Background. The genes involved in inner ear development and maintenance of the adult organ have yet to be fully characterized. Previous genetic analysis has emphasized the early development that gives rise to the otic vesicle. This study aimed to bridge the knowledge gap and identify candidate genes that are expressed as the auditory and vestibular sensory organs continue to grow and develop until the systems reach postmetamorphic maturity. Methods. Affymetrix microarrays were used to assess inner ear transcriptome profiles from three Xenopus laevis developmental ages where all eight endorgans comprise mechanosensory hair cells: larval stages 50 and 56, and the post-metamorphic juvenile. Pairwise comparisons were made between the three developmental stages and the resulting differentially expressed X. laevis Probe Set IDs (Xl-PSIDs) were assigned to four groups based on differential expression patterns. DAVID analysis was undertaken to impart functional annotation to the differentially regulated Xl-PSIDs. Results. Analysis identified 1510 candidate genes for differential gene expression in one or more pairwise comparison. Annotated genes not previously associated with inner ear development emerged from this analysis, as well as annotated genes with established inner ear function, such as oncomodulin, neurod1, and sp7. Notably, 36% of differentially expressed Xl-PSIDs were unannotated. Conclusions. Results draw attention to the complex gene regulatory patterns that characterize Xenopus inner ear development, and underscore the need for improved annotation of the X. laevis genome. Outcomes can be utilized to select candidate inner ear genes for functional analysis, and to promote Xenopus as a model organism for biomedical studies of hearing and balance.

Mechanical properties are essential for the biological activities of cells, and they have been shown to be affected by diseases. Therefore, accurate mechanical characterization is important for studying the cell lifecycle, cell-cell interactions, and disease diagnosis. While the cytoskeleton and actin cortex are typically the primary structural stiffness contributors in most live cells, oocytes possess an additional extracellular layer known as the vitelline membrane (VM), or envelope, which can significantly impact their overall mechanical properties. In this study, we utilized nanoindentation via an atomic force microscope to measure the Young's modulus of Xenopus laevis oocytes at different force setpoints and explored the influence of the VM by conducting measurements on oocytes with the membrane removed. The findings revealed that the removal of VM led to a significant decrease in the apparent Young's modulus of the oocytes, highlighting the pivotal role of the VM as the main structural component responsible for the oocyte's shape and stiffness. Furthermore, the mechanical behavior of VM was investigated through finite element (FE) simulations of the nanoindentation process. FE simulations with the VM Young's modulus in the range 20-60 MPa resulted in force-displacement curves that closely resemble experimental in terms of shape and maximum force for a given indentation depth.

Our team of graduate students and faculty developed a viscoelasticity workshop for middle and high school students as part of a summer program (NM Prep Academy) in the College of Engineering at NMSU, a Hispanic-serving institution. Academy activities have the goal of building interest and readiness for STEM careers and entry to college. We united our expertise in mechanical engineering, computational chemistry, and neuroscience, to develop content that empowered students with understanding of viscoelastic properties in materials, fostered their ability to apply this knowledge in real-world contexts, and inspired their curiosity about materials science and engineering.

Throughout their lifespan cells are constantly subjected to mechanical forces. Numerous studies have been conducted to measure mechanical properties of animal cells and understand their effects of the cells’ biological processes. However, the range of the apparent Young's moduli reported in the literature spans three orders of magnitude from 0.1 kPa to 100 kPa, and no clear explanation is given for the source of variation. One of the commonly used experimental techniques to measure material properties at nano- and microscale is nanoindentation. Nanoindentation works by applying a force on a sample via a small probe and recording a force-displacement curve. Probes with different tip shapes and sizes are available. However, there is little guidance on the appropriate choice of probe geometry and size for measurement of properties of animal cells. Eukaryotic cells have a complex structure including a membrane, an actin cortex, a cytoplasm, a nucleus and a cytoskeleton which consists of microfilaments, intermediate filaments and microtubules. These components have different mechanical properties contributing to the overall properties of cells. This work focuses on the effect of tip size and indentation depth on the apparent Young's modulus of Xenopus laevis oocytes measuring approximately 1 mm in diameter. Live cell fluorescent labeling using Lifeact-EGFP and confocal microscopy was used to measure the thickness of the oocyte actin cortex which is expected to have a significant contribution to the cell's stiffness. Apparent mechanical properties were measured using the JPK NanoWizard 4 BioAFM system. Indentations 5, 100 and 400 nm deep using probes with spherical tips of 0.1, 1, and 10 μm radii were performed. Contributions of the membrane and the actin cortex on the overall mechanical properties of the oocytes are analyzed.

