Guest Editorial: Critical Thinking in Education and Research-Why and How?

"A responsible scientist is able to step back, critically reflect on his or her own doings, and also explain them to a wider audience. Critical thinking includes the ability to talk to people working in other areas, as well as the broader public. It has to be taught to students and fostered at the university level, and should be practiced in relation to one's own work …" Read more in the Guest Editorial.

Salivary Alpha-Amylase Correlates with Subjective Heat Pain Perception.

OBJECTIVE: Self-reports of pain are important for an adequate therapy. This is a problem with patients and infants who are restricted in providing an accurate verbal estimation of their pain. Reliable, real-time, economical, and non-invasive physiological correlates might contribute to a more comprehensive description of pain. Salivary alpha-amylase constitutes one candidate biomarker, which reflects predominantly sympathetic nervous system alterations under stressful conditions and can be measured non-invasively. The current study investigated the effects of acute heat pain on salivary alpha-amylase activity. METHODS: Heat pain tolerance was measured on the non-dominant forearm. Participants completed visual analog scales on pain intensity and unpleasantness. Saliva samples were collected directly after pain induction. SUBJECTS: Twenty-seven healthy volunteers were recruited for this study. RESULTS: While salivary alpha-amylase levels correlated positively with intensity and unpleasantness ratings in response to acute heat pain stimuli, there was no corresponding association with pain tolerance. CONCLUSIONS: Salivary alpha-amylase is suggested to be an indirect physiologic correlate of subjective heat pain perception. Future studies should address the role of salivary alpha-amylase depending on the origin of pain, the concerned tissue, and other pain assessment methods.

Scrutinizing MHC-I binding peptides and their limits of variation.

Designed peptides that bind to major histocompatibility protein I (MHC-I) allomorphs bear the promise of representing epitopes that stimulate a desired immune response. A rigorous bioinformatical exploration of sequence patterns hidden in peptides that bind to the mouse MHC-I allomorph H-2K(b) is presented. We exemplify and validate these motif findings by systematically dissecting the epitope SIINFEKL and analyzing the resulting fragments for their binding potential to H-2K(b) in a thermal denaturation assay. The results demonstrate that only fragments exclusively retaining the carboxy- or amino-terminus of the reference peptide exhibit significant binding potential, with the N-terminal pentapeptide SIINF as shortest ligand. This study demonstrates that sophisticated machine-learning algorithms excel at extracting fine-grained patterns from peptide sequence data and predicting MHC-I binding peptides, thereby considerably extending existing linear prediction models and providing a fresh view on the computer-based molecular design of future synthetic vaccines. The server for prediction is available at http://modlab-cadd.ethz.ch (SLiDER tool, MHC-I version 2012).

Naloxone modulates visual judgments of similarity but not dissimilarity.

Endogenous opioids have been implicated in mediating (placebo) analgesia, in reward processes, and in the regulation of socially relevant emotions. To explore their potential contributions to higher cognitive functions, we used a novel task with tachistoscopically presented (for 150 ms) pairs of meaningless figures. Healthy right-handed men judged the similarities and dissimilarities between the two figures on a visual analogue scale (VAS) in two separate runs. In a double-blind, between-subjects design, subjects were administered intravenously either 0.2-mg/kg naloxone or placebo 10 min prior to the task, and VAS judgments and response latencies were measured. We found a significant interaction between substance group and type of judgment: The magnitude of the similarity judgments was lower in the naloxone than in the placebo group, while dissimilarity judgments remained uninfluenced by the treatment. Reaction latencies and mood scores, assessed before and after substance administration, did not differ between the two groups, indicating that the findings did not rely on altered motor performance or motivation. We suggest that naloxone decreased the "similarity criterion" in comparative judgments, indicating its potentially modulatory effect on visual cognition. The task introduced here could be used for the implicit study and quantification of subtle affective-cognitive processes beyond the level of mere questionnaire data.

Pleasure-related analgesia activates opioid-insensitive circuits.

