Employing GeNorm, BestKeeper and Normfinder algorithms, this study identifies PSMB6, MDH1 and some more RG as sufficiently unregulated, thus expressed at stable levels, in hepatocyte-like cells in vitro. Inclusion of multiple RG, quenching occasional regulations of single RG, greatly stabilises gene expression level calculations from RT-qPCR data. To further enhance validity and reproducibility of relative RT-qPCR quantifications, the ΔCT calculation can be extended (e-ΔCT) by replacing the CT of a single RG in ΔCT with an averaged CT-value from multiple RG. The use of two or three RG - here identified suited for human hepatocyte-like cells - for normalisation with the straightforward e-ΔCT calculation, should improve reproducibility and robustness of comparative RT-qPCR-based gene expression analyses.Reference genes (RG) as sample internal controls for gene transcript level analyses by quantitative RT-PCR (RT-qPCR) must be stably expressed within the experimental range. A variety of  Also, of the 192 RT-qPCR-papers found, 91 analysed primary liver material from patients and 131 studies included hepatic cell lines, wherein HepG2 (59%) and Huh-7 (47%) cell lines were by far the most commonly used ones.A literature survey covering eight volumes of the journal HEPATOLOGY (years 2009–2012) for RT-qPCR-analyses of human hepatocyte-like cells – i.e. primary hepatocytes and hepatic cell lines - identified in vitro investigations mainly in the areas of basic, preclinical and pharmacological research - including metabolic assays, drug- and drug-toxicity testings, accompanied by optimisations of culture conditions towards enhanced cell maturity. Also, studies with infectious human hepatotropic agents are of major importance and more recently, in vitro gene therapeutic approaches using primary hepatocytes - requiring the issues of rapid dedifferentiation and almost complete absence of proliferation of primary hepatocytes in tissue culture to be overcome, possibly by the culture of proliferating hepatic stem cells and their subsequent maturation in the future.Hepatocytes and hepatoma-derived cell lines are used for a plethora of in vitro settings with commonly used hepatocyte-like cell types, we identified in this study several reference genes suited for RT-qPCR-analyses of hepatocyte-like cells. Furthermore we were able to derive from the ΔCT-method a simple calculation allowing for the inclusion of multiple RG to significantly strenghten data normalisation in comparative, not absolute, gene expression analyses - without the need for laborious generation of qPCR-standard curves.Investigating the expression stability of 22 widely used RG in experimental A more detailed description of materials and methods used can be found in in silico.Briefly, the expression stability of 22 widely used RG  consistently rank well in the subgroups, offering the option to use multiple RG for normalisation. Noteworthy, albeit not included in the systematic analyses presented here, PSMB1 and MDH1 are also almost entirely unregulated in the human liver progenitor cell line HepaRG The individual RG rankings , and theGAPDH varies significantly in all subgroups and settings, similar to observations also in other cell types Expression of the most commonly used All RG identified as suitable are measured in a CT-range above 20 , thus arIn many RT-qPCR applications relative changes of TG expression levels between experimental settings - and not absolute molecule numbers - are of interest. For such relative comparisons, the ΔCT method is most straightforward and often used PSMB6 and CYC1 Click here for additional data file.


Left ventricular non-compaction (LVNC) cardiomyopathy is characterized by a thin, compacted epimyocardial and a thick non-compacted, trabeculated endomyocardial layer. High-resolution cardiac magnetic resonance imaging (CMR) has been successfully used to distinguish myocardial trabeculation within the LV cavity. Usually, a compact-to-non-compact ratio of 2.3 on MRI is regarded pathological.Unfortunately, this one-dimensional measure is not well standardized and observer-dependent. Moreover, only scarce data exists on age and gender dependency of myocardial trabeculation in normal volunteersWe present a novel multi-slice measurement approach for myocardial trabeculations and sought to investigate age and gender dependencies on LV and RV myocardial trabeculations.In 120 male/female healthy volunteers divided into three age groups  a vector-ECG gated multi-slice short axis standard cine SSFP-sequence was used. Trabeculation volume was measured by drawing contours between compacted and non-compacted myocardium (outer solid line) as well as between edges of the trabeculation net and normal end-diastolic LV volume (inner dashed line) and normalized to body mass index (BMI). Papillary muscle were excluded. Data was compared using ANOVA (p<0.05 significant).).Results are presented in figure Interestingly, myocardial trabeculation is different between RV and LV. Though the LV trabeculation is independent from age for women and men, there is a significant decrease of trabeculation volume in the RV. Male volunteers always reveal higher trabeculation volumes for all age groups in both ventricles


Many types of neurons show spike rate adaptation, a gradual decrease in spiking activity following a sudden increase in stimulus intensity. This behavior is typically mediated by slow potassium currents through voltage-sensitive low-threshold or calcium-activated high-threshold channels, both of which are susceptible to cholinergic modulation . Such adBased on a population of adaptive exponential integrate-and-fire (aEIF) model neurons receiving noisy external and recurrent synaptic inputs we use the Fokker-Planck equation to compute spike rates and ISI distributions in the limit of a large adaptation timescale.We show that a subthreshold adaptation current or synaptic inhibition received from independent neurons  change the neuronal I-O curve subtractively. That is, both mechanisms increase the spike threshold. On the other hand, a spike-triggered adaptation current or inhibitory synaptic feedback (recurrent inhibition) change the I-O curve divisively, i.e., they reduce the neuronal gain, see Figure