Biological membranes are characterized by a dynamic, heterogeneous lipid composition that can vary by cell type and species origin. Lipids are estimated to comprise 50% of the plasma membrane by weight. A growing body of evidence supports the view that membrane proteins and their adjacent lipids interact in a bidirectional manner. For example, lipid-protein interactions can affect protein function and contribute to membrane organization, such as the formation of domains. The Xenopus oocyte is a well-established classical heterologous expression system for the functional analysis of ion channels and transporters, especially those prevalent in neural tissue.

ABSTRACTThe tubulin protein fulfills a variety of cellular functions that range from chromosomal separation to locomotion. Functional diversity is achieved through the expression of specific tubulin isotypes in different cell types or developmental time periods. Post-translational modifications (PTMs) of tubulin also are vital for specific intracellular tasks, such as binding and recruiting motor proteins. In neurons, the isotypic expression profile for tubulin is well characterized, and the importance of PTMs for proper neuronal function has gained recent attention due to their implication in neurodegenerative disorders. In contrast, the role of tubulin specializations in the specification of neural cell fate has received minimal attention and studies of tubulin PTMs and isotypes in neuroglia such as astrocytes are relatively few. To bridge this knowledge gap, we undertook an analysis of PTMs in neurons and astrocytes derived from the federally approved H9 hESC-derived human neural stem cell (hNSC) line. In hNSCs, basal cells can be directed to assume neural fate as neurons or astrocytes by specifying different media growth conditions. Immunocytochemical methods, fluorescent antibody probes, and confocal microscopy facilitated image acquisition of fluorescent signals from class III β-tubulin (βIII-tubulin), acetylated tubulin, and polyglutamylated tubulin. Fluorescent probe intensities were assessed with the ‘EBImage’ package for the statistical programming language R, and compared using Student’s t-tests. Qualitative analysis indicated that βIII-tubulin, acetylated tubulin, and polyglutamylated tubulin were expressed to some degree in basal hNSCs and their media-differentiated hNSC neuronal and astroglial progeny. In media-differentiated hNSC astrocyte progeny, quantification and statistical analysis of fluorescence probe intensity showed that acetylated tubulin/ βIII-tubulin ratios were greater than the ratio for polyglutamylated tubulin/ βIII-tubulin. These findings represent a snapshot of the dynamic and varied changes in the tubulin expression profile during the specification of neural cell fate. Results imply that investigations of tubulin PTMs have the potential to advance our understanding of the generation and regeneration of nervous tissue.

