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Purpose
The purpose of this work is to develop fast deliverable step and shoot IMRT technique. A reduction in the number of segments should theoretically be possible, whilst simultaneously maintaining plan quality, provided that the reduction is accompanied by an increased number of gantry angles. A benefit of this method is that the segment shaping could be performed during gantry motion, thereby reducing the delivery time. The aim was to find classes of such solutions whose plan quality can compete with conventional IMRT.
Materials/Methods
A planning study was performed. Step and shoot IMRT plans were created using direct machine parameter optimization (DMPO) as a reference. DMPO plans were compared to an IMRT variant having only one segment per angle ("2-Step Fast"). 2-Step Fast is based on a geometrical analysis of the topology of the planning target volume (PTV) and the organs at risk (OAR). A prostate/rectum case, spine metastasis/spinal cord, breast/lung and an artificial PTV/OAR combination of the ESTRO-Quasimodo phantom were used for the study. The composite objective value (COV), a quality score, and plan delivery time were compared. The delivery time for the DMPO reference plan and the 2-Step Fast IMRT technique was measured and calculated for two different linacs, a twelve year old Siemens Primus™ ("old" linac) and two Elekta Synergy™ "S" linacs ("new" linacs).
Results
2-Step Fast had comparable or better quality than the reference DMPO plan. The number of segments was smaller than for the reference plan, the number of gantry angles was between 23 and 34. For the modern linac the delivery time was always smaller than that for the reference plan. The calculated (measured) values showed a mean delivery time reduction of 21% (21%) for the new linac, and of 7% (3%) for the old linac compared to the respective DMPO reference plans. For the old linac, the data handling time per beam was the limiting factor for the treatment time reduction.
Conclusions
2-Step Fast plans are suited to reduce the delivery time, especially if the data handling time per beam is short. The plan quality can be retained or even increased for fewer segments provided more gantry angles are used.
Background
The World Health Organization (WHO) has recommended guidelines for a HIV drug resistance (HIVDR) survey for resource-limited countries. Eligibility criteria for patients include age below 25 years in order to focus on the prevalence of transmitted HIVDR (tHIVDR) in newly-infected individuals. Most of the participating sites across Africa have so far reported tHIVDR prevalences of below 5%. In this study we investigated whether the rate of HIVDR in patients <25 years is representative for HIVDR in the rest of the therapy-naïve population.
Methods and Findings
HIVDR was determined in 88 sequentially enrolled ART-naïve patients from Mwanza, Tanzania (mean age 35.4 years). Twenty patients were aged <25 years and 68 patients were aged 25–63 years. The frequency of HIVDR in the study population was 14.8% (95%; CI 0.072–0.223) and independent of NVP-resistance induced by prevention of mother-to-child transmission programs. Patients >25 years had a significantly higher HIVDR frequency than younger patients (19.1%; 95% CI 0.095–0.28) versus 0%, P = 0.0344). In 2 out of the 16 patients with HIVDR we found traces of antiretrovirals (ARVs) in plasma.
Conclusions
ART-naïve patients aged over 25 years exhibited significantly higher HIVDR than younger patients. Detection of traces of ARVs in individuals with HIVDR suggests that besides transmission, undisclosed misuse of ARVs may constitute a significant factor in the generation of the observed high HIVDR rate. The current WHO tHIVDR survey that is solely focused on the transmission of HIVDR and that excludes patients over 25 years of age may therefore result in substantial underestimation of the prevalence of HIVDR in the therapy-naïve population. Similar studies should be performed also in other areas to test whether the so far reported optimistic picture of low HIVDR prevalence in young individuals is really representative for the rest of the ART-naïve HIV-infected population.
Background
Epithelial surfaces such as the gastrointestinal mucosa depend on expression of antimicrobial peptides like cathelicidin for immune defence against pathogens. The mechanisms behind mucosal cathelicidin regulation are incompletely understood.
Methods
Cathelicidin expression was analysed in duodenal, antral and corpus/fundic mucosal biopsies from African and German patients. Additionally, cathelicidin expression was correlated with Helicobacter pylori (HP) infection and the inflammatory status of the mucosa.
Results
High cathelicidin transcript abundance was detected in duodenal biopsies from African subjects. On the contrary, cathelicidin mRNA expression was either undetectable or very low in tissue specimens from German patients. Also, in the antrum and corpus/fundus regions of the stomach significantly higher cathelicidin transcript levels were measured in Tanzanian compared to German patients. In gastric biopsies from African patients cathelicidin expression was increased in HP positive compared to HP negative subjects. Additionally, the inflammatory status measured by IL-8 expression correlated well with the HP infection status.
Conclusions
A higher duodenal and gastric cathelicidin expression in African (compared with European) individuals may be due to upregulation by antigenic stimulation and may confer a higher resistance against enteric infections.
Background
Neisseria meningitidis is a naturally transformable, facultative pathogen colonizing the human nasopharynx. Here, we analyze on a genome-wide level the impact of recombination on gene-complement diversity and virulence evolution in N. meningitidis. We combined comparative genome hybridization using microarrays (mCGH) and multilocus sequence typing (MLST) of 29 meningococcal isolates with computational comparison of a subset of seven meningococcal genome sequences.
Principal Findings
We found that lateral gene transfer of minimal mobile elements as well as prophages are major forces shaping meningococcal population structure. Extensive gene content comparison revealed novel associations of virulence with genetic elements besides the recently discovered meningococcal disease associated (MDA) island. In particular, we identified an association of virulence with a recently described canonical genomic island termed IHT-E and a differential distribution of genes encoding RTX toxin- and two-partner secretion systems among hyperinvasive and non-hyperinvasive lineages. By computationally screening also the core genome for signs of recombination, we provided evidence that about 40% of the meningococcal core genes are affected by recombination primarily within metabolic genes as well as genes involved in DNA replication and repair. By comparison with the results of previous mCGH studies, our data indicated that genetic structuring as revealed by mCGH is stable over time and highly similar for isolates from different geographic origins.
Conclusions
Recombination comprising lateral transfer of entire genes as well as homologous intragenic recombination has a profound impact on meningococcal population structure and genome composition. Our data support the hypothesis that meningococcal virulence is polygenic in nature and that differences in metabolism might contribute to virulence.
The Wuerzburg Hybridoma Library against the Drosophila brain represents a collection of around 200 monoclonal antibodies that bind to specific structures in the Drosophila brain. Here we describe the immunohistochemical staining patterns, the Western blot signals of one- and two-dimensional electrophoretic separation, and the mass spectrometric characterization of the target protein candidates recognized by the monoclonal antibodies aa2 and ab52 from the library. Analysis of a mutant of a candidate gene identified the Drosophila homolog of the Epidermal growth factor receptor Pathway Substrate clone 15 (Eps15) as the antigen for these two antibodies.
Peripheral T cell lymphomas (PTCLs) are associated with a poor prognosis due to often advanced disease at the time of diagnosis and due to a lack of efficient therapeutic options. Therefore, appropriate animal models of PTCL are vital to improve clinical management of this disease. Here, we describe a monoclonal CD8\(^+\) CD4\(^−\) αβ T cell receptor Vβ2\(^+\) CD28\(^+\) T cell lymphoma line, termed T8-28. T8-28 cells were isolated from an un-manipulated adult BALB/c mouse housed under standard pathogen-free conditions. T8-28 cells induced terminal malignancy upon adoptive transfer into syngeneic BALB/c mice. Despite intracellular expression of the cytotoxic T cell differentiation marker granzyme B, T8-28 cells appeared to be defective with respect to cytotoxic activity as read-out in vitro. Among the protocols tested, only addition of interleukin 2 in vitro could partially compensate for the in vivo micro-milieu in promoting growth of the T8-28 lymphoma cells.
Recent studies as well as theoretical models of error processing assign fundamental importance to the brain's dopaminergic system. Research about how the electrophysiological correlates of error processing—the error-related negativity (ERN) and the error positivity (Pe)—are influenced by variations of common dopaminergic genes, however, is still relatively scarce. In the present study, we therefore investigated whether polymorphisms in the DAT1 gene and in the DRD4 gene, respectively, lead to interindividual differences in these error processing correlates. One hundred sixty participants completed a version of the Eriksen Flanker Task while a 26-channel EEG was recorded. The task was slightly modified in order to increase error rates. During data analysis, participants were split into two groups depending on their DAT1 and their DRD4 genotypes, respectively. ERN and Pe amplitudes after correct responses and after errors as well as difference amplitudes between errors and correct responses were analyzed. We found a differential effect of DAT1 genotype on the Pe difference amplitude but not on the ERN difference amplitude, while the reverse was true for DRD4 genotype. These findings are in line with predictions from theoretical models of dopaminergic transmission in the brain. They furthermore tie results from clinical investigations of disorders impacting on the dopamine system to genetic variations known to be at-risk genotypes.
Interleukin-4 (IL-4) is an anti-inflammatory and analgesic cytokine that induces opioid receptor transcription. We investigated IL-4 knockout (ko) mice to characterize their pain behavior before and after chronic constriction injury (CCI) of the sciatic nerve as a model for neuropathic pain. We investigated opioid responsivity and measured cytokine and opioid receptor gene expression in the peripheral and central nervous system (PNS, CNS) of IL-4 ko mice in comparison with wildtype (wt) mice. Naïve IL-4 ko mice displayed tactile allodynia (wt: 0.45 g; ko: 0.18 g; p<0.001), while responses to heat and cold stimuli and to muscle pressure were not different. No compensatory changes in the gene expression of tumor necrosis factor-alpha (TNF), IL-1β, IL-10, and IL-13 were found in the PNS and CNS of naïve IL-4 ko mice. However, IL-1β gene expression was stronger in the sciatic nerve of IL-4 ko mice (p<0.001) 28 days after CCI and only IL-4 ko mice had elevated IL-10 gene expression (p = 0.014). Remarkably, CCI induced TNF (p<0.01), IL-1β (p<0.05), IL-10 (p<0.05), and IL-13 (p<0.001) gene expression exclusively in the ipsilateral spinal cord of IL-4 ko mice. The compensatory overexpression of the anti-inflammatory and analgesic cytokines IL-10 and IL-13 in the spinal cord of IL-4 ko mice may explain the lack of genotype differences for pain behavior after CCI. Additionally, CCI induced gene expression of μ, κ, and δ opioid receptors in the contralateral cortex and thalamus of IL-4 ko mice, paralleled by fast onset of morphine analgesia, but not in wt mice. We conclude that a lack of IL-4 leads to mechanical sensitivity; the compensatory hyperexpression of analgesic cytokines and opioid receptors after CCI, in turn, protects IL-4 ko mice from enhanced pain behavior after nerve lesion.
Testing Pollen of Single and Stacked Insect-Resistant Bt-Maize on In vitro Reared Honey Bee Larvae
(2011)
The ecologically and economic important honey bee (Apis mellifera) is a key non-target arthropod species in environmental risk assessment (ERA) of genetically modified (GM) crops. Honey bee larvae are directly exposed to transgenic products by the consumption of GM pollen. But most ERA studies only consider responses of adult bees, although Bt-proteins primarily affect the larval phases of target organisms. We adopted an in vitro larvae rearing system, to assess lethal and sublethal effects of Bt-pollen consumption in a standardized eco-toxicological bioassay. The effects of pollen from two Bt-maize cultivars, one expressing a single and the other a total of three Bt-proteins, on the survival and prepupae weight of honey bee larvae were analyzed. The control treatments included pollen from three non-transgenic maize varieties and of Heliconia rostrata. Three days old larvae were fed the realistic exposure dose of 2 mg pollen within the semi-artificial diet. The larvae were monitored over 120 h, until the prepupal stage, where larvae terminate feeding and growing. Neither single nor stacked Bt-maize pollen showed an adverse effect on larval survival and the prepupal weight. In contrast, feeding of H. rostrata pollen caused significant toxic effects. The results of this study indicate that pollen of the tested Bt-varieties does not harm the development of in vitro reared A. mellifera larvae. To sustain the ecosystem service of pollination, Bt-impact on A. mellifera should always be a crucial part of regulatory biosafety assessments. We suggest that our approach of feeding GM pollen on in vitro reared honey bee larvae is well suited of becoming a standard bioassay in regulatory risk assessments schemes of GM crops.
In Vivo Imaging of Stepwise Vessel Occlusion in Cerebral Photothrombosis of Mice by \(^{19}\)F MRI
(2011)
Background
\(^{19}\)F magnetic resonance imaging (MRI) was recently introduced as a promising technique for in vivo cell tracking. In the present study we compared \(^{19}\)F MRI with iron-enhanced MRI in mice with photothrombosis (PT) at 7 Tesla. PT represents a model of focal cerebral ischemia exhibiting acute vessel occlusion and delayed neuroinflammation.
Methods/Principal Findings
Perfluorocarbons (PFC) or superparamagnetic iron oxide particles (SPIO) were injected intravenously at different time points after photothrombotic infarction. While administration of PFC directly after PT induction led to a strong \(^{19}\)F signal throughout the entire lesion, two hours delayed application resulted in a rim-like \(^{19}\)F signal at the outer edge of the lesion. These findings closely resembled the distribution of signal loss on T2-weighted MRI seen after SPIO injection reflecting intravascular accumulation of iron particles trapped in vessel thrombi as confirmed histologically. By sequential administration of two chemically shifted PFC compounds 0 and 2 hours after illumination the different spatial distribution of the \(^{19}\)F markers (infarct core/rim) could be visualized in the same animal. When PFC were applied at day 6 the fluorine marker was only detected after long acquisition times ex vivo. SPIO-enhanced MRI showed slight signal loss in vivo which was much more prominent ex vivo indicative for neuroinflammation at this late lesion stage.
Conclusion
Our study shows that vessel occlusion can be followed in vivo by \(^{19}\)F and SPIO-enhanced high-field MRI while in vivo imaging of neuroinflammation remains challenging. The timing of contrast agent application was the major determinant of the underlying processes depicted by both imaging techniques. Importantly, sequential application of different PFC compounds allowed depiction of ongoing vessel occlusion from the core to the margin of the ischemic lesions in a single MRI measurement.
Background
Investigation of the expression of an intestinal stem cell marker in esophageal adenocarcinomas (EAC) with and without Barrett's Esophagus (BE), with respect to a cancer stem cell (CSC) hypothesis.
