconductors is negative due to the band bending at the electrode interface [p-type semiconductors (photocathodes) bend towards lower energies, which lead to a collection of negative charge at the interface. In 2012, Mozer et al. [et al. [The photocurrent response of NiO nanoparticles on FTO was measured in a photo-electrochemical cell (PEC), the chronoamperogram is shown in nterface . The banr et al.  produced [et al. , we can  [et al. .W 150,000 (Aldrich), Nickel Oxide Nanopowder (Aldrich), 95% Terpineol (Aldrich), 48% Ethyl cellulose (Aldrich).Ninety-five percent Nickel(II) acetylacetonate (Aldrich), Polyacyrlonitrile M2 was added and the mixture was magnetically stirred for 2 h at 50 C. The solution was then loaded into a plastic syringe equipped with a 23 G stainless steel needle. The needle was then connected to a high-voltage supply . The solution was fed at a rate of 1.0 mL/h using a syringe pump . A piece of flat aluminum foil was placed 10 cm below the tip of the needle as a collector plate. The voltage for the electro-spinning was set to 17 kV and was conducted in a controlled temperature environment (25 °C). The fibers were electro-spun until the solution was depleted before they were then sintered at 500 °C for 2 h, yielding black NiO nanofibers sheets.In a typical synthesis, a 5 mL solution of 12% PAN in DMF was made by slowly dissolving 0.560 g of PAN in 4.40 mL of DMF. Once dissolved, 0.250 g of Ni(AcAc)Following the same procedure as above 2 × 2 cm sheets of FTO  were placed directly under the needle on the aluminium foil. Conductive tape was used to form a connection between the foil and the FTO. Nanofibers were directly spun onto the FTO electrode, yielding a sticky green fiberous coating. The electrodes were then sintered at 500 °C for 2 h to remove the insulating polymer, yielding a monolayer of black NiO nanofibers on the surface of the FTO.2. Finally, the electrode was sintered at 450 °C for 30 min to remove all organics.NiO nanoparticle slurry was made according to literature ,28. Brie2 AM 1.5 radiation .These measurements used an Ag/AgCl reference electrode, platinum counter electrode and phosphate buffered saline (PBS) electrolyte (pH 7.4) in a photoelectrochemical cell (PEC). The PEC was illuminated with 100 mW/cmp-type nanophotocathode from NiO nanofibers from a simple electrospinning and sintering procedure. For the first time, p-type nanofibers have been electrospun onto a conductive FTO surface. While the surface coverage did not provide us with a reproducible photocurrent, future work is to explore other means of nanofiber surface deposition to increase photocurrent response.In conclusion, we have successfully fabricated a


Autopsy, histology and cytology have been and histology and cytology still are the main diagnostic tools in surgical pathology. During the last two decades molecular biology gradually has extended the diagnostic armamentarium. In tumor pathology molecular biology techniques are used to diagnose and subclassify tumors, predict response to therapies and identify therapeutic targets. Molecular pathology has evolved into a novel focus of clinical pathology and transforms the historically morphology based discipline. Traditional pathology and molecular pathology combine and guide tumor therapy. Pathologists diagnose neoplastic diseases from tissue biopsies, surgical specimens and cytology aspirates. They employ as standard methods light microscopy of hematoxylin and eosin stained tissue slides and Giemsa or Papanicolaou stained cytologic smears. During the last half century the conventional light microscopy has been supplemented by immunohistology with expanding panels of antibodies . The tasThe success of therapies that target genetic alterations has initially been demonstrated by imatinib treatment of chronic myeloid leukemia with BCR-ABL gene fusion . In soli7 bases) or the whole genome (≈3 × 109 bases).The development of molecular tumor subclassifications and targeted therapies was facilitated by an improved knowledge of genetic aberrations. Although cancer research has achieved great successes during the last decades, e. g. the identification of oncogenes and tumor suppressor genes, the genetic characterisation of tumors has gained a tremendous speed during the last 10 years by the development of next generation sequencing (NGS) . This tehttp://cancergenome.nih.gov/) and the International Cancer Genome Consortium . TCGA has studied more than 10,000 specimens of 33 different tumor entities on the DNA, RNA and epigenome level [https://gdc.cancer.gov/ and https://dcc.icgc.org/).The massive genomic characterisation of tumors has been led by the The Cancer Genome Atlas  has generated a catalogue of genetic variants from 2504 people of 26 populations in 5 continental regions with the aim to identify most of the polymorphisms that occur at a frequency of at least 1% [http://www.internationalgenome.org/home) continues the 1000 Genomes Project. A further valuable initiative for the interpretation of tumor genome sequences are the databases of Short Genetic Variations (dbSNP) and Genomic Structural Variations (dbVar) of the NCBI .It is often difficult to distinguish mutations from polymorphisms. Therefore efforts that identify polymorphisms are important for tumor diagnostics. The 1000 Genomes Project , which is released by dying normal or tumor cells into the blood . The anaCirculating tumor cells (CTC) are very scarce in the blood. Mostly less than 10 CTC are present in 1 ml blood in patients with metastasis . In compThe challenges in molecular pathology require a new type of pathologists. The pathologists of the future need to combine morphological methods with practical and theoretical knowledge in genetics, cell biology, biochemistry and bioinformatics. The university departments of pathology will have to fulfill a leading role in training surgical and molecular pathology. The establishment of molecular pathology requires space, equipment and personal. Their financing is particularly difficult for smaller institutes. Therefore not all institutes will be able to offer a broad spectrum of molecular pathology diagnostics. A concentration in larger institutes, mainly university departments, that serve as reference centers, is likely.Molecular pathology tests are mostly performed with formalin fixed and paraffin embedded tissues (FFPE). The advantage is that the tissue blocks generated for histology can also be used for molecular tests. A disadvantage is the fragmentation and chemical modification of DNA by formalin . Exome, The future will bring an increase in molecular pathology testing. The pace will be determined by the availability of effective targeted drugs. Gene panels will replace single gene analysis. Exome, transcriptome and perhaps epigenome analysis will be widely used and the applications of liquid biopsy will expand. Furthermore proteome analysis and in vitro drug susceptibility testing may become further tasks. Molecular pathology will transform the classic morphology-based pathology. Pathologists will become pilots for precision cancer therapy through their unique ability to combine morphological and molecular findings.


