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1.  Crystallization of the novel S-adenosyl-l-methionine-dependent C-methyltransferase CouO from Streptomyces rishiriensis and preliminary diffraction data analysis 
Recombinant Q9F8T9 protein from Streptomyces rishiriensis (CouO), an S-­adenosyl-l-methionine-dependent C-methyltransferase, has been successfully cloned, expressed and purified.
Recombinant Q9F8T9 protein from Streptomyces rishiriensis (CouO), an S-­adenosyl-l-methionine-dependent C-methyltransferase, has been successfully cloned, expressed and purified. CouO was crystallized from a single condition in the Morpheus crystallization screen. A vitrified crystal diffracted to 2.05 Å resolution and belonged to space group P21, with unit-cell parameters a = 33.02, b = 82.87, c = 76.77 Å, β = 96.93°.
PMCID: PMC3370914  PMID: 22684074
CouO; methyltransferases; S-adenosyl-l-methionine; Streptomyces rishiriensis
2.  The virus's tooth: cyanophages affect an African flamingo population in a bottom-up cascade 
The ISME Journal  2014;8(6):1346-1351.
Trophic cascade effects occur when a food web is disrupted by loss or significant reduction of one or more of its members. In East African Rift Valley lakes, the Lesser Flamingo is on top of a short food chain. At irregular intervals, the dominance of their most important food source, the cyanobacterium Arthrospira fusiformis, is interrupted. Bacteriophages are known as potentially controlling photoautotrophic bacterioplankton. In Lake Nakuru (Kenya), we found the highest abundance of suspended viruses ever recorded in a natural aquatic system. We document that cyanophage infection and the related breakdown of A. fusiformis biomass led to a dramatic reduction in flamingo abundance. This documents that virus infection at the very base of a food chain can affect, in a bottom-up cascade, the distribution of end consumers. We anticipate this as an important example for virus-mediated cascading effects, potentially occurring also in various other aquatic food webs.
PMCID: PMC4030235  PMID: 24430484
virus; bottom-up cascade; cyanophage; flamingo; food chain; soda lake
3.  Crystal Structure of an (R)-Selective ω-Transaminase from Aspergillus terreus 
PLoS ONE  2014;9(1):e87350.
Chiral amines are important building blocks for the synthesis of pharmaceutical products, fine chemicals, and agrochemicals. ω-Transaminases are able to directly synthesize enantiopure chiral amines by catalysing the transfer of an amino group from a primary amino donor to a carbonyl acceptor with pyridoxal 5′-phosphate (PLP) as cofactor. In nature, (S)-selective amine transaminases are more abundant than the (R)-selective enzymes, and therefore more information concerning their structures is available. Here, we present the crystal structure of an (R)-ω-transaminase from Aspergillus terreus determined by X-ray crystallography at a resolution of 1.6 Å. The structure of the protein is a homodimer that displays the typical class IV fold of PLP-dependent aminotransferases. The PLP-cofactor observed in the structure is present in two states (i) covalently bound to the active site lysine (the internal aldimine form) and (ii) as substrate/product adduct (the external aldimine form) and free lysine. Docking studies revealed that (R)-transaminases follow a dual binding mode, in which the large binding pocket can harbour the bulky substituent of the amine or ketone substrate and the α-carboxylate of pyruvate or amino acids, and the small binding pocket accommodates the smaller substituent.
PMCID: PMC3907554  PMID: 24498081
4.  The 2.5 Å Structure of the Enterococcus Conjugation Protein TraM resembles VirB8 Type IV Secretion Proteins* 
The Journal of Biological Chemistry  2012;288(3):2018-2028.
Background: Conjugative plasmid transfer is the prevalent means for spreading antibiotic resistance genes among bacteria.
Results: Surface exposure of transfer protein TraM from the Gram-positive (G+) plasmid pIP501 was confirmed, and its crystal structure was solved.
Conclusion: Structural relations to type IV secretion (T4S) proteins provide a novel classification scheme.
Significance: The novel classification will help elucidate structure-function relationships in G+ T4S systems.