After a wash-out period of six wks without any RAAS blocking agents or other therapy to lower proteinuria (untreated proteinuria (UP)), patients subsequently received indomethacin 75 mg BID for 4 wks (NSAID). Healthy subjects (n = 10) screened for kidney donation served as controls. Urine and plasma levels of total IgG, IgG4, KIM-1, beta-2-microglobulin, H-FABP, MCP-1 and NGAL were determined using ELISA. Following NSAID treatment, 24 h -urinary excretion of glomerular and proximal tubular damage markers was reduced in comparison with the period without anti-proteinuric treatment  may be used to lower therapy-resistant proteinuria. The potentially beneficial anti-proteinuric, tubulo-protective, and anti-inflammatory effects of NSAIDs may be offset by an increased risk of  side effects. We investigated the effect of indomethacin on urinary markers of glomerular and tubular damage and renal inflammation. We performed a post-hoc analysis of a prospective open-label crossover study in chronic kidney disease patients ( Reduction of proteinuria is a major therapy target to achieve long-term renoprotection in chronic kidney disease. To this end, blockade of the renin-angiotensin-aldosterone system (RAAS) ideally combined with a diuretic or dietary sodium restriction Before the era of RAAS inhibitors, NSAIDs were well known for a distinct antiproteinuric effect. Their use in nephrotic syndrome was supported by retrospective data suggesting that NSAIDs may retard progression of renal function loss Most renoprotective effects of NSAIDs are considered to be related to their effect on prostaglandin synthesis To further investigate the glomerular, tubular and anti-inflammatory effects of NSAIDs in CKD, we measured a panel of urinary renal damage markers representing different parts of the nephron in patients with overt proteinuria, before and after treatment with indomethacin. As a secondary aim we investigated whether pretreatment biomarker levels were associated with the anti-proteinuric response.The study was approved by the medical ethics committee of the University of Groningen, and all participants provided written informed consent. All clinical investigations have been conducted according to the principles expressed in the Declaration of Helsinki.This study is performed as a post-hoc analysis of a prospective open-label crossover study The detailed study protocol was published previously At each visit, after an overnight fast, blood was sampled, 24-h urine was collected and blood pressure was measured by an automatic device . Twentyfour-hour urine was checked for collection errors using the equation previously formulated by Ix et al Aliquots from 24 h -urine were stored at −80°C until damage marker analysis. After thawing, all urine samples were vortexed and subsequently centrifuged (14.000 rpm). The supernatant was used for measurements. Samples were diluted to obtain optimal concentration for measurement. All urinary biomarkers were determined in one run. As glomerular damage markers we measured total IgG and IgG4, as markers of proximal tubular damage kidney injury molecule-1 (KIM-1) and beta-2-microglobulin (B2M), as markers of distal tubular damage heart-fatty acid binding protein (H-FABP), and as markers of inflammation neutrophil gelatinase associated lipocalin (NGAL), monocyte chemotactic protein-1 (MCP-1), as documented previously The 24-h excretion of renal damage markers was calculated by multiplying the urinary biomarker concentration with the total 24 h urine volume. Fractional excretion of renal damage markers was calculated as:Data are expressed as median (interquartile range). Differences between healthy controls and CKD patients without anti-proteinuric treatment were analyzed by Mann Whitney non-parametric test. Differences between the study periods without anti-proteinuric treatment versus NSAID treatment, respectively, were analyzed by Wilcoxon paired non-parametric test. Associations between urinary biomarker excretion and proteinuria were studied using linear regression with proteinuria as an independent variable, and the individual renal damage markers as dependent variables. For these analyses, urinary biomarker excretions were logarithmically transformed. We also determined whether the change in biomarker excretion between the study period without anti-proteinuric treatment and NSAID (delta biomarker) was related to the anti-proteinuric response (delta proteinuria). Furthermore similar analysis were performed for the association between biomarker excretion during the period without anti-proteinuric therapy (i.e. before anti-proteinuric intervention) and the anti-proteinuric response, to evaluate which biomarker(s) would be able to predict the anti-proteinuric response. For all analyses, a two-sided p<0.05 was considered statistically significant.All calculations and analyses were performed using SPSS 18.0 for Windows .Baseline characteristics are given in In parallel with proteinuria, 24-h urinary excretions of the glomerular damage markers total IgG and IgG4 were strongly increased in CKD patients as compared to healthy controls. Anti-proteinuric treatment with indomethacin reduced urinary total IgG and IgG4 excretion . Plasma The two proximal tubular markers KIM-1 and B2M were elevated in CKD patients during the study period without anti-proteinuric treatment, as compared to healthy controls, and reduced by NSAID treatment . FractioWe subsequently investigated whether the anti-inflammatory drug indomethacin would affect renal inflammation, as reflected by urinary MCP-1 and NGAL excretion. Surprisingly, indomethacin did not reduce the 24 h -urinary excretions of MCP-1 or NGAL, both of which were higher in CKD patients than in healthy subjects . In plasThe relationship between urinary renal damage marker excretions and proteinuria was further investigated by regression analysis. Univariate analysis indicated that urinary excretions of total IgG , IgG4 , KIM-1 , B2M , and H-FABP  were significantly associated with proteinuria. As expected, delta urinary total IgG excretion  and delta urinary IgG4 excretion  were significantly associated with the anti-proteinuric response.Finally we studied whether renal damage marker excretion during the period without anti-proteinuric treatment (i.e. before anti-proteinuric therapy) would be associated with the anti-proteinuric response to indomethacin. Interestingly, urinary excretion of KIM-1  and MCP-1  before anti-proteinuric treatment was inversely associated with the anti-proteinuric response. After adjustment for age and genderonly the association between MCP-1 and the anti-proteinuric response remained significant . A scatter plot illustrating the relationship between pre-treatment MCP-1 levels and the anti-proteinuric response is presented in RAAS blockade combined with volume restriction is the therapy of choice for most CKD patients, but the anti-proteinuric response to this regimen is often incomplete The reduced urinary excretion of glomerular damage markers total IgG and IgG4 is in line with the known anti-proteinuric effect of indomethacin, and may be related to prostaglandin inhibition affecting renal hemodynamics (i.e. vasoconstriction of the afferent arterioles). Furthermore, both 24 h -urinary excretion and fractional excretion of the proximal tubular damage markers were reduced following treatment with an NSAID, and the distal tubular marker H-FABP showed a trend to reduction. This suggests that NSAIDs may have protective effects, rather than toxic effects, on the tubular epithelium. Tubulotoxic effects may be seen in rare cases, but in our study on average signs of reduction of tubular damage are observed, probably secondary to the anti-proteinuric effect of NSAID treatment. This is in concert with previously observed tubulo-protective effects of RAAS-related anti-proteinuric therapy in both clinical cohorts data submitted). Our finding that the extent of pre-treatment renal inflammation is independently associated with the anti-proteinuric response is also in line with preclinical studies Surprisingly, NSAID treatment had no effect on the 24 h-urinary excretion of inflammation markers MCP-1 or NGAL. In contrast, fractional excretion of MCP-1 even increased following NSAID treatment. This was unexpected given the anti-inflammatory properties of this drug. On the other hand, the dissociation of renoprotective effects from anti-inflammatory effects of NSAIDs is in line with preclinical studies Treatment with indomethacin induced a trend towards decreased creatinine clearance in our study, and although this reduction was relatively small, our study lacks power to exclude an effect on renal function. Previous studies demonstrated an anti-proteinuric response in the presence of a significant decline of GFR following NSAID treatment NSAIDs are nowadays rarely considered for anti-proteinuric therapy in CKD. This is for the large part explained by the availability of RAAS blockers with a more favorable potential, whereas NSAIDs may have tubulotoxic effects. Also, the reduction of creatinine clearance observed in up to 20% of patients treated with NSAIDs Our study has its limitations such as limited sample size and study duration, and the post-hoc design. Given the heterogeneity of the population with respect to the underlying type of renal disease, it may be surprising that significant reductions of glomerular and proximal tubular biomarkers were found. This suggests that irrespective of the underlying disease, one or more shared pathophysiologic pathways (including proteinuria) are reduced by NSAID treatment. The current findings cannot be extrapolated to patients with more severe renal disease, who may experience more side effects such as hyperkalemia or edema, or patients on background RAAS blockade. The latter category may not tolerate the combined regimen, and a previous study has questioned the additional efficacy of NSAID when give on top of maximum RAAS blockade as anti-proteinuric therapy In conclusion, NSAID treatment is associated with a change in urinary biomarker profile that suggests glomerulo- and tubulo-protective effects along with reduction of proteinuria. Our findings, although they cannot be extrapolated to a setting of  background RAAS blockade with optimal volume depletion, suggest that under specific conditions NSAIDs may be used to reduce proteinuria as well as glomerular and tubular damage markers in CKD. This may be relevant given the prognostic importance of tubulointerstitial damage for the progression of renal function loss Table S1Plasma levels of renal damage markers in CKD patients after a period with no anti-proteinuric treatment, and after NSAID treatment.(DOC)Click here for additional data file.


Encephalitis with anti-N-methyl-D-aspartate receptor antibodies (anti-NMDAR-Ab) is a rapid-onset encephalitis including psychosis, seizures, various movement disorders and autonomic system disturbances.We report a very unusual case of extensive myelitis associated with anti-NMDAR-Ab. MRI also revealed a hyperintense T2 lesion, non-suggestive of MS, which progressively extended, associated with periventricular gadolinium enhancement visualized on brain MRI. Ophthalmological evaluation showed subclinical right optic neuritis. The absence of anti-AQP4 antibody argued against neuromyelitis optica spectrum disorder. A slight psychomotor slowing prompted us to search for various causes of autoimmune encephalitis. Anti-NMDAR-Ab was found in cerebrospinal fluid.In patients with extensive myelitis who are seronegative for anti-AQP4 antibodies, and after other classical causes have been excluded, the hypothesis of atypical anti-NMDAR-Ab encephalitis should also be considered. Encephalitis with anti-N-methyl-D-aspartate receptor antibodies (anti-NMDAR-Ab) is a rapid-onset encephalitis including psychosis, seizures, various movement disorders and autonomic system disturbances. The physiopathology is based on immune-mediated neuronal dysfunction,2. We reA 65-year-old woman was admitted because of progressive paraparesis (June 2012). Six months before admission, she had presented with fever, chills, abdominal pain and unusual headache for one week with spontaneous recovery. Abdomen and brain MRI were normal at that time. Two months before admission, she described a progressive onset of walking difficulties. At the same time, constipation and dysuria were noted. At her admission, neurological examination noted walking difficulties related to moderate paraparesis, moderate superficial and deep sensory dysfunction of the lower limbs and urinary retention. A very slight dysfunction of mental processing without speech disturbance was noted and confirmed by her family. No seizures, dyskinesia, movement disorders, psychiatric symptoms or autonomic dysfunction were observed.3), mild increased protein concentration (0.91 g/L) and oligoclonal bands. PCR assays for herpesviridae in CSF were all negative. Ophthalmological examination showed delayed P100 latencies on the right side on visual evoked potentials and asymmetric global retinal nerve fibre layer (RNFL) thicknesses on optical coherence tomography (OCT) suggestive of asymptomatic right optic neuritis (ON). OCT analyses were both perfectly centred on the optic nerve head  enhancement  plus tapering oral corticosteroids (1 mg/kg). Brain and spinal cord MRIs follow-up (August 2012) showed the disappearance of the extensive spinal cord T2 lesion and stable cerebral T2 lesions. Biological follow-up of anti-NMDAR-Ab rates in CSF and serum was performed (Table Intravenous corticosteroids (1 g per day during 3 days plus 5 days) were given but the paraparesis worsened (bedridden patient). Brain and spinal cord MRIs follow-up (July 2012) showed an extension of T2 lesions without Gd enhancement  or NMO spectrum disorder (NMOSD). Although cognitive disturbances are described in NMO, we alsoProdromal symptoms such as headache and fever are common but we nEven though some correlations have been found between clinical status and anti-NMDAR-Ab titres in CSF, there is no reported correlation with anti-NMDAR-Ab titres in serum, and we therefore do not recommend a follow-up of anti-NMDAR-Ab titres in serum,14.Anti-NMDAR-Ab encephalitis has been described following massive brain damage induced by herpes simplex encephalitis (HSE),16. AntiWe can also discuss the hypothesis that our patient may have presented 2 auto-immune diseases concurrently: an NMOSD and an anti-NMDAR-Ab encephalitis. These are both Ab-mediated diseases and a comorbid association of auto-immune diseases is possible. Recently a case of NMO (seropositive anti-AQP4 Ab) following anti-NMDAR-Ab encephalitis has been described. As our In patients with extensive myelitis who are seronegative for anti-AQP4 Ab, and after other classical causes have been excluded, the hypothesis of atypical anti-NMDAR-Ab encephalitis should also be considered.Written informed consent was obtained from the husband of the patient for publication of this Case report and the accompanying images. A copy of the written consent is available for review by the editor of this journal.The authors declare that they have no competing interests.OO drafted the manuscript, participated to acquisition, analysis and interpretation of the data. GB participated to drafting and acquisition of data. JHod participated to acquisition of data and revised the manuscript for important intellectual concept. MG participated to acquisition of data and revised the manuscript for important intellectual concept. AL participated to acquisition of data and revised the manuscript for important intellectual concept. JHon revised the manuscript for important intellectual concept. HZ participated to analysis and interpretation of data and revised the manuscript for important intellectual concept. PV participated to acquisition of data and revised the manuscript for important intellectual concept. All authors read and approved the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/13/211/prepub


