Nuclear Factor-kappaB controls the reaggregation of 3D neurosphere cultures in vitro

The approach of reaggregation involves the regeneration and self-renewal of histotypical 3D spheres from isolated tissue kept in suspension culture. Reaggregated spheres can be used as tumour, genetic, biohybrid and neurosphere models. In addition the functional superiority of 3D aggregates over conventional 2D cultures developed the use of neurospheres for brain engineering of CNS diseases. Thus 3D aggregate cultures created enormous interest in mechanisms that regulate the formation of multicellular aggregates in vitro. Here we analyzed mechanisms guiding the development of 3D neurosphere cultures. Adult neural stem cells can be cultured as self-adherent clusters, called neurospheres. Neurospheres are characterised as heterogeneous clusters containing unequal stem cell sub-types. Tumour necrosis factor-alpha (TNF-alpha is one of the crucial inflammatory cytokines with multiple actions on several cell types. TNF-alpha strongly activates the canonical Nuclear Factor Kappa-B (NF- kappaB) pathway. In order to investigate further functions of TNF in neural stem cells (NSCs) we tested the hypothesis that TNF is able to modulate the motility and/or migratory behaviour of SVZ derived adult neural stem cells. We observed a significantly faster sphere formation in TNF treated cultures than in untreated controls. The very fast aggregation of isolated NSCs (<2h) is a commonly observed phenomenon, though the mechanisms of 3D neurosphere formation remain largely unclear. Here we demonstrate for the first time, increased aggregation and enhanced motility of isolated NSCs in response to the TNF-stimulus. Moreover, this phenomenon is largely dependent on activated transcription factor NF-kappaB. Both, the pharmacological blockade of NF-kappaB pathway by pyrrolidine dithiocarbamate (PDTC) or Bay11-7082 and genetic blockade by expression of a transdominant-negative super-repressor IkappaB-AA1 led to decreased aggregation.

Efficient animal-serum free 3D cultivation method for adult human neural crest-derived stem cell therapeutics

Due to their broad differentiation potential and their persistence into adulthood, human neural crest-derived stem cells (NCSCs) harbour great potential for autologous cellular therapies, which include the treatment of neurodegenerative diseases and replacement of complex tissues containing various cell types, as in the case of musculoskeletal injuries. The use of serum-free approaches often results in insufficient proliferation of stem cells and foetal calf serum implicates the use of xenogenic medium components. Thus, there is much need for alternative cultivation strategies. In this study we describe for the first time a novel, human blood plasma based semi-solid medium for cultivation of human NCSCs. We cultivated human neural crest-derived inferior turbinate stem cells (ITSCs) within a blood plasma matrix, where they revealed higher proliferation rates compared to a standard serum-free approach. Three-dimensionality of the matrix was investigated using helium ion microscopy. ITSCs grew within the matrix as revealed by laser scanning microscopy. Genetic stability and maintenance of stemness characteristics were assured in 3D cultivated ITSCs, as demonstrated by unchanged expression profile and the capability for self-renewal. ITSCs pre-cultivated in the 3D matrix differentiated efficiently into ectodermal and mesodermal cell types, particularly including osteogenic cell types. Furthermore, ITSCs cultivated as described here could be easily infected with lentiviruses directly in substrate for potential tracing or gene therapeutic approaches. Taken together, the use of human blood plasma as an additive for a completely defined medium points towards a personalisable and autologous cultivation of human neural crest-derived stem cells under clinical grade conditions.

Quantitative single-molecule localization microscopy combined with rule-based modeling reveals ligand-induced TNF-R1 reorganization toward higher-order oligomers

We report on the assembly of tumor necrosis factor receptor 1 (TNF-R1) prior to ligand activation and its ligand-induced reorganization at the cell membrane. We apply single-molecule localization microscopy to obtain quantitative information on receptor cluster sizes and copy numbers. Our data suggest a dimeric pre-assembly of TNF-R1, as well as receptor reorganization toward higher oligomeric states with stable populations comprising three to six TNF-R1. Our experimental results directly serve as input parameters for computational modeling of the ligand-receptor interaction. Simulations corroborate the experimental finding of higher-order oligomeric states. This work is a first demonstration how quantitative, super-resolution and advanced microscopy can be used for systems biology approaches at the single-molecule and single-cell level.