Conjugative plasmid transfer is the most important means of spreading antibiotic resistance and virulence genes among bacteria and therefore presents a serious threat to human health. The process requires direct cell-cell contact made possible by a multiprotein complex that spans cellular membranes and serves as a channel for macromolecular secretion. Thus far, well studied conjugative type IV secretion systems (T4SS) are of Gram-negative (G−) origin. Although many medically relevant pathogens (e.g., enterococci, staphylococci, and streptococci) are Gram-positive (G+), their conjugation systems have received little attention. This study provides structural information for the transfer protein TraM of the G+ broad host range Enterococcus conjugative plasmid pIP501. Immunolocalization demonstrated that the protein localizes to the cell wall. We then used opsonophagocytosis as a novel tool to verify that TraM was exposed on the cell surface. In these assays, antibodies generated to TraM recruited macrophages and enabled killing of pIP501 harboring Enteroccocus faecalis cells. The crystal structure of the C-terminal, surface-exposed domain of TraM was determined to 2.5 Å resolution. The structure, molecular dynamics, and cross-linking studies indicated that a TraM trimer acts as the biological unit. Despite the absence of sequence-based similarity, TraM unexpectedly displayed a fold similar to the T4SS VirB8 proteins from Agrobacterium tumefaciens and Brucella suis (G−) and to the transfer protein TcpC from Clostridium perfringens plasmid pCW3 (G+). Based on the alignments of secondary structure elements of VirB8-like proteins from mobile genetic elements and chromosomally encoded T4SS from G+ and G− bacteria, we propose a new classification scheme of VirB8-like proteins.
PMCID: PMC3548508  PMID: 23188825
Antibiotic Resistance; Bacterial Conjugation; Bacterial Pathogenesis; Crystal Structure; X-ray Crystallography; Conjugative Plasmid; Enteroccucus; Gram-positive; Type IV Secretion System; pIP501
5.  Quality of life after pertrochanteric femoral fractures treated with a gamma nail: a single center study of 62 patients 
Intramedullary nailing of pertrochanteric femoral fractures has grown in popularity over the past 2 decades likely because this procedure is associated with a low risk for postoperative morbidity and a fast recovery of function. The evaluation of outcomes associated with pertrochanteric nailing has mainly been based on objective measures. The purpose of the present study is to correlate patients’ health-related quality of life results after intramedullary nailing of pertrochanteric fractures with objective outcome measures.
We conducted a single-center study including 62 patients (mean age 80 ± 10 years) with pertrochanteric fractures treated with a Gamma 3 Nail. Health related quality of life was measured using the Short Form-36. These results were compared to both US and Austrian age and sex-adjusted population norms. The objective outcome measures studied at one year postoperatively included Harris Hip Score, range of motion, leg length, body mass index, neck-shaft angle and grade of osteoarthritis.
According to the Harris Hip Score 43 patients (67%) had excellent or good results. There was no significant difference in the average neck-shaft angle comparing affected hip to non-affected hip at 12 months postoperatively. The average osteoarthritis score, for both the injured and uninjured hip, did not differ significantly. We found significant differences between the bodily pain, social functioning and mental health subscales and two summary scores of the Short-Form 36 in comparison to Austrian population norms. Complication rate was 8%.
The results of this study confirm that intramedullary nailing with the use of a Gamma Nail is a safe treatment option for stable and unstable pertrochanteric fractures. Despite good functional and radiographic results we noticed a substantial fall off in patients’ quality of life up to 12 months after operation.
PMCID: PMC3518197  PMID: 23110648
6.  The structure of monoacylglycerol lipase from Bacillus sp. H257 reveals unexpected conservation of the cap architecture between bacterial and human enzymes 
Biochimica et Biophysica Acta  2012;1821(7):1012-1021.
Monoacylglycerol lipases (MGLs) catalyse the hydrolysis of monoacylglycerol into free fatty acid and glycerol. MGLs have been identified throughout all genera of life and have adopted different substrate specificities depending on their physiological role. In humans, MGL plays an integral part in lipid metabolism affecting energy homeostasis, signalling processes and cancer cell progression. In bacteria, MGLs degrade short-chain monoacylglycerols which are otherwise toxic to the organism. We report the crystal structures of MGL from the bacterium Bacillus sp. H257 (bMGL) in its free form at 1.2 Å and in complex with phenylmethylsulfonyl fluoride at 1.8 Å resolution. In both structures, bMGL adopts an α/β hydrolase fold with a cap in an open conformation. Access to the active site residues, which were unambiguously identified from the protein structure, is facilitated by two different channels. The larger channel constitutes the highly hydrophobic substrate binding pocket with enough room to accommodate monoacylglycerol. The other channel is rather small and resembles the proposed glycerol exit hole in human MGL. Molecular dynamics simulation of bMGL yielded open and closed states of the entrance channel and the glycerol exit hole. Despite differences in the number of residues, secondary structure elements, and low sequence identity in the cap region, this first structure of a bacterial MGL reveals striking structural conservation of the overall cap architecture in comparison with human MGL. Thus it provides insight into the structural conservation of the cap amongst MGLs throughout evolution and provides a framework for rationalising substrate specificities in each organism.