Vaccination of macaques with p55gagDNA failed to elicit cellular or humoral immune responses to the CE, while epitopes outside of the CE were immunogenic.Immunodominance and sequence diversity are major hurdles in the development of effective HIV vaccines. We tested the hypothesis that a vaccine candidate composed of strictly Conserved Elements (CE) of the HIV proteome excluding the variable regions would help overcome problems of viral sequence diversity and potential negative effects of immunodominance. Seven CE were identified in p24gagCE DNA plasmids were generated, providing potential epitopes found in >99% of all HIV-1 M group sequences. DNA vectors, optimized for gene expression were used to immunize mice and macaques by IM injection followed by in vivo electroporation.Two HIV p24gagCEvac DNAs elicited potent, cross-clade cellular and humoral immune responses. Highly cytotoxic CE-specific T cells, capable of Granzyme B production and degranulation, were generated. Importantly, boosting of the CEvac-primed macaques with p55gagDNA greatly augmented the CE-specific cellular responses (up to 10-fold) as well as humoral responses, despite the failure of p55gagDNA vaccine to induce de novo CE-specific responses. CEvac DNA prime-p55gagDNA boost in mice led to similar conclusions. Interestingly, mapping analysis showed differential increase of the CE-specific responses by the p55gagDNA boost, demonstrating changed hierarchy of CE responses in macaques.Vaccination with p24gagCEvac DNA overcame the problem of diversity by generating strong cross-clade Gag-specific immune responses, and of immunodominance, eliciting responses to subdominant but highly conserved elements, and also by broadening the p55gagDNA induced immunity. p55gagDNA did not induce de novo responses to the CE, but was able to significantly boost pre-existing CE-induced responses and alter the hierarchy of these responses. Translation of this concept into clinical trials may elicit cross-clade cellular immune responses against components of the viral proteome with limited capacity for immunological escape.Vaccination with the p24


It is difficult to longitudinally characterize cognitive impairment in amyotrophic lateral sclerosis (ALS) due to motor deficits, and existing instruments aren’t comparable with assessments in other dementias.The ALS Brief Cognitive Assessment (ALS-BCA) was validated in 70 subjects (37 with ALS) who also underwent detailed neuropsychological analysis. Cognitive predictors for poor survival were then analyzed in a longitudinal cohort of 171 ALS patients.The ALS-BCA was highly sensitive (90%) and specific (85%) for ALS-dementia (ALS-D). ALS-D patients had shorter overall survival, primarily due to the poor survival among ALS-D patients with disinhibited or apathetic behaviors after adjusting for demographic variables, ALS site of onset, medications, and supportive measures. ALS-D without behavioral changes was not a predictor of poor survival.ALS-D can present with or without prominent behavioral changes. Cognitive screening in ALS patients should focus on behavioral changes for prognosis, while non-behavioral cognitive impairments may impact quality of life without impacting survival. Amyotrohpic lateral scleorsis (ALS) is a progressive neurodegenerative disorder that affects motor and extra-motor systems, and shares the pathologic hallmark of neuronal inclusions immunoreactive to TAR-DNA binding protein of ∼43 kD (TDP-43) with many cases of frontotemporal degeneration (FTD). ALS patients can also develop mild cognitive impairment (ALS-CI) detectable on detailed neuropsychological analysis. Studies on ALS-CI and survival have generated conflicting findings, validation cohort included 37 ALS patients evaluated and longitudinally followed at the Emory ALS Center, and 43 healthy subjects longitudinally followed at the Emory Alzheimer’s Disease Research Center with normal cognition and behavior. All subjects independently underwent cognitive evaluation using the ALS-BCA and, on a separate day within 3 months, an in-depth neuro-cognitive evaluation consisting of structured neuropsychological analysis, language evaluation, and diagnostic formulation through a consensus mechanism. Written informed consents were obtained from all subjects or their legal representatives in the validation cohort. The longitudinal cohort consisted of 171 ALS patients serially followed at the Emory ALS Center on a regular interval (every 3–4 months), including 20 subjects from the validation cohort. There was no significant (p>.05) difference in age, disease duration, and cognitive performance between subjects from the validation cohort who were or were not part of the longitudinal analysis (data not shown). The ALS-BCA was administered as part of their clinical evaluation, and their ALS-BCA performance was retrospectively reviewed . As part of their initial clinical evaluation, all patients from the longitudinal cohort underwent structured interview for demographic information , neurological examination, functional assessment using the revised ALS Functional Rating Scale (ALSFRS-R), Two cohorts of subjects were included in the current study, and all were diagnosed with possible, probable, or definite ALS. The ALS-BCA is a 5-item assessment that evaluates a subject’s executive functions , frontally-mediated language function, delayed verbal recall, and behaviors common in FTD . Four itSubjects in the validation cohort underwent detailed neuropsychological analysis according to the procedures of the Alzheimer Disease Centers Uniform Data Set, All statistical analysis using de-identified data was performed in SPSS 20.0 . Baseline comparisons between subjects with normal cognition, ALS-CI, and ALS-D were performed by Chi-squared tests for categorical variables and analysis of variance for continuous variables. Sensitivity and specificity of ALS-BCA for ALS-CI and ALS-D were determined by receiver-operating characteristics (ROC) curve analysis. Survival analysis was performed first by Kaplan-Meier analysis for the effect of ALS-CI or ALS-D, with p<0.01 as a threshold for significance at the ALS-BCA subtest level to adjust for multiple comparisons. Tests linked to poor survival were then analyzed in a Cox proportional hazards analysis to adjust for contribution from other factors, with age, gender, disease duration, and bulbar- vs. limb-onset were entered as fixed variables in the first step, and abnormal ALS-BCA test, FEV, rilutek use, NPPV use, PEG use, and family history entered in a forward likelihood ratio fashion.In the validation cohort n = 70, , ALS-D sThe longitudinal cohort for survival analysis contained 171 consecutive ALS patients with a median follow-up of 9 months. At baseline, 25% of the ALS patients had impaired performance on two or more subtests (ALS-D), and 27% of the ALS patients had impaired performance on only one subtest ALS-CI, . Impairm61 ALS patients (36%) died during a median follow-up of 9 months. ALS-D patients had worse overall survival than non-demented ALS patients . Abnormal behavior (FBI score of 23 or more) was strongly associated with poor survival (p<0.001), but survival was not influenced by impaired performance in delayed free recall (p = 0.130), reverse digit span (p = 0.08), letter-guided fluency (0.185), or oral trails (p = 0.123). When only ALS-D patients were analyzed, patients with abnormal FBI scores had significantly worse survival than patients with normal behaviors. After this dichotomization, ALS-D with normal behavior was no longer associated with poor survival . This waA number of tools have been proposed for the diagnosis of ALS-D and ALS-CI given the overlap between ALS-D and FTD, but they serve primarily as screening instruments and are not in regular use in cognitive research. Here we modified a previously validated tool for FTD to derive the ALS-BCA, and demonstrated its high sensitivity and specificity for ALS-D in a large validation cohort. The applicability of ALS-BCA and the broader PBAC will enable the comparative analysis of cognitive decline between ALS and other motor-sparing disorders including FTD and AD. Furthermore, we prospectively used ALS-BCA in a larger longitudinal cohort to show ALS-D to be quite common, and the poor survival associated with ALS-D is primarily due to patients with abnormal behaviors while dementia without behavioral abnormalities did not significantly influence overall survival in ALS.Cognitive impairment in ALS has recently become a focus of detailed clinical and neuropsychological research. The phenotypic information derived from the ALS-BCA also enabled us to conclude that ALS-D patients with abnormal behaviors, but not ALS-D patients with normal behaviors, have the poorest survival. This differs qualitatively from prior studies which mostly found FTD to be associated with poor survival in ALS. For example, one such study The phenotypic and quantitative nature of ALS-BCA additionally allowed us to study the impact of ALS-CI on survival. In the longitudinal cohort, executive dysfunction accounted for approximately 75% of all ALS-CI. Similar to two prior studies, While the ALS-BCA has high diagnostic accuracy for ALS-D, this study has a number of limitations. The ALS-BCA is geared towards common deficits in FTD and AD, but does not contain subtests that evaluate parietal functions. While visual-spatial dysfunction is more common in FTD cases associated with tau pathology,