ABSTRACTBackgroundOtotoxic chemicals can impair the senses of hearing and balance in mammals through irreversible damage to the mechanosensory bundles of inner ear hair cells. Fish and amphibians are useful models for investigating ototoxicity because their inner ear hair cells, like those of mammals, are susceptible to damage by ototoxins. Moreover, amphibian mechanosensation is augmented by a lateral line organ on the body surface that comprises external mechanosensory hair cells. The lateral line hair cells are arranged in clusters (neuromasts) and are structurally and functionally similar to inner ear hair cells, but are more accessible for experimental manipulation. Herein, we implemented neuromasts of the amphibian (Xenopus)lateral line as an organ system for evaluating the effects of ototoxic chemicals, such as antibiotics, on mechanosensory hair cell bundles.MethodsWe examined the ultrastructure of larvalXenopus laevisneuromasts with scanning electron microscopy (SEM) after larvae were continuously exposed to ototoxic aminoglycoside antibiotics at sub-lethal concentrations (gentamicin; streptomycin; neomycin) for 72 hours.ResultsSEM images demonstrated that 72 hours of exposure to antibiotic concentrations greater than 25 µM reduced the hair cell bundle number in lateral line neuromasts.ConclusionTherapeutic drug studies will benefit from the incorporation of bioassay strategies that evaluate ototoxicity across multiple species including genera of amphibian origin such asXenopus. Our outcomes support the use of theXenopuslateral line for identification of potential ototoxic chemicals and suggest thatXenopusneuromast hair cell bundles can withstand antibiotic exposure. TheXenopusbioassay presented here can be incorporated into drug discovery methodology as a high-resolution phenotypic screen for ototoxic effects.Summary statementDamage to sensory cells of the inner ear by chemical agents such as antibiotics contributes to the growing global prevalence of disorders of hearing and balance. Our results demonstrate that theXenopuslateral line, in conjunction with SEM, affords an accessible organ system for otoxicity screens during the drug discovery pipeline.

Lipid biomolecules serve diverse physiological and structural functions and increasingly are being considered as candidate biomarkers for disease and aging. For example, previous studies have demonstrated that DESI-MS lipid profiles, in combination with multivariate statistics, can differentiate between neoplastic and normal tissue samples that have been characterized with histopathological methods. We are interested in developing experimental cell culture systems that address reproducibility standards and that more closely resemble the in vivo state (doi:10.7717/peerj.2829; DOI: 10.1007/s11064-017-2308-7). In this pilot study, we undertook a comparative lipidomic analysis of two human cancer cell lines (glioblastoma ATCC CCF-STG1; primary ductal carcinoma grade 3, ATCC HCC70) and an amphibian epithelial kidney cell line (ATCC A6). Our goals were twofold: 1) to determine whether these three cell types could be differentiated by interrogation of lipid composition, and 2) to leverage lipidomics to increase the reproducibility of our cell culture systems. Lipids were extracted according to the Bligh & Dyer protocol from cells cultured at the third passage to 95% confluence, then analyzed using Reverse-phase Ultra Performance Liquid Chromatography (Acquity Ultraperformance RP-UPLC) and Mass Spectrometry with positive electrospray ionization (Micromass QTOF Ultima). Lipidomic data were evaluated using the MS-DIAL data independent MS/MS deconvolution program with multivariate statistical analysis. Preliminary results showed alignment of 208 independent lipid species and uncovered differences in the relative abundance of lipids among the three cell lines. Outcomes support the view that lipid profiles vary between human breast, human brain, and amphibian kidney cell lines and suggest that the lipidome may be useful for cell line authentication. Future experiments will expand the scope of the comparative analysis by examining the trend ratio and quantity of individual lipid species, and by developing protocols for analysis of the lipid profiles of subcellular compartments.

Astrocytoma is an invasive carcinoma occurring in the nervous system and currently lacks effective treatment options. A deeper understanding of the mechanisms of tumorigenesis and tumor progression is needed in order to develop novel therapeutic strategies. Recent advances in in vitro culture systems have demonstrated that the use of three-dimensional (3D) culture models could be more relevant for this purpose as compared to monolayer or two-dimensional (2D) models due to their resemblance to in vivo cancer pathology. High-throughput techniques such as RNA sequencing, microarray analyses and cloning could provide useful insights into the relevance of these systems to the native tissue. Previous studies have reported RNA extraction protocols needed for such applications. We have modified these protocols to suit the isolation of total RNA from monolayer and hydrogel cultures of astrocytoma established using basement membrane matrix, Geltrex (TM). We have used this method to demonstrate the differences in the expression of genes involved in autophagy, a process deregulated in many cancer types, in monolayer and hydrogel cultures using quantitative polymerase chain reaction (qPCR). This protocol can be adopted by the researchers who wish to understand the molecular basis of gene expression in hydrogel cultures of normal as well as cancer cell lines.

