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Measurements of Young’s moduli are mostly evaluated using strong assumptions, such as sample homogeneity and isotropy. At the same time, descriptions of measurement parameters often lack detailed specifications. Many of these assumptions are, for soft hydrogels especially, not completely valid and the complexity of hydrogel microindentation demands more sophisticated experimental procedures in order to describe their elastic properties more accurately. We created an algorithm that automates indentation data analysis as a basis for the evaluation of large data sets with consideration of the influence of indentation depth on the measured Young’s modulus. The algorithm automatically determines the Young’s modulus in indentation regions where it becomes independent of the indentation depth and furthermore minimizes the error from fitting an elastic model to the data. This approach is independent of the chosen elastic fitting model and indentation device. With this, we are able to evaluate large amounts of indentation curves recorded on many different sample positions and can therefore apply statistical methods to overcome deviations due to sample inhomogeneities. To prove the applicability of our algorithm, we carried out a systematic analysis of how the indentation speed, indenter size and sample thickness affect the determination of Young’s modulus from atomic force microscope (AFM) indentation curves on polyacrylamide (PAAm) samples. We chose the Hertz model as the elastic fitting model for this proof of principle of our algorithm and found that all of these parameters influence the measured Young’s moduli to a certain extent. Hence, it is essential to clearly state the experimental parameters used in microindentation experiments to ensure reproducibility and comparability of data. [ABSTRACT FROM AUTHOR]

Lectins are a group of ubiquitous proteins which specifically recognize and reversibly bind sugar moieties of glycoprotein and glycolipid constituents on cell surfaces. The mutagenesis approach is often employed to characterize lectin binding properties. As lectins are not enzymes, it is not easy to perform a rapid specificity screening of mutants using chromogenic substrates. It is necessary to use different binding assays such as isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), microscale thermophoresis (MST), enzyme-linked lectin assays (ELLA), or glycan arrays for their characterization. These methods often require fluorescently labeled proteins (MST), highly purified proteins (SPR) or high protein concentrations (ITC). Mutant proteins may often exhibit problematic behaviour, such as poor solubility or low stability. Lectin-based cell agglutination is a simple and low-cost technique which can overcome most of these problems. In this work, a modified method of the agglutination of human erythrocytes and yeast cells with microscopy detection was successfully used for a specificity study of the newly prepared mutant lectin RS-IIL_A22S, which experimentally completed studies on sugar preferences of lectins in the PA-IIL family. Results showed that the sensitivity of this method is comparable with ITC, is able to determine subtle differences in lectin specificity, and works directly in cell lysates. The agglutination method with microscopy detection was validated by comparison of the results with results obtained by agglutination assay in standard 96-well microtiter plate format. In contrast to this assay, the microscopic method can clearly distinguish between hemagglutination and hemolysis. Therefore, this method is suitable for examination of lectins with known hemolytic activity as well as mutant or uncharacterized lectins, which could damage red blood cells. This is due to the experimental arrangement, which includes very short sample incubation time in combination with microscopic detection of agglutinates, that are easily observed by a small portable microscope. [ABSTRACT FROM AUTHOR]

Once in the ocean, plastics are rapidly colonized by complex microbial communities. Factors affecting the development and composition of these communities are still poorly understood. Additionally, whether there are plastic-type specific communities developing on different plastics remains enigmatic. We determined the development and succession of bacterial communities on different plastics under ambient and dim light conditions in the coastal Northern Adriatic over the course of two months using scanning electron microscopy and 16S rRNA gene analyses. Plastics used were low- and high-density polyethylene (LDPE and HDPE, respectively), polypropylene (PP) and polyvinyl chloride with two typical additives (PVC DEHP and PVC DINP). The bacterial communities developing on the plastics clustered in two groups; one group was found on PVC and the other group on all the other plastics and on glass, which was used as an inert control. Specific bacterial taxa were found on specific surfaces in essentially all stages of biofilm development and in both ambient and dim light conditions. Differences in bacterial community composition between the different plastics and light exposures were stronger after an incubation period of one week than at the later stages of the incubation. Under both ambient and dim light conditions, one part of the bacterial community was common on all plastic types, especially in later stages of the biofilm development, with families such as Flavobacteriaceae, Rhodobacteraceae, Planctomycetaceae and Phyllobacteriaceae presenting relatively high relative abundances on all surfaces. Another part of the bacterial community was plastic-type specific. The plastic-type specific fraction was variable among the different plastic types and was more abundant after one week of incubation than at later stages of the succession. [ABSTRACT FROM AUTHOR]

An organism’s ability to mount a physiological response to external stressors is fundamental to its interaction with the environment. Experimental exploration of these interactions benefits greatly from the ability to maintain tight control of the environment, even under conditions in which it would be normal for the subject to flee the stressor. Here we present a nematode research platform that pairs automated image acquisition and analysis with a custom microfluidic device. This platform enables tight environmental control in low-density, single-worm arenas, which preclude animal escape while still allowing a broad range of behavioral activities. The platform is easily scalable, with two 50 arena arrays per chip and an imaging capacity of 600 animals per scanning device. Validating the device using dietary, osmotic, and oxidative stress indicates that it should be of broad use as a research platform, including eventual adaptation for additional stressors, anthelmintic-drug screening, and toxicology studies. [ABSTRACT FROM AUTHOR]

Adhesive interactions of soft materials play an important role in nature and technology. Interaction energies can be quantified by determining contact areas of deformable microparticles with the help of reflection interference contrast microscopy (RICM). For high throughput screening of adhesive interactions, a method to automatically evaluate large amounts of interacting microparticles was developed. An image is taken which contains circular interference patterns with visual characteristics that depend on the probe’s shape due to its surface interaction. We propose to automatically detect radial profiles in images, and to measure the contact radius and size of the spherical probe, allowing the determination of particle-surface interaction energy in a simple and fast imaging and image analysis setup. To achieve this, we analyze the image gradient and we perform template matching that utilizes the physical foundations of reflection interference contrast microscopy. [ABSTRACT FROM AUTHOR]

Adherence of the microorganism to submerged solid surfaces leads to biofilm formation. Biofilm formation modifies the surfaces in favor of bacteria facilitating the survival of the bacteria under different stressed conditions. On the other hand, the formation of biofilm has a direct adverse economic impact in various industries and more importantly in medical practices. This adherence is the reason for the failure of many indwelling medical devices. Surface biofilm adhesion is the key to biofilm growth and stability. Hence this adhesion needs to be substantially lowered to inhibit biofilm stability. Both chemical and physical properties of the surface influence biofilm formation and modulating these properties can control this formation. In this study, we have investigated the effect of Hydrofluoric acid (HF), at a specific concentration as an etchant, on the surface morphology of substrates and the growth of biofilms of Pseudomonas aeruginosa. and Staphylococcus aureus. We find that the bacterial counts on the etched surfaces undergo a periodic increase and decrease. This, on one hand, shows the close correlation between the biofilm growth and the particular roughness scale, and on the other hand, explains the existing contradictory results regarding the effects of etching on substrate roughness and biofilm growth. We propose a simple model of a sequence of hole formation, hole expansion and etching away of the hole walls to form a new, comparatively smooth surface, coupled with the preferential accumulation of bacteria at the hole edges, to explain these periodicities. [ABSTRACT FROM AUTHOR]