NTR)-dependent motor neuron death. Ligand binding by NgR could result in subsequent engagement of p75NTR, and this association could preclude pro-apoptotic signalling by the latter. Understanding the intricate interplay among Nogo isoforms, NgR and p75NTR in ALS disease progression may provide important, therapeutically exploitable information.Nogo/reticulon (RTN)-4 has been strongly implicated as a disease marker for the motor neuron disease amyotrophic lateral sclerosis (ALS). Nogo isoforms, including Nogo-A, are ectopically expressed in the skeletal muscle of ALS mouse models and patients and their levels correlate with the disease severity. The notion of a direct involvement of Nogo-A in ALS aetiology is supported by the findings that Nogo-A deletion in mice reduces muscle denervation and prolongs survival, whereas overexpression of Nogo-A destabilizes motor nerve terminals and promotes denervation. Another intriguing, and somewhat paradoxical, recent finding revealed that binding of the Nogo-66 receptor (NgR) by either agonistic or antagonistic Nogo-66-derived peptides protects against p75 neurotrophin receptor (p75 Nogo/reticulon (RTN)-4 gene has several splice isoforms, the major ones being Nogo-A, B and C. The longest of these, Nogo-A/RTN-4A, is enriched in the brain and spinal cord [Since its molecular identification, the adult central nervous system (CNS) myelin-associated Nogo –3 has benal cord . Nogo-B/nal cord .Molecular dissection of Nogo-A has revealed three separate regions that have neurite growth inhibitory activity , 10. TheNTR[NTR[NTR is able to confer responsiveness to NgR ligand.NgR requires membrane-spanning co-receptors to transduce growth inhibitory signals. The first NgR co-receptor identified is p75NTR, 19. TAJNTR[NTR, 21. AnoNTR[NTR, may be Other than being recognized as forming a major signalling axis inhibiting neuronal regeneration upon CNS injury, Nogo and NgR have been implicated in other neurological disorders . Nogo-A SOD1) gene [Another neurological disorder with a strong implication of Nogo involvement is amyotrophic lateral sclerosis (ALS), one of the most common forms of motor neuron degenerative diseases in adults . LargelyD1) gene , 45.Recent findings have strongly implicated Nogo-A expression in the muscles as a potential biomarker for ALS –49, althet al.[SOD1 (G86R) mutant transgenic mouse model of ALS using a subtractive hybridization approach. The examination of the gastrocnemius muscle of these mice revealed an anomalous, ectopically elevated expression of the transcripts of the adult brain-specific isoform Nogo-A. Nogo-B transcripts were also moderately elevated. On the other hand, the transcript levels of the adult muscle-enriched isoform Nogo-C were reduced. Experimentally induced denervation also reduced Nogo-C levels, but did not result in the ectopic elevation in Nogo-A. Elevated Nogo-A levels, to varying degrees, as well as depressed Nogo-C expressions, were also found in the postmortem and biopsy samples from diagnosed ALS patients, but not amongst control patients.The first association of Nogo with ALS was reported by Dupuis et al.. The autA further study by the authors on ALS patients indicated that Nogo-A and Nogo-B expression was significantly correlated with the disease severity measured by the ALS functional rating scale . Nogo-A The above view has recently been challenged by other workers. Wojcik and colleagues reported that the muscle biopsies from patients with other forms of myopathy and peripheral neuropathies also exhibited elevated Nogo-A immunoreactivity within the denervated muscle fibers . AlthougNogo may only be a bystander whose level changes in ALS, but does not influence the disease. The potentially more interesting possibility is that Nogo has a direct or indirect role in ALS progression, and that this could be exploited in a therapeutic sense. Nogo-C is enriched in the adult muscles, but its physiological role in the muscle function is unknown. As Nogo knockout mice depleted of all Nogo isoforms –60 have et al. have recently provided evidence that the ectopic expression of Nogo-A in muscles contribute directly to the disease aetiology [SOD1 (G86R) mutant transgenic mice with a Nogo-A−/− mutant mice [G86R/Nogo-A−/− double-transgenic exhibited a moderate but significantly longer mean survival time compared to the G86R parent. These mice also have a higher number of motor neurons, reduced neuronal ubiquitin inclusions indicative of stress and a reduced expression of neuromuscular disease markers, acetyl-choline receptor and muscle-specific receptor of tyrosine kinase.Jokic etiology . The autant mice  and showThe authors have also asked if the ectopic overexpression of Nogo-A could induce a neuromuscular junction (NMJ) pathology. The muscle fibres transfected with a Nogo-A expression, constructed by electroporation, indeed exhibited aberrant synaptic structures and a significantly reduced postsynaptic structure size. Although it is unclear whether the levels of Nogo-A expression in these transient transfection experiments are comparable to those in ALS, and whether the overexpression of Nogo-B or Nogo-C has similar or different effects, these experiments suggest that the muscle fibre Nogo-A overexpression destabilizes NMJs. This destabilization may conceivably cause nerve terminal retraction and denervation, with consequential motor neuron death due to deprivation of target tissue neurotrophin, resulting in muscle atrophy. Synaptic destabilization by Nogo-A overexpression is, probably, not unique in NMJs and has also been observed in the cere-bellar Purkinje cells and their terminals .NTR. Dupuis and colleagues [SOD1 (G93A) mice (another transgenic ALS model). This rescue was abolished by phosphatidylinositide-phospholipase C (PI-PLC) treatment (which cleaves GPI-linked proteins like NgR), a soluble dominant-negative NgR fragment, as well as antisense oligonucleotide-mediated silencing of NgR expression. Furthermore, the subcutaneous administration of these peptides could extend the survival of the spinal motor neurons in vivo after the sciatic nerve axotomy.Not all types of neurons are susceptible to growth inhibition by Nogo-A. The susceptibility would, necessarily, require the presence of the relevant receptors and signalling pathway components. It is also clear from recent findings that myelin-associated inhibitors signal growth inhibition through cell type-specific mechanisms , 63. Anolleagues  showed tlleagues , an effelleagues . Both peNTR mediated deaths. Apparently, it does not matter if the NgR is bound by an agonist or an antagonist. In other words, whether downstream signalling that leads to growth cone collapse as well as neurite growth inhibition was activated may be irrelevant in this case. What may be necessary is for the ligand-bound NgR to engage p75NTR in a manner that precludes its apoptotic signalling. p75NTR has multiple binding partners [NTR's interaction with the ligand-bound NgR binding could preclude or displace its interaction with sortilin. This notion is augmented by an interesting aspect of NgR signalling that has emerged recently – it is modulated by cell surface prote-olytic processing of itself and its co-receptor. The ligand binding to the cerebellar neurons induces α- and γ-secretase-mediated proteolytic cleavage of p75NTR in a protein kinase C (PKC)-dependent manner [NTR. In cultured dorsal root ganglion (DRG) neurons, this cleavage could underlie the attenuating effect of neurotrophic factors such as fibroblast growth factor 2 (FGF2) on the axonal growth inhibited by CNS myelin-associated inhibitors [NTR away from the pro-apoptotic complexes, and its subsequent cleavage effectively precludes the pro-apoptotic signalling, this could be a part of the underlying mechanism of the motor neuron survival effects demonstrated by the Nogo-66-derived peptides. This speculation, however, needs to be confirmed experimentally.Although it is not yet shown if the administration of these peptides prolongs the motor neuron survival or general lifespan in ALS transgenic mice models, the findings outlined above are interesting and noteworthy in several aspects. First of all, NgR engagement by Nogo-66-derived peptides did not result in a general anti-apoptotic signalling, but rather in specifically countering p75partners –67, and partners , 69. It t manner . The TNFhibitors . If the NTR-mediated motor neuron death. This explanation is speculative but entirely plausible as Nogo-A has an additional neurite growth inhibitory domain (encoded by the Nogo-A-specific exon 3) that is not present in Nogo-B [That NgR ligand binding could enhance the survival of the motor neurons seems paradoxical in view of the earlier observations made by the same authors, which indicated that the absence of Nogo-A delays denervation, motor neuron death and disease survival . An expln Nogo-B . A deplecis form with NgR on axons, or synapses, affects the latter's interaction in trans form with ligands from an opposing cell. It is also unclear as to how neuronal Nogo-A affects the interaction of NgR with its other ligands.It should be noted that the targeted disruption of NgR did not enhance the spinal cord neuron regeneration in some models , and recNTR in normal and ALS disease settings would also be useful in other emerging non-CNS clinical conditions in which Nogo has been implicated, such as cardiomyopathy [Although Nogo-A's validity as a biomarker for ALS diagnosis/prognosis may need further confirmation, the above findings suggest Nogo and NgR could be potential targets for ALS adjunct therapy. This may be helped by the fact that the targeted deletion of both Nogo-A –60 and Nmyopathy .


