The cyto-linker and scaffolding protein "plectin" mis-localization leads to softening of cancer cells.

Discovering tools to prevent cancer progression requires understanding the fundamental differences between normal and cancer cells. More than a decade ago, atomic force microscopy (AFM) revealed cancer cells' softer body compared to their healthy counterparts. Here, we investigated the mechanism underlying the softening of cancerous cells in comparison with their healthy counterparts based on AFM high resolution stiffness tomography and 3D confocal microscopy. We showed microtubules (MTs) network in invasive ductal carcinoma cell cytoskeleton is basally located and segmented for around 400 nm from the cell periphery. Additionally, the cytoskeleton scaffolding protein plectin exhibits a mis-localization from the cytoplasm to the surface of cells in the carcinoma which justifies the dissociation of the MT network from the cell's cortex. Furthermore, the assessment of MTs' persistence length using a worm-like-chain (WLC) model in high resolution AFM images showed lower persistence length of the single MTs in ductal carcinoma compared to that in the normal state. Overall, these tuned mechanics support the invasive cells to ascertain more flexibility under compressive forces in small deformations. These data provide new insights into the structural origins of cancer aids in progression.

The Bats of Greece: An Updated Review of Their Distribution, Ecology and Conservation.

Bats of Greece have been studied since the second half of the 19th century. Their distribution and ecology, however, remain poorly understood. Conservation efforts for the protection of the roosting and foraging habitats of their populations in Greece are limited. To date, 35 bat species have been recorded from Greece. Four species (Eptesicus anatolicus, Plecotus auritus, Myotis brandtii and Rousettus aegyptiacus) have a limited distribution in the country and the presence of one species (Myotis mystacinus) requires verification. The present study summarises all existing knowledge and adds several hundred new records on the distribution of bats of Greece. Additionally, it provides a summary of new insights on various aspects of their roosting ecology, foraging habitat use, altitudinal distribution, winter activity and landscape characteristics around major roosts. Finally, it discusses the current research and conservation needs of Greek bats.

Self-Assembly of Polymer-Modified FePt Magnetic Nanoparticles and Block Copolymers.

The fabrication of nanocomposites containing magnetic nanoparticles is gaining interest as a model for application in small electronic devices. The self-assembly of block copolymers (BCPs) makes these materials ideal for use as a soft matrix to support the structural ordering of the nanoparticles. In this work, a high-molecular-weight polystyrene-b-poly(methyl methacrylate) block copolymer (PS-b-PMMA) was synthesized through anionic polymerization. The influence of the addition of different ratios of PMMA-coated FePt nanoparticles (NPs) on the self-assembled morphology was investigated using transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS). The self-assembly of the NPs inside the PMMA phase at low particle concentrations was analyzed statistically, and the negative effect of higher particle ratios on the lamellar BCP morphology became visible. The placement of the NPs inside the PMMA phase was also compared to theoretical descriptions. The magnetic addressability of the FePt nanoparticles inside the nanocomposite films was finally analyzed using bimodal magnetic force microscopy and proved the magnetic nature of the nanoparticles inside the microphase-separated BCP films.

Probing local lateral forces of focal adhesions and cell-cell junctions of living cells by torsional force spectroscopy.

The number and strength of mechanical connections of cells to their local environment can be indicative of their migration and invasion potential. Gaining direct access to the mechanical properties of individual connections and bringing them into a relationship with the state of disease, however, is a formidable task. Here, we present a method to directly sense focal adhesions and cell-cell contacts with a force sensor to quantify the lateral forces of their anchoring points. We found local lateral forces of 1.0-1.5 nN for focal adhesions and slightly higher values at the interfaces between cells where cell-cell contacts are located. Interestingly, a modified surface layer was observed exhibiting considerably reduced tip friction directly next to the area of a retracting cell edge on the substrate. We expect that this technique can improve the understanding of the relationship between mechanical properties of cell connections and the pathological state of cells in the future.

Torsional and lateral eigenmode oscillations for atomic resolution imaging of HOPG in air under ambient conditions.