Recent findings suggest that pain and pleasure share common neurochemical circuits, and studies in animals and humans show that opioid-mediated descending pathways can inhibit or facilitate pain. We explored the role of endogenous opioid neurotransmission in pleasure-related analgesia. µ-Opioidergic activity was blocked with 0.2 mg/kg naloxone to assess its effects on hedonic responses to pleasant emotional pictures (International Affective Picture System) and its modulating effects on heat pain tolerance. Naloxone did not alter subjective and autonomous reactions to pleasure induction or overall mood of participants. In addition, pleasure-related increases in pain tolerance persisted after reversal of endogenous µ-opioidergic neurotransmission. Subjective pain intensity and unpleasantness ratings increased after naloxone administration. These findings suggest that, in addition to opioid-sensitive circuits, mainly opioid-insensitive pain-modulating circuits are activated during pleasure-related analgesia.

Monitoring stage fright outside the laboratory: an example in a professional musician using wearable sensors.

We implemented and tested a wearable sensor system to measure patterns of stress responses in a professional musician under public performance conditions. Using this sensor system, we monitored the cellist's heart activity, the motion of multiple body parts, and their gradual changes during three repeated performances of a skill-demanding piece in front of a professional audience. From the cellist and her teachers, we collected stage fright self-reports and performance ratings that were related to our sensor data analysis results. Concomitant to changes in body motion and heart rate, the cellist perceived a reduction in stage fright. Performance quality was objectively improved, as technical playing errors decreased throughout repeated renditions. In particular, from performance 1 to 3, the wearable sensors measured a significant increase in the cellist's bowing motion dynamics of approximately 6% and a decrease in heart rate. Bowing motion showed a marginal correlation to the observed heart rate patterns during playing. The wearable system did not interfere with the cellist's performance, thereby allowing investigation of stress responses during natural public performances.

Placebo-mediated, Naloxone-sensitive suggestibility of short-term memory performance.

Physiological studies of placebo-mediated suggestion have been recently performed beyond their traditional clinical context of pain and analgesia. Various neurotransmitter systems and immunological modulators have been used in successful placebo suggestions, including Dopamine, Cholecystokinin and, most extensively, opioids. We adhered to an established conceptual framework of placebo research and used the µ-opioid-antagonist Naloxone to test the applicability of this framework within a cognitive domain (e.g. memory) in healthy volunteers. Healthy men (n=62, age 29, SD=9) were required to perform a task-battery, including standardized and custom-designed memory tasks, to test short-term recall and delayed recognition. Tasks were performed twice, before and after intravenous injection of either NaCl (0.9%) or Naloxone (both 0.15 mg/kg), in a double-blind setting. While one group was given neutral information (S-), the other was told that it might receive a drug with suspected memory-boosting properties (S+). Objective and subjective indexes of memory performance and salivary cortisol (as a stress marker) were recorded during both runs and differences between groups were assessed. Short-term memory recall, but not delayed recognition, was objectively increased after placebo-mediated suggestion in the NaCl-group. Naloxone specifically blocked the suggestion effect without interfering with memory performance. These results were not affected when changes in salivary cortisol levels were considered. No reaction time changes, recorded to uncover unspecific attentional impairment, were seen. Placebo-mediated suggestion produced a training-independent, objective and Naloxone-sensitive increase in memory performance. These results indicate an opioid-mediated placebo effect within a circumscribed cognitive domain in healthy volunteers.

Heat pain threshold and tolerance show no left-right perceptual differences at complementary sites of the human forearm.

Pain threshold and pain tolerance of heat noxious stimuli were assessed to determine whether they are equivalent when measured at three equidistant sites of both volar forearms. Heat pain threshold and tolerance were measured in 18 healthy volunteers using a standard stimulation device consisting of a thermode. Pain threshold and pain tolerance did not differ within and across forearm sites. Experimenters addressing heat pain threshold and tolerance in healthy volunteers may freely choose and change stimulation sites on both volar forearms, without the risk of confounding site effects on dependent variables. This data completes previous reports on side effects by analyzing the effect of site on the forearm for both heat pain threshold and tolerance. The absence of side and site effects may contribute to setting a more secure basis for assessments of laterality effects of painful stimulation.

The crystal structure of Aspergillus fumigatus cyclophilin reveals 3D domain swapping of a central element.

The crystal structure of Aspergillus fumigatus cyclophilin (Asp f 11) was solved by the multiwavelength anomalous dispersion method and was refined to a resolution of 1.85 A with R and R(free) values of 18.9% and 21.4%, respectively. Many cyclophilin structures have been solved to date, all showing the same monomeric conformation. In contrast, the structure of A. fumigatus cyclophilin reveals dimerization by 3D domain swapping and represents one of the first proteins with a swapped central domain. The domain-swapped element consists of two beta strands and a subsequent loop carrying a conserved tryptophan. The tryptophan binds into the active site, inactivating cis-trans isomerization. This might be a means of biological regulation. The two hinge loops leave the protein prone to misfolding. In this context, alternative forms of 3D domain swapping that can lead to N- or C-terminally swapped dimers, oligomers, and aggregates are discussed.