► First crystal structure of monoacylglycerol lipase from bacterial species. ► Small angle X-ray scattering shows that the protein is a monomer in solution. ► A large hydrophobic channel enables access of the substrate to the active site. ► Molecular dynamic simulations reveal open and closed states of the cap region. ► The cap architecture is conserved on a structural but not on a sequence level.
PMCID: PMC3790968  PMID: 22561231
MGL, monoacylglycerol lipase; bMGL, monoacylglycerol lipase from Bacillus sp. H257; hMGL, human monoacylglycerol lipase; PMSF, phenylmethylsulfonyl fluoride; SAXS, small angle X-ray scattering; RMSD, root mean square deviation; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; Monoacylglycerol lipase; Open conformation; Evolutionary conservation; X-ray crystallography; Molecular dynamics simulation; Small-angle X-ray scattering
7.  Genetic polymorphisms of 5-HTT and DAT but not COMT differentially affect verbal and visuospatial working memory functioning 
Working memory deficits are found in different psychiatric populations and are most pronounced in schizophrenia. There is preliminary evidence from pharmacological studies that the verbal and visuospatial subcomponents of working memory are subject to differential neurotransmitter modulation. Here, we investigated the impact of well-known polymorphisms of the dopamine transporter gene (SLC6A3, DAT) and the catechol-O-methyl-transferase gene (COMT) as well as the serotonin transporter gene (SLC6A4, 5-HTT) on these specific working memory subcomponents in a mixed sample of patients and healthy individuals. Twenty healthy subjects and 80 patients diagnosed with schizophrenia, bipolar I disorder, or obsessive-compulsive disorder underwent genotyping for the DAT variable number of tandem repeats (VNTR), the COMT val/met-, and the 5-HTT promoter length polymorphism (5-HTTLPR) and neuropsychological testing using a battery of well-characterized, brain circuit–specific working memory tasks. DAT genotype revealed a significant and selective effect on visuospatial working memory, while there was no effect on verbal working memory functioning. 5-HTT genotype, by contrast, exerted a significant and selective effect on verbal working memory task performance. COMT genotype did not show any influence on either working memory domain. The results of the present study provide evidence for a differential impact of genetic polymorphisms of the dopaminergic and serotonergic systems on verbal and visuospatial working memory functioning. Together with prior evidence suggesting the existence of subgroups of schizophrenia patients exhibiting isolated deficits in only one working memory domain, this finding further supports the idea of endophenotypically and pathophysiologically distinct subgroups of schizophrenia with implications for personalized therapeutic approaches.
PMCID: PMC3491187  PMID: 22454241
Genetics; Schizophrenia; Bipolar disorder; Working memory; Endophenotype; Neuroimaging
8.  A systematic experimental neuropsychological investigation of the functional integrity of working memory circuits in major depression 
Verbal and visuospatial working memory (WM) impairment is a well-documented finding in psychiatric patients suffering from major psychoses such as schizophrenia or bipolar affective disorder. However, in major depression (MDD) the literature on the presence and the extent of WM deficits is inconsistent. The use of a multitude of different WM tasks most of which lack process-specificity may have contributed to these inconsistencies. Eighteen MDD patients and 18 healthy controls matched with regard to age, gender and education were tested using process- and circuit-specific WM tasks for which clear brain-behaviour relationships had been established in prior functional neuroimaging studies. Patients suffering from acute MDD showed a selective impairment in articulatory rehearsal of verbal information in working memory. By contrast, visuospatial WM was unimpaired in this sample. There were no significant correlations between symptom severity and WM performance. These data indicate a dysfunction of a specific verbal WM system in acutely ill patients with MDD. As the observed functional deficit did not correlate with different symptom scores, further, longitudinal studies are required to clarify whether and how this deficit is related to illness acuity and clinical state of MDD patients.
PMCID: PMC3071944  PMID: 21063718
Major depression; Neurocognition; Cognitive endophenotype; Psychosis; Brain imaging
9.  Common Variation in ISL1 Confers Genetic Susceptibility for Human Congenital Heart Disease 
PLoS ONE  2010;5(5):e10855.