Materials and methods
Expression of a putative intestinal stem cell marker LgR5 was analyzed in esophageal cancer specimen (n = 70: 41 EAC with BE, 19 EAC without BE, and n = 10 esophageal squamous-cell carcinomas, ESCC) and in the adenocarcinoma cell line OE-33. Ki-67 and Cdx-2 were co-labelled with LgR5 in double staining experiments. Immunhistochemical expression results were confirmed by RT-PCR and correlated with tumor stage and five-year survival rates.
Results
LgR5was found expressed in 35 of 41 (85%) EAC with BE and in 16 of 19 (81%) EAC without BE. By contrast, LgR5 was not found to be expressed in ESCC. Quantification of immunolabeling showed 15% LgR5+ cells in EAC with BE, 32% LgR5+ cells in adjacent BE and 13% in EAC without BE. Immunofluorescence double staining experiments with LgR5 and Ki-67 revealed a subpopulation (~5%) of proliferating LgR+/Ki-67+ cells. On mRNA-level, expression of LgR5 was higher in BE in comparison to EAC (p = 0.0159). High levels of LgR5 expression in BE associated EAC were associated with poorer survival in univariate analysis.
Conclusion
The stem cell marker LgR5 is expressed in EAC, irrespective of association with BE, and appears to have negative impact on survival. The subset of proliferating LgR5+ cells (<5%) might resemble rapidly cycling CSCs, which needs to be substantiated in further investigations.
Background
Genome wide association studies reported two single nucleotide polymorphisms in ANK3 (rs9804190 and rs10994336) as independent genetic risk factors for bipolar disorder. Another SNP in ANK3 (rs10761482) was associated with schizophrenia in a large European sample. Within the debate on common susceptibility genes for schizophrenia and bipolar disorder, we tried to investigate common findings by analyzing association of ANK3 with schizophrenia, bipolar disorder and unipolar depression.
Methods
We genotyped three single nucleotide polymorphisms (SNPs) in ANK3 (rs9804190, rs10994336, and rs10761482) in a case-control sample of German descent including 920 patients with schizophrenia, 400 with bipolar affective disorder, 220 patients with unipolar depression according to ICD 10 and 480 healthy controls. Sample was further differentiated according to Leonhard's classification featuring disease entities with specific combination of bipolar and psychotic syndromes.
Results
We found no association of rs9804190 and rs10994336 with bipolar disorder, unipolar depression or schizophrenia. In contrast to previous findings rs10761482 was associated with bipolar disorder (p = 0.015) but not with schizophrenia or unipolar depression. We observed no association with disease entities according to Leonhard's classification.
Conclusion
Our results support a specific genetic contribution of ANK3 to bipolar disorder though we failed to replicate findings for schizophrenia. We cannot confirm ANK3 as a common risk factor for different diseases.
Background
"Negative affect" is one of the major migraine triggers. The aim of the study was to assess attentional biases for negative affective stimuli that might be related to migraine triggers in migraine patients with either few or frequent migraine and healthy controls.
Methods
Thirty-three subjects with frequent migraine (FM) or with less frequent episodic migraine, and 20 healthy controls conducted two emotional Stroop tasks in the interictal period. In task 1, general affective words and in task 2, pictures of affective faces (angry, neutral, happy) were used. For each task we calculated two emotional Stroop indices. Groups were compared using one-way ANOVAs.
Results
The expected attentional bias in migraine patients was not found. However, in task 2 the controls showed a significant attentional bias to negative faces, whereas the FM group showed indices near zero. Thus, the FM group responded faster to negative than to positive stimuli. The difference between the groups was statistically significant.
Conclusions
The findings in the FM group may reflect a learned avoidance mechanism away from affective migraine triggers.
Disorder-specific effects of polymorphisms at opposing ends of the Insulin Degrading Enzymegene
(2011)
Background
Insulin-degrading enzyme (IDE) is the ubiquitously expressed enzyme responsible for insulin and amyloid beta (Aβ) degradation. IDE gene is located on chromosome region 10q23-q25 and exhibits a well-replicated peak of linkage with Type 2 diabetes mellitus (T2DM). Several genetic association studies examined IDE gene as a susceptibility gene for Alzheimer's disease (AD), however with controversial results.
Methods
We examined associations of three IDE polymorphisms (IDE2, rs4646953; IDE7, rs2251101 and IDE9, rs1887922) with AD, Aβ42 plasma level and T2DM risk in the longitudinal Vienna Transdanube Aging (VITA) study cohort.
Results
The upstream polymorphism IDE2 was found to influence AD risk and to trigger the Aβ42 plasma level, whereas the downstream polymorphism IDE7 modified the T2DM risk; no associations were found for the intronic variant IDE9.
Conclusions
Based on our SNP and haplotype results, we delineate the model that IDE promoter and 3' untranslated region/downstream variation may have different effects on IDE expression, presumably a relevant endophenotype with disorder-specific effects on AD and T2DM susceptibility.
Background
Currently established methods to identify viable and non-viable cells of cyanobacteria are either time-consuming (eg. plating) or preparation-intensive (eg. fluorescent staining). In this paper we present a new and fast viability assay for unicellular cyanobacteria, which uses red chlorophyll fluorescence and an unspecific green autofluorescence for the differentiation of viable and non-viable cells without the need of sample preparation.
Results
The viability assay for unicellular cyanobacteria using red and green autofluorescence was established and validated for the model organism Synechocystis sp. PCC 6803. Both autofluorescence signals could be observed simultaneously allowing a direct classification of viable and non-viable cells. The results were confirmed by plating/colony count, absorption spectra and chlorophyll measurements. The use of an automated fluorescence microscope and a novel ImageJ based image analysis plugin allow a semi-automated analysis.
Conclusions
The new method simplifies the process of viability analysis and allows a quick and accurate analysis. Furthermore results indicate that a combination of the new assay with absorption spectra or chlorophyll concentration measurements allows the estimation of the vitality of cells.
Background:
Ventilation with high positive end-expiratory pressure (PEEP) can lead to hepatic dysfunction. The aim of this study was to investigate the hepatic effects of strategies using high airway pressures either in pressure-controlled ventilation (PCV) or in high-frequency oscillatory ventilation (HFOV) combined with an arteriovenous extracorporeal lung assist (ECLA).
Material/Methods:
Pietrain pigs underwent induction of lung injury by saline lavage. Ventilation was continued for 24 hours either as PCV with tidal volumes of 6 ml/kg and PEEP 3 cmH2O above the lower inflection point of the pressure-volume curve or as HFOV (≥12 Hz) with a mean tracheal airway pressure 3 cmH2O above the lower inflection point combined with arteriovenous ECLA (HFOV+ECLA). Fluids and norepinephrine stabilized the circulation. The indocyanine green plasma disappearance rate, serum bilirubin, aspartate aminotransferase, alanine aminotransferase, γ-glutamyltransferase, alkaline phosphatase, glutamate dehydrogenase, lactate dehydrogenase and creatine kinase were determined repeatedly. Finally, liver neutrophils were counted and liver cell apoptosis was assessed by terminal deoxynucleotidyl transferase nick end labeling (TUNEL).
Results:
Aspartate aminotransferase increased in the PCV group about three-fold and in the HFOV+ECLA group five-fold (p<0.001). Correspondingly, creatine kinase increased about two-fold and four-fold, respectively (p<0.001). Lactate dehydrogenase was increased in the HFOV+ECLA group (p<0.028). The number of neutrophils infiltrating the liver tissue and the apoptotic index were low.
Conclusions:
High airway pressure PCV and HFOV with ECLA in the treatment of lavage-induced lung injury in pigs did not cause liver dysfunction or damage. The detected elevation of enzymes might be of extrahepatic origin.
Scientific research is a process concerned with the creation, collective accumulation, contextualization, updating and maintenance of knowledge. Wikis provide an environment that allows to collectively accumulate, contextualize, update and maintain knowledge in a coherent and transparent fashion. Here, we examine the potential of wikis as platforms for scholarly publishing. In the hope to stimulate further discussion, the article itself was drafted on Species-ID – a wiki that hosts a prototype for wiki-based scholarly publishing – where it can be updated, expanded or otherwise improved.
Super-resolution fluorescence imaging based on inglemolecule localization relies critically on the availability of efficient processing algorithms to distinguish, identify, and localize emissions of single fluorophores. In multiple current applications, such as threedimensional, time-resolved or cluster imaging, high densities of fluorophore emissions are common. Here, we provide an analytic tool to test the performance and quality of localization microscopy algorithms and demonstrate that common algorithms encounter difficulties for samples with high fluorophore density. We demonstrate that, for typical single-molecule localization microscopy methods such as dSTORM and the commonly used rapidSTORM scheme, computational precision limits the acceptable density of concurrently active fluorophores to 0.6 per square micrometer and that the number of successfully localized fluorophores per frame is limited to 0.2 per square micrometer.
The limbic system and especially the amygdala have been identified as key structures in emotion induction and regulation. Recently research has additionally focused on the influence of prefrontal areas on emotion processing in the limbic system and the amygdala. Results from fMRI studies indicate that the prefrontal cortex (PFC) is involved not only in emotion induction but also in emotion regulation. However, studies using fNIRS only report prefrontal brain activation during emotion induction. So far it lacks the attempt to compare emotion induction and emotion regulation with regard to prefrontal activation measured with fNIRS, to exclude the possibility that the reported prefrontal brain activation in fNIRS studies are mainly caused by automatic emotion regulation processes. Therefore this work tried to distinguish emotion induction from regulation via fNIRS of the prefrontal cortex. 20 healthy women viewed neutral pictures as a baseline condition, fearful pictures as induction condition and reappraised fearful pictures as regulation condition in randomized order. As predicted, the view-fearful condition led to higher arousal ratings than the view-neutral condition with the reappraise-fearful condition in between. For the fNIRS results the induction condition showed an activation of the bilateral PFC compared to the baseline condition (viewing neutral). The regulation condition showed an activation only of the left PFC compared to the baseline condition, although the direct comparison between induction and regulation condition revealed no significant difference in brain activation. Therefore our study underscores the results of previous fNIRS studies showing prefrontal brain activation during emotion induction and rejects the hypothesis that this prefrontal brain activation might only be a result of automatic emotion regulation processes.
While there is only little transformation to the absolute power of the party-state to be detected, some grassroots democratic experiments, however, are receiving enormous attention of the world, especially village elections. Nevertheless, this preliminary exercise of democracy is widely characterized as a mixed bag of results. Since its first conduction, it has experienced immense development and bought great impact not only on different rural political institutions, but also on common mass villagers, as well as changes to the local governance. But at the same time, the limitations of the factual effectiveness of these elections can hardly be underestimated and such aspects as the standardization of electoral procedures are still to be further improved. Moreover, given the wide variations across Chinese countryside and the strong oppositions from all levels, the future of China’s village elections remain hard to gauge.
After a century of its retreat from political and social stages in East Asia, Confucianism eventually found its revival together with the economic industrialization in the region. The awakening consciousness of the traditional Confucian values leads to a reconsideration of their implication on a modern society. Despite the criticism on the actual relevance of Confucianism and modernization, there are precious elements within the Confucian values which provide the relevance of Confucianism to the future, such as an ethic of responsibility and the understanding of the humanistic meaning of life.
SMAC Mimetic BV6 Induces Cell Death in Monocytes and Maturation of Monocyte-Derived Dendritic Cells
(2011)
Background: Compounds mimicking the inhibitory effect of SMAC / DIABLO on X-linked inhibitor of apoptosis (XIAP) have been developed with the aim to achieve sensitization for apoptosis of tumor cells resistant due to deregulated XIAP expression. It turned out that SMAC mimetics also have complex effects on the NFkB system and TNF signaling. In view of the overwhelming importance of the NFkB transcription factors in the immune system, we analyzed here the effects of the SMAC mimetic BV6 on immune cells. Principal Findings: BV6 induced apoptotic and necrotic cell death in monocytes while T-cells, dendritic cells and macrophages were largely protected against BV6-induced cell death. In immature dendritic cells BV6 treatment resulted in moderate activation of the classical NFkB pathway, but it also diminished the stronger NFkB-inducing effect of TNF and CD40L. Despite its inhibitory effect on TNF- and CD40L signaling, BV6 was able to trigger maturation of immature DCs as indicated by upregulation of CD83, CD86 and IL12. Significance: The demonstrated effects of SMAC mimetics on immune cells may complicate the development of tumor therapeutic concepts based on these compounds but also arise the possibility to exploit them for the development of immune stimulatory therapies.
TNFR1 and TNFR2 regulate the extrinsic apoptotic pathway in myeloma cells by multiple mechanisms
(2011)
The huge majority of myeloma cell lines express TNFR2 while a substantial subset of them failed to show TNFR1 expression. Stimulation of TNFR1 in the TNFR1-expressing subset of MM cell lines had no or only a very mild effect on cellular viability. Surprisingly, however, TNF stimulation enhanced cell death induction by CD95L and attenuated the apoptotic effect of TRAIL. The contrasting regulation of TRAIL- and CD95L-induced cell death by TNF could be traced back to the concomitant NFjBmediated upregulation of CD95 and the antiapoptotic FLIP protein. It appeared that CD95 induction, due to its strength, overcompensated a rather moderate upregulation of FLIP so that the net effect of TNF-induced NFjB activation in the context of CD95 signaling is pro-apoptotic. TRAIL-induced cell death, however, was antagonized in response to TNF because in this context only the induction of FLIP is relevant. Stimulation of TNFR2 in myeloma cells leads to TRAF2 depletion. In line with this, we observed cell death induction in TNFR1-TNFR2-costimulated JJN3 cells. Our studies revealed that the TNF-TNF receptor system adjusts the responsiveness of the extrinsic apoptotic pathway in myeloma cells by multiple mechanisms that generate a highly context-dependent net effect on myeloma cell survival.
Enormous amounts of data are being generated by modern methods such as transcriptome or exome sequencing and microarray profiling. Primary analyses such as quality control, normalization, statistics and mapping are highly complex and need to be performed by specialists. Thereafter, results are handed back to biomedical researchers, who are then confronted with complicated data lists. For rather simple tasks like data filtering, sorting and cross-association there is a need for new tools which can be used by non-specialists. Here, we describe CrossQuery, a web tool that enables straight forward, simple syntax queries to be executed on transcriptome sequencing and microarray datasets. We provide deepsequencing data sets of stem cell lines derived from the model fish Medaka and microarray data of human endothelial cells. In the example datasets provided, mRNA expression levels, gene, transcript and sample identification numbers, GO-terms and gene descriptions can be freely correlated, filtered and sorted. Queries can be saved for later reuse and results can be exported to standard formats that allow copy-and-paste to all widespread data visualization tools such as Microsoft Excel. CrossQuery enables researchers to quickly and freely work with transcriptome and microarray data sets requiring only minimal computer skills. Furthermore, CrossQuery allows growing association of multiple datasets as long as at least one common point of correlated information, such as transcript identification numbers or GO-terms, is shared between samples. For advanced users, the object-oriented plug-in and event-driven code design of both server-side and client-side scripts allow easy addition of new features, data sources and data types.