The NIH Brain Research through Advancing Innovative Neurotechnologies ( BRAIN) Initiative is focused on developing new tools and neurotechnologies to transform our understanding of the brain, and neuroethics is an essential component of this research effort. Coordination with other brain projects around the world will help maximize success.

Triple negative breast cancer (TNBC) is a belligerent carcinoma that is unresponsive to targeted receptor therapies. Development of new treatment strategies would benefit from an expanded repertoire of in vitro cell culture systems, such as those that support tridimensional growth in the presence of hydrogel scaffolds. To this end, we established protocols for maintenance of the TNBC cell line HCC70 in monolayer culture and in a commercially available basement membrane matrix hydrogel. We evaluated the general morphology of cells grown in both conditions with light microscopy, and examined their subcellular organization using transmission electron microscopy (TEM). Phase contrast and confocal microscopy showed the prevalence of irregularly shaped flattened cells in monolayer cultures, while cells maintained in hydrogel organized into multi-layered spheroids. A quantitative ultrastructural analysis comparing cells from the two culture conditions revealed that cells that formed spheroids comprised a greater number of mitochondria, autophagic vacuoles and intercellular junctions than their monolayer counterparts, within the equivalent area of sampled tissue. These observations suggest that triple negative breast cancer cells in culture can alter their organelle content, as well as their morphology, in response to their microenvironment. Methods presented here may be useful for those who intend to image cell cultures with TEM, and for investigators who seek to implement diverse in vitro models in the search for therapeutic molecular targets for TNBC.

Neuroscience presents important neuroethical considerations. Human neuroscience demands focused application of the core research ethics guidelines set out in documents such as the Belmont Report. Various mechanisms, including institutional review boards (IRBs), privacy rules, and the Food and Drug

RNA sequencing offers a versatile platform for profiling biological samples. The novelty and flexibility of this technology has the potential to introduce technical variation at distinct points in the experimental workflow. We evaluated variation in RNA sequencing data acquired from commercially available cell lines cultured in our laboratory: human neural stem cells and normal human astrocytes. After normalizing data with three different methods, we used principal variance component analysis to estimate the contribution to technical variance from replicate cell lots, library preparations, and flow cells. Differentially expressed genes were evaluated using ANOVA analysis. Results indicate that the largest component of technical variance was library preparation. Moreover, comparative analysis of RNA sequencing data from the two cell types showed that the identification of differentially expressed genes and the contributions to variance are strongly influenced by the normalization method. Our results underscore the necessity for technical replication in RNA-seq experiments.

Biomaterial scaffolds have the potential to enhance neuronal development and regeneration. Understanding the genetic responses of astrocytes and neurons to biomaterials could facilitate the development of synthetic environments that enable the specification of neural tissue organization with engineered scaffolds. In this study, we used high throughput transcriptomic and imaging methods to determine the impact of a hydrogel, PuraMatrix™, on human glial cells in vitro. Parallel studies were undertaken with cells grown in a monolayer environment on tissue culture polystyrene. When the Normal Human Astrocyte (NHA) cell line is grown in a hydrogel matrix environment, the glial cells adopt a structural organization that resembles that of neuronal-glial cocultures, where neurons form clusters that are distinct from the surrounding glia. Statistical analysis of next generation RNA sequencing data uncovered a set of genes that are differentially expressed in the monolayer and matrix hydrogel environments. Functional analysis demonstrated that hydrogel-upregulated genes can be grouped into three broad categories: neuronal differentiation and/or neural plasticity, response to neural insult, and sensory perception. Our results demonstrate that hydrogel biomaterials have the potential to transform human glial cell identity, and may have applications in the repair of damaged brain tissue.

Program evaluation for funded projects typically is undertaken during the award lifetime, and immediately afterward, but only rarely is the enduring impact assessed after an extended period of time has lapsed and project funds have ended. Our ongoing research aims to assess the long term impact of the New Mexico Nanoscience Education Network (NMNEN; 2007-2011).

