The hazards of DAPI photoconversion: effects of dye, mounting media and fixative, and how to minimize the problem

Immunocytochemistry is a powerful tool for detection and visualization of specific molecules in living or fixed cells, their localization and their relative abundance. One of the most commonly used fluorescent DNA dyes in immunocytochemistry applications is 4′,6-diamidino-2-phenylindole dihydrochloride, known as DAPI. DAPI binds strongly to DNA and is used extensively for visualizing cell nuclei. It is excited by UV light and emits characteristic blue fluorescence. Here, we report a phenomenon based on an apparent photoconversion of DAPI that results in detection of a DAPI signal using a standard filter set for detection of green emission due to blue excitation. When a sample stained with DAPI only was first imaged with the green filter set (FITC/GFP), only a weak cytoplasmic autofluorescence was observed. Next, we imaged the sample with a DAPI filter set, obtaining a strong nuclear DAPI signal as expected. Upon reimaging the same samples with a FITC/GFP filter set, robust nuclear fluorescence was observed. We conclude that excitation with UV results in a photoconversion of DAPI that leads to detection of DAPI due to excitation and emission in the FITC/GFP channel. This phenomenon can affect data interpretation and lead to false-positive results when used together with fluorochrome-labeled nuclear proteins detected with blue excitation and green emission. In order to avoid misinterpretations, extra precaution should be taken to prepare staining solutions with low DAPI concentration and DAPI (UV excitation) images should be acquired after all other higher wavelength images. Of various DNA dyes tested, Hoechst 33342 exhibited the lowest photoconversion while that for DAPI and Hoechst 33258 was much stronger. Different fixation methods did not substantially affect the strength of photoconversion. We also suggest avoiding the use of mounting medium with high glycerol concentrations since glycerol showed the strongest impact on photoconversion. This photoconversion effect cannot be avoided even when using narrow bandpass filter sets.     

Overcoming Microstructural Limitations in Water Processed Organic Solar Cells by Engineering Customized Nanoparticulate Inks

The application of conjugated polymer and fullerene water‐based nanoparticles (NP) as ecofriendly inks for organic photovoltaics (OPVs) is reported. A low bandgap polymer diketopyrrolopyrrole–quinquethiophene (PDPP5T‐2) and the methanofullerene PC₇₁BM are processed into three types of nanoparticles: pristine fullerene NPs, pristine polymer NPs, and mixed polymer:fullerene NPs, allowing the formation of bulk heterojunction (BHJ) composites with different domain sizes. Mild thermal annealing is required to melt the nanospheres and enable the formation of interconnected pathways within mixed phases. This BHJ is accompanied by a shrinkage of film, whereas the more compact layers show enhanced mobility. Consistently reduced recombination and better performance are found for mixed NP, containing both, the polymer and the fullerene within a single NP. The optimized solar cell processed by ultrasmall NPs delivers a power conversion efficiency of about 3.4%. This is among the highest values reported for aqueous processed OPVs but still lacks performance compared to those being processed from halogenated solvents. Incomplete crystallization is identified as the main root for reduced efficiency. It is nevertheless believed that postprocessing does not cut attraction from printing aqueous organic NP inks as a trendsetting strategy for the reliable and ecofriendly production of organic solar cells.     

Biomechanical characterization and clinical implications of artificially induced toe-walking: differences between pure soleus, pure gastrocnemius and combination of soleus and gastrocnemius contractures

The purpose of this study was to characterize biomechanically three different toe-walking gait patterns, artificially induced in six neurologically intact subjects and to compare them to selected cases of pathological toe-walking. The subjects, equipped with lightweight mechanical exoskeleton with elastic ropes attached to the left leg's heel on one end and on shank and thigh on the other end in a similar anatomical locations where soleus and gastrocnemius muscles attach to skeleton, walked at speed of approximately 1m/s along the walkway under four experimental conditions: normal walking (NW), soleus contracture emulation (SOL), gastrocnemius contracture emulation (GAS) and emulation of both soleus and gastrocnemius contractures (SOLGAS). Reflective markers and force platform data were collected and ankle, knee and hip joint angles, moments and powers were calculated using inverse dynamic model for both legs. Characteristic peaks of averaged kinematic and kinetic patterns were compared among all four experimental conditions in one-way ANOVA. In the left leg SOL contracture mainly influenced the ankle angle trajectory, while GAS and SOLGAS contractures influenced the ankle and knee angle trajectories. GAS and SOLGAS contractures significantly increased ankle moment during midstance as compared to SOL contracture and NW. All three toe-walking experimental conditions exhibited significant power absorption in the ankle during loading response, which was absent in the NW condition, while during preswing significant decrease in power absorption as compared to NW was seen. In the knee joint SOL contracture diminished, GAS contracture increased while SOLGAS contracture approximately halved knee extensor moment during midstance as compared to NW. All three toe-walking experimental conditions decreased hip range of motion, hip flexor moment and power requirements during stance phase. Main difference in the right leg kinematic and kinetic patterns was seen in the knee moment trajectory, where significant increase in the knee extensor moment took place in terminal stance for GAS and SOLGAS experimental conditions as compared to SOL and NW. The kinetic trajectories under SOL and GAS experimental conditions were qualitatively compared to two selected clinical cases showing considerable similarity. This implies that distinct differences in kinetics between SOL, GAS and SOLGAS experimental conditions, as described in this paper, may be clinically relevant in determining the relative contribution of soleus and gastrocnemius muscles contractures to toe-walking in particular pathological gait.     