Prenatal stress (PS) has been shown to influence the development of the fetal brain and to increase the risk for the development of psychiatric disorders in later life. Furthermore, the variation of human serotonin transporter (5-HTT, SLC6A4) gene was suggested to exert a modulating effect on the association between early life stress and the risk for depression. In the present study, we used a 5-Htt6PS paradigm to investigate whether the effects of PS are dependent on the 5-Htt genotype. For this purpose, the effects of PS on cognition, anxiety- and depression-related behavior were examined using a maternal restraint stress paradigm of PS in C57BL6 wild-type (WT) and heterozygous 5-Htt deficient (5-Htt +/2) mice. Additionally, in female offspring, a genome-wide hippocampal gene expression profiling was performed using the Affymetrix GeneChipH Mouse Genome 430 2.0 Array. 5-Htt +/2 offspring showed enhanced memory performance and signs of reduced anxiety as compared to WT offspring. In contrast, exposure of 5-Htt +/2 mice to PS was associated with increased depressive-like behavior, an effect that tended to be more pronounced in female offspring. Further, 5-Htt genotype, PS and their interaction differentially affected the expression of numerous genes and related pathways within the female hippocampus. Specifically, MAPK and neurotrophin signaling were regulated by both the 5-Htt +/2 genotype and PS exposure, whereas cytokine and Wnt signaling were affected in a 5-Htt genotype6PS manner, indicating a gene6environment interaction at the molecular level. In conclusion, our data suggest that although the 5-Htt +/2 genotype shows clear adaptive capacity, 5-Htt +/2 mice –particularly females– at the same time appear to be more vulnerable to developmental stress exposure when compared to WT offspring. Moreover, hippocampal gene expression profiles suggest that distinct molecular mechanisms mediate the behavioral effects of the 5-Htt genotype, PS exposure, and their interaction.
Magnetic Resonance Imaging of Tumors Colonized with Bacterial Ferritin-Expressing Escherichia coli
(2011)
Background: Recent studies have shown that human ferritin can be used as a reporter of gene expression for magnetic resonance imaging (MRI). Bacteria also encode three classes of ferritin-type molecules with iron accumulation properties. Methods and Findings: Here, we investigated whether these bacterial ferritins can also be used as MRI reporter genes and which of the bacterial ferritins is the most suitable reporter. Bacterial ferritins were overexpressed in probiotic E. coli Nissle 1917. Cultures of these bacteria were analyzed and those generating highest MRI contrast were further investigated in tumor bearing mice. Among members of three classes of bacterial ferritin tested, bacterioferritin showed the most promise as a reporter gene. Although all three proteins accumulated similar amounts of iron when overexpressed individually, bacterioferritin showed the highest contrast change. By site-directed mutagenesis we also show that the heme iron, a unique part of the bacterioferritin molecule, is not critical for MRI contrast change. Tumor-specific induction of bacterioferritin-expression in colonized tumors resulted in contrast changes within the bacteria-colonized tumors. Conclusions: Our data suggest that colonization and gene expression by live vectors expressing bacterioferritin can be monitored by MRI due to contrast changes
The risk of developing thyroid cancer increases considerably after exposure to external or internal radiation, especially in children below the age of 10. After the Chernobyl reactor accident, the yearly incidence of childhood thyroid cancer in Belarus increased to approximately 40 per 1.000.000 in girls and to roughly 20 per 1.000.000 in boys compared to approximately 0.5 cases per 1.000.000 prior to the accident. Typically, young children with thyroid cancer after radiation exposure present in ≈95% of the cases as papillary cancers, in ≈50% as invasive tumors growing outside the thyroid capsule, in ≈65% with lymph node metastases and in ≈15% with distant metastases. A joint Belarusian-German project starting in April 1993 that combined treatment with surgery and radioiodine was organized in 237 selected children from Belarus who were exposed to the Chernobyl fallout and had advanced stages of thyroid cancer. The study group included 141 girls and 96 boys. Their median age at the time of the accident was 1.7 years; whereas the median age at the time of diagnosis was 12.4 years. With the exception of two cases with follicular histology, the majority of the patients had been diagnosed with papillary thyroid cancers. In 63%, the tumor had grown outside the thyroid capsule and invaded the tissue of the neck (pT4). Nearly all of the selected cases (96%) showed-up with lymph node metastases (pN1) and 43% of the patients with distant metastases mainly to the lungs (pM1). In 58% of the children, complete remissions of thyroid cancer could be achieved until December 31st 2010 and in 34% of the children, stable partial remissions; in the remaining 8% of the patients, partial remissions were observed. The risk of radiation-induced thyroid cancer increased considerably in children and adolescents who were affected by the Chernobyl reactor accident. In spite of the fact, that thyroid cancers in young children seem to behave more aggressively than in older patients, the results of combined treatment with thyroidectomy, radioiodine therapy and thyroid hormone replacement are excellent.
Four new tetromycin derivatives, tetromycins 1–4 and a previously known one, tetromycin B (5) were isolated from Streptomyces axinellae Pol001T cultivated from the Mediterranean sponge Axinella polypoides. Structures were assigned using extensive 1D and 2D NMR spectroscopy as well as HRESIMS analysis. The compounds were tested for antiparasitic activities against Leishmania major and Trypanosoma brucei, and for protease inhibition against several cysteine proteases such as falcipain, rhodesain, cathepsin L, cathepsin B, and viral proteases SARS-CoV Mpro, and PLpro. The compounds showed antiparasitic activities against T. brucei and time-dependent inhibition of cathepsin L-like proteases with Ki values in the low micromolar range.
Background: In a number of gram-positive bacteria, including Listeria, the general stress response is regulated by the alternative sigma factor B (SigB). Common stressors which lead to the activation of SigB and the SigB-dependent regulon are high osmolarity, acid and several more. Recently is has been shown that also blue and red light activates SigB in Bacillus subtilis. Methodology/Principal Findings: By qRT-PCR we analyzed the transcriptional response of the pathogen L. monocytogenes to blue and red light in wild type bacteria and in isogenic deletion mutants for the putative blue-light receptor Lmo0799 and the stress sigma factor SigB. It was found that both blue (455 nm) and red (625 nm) light induced the transcription of sigB and SigB-dependent genes, this induction was completely abolished in the SigB mutant. The blue-light effect was largely dependent on Lmo0799, proving that this protein is a genuine blue-light receptor. The deletion of lmo0799 enhanced the red-light effect, the underlying mechanism as well as that of SigB activation by red light remains unknown. Blue light led to an increased transcription of the internalin A/B genes and of bacterial invasiveness for Caco-2 enterocytes. Exposure to blue light also strongly inhibited swimming motility of the bacteria in a Lmo0799- and SigB-dependent manner, red light had no effect there. Conclusions/Significance: Our data established that visible, in particular blue light is an important environmental signal with an impact on gene expression and physiology of the non-phototrophic bacterium L. monocytogenes. In natural environments these effects will result in sometimes random but potentially also cyclic fluctuations of gene activity, depending on the light conditions prevailing in the respective habitat.
Using the particle-resolved aerosol model PartMC-MOSAIC, we simulate the heterogeneous oxidation of a monolayer of polycyclic aromatic hydrocarbons (PAHs) on soot particles in an urban atmosphere. We focus on the interaction of the major atmospheric oxidants (O3, NO2, OH, and NO3) with PAHs and include competitive co-adsorption of water vapour for a range of atmospheric conditions. For the first time detailed heterogeneous chemistry based on the P¨oschl-Rudich-Ammann (PRA) framework is modelled on soot particles with a realistic size distribution and a continuous range of chemical ages. We find PAH half-lives, 1/2, on the order of seconds during the night, when the PAHs are rapidly oxidised by the gas-surface reaction with NO3. During the day, 1/2 is on the order of minutes and determined mostly by the surface layer reaction of PAHs with adsorbed O3. Such short half-lives of surface-bound PAHs may lead to efficient conversion of hydrophobic soot into more hygroscopic particles, thus increasing the particles’ aerosol-cloud interaction potential. Despite its high reactivity OH appears to have a negligible effect on PAH degradation which can be explained by its very low concentration in the atmosphere. An increase of relative humidity (RH) from 30% to 80% increases PAH half-lives by up to 50%for daytime degradation and by up to 100% or more for nighttime degradation. Uptake coefficients, averaged over the particle population, are found to be relatively constant over time for O3 (2×10-7 to 2×10-6) and NO2 (5×10-6 to 10-5) at the different levels of NOx emissions and RH considered in this study. In contrast, those for OH and NO3 depend strongly on the surface concentration of PAHs. We do not find a significant influence of heterogeneous reactions on soot particles on the gas phase composition. The derived half-lives of surfacebound PAHs and the time and particle population averaged uptake coefficients for O3 and NO2 presented in this paper can be used as parameterisations for the treatment of heterogeneous chemistry in large-scale atmospheric chemistry models.
The outcomes of measurements on entangled quantum systems can be nonlocally correlated. However, while it is easy to write down toy theories allowing arbitrary nonlocal correlations, those allowed in quantum mechanics are limited. Quantum correlations cannot, for example, violate a principle known as macroscopic locality, which implies that they cannot violate Tsirelson’s bound. This paper shows that there is a connection between the strength of nonlocal correlations in a physical theory and the structure of the state spaces of individual systems. This is illustrated by a family of models in which local state spaces are regular polygons, where a natural analogue of a maximally entangled state of two systems exists. We characterize the nonlocal correlations obtainable from such states. The family allows us to study the transition between classical, quantum and super-quantum correlations by varying only the local state space. We show that the strength of nonlocal correlations—in particular whether the maximally entangled state violates Tsirelson’s bound or not— depends crucially on a simple geometric property of the local state space, known as strong self-duality. This result is seen to be a special case of a general theorem, which states that a broad class of entangled states in probabilistic theories—including, by extension, all bipartite classical and quantum states— cannot violate macroscopic locality. Finally, our results show that models exist that are locally almost indistinguishable from quantum mechanics, but can nevertheless generate maximally nonlocal correlations.
Background: During the last years, 19F-MRI and perfluorocarbon nanoemulsion (PFC) emerged as a powerful contrast agent based MRI methodology to track cells and to visualize inflammation. We applied this new modality to visualize deep tissue abscesses during acute and chronic phase of inflammation caused by Staphylococcus aureus infection. Methodology and Principal Findings: In this study, a murine thigh infection model was used to induce abscess formation and PFC or CLIO (cross linked ironoxides) was administered during acute or chronic phase of inflammation. 24 h after inoculation, the contrast agent accumulation was imaged at the site of infection by MRI. Measurements revealed a strong accumulation of PFC at the abscess rim at acute and chronic phase of infection. The pattern was similar to CLIO accumulation at chronic phase and formed a hollow sphere around the edema area. Histology revealed strong influx of neutrophils at the site of infection and to a smaller extend macrophages during acute phase and strong influx of macrophages at chronic phase of inflammation. Conclusion and Significance: We introduce 19F-MRI in combination with PFC nanoemulsions as a new platform to visualize abscess formation in a murine thigh infection model of S. aureus. The possibility to track immune cells in vivo by this modality offers new opportunities to investigate host immune response, the efficacy of antibacterial therapies and the influence of virulence factors for pathogenesis.
The present study investigated event-related brain potentials elicited by true and false negated statements to evaluate if discrimination of the truth value of negated information relies on conscious processing and requires higher-order cognitive processing in healthy subjects across different levels of stimulus complexity. The stimulus material consisted of true and false negated sentences (sentence level) and prime-target expressions (word level). Stimuli were presented acoustically and no overt behavioral response of the participants was required. Event-related brain potentials to target words preceded by true and false negated expressions were analyzed both within group and at the single subject level. Across the different processing conditions (word pairs and sentences), target words elicited a frontal negativity and a late positivity in the time window from 600–1000 msec post target word onset. Amplitudes of both brain potentials varied as a function of the truth value of the negated expressions. Results were confirmed at the single-subject level. In sum, our results support recent suggestions according to which evaluation of the truth value of a negated expression is a time- and cognitively demanding process that cannot be solved automatically, and thus requires conscious processing. Our paradigm provides insight into higher-order processing related to language comprehension and reasoning in healthy subjects. Future studies are needed to evaluate if our paradigm also proves sensitive for the detection of consciousness in non-responsive patients.
We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching. Finally, we discuss opportunities to combine fluorescent proteins with organic fluorophores for dual-color photoswitching microscopy.
Background: The etiology of secondary cancer in childhood cancer survivors is largely unclear. Exposure of normal somatic cells to radiation and/or chemotherapy can damage DNA and if not all DNA lesions are properly fixed, the mis-repair may lead to pathological consequences. It is plausible to assume that genetic differences, i.e. in the pathways responsible for cell cycle control and DNA repair, play a critical role in the development of secondary cancer. Methodology/Findings: To identify factors that may influence the susceptibility for second cancer formation, we recruited 20 individuals who survived a childhood malignancy and then developed a second cancer as well as 20 carefully matched control individuals with childhood malignancy but without a second cancer. By antibody microarrays, we screened primary fibroblasts of matched patients for differences in the amount of representative DNA repair-associated proteins. We found constitutively decreased levels of RAD9A and several other DNA repair proteins in two-cancer patients, compared to onecancer patients. The RAD9A protein level increased in response to DNA damage, however to a lesser extent in the twocancer patients. Quantification of mRNA expression by real-time RT PCR revealed lower RAD9A mRNA levels in both untreated and 1 Gy c-irradiated cells of two-cancer patients. Conclusions/Significance: Collectively, our results support the idea that modulation of RAD9A and other cell cycle arrest and DNA repair proteins contribute to the risk of developing a second malignancy in childhood cancer patients.
How do physico-chemical stimulus features, perception, and physiology relate? Given the multi-layered and parallel architecture of brains, the question specifically is where physiological activity patterns correspond to stimulus features and/ or perception. Perceived distances between six odour pairs are defined behaviourally from four independent odour recognition tasks. We find that, in register with the physico-chemical distances of these odours, perceived distances for 3-octanol and n-amylacetate are consistently smallest in all four tasks, while the other five odour pairs are about equally distinct. Optical imaging in the antennal lobe, using a calcium sensor transgenically expressed in only first-order sensory or only second-order olfactory projection neurons, reveals that 3-octanol and n-amylacetate are distinctly represented in sensory neurons, but appear merged in projection neurons. These results may suggest that within-antennal lobe processing funnels sensory signals into behaviourally meaningful categories, in register with the physico-chemical relatedness of the odours.