In vivo imaging studies indicate an increased presynaptic dopamine synthesis capacity in striatal terminals and cell bodies in the midbrain in schizophrenia; however, measures of the dopamine-synthesising enzyme, tyrosine hydroxylase (TH), have not identified consistent changes. We hypothesise that dopamine dysregulation in schizophrenia could result from changes in expression of dopamine synthesis enzymes, receptors, transporters or catabolic enzymes. Gene expression of 12 dopamine-related molecules was examined in post-mortem midbrain (28 antipsychotic-treated schizophrenia cases/29 controls) using quantitative PCR. TH and the synaptic dopamine transporter (DAT) proteins were examined in post-mortem midbrain (26 antipsychotic-treated schizophrenia cases per 27 controls) using immunoblotting. TH and aromatic acid decarboxylase (AADC) mRNA and TH protein were unchanged in the midbrain in schizophrenia compared with controls. Dopamine receptor D2 short, vesicular monoamine transporter (VMAT2) and DAT mRNAs were significantly decreased in schizophrenia, with no change in DRD3 mRNA, DRD3nf mRNA and DAT protein between diagnostic groups. However, DAT protein was significantly increased in putatively treatment-resistant cases of schizophrenia compared to putatively treatment-responsive cases. Midbrain monoamine oxidase A (MAOA) mRNA was increased, whereas MAOB and catechol-O-methyl transferase mRNAs were unchanged in schizophrenia. We conclude that, whereas some mRNA changes are consistent with increased dopamine action (decreased DAT mRNA), others suggest reduced dopamine action (increased MAOA mRNA) in the midbrain in schizophrenia. Here, we identify a molecular signature of dopamine dysregulation in the midbrain in schizophrenia that mainly includes gene expression changes of molecules involved in dopamine synthesis and in regulating the time course of dopamine action.The dopamine hypothesis of schizophrenia posits that increased subcortical dopamine underpins psychosis.  Two meta-analyses of 11 and 15 imaging studies, respectively, found significant elevations in dopamine synthesis capacity in the striatum in schizophrenia,10 and a further imaging study identified increased dopamine synthesis capacity in the midbrain.11 Here, we ask whether molecular evidence of presynaptic dopamine changes can be found in the substantia nigra in people with schizophrenia.Subcortical dopamine dysregulation is considered the final common pathway in the pathophysiology of schizophrenia.L-tyrosine to L-DOPA, which is then converted by aromatic acid decarboxylase (AADC) to dopamine.7 TH is regulated at transcriptional, translational and post-translational levels12 and, although in rodents electroconvulsive shock treatment does not change midbrain TH mRNA levels,13 regulation of midbrain TH mRNA does occur in response to pharmacological agents.15 Studies measuring TH in the midbrain of people with schizophrenia do not concur, with no change,17 increases18 and region-specific decreases19 reported in people with schizophrenia compared with controls. Although previously no change was found in AADC mRNA in the midbrain of schizophrenia patients,16 positron-emission tomography (PET) imaging studies have suggested increases in AADC activity in schizophrenia patients.11 Thus, further studies of TH and AADC within the basal ganglia are needed to determine whether molecular changes consistent with increased dopamine biosynthesis occur in schizophrenia.Dopamine neurotransmission has multiple steps of possible regulation. Tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis, converts 20 DRD2 and DRD3 isoforms are expressed in the dopamine neuron somatodendritic field in the substantia nigra and VTA and at presynaptic terminals.21 Presynaptic DRD2 and DRD3 full-length (referred to as DRD3) are autoreceptors, inhibiting dopamine neuron firing, dopamine release and terminating dopamine synthesis.26 Alternatively, spliced DRD3 variants that produce truncated proteins that do not bind dopamine have been identified, the most abundant being DRD3 non-functional (DRD3nf).28 DRD3nf modulates dopamine reception by forming heteromers with DRD2 and DRD3, redirecting dopamine-binding receptors into an intracellular compartment and thus reducing DRD3 and DRD2 cell surface action.29 DRD3 mRNA is decreased, whereas DRD3nf mRNA is increased in the cortex  of schizophrenia patients30- setting a precedent for testing whether altered DRD3 expression occurs in the midbrain in schizophrenia.Dopamine interacts with five G-protein-coupled receptors, dopamine receptor (DR) D2, DRD3 and DRD4 (inhibitory D2-like family) as well as DRD1 and DRD5 (excitatory D1-like family).31 However, gene expression of DRD2 isoforms, DRD3 or DRD3nf within the midbrain in schizophrenia, has not yet been examined.We hypothesised that changes in mRNA encoding presynaptic dopamine receptors may contribute to changes in dopamine regulation in the midbrain in schizophrenia. A study of post-mortem midbrain showed increased DRD2 receptor binding in substantia nigra from schizophrenia cases.32 and these molecules also represent a possible site of dysregulation in schizophrenia. The ability of psychoactive drugs to bring about psychotic-like states by binding to DAT or VMAT2  imply that alterations in dopamine transport can contribute to psychosis. Dopamine is catabolised in dopamine neurons and glial cells by monoamine oxidases (MAOs) and catechol-O-methyl transferase (COMT).36 Midbrain monoamine oxidase A (MAOA) and MAOB are expressed in human substantia nigra,37 and inhibition of MAOs contributes to amphetamine-induced psychosis.33 Thus, decreased synthesis of dopamine breakdown or reduced dopamine transport proteins in schizophrenia may contribute to hyperdopaminergia or to dopamine dysregulation.Dopamine action is terminated by reuptake from the synaptic cleft via the dopamine transporter (DAT) and dopamine is packaged into vesicles for release by vesicular monoamine transporter 2 (VMAT2),We ask whether gene expression of 12 dopamine-related molecules and/or protein expression of TH and DAT are changed in the substantia nigra from individuals who suffered from chronic schizophrenia compared with healthy individuals. We hypothesised that TH and AADC mRNA would be increased in the midbrain in schizophrenia with a corresponding increase in TH protein in the midbrain. Further, we hypothesised that DAT mRNA and protein would be decreased in the midbrain of people with schizophrenia compared with controls. We hypothesised that gene expression of other molecules involved in dopamine reception, transport or catabolism may contribute to dysregulation of dopamine neurotransmission, and thus may be changed in the midbrain in schizophrenia compared with controls.