Local gene delivery systems utilizing RNA interference technology are a promising approach for therapeutic applications where site-specific release of agents is desired. Polyelectrolyte multilayers (PEMs) can be constructed using the layer-by-layer (LbL) technique and serve as a depot for bioactive substances, which can then be released in a controlled manner. Multilayers of hyaluronic acid/poly(ethylenimine) HA/PEI were built up with different numbers of bilayers and PEI-siRNA particles were embedded in bioactive layers for gene silencing. The increase of the bilayers and the release of siRNA particles were demonstrated by fluorescence intensity measurement with a fluorescence reader. Two different LbL techniques were tested for the reduction of ICAM–1 expression in EA.hy926: PEM build-up by dipping or drying steps, respectively. Herein, the drying technique of the bioactive layers with ICAM siRNA mediated a significant reduction of the ICAM–1 expression from 3 to 24 bilayers. The fluorescent siRNA release study and the re-culturing of the HA/PEI films demonstrated a release of the transfection particles within the first hour. The advantage of dried built-up PEMs compared to a dried monolayer of PEI-siRNA particles with the same siRNA concentration was a significant higher amount of viable cells. [ABSTRACT FROM AUTHOR]

The biofilm-forming ability of Burkholderia pseudomallei is crucial for its survival in unsuitable environments and is correlated with antibiotic resistance and relapsing cases of melioidosis. Extracellular DNA (eDNA) is an essential component for biofilm development and maturation in many bacteria. The aim of this study was to investigate the eDNA released by B. pseudomallei during biofilm formation using DNase treatment. The extent of biofilm formation and quantity of eDNA were assessed by crystal-violet staining and fluorescent dye-based quantification, respectively, and visualized by confocal laser scanning microscopy (CLSM). Variation in B. pseudomallei biofilm formation and eDNA quantity was demonstrated among isolates. CLSM images of biofilms stained with FITC-ConA (biofilm) and TOTO-3 (eDNA) revealed the localization of eDNA in the biofilm matrix. A positive correlation of biofilm biomass with quantity of eDNA during the 2-day biofilm-formation observation period was found. The increasing eDNA quantity over time, despite constant living/dead ratios of bacterial cells during the experiment suggests that eDNA is delivered from living bacterial cells. CLSM images demonstrated that depletion of eDNA by DNase I significantly lessened bacterial attachment (if DNase added at 0 h) and biofilm developing stages (if added at 24 h) but had no effect on mature biofilm (if added at 45 h). Collectively, our results reveal that eDNA is released from living B. pseudomallei and is correlated with biofilm formation. It was also apparent that eDNA is essential during bacterial cell attachment and biofilm-forming steps. The depletion of eDNA by DNase may provide an option for the prevention or dispersal of B. pseudomallei biofilm. [ABSTRACT FROM AUTHOR]

Numerous technologies exist to detect circulating tumor cells (CTCs), although reports on cytological detection of CTCs remain limited. We recently developed a cytology-based CTC detection device using glass slides and light microscopy. In this study, we automated this previously manual device to improve its efficiency and cost effectiveness for clinical applications. We conducted a pilot study using this device to compare CTCs in peripheral blood (PB) and draining venous blood (DVB) from patients with colorectal cancer (CRC). The cytology-based automated CTC detection platform consisted of a disposable filtration device with a three-dimensional (3D) metal filter and multichannel automated CTC enrichment device. This platform allowed rapid and gentle filtration of CTCs and their efficient transfer from the filter to glass slides for subsequent Papanicolaou (Pap) and immunocytochemical (ICC) staining. Cytological diagnosis of CTCs was performed by observing permanent glass slide specimens by light microscopy. The current pilot clinical study enrolled CRC patients (n = 26) with stage I–IV tumors, who underwent surgery. PB was collected before surgery, and DVB was obtained from the mesenteric vein immediately after resection. Based on the CTC morphology obtained from PB and DVB samples, we proposed the following cytological criteria for the diagnosis of CTCs: pan-cytokeratin-positive, atypical cells with malignant morphological features identified by Pap staining. The numbers of CTCs defined by these criteria were significantly higher in DVB than PB from CRC patients (p<0.01), and the number of CTCs in DVB was increased significantly with stage progression (p<0.05). These results suggest that DVB may be another potential source of CTCs other than PB for liquid biopsies including downstream analysis. This automated cytology-based CTC detection device therefore provides a unique and powerful tool to investigate the significance of CTCs in CRC patients in a clinical setting. [ABSTRACT FROM AUTHOR]

In this study, we utilized a stainless steel (SUS304) plate for measuring the Raman scattering spectra of body fluid samples. Using this stainless steel plate, we recorded the Raman scattering spectra of 99.5% ethanol and human serum samples by performing irradiation with 785- and 1064-nm lasers. Raman scattering spectra with intensities equal to or greater than those reported previously were obtained. In addition, the Raman scattering spectra acquired using the 1064-nm laser were less influenced by autofluorescence than those obtained via use of the shorter-wavelength laser. Moreover, the shapes of the spectra did not show any dependence on integration time, and denaturation of the samples was minimal. Our method, based on 1064-nm laser and the stainless steel plate, provides performance equal to or better than the methods reported thus far for the measurement of Raman scattering spectra from liquid samples. This method can be employed to rapidly evaluate the components of serum in liquid form without using surface-enhanced Raman scattering. [ABSTRACT FROM AUTHOR]

Atmospheric methane is rapidly lost when it enters humid subterranean critical and vadose zones (e.g., air in soils and caves). Because methane is a source of carbon and energy, it can be consumed by methanotrophic methane-oxidizing bacteria. As an additional subterranean sink, it has been hypothesized that methane is oxidized by natural radioactivity-induced radiolysis that produces energetic ions and radicals, which then trigger abiotic oxidation and consumption of methane within a few hours. Using controlled laboratory experiments, we tested whether radiolysis could rapidly oxidize methane in sealed air with different relative humidities while being exposed to elevated levels of radiation (more than 535 kBq m-3) from radon isotopes 222Rn and 220Rn (i.e., thoron). We found no evidence that radiolysis contributed to methane oxidation. In contrast, we observed the rapid loss of methane when moist soil was added to the same apparatus in the absence of elevated radon abundance. Together, our findings are consistent with the view that methane oxidizing bacteria are responsible for the widespread observations of methane depletion in subterranean environments. Further studies are needed on the ability of microbes to consume trace amounts of methane in poorly ventilated caves, even though the trophic and energetic benefits become marginal at very low partial pressures of methane. [ABSTRACT FROM AUTHOR]

Microcontact printing has become a versatile soft lithography technique used to produce molecular micro- and nano-patterns consisting of a large range of different biomolecules. Despite intensive research over the last decade and numerous applications in the fields of biosensors, microarrays and biomedical applications, the large-scale implementation of microcontact printing is still an issue. It is hindered by the stamp-inking step that is critical to ensure a reproducible and uniform transfer of inked molecules over large areas. This is particularly important when addressing application such as cell microarray manufacturing, which are currently used for a wide range of analytical and pharmaceutical applications. In this paper, we present a large-scale and multiplexed microcontact printing process of extracellular matrix proteins for the fabrication of cell microarrays. We have developed a microfluidic inking approach combined with a magnetic clamping technology that can be adapted to most standard substrates used in biology. We have demonstrated a significant improvement of homogeneity of printed protein patterns on surfaces larger than 1 cm2 through the control of both the flow rate and the wetting mechanism of the stamp surface during microfluidic inking. Thanks to the reproducibility and integration capabilities provided by microfluidics, we have achieved the printing of three different adhesion proteins in one-step transfer. Selective cell adhesion and cell shape adaptation on the produced patterns were observed, showing the suitability of this approach for producing on-demand large-scale cell microarrays. [ABSTRACT FROM AUTHOR]