Congenital heart disease (CHD) is the most common birth abnormality and the etiology is unknown in the overwhelming majority of cases. ISLET1 (ISL1) is a transcription factor that marks cardiac progenitor cells and generates diverse multipotent cardiovascular cell lineages. The fundamental role of ISL1 in cardiac morphogenesis makes this an exceptional candidate gene to consider as a cause of complex congenital heart disease. We evaluated whether genetic variation in ISL1 fits the common variant–common disease hypothesis. A 2-stage case-control study examined 27 polymorphisms mapping to the ISL1 locus in 300 patients with complex congenital heart disease and 2,201 healthy pediatric controls. Eight genic and flanking ISL1 SNPs were significantly associated with complex congenital heart disease. A replication study analyzed these candidate SNPs in 1,044 new cases and 3,934 independent controls and confirmed that genetic variation in ISL1 is associated with risk of non-syndromic congenital heart disease. Our results demonstrate that two different ISL1 haplotypes contribute to risk of CHD in white and black/African American populations.
PMCID: PMC2877111  PMID: 20520780
10.  Patients with schizophrenia show deficits of working memory maintenance components in circuit-specific tasks 
Working memory (WM) deficits are a neuropsychological core finding in patients with schizophrenia and also supposed to be a potential endophenotype of schizophrenia. Yet, there is a large heterogeneity between different WM tasks which is partly due to the lack of process specificity of the tasks applied. Therefore, we investigated WM functioning in patients with schizophrenia using process- and circuit-specific tasks. Thirty-one patients with schizophrenia and 47 controls were tested with respect to different aspects of verbal and visuospatial working memory using modified Sternberg paradigms in a computer-based behavioural experiment. Total group analysis revealed significant impairment of patients with schizophrenia in each of the tested WM components. Furthermore, we were able to identify subgroups of patients showing different patterns of selective deficits. Patients with schizophrenia exhibit specific and, in part, selective WM deficits with indirect but conclusive evidence of dysfunctions of the underlying neural networks. These deficits are present in tasks requiring only maintenance of verbal or visuospatial information. In contrast to a seemingly global working memory deficit, individual analysis revealed differential patterns of working memory impairments in patients with schizophrenia.
PMCID: PMC2953632  PMID: 20169354
Neurocognitive functioning; Working memory; Schizophrenia
11.  Body Mass Index, Follicle-Stimulating Hormone and Their Predictive Value in In Vitro Fertilization 
Purpose:The objective was to explore whether body mass and day 3 follicle-stimulating hormone have predictive value on odds of pregnancy after in vitro fertilisation. Few studies show that obesity produces a variety of alterations in the reproductive system, and that women with an elevation of day 3 FSH have declining ovarian function.
Methods: The data of one-hundred-seventy-one women who underwent a standard regime of controlled ovarian hyperstimulation was analyzed with particular reference to variations in body mass and hormone levels.
Results: By raising BMI and FSH (mIU/mL) by one unit, the odds for pregnancy were decreased by the respective factors 0.84 (95% confidence interval 0.73–0.97) and 0.77 (95% confidence interval 0.59–1.00).
Conclusions: The results demonstrate that for the purpose of raising the odds of pregnancy BMI should be reduced. A low FSH value may cause the same effect. Nontheless, obesity and hormonal function may be independent risk factors for failure in assisted reproduction.
PMCID: PMC3455618  PMID: 15704518
BMI; body weight; FSH; IVF; pregnancy outcome
12.  Identification of promiscuous ene-reductase activity by mining structural databases using active site constellations 
Nature Communications  2014;5:4150.
The exploitation of catalytic promiscuity and the application of de novo design have recently opened the access to novel, non-natural enzymatic activities. Here we describe a structural bioinformatic method for predicting catalytic activities of enzymes based on three-dimensional constellations of functional groups in active sites (‘catalophores’). As a proof-of-concept we identify two enzymes with predicted promiscuous ene-reductase activity (reduction of activated C–C double bonds) and compare them with known ene-reductases, that is, members of the Old Yellow Enzyme family. Despite completely different amino acid sequences, overall structures and protein folds, high-resolution crystal structures reveal equivalent binding modes of typical Old Yellow Enzyme substrates and ligands. Biochemical and biocatalytic data show that the two enzymes indeed possess ene-reductase activity and reveal an inverted stereopreference compared with Old Yellow Enzymes for some substrates. This method could thus be a tool for the identification of viable starting points for the development and engineering of novel biocatalysts.
Enzymes are very efficient reaction catalysts, though taking advantage of this synthetically is hampered by their notorious specificity. Here, the authors identify important arrangements of active site residues and use structural bioinformatics to successfully predict enzyme activity.
PMCID: PMC4083419  PMID: 24954722
13.  Trans-ethnic genome-wide association study of colorectal cancer identifies a new susceptibility locus in VTI1A 
Nature communications  2014;5:4613.