How is the balance of a forecast ensemble affected by adaptive and non-adaptive localization schemes?

This paper investigates the effect on balance of a number of Schur product-type localization schemes which have been designed with the primary function of reducing spurious far-field correlations in forecast error statistics. The localization schemes studied comprise a non-adaptive scheme (where the moderation matrix is decomposed in a spectral basis), and two adaptive schemes, namely a simplified version of SENCORP (Smoothed ENsemble COrrelations Raised to a Power) and ECO-RAP (Ensemble COrrelations Raised to A Power). The paper shows, we believe for the first time, how the degree of balance (geostrophic and hydrostatic) implied by the error covariance matrices localized by these schemes can be diagnosed. Here it is considered that an effective localization scheme is one that reduces spurious correlations adequately but also minimizes disruption of balance (where the 'correct' degree of balance or imbalance is assumed to be possessed by the unlocalized ensemble). By varying free parameters that describe each scheme (e.g. the degree of truncation in the schemes that use the spectral basis, the 'order' of each scheme, and the degree of ensemble smoothing), it is found that a particular configuration of the ECO-RAP scheme is best suited to the convective-scale system studied. According to our diagnostics this ECO-RAP configuration still weakens geostrophic and hydrostatic balance, but overall this is less so than for other schemes.

Genome-wide mapping of 5-hydroxymethylcytosine in three rice cultivars reveals its preferential localization in transcriptionally silent transposable element genes

5-Hydroxymethylcytosine (5hmC), a modified form of cytosine that is considered the sixth nucleobase in DNA, has been detected in mammals and is believed to play an important role in gene regulation. In this study, 5hmC modification was detected in rice by employing a dot-blot assay, and its levels was further quantified in DNA from different rice tissues using liquid chromatography-multistage mass spectrometry (LC-MS/MS/MS). The results showed large intertissue variation in 5hmC levels. The genome-wide profiles of 5hmC modification in three different rice cultivars were also obtained using a sensitive chemical labelling followed by a next-generation sequencing method. Thousands of 5hmC peaks were identified, and a comparison of the distributions of 5hmC among different rice cultivars revealed the specificity and conservation of 5hmC modification. The identified 5hmC peaks were significantly enriched in heterochromatin regions,and mainly located in transposable element (TE) genes, especially around retrotransposons. The correlation analysis of 5hmC and gene expression data revealed a close association between 5hmC and silent TEs. These findings provide a resource for plant DNA 5hmC epigenetic studies and expand our knowledge of 5hmC modification.

Using the Technology Acceptance Model to explore community dwelling older adults’ perceptions of a 3D interior design application to facilitate pre-discharge home adaptations

Background In the UK occupational therapy pre-discharge home visits are routinely carried out as a means of facilitating safe transfer from the hospital to home. Whilst they are an integral part of practice, there is little evidence to demonstrate they have a positive outcome on the discharge process. Current issues for patients are around the speed of home visits and the lack of shared decision making in the process, resulting in less than 50 % of the specialist equipment installed actually being used by patients on follow-up. To improve practice there is an urgent need to examine other ways of conducting home visits to facilitate safe discharge. We believe that Computerised 3D Interior Design Applications (CIDAs) could be a means to support more efficient, effective and collaborative practice. A previous study explored practitioners perceptions of using CIDAs; however it is important to ascertain older adult’s views about the usability of technology and to compare findings. This study explores the perceptions of community dwelling older adults with regards to adopting and using CIDAs as an assistive tool for the home adaptations process. Methods Ten community dwelling older adults participated in individual interactive task-focused usability sessions with a customised CIDA, utilising the think-aloud protocol and individual semi-structured interviews. Template analysis was used to carry out both deductive and inductive analysis of the think-aloud and interview data. Initially, a deductive stance was adopted, using the three pre-determined high-level themes of the technology acceptance model (TAM): Perceived Usefulness (PU), Perceived Ease of Use (PEOU), Actual Use (AU). Inductive template analysis was then carried out on the data within these themes, from which a number of sub-thmes emerged. Results Regarding PU, participants believed CIDAs served as a useful visual tool and saw clear potential to facilitate shared understanding and partnership in care delivery. For PEOU, participants were able to create 3D home environments however a number of usability issues must still be addressed. The AU theme revealed the most likely usage scenario would be collaborative involving both patient and practitioner, as many participants did not feel confident or see sufficient value in using the application autonomously. Conclusions This research found that older adults perceived that CIDAs were likely to serve as a valuable tool which facilitates and enhances levels of patient/practitioner collaboration and empowerment. Older adults also suggested a redesign of the interface so that less sophisticated dexterity and motor functions are required. However, older adults were not confident, or did not see sufficient value in using the application autonomously. Future research is needed to further customise the CIDA software, in line with the outcomes of this study, and to explore the potential of collaborative application patient/practitioner-based deployment.