Rationale: Diabetes causes cardiac dysfunction, and understanding of its mechanism is still incomplete. One reason could be limitations in modeling disease conditions by current in vitro cardiomyocyte culture. Emerging evidence suggests that the mechanical properties of the microenvironment affect cardiomyocyte function. Nevertheless, the impact of high glucose on cardiomyocytes cultured on substrates whose stiffness matches that of the heart (approximately 15 kPa) is untested. Objective: To test the hypothesis that cardiomyocytes cultured in microenvironments that mimic the mechanical properties of those for cardiomyocytes in vivo may reproduce the pathophysiology characteristics of diabetic cardiomyocytes ex vivo, such as the morphological appearance, ROS accumulation, mitochondrial dysfunction, apoptosis and insulin-stimulated glucose uptake. Methods and results: Isolated neonatal rat cardiomyocytes were seeded on 15 kPa polyacrylamide (PAA) gels, whose stiffness mimics that of heart tissues, or on glass coverslips, which represent conventional culture devices but are unphysiologically stiff. Cells were then cultured at 5 mM glucose, corresponding to the normal blood glucose level, or at high glucose levels (10 to 25 mM). Cytoskeletal disorganization, ROS accumulation, attenuated mitochondrial membrane potential and attenuated ATP level caused by high glucose and their reversal by a ROS scavenger were prominent in cells on gels, but not in cells on coverslips. The lack of response to ROS scavenging could be attributable to enhanced apoptosis in cells on glass, shown by enhanced DNA fragmentation and higher caspase 3/7 activity in cells on glass coverslips. High-glucose treatment also downregulated GLUT4 expression and attenuated insulin-stimulated glucose uptake only in cells on 15 kPa gels. Conclusion: Our data suggest that a mechanically compliant microenvironment increases the susceptibility of primary cardiomyocytes to elevated glucose levels, which enables these cells to serve as an innovative model for diabetic heart research. [ABSTRACT FROM AUTHOR]

Jellyfish blooms are a significant environmental problem that is increasing and may be influenced by anthropocentric practices such as overfishing, pollution, eutrophication, translocation, climate change, and ocean acidification. Many jellyfish have unknown life cycles leading to these blooms. We describe for the first time, the life cycle of scyphozoan jellyfish Rhizostoma luteum from the planula to the young medusa stages, based on laboratory observations. We also provide a preliminary assessment of temperature related to life stages. Comparisons were made with early life history stages of its sibling species Rhizostoma pulmo and Rhizostoma octopus. The life cycle of R. luteum follows the general pattern of metagenesis of scyphozoans. Scyphistoma culture was maintained in filtered seawater at 17–17.5 °C, salinity 37 and light photoperiod (12:12 h light:dark). Scyphistomae were exposed to an experimental temperature descent for two days to test their survival capacity under severe winter conditions. Only one asexual reproduction mode was observed, which is employed for propagation, consisting of podocyst formation with excystment, subsequent development of scyphistoma, strobilation and liberation of viable ephyra. The development of the ephyra to metaephyra was photodocumented, reaching the metaephyra stage in approximately 21–25 days. Young medusae grow rapidly and maturity was reached after a 3-month post-liberation period with a mean bell diameter of 13.27 ± 2.26 cm and wet weight of 181.53 ± 53 g. The life cycle of R. luteum resembles that of its congeners, with the distinction that it has the unique features of being a brooding species (internal fertilisation with subsequent release of planulae) and under the conditions tested, the predominantly strobilation type observed was monodisc, and not polydisc as with the other two species in the genus Rhizostoma. As R. luteum shows sufficient requisite to form blooms if environmental circumstances change, it is important to understand its life cycle. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii is a cause of healthcare-associated infections. Although A. baumannii is an opportunistic pathogen, its infections are notoriously difficult to treat due to intrinsic and acquired antimicrobial resistance, often limiting effective therapeutic options. A. baumannii can survive for long periods in the hospital environment, particularly on inanimate surfaces. Such environments may act as a reservoir for cross-colonization and infection outbreaks and should be considered a substantial factor in infection control practices. Moreover, clothing of healthcare personnel and gadgets may play a role in the spread of nosocomial bacteria. A link between contamination of hospital surfaces and A. baumannii infections or between its persistence in the environment and its virulence has not yet been established. Bacteria under stress (i.e., long-term desiccation in hospital setting) could conserve factors that favor infection. To investigate whether desiccation and/or starvation may be involved in the ability of certain strains of A. baumannii to retain virulence factors, we have studied five well-characterized clinical isolates of A. baumannii for which survival times were determined under simulated hospital conditions. Despite a considerable reduction in the culturability over time (up to 88% depending on strain and the condition tested), some A. baumannii strains were able to maintain their ability to form biofilms after rehydration, addition of nutrients, and changing temperature. Also, after long-term desiccation, several clinical strains were able to grow in the presence of non-immune human serum as fine as their non-stressed homologs. Furthermore, we also show that the ability of bacterial strains to kill Galleria mellonella larvae does not change although A. baumannii cells were stressed by long-term starvation (up to 60 days). This means that A. baumannii can undergo a rapid adaptation to both the temperature shift and nutrients availability, conditions that can be easily found by bacteria in a new patient in the hospital setting. [ABSTRACT FROM AUTHOR]

Oxidative stress has been implicated in various disorders and controlling it would be important for healthy life. We have developed a new optical system for easily and accurately measuring oxidative stress in whole blood. It is optimized for simultaneously detecting reactive oxygen species (ROS) and highly reactive ROS (hROS), elicited mostly by white blood cells in a few microliters of blood. Results obtained by using this system show at least four important findings. 1) chemiluminescence of MCLA was confirmed to be attributable to O2-•. 2) PMA-stimulated cells released O2-• longer and more slowly than fMLP-stimulated ones. 3) fluorescence produced by APF oxidation was confirmed to be attributable to hROS, mostly OCl-, produced by myeloperoxidase. 4) the generation of OCl- was found to be a slower process than the O2-• generation. We also conducted pilot studies of oxidative stress in healthy volunteers. [ABSTRACT FROM AUTHOR]