Development and validation of a capillary electrophoresis method for the characterization of herpes simplex virus type 1 (HSV-1) thymidine kinase substrates and inhibitors.

A fast, convenient capillary electrophoresis (CE) method was developed for monitoring the enzymatic reaction of herpes simplex virus type 1 thymidine kinase (HSV-1 TK). The reaction was performed in a test tube followed by quantitative analysis of the products. The optimized CE conditions were as follows: polyacrylamide-coated capillary (20 cm effective length x 50 microm), electrokinetic injection for 30s, 50 mM phosphate buffer at pH 6.5, constant current of -60 microA, UV detection at 210 nm, UMP or cAMP were used as internal standards. Phosphorylated products eluted within less than 7 min. The limits of detection were 0.36 microM for dTMP and 0.86 microM for GMP. The method was used to study enzyme kinetics, and to investigate alternative substrates and inhibitors.

Patupilone acts as radiosensitizing agent in multidrug-resistant cancer cells in vitro and in vivo.

Interference with microtubule function is a promising antitumoral concept. Paclitaxel is a clinically validated tubulin-targeting agent; however, treatment with paclitaxel is often limited by taxane-related toxicities and is ineffective in tumors with multidrug-resistant cells. Patupilone (EPO906, epothilone B) is a novel non-taxane-related microtubule-stabilizing natural compound that retains full activity in multidrug-resistant tumors and is clinically less toxic than paclitaxel. Here we have investigated the effect of combined treatment with ionizing radiation and patupilone or paclitaxel in the P-glycoprotein-overexpressing, p53-mutated human colon adenocarcinoma cell line SW480 and in murine, genetically defined E1A/ras-transformed paclitaxel-sensitive embryo fibroblasts. Patupilone and paclitaxel alone and in combination with ionizing radiation reduced the proliferative activity of the E1A/ras-transformed cell line with similar potency in the sub and low nanomolar range. SW480 cells were only sensitive to patupilone, and combined treatment with low-dose patupilone (0.1 nmol/L) followed by clinically relevant doses of ionizing radiation (2 and 5 Gy) resulted in a supra-additive cytotoxic effect. Inhibition of the drug efflux protein P-glycoprotein with verapamil resensitized SW480 cells to treatment with low doses of paclitaxel alone and in combination with IR. In tumor xenografts derived from SW480 cells a minimal treatment regimen with patupilone and fractionated irradiation (1 x 2 mg/kg plus 4 x 3 Gy) resulted in an at least additive tumor response with extended tumor growth arrest. Analysis by flow cytometry in vitro revealed an apoptosis- and G(2)-M-independent mode of radiosensitization by patupilone. Interestingly though, a transient accumulation of cells in S phase was observed on combined treatment.Overall, patupilone might be a promising alternative in paclitaxel-resistant, P-glycoprotein-overexpressing tumors for a combined treatment regimen using ionizing radiation and a microtubule inhibitor.

The crystal structure of PfFabZ, the unique beta-hydroxyacyl-ACP dehydratase involved in fatty acid biosynthesis of Plasmodium falciparum.

The unique beta-hydroxyacyl-ACP dehydratase in Plasmodium falciparum, PfFabZ, is involved in fatty acid biosynthesis and catalyzes the dehydration of beta-hydroxy fatty acids linked to acyl carrier protein. The structure was solved by single anomalous dispersion (SAD) phasing using a quick-soaking experiment with potassium iodide and refined to a resolution of 2.1 A. The crystal structure represents the first structure of a Plasmodium beta-hydroxyacyl-ACP dehydratase with broad substrate specificity. The asymmetric unit contains a hexamer that appears as a trimer of dimers. Each dimer shows the known "hot dog" fold that has been observed in only a few other protein structures. Each of the two independent active sites in the dimer is formed by equal contributions from both subunits. The active site is mainly hydrophobic and looks like an L-shaped tunnel. The catalytically important amino acids His 133 and Glu 147' (from the other subunit), together with His98', form the only hydrophilic site in this tunnel. The inner end of the active site tunnel is closed by the phenyl ring of Phe 169, which is located in a flexible, partly visible loop. In order to explain the acceptance of substrates longer than ~C-7, the phenyl ring must move away to open the tunnel. The present structure supports an enzymatic mechanism consisting of an elimination reaction catalyzed by His 133 and Glu147'. 3-decynoyl-N-acetylcysteamine, an inhibitor known to interact with the E. coli dehydratase/isomerase, turned out to interact covalently with PfFabZ. A first model of PfFabZ with this potent inhibitor is presented.