Overexpression of the oncogene ERG (ETS-related gene) is an adverse prognostic factor in acute myeloid and T-cell lymphoblastic leukemia (AML and T-ALL). We hypothesize that ERG overexpression is associated with primary drug resistance thereby influencing the outcome in leukemia. We previously reported a cell-line based model of ERG overexpression which induced a potentially chemo-resistant spindle shape cell type. Herein, we report a specific transcriptional gene signature for the observed spindle shaped morphology. Genes significantly over-expressed after ERG induction strongly resembled adhesive mesenchymal-like genes that included integrins , CD44, and CD24. Interestingly, the mesenchymal-like signature was accompanied by the repression of DNA chromatin remodeling and DNA repair genes, such as CHEK1, EZH2, SUZ12, and DNMT3a. The ERG-induced mesenchymal-like signature positively correlated with TMPRSS2-ERG prostate tissues and invasive breast cancer mRNA expression datasets reflecting a general ERG-driven pattern of malignancy. Furthermore, inhibitors modulating ERG druggable pathways WNT, PKC, and AKT, and chemotherapeutic agent cytarabine revealed ERG-induced drug resistance. In particular, PKC412 treatment enhanced proliferative rates and promoted spindle shape formation in ERG-induced cells. Nilotinib and dasatinib were effective at abolishing ERG-induced cells. Moreover, ERG overexpression also led to an increase in double strand breaks. This report provides mechanistic clues into ERG-driven drug resistance in the poor prognostic group of high ERG expressers, provides insight to improved drug targeted therapies, and provides novel markers for a mesenchymal-like state in acute leukemia. The oncogene ERG belongs to an evolutionary related group of ETS DNA binding proteins and directs gene expression in hematopoietic processes establishing definitive hematopoiesis, maintaining the stem cell pool and promCurrent chemotherapy regimens are insufficient for high-risk acute leukemia patients characterized by high ERG expression. For instance, in AML, the cumulative incidence of relapse in high ERG expressers was 81%, in comparison to only 33% in low ERG expressers at 5-years. SimilarDue to the high incidence of TMPRSS2-ERG fusion in prostate cancer, recent studies have mainly focused on mapping ERG signaling networks in prostate. These networks comprise a diaspora of functions that show a role for ERG in the regulation of extracellular matrix through the plasminogen activator pathway, upregulWhile it is unarguable that ERG overexpression is involved in oncogenesis of leukemia and prostate cancers, much less is clear as to how ERG signaling mediates drug resistance. Emerging reports describe EMT in tumor progression as a mechanism for cell proliferative and survival advantages. EMT is We previously reported that prolonged ERG overexpression induced leukemia cells to adhere and develop bi-directional protrusions (spindle shaped cells) Fig. . This moTable 1Significantly over-expressed and under-expressed genes were uploaded and analyzed with DAVID Bioinformatics. Listed below are genes that signficantly cluster to the respective gene ontology functions. P-Value is included for significance.We then used Oncomine, a microarray cancer database, to assess whether ERG-induced gene signature generated from the K562 pTRE-ERG leukemia cell line was disease-relevant. 128 over-expressed genes were uploaded to Oncomine platform. ERG-induced gene signature positively correlated with three invasive and metastatic cancer mRNA expression dataseis: 1) Grasso dataset prostate tissues habouring ERG rearrangement versus no ERG rearrangement, 2) GrasRecently, we reported that ERG overexpression conferred resistance to the multi-kinase inhibitors TKI258 and sorafinib. A panelGiven the survival properties of ERG overexpression, we examined the proliferative properties of ERG-induced cells. CFSE labeled ERG-induced cells displayed a 2-fold proliferative advantage over non-induced cells at 72-hours Figure . ProlifeWe also investigated ERG-overexpressing cells treated with PKC412, to test the effect of drug resistance in the presence of HS-5 and AML MSCs. Untreated ERG DsRed positive cells were expanded in the presence of HS-5 (10%) but not in the presence of AML MSCs. However, ERG-induced cells (DsRed positive) treated with PKC412 expanded by 40% in co-culture with HS-5 and 20% with AML MSCs Figure . Thus, EDue to the enhanced survival of ERG-induced cells, we sought to identify potential adhesion molecules induced by ERG overexpression that would regulate cell-to-cell contacts. A key regulator of mesenchymal-like properties is CD44, a major adhesion molecule for homing, cell migration, and signal transduction. DeregulCurrent evidence supports the notion that, in cancer. MET (as opposed to EMT) is turned on in malignant cells to promote cell proliferation, migration, and evasion from apoptosis. Genome-Due to the severe downregulation of DNA repair gene expression in ERG overexpressing cells Table  and in pHerein we show that in leukemia cells ERG overexpression promotes a mesenchymal-like gene expression signature that includes: CD24, CD44, ITGA10, ITGB5, ITGB3, ITGA2B and the morphogenic-associated genes, such as SHANK3, TYROBP. These results are in line with the positive correlation of ERG-induced mesenchymal-like gene signature in leukemia with prostate carcinoma and chemotherapy-treated invasive breast tissues that validated our model system. We propose that, irrespective of cell type, high ERG expressing cells may initiate a malignant phenotype by promoting adhesion thereby enhancing the likelihood of escaping chemotherapeutic agents.Through fluorescent tracking, we have also demonstrated that ERG overexpression in leukemia cells confers proliferative growth advantage at twice the rate of non-induced cells, whilst mediating the development of spindle shape morphology. In our stable phenotypic model of ERG overexpression, these proliferative features allowed for an even greater ERG-induced cell expansion in co-culture with the cell line HS-5 and in co-culture with primary bone marrow derived stroma from AML. Thus, enhanced adhesion of ERG overexpressing cells to the stromal bed may exploit direct interactions with stroma cells in order to increase cell survival. In the presence of cytotoxic drugs, the percentage of ERG-induced cells treated with PKC412 was enhanced, although the rates of proliferation were not altered (data not shown). In addition the drug resistance promoted directly by ERG, stroma may provide additional apoptotic protection from cytotoxic drugs.ERG-induced leukemia cells treated with other PKC or WNT targeting drugs, such as LY294002, rapamycin and ICG-001, promoted expansion of ERG-induced cells rather than inducing apoptosis. This indicates that apoptotic evasion is another feature of ERG-induced survival, particularly under cytotoxic stress, possibly through the inhibition of PKC or WNT signaling pathways. Hence, it seems unlikely that targeted therapy through these drugs will benefit high ERG expressers. Conversely, nilotinib, PD325901, 5-azacytidine, dasatanib, were effective at depleting DsRed ERG positive cells. Of the agents screened, nilotinib induced apoptosis most effectively in ERG-induced cells.Our treatment with a CD44 blocking antibody completely abrogated the spindle shape phenotype, with minimal apoptotic effects. Due to antigen specificity, blocking monoclonal antibodies have proven to be effective when combined with chemotherapy, such as rituximab for B-Cell lymphoma, trastuzumab for HER2-positive cancer, cetuximab for colorectal carcinoma, and alemtuzumab for chronic lymphocytic leukemia. CD44, in particular, is believed to prevent homing of leukemia stem cells to the bone marrow, thus representing an attractive target for AML therapy. This apThe acquisition of drug resistance though activation of ERK1/2 is a common feature of oncogenes. ERK1/2 and downstream targets are regulated by ERG overexpression in the normal prostate cell line RWPE-1 through the ERG target PLAU. Our resIn prostate cancer, genomic mutations of DNA repair-associated genes, such as polymerase iota, were positively correlated with TMPRSS2-ERG positive tumors, which aIn conclusion, our K562 ERG-induced cell model system unraveled an ERG mesenchymal-like gene signature that overrides disease entities and reflects a general pattern of ERG-driven malignancies. We highlight, a number of changes in gene transcription in key pathways responsible for drug resistance, and provide novel targets for further validation and development in order to achieve custom tailored treatment strategies for the high ERG subgroup of leukemia.Cultures of K562 pTRE-ERG doxycycline (DOX) inducible clones were grown in 10% FCS RPMI and seeded at 1×105 cells/ml, as previously described. K562 pTFor co-culture experiments, the HS-5 stroma original cell line was purchased from ATCC and grown in 20% FCS RPMI to 80% confluence. Stromal layers were seeded one day prior to co-culture. ERG-inducible cells were stimulated for an additional 24 hours with DOX and combined with HS-5 stroma in 10% FCS RPMI for an additional 24 hour period before the addition of drugs. ERG-inducible cells and stroma were then co-cultured for an additional 4 days with and without DOX to allow cell morphology changes to take place.For the counting of spindle shaped cells, K562 pTRE-ERG CL9 and CL46 cells were seeded onto culture dishes with 2 mm grid plates and stimulated with DOX for six days. Two independent experiments with blind labels in duplicate were performed for each experimental group. Rabbit monoclonal anti-CD44  and anti-mouse IgG  were used in cell adhesion experiments at 1:500 dilution.http://david.abcc.ncifcrf.gov), which was employed to group and define enriched gene ontology terms[Three independent stably transfected clones  of K562 pTRE-ERG cells were grown in the presence and absence of DOX (ERG-induced and non-induced) for long-term culture (8 days). Total RNA was extracted from ERG-induced and non-induced cells and hybridized using Affymetrix U133-plus chip. Data analysis was performed with Gene Spring 4.2 (Agilent Technologies). Differential expression of genes from biological triplicates  that displayed an over- and under-expression of 2-fold or greater between ERG-induced and non-induced cells were used for further analysis. Microarray data have been deposited in the Gene Expression Omnibus Database. To determine enriched biological terms, over- and under-expressed candidate gene lists were uploaded to the DAVID Bioinformatics server (ogy terms.www.oncomine.org). Co-expression of ERG over-expressed and under-expressed genes were compared with leukemia, breast and prostate dataseis which were obtained directly through the Oncomine 3.0 software.Differential microarray dataset obtained from clones CL4, CL46, and CL9 (+/− DOX) were uploaded to the Oncomine Cancer Profiling Database (Quantitative RT PCR was conducted with Sybr green master mix (Fermentas) and analyses were conducted as previously described. RelativTo harvest mesenchymal stroma cells, the mononuclear fraction of bone marrow was cultured as previously described. InformeLive ERG-inducible adherent cells were stained with primary rabbit polyclonal anti-WNT11  and secondary anti-rabbit IgG (Alexa Fluor 488 conjugated) antibodies. Annexin V-FITC  staining was carried out according to manufacturer's instructions. Carboxyfluorescein diacetate succinimidyl ester, CFSE  was used at 1:400 dilution and staining was conducted according to manufacturer's instructions.To capture endogenous levels of pan ERK and ERK (pT202/pY204), ERG-induced cells were DOX stimulated for three days followed by 24-hour drug treatment (Ara-C or PKC412). Briefly, for intracellular staining, K562 pTRE-ERG cells (5x105 cells/ml) were fixed with 150 μl Phosflow fix buffer I (BD biosciences) at 37°C, washed with PBS, resuspended in 200 ul cold methanol for 10 min, blocked by PBS with 1% BSA, and labeled with the primary antibody (1:100 dilution) for 15min at 4°C. To detect endogenous ERK levels the a-mouse pan ERK and phospho Alexa a-mouse 647 (pT202/pY204) antibodies were used. When required, secondary antibody, anti-mouse IgG (Alexa Fluor 488 conjugated), was used for labeling. The three antibodies were purchased from Cell Signaling . The labeled cells were measured by flow cytometry and analysed using Flowjo software. All flow cytometry techniques were conducted on a Becton Dickinson FACSCalibur instrument.rsbweb.nih.gov/IJ).Double strand DNA breakage was measured through the neutral comet assay. ERG-ind