Spanish as a World Language: The Interplay of Globalized Localization and Localized Globalization.

This article argues that two movements in constant interplay operate within the historical trajectory of the Spanish language: the localization that becomes globalized and the globalization that becomes localized. Equally, this article illustrates how, at the same time that Spanish is expanding in the world, new idiosyncratic and localized forms of the language are emerging. This article deals with the issues of standardization and language ideology, language contact, and redefinition of identities. The article focuses on three geographic loci: Spain, where Spanish opposes Catalan, Basque, and Galician; the United States, where migrants' Spanish dialects converge and confront English and each other; and finally, Latin America, where Spanish is in contact with Portuguese, indigenous, and Afro-Hispanic languages. The concepts that structure the discussion explain both language expansion and contraction as well as the conflict and constant negotiation between a language's standardized forms and its regional and social varieties.

Neuronal-glial populations form functional networks in a biocompatible 3D scaffold

Monolayers of neurons and glia have been employed for decades as tools for the study of cellular physiology and as the basis for a variety of standard toxicological assays. A variety of three dimensional (3D) culture techniques have been developed with the aim to produce cultures that recapitulate desirable features of intact. In this study, we investigated the effect of preparing primary mouse mixed neuron and glial cultures in the inert 3D scaffold, Alvetex. Using planar multielectrode arrays, we compared the spontaneous bioelectrical activity exhibited by neuroglial networks grown in the scaffold with that seen in the same cells prepared as conventional monolayer cultures. Two dimensional (monolayer; 2D) cultures exhibited a significantly higher spike firing rate than that seen in 3D cultures although no difference was seen in total signal power (<50 Hz) while pharmacological responsiveness of each culture type to antagonism of GABAAR, NMDAR and AMPAR was highly comparable. Interestingly, correlation of burst events, spike firing and total signal power (<50 Hz) revealed that local field potential events were associated with action potential driven bursts as was the case for 2D cultures. Moreover, glial morphology was more physiologically normal in 3D cultures. These results show that 3D culture in inert scaffolds represents a more physiologically normal preparation which has advantages for physiological, pharmacological, toxicological and drug development studies, particularly given the extensive use of such preparations in high throughput and high content systems.

Combining 3D and 2D for less constrained periocular recognition

Periocular recognition has recently become an active topic in biometrics. Typically it uses 2D image data of the periocular region. This paper is the first description of combining 3D shape structure with 2D texture. A simple and effective technique using iterative closest point (ICP) was applied for 3D periocular region matching. It proved its strength for relatively unconstrained eye region capture, and does not require any training. Local binary patterns (LBP) were applied for 2D image based periocular matching. The two modalities were combined at the score-level. This approach was evaluated using the Bosphorus 3D face database, which contains large variations in facial expressions, head poses and occlusions. The rank-1 accuracy achieved from the 3D data (80%) was better than that for 2D (58%), and the best accuracy (83%) was achieved by fusing the two types of data. This suggests that significant improvements to periocular recognition systems could be achieved using the 3D structure information that is now available from small and inexpensive sensors.