Structure of a type II thymidine kinase with bound dTTP.

The structure of human cytosolic thymidine kinase in complex with its feedback inhibitor 2'-deoxythymidine-5'-triphosphate was determined. This structure is the first representative of the type II thymidine kinases found in several pathogens. The structure deviates strongly from the known structures of type I thymidine kinases such as the Herpes simplex enzyme. It contains a zinc-binding domain with four cysteines complexing a structural zinc ion. Interestingly, the backbone atoms of the type II enzyme bind thymine via hydrogen-bonds, in contrast to type I, where side chains are involved. This results in a specificity difference exploited for antiviral therapy. The presented structure will foster the development of new drugs and prodrugs for numerous therapeutic applications.

Bovine pancreatic polypeptide (bPP) undergoes significant changes in conformation and dynamics upon binding to DPC micelles.

The pancreatic polypeptide (PP), a 36-residue, C-terminally amidated polypeptide hormone is a member of the neuropeptide Y (NPY) family. Here, we have studied the structure and dynamics of bovine pancreatic polypeptide (bPP) when bound to DPC-micelles as a membrane-mimicking model as well as the dynamics of bPP in solution. The comparison of structure and dynamics of bPP in both states reveals remarkable differences. The overall correlation time of 5.08ns derived from the 15N relaxation data proves unambiguously that bPP in solution exists as a dimer. Therein, intermolecular as well as intramolecular hydrophobic interactions from residues of both the amphiphilic helix and of the back-folded N terminus contribute to the stability of the PP fold. The overall rigidity is well-reflected in positive values for the heteronuclear NOE for residues 4-34. The membrane-bound species displays a partitioning into a more flexible N-terminal region and a well-defined alpha-helical region comprising residues 17-31. The average RMSD value for residues 17-31 is 0.22(+/-0.09)A. The flexibility of the N terminus is compatible with negative values of the heteronuclear NOE observed for the N-terminal residues 4-12 and low values of the generalized order parameter S(2). The membrane-peptide interface was investigated by micelle-integrating spin-labels and H,2H exchange measurements. It is formed by those residues which make contacts between the C-terminal alpha-helix and the polyproline helix. In contrast to pNPY, also residues from the N terminus display spatial proximity to the membrane interface. Furthermore, the orientation of the C terminus, that presumably contains residues involved in receptor binding, is different in the two environments. We speculate that this pre-positioning of residues could be an important requirement for receptor activation. Moreover, we doubt that the PP fold is of functional relevance for binding at the Y(4) receptor.

Random chemistry as a new tool for the generation of small compound libraries: development of a new acetylcholinesterase inhibitor.

Random chemistry, the serendipitous generation of small compound libraries by gamma-irradiation of source compounds, presents a methodology providing reassembled and rearranged structures. The gamma-irradiation was applied to generate new acetylcholinesterase (AChE) inhibitors. The bioassay-guided fractionation as a deconvolution strategy was employed to analyze gained product mixture. The structure of the new highly potent AChE inhibitor, 9-amino-5,6,7,8-tetrahydroacridin-4yl)methanol (1), was elucidated by NMR spectroscopy and ESI (tandem) mass spectrometry.

Binding mode prediction of cytochrome p450 and thymidine kinase protein-ligand complexes by consideration of water and rescoring in automated docking.

The popular docking programs AutoDock, FlexX, and GOLD were used to predict binding modes of ligands in crystallographic complexes including X-ray water molecules or computationally predicted water molecules. Isoenzymes of two different enzyme systems were used, namely cytochromes P450 (n = 19) and thymidine kinases (n = 19) and three different "water" scenarios: i.e., docking (i) into water-free active sites, (ii) into active sites containing crystallographic water molecules, and (iii) into active sites containing water molecules predicted by a novel approach based on the program GRID. Docking accuracies were determined in terms of the root-mean-square deviation (RMSD) accuracy and, newly defined, in terms of the ligand catalytic site prediction (CSP) accuracy. Consideration of both X-ray and predicted water molecules and the subsequent pooling and rescoring of all solutions (generated by all three docking programs) with the SCORE scoring function significantly improved the quality of prediction of the binding modes both in terms of RMSD and CSP accuracy.