Background: Anxiety is a heterogeneous behavioral domain playing a role in a variety of neuropsychiatric diseases. While anxiety is the cardinal symptom in disorders such as panic disorder, co-morbid anxious behavior can occur in a variety of diseases. Stiff person syndrome (SPS) is a CNS disorder characterized by increased muscle tone and prominent agoraphobia and anxiety. Most patients have high-titer antibodies against glutamate decarboxylase (GAD) 65. The pathogenic role of these autoantibodies is unclear. Methodology/Principal Findings: We re-investigated a 53 year old woman with SPS and profound anxiety for GABA-A receptor binding in the amygdala with (11)C-flumazenil PET scan and studied the potential pathogenic role of purified IgG from her plasma filtrates containing high-titer antibodies against GAD 65. We passively transferred the IgG fraction intrathecally into rats and analyzed the effects using behavioral and in vivo electrophysiological methods. In cell culture, we measured the effect of patient IgG on GABA release from hippocampal neurons. Repetitive intrathecal application of purified patient IgG in rats resulted in an anxious phenotype resembling the core symptoms of the patient. Patient IgG selectively bound to rat amygdala, hippocampus, and frontal cortical areas. In cultured rat hippocampal neurons, patient IgG inhibited GABA release. In line with these experimental results, the GABA-A receptor binding potential was reduced in the patient’s amygdala/hippocampus complex. No motor abnormalities were found in recipient rats. Conclusion/Significance: The observations in rats after passive transfer lead us to propose that anxiety-like behavior can be induced in rats by passive transfer of IgG from a SPS patient positive for anti-GAD 65 antibodies. Anxiety, in this case, thus may be an antibody-mediated phenomenon with consecutive disturbance of GABAergic signaling in the amygdala region.
Background: Pre- and early clinical studies on patients with autoimmune diseases suggested that induction of regulatory T(Treg) cells may contribute to the immunosuppressive effects of glucocorticoids(GCs). Objective: We readdressed the influence of GC therapy on Treg cells in immunocompetent human subjects and naı¨ve mice. Methods: Mice were treated with increasing doses of intravenous dexamethasone followed by oral taper, and Treg cells in spleen and blood were analyzed by FACS. Sixteen patients with sudden hearing loss but without an inflammatory disease received high-dose intravenous prednisolone followed by stepwise dose reduction to low oral prednisolone. Peripheral blood Treg cells were analyzed prior and after a 14 day GC therapy based on different markers. Results: Repeated GC administration to mice for three days dose-dependently decreased the absolute numbers of Treg cells in blood (100 mg dexamethasone/kg body weight: 2.861.86104 cells/ml vs. 336116104 in control mice) and spleen (dexamethasone: 2.861.96105/spleen vs. 956226105/spleen in control mice), which slowly recovered after 14 days taper in spleen but not in blood. The relative frequency of FOXP3+ Treg cells amongst the CD4+ T cells also decreased in a dose dependent manner with the effect being more pronounced in blood than in spleen. The suppressive capacity of Treg cells was unaltered by GC treatment in vitro. In immunocompetent humans, GCs induced mild T cell lymphocytosis. However, it did not change the relative frequency of circulating Treg cells in a relevant manner, although there was some variation depending on the definition of the Treg cells (FOXP3+: 4.061.5% vs 3.461.5%*; AITR+: 0.660.4 vs 0.560.3%, CD127low: 4.061.3 vs 5.063.0%* and CTLA4+: 13.8611.5 vs 15.6612.5%; * p,0.05). Conclusion: Short-term GC therapy does not induce the hitherto supposed increase in circulating Treg cell frequency, neither in immunocompetent humans nor in mice. Thus, it is questionable that the clinical efficacy of GCs is achieved by modulating Treg cell numbers.
no abstract available
In the model plant Arabidopsis thaliana, more than 2000 genes are estimated to encode transcription factors (TFs), which clearly emphasizes the importance of transcriptional control. Although genomic approaches have generated large TF open reading frame (ORF) collections, only a limited number of these genes is functionally characterized, yet. This review evaluates strategies and methods to identify TF functions. In particular, we focus on two recently developed TF screening platforms, which make use of publically available GATEWAY®-compatible ORF collections. (1) The Arabidopsis thaliana TF ORF over-Expression (AtTORF-Ex) library provides pooled collections of transgenic lines over-expressing HA-tagged TF genes, which are suited for screening approaches to define TF functions in stress defense and development. (2) A high-throughput microtiter plate based protoplast trans activation (PTA) system has been established to screen for TFs which are regulating a given promoter:Luciferase construct in planta.
Dendritic cell tumors are extremely rare neoplasms arising from antigen-presenting cells of the immune system. We report a case of a 69-year-old man with an unremarkable medical history who presented with a 2-months history of a gradually enlarging painless, firm, mobile, 2 × 2-cm swelling at the caudal pole of the left parotid gland without systemic symptoms. Histologically, the tumor consisted of a spindle cell proliferation in an intraparotideal lymph node. Based on the histopathologic, immunohistochemical and electron microscopic findings, a dendritic cell tumor, not otherwise specified (NOS) in an intraparotideal lymph node was diagnosed. The patient underwent complete tumor resection, and is currently free of disease, 2 years after surgery. These extremely rare tumors must be distinguished from other more common tumors in the salivary glands. Awareness that dendritic cell tumors may occur in this localization, careful histologic evaluation and ancillary immunohistochemical and electron microscopical analyses should allow for recognition of this entity. Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1614859498581601.
Over the last years, evidence has accumulated suggesting that by systematically reducing the amount of dietary carbohydrates (CHOs) one could suppress, or at least delay, the emergence of cancer, and that proliferation of already existing tumor cells could be slowed down. This hypothesis is supported by the association between modern chronic diseases like the metabolic syndrome and the risk of developing or dying from cancer. CHOs or glucose, to which more complex carbohydrates are ultimately digested, can have direct and indirect effects on tumor cell proliferation: first, contrary to normal cells, most malignant cells depend on steady glucose availability in the blood for their energy and biomass generating demands and are not able to metabolize significant amounts of fatty acids or ketone bodies due to mitochondrial dysfunction. Second, high insulin and insulin-like growth factor (IGF)-1 levels resulting from chronic ingestion of CHO-rich Western diet meals, can directly promote tumor cell proliferation via the insulin/IGF1 signaling pathway. Third, ketone bodies that are elevated when insulin and blood glucose levels are low, have been found to negatively affect proliferation of different malignant cells in vitro or not to be usable by tumor cells for metabolic demands, and a multitude of mouse models have shown antitumorigenic properties of very low CHO ketogenic diets. In addition, many cancer patients exhibit an altered glucose metabolism characterized by insulin resistance and may profit from an increased protein and fat intake. In this review, we address the possible beneficial effects of low CHO diets on cancer prevention and treatment. Emphasis will be placed on the role of insulin and IGF1 signaling in tumorigenesis as well as altered dietary needs of cancer patients.
Bacterial glucuronidase as general marker for oncolytic virotherapy or other biological therapies
(2011)
Background: Oncolytic viral tumor therapy is an emerging field in the fight against cancer with rising numbers of clinical trials and the first clinically approved product (Adenovirus for the treatment of Head and Neck Cancer in China) in this field. Yet, until recently no general (bio)marker or reporter gene was described that could be used to evaluate successful tumor colonization and/or transgene expression in other biological therapies. Methods: Here, a bacterial glucuronidase (GusA) encoded by biological therapeutics (e.g. oncolytic viruses) was used as reporter system. Results: Using fluorogenic probes that were specifically activated by glucuronidase we could show 1) preferential activation in tumors, 2) rena l excretion of the activated fluorescent compounds and 3) reproducible detection of GusA in the serum of oncolytic vaccinia virus treated, tumor bearing mice in several tumor models. Time course studies revealed that reliable differentiation between tumor bearing and healthy mice can be done as early as 9 days post injection of the virus. Regarding the sensitivity of the newly developed assay system, we could show that a single infected tumor cell could be reliably detected in this assay. Conclusion: GusA therefore has the potential to be used as a general marker in the preclinical and clinical evaluation of (novel) biological therapies as well as being useful for the detection of rare cells such as circulating tumor cells
Retinoic acid pathway activity in Wilms tumors and characterization of biological responses in vitro
(2011)
Background: Wilms tumor (WT) is one of the most common malignancies in childhood. With current therapy protocols up to 90% of patients can be cured, but there is still a need to improve therapy for patients with aggressive WT and to reduce treatment intensity where possible. Prior data suggested a deregulation of the retinoic acid (RA) signaling pathway in high-risk WT, but its mode of action remained unclear. Results: The association of retinoid signaling and clinical parameters could be validated in a large independent tumor set, but its relevance in primary nephrectomy tumors from very young children may be different. Reduced RA pathway activity and MYCN overexpression were found in high risk tumors as opposed to tumors with low/ intermediate risk, suggesting a beneficial impact of RA especially on advanced WT. To search for possible modes of action of retinoids as novel therapeutic options, primary tumor cell cultures were treated in vitro with all-trans-RA (ATRA), 9cis-RA, fenretinide and combinations of retinoids and a histone deacetylase (HDAC) inhibitor. Genes deregulated in high risk tumors showed opposite changes upon treatment suggesting a positive effect of retinoids. 6/7 primary cultures tested reduced proliferation, irrespective of prior RA signaling levels. The only variant culture was derived from mesoblastic nephroma, a distinct childhood kidney neoplasm. Retinoid/HDAC inhibitor combinations provided no synergistic effect. ATRA and 9cis-RA induced morphological changes suggestive of differentiation, while fenretinide induced apoptosis in several cultures tested. Microarray analysis of ATRA treated WT cells revealed differential expression of many genes involved in extracellular matrix formation and osteogenic, neuronal or muscle differentiation. The effects documented appear to be reversible upon drug withdrawal, however. Conclusions: Altered retinoic acid signaling has been validated especially in high risk Wilms tumors. In vitro testing of primary tumor cultures provided clear evidence of a potential utility of retinoids in Wilms tumor treatment based on the analysis of gene expression, proliferation, differentiation and apoptosis.
Background: Hybridization can have complex effects on evolutionary dynamics in ants because of the combination of haplodiploid sex-determination and eusociality. While hybrid non-reproductive workers have been found in a range of species, examples of gene-flow via hybrid queens and males are rare. We studied hybridization in East African army ants (Dorylus subgenus Anomma) using morphology, mitochondrial DNA sequences, and nuclear microsatellites. Results: While the mitochondrial phylogeny had a strong geographic signal, different species were not recovered as monophyletic. At our main study site at Kakamega Forest, a mitochondrial haplotype was shared between a “Dorylus molestus-like” and a “Dorylus wilverthi-like” form. This pattern is best explained by introgression following hybridization between D. molestus and D. wilverthi. Microsatellite data from workers showed that the two morphological forms correspond to two distinct genetic clusters, with a significant proportion of individuals being classified as hybrids. Conclusions: We conclude that hybridization and gene-flow between the two army ant species D. molestus and D. wilverthi has occurred, and that mating between the two forms continues to regularly produce hybrid workers. Hybridization is particularly surprising in army ants because workers have control over which males are allowed to mate with a young virgin queen inside the colony.
In common with most viruses, measles virus (MV) relies on the integrity of the cytoskeleton of its host cells both with regard to efficient replication in these cells, but also retention of their motility which favors viral dissemination. It is, however, the surface interaction of the viral glycoprotein (gp) complex with receptors present on lymphocytes and dendritic cells (DCs), that signals effective initiation of host cell cytoskeletal dynamics. For DCs, these may act to regulate processes as diverse as viral uptake and sorting, but also the ability of these cells to successfully establish and maintain functional immune synapses (IS) with T cells. In T cells, MV signaling causes actin cytoskeletal paralysis associated with a loss of polarization, adhesion and motility, which has been linked to activation of sphingomyelinases and subsequent accumulation of membrane ceramides. MV modulation of both DC and T cell cytoskeletal dynamics may be important for the understanding of MV immunosuppression at the cellular level.
Streptococcus pneumoniae is a common pathogen that causes various infections, such as sepsis and meningitis. A major pathogenic factor of S. pneumoniae is the cholesterol-dependent cytolysin, pneumolysin. It produces cell lysis at high concentrations and apoptosis at lower concentrations. We have shown that sublytic amounts of pneumolysin induce small GTPase-dependent actin cytoskeleton reorganization and microtubule stabilization in human neuroblastoma cells that are manifested by cell retraction and changes in cell shape. In this study, we utilized a live imaging approach to analyze the role of pneumolysin’s pore-forming capacity in the actin-dependent cell shape changes in primary astrocytes. After the initial challenge with the wild-type toxin, a permeabilized cell population was rapidly established within 20–40 minutes. After the initial rapid permeabilization, the size of the permeabilized population remained unchanged and reached a plateau. Thus, we analyzed the non-permeabilized (non-lytic) population, which demonstrated retraction and shape changes that were inhibited by actin depolymerization. Despite the non-lytic nature of pneumolysin treatment, the toxin’s lytic capacity remained critical for the initiation of cell shape changes. The non-lytic pneumolysin mutants W433F-pneumolysin and delta6-pneumolysin, which bind the cell membrane with affinities similar to that of the wild-type toxin, were not able to induce shape changes. The initiation of cell shape changes and cell retraction by the wild-type toxin were independent of calcium and sodium influx and membrane depolarization, which are known to occur following cellular challenge and suggested to result from the ion channel-like properties of the pneumolysin pores. Excluding the major pore-related phenomena as the initiation mechanism of cell shape changes, the existence of a more complex relationship between the pore-forming capacity of pneumolysin and the actin cytoskeleton reorganization is suggested.
Reconstruction of the extensor mechanism is essential for good extremity function after endoprosthetic knee replacement following tumor resection. Only a few biological methods have been able to reliably restore a functional extensor mechanism, but they are often associated with significant complication rates. Reattachment of the patellar tendon to the prosthesis using an alloplastic patellar ligament (Trevira cord) can be an appropriate alternative. In vivo and in vitro studies have already shown that complete fibrous ingrowth in polyethylene chords can be seen after a period of six months. However, until now, no biomechanical study has shown the efficacy of an alloplastic cord and its fixation device in providing sufficient stability and endurance in daily life-activity until newly formed scar tissue can take over this function. In a special test bench developed for this study, different loading regimes were applied to simulate loads during everyday life. Failure loads and failuremodes were evaluated. The properties of the cord were compared before and after physiological conditioning. It was shown that rubbing was the mode of failure under dynamic loading. Tensile forces up to 2558N did not result in material failure. Thus, using an artificial cord together with this fixation device, temporary sufficient stable fixation can be expected.