3D printing of biocompatible supramolecular polymers and their composites

A series of polymers capable of self-assembling into infinite networks via supramolecular interactions have been designed, synthesized, and characterized for use in 3D printing applications. The biocompatible polymers and their composites with silica nanoparticles were successfully utilized to deposit both simple cubic structures, as well as a more complex twisted pyramidal feature. The polymers were found to be not toxic to a chondrogenic cell line, according to ISO 10993-5 and 10993-12 standard tests and the cells attached to the supramolecular polymers as demonstrated by confocal microscopy. Silica nanoparticles were then dispersed within the polymer matrix, yielding a composite material which was optimized for inkjet printing. The hybrid material showed promise in preliminary tests to facilitate the 3D deposition of a more complex structure.

Intracellular trafficking, localization, and mobilization of platelet-borne thiol isomerases

OBJECTIVE: Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including protein disulfide isomerase and ERp57, are mobilized to the surface of activated platelets, where they influence platelet aggregation, blood coagulation, and thrombus formation. In this study, we examined the synthesis and trafficking of thiol isomerases in megakaryocytes, determined their subcellular localization in platelets, and identified the cellular events responsible for their movement to the platelet surface on activation. APPROACH AND RESULTS: Immunofluorescence microscopy imaging was used to localize protein disulfide isomerase and ERp57 in murine and human megakaryocytes at various developmental stages. Immunofluorescence microscopy and subcellular fractionation analysis were used to localize these proteins in platelets to a compartment distinct from known secretory vesicles that overlaps with an inner cell-surface membrane region defined by the endoplasmic/sarcoplasmic reticulum proteins calnexin and sarco/endoplasmic reticulum calcium ATPase 3. Immunofluorescence microscopy and flow cytometry were used to monitor thiol isomerase mobilization in activated platelets in the presence and absence of actin polymerization (inhibited by latrunculin) and in the presence or absence of membrane fusion mediated by Munc13-4 (absent in platelets from Unc13dJinx mice). CONCLUSIONS: Platelet-borne thiol isomerases are trafficked independently of secretory granule contents in megakaryocytes and become concentrated in a subcellular compartment near the inner surface of the platelet outer membrane corresponding to the sarco/endoplasmic reticulum of these cells. Thiol isomerases are mobilized to the surface of activated platelets via a process that requires actin polymerization but not soluble N-ethylmaleimide-sensitive fusion protein attachment receptor/Munc13-4-dependent vesicular-plasma membrane fusion.

Environmental factors and features that influence visual search in a 3D WIMP interface

The challenge of moving past the classic Window Icons Menus Pointer (WIMP) interface, i.e. by turning it ‘3D’, has resulted in much research and development. To evaluate the impact of 3D on the ‘finding a target picture in a folder’ task, we built a 3D WIMP interface that allowed the systematic manipulation of visual depth, visual aides, semantic category distribution of targets versus non-targets; and the detailed measurement of lower-level stimuli features. Across two separate experiments, one large sample web-based experiment, to understand associations, and one controlled lab environment, using eye tracking to understand user focus, we investigated how visual depth, use of visual aides, use of semantic categories, and lower-level stimuli features (i.e. contrast, colour and luminance) impact how successfully participants are able to search for, and detect, the target image. Moreover in the lab-based experiment, we captured pupillometry measurements to allow consideration of the influence of increasing cognitive load as a result of either an increasing number of items on the screen, or due to the inclusion of visual depth. Our findings showed that increasing the visible layers of depth, and inclusion of converging lines, did not impact target detection times, errors, or failure rates. Low-level features, including colour, luminance, and number of edges, did correlate with differences in target detection times, errors, and failure rates. Our results also revealed that semantic sorting algorithms significantly decreased target detection times. Increased semantic contrasts between a target and its neighbours correlated with an increase in detection errors. Finally, pupillometric data did not provide evidence of any correlation between the number of visible layers of depth and pupil size, however, using structural equation modelling, we demonstrated that cognitive load does influence detection failure rates when there is luminance contrasts between the target and its surrounding neighbours. Results suggest that WIMP interaction designers should consider stimulus-driven factors, which were shown to influence the efficiency with which a target icon can be found in a 3D WIMP interface.