Pituitary endocrine cells are supplied by Sox2-expressing stem/progenitor cells in the anterior lobe of the adult pituitary. In relation to their microenvironment (“niche”), SOX2-positive cells exist in two types of niches; the marginal cell layer-niche and the parenchymal-niche. Recently, we isolated dense stem/progenitor cell clusters from the parenchymal-niche as parenchymal stem/progenitor cell (PS)-clusters. We classified these PS-clusters into three subtypes based on differences in S100β-expression (S100β-positive, -negative, and -mixed type), and reported that S100β-positive PS-clusters exhibited the capacity for differentiation into endocrine cells under 3-dimensional cultivation system. In the present study, we further characterized S100β-positive PS-clusters using an in vitro 2-dimensional cultivation system. The results demonstrated that S100β-positive PS-clusters in the 2-dimensional cultivation system proliferated more actively than S100β-negative clusters. Moreover, in 2-dimensional cultivation conditions, S100β-positive PS-clusters showed differentiation capacity into non-endocrine cells (Myogenin-, αSMA-, NG2-, or SOX17-positive cells) but not into endocrine cells, whereas S100β-negative PS-clusters did not. Collectively, PS-clusters were heterogeneous, exhibiting different proliferation and differentiation properties based on the difference in S100β-expression. Specifically, a part of SOX2-positive cells in the parenchymal-niche had capacities for differentiation into non-endocrine cells, and S100β-positive PS-clusters may be in more progressive stages toward differentiation than S100β-negative clusters. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei is the causative agent of melioidosis and regarded as a bioterrorism threat. It can adapt to the nutrient-limited environment as the bacteria can survive in triple distilled water for 16 years. Moreover, B. pseudomallei exhibits intrinsic resistance to diverse groups of antibiotics in particular while growing in biofilms. Recently, nutrient-limited condition influenced both biofilm formation and ceftazidime (CAZ) tolerance of B. pseudomallei were found. However, there is no information about how nutrient-limitation together with antibiotics used in melioidosis treatment affects the structure of the biofilm produced by B. pseudomallei. Moreover, no comparative study to investigate the biofilm architectures of B. pseudomallei and the related B. thailandensis under different nutrient concentrations has been reported. Therefore, this study aims to provide new information on the effects of four antibiotics used in melioidosis treatment, viz. ceftazidime (CAZ), imipenem (IMI), meropenem (MEM) and doxycycline (DOX) on biofilm architecture of B. pseudomallei and B. thailandensis with different nutrient concentrations under static and flow conditions using confocal laser scanning microscopy. Impact of nutritional stress on drug susceptibility of B. pseudomallei and B. thailandensis grown planktonically or as biofilm was also evaluated. The findings of this study indicate that nutrient-limited environment enhanced survival of B. pseudomallei in biofilm after exposure to the tested antibiotics. The shedding planktonic B. pseudomallei and B. thailandensis were also found to have increased CAZ tolerance in nutrient-limited environment. However, killing activities of MEM and IMI were stronger than CAZ and DOX on B. pseudomallei and B. thailandensis both in planktonic cells and in 2-day old biofilm. In addition, MEM and IMI were able to inhibit B. pseudomallei and B. thailandensis biofilm formation to a larger extend compared to CAZ and DOX. Differences in biofilm architecture were observed for biofilms grown under static and flow conditions. Under static conditions, biofilms grown in full strength modified Vogel and Bonner’s medium (MVBM) showed honeycomb-like architecture while a knitted-like structure was observed under limited nutrient condition (0.1×MVBM). Under flow conditions, biofilms grown in MVBM showed a multilayer structure while merely dispersed bacteria were found when grown in 0.1×MVBM. Altogether, this study provides more insight on the effect of four antibiotics against B. pseudomallei and B. thailandensis in biofilm under different nutrient and flow conditions. Since biofilm formation is believed to be involved in disease relapse, MEM and IMI may be better therapeutic options than CAZ for melioidosis treatment. [ABSTRACT FROM AUTHOR]

Zebrafish are a valuable model organism in biomedical research. Their rapid development, ability to model human diseases, utility for testing genetic variants identified from next-generation sequencing, amenity to CRISPR mutagenesis, and potential for therapeutic compound screening, has led to their wide-spread adoption in diverse fields of study. However, their power for large-scale screens is limited by the absence of automated genotyping tools for live animals. This constrains potential drug screen options, limits analysis of embryonic and larval phenotypes, and requires raising additional animals to adulthood to ensure obtaining an animal of the desired genotype. Our objective was to develop an automated system that would rapidly obtain cells and DNA from zebrafish embryos and larvae for genotyping, and that would keep the animals alive. We describe the development, testing, and validation of a zebrafish embryonic genotyping device, termed “ZEG” (Zebrafish Embryo Genotyper). Using microfluidic harmonic oscillation of the animal on a roughened glass surface, the ZEG is able to obtain genetic material (cells and DNA) for use in genotyping, from 24 embryos or larvae simultaneously in less than 10 minutes. Loading and unloading of the ZEG is performed manually with a standard pipette tip or transfer pipette. The obtained genetic material is amplified by PCR and can be used for subsequent analysis including sequencing, gel electrophoresis, or high-resolution melt-analysis. Sensitivity of genotyping and survival of animals are both greater than 90%. There are no apparent effects on body morphology, development, or motor behavior tests. In summary, the ZEG device enables rapid genotyping of live zebrafish embryos and larvae, and animals are available for downstream applications, testing, or raising. [ABSTRACT FROM AUTHOR]

The nematode Caenorhabditis elegans is an important model organism for biomedical research and genetic studies relevant to human biology and disease. Such studies are often based on high-resolution imaging of dynamic biological processes in the worm body tissues, requiring well-immobilized and physiologically active animals in order to avoid movement-related artifacts and to obtain meaningful biological information. However, existing immobilization methods employ the application of either anesthetics or servere physical constraints, by using glue or specific microfluidic on-chip mechanical structures, which in some cases may strongly affect physiological processes of the animals. Here, we immobilize C. elegans nematodes by taking advantage of a biocompatible and temperature-responsive hydrogel-microbead matrix. Our gel-based immobilization technique does not require a specific chip design and enables fast and reversible immobilization, thereby allowing successive imaging of the same single worm or of small worm populations at all development stages for several days. We successfully demonstrated the applicability of this method in challenging worm imaging contexts, in particular by applying it for high-resolution confocal imaging of the mitochondrial morphology in worm body wall muscle cells and for the long-term quantification of number and size of specific protein aggregates in different C. elegans neurodegenerative disease models. Our approach was also suitable for immobilizing other small organisms, such as the larvae of the fruit fly Drosophila melanogaster and the unicellular parasite Trypanosoma brucei. We anticipate that this versatile technique will significantly simplify biological assay-based longitudinal studies and long-term observation of small model organisms. [ABSTRACT FROM AUTHOR]

Successfully cryopreserving cells adhered to a substrate would facilitate the growth of a vital confluent cell culture after thawing while dramatically shortening the post-thaw culturing time. Herein we propose a controlled slow cooling method combining initial directional freezing followed by gradual cooling down to -80°C for robust preservation of cell monolayers adherent to a substrate. Using computer controlled cryostages we examined the effect of cooling rates and dimethylsulfoxide (DMSO) concentration on cell survival and established an optimal cryopreservation protocol. Experimental results show the highest post-thawing viability for directional ice growth at a speed of 30 μm/sec (equivalent to freezing rate of 3.8°C/min), followed by gradual cooling of the sample with decreasing rate of 0.5°C/min. Efficient cryopreservation of three widely used epithelial cell lines: IEC-18, HeLa, and Caco-2, provides proof-of-concept support for this new freezing protocol applied to adherent cells. This method is highly reproducible, significantly increases the post-thaw cell viability and can be readily applied for cryopreservation of cellular cultures in microfluidic devices. [ABSTRACT FROM AUTHOR]

The accuracy of quantitative stereological analysis tools such as the (physical) disector method substantially depends on the precise determination of the thickness of the analyzed histological sections. One conventional method for measurement of histological section thickness is to re-embed the section of interest vertically to its original section plane. The section thickness is then measured in a subsequently prepared histological section of this orthogonally re-embedded sample. However, the orthogonal re-embedding (ORE) technique is quite work- and time-intensive and may produce inaccurate section thickness measurement values due to unintentional slightly oblique (non-orthogonal) positioning of the re-embedded sample-section. Here, an improved ORE method is presented, allowing for determination of the factual section plane angle of the re-embedded section, and correction of measured section thickness values for oblique (non-orthogonal) sectioning. For this, the analyzed section is mounted flat on a foil of known thickness (calibration foil) and both the section and the calibration foil are then vertically (re-)embedded. The section angle of the re-embedded section is then calculated from the deviation of the measured section thickness of the calibration foil and its factual thickness, using basic geometry. To find a practicable, fast, and accurate alternative to ORE, the suitability of spectral reflectance (SR) measurement for determination of plastic section thicknesses was evaluated. Using a commercially available optical reflectometer (F20, Filmetrics®, USA), the thicknesses of 0.5 μm thick semi-thin Epon (glycid ether)-sections and of 1–3 μm thick plastic sections (glycolmethacrylate/ methylmethacrylate, GMA/MMA), as regularly used in physical disector analyses, could precisely be measured within few seconds. Compared to the measured section thicknesses determined by ORE, SR measures displayed less than 1% deviation. Our results prove the applicability of SR to efficiently provide accurate section thickness measurements as a prerequisite for reliable estimates of dependent quantitative stereological parameters. [ABSTRACT FROM AUTHOR]