α=0.05)38 and tissue availability. Substantia nigra was excised from midbrain cryostat-generated 60 μm slices based on TH immunolabelling of adjacent 14 μm slide-mounted sections . Hemisected fresh frozen midbrain tissue neuroanatomically matched at the level of the oculomotor nerve from 30 schizophrenia cases and 30 control individuals (14 μm sections mounted on gelatin-coated glass slides and adjacent 60 μm sections collected between wax paper) were provided by the New South Wales Brain Tissue Resource Centre. Sample size was selected based on previous post-mortem studies  was used as an indicator of possible treatment resistance versus all other antipsychotics at time of death (n=21–19). Antipsychotic medication was converted to chlorpromazine (CPZ) equivalents 42 (n=22) or more prevalent negative symptoms (n=7) with one unknown. Toxicology screening of control cases at time of post-mortem revealed nothing (n=11), diazepam (n=1), cannabis (n=1), paracetamol (n=7), codeine (n=3) and blood pressure medication (n=2), and some (n=5) had no toxicology screen with cardiac failure recorded as cause of death. Toxicology screening of schizophrenia cases revealed no comorbid substance use (n=13), methadone and diazepam (n=1), diazepam and insulin (n=1), diazepam, pethidine, codeine and paracetamol (n=1), codeine, diazepam and morphine (n=1), paracetamol only (n=4), a non-steroidal anti-inflammatory (n=1), morphine (n=1) codeine (n=1) and ibuprofen (n=1) and screening not available (n=5). Alcohol consumption at time of death in controls and schizophrenia cases was as follows: nil (n=1–0), <20 g per day (n=17–22), 20–50 g per day (n=6–3), 50–80 g per day (n=1–0), >80 g per day (1–3) and unknown (n=4–2). A history of depression symptoms during lifetime was identified in eight schizophrenia cases (six were treated with serotonin-selective reuptake inhibitors (SSRIs) and two with tricyclic antidepressants) and in one control  and the final midbrain protein cohort comprised 26 schizophrenia cases and 27 controls . In both cohorts, diagnostic groups were matched for age, gender and post-mortem interval (PMI), and in the mRNA cohort diagnostic groups were matched for RNA integrity number (RIN)39 . All sch dose)42 . The sch43 Frozen slides (14 μm) were thawed at room temperature and fixed in 4% paraformaldehyde in phosphate-buffered saline . After rinsing, endogenous peroxidase activity was blocked using 30% MeOH and 3% H2O2 (20 min), and slides were rinsed again and blocked with 10% normal horse serum  for 1 h. Anti-TH mouse primary antibody was applied overnight at 4 °C at a 1:1000 dilution . After rinsing, the slides were incubated with a horse anti-mouse IgG-biotinylated secondary antibody  at a 1:500 dilution  followed by rinsing and incubation with the avidin–biotin–peroxidase complex  and then application of 3,3′-diaminobenzidine solution  to visualize TH immunoreactivity (2 min). The slides were rinsed, dehydrated, stained with Nissl (5 min exposure to 0.02% thionin) and cover-slipped. Slides were visualised on a Nikon Eclipse 80i light microscope. TH immunohistochemistry was scored  based on overall TH staining intensity (including cell bodies and fibres) for the area of the midbrain equivalent to the area excised for homogenisation  and TaqMan gene expression assays . The following inventoried Taqman assays were used to measure the gene expression of three housekeeper genes . The following inventoried TaqMan assays were used to measure gene expression of dopamine-related mRNAs: TH, Hs00165941; AADC, Hs01105048; MAOA, Hs00165140; MAOB, Hs00168533; COMT, Hs00241349; DAT, Hs00997364; VMAT2, Hs00996835; DRD2S, Hs01014210, DRD3, Hs00949496 and DRD3nf, Hs00945868. Custom probes were designed for DRD2L (AIHSPG8) and DRD2longer (AII1NNG) based on published sequences.45Total RNA from substantia nigra samples was extracted with Trizol . RNA quality was determined using the Agilent Bioanalyzer 2100  and samples with low RIN were removed from gene expression analysis (one control and two schizophrenia cases). Complementary DNA was synthesised using the Superscript III First Strand Synthesis Kit (Life Technologies). Quantitative real-time PCR (qPCR) analysis was conducted as reported previously46Serial dilutions of pooled complementary DNA from all samples were included on every qPCR plate for quantitation of sample expression by the relative standard curve method. All qPCR reactions were performed in triplicate. The SDS 2.4 software  was employed to analyse the qPCR data. Expression levels of the triplicate means of sample expression were normalised to the geomean of the housekeepers.47 Midbrain samples were homogenised (0.1 M Tris (pH 7.5), 50% glycerol, proteinase inhibitor cocktail and aprotinin (0.015 mM), all Sigma-Aldrich) using a handheld electric homogeniser . Proteins were quantified using the Bradford protein assay (Sigma-Aldrich). An aliquot of each sample was pooled and run in duplicate on all gels for standardisation between immunoblots . Standard curves (0.5–15 μg protein) were run to determine the linear range of expression. Protein concentrations loaded for TH and DAT detection for two western runs/protein were 3 and 10 μg per sample for TH and DAT, respectively. Proteins were separated by SDS-PAGE (10% acrylamide) and transferred to nitrocellulose membranes . Membranes were blocked in 5% non-fat milk  and incubated in primary antibody overnight at 4 °C. Primary antibodies were mouse anti-TH  and rabbit anti-DAT . Secondary antibodies were horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit . Bands were visualised using chemiluminescence  and captured on a Chemidoc XRS system (Bio-Rad). Band densities were determined using Quantity One Software  and expressed numerically as quantity values (mm × intensity). All membranes were re-probed with mouse anti-β-actin  as a loading control.Western blotting was performed as previously described.χ2-test was used to determine the relationship between diagnosis and TH intensity scores determined from immunohistochemistry. The Grubb’s test  was used to exclude group outliers  in genes or proteins of interest after normalising to housekeepers. Independent-sample two-tailed t-tests were used to test for differences in mRNA and protein cohort demographics . A  and RIN ) and indThe relative intensity of protein bands  was divided by the internal control for standardisation between blots. The relative intensity of TH and DAT bands was then normalised to the corresponding β-actin band relative intensity and tested for normality. To achieve a normal distribution, TH and DAT protein data underwent log10 transformations. The levels of the mRNAs of interest were normalised to the geometric mean (referred to as geomean3) of the housekeeper mRNAs  and the relative gene expression of each gene was tested for normality (Shapiro–Wilk test). Data that were not normally distributed were transformed. Monoamine oxidase and DRD3nf mRNA underwent log10 transformation and TH mRNA underwent a square root transformation.If normality was not achieved after transformation, as in the case of COMT mRNA, the non-normally distributed mRNA data were analysed using the Mann–Whitney test. Spearman’s correlations for COMT mRNA were performed with continuous demographic variables .t-test was used to detect diagnostic differences.For all normally