Maternal dietary Alpine butter intake affects human milk: fatty acids and conjugated linoleic acid isomers.

Consumption of CLA by lactating women affects the composition of their milk, but the pattern of the different CLA isomers is still unknown. We determined the effects of short maternal supplementation with CLA-rich Alpine butter on the occurrence of FA and CLA isomers in human milk. In an open randomized controlled study with a two-period cross-over design, milk FA and CLA isomer concentrations were measured on postpartum days > or = 20 in two parallel groups of lactating women before, during, and after consumption of defined quantities of Alpine butter or margarine with comparable fat content (10 d of butter followed by 10 d of margarine for one group, and vice versa in the other). In the 16 women who completed the study (8/group), Alpine butter supplementation increased the C16 and C18 FA, the sum of saturated FA, the 18:1 trans FA, and the trans FA with CLA. The CLA isomer 18:2 c9,t11 increased by 49.7%. Significant increases were also found for the isomers t9,t11, t7,c9, t11,c13, and t8,c10 18:2. The remaining nine of the total 14 detectable isomers showed no changes, and concentrations were <5 mg/100 g fat. A breastfeeding mother can therefore modulate the FA/CLA supply of her child by consuming Alpine butter. Further studies will show whether human milk containing this FA and CLA isomer pattern acts as a functional food for newborns.

How much reproducibility do we need in human and veterinary pathology?

In diagnostic and research reports as well as text-books of human and veterinary pathology repeatability, reproducibility, inter- and intra-observer variation are mentioned rarely as a problem in preparing diagnosis from macroscopic and/or microscopic samples and discussed inconsistently. However, optimal care and restoration of health for a patient are dependent on reliability of diagnosis, therapy, prognosis and prophylaxis. This requires for all tests and procedures a maximal repeatability and reproducibility, a sensitivity and specificity of 85-95% for procedures and methodologies and a comparison of results procedures and methodologies to a gold standard. Looking at the various steps on the road to diagnosis in pathology this is influenced by a series of laboratory steps preparing tissue samples but most importantly reproducibility depends on the handling of visual information in the central nervous system of the individual diagnostician. Thus reproducibility in this context has to be divided into at least three levels: individual (epistemological, organoleptic, inter- and intra-observer variation, and formal/technological- and normative reproducibility). The aim of the present manuscript is to stimulate the reflection among the pathology experts on this most important topic.

Quality of Animal Experiments in Anti-Angiogenic Cancer Drug Development--A Systematic Review.

Translation from preclinical animal research to clinical bedside has proven to be difficult to impossible in many fields of research (e.g. acute stroke, ALS and HIV vaccination development) with oncology showing particularly low translation rates (5% vs. 20% for cardiovascular diseases). Several investigations on published preclinical animal research have revealed that apart from plain species differences, translational problems can arise from low study quality (e.g. study design) or non-representative experimental conditions (e.g. treatment schedule). This review assessed the published experimental circumstances and quality of anti-angiogenic cancer drug development in 232 in vivo studies. The quality of study design was often insufficient; at least the information published about the experiments was not satisfactory in most cases. There was no quality improvement over time, with the exception of conflict of interest statements. This increase presumably arose mainly because journal guidelines request such statements more often recently. Visual inspection of data and a cluster analysis confirmed a trend described in literature that low study quality tends to overestimate study outcome. It was also found that experimental outcome was more favorable when a potential drug was investigated as the main focus of a study, compared to drugs that were used as comparison interventions. We assume that this effect arises from the frequent neglect of blinding investigators towards treatment arms and refer to it as hypothesis bias. In conclusion, the reporting and presumably also the experimental performance of animal studies in drug development for oncology suffer from similar shortcomings as other fields of research (such as stroke or ALS). We consider it necessary to enforce experimental quality and reporting that corresponds to the level of clinical studies. It seems that only clear journal guidelines or guidelines from licensing authorities, where failure to fulfill prevents publication or experimental license, can help to improve this situation.