Psychrotroph microorganisms have developed cellular mechanisms to cope with cold stress. Cell envelopes are key components for bacterial survival. Outer membrane is a constituent of Gram negative bacterial envelopes, consisting of several components, such as lipopolysaccharides (LPS). In this work we investigated the relevance of envelope characteristics for cold adaptation in the Antarctic bacterium Pseudomonas extremaustralis by analyzing a mini Tn5 wapH mutant strain, encoding a core LPS glycosyltransferase. Our results showed that wapH strain is impaired to grow under low temperature but not for cold survival. The mutation in wapH, provoked a strong aggregative phenotype and modifications of envelope nanomechanical properties such as lower flexibility and higher turgor pressure, cell permeability and surface area to volume ratio (S/V). Changes in these characteristics were also observed in the wild type strain grown at different temperatures, showing higher cell flexibility but lower turgor pressure under cold conditions. Cold shock experiments indicated that an acclimation period in the wild type is necessary for cell flexibility and S/V ratio adjustments. Alteration in cell-cell interaction capabilities was observed in wapH strain. Mixed cells of wild type and wapH strains, as well as those of the wild type strain grown at different temperatures, showed a mosaic pattern of aggregation. These results indicate that wapH mutation provoked marked envelope alterations showing that LPS core conservation appears as a novel essential feature for active growth under cold conditions. [ABSTRACT FROM AUTHOR]

The interaction between actin filaments and myosin molecular motors is a power source of a variety of cellular functions including cell division, cell motility, and muscular contraction. In vitro motility assay examines actin filaments interacting with myosin molecules that are adhered to a substrate (e.g., glass surface). This assay has been the standard method of studying the molecular mechanisms of contraction under an optical microscope. While the force generation has been measured through an optically trapped bead to which an actin filament is attached, a force vector vertical to the glass surface has been largely ignored with the in vitro motility assay. The vertical vector is created by the gap (distance) between the trapped bead and the glass surface. In this report, we propose a method to estimate the angle between the actin filament and the glass surface by optically determining the gap size. This determination requires a motorized stage in a standard epi-fluorescence microscope equipped with optical tweezers. This facile method is applied to force measurements using both pure actin filaments, and thin filaments reconstituted from actin, tropomyosin and troponin. We find that the angle-corrected force per unit filament length in the active condition (pCa = 5.0) decreases as the angle between the filament and the glass surface increases; i.e. as the force in the vertical direction increases. At the same time, we demonstrate that the force on reconstituted thin filaments is approximately 1.5 times larger than that on pure actin filaments. The range of angles we tested was between 11° and 36° with the estimated measurement error less than 6°. These results suggest the ability of cytoplasmic tropomyosin isoforms maintaining actomyosin active force to stabilize cytoskeletal architecture. [ABSTRACT FROM AUTHOR]

Polyacrylamide gels functionalized with extracellular matrix proteins are commonly used as cell culture platforms to evaluate the combined effects of extracellular matrix composition, cell geometry and substrate rigidity on cell physiology. For this purpose, protein transfer onto the surface of polyacrylamide hydrogels must result in geometrically well-resolved micropatterns with homogeneous protein distribution. Yet the outcomes of micropatterning methods have not been pairwise evaluated against these criteria. We report a high-fidelity photoresist lift-off patterning method to pattern ECM proteins on polyacrylamide hydrogels with elastic moduli ranging from 5 to 25 kPa. We directly compare the protein transfer efficiency and pattern geometrical accuracy of this protocol to the widely used microcontact printing method. Lift-off patterning achieves higher protein transfer efficiency, increases pattern accuracy, increases pattern yield, and reduces variability of these factors within arrays of patterns as it bypasses the drying and transfer steps of microcontact printing. We demonstrate that lift-off patterned hydrogels successfully control cell size and shape and enable long-term imaging of actin intracellular structure and lamellipodia dynamics when we culture epithelial cells on these substrates. [ABSTRACT FROM AUTHOR]

IQGAP1 interacts with a number of binding partners through a calponin homology domain (CHD), a WW motif, IQ repeats, a Ras GAP-related domain (GRD), and a conserved C-terminal (CT) domain. Among various biological and cellular functions, IQGAP1 is known to play a role in actin cytoskeleton dynamics during membrane ruffling and lamellipodium protrusion. In addition, phosphorylation near the CT domain is thought to control IQGAP1 activity through regulation of intramolecular interaction. In a previous study, we discovered that IQGAP1 preferentially localizes to retracting areas in B16F10 mouse melanoma cells, not areas of membrane ruffling and lamellipodium protrusion. Nothing is known of the domains needed for retraction localization and very little is known of IQGAP1 function in the actin cytoskeleton of melanoma cells. Thus, we examined localization of IQGAP1 mutants to retracting areas, and characterized knock down phenotypes on tissue culture plastic and physiologic-stiffness hydrogels. Localization of IQGAP1 mutants (S1441E/S1443D, S1441A/S1443A, ΔCHD, ΔGRD or ΔCT) to retracting and protruding cell edges were measured. In retracting areas there was a decrease in S1441A/S1443A, ΔGRD and ΔCT localization, a minor decrease in ΔCHD localization, and normal localization of the S1441E/S1443D mutant. In areas of cell protrusion just behind the lamellipodium leading edge, we surprisingly observed both ΔGRD and ΔCT localization, and increased number of microtubules. IQGAP1 knock down caused loss of cell polarity on laminin-coated glass, decreased proliferation on tissue culture polystyrene, and abnormal spheroid growth on laminin-coated hydrogels. We propose that the GRD and CT domains regulate IQGAP1 localization to retracting actin networks to promote a tumorigenic role in melanoma cells. [ABSTRACT FROM AUTHOR]

Background: Treatment of advanced cutaneous melanoma remains challenging, and new data on melanoma biology are required. The most widely accepted criteria for the prognostic evaluation of melanoma are histopathological and clinical parameters, and the identification of additional tumor markers is thus of paramount importance. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI), an important tool in cancer research, is useful for unraveling the molecular profile of melanoma. Methodology/Principal findings: In this report, we used the melanoma-bearing Libechov minipig (MeLiM), a unique animal model that allows observation of the complete spontaneous regression of invasive cutaneous melanoma, to investigate i) the differences between melanoma and healthy skin protein profiles and ii) the proteins potentially involved in spontaneous regression. The MeLiM tissues were cryosected, histologically characterized, analyzed by MALDI MSI, and immunohistologically stained. Multivariate statistical analyses of the MALDI MSI data revealed ten relevant m/z ions, of which the expression levels varied significantly among the studied MeLiM tissues. These ion peaks were used to create mass ion images/maps and visualize the differences between tumor and healthy skin specimens, as well as among histologically characterized tissue regions. Conclusions/Significance: Protein profiles comprising ten statistically significant mass ion peaks useful for differentiating cutaneous melanoma and healthy skin tissues were determined. Peaks at m/z 3044, 6011, 6140 and 10180 were overexpressed in melanoma compared with healthy skin tissue. More specifically, m/z 6140 was expressed at significantly (p < 0.05) higher levels in normally growing melanoma regions than in regions with early and late spontaneous regression. This study demonstrates the clinical utility of MALDI MSI for the analysis of tissue cryosections at a molecular level. [ABSTRACT FROM AUTHOR]