Combined in-plane and out-of-plane multifrequency atomic force microscopy techniques have been demonstrated to be important tools to decipher spatial differences of sample surfaces at the atomic scale. The analysis of physical properties perpendicular to the sample surface is routinely achieved from flexural cantilever oscillations, whereas the interpretation of in-plane sample properties via force microscopy is still challenging. Besides the torsional oscillation, there is the additional option to exploit the lateral oscillation of the cantilever for in-plane surface analysis. In this study, we used different multifrequency force microscopy approaches to attain better understanding of the interactions between a super-sharp tip and an HOPG surface focusing on the discrimination between friction and shear forces. We found that the lateral eigenmode is suitable for the determination of the shear modulus whereas the torsional eigenmode provides information on local friction forces between tip and sample. Based on the results, we propose that the full set of elastic constants of graphite can be determined from combined in-plane and out-of-plane multifrequency atomic force microscopy if ultrasmall amplitudes and high force constants are used.

Micro-sized open-source and low-cost GPS loggers below 1 g minimise the impact on animals while collecting thousands of fixes.

GPS-enabled loggers have been proven as valuable tools for monitoring and understanding animal movement, behaviour and ecology. While the importance of recording accurate location estimates is well established, deployment on many, especially small species, has been limited by logger mass and cost. We developed an open-source and low-cost 0.65 g GPS logger with a simple smartphone-compatible user interface, that can record more than 10,000 GPS fixes on a single 30 mAh battery charge (resulting mass including battery: 1.3 g). This low-budget 'TickTag' (currently 32 USD) allows scientists to scale-up studies while becoming a 'wearable' for larger animals and simultaneously enabling high-definition studies on small animals. Tests on two different species (domestic dog, Canis lupus familiaris and greater mouse-eared bats, Myotis myotis) showed that our combination of optimised hardware design and software-based recording strategies increases the number of achievable GPS fixes per g device mass compared to existing micro-sized solutions. We propose that due to the open-source access, as well as low cost and mass, the TickTag fills a technological gap in wildlife ecology and will open up new possibilities for wildlife research and conservation.

Standardization of molecular monitoring of CML: results and recommendations from the European treatment and outcome study.

Standardized monitoring of BCR::ABL1 mRNA levels is essential for the management of chronic myeloid leukemia (CML) patients. From 2016 to 2021 the European Treatment and Outcome Study for CML (EUTOS) explored the use of secondary, lyophilized cell-based BCR::ABL1 reference panels traceable to the World Health Organization primary reference material to standardize and validate local laboratory tests. Panels were used to assign and validate conversion factors (CFs) to the International Scale and assess the ability of laboratories to assess deep molecular response (DMR). The study also explored aspects of internal quality control. The percentage of EUTOS reference laboratories (n = 50) with CFs validated as optimal or satisfactory increased from 67.5% to 97.6% and 36.4% to 91.7% for ABL1 and GUSB, respectively, during the study period and 98% of laboratories were able to detect MR4.5 in most samples. Laboratories with unvalidated CFs had a higher coefficient of variation for BCR::ABL1IS and some laboratories had a limit of blank greater than zero which could affect the accurate reporting of DMR. Our study indicates that secondary reference panels can be used effectively to obtain and validate CFs in a manner equivalent to sample exchange and can also be used to monitor additional aspects of quality assurance.

Conceptual Framework for Dislocation-Modified Conductivity in Oxide Ceramics Deconvoluting Mesoscopic Structure, Core, and Space Charge Exemplified for SrTiO3.

The introduction of dislocations is a recently proposed strategy to tailor the functional and especially the electrical properties of ceramics. While several works confirm a clear impact of dislocations on electrical conductivity, some studies raise concern in particular when expanding to dislocation arrangements beyond a geometrically tractable bicrystal interface. Moreover, the lack of a complete classification on pertinent dislocation characteristics complicates a systematic discussion and hampers the design of dislocation-modified electrical conductivity. We proceed by mechanically introducing dislocations with three different mesoscopic structures into the model material single-crystal SrTiO3 and extensively characterizing them from both a mechanical as well as an electrical perspective. As a final result, a deconvolution of mesoscopic structure, core structure, and space charge enables us to obtain the complete picture of the effect of dislocations on functional properties, focusing here on electric properties.

Integration of the ImageJ Ecosystem in the KNIME Analytics Platform.