Effect of hydrophobic mutations in the H2-H3 subdomain of prion protein on stability and conversion in vitro and in vivo

Prion diseases are fatal neurodegenerative diseases, which can be acquired, sporadic or genetic, the latter being linked to mutations in the gene encoding prion protein. We have recently described the importance of subdomain separation in the conversion of prion protein (PrP). The goal of the present study was to investigate the effect of increasing the hydrophobic interactions within the H2-H3 subdomain on PrP conversion. Three hydrophobic mutations were introduced into PrP. The mutation V209I associated with human prion disease did not alter protein stability or in vitro fibrillization propensity of PrP. The designed mutations V175I and T187I on the other hand increased protein thermal stability. V175I mutant fibrillized faster than wild-type PrP. Conversion delay of T187I was slightly longer, but fluorescence intensity of amyloid specific dye thioflavin T was significantly higher. Surprisingly, cells expressing V209I variant exhibited inefficient proteinase K resistant PrP formation upon infection with 22L strain, which is in contrast to cell lines expressing wild-type, V175I and T187I mPrPs. In agreement with increased ThT fluorescence at the plateau T187I expressing cell lines accumulated an increased amount of the proteinase K-resistant prion protein. We showed that T187I induces formation of thin fibrils, which are absent from other samples. We propose that larger solvent accessibility of I187 in comparison to wild-type and other mutants may interfere with lateral annealing of filaments and may be the underlying reason for increased conversion efficiency.     

Decoding the mechanisms of gait generation in salamanders by combining neurobiology, modeling and robotics

Vertebrate animals exhibit impressive locomotor skills. These locomotor skills are due to the complex interactions between the environment, the musculo-skeletal system and the central nervous system, in particular the spinal locomotor circuits. We are interested in decoding these interactions in the salamander, a key animal from an evolutionary point of view. It exhibits both swimming and stepping gaits and is faced with the problem of producing efficient propulsive forces using the same musculo-skeletal system in two environments with significant physical differences in density, viscosity and gravitational load. Yet its nervous system remains comparatively simple. Our approach is based on a combination of neurophysiological experiments, numerical modeling at different levels of abstraction, and robotic validation using an amphibious salamander-like robot. This article reviews the current state of our knowledge on salamander locomotion control, and presents how our approach has allowed us to obtain a first conceptual model of the salamander spinal locomotor networks. The model suggests that the salamander locomotor circuit can be seen as a lamprey-like circuit controlling axial movements of the trunk and tail, extended by specialized oscillatory centers controlling limb movements. The interplay between the two types of circuits determines the mode of locomotion under the influence of sensory feedback and descending drive, with stepping gaits at low drive, and swimming at high drive.     

Identification and Prediction of Diabetic Sensorimotor Polyneuropathy Using Individual and Simple Combinations of Nerve Conduction Study Parameters

Objective

Evaluation of diabetic sensorimotor polyneuropathy (DSP) is hindered by the need for complex nerve conduction study (NCS) protocols and lack of predictive biomarkers. We aimed to determine the performance of single and simple combinations of NCS parameters for identification and future prediction of DSP.

Materials and Methods

406 participants (61 with type 1 diabetes and 345 with type 2 diabetes) with a broad spectrum of neuropathy, from none to severe, underwent NCS to determine presence or absence of DSP for cross-sectional (concurrent validity) analysis. The 109 participants without baseline DSP were re-evaluated for its future onset (predictive validity). Performance of NCS parameters was compared by area under the receiver operating characteristic curve (AROC).