Aqueous Viscum album L. extracts (VAE) are widely used in complementary cancer therapies. Due to their low solubility, triterpene acids, which are known to possess anti-cancer properties, do not occur in aqueous extracts in significant amounts. Using cyclodextrins it was possible to solubilize mistletoe triterpene acids and to determine the anti-cancer properties in different acute lymphoblastic (ALL) and myeloid leukemia cell lines (AML).in vitro. Apoptosis was determined using mitochondrial membrane potential measurement, Annexin/PI, Western blot analysis and caspase assays. A C.B-17/SCID model of pre-B ALL/NALM-6 was used to test efficacy and mechanisms of treatment with lectin- and triterpene-containing preparations in vivo.The experimental extracts contain either mistletoe lectin-I and viscotoxins (viscum) or solubilized oleanolic- and betulinic acids (TT) and more interestingly, a combination thereof (viscumTT). The cytotoxicity of increasing concentrations of VAE preparations was tested in NALM-6, U937 and HL-60 cells in vivo experiment showed that treatment of mice with the viscumTT combination prolonged the mean survival significantly compared control group.All three cell lines have shown distinct apoptosis induction for viscum, TT and viscumTT. However, differences between ALL and AML cell lines toward the lectin and triterpene acids sensitivity were observed. Annexin/PI and mitochondrial membrane potential assays indicated that dose-dependent induction of apoptosis was the main mechanism. The combination (viscumTT) of lectin- (viscum) and triterpene acids-containing (TT) extracts showed the strongest apoptosis induction. Furthermore, caspase activity demonstrated that these extracts are able to induce apoptosis via caspase-8 and -9 dependent pathways. The Taken together, we were able to show that this new formulation “viscumTT” of aqueous mistletoe extracts and triterpene acids can induce apoptosis in leukemia cells via the intrinsic and extrinsic signaling pathways. Based on these data we believe that Viscum album L. extracts containing triterpene acids may possess impressive therapeutic potential.


Psoriasis is a common inflammatory skin disease with a multifactorial genetic basis. A dysregulated interplay between keratinocytes and infiltrating immune cells underlies the cutaneous inflammation in psoriasis. Keratinocytes are important producers of antimicrobial peptides such as hBD-2 and LL37 and cytokines such as TNF-alpha, which are essential elements in this process of cell-cell communication . RecentlAn optimized cytokine mix was used to induce a psoriatic phenotype starting from normal human keratinocytes. Complexes of siRNA or anti-miRNA and SECosomes were made and validated prior to transfection. 24h post-tranfection, qPCR analysis was performed to evaluate mRNA expression levels.Transfection experiments with the complexes showed a stable knockdown efficiency of more than 80% of hBD-2, LL37, TNF-alpha and miR-203 mRNA.in vitro work we prepared and characterized siRNA and anti-miRNA complexes with SECosomes against hBD-2, LL37, TNF-alpha and miR-203 respectively. These complexes efficiently knock-down the targeted genes with concomitant downregulation of the associated proteins. Hereafter we will test the therapeutic applicability of our complexes in xenografted psoriatic skin by topical application.In this


Bactrocera oleae, is the major arthropod pest of commercial olive production, causing extensive damage to olive crops worldwide. Current control techniques rely on spraying of chemical insecticides. The sterile insect technique (SIT) presents an alternative, environmentally friendly and species-specific method of population control. Although SIT has been very successful against other tephritid pests, previous SIT trials on olive fly have produced disappointing results. Key problems included altered diurnal mating rhythms of the laboratory-reared insects, resulting in asynchronous mating activity between the wild and released sterile populations, and low competitiveness of the radiation-sterilised mass-reared flies. Consequently, the production of competitive, male-only release cohorts is considered an essential prerequisite for successful olive fly SIT.The olive fruit fly, ®), providing highly penetrant female-specific lethality, dominant fluorescent marking, and genetic sterility. We found that males of the lead strain, OX3097D-Bol, 1) are strongly sexually competitive with wild olive flies, 2) display synchronous mating activity with wild females, and 3) induce appropriate refractoriness to wild female re-mating. Furthermore, we showed, through a large proof-of-principle experiment, that weekly releases of OX3097D-Bol males into stable populations of caged wild-type olive fly could cause rapid population collapse and eventual eradication.We developed a set of conditional female-lethal strains of olive fly (named Release of Insects carrying a Dominant Lethal; RIDLThe observed mating characteristics strongly suggest that an approach based on the release of OX3097D-Bol males will overcome the key difficulties encountered in previous olive fly SIT attempts. Although field confirmation is required, the proof-of-principle suppression and elimination of caged wild-type olive fly populations through OX3097D-Bol male releases provides evidence for the female-specific RIDL approach as a viable method of olive fly control. We conclude that the promising characteristics of OX3097D-Bol may finally enable effective SIT-based control of the olive fly. Bactrocera oleae (Rossi) (Diptera: Tephritidae), is the major insect pest of olives. Females typically lay a single egg per olive [The olive fly, er olive , injecteer olive . Controler olive , there iCeratitis capitata (Medfly) males used for SIT [The sterile insect technique (SIT) is an environmentally friendly and species-specific method of pest control based on the release of large numbers of sterilised insects . Competi for SIT .®  system is a transgene-based derivative of SIT [The RIDLe of SIT -14, one e of SIT -17. Encoe of SIT .We report the development of the first transgenic control strains for the olive fly. The lead strain, OX3097D-Bol, provides highly penetrant, dominant, female-specific lethality when reared in the absence of a transgene repressor. Using a series of behavioral studies, we found that OX3097D-Bol males show fully synchronous and strongly competitive mating behavior with wild olive flies, and display no reduction in their ability to induce refractoriness to wild female re-mating. This is a more stringent test than using laboratory wild-type flies, which may have co-adapted to laboratory rearing conditions. Furthermore, we describe results from experiments showing the ability of periodic releases of OX3097D-Bol males to suppress large caged wild-type olive fly populations.et al., unpublished data), female lethality occurred at the early pupal stage. Analysis of transgene-derived transcripts indicated that the alternative splicing pattern of the Cctra intron in OX3097D-Bol olive fly is equivalent to that in its native context [Six transgenic lines were generated carrying the OX3097 transgene Figure . Of the  context ,18,19  = 1, likelihood ratio test for goodness of fit, n = 406). Nonetheless, this near-equal outcome far exceeds established thresholds for SIT [P = 0.44, d.f. = 1, circular statistics F-test), suggesting synchronicity in mating activity between OX3097D-Bol males and wild flies.Caged mating competitiveness tests challenging homozygous OX3097D-Bol males to compete with wild males for copulations with wild females were performed based on established guidelines , using w for SIT . CopulatP = 0.7, d.f. = 1, χ2 test), or second-mate genotype were seen   Figure .3) cages in a greenhouse at the University of Crete. At week 13 from first establishment, approximately 1,600 OX3097D-Bol male pupae were introduced per week. Population density was monitored by measuring egg production . It is possible that the use of genetically modified arthropods to protect the crop in a sustainable way, which leaves little or no residue in the food product (here olives or olive oil) might be more acceptable in the EU than food products that are produced directly from genetically modified organisms. For comparison, the Standing Committee on the Food Chain and Animal Health stated that foods and feeds produced by fermentation with genetically modified microorganisms that are not present in the final product are "excluded from the scope of regulations" . AdditioUsing modern genetic engineering techniques, we have developed OX3097D-Bol, an fsRIDL olive fly strain that incorporates engineered phenotypes, directly addressing problems encountered in previous unsuccessful olive fly SIT trials . OX3097DThe good mating characteristics of OX3097D-Bol males seen in this study, including high sexual competitiveness, photo-period compatibility, and efficient induction of re-mating refractoriness in wild females, suggest a strong potential for application in a SIT control programme. Although the results presented here need field confirmation, the proof-of-principle suppression and elimination of caged wild-type olive fly populations through OX3097D-Bol male releases provides evidence for the fsRIDL approach as a viable method for olive fly control. Independently, significant advances have been achieved in olive fly mass-rearing ,29 and rB. oleae strain were micro-injected with the OX3097 plasmid and piggyBac mRNA. as described by Koukidou et al. [0 adults. After back-crossing pools of five male survivors with ten wild-type females, and five female survivors with five wild-type males, six OX3097 lines were isolated (transformation efficiency ~4%). Transgenics were outcrossed for five generations to the Argov wild-type strain (Israel), and these outcrossed Argov derivatives were used for all experiments. The Argov strain was derived from field collections of wild male olive fly in Israel, outcrossed to Democritus females. To develop a homozygous strain, a pool of homozygous and heterozygous OX3097D-Bol adults was generated by crossing OX3097D-Bol heterozygotes. DNA from these parents was analysed by PCR using primers (5'-CCTGCGTTTGGAGATGACGAAATC-3' and 5'-CTTACATATAGAGCAGTGCGCTCACATG-3') that anneal to genomic sites flanking the insertion site producing a WT (no insertion) amplicon were discarded. Homozygotes were thereby identified, and a homozygous line was developed from 15 female and 13 male founder flies. The key phenotypic properties identified in OX3097D-Bol heterozygotes  u et al. . This res Figure  were alsMale and female tTAV ,34 trans2 and contained a large olive branch) in greenhouse facilities at the University of Crete under natural light. Adult male OX3097D-Bol flies were obtained from larvae reared in the absence of tetracycline ("off-tet") at low density . Wild pupae were recovered from infested olives gathered from olive orchards near to the University of Crete. Each mating test used 50 OX3097D-Bol males, 50 wild males, and 50 wild females. Mated males were scored for the presence of the DsRed2 fluorescent marker by epifluorescence microscopy [Mating competitiveness tests were carried out in accordance with the United Nations Food and Agriculture Organization (FAO)/IAEA/United States Department of Agriculture (USDA) guidelines  in semi-croscopy ,33  over a 12-week period by introducing a fixed number of pupae to each cage weekly . To assess egg production rates and to sustain caged olive fly populations, four ceresin-wax cones (combined surface area approximately 550 cm2) were added to each cage daily for female oviposition. Pupal additions for the first 4 weeks during population establishment originated from a wild-type stock colony; thereafter, experimental cages were self-sufficient. Eggs were collected from the cages and counted daily. On week 12, the experimental cages were randomly divided into two RIDL-treatment and two no-treatment (control) cages. From week 13 onwards, RIDL-treatment cages received weekly additions of 1,600 OX3097D-Bol male pupae reared off-tet . Once the OX3097D-Bol introductions began, pupal introductions to the RIDL-treatment cages were proportional to the cage's respective rate of egg production, with the control cages providing a coefficient of weekly egg production to pupal return. Numbers of females in a cage were monitored by collecting and counting dead females. The ratio of RIDL heterozygous to wild-type pupae returning to RIDL-treatment cages was monitored by fluorescence microscopy . Larvae were screened only after the returning population was separated, to remove the possibility of bias in selecting individual flies for reintroduction to the cage populations.The protocol for the caged suppression experiment was based on that of Wise de Valdez et al. . Stable F-test with the statistical software Oriana for Windows . The Pearson's χ2 test was used to compare numbers of re-mated to non-re-mated females for both initial mating combinations. The same test was used to compare initial and second-mate choice. Unless otherwise stated, all statistical tests were performed using SPSS for Windows , with significance set at P < 0.05.Comparison of mating competitiveness between OX3097D-Bol and wild males was performed using a likelihood ratio test for goodness of fit with numbers of successfully mated males from both genotypes pooled across experiments, and compared against a theoretical 1:1 genotype ratio expected in random mating. Differences in copulation initiation time between the two possible mating combinations  were analysed using a circular statistics Ceratitis capitata transformer intron; fsRIDL: female-specific Release of Insects carrying a Dominant Lethal; PR: post-first-release; RIDL: Release of Insects carrying a Dominant Lethal; SIT: Sterile Insect Technique.CcTra intron: TA, MK, PK, HFG and LA are employees or students of Oxitec Ltd and have employment, stipend and/or equity interest in Oxitec. Oxitec and the University of Oxford have intellectual property related to the subject matter of this paper. All other authors declare no interest.TA and MK created the transgenic olive fly lines. TA conducted the molecular analysis TA, MK, PR, carried out mating tests and suppression trial. HG, JV and AE provided advice, LA conceived the project, and MK supervised the project. TA and LA wrote the paper. All authors discussed results and commented on the manuscript