The cytoskeletal protein tubulin plays an integral role in the functional specialization of many cell types. In the central nervous system, post-translational modifications and the expression of specific tubulin isotypes in neurons have been analyzed in greater detail than in their astrocytic counterparts. In this study, we characterized post-translational specifications of tubulin in human astrocytes using the normal human astrocyte (NHA; Lonza) commercial cell line of fetal origin. Immunocytochemical techniques were implemented in conjunction with confocal microscopy to image class III β-tubulin (βIII-tubulin), acetylated tubulin, and polyglutamylated tubulin using fluorescent antibody probes. Fluorescent probe intensity differences and colocalization were quantitatively assessed with the ‘EBImage’ package for the statistical programming language R. Colocalization analysis revealed that, although both acetylated tubulin and polyglutamylated tubulin showed a high degree of correlation with βIII-tubulin, the correlation with acetylated tubulin was stronger. Quantification and statistical analysis of fluorescence intensity demonstrated that the fluorescence probe intensity ratio for acetylated tubulin/βIII-tubulin was greater than the ratio for polyglutamylated tubulin/βIII-tubulin. The open source GEODATA set GSE819950, comprising RNA sequencing data for the NHA cell line, was mined for the expression of enzymes responsible for tubulin modifications. Our analysis uncovered greater expression at the mRNA level for enzymes reported to function in acetylation and deacetylation as compared to enzymes implicated in glutamylation and deglutamylation. Taken together, the results represent a step toward unraveling the tubulin isotypic expression profile and post-translational modification patterns in astrocytes during human brain development.

Malignant astrocytomas are aggressive cancers of glial origin that can develop into invasive brain tumors. The disease has poor prognosis and high recurrence rate. Astrocytoma cell lines of human origin are an important tool in the experimental pathway from bench to bedside because they afford a convenient intermediate system for in vitro analysis of brain cancer pathogenesis and treatment options. We undertook the current study to determine whether hydrogel culture methods could be adapted to support the growth of astrocytoma cell lines, thereby facilitating a system that may be biologically more similar to in vivo tumor tissue. Our experimental protocols enabled maintenance of Grade IV astrocytoma cell lines in conventional monolayer culture and in the extracellular matrix hydrogel, Geltrex ™ . Light and fluorescence microscopy showed that hydrogel environments promoted cellular reorganization from dispersed cells into multilayered aggregates. Transmission electron microscopy revealed the prevalence of autophagy and nuclear membrane distortions in both culture systems. Analysis of microarray Gene Expression Omnibus (GEO) DataSets highlighted expression of genes implicated in pathways for cancer progression and autophagy. A pilot quantitative polymerase chain reaction (qPCR) analysis of the autophagic biomarkers, Beclin 1 (BECN1) and microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B), with two reference genes (beta actin, ACTB; glyceraldehyde 3-phosphate dehydrogenase, GAPDH), uncovered a relative increase of BECN1 and LC3B in hydrogel cultures of astrocytoma as compared to the monolayer. Taken together, results establish that ultrastructural and molecular characteristics of autophagy are features of this astrocytoma cell line, and that hydrogel culture systems can afford novel opportunities for in vitro studies of glioma.