Results

At baseline there were 246 (60%) Prevalent Cases. After 3.9 years mean follow-up, 25 (23%) of the 109 Prevalent Controls that were followed became Incident DSP Cases. Threshold values for peroneal conduction velocity and sural amplitude potential best identified Prevalent Cases (AROC 0.90 and 0.83, sensitivity 80 and 83%, specificity 89 and 72%, respectively). Baseline tibial F-wave latency, peroneal conduction velocity and the sum of three lower limb nerve conduction velocities (sural, peroneal, and tibial) best predicted 4-year incidence (AROC 0.79, 0.79, and 0.85; sensitivity 79, 70, and 81%; specificity 63, 74 and 77%, respectively).

Discussion

Individual NCS parameters or their simple combinations are valid measures for identification and future prediction of DSP. Further research into the predictive roles of tibial F-wave latencies, peroneal conduction velocity, and sum of conduction velocities as markers of incipient nerve injury is needed to risk-stratify individuals for clinical and research protocols.     

Fossil lizard from central Europe resolves the origin of large body size and herbivory in giant Canary Island lacertids

The endemic Canary Island lizard clade Gallotia, which includes the largest members of Europe's dominant reptile group, Lacertidae, is one of the classic examples of insular gigantism. For the first time we use fossil data to test the evolutionary reasons for the association between gigantism and herbivory. We describe an almost completely preserved skeleton of J anosikia ulmensis comb. nov. from the early Miocene of Ulm, Germany (MN 2a, ～ 22 Mya). We show that this species and Oligocene Pseudeumeces cadurcensis (Filhol, 1877) are in fact crown lacertids, and the first known pre‐Quaternary record of the total clade of Gallotia. Pseudeumeces confirms the early origin of crown Lacertidae in the Palaeogene of Europe. More importantly, these fossil taxa show that large body size was already achieved on the European mainland by the early Miocene. Furthermore, Pseudeumeces and Janosikia were faunivorous, thus demonstrating that insularity, not large body size, was crucial to the evolution of herbivory in this lineage. Body size change in Gallotia was more complex than previously thought, encompassing size increase [e.g. in the extinct Gallotia goliath (Mertens, 1942)], but more commonly involving miniaturization. The physical environment may play a crucial role in modulating the evolution of body size in this natural laboratory.     

Biomedical question answering using semantic relations

Background

The proliferation of the scientific literature in the field of biomedicine makes it difficult to keep abreast of current knowledge, even for domain experts. While general Web search engines and specialized information retrieval (IR) systems have made important strides in recent decades, the problem of accurate knowledge extraction from the biomedical literature is far from solved. Classical IR systems usually return a list of documents that have to be read by the user to extract relevant information. This tedious and time-consuming work can be lessened with automatic Question Answering (QA) systems, which aim to provide users with direct and precise answers to their questions. In this work we propose a novel methodology for QA based on semantic relations extracted from the biomedical literature.

Results

We extracted semantic relations with the SemRep natural language processing system from 122,421,765 sentences, which came from 21,014,382 MEDLINE citations (i.e., the complete MEDLINE distribution up to the end of 2012). A total of 58,879,300 semantic relation instances were extracted and organized in a relational database. The QA process is implemented as a search in this database, which is accessed through a Web-based application, called SemBT (available at http://sembt.mf.uni-lj.si). We conducted an extensive evaluation of the proposed methodology in order to estimate the accuracy of extracting a particular semantic relation from a particular sentence. Evaluation was performed by 80 domain experts. In total 7,510 semantic relation instances belonging to 2,675 distinct relations were evaluated 12,083 times. The instances were evaluated as correct 8,228 times (68%).

Conclusions

In this work we propose an innovative methodology for biomedical QA. The system is implemented as a Web-based application that is able to provide precise answers to a wide range of questions. A typical question is answered within a few seconds. The tool has some extensions that make it especially useful for interpretation of DNA microarray results.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0365-3) contains supplementary material, which is available to authorized users.     

Outcome of the first electron microscopy validation task force meeting

This Meeting Review describes the proceedings and conclusions from the inaugural meeting of the Electron Microscopy Validation Task Force organized by the Unified Data Resource for 3DEM (http://www.emdatabank.org) and held at Rutgers University in New Brunswick, NJ on September 28 and 29, 2010. At the workshop, a group of scientists involved in collecting electron microscopy data, using the data to determine three-dimensional electron microscopy (3DEM) density maps, and building molecular models into the maps explored how to assess maps, models, and other data that are deposited into the Electron Microscopy Data Bank and Protein Data Bank public data archives. The specific recommendations resulting from the workshop aim to increase the impact of 3DEM in biology and medicine.