distributed mRNA and protein measures, Pearson’s correlations were performed with continuous demographic variables ). For all mRNA and protein measures, Spearman’s correlations were conducted with antipsychotic drug measurements and duration of illness in the schizophrenia group. If a correlation was detected, analysis of covariance was used to test for diagnostic differences between controls and schizophrenia cases. Factorial analysis of variance was used to determine the effect of smoking status at time of death and agonal state on gene and protein expression in the diagnostic groups. If an effect was detected, this was included as a fixed factor in the analysis of covariance. If no correlations with demographic variables or effect of smoking or agonal state were detected, an independent sample two-tailed P⩽0.05.Relative gene expression and relative protein expression data were converted to a percentage of the control group mean  and graphed as mean±s.e.m. Statistical significance was set at t-tests were used to explore differences in gene or protein expression in the schizophrenia group based on antipsychotic treatment, symptoms and depression, and statistical significance was set at P⩽0.05. We highlight that this analysis is exploratory as the group sizes are modest and no corrections are made for multiple analyses.The effect of antipsychotics on gene and protein expression was explored in three ways. First, correlation analyses of mRNAs and proteins with CPZ equivalents (described above) were performed. Second, comparisons between schizophrenia cases divided into those treated with mostly first-generation antipsychotics and those treated with mostly second-generation antipsychotics. Third, comparisons were made between cases using clozapine at time of death (possible treatment resistance) versus those on antipsychotics other than clozapine at time of death, although we acknowledge that not taking clozapine does not rule out treatment resistance. In addition, the effect of mostly positive or mostly negative symptoms and presence of depression symptoms during lifetime or no depression symptoms were explored in the schizophrenia cases. Student’s two-tailed The relationships between demographic variables were assessed, and detailed statistical data are shown in r=0.458, P<0.0001, n=57) and gene expression of all three housekeepers individually. None of the housekeeper genes or the geomean3 correlated with PMI. Brain pH and PMI were not correlated with relative intensity of β-actin protein bands. All housekeeper genes and geomean3  showed strong positive correlations with RNA quality as determined by RIN, in the full cohort and by diagnostic group.The relationship between demographic variables and housekeeper mRNAs and β-actin protein were assessed. Detailed statistical analysis is reported in Most genes of interest in the mRNA expression cohort positively correlated with both RIN and pH, except for DRD2longer, which only correlated with RIN and AADC, DRD3nf and DRD3 mRNAs, which only correlated with pH . Only THP=0.395) levels in the substantia nigra between control and schizophrenia brains were identified by qPCR  intensity of TH immunolabeling in the substantia nigra and diagnosis . A TH and β-actin band were detected at the expected molecular weights, ~59 and ~42 kDa, respectively, in all samples (P=0.584) was detected by western blotting .No significant difference in TH mRNA  in substantia nigra from schizophrenia relative to controls; however, this difference did not reach statistical significance , DRD2L mRNA by 29%  and DRD2Longer mRNA by 22% .Although DRD3 was decreased by 26.76% and DRD3nf was decreased by 23.20% in substantia nigra of people with schizophrenia compared with controls, neither decrease reached statistical significance , but no diagnostic changes were found in the levels of MAOB mRNA  or COMT mRNA  and also a significant 37% decrease in the levels of VMAT2 mRNA  in the substantia nigra of schizophrenia cases when compared with controls . No significant difference in DAT protein expression was detected between control and schizophrenia . In contrast, we detected a 72.5% increase in DAT protein expression in cases on clozapine compared with cases treated with other antipsychotics . There were no changes in gene or protein expression when schizophrenia cases that received mostly first-generation antipsychotics were compared with schizophrenia cases that received mostly second-generation antipsychotics .None of the dopamine-related genes or proteins of interest measured in the substantia nigra from schizophrenia cases correlated significantly with measures of antipsychotic use or duration of illness from either the mRNA or protein expression cohorts . In addit=−2.188, df=20, P=0.041). There were no changes in any other gene or protein expression based on symptoms. No gene or protein of interest varied significantly in the schizophrenia group based on the presence of depression symptoms within their lifetime (data not shown).When schizophrenia cases displaying mostly positive or negative symptoms were compared, there was a significant 56.5% increase in DRD3 mRNA in the primarily positive symptoms group compared with those people with schizophrenia displaying mostly negative symptoms 50 are important for TH level and activity. Midbrain TH mRNA and protein levels were highly correlated, thus suggesting that our measurements of human TH are accurate and valid. However, we measured only one TH mRNA transcript (pan) and four TH mRNA transcripts have been identified in human brain.52 Thus, changes in individual TH transcripts and their corresponding protein isoforms may have been missed in our study. Additionally, our study did not compare subregions (VTA versus substantia nigra) or dorsal or ventral distribution within these regions and a recent study identified reductions in TH protein in subregions of the midbrain in schizophrenia.19Whereas midbrain TH mRNA and protein levels were unchanged between schizophrenia and controls, we observed a greater range of values in schizophrenia compared with controls in both mRNA and protein, perhaps reflecting the contradictory evidence for changes (both increases and decreases) found in previous studies.53 Thus, increased dopamine synthesis capacity via AADC appears to be changed as a consequence of schizophrenia and may not be only secondary to antipsychotic treatment. In contrast, PET imaging studies in schizophrenia patients, who were antipsychotic-naive or had not received treatment in the previous 6 months and subsequently treated subchronically (20–45 days) with haloperidol (a first-generation antipsychotic with high affinity for DRD2), showed a downregulation of this AADC-dependent step.54 Contrary to our hypothesis, but in line with the study by Grunder et al.,54 we found a trend for a decrease (22.9%) in AADC mRNA levels in the midbrain of patients with schizophrenia that suggests that there could be less synthesis of AADC in chronic, medicated patients. In rodent whole-brain homogenates, haloperidol and