Necator americanus, a haematophagous hookworm parasite, infects ~10% of the world’s population and is considered to be a significant public health risk. Its lifecycle has distinct stages, permitting its successful transit from the skin via the lungs (L3) to the intestinal tract (L4 maturing to adult). It has been hypothesised that the L3 larval sheath, which is shed during percutaneous infection (exsheathment), diverts the immune system to allow successful infection and reinfection in endemic areas. However, the physicochemical properties of the L3 larval cuticle and sheath, which are in direct contact with the skin and its immune defences, are unknown. In the present study, we controlled exsheathment, to characterise the sheath and underlying cuticle surfaces in situ, using atomic force microscopy (AFM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). AFM revealed previously unseen surface area enhancing nano-annuli exclusive to the sheath surface and confirmed greater adhesion forces exist between cationic surfaces and the sheath, when compared to the emergent L3 cuticle. Furthermore, ToF-SIMS elucidated different chemistries between the surfaces of the cuticle and sheath which could be of biological significance. For example, the phosphatidylglycerol rich cuticle surface may support the onward migration of a lubricated infective stage, while the anionic and potentially immunologically active heparan sulphate rich deposited sheath could result in the diversion of immune defences to an inanimate antigenic nidus. We propose that our initial studies into the surface analysis of this hookworm provides a timely insight into the physicochemical properties of a globally important human pathogen at its infective stage and anticipate that the development and application of this analytical methodology will support translation of these findings into a biological context. [ABSTRACT FROM AUTHOR]

The limits of life of aerobic microorganisms are well understood, but the responses of anaerobic microorganisms to individual and combined extreme stressors are less well known. Motivated by an interest in understanding the survivability of anaerobic microorganisms under Martian conditions, we investigated the responses of a new isolate, Yersinia intermedia MASE-LG-1 to individual and combined stresses associated with the Martian surface. This organism belongs to an adaptable and persistent genus of anaerobic microorganisms found in many environments worldwide. The effects of desiccation, low pressure, ionizing radiation, varying temperature, osmotic pressure, and oxidizing chemical compounds were investigated. The strain showed a high tolerance to desiccation, with a decline of survivability by four orders of magnitude during a storage time of 85 days. Exposure to X-rays resulted in dose-dependent inactivation for exposure up to 600 Gy while applied doses above 750 Gy led to complete inactivation. The effects of the combination of desiccation and irradiation were additive and the survivability was influenced by the order in which they were imposed. Ionizing irradiation and subsequent desiccation was more deleterious than vice versa. By contrast, the presence of perchlorates was not found to significantly affect the survival of the Yersinia strain after ionizing radiation. These data show that the organism has the capacity to survive and grow in physical and chemical stresses, imposed individually or in combination that are associated with Martian environment. Eventually it lost its viability showing that many of the most adaptable anaerobic organisms on Earth would be killed on Mars today. [ABSTRACT FROM AUTHOR]

One hundred and forty-one glass fragments from medieval Ciudad de Vascos (Toledo, Spain) were analysed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The glasses fall into three types according to the fluxing agents used: mineral natron, soda-rich plant ash, and a combination of soda ash and lead. The natron glasses can be assigned to various established primary production groups of eastern Mediterranean provenance. Different types of plant ash glasses indicate differences in the silica source as well as the plant ash component, reflecting changing supply mechanisms. While the earlier plant ash groups can be related to Islamic glasses from the Near East, both in terms of typology and composition, the chemical signature of the later samples appear to be specific to glass from the Iberian Peninsula. This has important implications for our understanding of the emerging glass industry in Spain and the distribution patterns of glass groups and raw materials. The plant ash that was used for the Vascos glasses is rich in soda with low levels of potash, similar to ash produced in the eastern Mediterranean. It could therefore be possible that Levantine plant ash was imported and used in Islamic period glass workshops in Spain. Unlike central and northern Europe where an independent glass industry based on potassium-rich wood ash developed during the Carolingian period, the prevalence of soda ash and soda ash lead glass on the Iberian Peninsula indicates its commercial and technological interconnection with the Islamic east. Our study thus traces several stages leading to the development of a specifically Spanish primary glassmaking industry. [ABSTRACT FROM AUTHOR]

A host of animals build architectural constructions. Such constructions frequently vary with environmental and individual/colony conditions, and their architecture directly influences behavior and fitness. The nests of ant colonies drive and enable many of their collective behaviors, and as such are part of their ‘extended phenotype’. Since ant colonies have been recently shown to differ in behavior and life history strategy, we ask whether colonies differ in another trait: the architecture of the constructions they create. We allowed Temnothorax rugatulus rock ants, who create nests by building walls within narrow rock gaps, to repeatedly build nest walls in a fixed crevice but under two environmental conditions. We find that colonies consistently differ in their architecture across environments and over nest building events. Colony identity explained 12–40% of the variation in nest architecture, while colony properties and environmental conditions explained 5–20%, as indicated by the condition and marginal R2 values. When their nest boxes were covered, which produced higher humidity and lower airflow, colonies built thicker, longer, and heavier walls. Colonies also built more robust walls when they had more brood, suggesting a protective function of wall thickness. This is, to our knowledge, the first study to explicitly investigate the repeatability of nestbuilding behavior in a controlled environment. Our results suggest that colonies may face tradeoffs, perhaps between factors such as active vs. passive nest defense, and that selection may act on individual construction rules as a mechanisms to mediate colony-level behavior. [ABSTRACT FROM AUTHOR]

In ants, dispersal strategies and morphology of female sexuals are generally linked to the mode of colony founding. In species using long-range dispersal tactics, queen/worker dimorphism is generally high and young queens are able to initiate new colonies by themselves, using their metabolic reserves. By contrast, in species using short-range dispersal strategies, queen/worker dimorphism is generally low and, due to their limited metabolic reserves, queens have lost the capacity to raise their brood alone and to found their colony independently. Moreover, polygyny is also often associated with short-range dispersal strategies, although the relationship between the number of queens and the dispersal strategy in ants is not clear-cut. Here, dispersal strategies were investigated in C. pygmaea, a highly polygynous and polydomous ant species from northeastern Brazil. Field observations and laboratory experiments show that this ant exhibits a suite of traits that are more commonly associated with long-range dispersal and independent colony foundation: functional wings in both males and females, high queen/worker dimorphism, strong weight loss in mature queens, nuptial flights and, in the lab, ability of young queens to found new colonies in haplometrotic conditions. On the other hand, this species shows a high degree of polygyny with a strong seasonal component, and, at least under laboratory conditions, mature queens seem able to develop propagules if they are accompanied by at least 10 workers. These features strongly suggest that (1) some of the gynes do not engage in a long-range dispersal but become new queens in their mother colony and (2) that budding events are possible in this species. We therefore speculate that C. pygmaea has a dual dispersal strategy probably related to environmental conditions: some gynes engage in long-range dispersal followed by independent colony foundation at the beginning of rainy season, while others mate in the parental colony and are re-adopted leading to high polygyny. During the rainy season, budding events can lead to colony extension and increased polydomy. Polydomy is commonly thought to improve resource discovery and exploitation through decentralized foraging behavior, a significant advantage during the rainy season when food ressources (mainly floral/extrafloral nectaries and hemipteran honeydew) are more abundant and when colony needs for food supplies are highest. [ABSTRACT FROM AUTHOR]