Open-source software tools are often used for analysis of scientific image data due to their flexibility and transparency in dealing with rapidly evolving imaging technologies. The complex nature of image analysis problems frequently requires many tools to be used in conjunction, including image processing and analysis, data processing, machine learning and deep learning, statistical analysis of the results, visualization, correlation to heterogeneous but related data, and more. However, the development, and therefore application, of these computational tools is impeded by a lack of integration across platforms. Integration of tools goes beyond convenience, as it is impractical for one tool to anticipate and accommodate the current and future needs of every user. This problem is emphasized in the field of bioimage analysis, where various rapidly emerging methods are quickly being adopted by researchers. ImageJ is a popular open-source image analysis platform, with contributions from a global community resulting in hundreds of specialized routines for a wide array of scientific tasks. ImageJ's strength lies in its accessibility and extensibility, allowing researchers to easily improve the software to solve their image analysis tasks. However, ImageJ is not designed for development of complex end-to-end image analysis workflows. Scientists are often forced to create highly specialized and hard-to-reproduce scripts to orchestrate individual software fragments and cover the entire life-cycle of an analysis of an image dataset. KNIME Analytics Platform, a user-friendly data integration, analysis, and exploration workflow system, was designed to handle huge amounts of heterogeneous data in a platform-agnostic, computing environment and has been successful in meeting complex end-to-end demands in several communities, such as cheminformatics and mass spectrometry. Similar needs within the bioimage analysis community led to the creation of the KNIME Image Processing extension which integrates ImageJ into KNIME Analytics Platform, enabling researchers to develop reproducible and scalable workflows, integrating a diverse range of analysis tools. Here we present how users and developers alike can leverage the ImageJ ecosystem via the KNIME Image Processing extension to provide robust and extensible image analysis within KNIME workflows. We illustrate the benefits of this integration with examples, as well as representative scientific use cases.

Connecting High School Students With Nature - How Different Guided Tours in the Zoo Influence the Success of Extracurricular Educational Programs.

Zoos attract millions of visitors every year, many of whom are schoolchildren. For this reason, zoos are important institutions for the environmental education of future generations. Empirical studies on the educational impact of environmental education programs in zoos are still rare. To address this issue, we conducted two studies: In study 1, we investigated students' interests in different biological topics, including zoos (n = 1,587). Data analysis of individual topics revealed large differences of interest, with advanced students showing less interest in zoos. In study 2, we invited school classes of this age group to visit different guided tours at the zoo and tested connection to nature before and after each educational intervention (n = 608). The results showed that the guided tours are an effective tool to raise students' connection to nature. Add-on components have the potential to further promote connection to nature. The education programs are most effective with students with a low initial nature connection.

Patterns of mtDNA introgression suggest population replacement in Palaearctic whiskered bat species.

Secondary contacts can play a major role in the evolutionary histories of species. Various taxa diverge in allopatry and later on come into secondary contact during range expansions. When they meet, their interactions and the extent of gene flow depend on the level of their ecological differentiation and the strength of their reproductive isolation. In this study, we present the multilocus phylogeography of two cryptic whiskered bat species, Myotis mystacinus and M. davidii, with a particular focus on their putative sympatric zone. Our findings suggest that M. mystacinus and M. davidii evolved in allopatry and came into secondary contact during range expansions. Individuals in the area of secondary contact, in Anatolia and the Balkans, have discordant population assignments based on the mitochondrial and the nuclear datasets. These observed patterns suggest that the local M. mystacinus populations hybridized with expanding M. davidii populations, which resulted in mitochondrial introgression from the former. In the introgression area, M. mystacinus individuals with concordant nuclear and mitochondrial genotypes were identified in relatively few locations, suggesting that the indigenous populations might have been largely replaced by invading M. davidii. Changing environmental conditions coupled with ecological competition is the likely reason for this replacement. Our study presents one possible example of a historical population replacement that was captured in phylogeographic patterns.

Carcinomas with Occult Metastasis Potential: Diagnosis/Prognosis Accuracy Improvement by Means of Force Spectroscopy.

