Comparative transcriptomics indicate changes in cell wall organization and stress response in seedlings during spaceflight.

PREMISE OF THE STUDY: Plants will play an important role in the future of space exploration as part of bioregenerative life support. Thus, it is important to understand the effects of microgravity and spaceflight on gene expression in plant development. METHODS: We analyzed the transcriptome of Arabidopsis thaliana using the Biological Research in Canisters (BRIC) hardware during Space Shuttle mission STS-131. The bioinformatics methods used included RMA (robust multi-array average), MAS5 (Microarray Suite 5.0), and PLIER (probe logarithmic intensity error estimation). Glycome profiling was used to analyze cell wall composition in the samples. In addition, our results were compared to those of two other groups using the same hardware on the same mission (BRIC-16). KEY RESULTS: In our BRIC-16 experiments, we noted expression changes in genes involved in hypoxia and heat shock responses, DNA repair, and cell wall structure between spaceflight samples compared to the ground controls. In addition, glycome profiling supported our expression analyses in that there was a difference in cell wall components between ground control and spaceflight-grown plants. Comparing our studies to those of the other BRIC-16 experiments demonstrated that, even with the same hardware and similar biological materials, differences in results in gene expression were found among these spaceflight experiments. CONCLUSIONS: A common theme from our BRIC-16 space experiments and those of the other two groups was the downregulation of water stress response genes in spaceflight. In addition, all three studies found differential regulation of genes associated with cell wall remodeling and stress responses between spaceflight-grown and ground control plants.

Remodeling of peripheral nerve ensheathment during the larval-to-adult transition in Drosophila.

Over the course of a 4-day period of metamorphosis, the Drosophila larval nervous system is remodeled to prepare for adult-specific behaviors. One example is the reorganization of peripheral nerves in the abdomen, where five pairs of abdominal nerves (A4-A8) fuse to form the terminal nerve trunk. This reorganization is associated with selective remodeling of four layers that ensheath each peripheral nerve. The neural lamella (NL), is the first to dismantle; its breakdown is initiated by 6 hours after puparium formation, and is completely removed by the end of the first day. This layer begins to re-appear on the third day of metamorphosis. Perineurial glial (PG) cells situated just underneath the NL, undergo significant proliferation on the first day of metamorphosis, and at that stage contribute to 95% of the glial cell population. Cells of the two inner layers, Sub-Perineurial Glia (SPG) and Wrapping Glia (WG) increase in number on the second half of metamorphosis. Induction of cell death in perineurial glia via the cell death gene reaper and the Diptheria toxin (DT-1) gene, results in abnormal bundling of the peripheral nerves, suggesting that perineurial glial cells play a role in the process. A significant number of animals fail to eclose in both reaper and DT-1 targeted animals, suggesting that disruption of PG also impacts eclosion behavior. The studies will help to establish the groundwork for further work on cellular and molecular processes that underlie the co-ordinated remodeling of glia and the peripheral nerves they ensheath. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1144-1160, 2017.

p28 dynein light chains and ciliary motility in Tetrahymena thermophila.

Dynein light chains are required for the assembly of axonemal dyneins into cilia and flagella. Most organisms express a single p28 dynein light chain and four to nine one-headed inner arm dynein heavy chains. In contrast, Tetrahymena encodes three p28 dynein light chain genes (p28A, p28B, and p28C) and 18 one-headed inner arm dynein heavy chains. In this article it is shown that mutations in p28A and p28B affected both beat frequency and waveform of cilia, while mutations in p28C affected only ciliary beat frequency. A similar set of dynein heavy chains were affected in both p28AKO and p28BKO, but a distinct set of heavy chains was affected in p28CKO. The results suggested that the p28s have non-redundant functions in Tetrahymena and that p28C was associated with a different set of dynein heavy chains than were p28A and p28B.

Prospect of Stem Cells in Bone Tissue Engineering: A Review.

Mesenchymal stem cells (MSCs) have been the subject of many studies in recent years, ranging from basic science that looks into MSCs properties to studies that aim for developing bioengineered tissues and organs. Adult bone marrow-derived mesenchymal stem cells (BM-MSCs) have been the focus of most studies due to the inherent potential of these cells to differentiate into various cell types. Although, the discovery of induced pluripotent stem cells (iPSCs) represents a paradigm shift in our understanding of cellular differentiation. These cells are another attractive stem cell source because of their ability to be reprogramed, allowing the generation of multiple cell types from a single cell. This paper briefly covers various types of stem cell sources that have been used for tissue engineering applications, with a focus on bone regeneration. Then, an overview of some recent studies making use of MSC-seeded 3D scaffold systems for bone tissue engineering has been presented. The emphasis has been placed on the reported scaffold properties that tend to improve MSCs adhesion, proliferation, and osteogenic differentiation outcomes.

Morphometric analyses of petioles of seedlings grown in a spaceflight experiment.