The wardrobe malfunction—an unanticipated exposure of bodily parts in the public—has become a prevailing issue in concerts, shows and other celebrity events that is reliably reported by the media. The internet as the fastest source for celebrity gossip allows measuring the impact of such wardrobe malfunctions on the public in-terest in a celebrity. This measurement in turn allows conclusions about intention, motivation, and internet be-haviour of a wide variety of internet users. The present study exemplifies the use of an innovative non-reactive measure of active interest—the Search Volume Index—to assess the impact of a variety of internet-related phe-nomena, including wardrobe malfunctions. Results indicate that interest in a celebrity increases immediately af-ter such an event and stays at a high level for about three weeks (the wardrobe plateau). This special form of ce-lebrity gossip thus meets a constant interest of a substantial proportion of internet users.
Binding of proteins to DNA is usually considered 1D with one protein bound to one DNA molecule. In principle, proteins with multiple DNA binding domains could also bind to and thereby cross-link different DNA molecules. We have investigated this possibility using high-mobility group A1 (HMGA1) proteins, which are architectural elements of chromatin and are involved in the regulation of multiple DNA-dependent processes. Using direct stochastic optical reconstruction microscopy (dSTORM), we could show that overexpression of HMGA1a-eGFP in Cos-7 cells leads to chromatin aggregation. To investigate if HMGA1a is directly responsible for this chromatin compaction we developed a DNA cross-linking assay. We were able to show for the first time that HMGA1a can cross-link DNA directly. Detailed analysis using point mutated proteins revealed a novel DNA cross-linking domain. Electron microscopy indicates that HMGA1 proteins are able to create DNA loops and supercoils in linearized DNA confirming the cross-linking ability of HMGA1a. This capacity has profound implications for the spatial organization of DNA in the cell nucleus and suggests cross-linking activities for additional nuclear proteins.
Background: The role of serotonin (5-hydroxytrptamine, 5-HT) in the modulation of pain has been widely studied. Previous work led to the hypothesis that 5-hydroxyindolacetic acid (5-HIAA), a main metabolite of serotonin, might by itself influence pain thresholds. Results: In the present study, we investigated the role of 5-HIAA in inflammatory pain induced by intraplantar injection of complete Freund’s adjuvant (CFA) into the hind paw of mice. Wild-type mice were compared to mice deficient of the 5-HT transporter (5-HTT-/- mice) using behavioral tests for hyperalgesia and high-performance liquid chromatography (HPLC) to determine tissue levels of 5-HIAA. Wild-type mice reproducibly developed thermal hyperalgesia and paw edema for 5 days after CFA injection. 5-HTT-/- mice treated with CFA had reduced thermal hyperalgesia on day 1 after CFA injection and normal responses to heat hereafter. The 5-HIAA levels in spinal cord and sciatic nerve as measured with HPLC were lower in 5-HTT-/- mice than in wild-type mice after CFA injection. Pretreatment of wild-type mice with intraperitoneal injection of para-chlorophenylalanine (p-CPA), a serotonin synthesis inhibitor, resulted in depletion of the 5-HIAA content in spinal cord and sciatic nerve and decrease in thermal hyperalgesia in CFA injected mice. The application of exogenous 5-HIAA resulted in potentiation of thermal hyperalgesia induced by CFA in 5-HTT-/- mice and in wild-type mice pretreated with p- CPA, but not in wild-type mice without p-CPA pretreatment. Further, methysergide, a broad-spectrum serotonin receptor antagonist, had no effect on 5-HIAA-induced potentiation of thermal hyperalgesia in CFA-treated wildtype mice. Conclusion: Taken together, the present results suggest that 5-HIAA plays an important role in modulating peripheral thermal hyperalgesia in CFA induced inflammation, probably via a non-serotonin receptor mechanism.
Early onset sepsis due to group B streptococcus leads to neonatal morbidity, increased mortality, and long-term neurological deficencies. Interaction between septicemic GBS and confluent monolayers of human coronary artery endothelial cells (HCAECs) was analyzed by genome wide expression profiling. In total, 124 genes were differentially expressed (89 upregulated, 35 downregulated) based on a more than 3-fold difference to control HCAEC. Regulated genes are involved in apoptosis, hemostasis, oxidative stress response, infection, and inflammation. Regulation of selected genes and proteins identified in the gene array analysis was confirmed by Real-time RT-PCR assay (granulocy te chemotactic protein 2), ELISA (urokinase, cyclooxygenase 2, granulocyte chemotactic protein 1), and western blotting (Heme oxygenase1, BCL2 interacting protein) at various time points between 4 and 24 hours. These results indicate that GBS infection might influence signalling pathways leading to impaired function of the innate immune system and hemorrhagic and inflammatory complications during GBS sepsis.
Background: Intraarticular distal radius fractures can be treated with many methods. While internal fixation with angle stable implants has become increasingly popular, the use of bone graft substitutes has also been recommended to address comminution zones and thus increase stability. Whether a combination of both methods will improve clinical outcomes was the purpose of the study Methods: The study was thus conducted as a prospective randomized clinical trial. 39 patients with unilateral, intraarticular fractures of the distal radius were included and randomized to 2 groups, one being treated with internal fixation only, while the second group received an additional bone graft substitute. Results: There was no statistical significance between both groups in functional and radiological results. The occurrence of complications did also not show statistical significance. Conclusions: No advantage of additional granular bone graft substitutes could be seen in this study. Granular bone graft substitutes do not seem to provide extra stability if dorsal angle stable implants are used. Dorsal plates have considerable complication rates such as extensor tendon ruptures and development of CRPS.
Background: Investigation of the expression of an intestinal stem cell marker in esophageal adenocarcinomas (EAC) with and without Barrett’s Esophagus (BE), with respect to a cancer stem cell (CSC) hypothesis. Materials and methods: Expression of a putative intestinal stem cell marker LgR5 was analyzed in esophageal cancer specimen (n = 70: 41 EAC with BE, 19 EAC without BE, and n = 10 esophageal squamous-cell carcinomas, ESCC) and in the adenocarcinoma cell line OE-33. Ki-67 and Cdx-2 were co-labelled with LgR5 in double staining experiments. Immunhistochemical expression results were confirmed by RT-PCR and correlated with tumor stage and five-year survival rates. Results: LgR5was found expressed in 35 of 41 (85%) EAC with BE and in 16 of 19 (81%) EAC without BE. By contrast, LgR5 was not found to be expressed in ESCC. Quantification of immunolabeling showed 15% LgR5+ cells in EAC with BE, 32% LgR5+ cells in adjacent BE and 13% in EAC without BE. Immunofluorescence double staining experiments with LgR5 and Ki-67 revealed a subpopulation (~5%) of proliferating LgR+/Ki-67+ cells. On mRNAlevel, expression of LgR5 was higher in BE in comparison to EAC (p = 0.0159). High levels of LgR5 expression in BE associated EAC were associated with poorer survival in univariate analysis. Conclusion: The stem cell marker LgR5 is expressed in EAC, irrespective of association with BE, and appears to have negative impact on survival. The subset of proliferating LgR5+ cells (<5%) might resemble rapidly cycling CSCs, which needs to be substantiated in further investigations.
Background: Xenobiotics represent an environmental stress and as such are a source for antibiotics, including the isoquinoline (IQ) compound IQ-143. Here, we demonstrate the utility of complementary analysis of both host and pathogen datasets in assessing bacterial adaptation to IQ-143, a synthetic analog of the novel type N,C-coupled naphthyl-isoquinoline alkaloid ancisheynine. Results: Metabolite measurements, gene expression data and functional assays were combined with metabolic modeling to assess the effects of IQ-143 on Staphylococcus aureus, Staphylococcus epidermidis and human cell lines, as a potential paradigm for novel antibiotics. Genome annotation and PCR validation identified novel enzymes in the primary metabolism of staphylococci. Gene expression response analysis and metabolic modeling demonstrated the adaptation of enzymes to IQ-143, including those not affected by significant gene expression changes. At lower concentrations, IQ-143 was bacteriostatic, and at higher concentrations bactericidal, while the analysis suggested that the mode of action was a direct interference in nucleotide and energy metabolism. Experiments in human cell lines supported the conclusions from pathway modeling and found that IQ-143 had low cytotoxicity. Conclusions: The data suggest that IQ-143 is a promising lead compound for antibiotic therapy against staphylococci. The combination of gene expression and metabolite analyses with in silico modeling of metabolite pathways allowed us to study metabolic adaptations in detail and can be used for the evaluation of metabolic effects of other xenobiotics.
Mammalian Sun1 belongs to an evolutionarily conserved family of inner nuclear membrane proteins, which are known as SUN domain proteins. SUN domain proteins interact with KASH domain partners to form bridging complexes, so-called LINC complexes, that physically connect the nuclear interior to the cytoskeleton. LINC complexes are critical for nuclear integrity and play fundamental roles in nuclear positioning, shaping and movement. The mammalian genome codes for at least five different SUN domain proteins used for the formation of a number of different LINC complexes. Recently, we reported on the identification of everal Sun1 isoforms, which tremendously enlarges the alternatives to form functional LINC complexes. We now confirmed that Sun1 actually exists in at least seven distinct splice variants. Besides that, we observed that expression of individual Sun1 isoforms remarkably depends on the cell type, suggesting a cell type-specific adaption of Sun1 dependent LINC complexes to specific cellular and physiological requirements.
We herein present a patient with delayed-type allergic hypersensitivity against prilocaine leading to spreading eczematous dermatitis after subcutaneous injections for local anesthesia with prilocaine. Prilocaine allergy was proven by positive skin testing and subcutaneous provocation, whereas the evaluation of other local anesthetics - among them lidocaine, articaine and mepivacaine - did not exhibit any evidence for cross-reactivity. Interestingly, our patient repeatedly tolerated strictly deep subcutaneous injection of prilocaine in provocation testing while patch and superficial subcutaneous application mounted strong allergic responses. We hypothesize, that lower DC density in deeper cutaneous compartments and/or different DC subsets exhibiting distinct functional immunomodulatory properties in the various layers of the skin may confer to the observed absence of clinical reactivity against prilocaine after deep subcutaneous injection. The term compartment allergy indicates that the route of allergen administration together with the targeted immunologic environment orchestrates on the immunologic outcome: overt T-cell mediated allergy or clinical tolerance.
Popliteal cysts, or Baker cysts, are considered rare in children and may exhibit particular features, as compared with adults. We studied data from80 paediatric patients with 55 Baker cysts, examined over a period of 7 years, and correlated clinical presentation with findings on ultrasonography and MRI. Prevalence of popliteal cysts was 57% in arthritic knees, 58% with hypermobility syndrome, and 28% without risk factors. Only one patient had a trauma history and showed an ipsilateral cyst. Mean cyst volume was 3.4 mL; cysts were larger in boys. Patients with arthritis had echogenic cysts in 53%. Cyst communication with the joint space was seen in 64% on ultrasonography and 86% on MRI. In conclusion, Baker cysts are a common finding in a clinically preselected paediatric population. Children with Baker cysts should be assessed for underlying arthritis and inherited joint hypermobility, while sporadic Baker cysts appear to be common, as well.
Background: Tumor patients exhibit an increased peripheral demand of fatty acids and protein. Contrarily, tumors utilize glucose as their main source of energy supply. Thus, a diet supplying the cancer patient with sufficient fat and protein for his demands while restricting the carbohydrates (CHO) tumors thrive on, could be a helpful strategy in improving the patients’ situation. A ketogenic diet (KD) fulfills these requirements. Therefore, we performed a pilot study to investigate the feasibility of a KD and its influence on the quality of life of patients with advanced metastatic tumors. Methods: Sixteen patients with advanced metastatic tumors and no conventional therapeutic options participated in the study. The patients were instructed to follow a KD (less than 70 g CHO per day) with normal groceries and were provided with a supply of food additives to mix a protein/fat shake to simplify the 3-month intervention period. Quality of life [assessed by EORTC QLQ-C30 (version 2)], serum and general health parameters were determined at baseline, after every two weeks of follow-up, or after drop out. The effect of dietary change on metabolism was monitored daily by measuring urinary ketone bodies. Results: One patient did not tolerate the diet and dropped out within 3 days. Among those who tolerated the diet, two patients died early, one stopped after 2 weeks due to personal reasons, one felt unable to stick to the diet after 4 weeks, one stopped after 6 and two stopped after 7 and 8 weeks due to progress of the disease, one had to discontinue after 6 weeks to resume chemotherapy and five completed the 3 month intervention period. These five and the one who resumed chemotherapy after 6 weeks report an improved emotional functioning and less insomnia, while several other parameters of quality of life remained stable or worsened, reflecting their very advanced disease. Except for temporary constipation and fatigue, we found no severe adverse side effects, especially no changes in cholesterol or blood lipids. Conclusions: These pilot data suggest that a KD is suitable for even advanced cancer patients. It has no severe side effects and might improve aspects of quality of life and blood parameters in some patients with advanced metastatic tumors.
Background: Although literature provides support for cognitive behavioral therapy (CBT) as an efficacious intervention for social phobia, more research is needed to improve treatments for children. Methods: Forty four Caucasian children (ages 8-14) meeting diagnostic criteria of social phobia according to the Diagnostic and Statistical Manual of Mental Disorders (4th ed.; APA, 1994) were randomly allocated to either a newly developed CBT program focusing on cognition according to the model of Clark and Wells (n = 21) or a wait-list control group (n = 23). The primary outcome measure was clinical improvement. Secondary outcomes included improvements in anxiety coping, dysfunctional cognitions, interaction frequency and comorbid symptoms. Outcome measures included child report and clinican completed measures as well as a diagnostic interview. Results: Significant differences between treatment participants (4 dropouts) and controls (2 dropouts) were observed at post test on the German version of the Social Phobia and Anxiety Inventory for Children. Furthermore, in the treatment group, significantly more children were free of diagnosis than in wait-list group at post-test. Additional child completed and clinician completed measures support the results. Discussion: The study is a first step towards investigating whether CBT focusing on cognition is efficacious in treating children with social phobia. Future research will need to compare this treatment to an active treatment group. There remain the questions of whether the effect of the treatment is specific to the disorder and whether the underlying theoretical model is adequate. Conclusion: Preliminary support is provided for the efficacy of the cognitive behavioral treatment focusing on cognition in socially phobic children. Active comparators should be established with other evidence-based CBT programs for anxiety disorders, which differ significantly in their dosage and type of cognitive interventions from those of the manual under evaluation (e.g. Coping Cat).