The genetic basis of sporadic colorectal cancer (CRC) is not well explained by known risk polymorphisms. Here we perform a meta-analysis of two genome-wide association studies in 2,627 cases and 3,797 controls of Japanese ancestry and 1,894 cases and 4,703 controls of African ancestry, to identify genetic variants that contribute to CRC susceptibility. We replicate genome-wide statistically significant associations (P < 5×10−8) in 16,823 cases and 18,211 controls of European ancestry. This study reveals a new pan-ethnic CRC risk locus at 10q25 (rs12241008, intronic to VTI1A; P=1.4×10−9), providing additional insight into the etiology of CRC and highlighting the value of association mapping in diverse populations.
PMCID: PMC4180879  PMID: 25105248
14.  Heterogeneity in Choice Inconsistencies Among the Elderly: Evidence from Prescription Drug Plan Choice 
The American economic review  2011;101(3):377-381.
This paper investigates the degree to which choice inconsistencies documented in the context of Medicare Part D plan choice vary across consumers and geographic regions. Our main finding is that there is surprisingly little variation: regardless of age, gender, predicted drug expenditures or the predictability of drug demand consumers underweight out of pocket costs relative to premiums and fail to consider the individualized consequences of plan characteristics; as a result, they frequently choose plans which are dominated in the sense that an alternative plan provides better risk protection at a lower cost. We find limited evidence that the sickest individuals had more difficulty with plan choice, and we document that much of the variation in potential cost savings across states comes from variation in choice sets, not variation in consumers’ ability to choose.
PMCID: PMC4319794
15.  Apes have culture but may not know that they do 
There is good evidence that some ape behaviors can be transmitted socially and that this can lead to group-specific traditions. However, many consider animal traditions, including those in great apes, to be fundamentally different from human cultures, largely because of lack of evidence for cumulative processes and normative conformity, but perhaps also because current research on ape culture is usually restricted to behavioral comparisons. Here, we propose to analyze ape culture not only at the surface behavioral level but also at the underlying cognitive level. To this end, we integrate empirical findings in apes with theoretical frameworks developed in developmental psychology regarding the representation of tools and the development of metarepresentational abilities, to characterize the differences between ape and human cultures at the cognitive level. Current data are consistent with the notion of apes possessing mental representations of tools that can be accessed through re-representations: apes may reorganize their knowledge of tools in the form of categories or functional schemes. However, we find no evidence for metarepresentations of cultural knowledge: apes may not understand that they or others hold beliefs about their cultures. The resulting Jourdain Hypothesis, based on Molière’s character, argues that apes express their cultures without knowing that they are cultural beings because of cognitive limitations in their ability to represent knowledge, a determining feature of modern human cultures, allowing representing and modifying the current norms of the group. Differences in metarepresentational processes may thus explain fundamental differences between human and other animals’ cultures, notably limitations in cumulative behavior and normative conformity. Future empirical work should focus on how animals mentally represent their cultural knowledge to conclusively determine the ways by which humans are unique in their cultural behavior.
PMCID: PMC4319388
animal culture; comparative cognition; field experiments; cultural mind; metarepresentation
16.  Increasing the inspiratory time and I:E ratio during mechanical ventilation aggravates ventilator-induced lung injury in mice 
Critical Care  2015;19(1):23.
Lung-protective ventilation reduced acute respiratory distress syndrome (ARDS) mortality. To minimize ventilator-induced lung injury (VILI), tidal volume is limited, high plateau pressures are avoided, and positive end-expiratory pressure (PEEP) is applied. However, the impact of specific ventilatory patterns on VILI is not well defined. Increasing inspiratory time and thereby the inspiratory/expiratory ratio (I:E ratio) may improve oxygenation, but may also be harmful as the absolute stress and strain over time increase. We thus hypothesized that increasing inspiratory time and I:E ratio aggravates VILI.
VILI was induced in mice by high tidal-volume ventilation (HVT 34 ml/kg). Low tidal-volume ventilation (LVT 9 ml/kg) was used in control groups. PEEP was set to 2 cm H2O, FiO2 was 0.5 in all groups. HVT and LVT mice were ventilated with either I:E of 1:2 (LVT 1:2, HVT 1:2) or 1:1 (LVT 1:1, HVT 1:1) for 4 hours or until an alternative end point, defined as mean arterial blood pressure below 40 mm Hg. Dynamic hyperinflation due to the increased I:E ratio was excluded in a separate group of animals. Survival, lung compliance, oxygenation, pulmonary permeability, markers of pulmonary and systemic inflammation (leukocyte differentiation in lung and blood, analyses of pulmonary interleukin-6, interleukin-1β, keratinocyte-derived chemokine, monocyte chemoattractant protein-1), and histopathologic pulmonary changes were analyzed.