Triple negative breast cancer (TNBC) is characterized by the lack of receptors for estrogen (ER), progesterone (PR) and human epidermal growth factor (HER2). Reports suggest that cancer cells maintained in three‐dimensional (3D) cultures are biologically more similar than monolayer cultures to in vivo tumor tissue. We undertook the current study to enable design of a 3D platform that may afford success in developing interventions to treat TNBC. To this end, we determined the morphological impact of growing a triple negative breast cancer cell line, HCC70 in monolayer culture on tissue culture polystyrene, as compared with growth in 3D culture using extracellular matrix Geltrex™. When we analyzed the morphology of cells in culture at the resolution of light microscopy using phase contrast and laser confocal imaging methods, we found that monolayer cells appeared flat, adhered to the polystyrene surface, and formed clusters with neighboring cells. In contrast, matrix‐organized cells formed compact spheroids. Ultra‐structural characterization was achieved using quantitative analysis of images captured with transmission electron microscopy. Our results showed that the 3D cultures had a higher number of mitochondria, autophagic vacuoles and intercellular junctions than those present in monolayer cultures. In addition, 3D cultures had membrane protrusions which were not present in monolayer cultures; we hypothesize that these structures may play an important role in matrix invasion. Our results demonstrate that the morphology of triple negative breast cancer cells is influenced by the surrounding environment. Our results suggest that 3D culture systems may provide a useful alternative platform for testing anti‐cancer treatments in triple negative breast cancer cell lines as compared to monolayer culture systems.Support or Funding InformationNIH grant P50GM068762 and the New Mexico State University's (NMSU) Manasse Endowment to Dr. Elba Serrano.

Both in the classroom and in the broader educational space we know as the Web, we are entering an Era of Annotation. While there is nothing new about the age-old learning practice of annotation, social reading and commenting platforms have recently emerged that allow students to better engage with each other and the texts they study online. Digital annotation enables students to explore independent lines of inquiry more dynamically, but also to collaborate in powerful new forms of knowledge production.This paper will provide an introduction to Hypothes. is, an open source platform for web annotation that asserts no rights over the content generated by individuals and can be tailored to the language and learning goals of the instructor. Hypothes. is promotes open collaboration and scholarship in STEM educational settings by allowing students and teachers to interact over online documents. The software provides an easy-to-use interactive layer that can be launched from any web browser and allows inline annotation of web documents, including PDF's. Users can highlight text and add notes, including tags. Unlike scribbles or underlining on textbooks, annotations are presented as an interactive overlay to the original text. Annotations may be turned off, shared, replied to and searched. Annotations can be either public-that is, anyone with Hypothes. is active can see the annotations-or private. Users may annotate as individuals or as part of annotation groups.Over the past year, Hypothes. is has been used in over 100 classrooms as a core teaching technology. Students have generated thousands of annotations in their exploration of texts from works of literature to scientific articles. Teachers have incorporated online collaborative annotation into assignments aimed at enhancing scientific literacy and building critical reasoning skills. Our feedback suggests that student annotation of class assignments is a powerful means to promote critical thinking, prepare students for class and for working in teams. To date, the majority of use has been in the humanities; preliminary explorations of Hypothes. is in STEM areas suggests, however, that the use of web-based annotation will have a similar impact in these fields as well. In addition to classroom use, organizations such as the American Association for the Advancement of Science are producing annotated scientific articles to be used in the classroom. We further propose that web annotation tools such as Hypothes. is have the potential to link classrooms and students separated by distance and national boundaries to foster the training of the next generation of STEM students in a global environment.

Stomatal opening in response to light has a component that matches the absorption spectrum of chloro- phyll; however, the intervening sensory transduction steps are not well understood. To study this process, we illuminated Vici faba guard cell protoplasts with red light and simulta- neously recorded current flow across the plasma membrane, utilizing the patch clamp technique in the whole cell config- uration. We report evidence that under voltage clamp condi- tions, red light (1 mmol of photonsm-2s-1) stimulated an outward current. This response required ATP (2.5 mM) and orthophosphate (1 mM) at the cytoplasmic side of the mem- brane. Both red-light-stimulated currents and currents acti- vated in the dark by the proton pump agonist fusicoccin (10 FM) were abolished by the protonophore carbonylcyanide m-chlorophenylhydrazone at 10 pM, indicating that these responses were carried by protons. Pump currents were inhibited by orthovanadate applied to the cytoplasmic side of the membrane (50% inhibition at 3.5 ,M), implicating a H ' -ATPase. Elimination of the current by the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, in the pres- ence of saturating concentrations of ATP, pointed to a require- ment for photosyntheticaly active chloroplasts. We conclude that red light stimulates an electrogenic proton pump at the plasmalemma of Vicia guard cells and that chloroplasts mod- ulate this response.