loxapine treatments increased AADC mRNA, whereas the second-generation antipsychotic sulpiride (a DRD2 and DRD3 antagonist) did not.55 The differential effects of distinct antipsychotics on AADC gene expression may reduce the ability to detect a consistent change in AADC in post-mortem tissue and exposure to antipsychotics may mask potential changes in AADC in schizophrenia. It is proposed that some schizophrenia patients do not respond to antipsychotic treatment as they do not exhibit the elevation in dopamine synthesis capacity that is typically associated with the disorder.56 However, using clozapine at the time of death as a proxy for possible treatment resistance40 did not reveal a difference in AADC gene expression. Interestingly, there was an indication of a negative correlation with the last CPZ equivalent dose and AADC mRNA, suggesting that in our study, the AADC mRNA reductions may be due to antipsychotic treatment.Increased activity of the second step in the dopamine synthesis pathway has been identified in schizophrenia. PET studies measuring 18F-DOPA uptake and conversion to 18F-dopamine show that the dopamine biosynthetic step that is AADC-dependent is elevated in people at ultrahigh risk for psychosis.58 however, imaging studies indicated no change in striatal DAT binding in schizophrenia patients.60 Similar binding studies have not been carried out in the midbrain of schizophrenia patients in whom dopamine can be released in the somatodendritic field to regulate dopamine neuron activity via feedback inhibition. Further work including study of DAT protein, binding and activity, both in post-mortem and PET imaging studies in the brains of people with schizophrenia  compared with controls, are needed to more fully characterize the anatomical sites of DAT abnormalities, especially in the midbrain, and to better understand the implications of our current findings. This is, however, to our knowledge, the first study to implicate reduced midbrain DAT gene expression in the pathophysiology of schizophrenia, highlighting a putative major dysregulation of DAT.As a major finding of this study, we found a 45% decrease in DAT mRNA in schizophrenia. This may seem at odds with our lack of ability to detect a change in DAT protein levels in the substantia nigra in schizophrenia. The lack of diagnostic change in DAT protein could suggest that DAT mRNA changes may not have an impact on protein levels in midbrain or that the protein function/stability may be altered causing feedback changes on DAT transcription. Alternatively, there may be increased translation of DAT protein or decreased DAT utilisation and breakdown, either of which could result in no change in steady-state DAT protein levels with a decrease in DAT mRNA. However, we report a decrease in DAT protein in cases treated with antipsychotics other than clozapine  relative to those treated with clozapine —such differences may contribute to the difficulty in detecting a diagnostic change in DAT protein expression overall and suggest that posttranscriptional regulation of DAT may vary with clinical state. Alternatively, it is possible that the decrease in DAT mRNA may be reflected in DAT protein changes in either the dorsal or ventral striatum or in cortical sites, which were not examined in this study. As dopamine action in the striatum is primarily terminated by dopamine reuptake from the synaptic cleft by DAT, less DAT levels or DAT action leading to slower termination of dopamine neurotransmission could contribute to hyperdopaminergia in schizophrenia. In the striatum, post-mortem studies report reduced levels of DAT binding in schizophrenia;61 A decrease in VMAT2 gene expression in the substantia nigra may contribute to reduced VMAT2 protein and suggests less efficient vesicular packaging of dopamine; however, less VMAT2 action can contribute to hyperdopaminergia in certain contexts as inhibition of VMAT2 contributes to amphetamine-induced psychosis.34 In contrast to our study, VMAT2 binding was found to be increased in the ventral brainstem in a PET imaging study in schizophrenia;61 however, this apparent difference may be due to regional differences in the expression of dopamine molecules in the brainstem. These observations set the stage for designing experimental systems that would mimic the putative state of transporter abnormalities found in schizophrenia and could serve as a platform for testing the impact of novel therapies.In addition to a reduction in the mRNA encoding the main dopamine transporter localized to the outer cell membrane, we also find reduced mRNA encoding dopamine transporter localized to the vesicles  in schizophrenia, VMAT2 mRNA does not appear to be regulated by antipsychotics in our study or in others.62 Serotonin released in the substantia nigra by dorsal raphe serotonergic fibres results in activation of 5-HT2 receptors and subsequent inhibition of dopamine neuron firing.63 Therefore, more MAOA synthesis/activity in the midbrain in schizophrenia could lead to increased serotonin breakdown and reduced inhibitory modulation of dopamine neurons by serotonin, thus contributing to the dopamine dysregulation. Increased cortical COMT activity, inferred from genetic changes, was linked to increased midbrain TH mRNA in normal subjects;64 however, we find no change in midbrain COMT mRNA levels in people with schizophrenia compared with controls, supporting previous studies in other brain regions finding that changes in COMT mRNA levels do not appear to be a predominant mechanism associated with the pathophysiology of schizophrenia.67In addition to changes in dopamine synthesis, transporter and receptor mRNAs, we found changes in mRNA for one catabolic enzyme, MAOA, that can also terminate dopamine action. If there is an increase in dopamine in the synaptic cleft in people with schizophrenia, we suggest that it is possible that the increase in MAOA mRNA may reflect a compensatory response to balance extracellular dopamine levels. However, MAOA is also involved in the breakdown of other neurotransmitters including serotonin and norepinephrine, and MAOA variants are implicated in psychiatric disorders.68 Our gene expression findings are in contrast to a previous post-mortem study showing increased DRD2 receptor binding in midbrain homogenates from drug-naive and drug-treated schizophrenia cases compared with controls;31 thus, we speculate that the changes we find in DRD2 mRNA may result in less DRD2 protein in the presynaptic terminal rather than in the midbrain. The binding of dopamine to the DRD2S autoreceptor on the presynaptic terminal results in the inhibition of dopamine synthesis and release and, thus, a decrease in dopamine neurotransmission.25 We speculate that if the decrease in DRD2S gene expression is reflected in less DRD2S protein expression in the terminal field of the striatum in schizophrenia, this could contribute to an increase in striatal dopamine neurotransmission. In contrast to our evidence of decreased DRD2, we and others, find increased DRD2S mRNA in DLPFC in