Introduction: Porcine thrombi are employed routinely in preclinical models of ischemic stroke. In this study, we examined the differential lytic susceptibility of porcine and human whole blood clots with and without the use of microbubbles and ultrasound (US) as an adjuvant. Materials and methods: An in vitro system equipped with time-lapse microscopy was used to evaluate recombinant tissue-plasminogen activator (rt-PA) lysis of porcine and human clots in the same species or cross species plasma. Human and porcine whole blood clots were treated with rt-PA and an echo contrast agent, Definity®, and exposed to intermittent 120 kHz US. Results and conclusions: The rt-PA lytic efficacy observed for porcine clots in porcine plasma was 22 times lower than for human clots in human plasma reported previously. Further, porcine clots did not exhibit increased lysis with adjuvant Definity® and US exposure. However, the rt-PA lytic susceptibility of the porcine clots in human plasma was similar to that of human clots in human plasma. Human clots perfused with porcine plasma did not respond to rt-PA, but adjuvant use of Definity® and US enhanced lysis. These results reveal considerable differences in lytic susceptibility of porcine clots and human clots to rt-PA. The use of porcine clot models to test new human thrombolytic therapies may necessitate modulation of coagulation and thrombolytic factors to reflect human hemostasis accurately. [ABSTRACT FROM AUTHOR]

The extracellular matrix (ECM) is a major regulator of cell behavior. Recent studies have indicated the importance of the physical properties of the ECM, including its stiffness, for cell migration and differentiation. Using actomyosin-generated forces, cells pull the ECM and sense stiffness via cell-ECM adhesion structures called focal adhesions (FAs). Vinculin, an actin-binding FA protein, has emerged as a major player in FA-mediated mechanotransduction. Although vinculin is important for sensing ECM stiffness, the role of vinculin binding to actin in the ECM stiffness-mediated regulation of vinculin behavior remains unknown. Here, we show that an actin binding-deficient mutation disrupts the ECM stiffness-dependent regulation of CSB (cytoskeleton stabilization buffer) resistance and the stable localization of vinculin. These results suggest that the vinculin-actin interaction participates in FA-mediated mechanotransduction. [ABSTRACT FROM AUTHOR]

Detection and identification of microbial species are crucial in a wide range of industries, including production of beverages, foods, cosmetics, and pharmaceuticals. Traditionally, colony formation and its morphological analysis (e.g., size, shape, and color) with a naked eye have been employed for this purpose. However, such a conventional method is time consuming, labor intensive, and not very reproducible. To overcome these problems, we propose a novel method that detects microcolonies (diameter 10–500 μm) using a lensless imaging system. When comparing colony images of five microorganisms from different genera (Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans), the images showed obvious different features. Being closely related species, St. aureus and St. epidermidis resembled each other, but the imaging analysis could extract substantial information (colony fingerprints) including the morphological and physiological features, and linear discriminant analysis of the colony fingerprints distinguished these two species with 100% of accuracy. Because this system may offer many advantages such as high-throughput testing, lower costs, more compact equipment, and ease of automation, it holds promise for microbial detection and identification in various academic and industrial areas. [ABSTRACT FROM AUTHOR]

The net interaction between a probe tip coated with bovine serum albumin (BSA) protein and a flat substrate coated with poly(ethylene oxide) (PEO) polymer was measured directly on approach in water and electrolyte solutions using AFM. The approach force curve between the two surfaces was monotonically repulsive in water and in electrolyte solutions. At pH ~5, slightly above the isoelectric point (pI) of BSA, and at large distances, the force was dominated by electrostatic repulsion between the oxygen atoms of the incoming protein with those belonging to the ether groups of PEO. Such repulsive force and range decreased in NaCl. Under physiological conditions, pH 6, BSA is definitely charged and the electrostatic repulsion with ether groups in PEO appears at larger separation distances. Interestingly, at pH 4, below the pI of BSA, the repulsion decreased because of an attractive, although weak, electrostatic force that appeared between the ether groups in PEO and the positively charged amino groups of BSA. However, for all solution conditions, once compression of PEO begun, the net repulsion was always dominated by short-range polymeric steric repulsion and repulsive enthalpy penalties for breaking PEO-water bonds. Results suggest that PEO in mushroom conformation may also be effective in reducing biofouling. [ABSTRACT FROM AUTHOR]

Cell polarization toward an attractant is influenced by both physical and chemical factors. Most existing mathematical models are based on reaction-diffusion systems and only focus on the chemical process occurring during cell polarization. However, membrane tension has been shown to act as a long-range inhibitor of cell polarization. Here, we present a cell polarization model incorporating the interplay between Rac GTPase, filamentous actin (F-actin), and cell membrane tension. We further test the predictions of this model by performing single cell measurements of the spontaneous polarization of cancer stem cells (CSCs) and non-stem cancer cells (NSCCs), as the former have lower cell membrane tension. Based on both our model and the experimental results, cell polarization is more sensitive to stimuli under low membrane tension, and high membrane tension improves the robustness and stability of cell polarization such that polarization persists under random perturbations. Furthermore, our simulations are the first to recapitulate the experimental results described by Houk et al., revealing that aspiration (elevation of tension) and release (reduction of tension) result in a decrease in and recovery of the activity of Rac-GTP, respectively, and that the relaxation of tension induces new polarity of the cell body when a cell with the pseudopod-neck-body morphology is severed. [ABSTRACT FROM AUTHOR]

Whole slide imaging (WSI) is a useful tool for multi-modal imaging, and in our work, we have often combined WSI with darkfield microscopy. However, traditional darkfield microscopy cannot use a single condenser to support high- and low-numerical-aperture objectives, which limits the modality of WSI. To overcome this limitation, we previously developed a darkfield internal reflection illumination (DIRI) microscope using white light-emitting diodes (LEDs). Although the developed DIRI is useful for biological applications, substantial problems remain to be resolved. In this study, we propose a novel illumination technique called color DIRI. The use of three-color LEDs dramatically improves the capability of the system, such that color DIRI (1) enables optimization of the illumination color; (2) can be combined with an oil objective lens; (3) can produce fluorescence excitation illumination; (4) can adjust the wavelength of light to avoid cell damage or reactions; and (5) can be used as a photostimulator. These results clearly illustrate that the proposed color DIRI can significantly extend WSI modalities for biological applications. [ABSTRACT FROM AUTHOR]

Although it is well known that phospholipids self-assemble on hydrophilic plasma-oxidized PMDS surfaces (ox-PDMS) to form cell membrane mimetic bilayers, the temporal stability of phospholipid membranes on these surfaces is unknown. Here we report that phospholipid bilayers remain stable on solvent-cleaned ox-PDMS for at least 132 hours after preparation. Absent solvent cleaning, the bilayers were stable for only 36 hours. We characterized the phospholipid bilayers, i) through quantitative comparative analysis of the fluorescence intensity of phospholipid bilayers on ox-PDMS and phospholipid monolayers on native PDMS and, ii) through measurements of the diffusive mobility of the lipids through fluorescence recovery after photobleaching (FRAP). The fluorescence intensity of the phospholipid layer remained consistent with that of a bilayer for 132 hours. The evolution of the diffusive mobility of the phospholipids in the bilayer on ox-PDMS over time was similar to lipids in control bilayers prepared on glass surfaces. Solvent cleaning was essential for the long-term stability of the bilayers on ox-PDMS. Without cleaning in acetone and isopropanol, phospholipid bilayers prepared on ox-PDMS surfaces peeled off in large patches within 36 hours. Importantly, we find that phospholipid bilayers supported on solvent-cleaned ox-PDMS were indistinguishable from phospholipid bilayers supported on glass for at least 36 hours after preparation. Our results provide a link between the two common surfaces used to prepare in vitro biomimetic phospholipid membranes—i) glass surfaces used predominantly in fundamental biophysical experiments, for which there is abundant physicochemical information, with ii) ox-PDMS, the dominant material used in practical, applications-oriented systems to build micro-devices, topographically-patterned surfaces, and biosensors where there is a dearth of information. [ABSTRACT FROM AUTHOR]