Cerebral malaria (CM) is a lethal complication of Plasmodium falciparum infections. In the Plasmodium berghei ANKA (PbA) murine model, CM is associated with marked brain inflammation, increased expression of endothelial cell adhesion molecules and leukocyte and platelet accumulation in brain vessels, causing vascular occlusion and decreased blood flow, damaging the endothelium and leading to blood-brain barrier breakdown, leakage and hemorrhages. Exogenous nitric oxide (NO) administration largely prevents the syndrome. Here we evaluated whether the mechanism of action of NO in preventing murine CM is related to its anti-inflammatory properties and to protection of the endothelium.C57Bl/6 mice infected with PbA were treated twice a day with saline or dipropylenetriamineNONOate (DPTA-NO). Endothelial cell adhesion molecule  expression in brain tissue on day 6 of infection was assessed in both groups by western blot. For intravital microscopy studies, DPTA-NO-treated and saline-treated mice with a previously implanted closed cranial window were injected with albumin-FITC, anti-CD45-TxR and anti-CD41-FITC antibodies on day 6 of infection for quantification of albumin leakage, leukocyte and platelet adherence in pial vessels.PbA-infected mice treated with the NO-donor DPTA-NO showed decreased expression of ICAM-1 and P-selectin, but not VCAM-1, in the brain, compared to saline-treated mice. DPTA-NO treatment also decreased the number of adherent leukocytes and platelets in pial vessels, particularly in venules 30-50 μm in diameter, decreased inflammatory vascular resistance and prevented the occurrence of arteriolar and venular albumin leakage observed in saline-treated PbA-infected mice, as assessed by intravital microscopy.These results indicate that the protective effect of exogenous NO on murine CM is associated with decreased brain vascular expression of inflammatory markers resulting in attenuated endothelial junction damage and facilitating blood flow. Cerebral malaria (CM), a complication of malaria infection by Plasmodium falciparum, is a leading cause of mortality and neurological impairment in endemic areas, with an estimated 1 million deaths every year -8 as welEndothelial dysfunction by different mechanisms such as NO depletion can contribute to vascular inflammation. It has been shown that low NO bioavailability or NO inhibition can induce the expression of eCAMs -21, platIn the present study, we show that NO supplementation acts by decreasing eCAM expression in the brain leading to decreased adherence of leukocytes and platelets in pial vessels and preventing vascular leakage in arterioles and venules.6 Plasmodium berghei ANKA parasites expressing the green fluorescent protein . Parasitemia, body weight, rectal temperature and clinical status  were inoculated intraperitoneally (IP) with 1 × 10escribed , were moOn day 6 of infection, saline-treated and DPTA-NO-treated PbA-infected mice, as well as uninfected control mice, were euthanized with 100 mg/kg of euthasol IP, perfused with saline and the brains collected and frozen at -80°C until processing. Brains were defrosted and homogenized in lysis buffer , on ice for 30 min than centrifuged at 14.000 g for 20 min, the supernatant was diluted and proteins were resolved by sodium dodecyl-sulphate polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes (Milipore). Membranes were blocked and probed with antibodies to intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-selectin or E-selectin . Detection was performed with anti-IgG secondary antibodies conjugated to peroxidase and reaction was developed with Super Signal West Pico Chemiluminescence Substrate (Thermo Scientific). After development, antibodies were unbound using Restore Western Blot Stripping buffer (Thermo Scientific), re-incubated with antibodies to GAPDH (V-18) (Santa Cruz) and reaction was developed by chemiluminescence. The band intensities were determined after scanning the radiographic films and image analysis using ImageJ (NIH) software, and eCAM expression was normalized in relation to GAPDH expression.The closed cranial window model was used as previously described ,28. AfteStatistical analyses were performed using analysis of variance (ANOVA) with Dunnett's post-hoc analysis to compare eCAM expression, leukocyte and platelet adherence and vascular leakage in uninfected, saline-treated and DPTA-NO-treated PbA-infected mice. Correlations were established using the Spearmann rank test. All statistics were performed using the Graphpad Prism software . A p value < 0.05 was considered significant.In the eCAM expression studies, 79% of the saline-treated mice n = 14) showed clinical signs of CM on day 6 of infection, against 29% of the DPTA-NO-treated mice (n = 14). All mice were euthanized regardless of CM expression and brains were collected. The course of parasitemia was similar in both groups . Saline-treated PbA-infected mice showed increased expression of ICAM-1, VCAM-1 and P-selectin, but not E-selectin  and platelet (r2 = 0.15) adherence, but not albumin leakage (r2 = 0.03), were significantly correlated with increasing venular diameters. Vessels with more leukocytes also presented more platelets, this correlation being also present in DPTA-NO-treated mice despite the lower numbers of adherent cells. Platelet adherence (r2 = 0.49), but not leakage (r2 = 0.01), was significantly correlated with leukocyte adherence in venules. No correlation was found between leukocyte or platelet adherence and leakage (r2 = 0.06).We asked whether vessel size would affect leukocyte/platelet adherence and intensity of leakage in saline-treated PbA-infected mice, and whether venules presenting higher numbers of adherent leukocytes would also present higher platelet adherence and more intense leakage. Leukocyte (rDPTA-NO treatment affected the distribution of adherent leukocytes at different microvessel diameters. While in saline-treated animals the number of adherent leukocytes increased with increasing venular diameter, in DPTA-NO-treated mice the distribution of adherent leukocytes was relatively homogeneous in microvessels of different diameters Figure . These rTo analyze the effect of leukocyte accumulation on venular resistance to blood flow, venules were categorized in three groups based on diameter: 20-30 μm, 30-40 μm and 40-50 μm. Figure Murine cerebral malaria is associated with increased expression of eCAMs as well as leukocyte and platelet accumulation in brain vessels leading to microvascular damage and BBB breakdown ,30. HereStudies of eCAM expression in human and murine CM consistently show an increased expression of ICAM-1, whereas the results for other eCAMs are variable. In cases of fatal human CM, the picture that emerges is of marked increases in ICAM-1, significant but less pronounced increases in VCAM-1 and E-selectin and no changes in P-selectin expression in the brain -33. In mLow NO bioavailability plays a role in murine CM pathogenesis  and it cIn saline-treated PbA-infected mice, the venular sites with higher accumulation of leukocytes presented higher platelet counts. Because there were also direct correlations between the number of adherent leukocytes/platelets and venular diameters, this finding indicates that the differential quantitative accumulation was due basically to the larger available area for binding in larger vessels rather than to eventual differential expression of eCAMs in different vascular beds, although the latter was not directly assessed and cannot be ruled out. This is in accordance with previous findings by Sun and colleagues regarding platelet adherence during murine CM . These rSeveral studies have shown that murine CM is associated with BBB breakdown with consequent vascular leakage ,35. HereIn summary, exogenous NO supplementation significantly decreases brain vascular inflammation during PbA infection by decreasing the expression of eCAMs and consequently decreasing recruitment of leukocytes and platelets, which eventually results in decreased vascular dysfunction and damage.The authors declare that they have no competing interests.GMZ infected and treated the mice and performed the eCAM expression studies, analyzed and interpreted data and wrote the manuscript; PC performed the intravital microscopy studies, analyzed and interpreted data and performed statistical analysis; WB participated in the intravital microscopy experiments; JAF analyzed and interpreted data; LJMC designed research, analyzed and interpreted data and wrote the manuscript. All authors have read and approved the final version of this manuscript.