schizophrenia,69 indicating potential region-specific alterations of this receptor splice variant mRNA in schizophrenia. Although DRD2L and DRD2longer are traditionally thought of as postsynaptic receptors found at terminal sites, we have readily measured mRNA of both of these isoforms in the human midbrain and report decreases in these isoforms in schizophrenia. This suggests that these potential decreases in DRD2 isoforms (short and long) may contribute to presynaptic or dendritic pathophysiology in schizophrenia. Although we did not identify a diagnostic difference in DRD3 or DRD3nf mRNA, schizophrenia cases exhibiting more positive symptoms had a 56% increase in DRD3 mRNA compared with those with more negative symptoms. Thus, increased DRD3 mRNA and possibly protein may also contribute to dopamine dysregulation in some people with schizophrenia.We find decreased DRD2S mRNA in the substania nigra in schizophrenia in our cohort and DRD2S mRNA levels do not correlate with antipsychotic measurements. This supports previous studies showing that treatment with antipsychotics did not change DRD2 mRNA in rodent cortex or striatum.39 However, these studies provide a basis for interpreting changes seen in in vivo studies in schizophrenia patients and the putative clues they provide to pathophysiology, and potential therapeutic targets beyond DRD2 antagonism can be followed up in preclinical models of dopamine dysregulation.In sum, we find evidence of changes in gene expression of multiple molecules that may act individually or in combination to contribute to the dopaminergic system dysregulation identified in people with schizophrenia (see We conclude that, in a demographically well-matched and characterised post-mortem midbrain cohort, gene expression of multiple dopamine-regulating molecules are changed in the substantia nigra in schizophrenia and that changes in autoreception, transport and possible catabolism together may contribute to the dopamine dysregulation in schizophrenia . This st


To evaluate tacrolimus as therapeutic option for diabetic nephropathy (DN) based on molecular profile and network-based molecular model comparisons.We generated molecular models representing pathophysiological mechanisms of DN and tacrolimus mechanism of action (MoA) based on literature derived data and transcriptomics datasets. Shared enriched molecular pathways were identified based on both model datasets. A newly generated transcriptomics dataset studying the effect of tacrolimus on mesangial cells in vitro was added to identify mechanisms in DN pathophysiology. We searched for features in interference between the DN molecular model and the tacrolimus MoA molecular model already holding annotation evidence as diagnostic or prognostic biomarker in the context of DN.Thirty nine molecular features were shared between the DN molecular model, holding 252 molecular features and the tacrolimus MoA molecular model, holding 209 molecular features, with six additional molecular features affected by tacrolimus in mesangial cells. Significantly affected molecular pathways by both molecular model sets included cytokine-cytokine receptor interactions, adherens junctions, TGF-beta signaling, MAPK signaling, and calcium signaling. Molecular features involved in inflammation and immune response contributing to DN progression were significantly downregulated by tacrolimus , interleukin 4, or interleukin 10). On the other hand, pro-fibrotic stimuli being detrimental to renal function were induced by tacrolimus like the transforming growth factor beta 1 (TGFB1), endothelin 1 (EDN1), or type IV collagen alpha 1 (COL4A1).Patients with DN and elevated TNF levels might benefit from tacrolimus treatment regarding maintaining GFR and reducing inflammation. TGFB1 and EDN1 are proposed as monitoring markers to assess degree of renal damage. Next to this stratification approach, the use of drug combinations consisting of tacrolimus in addition to ACE inhibitors, angiotensin receptor blockers, TGFB1- or EDN1-receptor antagonists might warrant further studies. Tacrolimus is a powerful immunosuppressive drug belonging to the group of calcineurin inhibitors (CNIs), which was introduced in clinical use to tackle organ rejection in solid organ transplantation . TacroliTacrolimus is used for the treatment of lupus nephritis and also a clinical trial in the context of IgA glomerulonephritis has recently been conducted NCT0122445]6]. [6]. 5][6. [6]. 5]In this study we investigated the effects of tacrolimus on pathophysiological mechanisms of diabetic nephropathy on the level of in-silico constructed mechanism of action and disease pathophysiology molecular models respectively. We furthermore searched for molecular markers showing the potential for identifying the cohort of diabetic nephropathy patients benefitting the most from the immunosuppressive properties of tacrolimus.Molecular features affected by tacrolimus were extracted from scientific literature taking into consideration genes linked via NCBI gene2pubmed associations to publications which were annotated with the MeSH term “tacrolimus”. In addition deregulated transcripts were extracted from two published transcriptomics studies from Kern and colleagues as well as Maluf and colleagues focusing on the effect of tacrolimus on renal tissue, in particular on renal fibroblasts and post-kidney transplants, respectively 12]. Tra. Tra12].Construction of a diabetic nephropathy molecular model followed the same procedure as construction of the tacrolimus MoA model using as input features associated via gene2pubmed to publications holding “diabetic nephropathies” as major MeSH term.A molecular pathway enrichment analysis was conducted for the two constructed molecular models using the Database for Annotation, Visualization and Integrated Discovery (DAVID) version 6.7 . Results2 in air. Cells were seeded onto six-well cell culture plates (Greiner Bio one) and were used for experiments at the fifth passage. Confluent cells were cultured in serum free media and FK 506 (Abcam) dissolved in DMSO (Sigma) in a final concentration of 1 μM for 24h. The control group was treated in serum free media and DMSO only and a total of four independent experiments were conducted. Trizol® and chloroform reagent was used to extract RNA (Invitrogen). The RNA pellet was dissolved in 10 μl sterile water and stored at –80°C.Primary human mesangial cells  were cultured in MCDB-131 media (Gibco-invitrogen), supplemented with 10% FBS, 100U penicillin/ml 100ug/ml streptomycin and 2mmol L-Glutamine  at 37°C in humid atmosphere with 5%COhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE84908).Total RNA from human mesangial cells was reverse transcribed, labelled and hybridised to Affymetrix Human Gene (HuGene) 2.0 ST Array (Affymetrix) according to the manufacturer’s protocol. The oligo R package was used for pre-processing and normalization of expression data. The statistical analysis of microarrays (SAM) method available in the TIGR MultiExperimentViewer was used in order to identify differentially