Accurate diagnosis of cancer stage is inevitable for the following prognosis in patients struggling with these lesions to promote their health and survival rate. Previous studies on survival rate statistics show, in some cases, failure in cancer stage surveys in which metastasis or recurrence of the disease was not accurately prognosed. Morphology study of cancer cells advances the understanding about cancer behavior and its progression, in which, in our previous study on invasive cancer cells, fewer formations of cytoskeleton components compared to their counterparts was observed. Here it is shown that carcinomas with an occult propensity of metastasis depict a number of poorly differentiated cells with decreased amounts of cytoskeleton components in a near-well differentiated population. Force spectroscopy in conjunction with fluorescence microscopy of lung cancer, liver hepatoma, and melanoma provides a general view of these cells' architecture, leading to the conclusion that the scarce abnormal-shaped cells with low formation of structural filaments convey the high risk of metastatic potential of the tumor. The results demonstrate that force spectroscopy complements conventional diagnostic approaches by an accurate cytoskeleton assessment and can improve the following prognosis in epithelial cancers with occult metastasis risk.

Development of a neural rosette formation assay (RoFA) to identify neurodevelopmental toxicants and to characterize their transcriptome disturbances.

The first in vitro tests for developmental toxicity made use of rodent cells. Newer teratology tests, e.g. developed during the ESNATS project, use human cells and measure mechanistic endpoints (such as transcriptome changes). However, the toxicological implications of mechanistic parameters are hard to judge, without functional/morphological endpoints. To address this issue, we developed a new version of the human stem cell-based test STOP-tox(UKN). For this purpose, the capacity of the cells to self-organize to neural rosettes was assessed as functional endpoint: pluripotent stem cells were allowed to differentiate into neuroepithelial cells for 6 days in the presence or absence of toxicants. Then, both transcriptome changes were measured (standard STOP-tox(UKN)) and cells were allowed to form rosettes. After optimization of staining methods, an imaging algorithm for rosette quantification was implemented and used for an automated rosette formation assay (RoFA). Neural tube toxicants (like valproic acid), which are known to disturb human development at stages when rosette-forming cells are present, were used as positive controls. Established toxicants led to distinctly different tissue organization and differentiation stages. RoFA outcome and transcript changes largely correlated concerning (1) the concentration-dependence, (2) the time dependence, and (3) the set of positive hits identified amongst 24 potential toxicants. Using such comparative data, a prediction model for the RoFA was developed. The comparative analysis was also used to identify gene dysregulations that are particularly predictive for disturbed rosette formation. This 'RoFA predictor gene set' may be used for a simplified and less costly setup of the STOP-tox(UKN) assay.

pH-depended protein shell dis- and reassembly of ferritin nanoparticles revealed by atomic force microscopy.

Ferritin, a protein that is present in the human body for a controlled iron storage and release, consists of a ferrihydrite core and a protein shell. Apoferritin, the empty shell of ferritin, can be modified to carry tailored properties exploitable for targeted and direct drug delivery. This protein shell has the ability to dis- and reassemble depending on the pH value of the liquid environment and can thus be filled with the desired substance. Here we observed the dis- and reassembly process of the protein shell of ferritin and apoferritin in situ and in real space using atomic force microscopy. Ferritin and apoferritin nanoparticles adsorbed on a mica substrate exhibited a change in their size by varying the pH value of the surrounding medium. Lowering the pH value of the solution led to a decrease in size of the nanoparticles whereas a successive increase of the pH value increased the particle size again. The pH dependent change in size could be related to the dis- and reassembling of the protein shell of ferritin and apoferritin. Supplementary imaging by bimodal magnetic force microscopy of ferritin molecules accomplished in air revealed a polygonal shape of the core and a three-fold symmetry of the protein shell providing valuable information about the substructure of the nanoparticles.

Nanomechanical sub-surface mapping of living biological cells by force microscopy.