Background: Parkinson’s disease (PD) is characterized at the cellular level by a destruction of neuromelanin (NM)-containing dopaminergic cells and a profound reduction in striatal dopamine. It has been shown recently that antimelanin antibodies are increased in sera of Parkinson patients, suggesting that NM may act as an autoantigen. In this study we tested whether NM is being recognized by dendritic cells (DCs), the major cell type for inducing Tand B-cell responses in vivo. This recognition of NM by DCs is a prerequisite to trigger an adaptive autoimmune response directed against NM-associated structures. Results: Murine DCs were treated with NM of substantia nigra (SN) from human subjects or with synthetic dopamine melanin (DAM). DCs effectively phagocytized NM and subsequently developed a mature phenotype (CD86high/MHCIIhigh). NM-activated DCs secreted the proinflammatory cytokines IL-6 and TNF-a. In addition, they potently triggered T cell proliferation in a mixed lymphocyte reaction, showing that DC activation was functional to induce a primary T cell response. In contrast, DAM, which lacks the protein and lipid components of NM but mimics the dopamine-melanin backbone of NM, had only very little effect on DC phenotype and function. Conclusions: NM is recognized by DCs in vitro and triggers their maturation. If operative in vivo, this would allow the DC-mediated transport and presentation of SN antigens to the adaptive immune system, leading to autoimmmunity in susceptible individuals. Our data provide a rationale for an autoimmune-based pathomechanism of PD with NM as the initial trigger.
The pathogenic yeast Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis, by the overexpression of genes encoding multidrug efflux pumps or ergosterol biosynthesis enzymes. Zinc cluster transcription factors play a central role in the transcriptional regulation of drug resistance. Mrr1 regulates the expression of the major facilitator MDR1, Tac1 controls the expression of the ABC transporters CDR1 and CDR2, and Upc2 regulates ergosterol biosynthesis (ERG) genes. Gain-of-function mutations in these transcription factors result in constitutive overexpression of their target genes and are responsible for fluconazole resistance in many clinical C. albicans isolates. The transcription factor Ndt80 contributes to the drug-induced upregulation of CDR1 and ERG genes and also binds to the MDR1 and CDR2 promoters, suggesting that it is an important component of all major transcriptional mechanisms of fluconazole resistance. However, we found that Ndt80 is not required for the induction of MDR1 and CDR2 expression by inducing chemicals. CDR2 was even partially derepressed in ndt80D mutants, indicating that Ndt80 is a repressor of CDR2 expression. Hyperactive forms of Mrr1, Tac1, and Upc2 promoted overexpression of MDR1, CDR1/CDR2, and ERG11, respectively, with the same efficiency in the presence and absence of Ndt80. Mrr1- and Tac1-mediated fluconazole resistance was even slightly enhanced in ndt80D mutants compared to wild-type cells. These results demonstrate that Ndt80 is dispensable for the constitutive overexpression of Mrr1, Tac1, and Upc2 target genes and the increased fluconazole resistance of strains that have acquired activating mutations in these transcription factors.
Background: To perform a systematic review and meta-analysis on cytokine levels in patients with fibromyalgia syndrome (FMS). Methods: Through December 2010 we systematically reviewed the databases PubMed, MEDLINE, and PsycINFO and screened the reference lists of 22 review articles for suitable original articles. Original articles investigating cytokines in patients with FMS were included. Data were extracted by two independent authors. Differences of the cytokine levels of FMS patients and controls were summarized by standardized mean differences (SMD) using a random effects model. Study quality was assessed applying methodological scores: modified Center of Evidence Based Medicine, Newcastle-Ottawa-Scale, and Würzburg Methodological Quality Score. Results: Twenty-five articles were included investigating 1255 FMS patients and 800 healthy controls. Data of 13/25 studies entered meta-analysis. The overall methodological quality of studies was low. The results of the majority of studies were not comparable because methods, investigated material, and investigated target cytokines differed. Systematic review of the selected 25 articles revealed that FMS patients had higher serum levels of interleukin (IL)-1 receptor antagonist, IL-6, and IL-8, and higher plasma levels of IL-8. Meta-analysis of eligible studies showed that FMS patients had higher plasma IL-6 levels compared to controls (SMD = -0.34 [-0.64, -0.03] 95% CI; p = 0.03). The majority of investigated cytokines were not different between patients and controls. Conclusions: The pathophysiological role of cytokines in FMS is still unclear. Studies of higher quality and with higher numbers of subjects are needed.
Background: Hemostasis is a critical and active function of the blood mediated by platelets. Therefore, the prevention of pathological platelet aggregation is of great importance as well as of pharmaceutical and medical interest. Endogenous platelet inhibition is predominantly based on cyclic nucleotides (cAMP, cGMP) elevation and subsequent cyclic nucleotide-dependent protein kinase (PKA, PKG) activation. In turn, platelet phosphodiesterases (PDEs) and protein phosphatases counterbalance their activity. This main inhibitory pathway in human platelets is crucial for countervailing unwanted platelet activation. Consequently, the regulators of cyclic nucleotide signaling are of particular interest to pharmacology and therapeutics of atherothrombosis. Modeling of pharmacodynamics allows understanding this intricate signaling and supports the precise description of these pivotal targets for pharmacological modulation. Results: We modeled dynamically concentration-dependent responses of pathway effectors (inhibitors, activators, drug combinations) to cyclic nucleotide signaling as well as to downstream signaling events and verified resulting model predictions by experimental data. Experiments with various cAMP affecting compounds including antiplatelet drugs and their combinations revealed a high fidelity, fine-tuned cAMP signaling in platelets without crosstalk to the cGMP pathway. The model and the data provide evidence for two independent feedback loops: PKA, which is activated by elevated cAMP levels in the platelet, subsequently inhibits adenylyl cyclase (AC) but as well activates PDE3. By multi-experiment fitting, we established a comprehensive dynamic model with one predictive, optimized and validated set of parameters. Different pharmacological conditions (inhibition, activation, drug combinations, permanent and transient perturbations) are successfully tested and simulated, including statistical validation and sensitivity analysis. Downstream cyclic nucleotide signaling events target different phosphorylation sites for cAMP- and cGMP-dependent protein kinases (PKA, PKG) in the vasodilator-stimulated phosphoprotein (VASP). VASP phosphorylation as well as cAMP levels resulting from different drug strengths and combined stimulants were quantitatively modeled. These predictions were again experimentally validated. High sensitivity of the signaling pathway at low concentrations is involved in a fine-tuned balance as well as stable activation of this inhibitory cyclic nucleotide pathway. Conclusions: On the basis of experimental data, literature mining and database screening we established a dynamic in silico model of cyclic nucleotide signaling and probed its signaling sensitivity. Thoroughly validated, it successfully predicts drug combination effects on platelet function, including synergism, antagonism and regulatory loops.
Background: Data on complications in children with seasonal influenza virus infection are limited. We initiated a nation-wide three-year surveillance of children who were admitted to a paediatric intensive care unit (PICU) with severe seasonal influenza. Methods: From October 2005 to July 2008, active surveillance was performed using an established reporting system for rare diseases (ESPED) including all paediatric hospitals in Germany. Cases to be reported were hospitalized children < 17 years of age with laboratory-confirmed influenza treated in a PICU or dying in hospital. Results: Twenty severe influenza-associated cases were reported from 14 PICUs during three pre-pandemic influenza seasons (2005-2008). The median age of the patients (12 males/8 females) was 7.5 years (range 0.1-15 years). None had received vaccination against influenza. In 14 (70%) patients, the infection had been caused by influenza A and in five (25%) by influenza B; in one child (5%) the influenza type was not reported. Patients spent a median of 19 (IQR 12-38) days in the hospital and a median of 11 days (IQR 6-18 days) in the PICU; 10 (50%) needed mechanical ventilation. Most frequent diagnoses were influenza-associated pneumonia (60%), bronchitis / bronchiolitis (30%), encephalitis / encephalopathy (25%), secondary bacterial pneumonia (25%), and ARDS (25%). Eleven (55%) children had chronic underlying medical conditions, including 8 (40%) with chronic pulmonary diseases. Two influenza A- associated deaths were reported: i) an 8-year old boy with pneumococcal encephalopathy following influenza infection died from cerebral edema, ii) a 14-year-old boy with asthma bronchiale, cardiac malformation and Addison’s disease died from cardiac and respiratory failure. For nine (45%) patients, possibly permanent sequelae were reported (3 neurological, 3 pulmonary, 3 other sequelae). Conclusions: Influenza-associated pneumonia and secondary bacterial infections are relevant complications of seasonal influenza in Germany. The incidence of severe influenza cases in PICUs was relatively low. This may be either due to the weak to moderate seasonal influenza activity during the years 2005 to 2008 or due to underdiagnosis of influenza by physicians. Fifty % of the observed severe cases might have been prevented by following the recommendations for vaccination of risk groups in Germany.
Background: Gene function analysis of the obligate intracellular bacterium Chlamydia pneumoniae is hampered by the facts that this organism is inaccessible to genetic manipulations and not cultivable outside the host. The genomes of several strains have been sequenced; however, very little information is available on the gene structure and transcriptome of C. pneumoniae. Results: Using a differential RNA-sequencing approach with specific enrichment of primary transcripts, we defined the transcriptome of purified elementary bodies and reticulate bodies of C. pneumoniae strain CWL-029; 565 transcriptional start sites of annotated genes and novel transcripts were mapped. Analysis of adjacent genes for cotranscription revealed 246 polycistronic transcripts. In total, a distinct transcription start site or an affiliation to an operon could be assigned to 862 out of 1,074 annotated protein coding genes. Semi-quantitative analysis of mapped cDNA reads revealed significant differences for 288 genes in the RNA levels of genes isolated from elementary bodies and reticulate bodies. We have identified and in part confirmed 75 novel putative non-coding RNAs. The detailed map of transcription start sites at single nucleotide resolution allowed for the first time a comprehensive and saturating analysis of promoter consensus sequences in Chlamydia. Conclusions: The precise transcriptional landscape as a complement to the genome sequence will provide new insights into the organization, control and function of genes. Novel non-coding RNAs and identified common promoter motifs will help to understand gene regulation of this important human pathogen.
Background: “Negative affect” is one of the major migraine triggers. The aim of the study was to assess attentional biases for negative affective stimuli that might be related to migraine triggers in migraine patients with either few or frequent migraine and healthy controls. Methods: Thirty-three subjects with frequent migraine (FM) or with less frequent episodic migraine, and 20 healthy controls conducted two emotional Stroop tasks in the interictal period. In task 1, general affective words and in task 2, pictures of affective faces (angry, neutral, happy) were used. For each task we calculated two emotional Stroop indices. Groups were compared using one-way ANOVAs. Results: The expected attentional bias in migraine patients was not found. However, in task 2 the controls showed a significant attentional bias to negative faces, whereas the FM group showed indices near zero. Thus, the FM group responded faster to negative than to positive stimuli. The difference between the groups was statistically significant. Conclusions: The findings in the FM group may reflect a learned avoidance mechanism away from affective migraine triggers.
As pattern recognition receptor on dendritic cells (DCs), DC-SIGN binds carbohydrate structures on its pathogen ligands and essentially determines host pathogen interactions because it both skews T cell responses and enhances pathogen uptake for cis infection and/or T cell trans-infection. How these processes are initiated at the plasma membrane level is poorly understood. We now show that DC-SIGN ligation on DCs by antibodies, mannan or measles virus (MV) causes rapid activation of neutral and acid sphingomyelinases followed by accumulation of ceramides in the outer membrane leaflet. SMase activation is important in promoting DC-SIGN signaling, but also for enhancement of MV uptake into DCs. DCSIGN-dependent SMase activation induces efficient, transient recruitment of CD150, which functions both as MV uptake receptor and microbial sensor, from an intracellular Lamp-1+ storage compartment shared with acid sphingomyelinase (ASM) within a few minutes. Subsequently, CD150 is displayed at the cell surface and co-clusters with DC-SIGN. Thus, DCSIGN ligation initiates SMase-dependent formation of ceramide-enriched membrane microdomains which promote vertical segregation of CD150 from intracellular storage compartments along with ASM. Given the ability to promote receptor and signalosome co-segration into (or exclusion from) ceramide enriched microdomains which provide a favorable environment for membrane fusion, DC-SIGN-dependent SMase activation may be of general importance for modes and efficiency of pathogen uptake into DCs, and their routing to specific compartments, but also for modulating T cell responses.
Background: Successful cooperation depends on reliable identification of friends and foes. Social insects discriminate colony members (nestmates/friends) from foreign workers (non-nestmates/foes) by colony-specific, multi-component colony odors. Traditionally, complex processing in the brain has been regarded as crucial for colony recognition. Odor information is represented as spatial patterns of activity and processed in the primary olfactory neuropile, the antennal lobe (AL) of insects, which is analogous to the vertebrate olfactory bulb. Correlative evidence indicates that the spatial activity patterns reflect odor-quality, i.e., how an odor is perceived. For colony odors, alternatively, a sensory filter in the peripheral nervous system was suggested, causing specific anosmia to nestmate colony odors. Here, we investigate neuronal correlates of colony odors in the brain of a social insect to directly test whether they are anosmic to nestmate colony odors and whether spatial activity patterns in the AL can predict how odor qualities like ‘‘friend’’ and ‘‘foe’’ are attributed to colony odors. Methodology/Principal Findings: Using ant dummies that mimic natural conditions, we presented colony odors and investigated their neuronal representation in the ant Camponotus floridanus. Nestmate and non-nestmate colony odors elicited neuronal activity: In the periphery, we recorded sensory responses of olfactory receptor neurons (electroantennography), and in the brain, we measured colony odor specific spatial activity patterns in the AL (calcium imaging). Surprisingly, upon repeated stimulation with the same colony odor, spatial activity patterns were variable, and as variable as activity patterns elicited by different colony odors. Conclusions: Ants are not anosmic to nestmate colony odors. However, spatial activity patterns in the AL alone do not provide sufficient information for colony odor discrimination and this finding challenges the current notion of how odor quality is coded. Our result illustrates the enormous challenge for the nervous system to classify multi-component odors and indicates that other neuronal parameters, e.g., precise timing of neuronal activity, are likely necessary for attribution of odor quality to multi-component odors.