regulated transcripts between tacrolimus treated and untreated cells 18]. Tra. Tra18].The two constructed molecular models, namely the one representing tacrolimus MoA and the one reflecting diabetic nephropathy pathophysiology on a molecular network level were compared to each other with respect to molecular feature overlap as previously described . DeregulBiomarker evidence for diabetic nephropathy diagnosis and prognosis was gathered from scientific literature for molecular features in the diabetic nephropathies molecular model being affected by tacrolimus. Therefore molecular features were extracted from publications annotated with the major MeSH term “diabetic nephropathies” as well as the MeSH terms “biological markers” OR “genetic markers” neglecting profiling studies by excluding publications annotated with one of the following MeSH terms: “gene expression studies”, “microarray analysis”, “proteomics”, “metabolomics”, or “genome-wide association study”. Publications however had to be either of publication type “clinical trial” or annotated with “humans” or “disease models, animal”. Prognostic markers were extracted from publications further holding the MeSH term “prognosis”, whereas diagnostic markers were extracted from publications annotated either with “diagnosis”, “diagnosis, differential”, “early diagnosis”, or “diabetic nephropathies/diagnosis”. Next to considering molecular features assigned via gene2pubmed to the respective marker papers, we mined for molecular features using their respective gene names and considered perfect matches as hits.calcium signaling with 28 molecular features out of the 252 being assigned to this pathway . A set of signaling cascades were among the list of enriched molecular pathways like e.g. Adipocytokine signaling , MAPK signaling , TGF-beta signaling , or PPAR signaling . Next to these signaling cascades, molecular pathways linked to inflammation and immune response were enriched like for example cytokine-cytokine receptor interactions , Toll-like receptor signaling , or chemokine signaling . In addition the renin-angiotensin system  as well as the complement and coagulation system  were enriched with molecular features from the constructed DN molecular model.583 molecular features could be extracted from scientific literature being associated with diabetic nephropathy. The constructed molecular model for diabetic nephropathies in total held 252 molecular features in 15 process units ranging in size from 3 to 58 molecular features. The top enriched molecular pathway of the 17 identified molecular pathways was The tacrolimus MoA molecular model held 209 molecular features assigned to 19 process units ranging in size from 3 to 38 molecular features. From the 785 unique protein coding genes forming the input for model construction, 582 protein-coding genes originated from the two transcriptomics studies with 217 genes coming from literature mining for tacrolimus-associated molecular features. Five of the enriched molecular pathways in DN were also significantly enriched based on the set of molecular features in the tacrolimus MoA molecular model, namely cytokine-cytokine receptor interactions, adherens junctions, TGF-beta signaling, MAPK signaling, as well as calcium signaling .The use of heterogeneous data sets for constructing the tacrolimus MoA molecular model could be seen very critically but can also be seen from a positive angle. With input data coming from fibroblasts as well as from post-transplant kidneys it is not possible to reconstruct cell-specific molecular pathways, but on the other hand it allows assessing the effect of tacrolimus on kidney as a whole. Following this line we furthermore assessed the effect of tacrolimus on mesangial cells using gene expression profiling.21534 transcripts remained after preprocessing and normalization steps which were used for hierarchical cluster analysis in order to see whether there was an effect of tacrolimus on mesangial cells in culture. 1—Pearson correlation and average linkage were used as distance measure and linkage rule. In the cluster dendrogram, a clear separation between the tacrolimus-treated and the untreated control samples was detectable although absolute differences in gene expression between samples were only minor. Nevertheless 222 differentially regulated transcripts were identified using the SAM with a false discovery rate of lower than 5%. 154 transcripts were upregulated after tacrolimus treatment with 68 being downregulated. The deregulated transcripts could be mapped to 204 unique protein coding genes on the level of Ensembl Gene IDs with 141 upregulated and 63 downregulated genes.39 of the 252 individual molecular features of the DN molecular model were also part of the tacrolimus MoA molecular model thus being affected by tacrolimus. In addition six further molecular features of the DN molecular model could be identified as deregulated after tacrolimus treatment in mesangial cells .Three molecular features had annotation evidence as marker for diabetic nephropathy, with TNF being reported as diagnostic marker for DN and TGFB1 and EDN1 showing evidence as prognostic marker for DN.A set of molecular features involved in inflammation and immune response like the tumor necrosis factor alpha (TNF), interleukins 4 and 10 , or the heat shock protein family A (Hsp70) member 1B (HSPA1B) were all downregulated by tacrolimus.Inflammation is recognized as a major factor in diabetic nephropathy 21], sub, sub21],TNF was previously reported as biomarker for diagnosing diabetic nephropathy and also proposed as potential drug target in order to slow disease progression . Next toThe downside of tacrolimus on renal tissue becomes evident when looking at upregulated molecular features in the DN molecular model . EspeciaAnother pro-fibrotic molecule, namely endothelin 1 (EDN1), was upregulated by tacrolimus in mesangial cells, thus reinforcing the finding that tacrolimus rather activates than inhibits TGF-beta signaling .Based on these findings further studies are warranted stratifying DN patients based on TNF levels in order to identify patients being prone for treatment with low-dose tacrolimus 44]. TGF. TGF44].A further strategy might be to use drugs that block detrimental effects induced by tacrolimus like e.g. blocking TGF-beta signaling cascade with TGFB1 antibodies or using endothelin 1 receptor antagonists which are currently in clinical testing in the context of diabetic nephropathy. Such strategies were already proposed as alternative to conventional strategies of calcineurin inhibitor avoidance, withdrawal, or minimization in the context of renal transplantation . The endInflammatory mechanisms, and in particular the tumor necrosis factor signaling cascade, contributing to progression of diabetic nephropathy might be counterbalanced by tacrolimus treatment. Using TNF as predictive marker and TGFB1 and EDN1 as monitoring markers might be a valuable strategy finding the subset of diabetic nephropathy patients benefitting the most from tacrolimus treatment.