Gravity is a constant unidirectional stimulus on Earth, and gravitropism in plants involves three phases: perception, transduction, and response. In shoots, perception takes place within the endodermis. To investigate the cellular machinery of perception in microgravity, we conducted a spaceflight study with Arabidopsis thaliana seedlings, which were grown in microgravity in darkness using the Biological Research in Canisters (BRIC) hardware during space shuttle mission STS-131. In the 14-day-old etiolated plants, we studied seedling development and the morphological parameters of the endodermal cells in the petiole. Seedlings from the spaceflight experiment (FL) were compared to a ground control (GC), which both were in the BRIC flight hardware. In addition, to assay any potential effects from growth in spaceflight hardware, we performed another control by growing seedlings in Petri dishes in standard laboratory conditions (termed the hardware control, HC). Seed germination was significantly lower in samples grown in flight hardware (FL, GC) compared to the HC. In terms of cellular parameters of endodermal cells, the greatest differences also were between seedlings grown in spaceflight hardware (FL, GC) compared to those grown outside of this hardware (HC). Specifically, the endodermal cells were significantly smaller in seedlings grown in the BRIC system compared to those in the HC. However, a change in the shape of the cell, suggesting alterations in the cell wall, was one parameter that appears to be a true microgravity effect. Taken together, our results suggest that caution must be taken when interpreting results from the increasingly utilized BRIC spaceflight hardware system and that it is important to perform additional ground controls to aid in the analysis of spaceflight experiments.

Solvent-free polymer/bioceramic scaffolds for bone tissue engineering: fabrication, analysis, and cell growth.

This study examines the potential use of porous polycaprolactone (PCL) and polycaprolocatone/hydroxyapatite (PCL/HA) scaffolds fabricated through melt molding and porogen leaching for bone tissue engineering. While eliminating organic solvents is desirable, the process steps proposed in this study for uniformly dispersing HA particles (~5 µm in size) within the scaffold can also contribute to homogeneous properties for these porous composites. Poly(ethylene oxide) (PEO) was chosen as a porogen due to its similar density and melting point as PCL. Pore size of the scaffold was controlled by limiting the size of PCL and PEO particles used in fabrication. The percent of HA in the fabricated scaffolds was quantified by thermogravimetric analysis (TGA). Mechanical testing was used to compare the modulus of the scaffolds to that of bone, and the pore size distribution was examined with microcomputed tomography (µCT). Scanning electron microscopy (SEM) was used to examine the effect on scaffold morphology caused by the addition of HA particles. Both µCT and SEM results showed that HA could be incorporated into PCL scaffolds without negatively affecting scaffold morphology or pore formation. Energy-dispersive X-ray spectroscopy (EDS) and elemental mapping demonstrated a uniform distribution of HA within PCL/HA scaffolds. Murine calvaria-derived MC3T3-E1 cells were used to determine whether cells could attach on scaffolds and grow for up to 21 days. SEM images revealed an increase in cell attachment with the incorporation of HA into the scaffolds. Similarly, DNA content analysis showed a higher cell adhesion to PCL/HA scaffolds.

Dynein-2 affects the regulation of ciliary length but is not required for ciliogenesis in Tetrahymena thermophila.

Eukaryotic cilia and flagella are assembled and maintained by the bidirectional intraflagellar transport (IFT). Studies in alga, nematode, and mouse have shown that the heavy chain (Dyh2) and the light intermediate chain (D2LIC) of the cytoplasmic dynein-2 complex are essential for retrograde intraflagellar transport. In these organisms, disruption of either dynein-2 component results in short cilia/flagella with bulbous tips in which excess IFT particles have accumulated. In Tetrahymena, the expression of the DYH2 and D2LIC genes increases during reciliation, consistent with their roles in IFT. However, the targeted elimination of either DYH2 or D2LIC gene resulted in only a mild phenotype. Both knockout cell lines assembled motile cilia, but the cilia were of more variable lengths and less numerous than wild-type controls. Electron microscopy revealed normally shaped cilia with no swelling and no obvious accumulations of material in the distal ciliary tip. These results demonstrate that dynein-2 contributes to the regulation of ciliary length but is not required for ciliogenesis in Tetrahymena.

The adult abdominal neuromuscular junction of Drosophila: a model for synaptic plasticity.

During its life cycle, Drosophila makes two sets of neuromuscular junctions (NMJs), embryonic/larval and adult, which serve distinct stage-specific functions. During metamorphosis, the larval NMJs are restructured to give rise to their adult counterparts, a process that is integrated into the overall remodeling of the nervous system. The NMJs of the prothoracic muscles and the mesothoracic dorsal longitudinal (flight) muscles have been previously described. Given the diversity and complexity of adult muscle groups, we set out to examine the less complex abdominal muscles. The large bouton sizes of these NMJs are particularly advantageous for easy visualization. Specifically, we have characterized morphological attributes of the ventral abdominal NMJ and show that an embryonic motor neuron identity gene, dHb9, is expressed at these adult junctions. We quantified bouton numbers and size and examined the localization of synaptic markers. We have also examined the formation of boutons during metamorphosis and examined the localization of presynaptic markers at these stages. To test the usefulness of the ventral abdominal NMJs as a model system, we characterized the effects of altering electrical activity and the levels of the cell adhesion molecule, FasciclinII (FasII). We show that both manipulations affect NMJ formation and that the effects are specific as they can be rescued genetically. Our results indicate that both activity and FasII affect development at the adult abdominal NMJ in ways that are distinct from their larval and adult thoracic counterparts