LVT 1:2 or LVT 1:1 did not result in VILI, and all individuals survived the ventilation period. HVT 1:2 decreased lung compliance, increased pulmonary neutrophils and cytokine expression, and evoked marked histologic signs of lung injury. All animals survived. HVT 1:1 caused further significant worsening of oxygenation, compliance and increased pulmonary proinflammatory cytokine expression, and pulmonary and blood neutrophils. In the HVT 1:1 group, significant mortality during mechanical ventilation was observed.
According to the “baby lung” concept, mechanical ventilation-associated stress and strain in overinflated regions of ARDS lungs was simulated by using high tidal-volume ventilation. Increase of inspiratory time and I:E ratio significantly aggravated VILI in mice, suggesting an impact of a “stress/strain × time product” for the pathogenesis of VILI. Thus increasing the inspiratory time and I:E ratio should be critically considered.
Electronic supplementary material
The online version of this article (doi:10.1186/s13054-015-0759-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4336519
17.  The Collective Benefits of Feeling Good and Letting Go: Positive Emotion and (dis)Inhibition Interact to Predict Cooperative Behavior 
PLoS ONE  2015;10(1):e0117426.
Cooperation is central to human existence, forming the bedrock of everyday social relationships and larger societal structures. Thus, understanding the psychological underpinnings of cooperation is of both scientific and practical importance. Recent work using a dual-process framework suggests that intuitive processing can promote cooperation while deliberative processing can undermine it. Here we add to this line of research by more specifically identifying deliberative and intuitive processes that affect cooperation. To do so, we applied automated text analysis using the Linguistic Inquiry and Word Count (LIWC) software to investigate the association between behavior in one-shot anonymous economic cooperation games and the presence inhibition (a deliberative process) and positive emotion (an intuitive process) in free-response narratives written after (Study 1, N = 4,218) or during (Study 2, N = 236) the decision-making process. Consistent with previous results, across both studies inhibition predicted reduced cooperation while positive emotion predicted increased cooperation (even when controlling for negative emotion). Importantly, there was a significant interaction between positive emotion and inhibition, such that the most cooperative individuals had high positive emotion and low inhibition. This suggests that inhibition (i.e., reflective or deliberative processing) may undermine cooperative behavior by suppressing the prosocial effects of positive emotion.
PMCID: PMC4308081  PMID: 25625722
18.  Overview about the localization of nanoparticles in tissue and cellular context by different imaging techniques 
The increasing interest and recent developments in nanotechnology pose previously unparalleled challenges in understanding the effects of nanoparticles on living tissues. Despite significant progress in in vitro cell and tissue culture technologies, observations on particle distribution and tissue responses in whole organisms are still indispensable. In addition to a thorough understanding of complex tissue responses which is the domain of expert pathologists, the localization of particles at their sites of interaction with living structures is essential to complete the picture. In this review we will describe and compare different imaging techniques for localizing inorganic as well as organic nanoparticles in tissues, cells and subcellular compartments. The visualization techniques include well-established methods, such as standard light, fluorescence, transmission electron and scanning electron microscopy as well as more recent developments, such as light and electron microscopic autoradiography, fluorescence lifetime imaging, spectral imaging and linear unmixing, superresolution structured illumination, Raman microspectroscopy and X-ray microscopy. Importantly, all methodologies described allow for the simultaneous visualization of nanoparticles and evaluation of cell and tissue changes that are of prime interest for toxicopathologic studies. However, the different approaches vary in terms of applicability for specific particles, sensitivity, optical resolution, technical requirements and thus availability, and effects of labeling on particle properties. Specific bottle necks of each technology are discussed in detail. Interpretation of particle localization data from any of these techniques should therefore respect their specific merits and limitations as no single approach combines all desired properties.
PMCID: PMC4311646
fluorescence lifetime imaging; fluorescence microscopy; histopathology; light microscopic autoradiography; structured illumination microscopy
19.  Tailoring the Antibody Response to Aggregated Aß Using Novel Alzheimer-Vaccines 
PLoS ONE  2015;10(1):e0115237.