Atomic force microscopy allows for the nanomechanical surface characterization of a multitude of types of materials with highest spatial precision in various relevant environments. In recent years, researchers have refined this methodology to analyze living biological materials in vitro. The atomic force microscope thus has become an essential instrument for the (in many cases) non-destructive, high-resolution imaging of cells and visualization of their dynamic mechanical processes. Mapping force versus distance curves and the local evaluation of soft samples allow the operator to "see" beneath the sample surface and to capture the local mechanical properties. In this work, we combine atomic force microscopy with fluorescence microscopy to investigate cancerous epithelial breast cells in culture medium. With unprecedented spatial resolution, we provide tomographic images for the local elasticity of confluent layers of cells. For these particular samples, a layer of higher elastic modulus located directly beneath the cell membrane in comparison with the average elastic properties was observed. Strikingly, this layer appears to be perforated at unique locations of the sample surface of weakest mechanical properties where distinct features were visible permitting the tip to indent farthest into the cell's volume. We interpret this layer as the cell membrane mechanically supported by the components of the cytoskeleton that is populated with sites of integral membrane proteins. These proteins act as breaking points for the indenter thus explaining the mechanical weakness at these locations. In contrast, the highest mechanical strength of the cell was found at locations of the cell cores as cross-checked by fluorescence microscopy images of staining experiments, in particular at nucleoli sites as the cumulative elastic modulus there comprises cytoskeletal features and the tight packing ribosomal DNA of the cell.

Fluoropolymer-Containing Opals and Inverse Opals by Melt-Shear Organization.

The preparation of highly ordered colloidal architectures has attracted significant attention and is a rapidly growing field for various applications, e.g., sensors, absorbers, and membranes. A promising technique for the preparation of elastomeric inverse opal films relies on tailored core/shell particle architectures and application of the so-called melt-shear organization technique. Within the present work, a convenient route for the preparation of core/shell particles featuring highly fluorinated shell materials as building blocks is described. As particle core materials, both organic or inorganic (SiO2) particles can be used as a template, followed by a semi-continuous stepwise emulsion polymerization for the synthesis of the soft fluoropolymer shell material. The use of functional monomers as shell-material offers the possibility to create opal and inverse opal films with striking optical properties according to Bragg's law of diffraction. Due to the presence of fluorinated moieties, the chemical resistance of the final opals and inverse opals is increased. The herein developed fluorine-containing particle-based films feature a low surface energy for the matrix material leading to good hydrophobic properties. Moreover, the low refractive index of the fluoropolymer shell compared to the core (or voids) led to excellent optical properties based on structural colors. The herein described fluoropolymer opals and inverse opals are expected to pave the way toward novel functional materials for application in fields of coatings and optical sensors.

Structural analysis of healthy and cancerous epithelial-type breast cells by nanomechanical spectroscopy allows us to obtain peculiarities of the skeleton and junctions.

The transition of healthy epithelial cells to carcinoma is associated with an alteration in the structure and organization of the cytoskeleton of the cells. A comparison of the mechanical properties of cancerous and healthy cells indicated a higher deformability of the cancer cells based on averaging the mechanical properties of single cells. However, the exact reason for softening of the cancerous cells compared to their counterparts remains unclear. Here, we focused on nanomechanical spectroscopy of healthy and cancerous ductal epithelial-type breast cells by means of atomic force microscopy with high lateral and depth precision. As a result, based on atomic force microscopy measurements formation of significantly fewer microtubules in cancerous cells which was observed in our study is most likely one of the main causes for the overall change in mechanical properties without any phenotypic shift. Strikingly, in a confluent layer of invasive ductal carcinoma cells, we observed the formation of cell-cell junctions that have the potential for signal transduction among neighboring cells such as desmosomes and adherens junctions. This increases the possibility of cancerous cell collaboration in malignancy, infiltration or metastasis phenomena.

Integrative genomic analysis reveals cancer-associated mutations at diagnosis of CML in patients with high-risk disease.