Tetranychusurticae), on tomato plants, Solanumlycopersicum. Twospotted spider mites showed increased preference for and fecundity on TSWV-infected plants compared to mock-inoculated plants. Transcriptome profiles of TSWV-infected plants indicated significant up-regulation of salicylic acid (SA)-related genes, but no apparent down-regulation of jasmonic acid (JA)-related genes which could potentially confer induced resistance against TSM. This suggests that there was no antagonistic crosstalk between the signaling pathways to influence the interaction between TSWV and spider mites. In fact, SA- and JA-related genes were up-regulated when plants were challenged with both TSWV and the herbivore. TSWV infection resulted in down-regulation of cell wall-related genes and photosynthesis-associated genes, which may contribute to host plant susceptibility. There was a three-fold increase in total free amino acid content in virus-infected plants compared to mock-inoculated plants. Total free amino acid content is critical for arthropod nutrition and may, in part, explain the apparent positive indirect effect of TSWV on spider mites. Taken together, these data suggest that the mechanism(s) of increased host suitability of TSWV-infected plants to non-vector herbivores is complex and likely involves several plant biochemical processes. The interaction between plant viruses and non-vector arthropod herbivores is poorly understood. However, there is accumulating evidence that plant viruses can impact fitness of non-vector herbivores. In this study, we used oligonucleotide microarrays, phytohormone, and total free amino acid analyses to characterize the molecular mechanisms underlying the interaction between Tomato spotted wilt virus (TSWV) and a non-vector arthropod, twospotted spider mite ( Spodopteraexigua caterpillars was greater on TMV-infected tomato plants than on control plants /ErefΔCtref[control-treatment] where E refers to the PCR primer efficiency for target or internal reference (EF1) genes and ΔCt is the difference in Ct-values  between a treatment and mock control. The average Ct value (n = 3) obtained for the mock control for each target and reference gene was determined and used in the RER calculations. To estimate virus titer, the normalized abundance of TSWV N RNA (genomic and transcript RNA) was calculated using the Pfaffl inverse equation [refCtref / ENCtN as described previously [The relative abundance of target RNA was determined for each treatment compared to the mock control. The relative expression ratio (RER) equation  was calcequation : ErefCteviously .10-transformed phytohormone contents  in Minitab using a GLM that included Treatment and Biological Replicate as two fixed factors and their interaction term and greenhouse Block as the random factor. The analyses revealed no apparent main effect or treatment interaction for any of the phytohormones measured due to biological replicate; therefore, data obtained for the three biological replicates were pooled and one-way ANOVA was performed to determine the main effect of treatment on phytohormone contents. Pairwise treatment comparisons were performed using Tukey’s family error rate (P< 0.05).Plant hormones evaluated included salicylic acid (SA), jasmonic acid (JA), jasmonyl-isoleucine (JA-IL), and 12-oxo-phytodienoic acid (OPDA). Frozen tomato leaflet samples were sent to The Donald Danforth Proteomics & Mass Spectrometry Facility, St. Louis, MO for chemical extraction and liquid chromatography–electrospray tandem mass spectrometry using methods described by Pan and co-authors . Analysig for 5 min. The dry residue was dissolved in 2.5 ml of 0.1 N HCl and kept at -20°C until assayed. Colorimetric procedures were adapted to Technicon Autoanalyzer II for simultaneous determination of total free amino acid content based on an internal standard (leucine) in plant tissue samples modified from [Total free amino acid content in tomato leaf tissue samples was analyzed using a protocol described by Fahaam and co-authors  with a fied from . Total fTable S1Differentially-expressed genes in tomato plants systemically-infected with TSWV and/or infested with TSM.Statistically significant .(XLSX)Click here for additional data file.Table S2Differentially-expressed genes in tomato plants systemically-infected with TSWV.Statistically significant .(XLSX)Click here for additional data file.Table S3Differentially-expressed genes in tomato plants infested with TSM.Statistically significant .(XLSX)Click here for additional data file.Table S4Differentially-expressed genes in tomato plants systemically-infected with TSWV and infested with TSM.Statistically significant .(XLSX)Click here for additional data file.


EBNA1 mRNA, thereby suppressing the production of antigenic peptides that the human immune system recognises as foreign. Here, Blondel et al. establish a yeast-based assay that recapitulates GAr-mediated suppression of EBNA1 mRNA translation and use it to identify doxorubicin and its active analogues as compounds that specifically interfere with GAr-mediated suppression of translation. Importantly, in mammalian cells, doxorubicin and its analogues stimulate EBNA1 expression in a GAr-dependent manner and overcome the GAr-dependent restriction of antigen presentation. Together, these results validate the yeast-based assay as an effective cell-based screening approach for compounds that interfere with EBV immune evasion and that might, therefore, provide treatments for EBV-related diseases, including cancer. Page 435Latent infection with Epstein-Barr virus (EBV) is associated with several human cancers, including nasopharyngeal cancer. During latent EBV infections, the glycine-alanine repeat (GAr) domain of the EBV nuclear antigen-1 (EBNA1) inhibits translation of


To analyze the effectiveness of pre-study questionnaires in identifying at-risk patients as well as estimate the prevalence of chronic kidney disease (CKD), nephrogenic systemic fibrosis (NSF) risk factors, and other co-morbidities in patients scheduled to undergo magnetic resonance angiography (MRA) gadonlinium-based contrast agents (GBCA) studies.Nephrogenic systemic fibrosis (NSF) is a rare, but serious, disease that is highly linked to the use of GBCAs in patients with renal insufficiency. Identification of these patients is considered vital prior to performing any such study. Thus, many institutions have administered pre-study questionnaires to patients in efforts to determine the degree of any pre-existing renal disease. However, identification of patients with renal dysfunction using this method has proven challenging due to many patients denying any history of renal disease on patient questionnaires, despite having low glomerular filtration rate (GFR) values. As a result, some institutions have instituted same-day point of care (POC) serum creatinine testing to estimate GFR values for all patients scheduled to undergo GBCA studies. In this study, we sought to quantify the effectiveness of these questionnaires relative to same-day POC data in identifying patients with renal insufficiency.This study was based upon a HIPAA-compliant and IRB-approved retrospective review of patients’ records, and informed consent of patients was not required. Patent demographics, co-morbidities, contrast type, POC serum creatinine values, and responses to pre-study questionnaires regarding knowledge of patients’ history of renal dysfunction were recorded for all patients undergoing a lower extremity magnetic resonance angiography exam in a single year. Patients were compared with respect to demographic, co-morbidity, and contrast data according to their CKD status. Patient questionnaire results were also compared to their POC-based CKD classification.Of 199 total patients, 72 patients had stage 3 CKD, 6 patients had stage 4 CKD, and 7 patients had stage 5 CKD. Co-morbidities including transplant status, presence of diabetes, hypertension, coronary artery disease, and smoking status showed significant differences among the CKD groups. Only 6.9% of stage 3 patients were aware of any history of renal dysfunction, whereas 50% of stage 4 and 100% of stage 5 patients admitted any history of renal dysfunction via questinonnaires (Figure A pre-study questionnaire alone cannot be relied upon in screening at-risk patients, and POC creatinine testing can more reliably detect renal dysfunction in patients scheduled to undergo MRA GBCA studies.