Recent evidence suggests Alzheimer-Disease (AD) to be driven by aggregated Aß. Capitalizing on the mechanism of molecular mimicry and applying several selection layers, we screened peptide libraries for moieties inducing antibodies selectively reacting with Aß-aggregates. The technology identified a pool of peptide candidates; two, AFFITOPES AD01 and AD02, were assessed as vaccination antigens and compared to Aβ1-6, the targeted epitope. When conjugated to Keyhole Limpet Hemocyanin (KLH) and adjuvanted with aluminum, all three peptides induced Aß-targeting antibodies (Abs). In contrast to Aß1-6, AD01- or AD02-induced Abs were characterized by selectivity for aggregated forms of Aß and absence of reactivity with related molecules such as Amyloid Precursor Protein (APP)/ secreted APP-alpha (sAPPa). Administration of AFFITOPE-vaccines to APP-transgenic mice was found to reduce their cerebral amyloid burden, the associated neuropathological alterations and to improve their cognitive functions. Thus, the AFFITOME-technology delivers vaccines capable of inducing a distinct Ab response. Their features may be beneficial to AD-patients, a hypothesis currently tested within a phase-II-study.
PMCID: PMC4303436  PMID: 25611858
20.  Molecular Basis for SMC Rod Formation and Its Dissolution upon DNA Binding 
Molecular Cell  2015;57(2):290-303.
SMC condensin complexes are central modulators of chromosome superstructure in all branches of life. Their SMC subunits form a long intramolecular coiled coil, which connects a constitutive “hinge” dimerization domain with an ATP-regulated “head” dimerization module. Here, we address the structural arrangement of the long coiled coils in SMC complexes. We unequivocally show that prokaryotic Smc-ScpAB, eukaryotic condensin, and possibly also cohesin form rod-like structures, with their coiled coils being closely juxtaposed and accurately anchored to the hinge. Upon ATP-induced binding of DNA to the hinge, however, Smc switches to a more open configuration. Our data suggest that a long-distance structural transition is transmitted from the Smc head domains to regulate Smc-ScpAB’s association with DNA. These findings uncover a conserved architectural theme in SMC complexes, provide a mechanistic basis for Smc’s dynamic engagement with chromosomes, and offer a molecular explanation for defects in Cornelia de Lange syndrome.
Graphical Abstract
•Prokaryotic Smc-ScpAB complexes form rod-like structures•Binding of ATP and DNA induces a rod-to-ring transition in prokaryotic condensin•The condensin hinge is rigidly anchored to its coiled coil•The rod-like conformation is a conserved feature of SMC protein dimers
Soh et al. show that the rod-like conformation is a conserved architectural scheme of SMC complexes. Upon ATP-induced binding to DNA, the juxtaposed coiled coils of prokaryotic Smc-ScpAB adopt an open conformation to expose a DNA binding site at the inner surface of the hinge domain.
PMCID: PMC4306524  PMID: 25557547
21.  Relative survival of patients with non-malignant central nervous system tumours: a descriptive study by the Austrian Brain Tumour Registry 
British Journal of Cancer  2013;110(2):286-296.
Unlike malignant primary central nervous system (CNS) tumours outcome data on non-malignant CNS tumours are scarce. For patients diagnosed from 1996 to 2002 5-year relative survival of only 85.0% has been reported. We investigated this rate in a contemporary patient cohort to update information on survival.
We followed a cohort of 3983 cases within the Austrian Brain Tumour Registry. All patients were newly diagnosed from 2005 to 2010 with a histologically confirmed non-malignant CNS tumour. Vital status, cause of death, and population life tables were obtained by 31 December 2011 to calculate relative survival.
Overall 5-year relative survival was 96.1% (95% CI 95.1–97.1%), being significantly lower in tumours of borderline (90.2%, 87.2–92.7%) than benign behaviour (97.4%, 96.3–98.3%). Benign tumour survival ranged from 86.8 for neurofibroma to 99.7% for Schwannoma; for borderline tumours survival rates varied from 83.2 for haemangiopericytoma to 98.4% for myxopapillary ependymoma. Cause of death was directly attributed to the CNS tumour in 39.6%, followed by other cancer (20.4%) and cardiovascular disease (15.8%).
The overall excess mortality in patients with non-malignant CNS tumours is 5.5%, indicating a significant improvement in survival over the last decade. Still, the remaining adverse impact on survival underpins the importance of systematic registration of these tumours.
PMCID: PMC3899758  PMID: 24253501
relative survival; CNS tumour; benign behaviour; borderline behaviour; non-malignant
22.  Contributions of thrombin targets to tissue factor-dependent metastasis in hyperthrombotic mice 
Tumor cell tissue factor (TF)-initiated coagulation supports hematogenous metastasis by fibrin formation, platelet activation, and monocyte/macrophage recruitment. Recent studies identified host anticoagulant mechanisms as a major impediment for successful hematogenous tumor cell metastasis.