The diversity of species is striking, but can be far exceeded by the chemical diversity of compounds collected, produced or used by them. Here, we relate the specificity of plant-consumer interactions to chemical diversity applying a comparative network analysis to both levels. Chemical diversity was explored for interactions between tropical stingless bees and plant resins, which bees collect for nest construction and to deter predators and microbes. Resins also function as an environmental source for terpenes that serve as appeasement allomones and protection against predators when accumulated on the bees’ body surfaces. To unravel the origin of the bees’ complex chemical profiles, we investigated resin collection and the processing of resin-derived terpenes. We therefore analyzed chemical networks of tree resins, foraging networks of resin collecting bees, and their acquired chemical networks. We revealed that 113 terpenes in nests of six bee species and 83 on their body surfaces comprised a subset of the 1,117 compounds found in resins from seven tree species. Sesquiterpenes were the most variable class of terpenes. Albeit widely present in tree resins, they were only found on the body surface of some species, but entirely lacking in others. Moreover, whereas the nest profile of Tetragonula melanocephala contained sesquiterpenes, its surface profile did not. Stingless bees showed a generalized collecting behavior among resin sources, and only a hitherto undescribed species-specific ‘‘filtering’’ of resin-derived terpenes can explain the variation in chemical profiles of nests and body surfaces fromdifferent species. The tight relationship between bees and tree resins of a large variety of species elucidates why the bees’ surfaces contain a much higher chemodiversity than other hymenopterans.
Background: The World Health Organization (WHO) has recommended guidelines for a HIV drug resistance (HIVDR) survey for resource-limited countries. Eligibility criteria for patients include age below 25 years in order to focus on the prevalence of transmitted HIVDR (tHIVDR) in newly-infected individuals. Most of the participating sites across Africa have so far reported tHIVDR prevalences of below 5%. In this study we investigated whether the rate of HIVDR in patients ,25 years is representative for HIVDR in the rest of the therapy-naive population. Methods and Findings: HIVDR was determined in 88 sequentially enrolled ART-naive patients from Mwanza, Tanzania (mean age 35.4 years). Twenty patients were aged, 25 years and 68 patients were aged 25–63 years. The frequency of HIVDR in the study population was 14.8% (95%; CI 0.072–0.223) and independent of NVP-resistance induced by prevention of mother-to-child transmission programs. Patients .25 years had a significantly higher HIVDR frequency than younger patients (19.1%; 95% CI 0.095–0.28) versus 0%, P = 0.0344). In 2 out of the 16 patients with HIVDR we found traces of antiretrovirals (ARVs) in plasma. Conclusions: ART-naive patients aged over 25 years exhibited significantly higher HIVDR than younger patients. Detection of traces of ARVs in individuals with HIVDR suggests that besides transmission, undisclosed misuse of ARVs may constitute a significant factor in the generation of the observed high HIVDR rate. The current WHO tHIVDR survey that is solely focused on the transmission of HIVDR and that excludes patients over 25 years of age may therefore result in substantial underestimation of the prevalence of HIVDR in the therapy-naive population. Similar studies should be performed also in other areas to test whether the so far reported optimistic picture of low HIVDR prevalence in young individuals is really representative for the rest of the ART-naive HIV-infected population.
Background: Inactivation of the p53 pathway that controls cell cycle progression, apoptosis and senescence, has been proposed to occur in virtually all human tumors and p53 is the protein most frequently mutated in human cancer. However, the mutational status of p53 in melanoma is still controversial; to clarify this notion we analysed the largest series of melanoma samples reported to date. Methodology/Principal Findings: Immunohistochemical analysis of more than 180 melanoma specimens demonstrated that high levels of p53 are expressed in the vast majority of cases. Subsequent sequencing of the p53 exons 5–8, however, revealed only in one case the presence of a mutation. Nevertheless, by means of two different p53 reporter constructs we demonstrate transcriptional inactivity of wild type p53 in 6 out of 10 melanoma cell lines; the 4 other p53 wild type melanoma cell lines exhibit p53 reporter gene activity, which can be blocked by shRNA knock down of p53. Conclusions/Significance: In melanomas expressing high levels of wild type p53 this tumor suppressor is frequently inactivated at transcriptional level.
Organic farming is one of the most successful agri-environmental schemes, as humans benefit from high quality food, farmers from higher prices for their products and it often successfully protects biodiversity. However there is little knowledge if organic farming also increases ecosystem services like pest control. We assessed 30 triticale fields (15 organic vs. 15 conventional) and recorded vascular plants, pollinators, aphids and their predators. Further, five conventional fields which were treated with insecticides were compared with 10 non-treated conventional fields. Organic fields had five times higher plant species richness and about twenty times higher pollinator species richness compared to conventional fields. Abundance of pollinators was even more than one-hundred times higher on organic fields. In contrast, the abundance of cereal aphids was five times lower in organic fields, while predator abundances were three times higher and predator-prey ratios twenty times higher in organic fields, indicating a significantly higher potential for biological pest control in organic fields. Insecticide treatment in conventional fields had only a short-term effect on aphid densities while later in the season aphid abundances were even higher and predator abundances lower in treated compared to untreated conventional fields. Our data indicate that insecticide treatment kept aphid predators at low abundances throughout the season, thereby significantly reducing top-down control of aphid populations. Plant and pollinator species richness as well as predator abundances and predator-prey ratios were higher at field edges compared to field centres, highlighting the importance of field edges for ecosystem services. In conclusion organic farming increases biodiversity, including important functional groups like plants, pollinators and predators which enhance natural pest control. Preventative insecticide application in conventional fields has only short-term effects on aphid densities but long-term negative effects on biological pest control. Therefore conventional farmers should restrict insecticide applications to situations where thresholds for pest densities are reached.
Understanding a complex network’s structure holds the key to understanding its function. The physics community has contributed a multitude of methods and analyses to this cross-disciplinary endeavor. Structural features exist on both the microscopic level, resulting from differences between single node properties, and the mesoscopic level resulting from properties shared by groups of nodes. Disentangling the determinants of network structure on these different scales has remained a major, and so far unsolved, challenge. Here we show how multiscale generative probabilistic exponential random graph models combined with efficient, distributive message-passing inference techniques can be used to achieve this separation of scales, leading to improved detection accuracy of latent classes as demonstrated on benchmark problems. It sheds new light on the statistical significance of motif-distributions in neural networks and improves the link-prediction accuracy as exemplified for gene-disease associations in the highly consequential Online Mendelian Inheritance in Man database.
Honeybee foragers frequently fly several kilometres to and from vital resources, and communicate those locations to their nest mates by a symbolic dance language. Research has shown that they achieve this feat by memorizing landmarks and the skyline panorama, using the sun and polarized skylight as compasses and by integrating their outbound flight paths. In order to investigate the capacity of the honeybees’ homing abilities, we artificially displaced foragers to novel release spots at various distances up to 13 km in the four cardinal directions. Returning bees were individually registered by a radio frequency identification (RFID) system at the hive entrance. We found that homing rate, homing speed and the maximum homing distance depend on the release direction. Bees released in the east were more likely to find their way back home, and returned faster than bees released in any other direction, due to the familiarity of global landmarks seen from the hive. Our findings suggest that such large scale homing is facilitated by global landmarks acting as beacons, and possibly the entire skyline panorama.
Control of host cell death is of paramount importance for the survival and replication of obligate intracellular bacteria. Among these, human pathogenic Chlamydia induces the inhibition of apoptosis in a variety of different host cells by directly interfering with cell death signaling. However, the evolutionary conservation of cell death regulation has not been investigated in the order Chlamydiales, which also includes Chlamydia-like organisms with a broader host spectrum. Here, we investigated the apoptotic response of human cells infected with the Chlamydia-like organism Simkania negevensis (Sn). Simkania infected cells exhibited strong resistance to apoptosis induced by intrinsic stress or by the activation of cell death receptors. Apoptotic signaling was blocked upstream of mitochondria since Bax translocation, Bax and Bak oligomerisation and cytochrome c release were absent in these cells. Infected cells turned on pro-survival pathways like cellular Inhibitor of Apoptosis Protein 2 (cIAP-2) and the Akt/PI3K pathway. Blocking any of these inhibitory pathways sensitized infected host cell towards apoptosis induction, demonstrating their role in infection-induced apoptosis resistance. Our data support the hypothesis of evolutionary conserved signaling pathways to apoptosis resistance as common denominators in the order Chlamydiales.
Acellular Pertussis Booster in Adolescents Induces Th1 and Memory CD8+ T Cell Immune Response
(2011)
In a number of countries, whole cell pertussis vaccines (wcP) were replaced by acellular vaccines (aP) due to an improved reactogenicity profile. Pertussis immunization leads to specific antibody production with the help of CD4+ T cells. In earlier studies in infants and young children, wcP vaccines selectively induced a Th1 dominated immune response, whereas aP vaccines led to a Th2 biased response. To obtain data on Th1 or Th2 dominance of the immune response in adolescents receiving an aP booster immunization after a wcP or aP primary immunization, we analyzed the concentration of Th1 (IL-2, TNF-a, INF-c) and Th2 (IL-4, IL-5, IL-10) cytokines in supernatants of lymphocyte cultures specifically stimulated with pertussis antigens. We also investigated the presence of cytotoxic T cell responses against the facultative intracellular bacterium Bordetella pertussis by quantifying pertussis-specific CD8+ T cell activation following the aP booster immunization. Here we show that the adolescent aP booster vaccination predominantly leads to a Th1 immune response based on IFNgamma secretion upon stimulation with pertussis antigen, irrespective of a prior whole cell or acellular primary vaccination. The vaccination also induces an increase in peripheral CD8+CD69+ activated pertussis-specific memory T cells four weeks after vaccination. The Th1 bias of this immune response could play a role for the decreased local reactogenicity of this adolescent aP booster immunization when compared to the preceding childhood acellular pertussis booster. Pertussis-specific CD8+ memory T cells may contribute to protection against clinical pertussis.
No abstract avDendritic cells (DC) are the most important antigen presenting cells and play a pivotal role in host immunity to infectious agents by acting as a bridge between the innate and adaptive immune systems. Monocyte-derived immature DCs (iDC) were infected with viable resting conidia of Aspergillus fumigatus (Af293) for 12 hours at an MOI of 5; cells were sampled every three hours. RNA was extracted from both organisms at each time point and hybridised to microarrays. iDC cell death increased at 6 h in the presence of A. fumigatus which coincided with fungal germ tube emergence; .80% of conidia were associated with iDC. Over the time course A. fumigatus differentially regulated 210 genes, FunCat analysis indicated significant up-regulation of genes involved in fermentation, drug transport, pathogenesis and response to oxidative stress. Genes related to cytotoxicity were differentially regulated but the gliotoxin biosynthesis genes were down regulated over the time course, while Aspf1 was up-regulated at 9 h and 12 h. There was an up-regulation of genes in the subtelomeric regions of the genome as the interaction progressed. The genes up-regulated by iDC in the presence of A. fumigatus indicated that they were producing a pro-inflammatory response which was consistent with previous transcriptome studies of iDC interacting with A. fumigatus germ tubes. This study shows that A. fumigatus adapts to phagocytosis by iDCs by utilising genes that allow it to survive the interaction rather than just up-regulation of specific virulence genes.
Background: Acquisition of information about food sources is essential for animals that forage collectively like social insects. Foragers deliver two commodities to the nest, food and information, and they may favor the delivery of one at the expenses of the other. We predict that information needs should be particularly high at the beginning of foraging: the decision to return faster to the nest will motivate a grass-cutting ant worker to reduce its loading time, and so to leave the source with a partial load. Principal Findings: Field results showed that at the initial foraging phase, most grass-cutting ant foragers (Acromyrmex heyeri) returned unladen to the nest, and experienced head-on encounters with outgoing workers. Ant encounters were not simply collisions in a probabilistic sense: outgoing workers contacted in average 70% of the returning foragers at the initial foraging phase, and only 20% at the established phase. At the initial foraging phase, workers cut fragments that were shorter, narrower, lighter and tenderer than those harvested at the established one. Foragers walked at the initial phase significantly faster than expected for the observed temperatures, yet not at the established phase. Moreover, when controlling for differences in the fragment-size carried, workers still walked faster at the initial phase. Despite the higher speed, their individual transport rate of vegetable tissue was lower than that of similarly-sized workers foraging later at the same patch. Conclusions/Significance: At the initial foraging phase, workers compromised their individual transport rates of material in order to return faster to the colony. We suggest that the observed flexible cutting rules and the selection of partial loads at the beginning of foraging are driven by the need of information transfer, crucial for the establishment and maintenance of a foraging process to monopolize a discovered resource.
Background: Patterns that arise from an ecological process can be driven as much from the landscape over which the process is run as it is by some intrinsic properties of the process itself. The disentanglement of these effects is aided if it possible to run models of the process over artificial landscapes with controllable spatial properties. A number of different methods for the generation of so-called ‘neutral landscapes’ have been developed to provide just such a tool. Of these methods, a particular class that simulate fractional Brownian motion have shown particular promise. The existing methods of simulating fractional Brownian motion suffer from a number of problems however: they are often not easily generalisable to an arbitrary number of dimensions and produce outputs that can exhibit some undesirable artefacts. Methodology: We describe here an updated algorithm for the generation of neutral landscapes by fractional Brownian motion that do not display such undesirable properties. Using Monte Carlo simulation we assess the anisotropic properties of landscapes generated using the new algorithm described in this paper and compare it against a popular benchmark algorithm. Conclusion/Significance: The results show that the existing algorithm creates landscapes with values strongly correlated in the diagonal direction and that the new algorithm presented here corrects this artefact. A number of extensions of the algorithm described here are also highlighted: we describe how the algorithm can be employed to generate landscapes that display different properties in different dimensions and how they can be combined with an environmental gradient to produce landscapes that combine environmental variation at the local and macro scales.
A Candidate Approach Implicates the Secreted Salmonella Effector Protein SpvB in P-Body Disassembly
(2011)
P-bodies are dynamic aggregates of RNA and proteins involved in several post-transcriptional regulation processes. Pbodies have been shown to play important roles in regulating viral infection, whereas their interplay with bacterial pathogens, specifically intracellular bacteria that extensively manipulate host cell pathways, remains unknown. Here, we report that Salmonella infection induces P-body disassembly in a cell type-specific manner, and independently of previously characterized pathways such as inhibition of host cell RNA synthesis or microRNA-mediated gene silencing. We show that the Salmonella-induced P-body disassembly depends on the activation of the SPI-2 encoded type 3 secretion system, and that the secreted effector protein SpvB plays a major role in this process. P-body disruption is also induced by the related pathogen, Shigella flexneri, arguing that this might be a new mechanism by which intracellular bacterial pathogens subvert host cell function.