Genomic events associated with poor outcome in chronic myeloid leukemia (CML) are poorly understood. We performed whole-exome sequencing, copy-number variation, and/or RNA sequencing for 65 patients to discover mutations at diagnosis and blast crisis (BC). Forty-six patients with chronic-phase disease with the extremes of outcome were studied at diagnosis. Cancer gene variants were detected in 15 (56%) of 27 patients with subsequent BC or poor outcome and in 3 (16%) of 19 optimal responders (P = .007). Frequently mutated genes at diagnosis were ASXL1, IKZF1, and RUNX1 The methyltransferase SETD1B was a novel recurrently mutated gene. A novel class of variant associated with the Philadelphia (Ph) translocation was detected at diagnosis in 11 (24%) of 46 patients comprising fusions and/or rearrangement of genes on the translocated chromosomes, with evidence of fragmentation, inversion, and imperfect sequence reassembly. These were more frequent at diagnosis in patients with poor outcome: 9 (33%) of 27 vs 2 (11%) of 19 optimal responders (P = .07). Thirty-nine patients were tested at BC, and all had cancer gene variants, including ABL1 kinase domain mutations in 58%. However, ABL1 mutations cooccurred with other mutated cancer genes in 89% of cases, and these predated ABL1 mutations in 62% of evaluable patients. Gene fusions not associated with the Ph translocation occurred in 42% of patients at BC and commonly involved fusion partners that were known cancer genes (78%). Genomic analysis revealed numerous relevant variants at diagnosis in patients with poor outcome and all patients at BC. Future refined biomarker testing of specific variants will likely provide prognostic information to facilitate a risk-adapted therapeutic approach.

Effect of ABCG2, OCT1, and ABCB1 (MDR1) Gene Expression on Treatment-Free Remission in a EURO-SKI Subtrial.

INTRODUCTION: Tyrosine kinase inhibitors (TKIs) can safely be discontinued in chronic myeloid leukemia (CML) patients with sustained deep molecular response. ABCG2 (breast cancer resistance protein), OCT1 (organic cation transporter 1), and ABCB1 (multidrug resistance protein 1) gene products are known to play a crucial role in acquired pharmacogenetic TKI resistance. Their influence on treatment-free remission (TFR) has not yet been investigated. MATERIALS AND METHODS: RNA was isolated on the last day of TKI intake from peripheral blood leukocytes of 132 chronic phase CML patients who discontinued TKI treatment within the European Stop Tyrosine Kinase Inhibitor Study trial. Plasmid standards were designed including subgenic inserts of OCT1, ABCG2, and ABCB1 together with GUSB as reference gene. For expression analyses, quantitative real-time polymerase chain reaction was used. Multiple Cox regression analysis was performed. In addition, gene expression cutoffs for patient risk stratification were investigated. RESULTS: The TFR rate of 132 patients, 12 months after TKI discontinuation, was 54% (95% confidence interval [CI], 46%-62%). ABCG2 expression (‰) was retained as the only significant variable (P = .02; hazard ratio, 1.04; 95% CI, 1.01-1.07) in multiple Cox regression analysis. Only for the ABCG2 efflux transporter, a significant cutoff was found (P = .04). Patients with an ABCG2/GUSB transcript level >4.5‰ (n = 93) showed a 12-month TFR rate of 47% (95% CI, 37%-57%), whereas patients with low ABCG2 expression (≤4.5‰; n = 39) had a 12-month TFR rate of 72% (95% CI, 55%-82%). CONCLUSION: In this study, we investigated the effect of pharmacogenetics in the context of a CML treatment discontinuation trial. The transcript levels of the efflux transporter ABCG2 predicted TFR after TKI discontinuation.

From SOMDA to application - integration strategies in the OR.NET demonstration sites.

The effective development and dissemination of the open integration for the next generation of operating rooms require a comprehensive testing environment. In this paper, we present the various challenges to be addressed in demonstration applications, and we discuss the implementation approach, the foci of the demonstration sites and the evaluation efforts. Overall, the demonstrator setups have proven the feasibility of the service-oriented medical device architecture (SOMDA) and real-time approaches with a large variety of example applications. The applications demonstrate the potentials of open device interoperability. The demonstrator implementations were technically evaluated as well as discussed with many clinicians from various disciplines. However, the evaluation is still an ongoing research at the demonstration sites. Technical evaluation focused on the properties of a network of medical devices, latencies in data transmission and stability. A careful evaluation of the SOMDA design decisions and implementations are essential to a safe and reliable interoperability of integrated medical devices and information technology (IT) system in the especially critical working environment. The clinical evaluation addressed the demands of future users and stakeholders, especially surgeons, anesthesiologists, scrub nurses and hospital operators. The opinions were carefully collected to gain further insights into the potential benefits of the technology and pitfalls in future work.