Here we address mechanisms that contribute to enhanced metastasis in hyperthrombotic mice with functional thrombomodulin deficiency (TMPro mice).
Pharmacological and genetic approaches were combined to characterize relevant thrombin targets in a mouse model of experimental hematogenous metastasis.
TF-dependent, but contact pathway-independent syngeneic breast cancer metastasis was associated with marked platelet hyper-reactivity and formation of leukocyte-platelet aggregates in immune-competent TMPro mice. Blockade of CD11b or genetic deletion of platelet glycoprotein Ibα excluded contributions of these receptors to enhanced platelet-dependent metastasis in hyperthrombotic mice. Mice with very low levels of the endothelial protein C receptor (EPCR) did not phenocopy the enhanced metastasis seen in TMPro mice. Genetic deletion of the thrombin receptor PAR1 or endothelial thrombin signaling targets alone did not diminish enhanced metastasis in TMPro mice. Combined deficiency of PAR1 on tumor cells and the host reduced metastasis in TMPro mice.
Metastasis in the hyperthrombotic TMPro mouse model is mediated by platelet hyper-reactivity and contributions of PAR1 signaling on tumor and host cells.
PMCID: PMC3947224  PMID: 24175924
Tissue Factor; Thrombin; Hypercoagulability; Metastasis; Platelets
23.  Trans-Splicing Improvement by the Combined Application of Antisense Strategies 
Spliceosome-mediated RNA trans-splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA trans-splicing molecules (RTMs) are designed to induce a specific trans-splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM trans-splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs), presuming to improve the trans-splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB). We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG), lead to a significant increase of the RNA trans-splicing efficiency. Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.
PMCID: PMC4307297  PMID: 25569093
RNA trans-splicing; antisense oligonucleotides; RNA repair; genetic diseases
24.  Human papillomavirus is not associated with colorectal cancer in a large international study 
Cancer causes & control : CCC  2010;21(5):737-743.
Objective of the study
Recent publications have reported an association between colon cancer and human papillomaviruses (HPV), suggesting that HPV infection of the colonic mucosa may contribute to the development of colorectal cancer.
The GP5+/GP6+ PCR reverse line blot method was used for detection of 37 types of human papilloma-virus (HPV) in DNA from paraffin-embedded or frozen tissues from patients with colorectal cancer (n = 279) and normal adjacent tissue (n = 30) in three different study populations, including samples from the United States (n = 73), Israel (n = 106) and Spain (n = 100). Additionally, SPF10 PCR was run on all samples (n = 279) and the Innogenetics INNO-LiPA assay was performed on a subset of samples (n = 15).
All samples were negative for all types of HPV using both the GP5+/GP6+ PCR reverse line blot method and the SPF10 INNO-LiPA method.
We conclude that HPV types associated with malignant transformation do not meaningfully contribute to adenocarcinoma of the colon.
PMCID: PMC4269349  PMID: 20087645
Human papillomavirus; Colorectal cancer; International study
25.  Transcriptome Profiling Identifies HMGA2 as a Biomarker of Melanoma Progression and Prognosis 
The genetic alterations contributing to melanoma pathogenesis are incompletely defined, and few independent prognostic features have been identified beyond the clinicopathological characteristics of the primary tumor. We used transcriptome profiling of 46 primary melanomas, 12 melanoma metastases, and 16 normal skin (N) samples to find genes associated with melanoma development and progression. Results were confirmed using immunohistochemistry and real-time PCR and replicated in an independent set of 330 melanomas using AQUA analysis of tissue microarray (TMA). Transcriptome profiling revealed that transcription factor HMGA2, previously unrecognized in melanoma pathogenesis, is significantly upregulated in primary melanoma and metastases (P-values=1.2 × 10−7 and 9 × 10−5) compared with N. HMGA2 overexpression is associated with BRAF/NRAS mutations (P=0.0002). Cox proportional hazard regression model and log-rank test showed that HMGA2 is independently associated with disease-free survival (hazard ratio (HR)=6.3, 95% confidence interval (CI)= 1.8–22.3, P=0.004), overall survival (OS) (stratified log-rank P=0.008), and distant metastases–free survival (HR=6.4, 95% CI=1.4–29.7, P=0.018) after adjusting for American Joint Committee on Cancer (AJCC) stage and age at diagnosis. Survival analysis in an independent replication TMA of 330 melanomas confirmed the association of HMGA2 expression with OS (P=0.0211). Our study implicates HMGA2 in melanoma progression and demonstrates that HMGA2 overexpression can serve as an independent predictor of survival in melanoma.
PMCID: PMC4267221  PMID: 23633021

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