Fanconi Anemia Core Complex Gene Promoters Harbor Conserved Transcription Regulatory Elements
(2011)
The Fanconi anemia (FA) gene family is a recent addition to the complex network of proteins that respond to and repair certain types of DNA damage in the human genome. Since little is known about the regulation of this novel group of genes at the DNA level, we characterized the promoters of the eight genes (FANCA, B, C, E, F, G, L and M) that compose the FA core complex. The promoters of these genes show the characteristic attributes of housekeeping genes, such as a high GC content and CpG islands, a lack of TATA boxes and a low conservation. The promoters functioned in a monodirectional way and were, in their most active regions, comparable in strength to the SV40 promoter in our reporter plasmids. They were also marked by a distinctive transcriptional start site (TSS). In the 59 region of each promoter, we identified a region that was able to negatively regulate the promoter activity in HeLa and HEK 293 cells in isolation. The central and 39 regions of the promoter sequences harbor binding sites for several common and rare transcription factors, including STAT, SMAD, E2F, AP1 and YY1, which indicates that there may be cross-connections to several established regulatory pathways. Electrophoretic mobility shift assays and siRNA experiments confirmed the shared regulatory responses between the prominent members of the TGF-b and JAK/STAT pathways and members of the FA core complex. Although the promoters are not well conserved, they share region and sequence specific regulatory motifs and transcription factor binding sites (TBFs), and we identified a bi-partite nature to these promoters. These results support a hypothesis based on the co-evolution of the FA core complex genes that was expanded to include their promoters.
In the initial phase of development of fish embryos, a prominent and critical event is the midblastula transition (MBT). Before MBT cell cycle is rapid, highly synchronous and zygotic gene transcription is turned off. Only during MBT the cell cycle desynchronizes and transcription is activated. Multiple mechanisms, primarily the nucleocytoplasmic ratio, are supposed to control MBT activation. Unexpectedly, we find in the small teleost fish medaka (Oryzias latipes) that at very early stages, well before midblastula, cell division becomes asynchronous and cell volumes diverge. Furthermore, zygotic transcription is extensively activated already after the 64-cell stage. Thus, at least in medaka, the transition from maternal to zygotic transcription is uncoupled from the midblastula stage and not solely controlled by the nucleocytoplasmic ratio.
The synaptonemal complex (SC) is a proteinaceous, meiosis-specific structure that is highly conserved in evolution. During meiosis, the SC mediates synapsis of homologous chromosomes. It is essential for proper recombination and segregation of homologous chromosomes, and therefore for genome haploidization. Mutations in human SC genes can cause infertility. In order to gain a better understanding of the process of SC assembly in a model system that would be relevant for humans, we are investigating meiosis in mice. Here, we report on a newly identified component of the murine SC, which we named SYCE3. SYCE3 is strongly conserved among mammals and localizes to the central element (CE) of the SC. By generating a Syce3 knockout mouse, we found that SYCE3 is required for fertility in both sexes. Loss of SYCE3 blocks synapsis initiation and results in meiotic arrest. In the absence of SYCE3, initiation of meiotic recombination appears to be normal, but its progression is severely impaired resulting in complete absence of MLH1 foci, which are presumed markers of crossovers in wild-type meiocytes. In the process of SC assembly, SYCE3 is required downstream of transverse filament protein SYCP1, but upstream of the other previously described CE–specific proteins. We conclude that SYCE3 enables chromosome loading of the other CE–specific proteins, which in turn would promote synapsis between homologous chromosomes.
Background: We evaluated the effect of insulin stimulation and dietary changes on myocardial, skeletal muscle and brain [18F]-fluorodeoxyglucose (FDG) kinetics and uptake in vivo in intact mice. Methods: Mice were anesthetized with isoflurane and imaged under different conditions: non-fasted (n = 7; "controls"), non-fasted with insulin (2 IU/kg body weight) injected subcutaneously immediately prior to FDG (n = 6), fasted (n = 5), and fasted with insulin injection (n = 5). A 60-min small-animal PET with serial blood sampling and kinetic modeling was performed. Results: We found comparable FDG standardized uptake values (SUVs) in myocardium in the non-fasted controls and non-fasted-insulin injected group (SUV 45-60 min, 9.58 ± 1.62 vs. 9.98 ± 2.44; p = 0.74), a lower myocardial SUV was noted in the fasted group (3.48 ± 1.73; p < 0.001). In contrast, the FDG uptake rate constant (Ki) for myocardium increased significantly by 47% in non-fasted mice by insulin (13.4 ± 3.9 ml/min/100 g vs. 19.8 ± 3.3 ml/min/100 g; p = 0.030); in fasted mice, a lower myocardial Ki as compared to controls was observed (3.3 ± 1.9 ml/min/100 g; p < 0.001). Skeletal muscle SUVs and Ki values were increased by insulin independent of dietary state, whereas in the brain, those parameters were not influenced by fasting or administration of insulin. Fasting led to a reduction in glucose metabolic rate in the myocardium (19.41 ± 5.39 vs. 3.26 ± 1.97 mg/min/100 g; p < 0.001), the skeletal muscle (1.06 ± 0.34 vs. 0.34 ± 0.08 mg/min/100 g; p = 0.001) but not the brain (3.21 ± 0.53 vs. 2.85 ± 0.25 mg/min/100 g; p = 0.19). Conclusions: Changes in organ SUVs, uptake rate constants and metabolic rates induced by fasting and insulin administration as observed in intact mice by small-animal PET imaging are consistent with those observed in isolated heart/muscle preparations and, more importantly, in vivo studies in larger animals and in humans. When assessing the effect of insulin on the myocardial glucose metabolism of non-fasted mice, it is not sufficient to just calculate the SUV - dynamic imaging with kinetic modeling is necessary.
Background: Genome wide association studies reported two single nucleotide polymorphisms in ANK3 (rs9804190 and rs10994336) as independent genetic risk factors for bipolar disorder. Another SNP in ANK3 (rs10761482) was associated with schizophrenia in a large European sample. Within the debate on common susceptibility genes for schizophrenia and bipolar disorder, we tried to investigate common findings by analyzing association of ANK3 with schizophrenia, bipolar disorder and unipolar depression. Methods: We genotyped three single nucleotide polymorphisms (SNPs) in ANK3 (rs9804190, rs10994336, and rs10761482) in a case-control sample of German descent including 920 patients with schizophrenia, 400 with bipolar affective disorder, 220 patients with unipolar depression according to ICD 10 and 480 healthy controls. Sample was further differentiated according to Leonhard’s classification featuring disease entities with specific combination of bipolar and psychotic syndromes. Results: We found no association of rs9804190 and rs10994336 with bipolar disorder, unipolar depression or schizophrenia. In contrast to previous findings rs10761482 was associated with bipolar disorder (p = 0.015) but not with schizophrenia or unipolar depression. We observed no association with disease entities according to Leonhard’s classification. Conclusion: Our results support a specific genetic contribution of ANK3 to bipolar disorder though we failed to replicate findings for schizophrenia. We cannot confirm ANK3 as a common risk factor for different diseases.
Varicella-zoster virus infections in immunocompromised patients - a single centre 6-years analysis
(2011)
Background: Infection with varicella-zoster virus (VZV) contemporaneously with malignant disease or immunosuppression represents a particular challenge and requires individualized decisions and treatment. Although the increasing use of varicella-vaccines in the general population and rapid initiation of VZVimmunoglobulins and acyclovir in case of exposure has been beneficial for some patients, immunocompromised individuals are still at risk for unfavourable courses. Methods: In this single center, 6-year analysis we review incidence, hospitalization and complication rates of VZVinfections in our center and compare them to published data. Furthermore, we report three instructive cases. Results: Hospitalization rate of referred children with VZV-infections was 45%, among these 17% with malignancies and 9% under immunosuppressive therapy. Rate of complications was not elevated in these two high-risk cohorts, but one ALL-patient died due to VZV-related complications. We report one 4-year old boy with initial diagnosis of acute lymphoblastic leukemia who showed a rapidly fatal outcome of his simultaneous varicella-infection, one 1.8-year old boy with an identical situation but a mild course of his disease, and an 8.5-year old boy with a steroiddependent nephrotic syndrome. This boy developed severe hepatic involvement during his varicella-infection but responded to immediate withdrawl of steroids and administration of acyclovir plus single-dose cidofovir after nonresponse to acyclovir after 48 h. Conclusion: Our data show that patients with malignant diseases or immunosuppressive therapy should be hospitalized and treated immediately with antiviral agents. Despite these measures the course of VZV-infections can be highly variable in these patients. We discuss aids to individual decision-making for these difficult situations.
Background: Neuropathic pain must be correctly diagnosed for optimal treatment. The questionnaire named Neuropathic Pain Symptom Inventory (NPSI) was developed in its original French version to evaluate the different symptoms of neuropathic pain. We hypothesized that the NPSI might also be used to differentiate neuropathic from non-neuropathic pain. Methods: We translated the NPSI into German using a standard forward-backward translation and administered it in a case-control design to patients with neuropathic (n = 68) and non-neuropathic pain (headache and osteoarthritis, n = 169) to validate it and to analyze its discriminant properties, its sensitivity to change, and to detect neuropathic pain subgroups with distinct profiles. Results: Using a sum score (the NPSI-G score), we found sensitivity to change (r between 0.37 and 0.5 for pain items of the graded chronic pain scale) and could distinguish between neuropathic and other pain on a group basis, but not for individual patients. Post hoc development of a discriminant score with optimized diagnostic properties to distinguish neuropathic pain from non-neuropathic pain resulted in an instrument with high sensitivity (91%) and acceptable specificity (70%). We detected six different pain profiles in the patient group with neuropathic pain; three profiles were found to be distinct. Conclusions: The NPSI-G potentially combines the properties of a diagnostic tool and an instrument to identify subtypes of neuropathic pain.
Background: Specific cell targeting is an important, yet unsolved problem in bacteria-based therapeutic applications, like tumor or gene therapy. Here, we describe the construction of a novel, internalin A and B (InlAB)-deficient Listeria monocytogenes strain (Lm-spa+), which expresses protein A of Staphylococcus aureus (SPA) and anchors SPA in the correct orientation on the bacterial cell surface. Results: This listerial strain efficiently binds antibodies allowing specific interaction of the bacterium with the target recognized by the antibody. Binding of Trastuzumab (Herceptin®) or Cetuximab (Erbitux®) to Lm-spa+, two clinically approved monoclonal antibodies directed against HER2/neu and EGFR/HER1, respectively, triggers InlABindependent internalization into non-phagocytic cancer cell lines overexpressing the respective receptors. Internalization, subsequent escape into the host cell cytosol and intracellular replication of these bacteria are as efficient as of the corresponding InlAB-positive, SPA-negative parental strain. This specific antibody/receptormediated internalization of Lm-spa+ is shown in the murine 4T1 tumor cell line, the isogenic 4T1-HER2 cell line as well as the human cancer cell lines SK-BR-3 and SK-OV-3. Importantly, this targeting approach is applicable in a xenograft mouse tumor model after crosslinking the antibody to SPA on the listerial cell surface. Conclusions: Binding of receptor-specific antibodies to SPA-expressing L. monocytogenes may represent a promising approach to target L. monocytogenes to host cells expressing specific receptors triggering internalization.
Background: In principle, the elimination of malignancies by oncolytic virotherapy could proceed by different mechanisms - e.g. tumor cell specific oncolysis, destruction of the tumor vasculature or an anti-tumoral immunological response. In this study, we analyzed the contribution of these factors to elucidate the responsible mechanism for regression of human breast tumor xenografts upon colonization with an attenuated vaccinia virus (VACV). Methods: Breast tumor xenografts were analyzed 6 weeks post VACV infection (p.i.; regression phase) by immunohistochemistry and mouse-specific expression arrays. Viral-mediated oncolysis was determined by tumor growth analysis combined with microscopic studies of intratumoral virus distribution. The tumor vasculature was morphologically characterized by diameter and density measurements and vessel functionality was analyzed by lectin perfusion and extravasation studies. Immunological aspects of viral-mediated tumor regression were studied in either immune-deficient mouse strains (T-, B-, NK-cell-deficient) or upon cyclophosphamide-induced immunosuppression (MHCII+-cell depletion) in nude mice. Results: Late stage VACV-infected breast tumors showed extensive necrosis, which was highly specific to cancer cells. The tumor vasculature in infected tumor areas remained functional and the endothelial cells were not infected. However, viral colonization triggers hyperpermeability and dilatation of the tumor vessels, which resembled the activated endothelium in wounded tissue. Moreover, we demonstrated an increased expression of genes involved in leukocyte-endothelial cell interaction in VACV-infected tumors, which orchestrate perivascular inflammatory cell infiltration. The immunohistochemical analysis of infected tumors displayed intense infiltration of MHCII-positive cells and colocalization of tumor vessels with MHCII+/CD31+ vascular leukocytes. However, GI-101A tumor growth analysis upon VACV-infection in either immunosuppressed nude mice (MHCII+-cell depleted) or in immune-deficient mouse strains (T-, B-, NK-cell-deficient) revealed that neither MHCII-positive immune cells nor T-, B-, or NK cells contributed significantly to VACV-mediated tumor regression. In contrast, tumors of immunosuppressed mice showed enhanced viral spreading and tumor necrosis. Conclusions: Taken together, these results indicate that VACV-mediated oncolysis is the primary mechanism of tumor shrinkage in the late regression phase. Neither the destruction of the tumor vasculature nor the massive VACV-mediated intratumoral inflammation was a prerequisite for tumor regression. We propose that approaches to enhance viral replication and spread within the tumor microenvironment should improve therapeutical outcome.
181 names of Hieracium species going back to original herbarium material of Alexis Jordan or Alexandre Boreau are lectotypified, 27 are neotypified. The study is based on herbarium specimens of the Université Catholique de Lyon (LY) and Ville d’Angers (ANG), Martrin-Donos’s herbarium at the Institut Botanique de Montpellier (MPUTarn) and Arvet-Touvet’s herbarium at the Musée d’Histoire Naturelle de Grenoble (GRM-AT). The type specimens are illustrated by photographs of the entire herbarium sheets with some detail views of flower heads and leaves. Usual nomenclatural synonyms are given for each taxon.