Pollen tubes are used as a model in the study of plant morphogenesis, cellular differentiation, cell wall biochemistry, biomechanics, and intra- and intercellular signaling. For a “systems-understanding” of the bio-chemo-mechanics of tip-polarized growth in pollen tubes, the need for a versatile, experimental assay platform for quantitative data collection and analysis is critical. We introduce a Lab-on-a-Chip (LoC) concept for high-throughput pollen germination and pollen tube guidance for parallelized optical and mechanical measurements. The LoC localizes a large number of growing pollen tubes on a single plane of focus with unidirectional tip-growth, enabling high-resolution quantitative microscopy. This species-independent LoC platform can be integrated with micro-/nano-indentation systems, such as the cellular force microscope (CFM) or the atomic force microscope (AFM), allowing for rapid measurements of cell wall stiffness of growing tubes. As a demonstrative example, we show the growth and directional guidance of hundreds of lily (Lilium longiflorum) and Arabidopsis (Arabidopsis thaliana) pollen tubes on a single LoC microscopy slide. Combining the LoC with the CFM, we characterized the cell wall stiffness of lily pollen tubes. Using the stiffness statistics and finite-element-method (FEM)-based approaches, we computed an effective range of the linear elastic moduli of the cell wall spanning the variability space of physiological parameters including internal turgor, cell wall thickness, and tube diameter. We propose the LoC device as a versatile and high-throughput phenomics platform for plant reproductive and development biology using the pollen tube as a model. [ABSTRACT FROM AUTHOR]

We previously showed that the females of the mushroom sciarid, Lycoriella ingenua (Dufour, 1839) (Diptera: Sciaridae), one of the most severe pests of the cultivated white button mushroom, Agaricus bisporus (J.E. Lange) Emil J. Imbach (Agaricales: Agaricaceae), are attracted to the mushroom compost that mushrooms are grown on and not to the mushrooms themselves. We also showed that females are attracted to the parasitic green mold, Trichoderma aggressivum. In an attempt to identify what is in the mushroom compost that attracts female L. ingenua, we isolated several species of fungi from adult males and females, third instar larvae, and mushroom compost itself. We then analyzed the attraction of females to these substrates using a static-flow two choice olfactometer, as well as their oviposition tendencies in another type of assay under choice and no-choice conditions. We also assessed the survival of larvae to adulthood when first instar larvae were placed on each of the isolated fungal species. We found that female flies were attracted most to the mycoparasitic green mold, T. aggressivum, to Penicilium citrinum isolated from adult female bodies, and to Scatylidium thermophilium isolated from the mushroom compost. Gravid female flies laid the most eggs on T. aggressivum, Aspergillus flavus isolated from third instar larval frass, Aspergillus fumigatus isolated from adult male bodies, and on P. citrinum. This egg-laying trend remained consistent under no-choice conditions as females aged. First instar larvae developed to adulthood only on S. thermophilium and Chaetomium sp. isolated from mushroom compost, and on P. citrinum. Our results indicate that the volatiles from a suite of different fungal species act in tandem in the natural setting of mushroom compost, with some first attracting gravid female flies and then others causing them to oviposit. The ecological context of these findings is important for creating an optimal strategy for using possible semiochemicals isolated from these fungal species to better monitor and control this pestiferous mushroom fly species. [ABSTRACT FROM AUTHOR]

Background: Tumor proliferation speed, most commonly assessed by counting of mitotic figures in histological slide preparations, is an important biomarker for breast cancer. Although mitosis counting is routinely performed by pathologists, it is a tedious and subjective task with poor reproducibility, particularly among non-experts. Inter- and intraobserver reproducibility of mitosis counting can be improved when a strict protocol is defined and followed. Previous studies have examined only the agreement in terms of the mitotic count or the mitotic activity score. Studies of the observer agreement at the level of individual objects, which can provide more insight into the procedure, have not been performed thus far. Methods: The development of automatic mitosis detection methods has received large interest in recent years. Automatic image analysis is viewed as a solution for the problem of subjectivity of mitosis counting by pathologists. In this paper we describe the results from an interobserver agreement study between three human observers and an automatic method, and make two unique contributions. For the first time, we present an analysis of the object-level interobserver agreement on mitosis counting. Furthermore, we train an automatic mitosis detection method that is robust with respect to staining appearance variability and compare it with the performance of expert observers on an “external” dataset, i.e. on histopathology images that originate from pathology labs other than the pathology lab that provided the training data for the automatic method. Results: The object-level interobserver study revealed that pathologists often do not agree on individual objects, even if this is not reflected in the mitotic count. The disagreement is larger for objects from smaller size, which suggests that adding a size constraint in the mitosis counting protocol can improve reproducibility. The automatic mitosis detection method can perform mitosis counting in an unbiased way, with substantial agreement with human experts. [ABSTRACT FROM AUTHOR]

We report the identification, synthesis, and field bioassay of a novel attractant pheromone produced by males of Eburodacrys vittata (Blanchard), a South American cerambycid beetle in the subfamily Cerambycinae. Headspace volatiles from males contained a sex-specific compound, identified as 10-methyldodecanal. In a field bioassay conducted in Brazil, significant numbers of males and females were caught in traps baited with synthesized racemic 10-methyldodecanal, consistent with the aggregation-sex pheromones produced by males of many cerambycine species. This compound represents a new structural class of cerambycid pheromones, and it is the first pheromone identified for a species in the tribe Eburiini. [ABSTRACT FROM AUTHOR]

Saliva crystallization was previously studied in both humans and animals with various results. The study aimed to confirm of the presence of saliva crystallization in female Bornean orangutans (Pongo pygmaeus), to evaluate the quality of samples which were collected from animals and processed by keepers, and to test preliminarily if the saliva crystallization could be connected with menstrual cycle and could serve as a cheap, quick and simple method for the basic monitoring of their reproductive status. The research was carried out from September 2014 to January 2015. Sampling of saliva was done in three female orangutans from three zoological gardens (Dvur Kralove, Usti nad Labem, Bojnice) daily, mostly by tongue prints on glass slides with ground edges or by sampling directly from the mouth using plastic spoons from which the saliva was transferred onto glass slides. Samples were evaluated by light microscopy with ×400 magnification. The quality of the sample and type of crystallization was assessed for two different approaches. In total, 246 samples were evaluated. We confirmed the presence of saliva crystallization in orangutans. The quality of samples was variable however acceptable. Unfortunately, it was impossible to detect exact fertile period in two females. However in one orangutan female, when the crystallization was evaluated by the approach typically used in humans, we discovered that saliva crystallization during the fertile period significantly differed from saliva crystallization in the non-fertile period. This points out the possibility of using saliva crystallization for detection of the fertile period in orangutans. However, further research was recommended. [ABSTRACT FROM AUTHOR]