The BRadykinesia Akinesia INcordination (BRAIN) test is an online keyboard-tapping test previously validated as a sensitive tool for detecting signs of Parkinson’s disease.To determine whether the BRAIN test can measure disability in MS and identify the presence of pyramidal or cerebellar dysfunction.Kinesia scores , akinesia times  and incoordination scores  were calculated in 39 MS patients. These were correlated against the Expanded Disability Status Scale (EDSS) scores, pyramidal and cerebellar functional system scores and 9-hole peg test scores.r = − 0.594, p < 0.001), AT  and IS . 9-HPT scores strongly correlated with KS . Pyramidal scores correlated with KS . Cerebellar scores correlated with KS , AT  and IS . Receiver operating characteristic curves demonstrate that KS can distinguish between the presence or absence of pyramidal and cerebellar dysfunction with area under curve 0.840 (p < 0.001) and 0.829 (p < 0.001), respectively.EDSS correlated with KS  contains supplementary material, which is available to authorized users. A variety of outcome measures are available to assess upper limb function in multiple sclerosis (MS) . The 9-hThe BRadykinesia Akinesia INcordination (BRAIN) test is a computer keyboard-tapping test that was developed to assess upper limb function in neurological disease. It has been validated as a sensitive tool for detecting signs of Parkinson’s disease and can be completed in the outpatient department, at home and in clinical trials using online software , 9.The test involves alternately tapping keys at the opposite end of a keyboard using one finger. Subjects perform this as fast and accurately as possible during a 30 s period and this is then repeated with the other hand to derive several scores. The Kinesia Score (KS) measures the number of taps in 30 s, the akinesia time (AT) measures the mean dwell time on each key in milliseconds and the incoordination score (IS) measures the variance of the time interval between keystrokes in milliseconds.r = − 0.53, p < 0.001) [r = 0.27, p = 0.03 and r = 0.28, p = 0.03 respectively). A study using an earlier version of the BRAIN test in 12 patients with cerebellar dysfunction from a range of aetiologies found that KS was correlated to the score on the  Cerebellar Disease Rating Scale  [r = − 0.55, p = 0.06 and r = 0.30, p = 0.30, respectively).In Parkinson’s disease, KS has been shown to be significantly lower than controls and is correlated to the severity of motor dysfunction, as measured by the UPDRS motor score (Spearman’s < 0.001) . In the  = 0.03) . AT and r = − 0.59, p < 0.001). The BRAIN test had not previously been studied in MS patients. In the present study, we aimed to confirm whether the BRAIN test could be used to measure disability in MS and to further characterise how KS, AT and IS vary in patients with pyramidal and cerebellar dysfunction.Preliminary (unpublished) data from our group derived from a service development study using fampradine in MS patients showed that KS correlated strongly with EDSS  score by a trained clinician . In deteParticipants undertook the BRAIN test following the EDSS assessment. They were seated at a desktop computer with a keyboard and followed on-screen test instructions from the BRAIN test website. This included being asked to alternately tap the ‘s’ and ‘;’ on the keyboard as fast and as accurately as possible during a 30 s period. They performed the test once using each hand and received no assistance during the test. Raw data from the BRAIN test website was used to determine the KS, AT and IS for each hand. The mean KS, mean AT and mean IS for each subject was then calculated. Subjects were also asked to perform the 9-hole peg test (9HPT). They were given standardised instructions and the scores were calculated, as previously described . The 9HPZ test with Fisher’s r-to-z transformation. Linear regression analysis was used to determine the relationship between BRAIN tests and EDSS scores. The t test and Mann–Whitney test were used to compare BRAIN test scores in those patients with or without pyramidal or cerebellar dysfunction and receiver operating characteristic (ROC) curves were used to compare the ability of BRAIN test scores to distinguish patients with or without any pyramidal or cerebellar dysfunction. The pre-determined significance level for all calculations was p = 0.01. All analyses were performed using GraphPad Prism version 6 for Mac.Descriptive statistics for these variables were calculated. For continuous variables, means were reported if the data were normally distributed (assessed using the Shapiro–Wilks test) and medians were reported if not normally distributed. Associations between BRAIN test, EDSS, FS and 9HPT scores were estimated using Spearman’s rank correlation coefficient for non-normally distributed data. These correlation coefficients were compared using Steiger’s The study was approved by the Queen Square Research Ethics Committee (09/H0716/48) and has, therefore, been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. All participants provided written informed consent prior to inclusion in the study.Assessments were performed on 39 patients with MS; three participants declined to undertake the 9-HPT and a further three did not complete the task within a minute and were assigned a value of 60 s. The mean age was 43.2 years (range 23–70), 74.4% of participants were female and 89.7% were right-handed.2 . The median 9-HPT score was 0.047 s−1 (range 0.024–0.065).The median disease duration was 4 years and the median EDSS score was 3 (range 0–6.5). The median pyramidal FS score was 2 (range 0–3) and median cerebellar FS score was 1 (range 0–4). The mean KS was 55.6 taps (range 21.5–87.5), the median AT was 96.5 ms (range 50.0–210.0) and the median IS was 10,780 msr = − 0.594, p < 0.001), AT  and IS  scores with EDSS were not significantly different from each other . The distribution of KS according to EDSS is shown in Fig. The EDSS correlated with KS, AT and IS. The correlation coefficients for KS . As expected, the 9-HPT scores correlated with EDSS . There was no significant difference between the correlation coefficients for 9-HPT and KS with EDSS .The 9-HPT scores strongly correlated with KS (p < 0.001) and KS correlated to the pyramidal score . AT and IS were not significantly different in patients with pyramidal dysfunction and did not correlate with pyramidal score, as shown in Table Pyramidal dysfunction was associated with abnormal KS. The mean KS was lower in those patients with any pyramidal dysfunction compared to those without  and KS correlated to the cerebellar score . The median AT was higher in those patients with any cerebellar dysfunction compared to those without  and AT correlated to the cerebellar score . The median IS was higher in those patients with any cerebellar dysfunction compared to those without  and IS correlated to the cerebellar score . The Spearman’s correlation coefficients and p values for KS, AT and IS with pyramidal and cerebellar scores are shown in Table Cerebellar dysfunction was associated with abnormal KS, AT and IS. The mean KS was lower in those patients with cerebellar dysfunction compared to those without  and cerebellar dysfunction with an AUC of 0.829 (p < 0.001). AT and IS can distinguish between the presence or absence of cerebellar dysfunction with an area under curve (AUC) of 0.723 (p = 0.007) and 0.745 (p = 0.009), respectively, and as shown in Table The BRAIN test distinguished between the presence or absence of pyramidal and cerebellar dysfunction. ROC curve analysis shows that KS can distinguish between the presence or absence of pyramidal dysfunction with an area under curve (AUC) of 0.840  and to a lesser degree AT  and IS (r = 0.28 and p = 0.03) [Analysis of BRAIN test scores in MS patients with or without pyramidal and cerebellar dysfunction, in conjunction with the correlations of BRAIN test scores to FS scores, demonstrated important differences between specific BRAIN test scores. Pyramidal dysfunction is associated with abnormal KS alone and the severity of pyramidal dysfunction correlates with KS. In contrast, cerebellar dysfunction was associated with abnormal KS, AT and IS and the severity of cerebellar dysfunction correlates with each of these scores. The caveat to these observations is that a large proportion of participants had both pyramidal and cerebellar dysfunction. In the aforementioned study, parkinsonism was associated with abnormal KS, AT and IS and the severity of parkinsonism (as measured by UPDRS) was correlated with KS Below is the link to the electronic supplementary material.