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At least 163 records · Page 9

A systems genetic analysis identifies putative mechanisms and candidate genes regulating vessel traits in poplar wood

Wood is the water conducting tissue of tree stems. Like most angiosperm trees, poplar wood contains water-conducting vessel elements whose functional properties affect water transport and growth rates, as well as susceptibility to embolism and hydraulic failure during water stress and drought. Here we used a unique hybrid poplar pedigree carrying genomically characterized chromosomal insertions and deletions to undertake a systems genomics analysis of vessel traits. We assayed gene expression in wood forming tissues from clonal replicates of genotypes covering dosage quantitative trait loci with insertions and deletions, genotypes with extreme vessel trait phenotypes, and control genotypes. A gene co-expression analysis was used to assign genes to modules, which were then used in integrative analyses to identify modules associated with traits, to identify putative molecular and cellular processes associated with each module, and finally to identify candidate genes using multiple criteria including dosage responsiveness. These analyses identified known processes associated with vessel traits including stress response, abscisic acid and cell wall biosynthesis, and in addition identified previously unexplored processes including cell cycle and protein ubiquitination. We discuss our findings relative to component processes contributing to vessel trait variation including signaling, cell cycle, cell expansion, and cell differentiation.

59 BASIC BIOLOGICAL SCIENCES↗

Characterization of protein redox dynamics induced during light-to-dark transitions and nutrient limitation in cyanobacteria

Protein redox chemistry constitutes a major void in knowledge pertaining to photoautotrophic system regulation and signaling processes. We have employed a chemical biology approach to analyze redox sensitive proteins in live Synechococcus sp. PCC 7002 cells in both light and dark periods, and to understand how cellular redox balance is disrupted during nutrient perturbation. The present work identified several novel putative redox-sensitive proteins that are involved in the generation of reductant, macromolecule synthesis, and carbon flux through central metabolic pathways, and may be involved in cell signaling and response mechanisms. Furthermore, our research suggests that dynamic redox changes in response to specific nutrient limitations contribute to the regulatory changes driven by a shift from light to dark. Taken together, these results contribute to the high-level understanding of post-translational mechanisms regulating flux distributions and therefore present potential metabolic engineering targets for redirecting carbon towards biofuel precursors.

Environmental Molecular Sciences Laboratory↗

Protein Kinases Possibly Mediate Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

Basic cellular functions such as electrolyte concentration, cell growth rate, glucose utilization, bone formation, response to growth stimulation, and exocytosis are modified in microgravity. These studies indicate that microgravity affects a number of physiological systems and included in this are cell signaling mechanisms. Rijken and coworkers performed growth factor studies that showed PKC signaling and actin microfilament organization appears to be sensitive to microgravity, suggesting that the inhibition of signal transduction by microgravity may be related to alterations in actin microfilament organization. However, similar studies have not been done for vascular cells. Vascular endothelial cells play critical roles in providing nutrients to organ and tissues and in wound repair. The major deterrent to ground-based microgravity studies is that it is impossible to achieved true microgravity for longer than a few minutes on earth. Hence, it has not been possible to conduct prolonged microgravity studies except for two models that simulate certain aspects of microgravity. However, hypergravity is quite easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell lines while decreasing cell motility and slowing liver regeneration following partial hepatectomy, These studies indicate the hypergravity also alters the behavior of most cells. Several investigators have shown that hypergravity affects the activation of several protein kinases (PKs) in cells. In this study, we investigated whether hypergravity alters the expression of f-actin by bovine aortic endothelial cells (BAECs) and the role of PK's (calmodulin 11 dependent, PKA and PKC) as mediators of these effects.

Love, Felisha D.↗

Microsurgical removal of epidermal and cortical cells: evidence that the gravitropic signal moves through the outer cell layers in primary roots of maize

There is general agreement that during root gravitropism some sort of growth-modifying signal moves from the cap to the elongation zone and that this signal ultimately induces the curvature that leads to reorientation of the root. However, there is disagreement regarding both the nature of the signal and the pathway of its movement from the root cap to the elongation zone. We examined the pathway of movement by testing gravitropism in primary roots of maize (Zea mays L.) from which narrow (0.5 mm) rings of epidermal and cortical tissue were surgically removed from various positions within the elongation zone. When roots were girdled in the apical part of the elongation zone gravitropic curvature occurred apical to the girdle but not basal to the girdle. Filling the girdle with agar allowed curvature basal to the girdle to occur. Shallow girdles, in which only two or three cell layers (epidermis plus one or two cortical cell layers) were removed, prevented or greatly delayed gravitropic curvature basal to the girdle. The results indicate that the gravitropic signal moves basipetally through the outermost cell layers, perhaps through the epidermis itself.

NASA Discipline Number 29-20↗

Targeted Quantification of Protein Phosphorylation and Its Contributions towards Mathematical Modeling of Signaling Pathways

Post-translational modifications (PTMs) are key regulatory mechanisms that can control protein function. Of these, phosphorylation is the most common and widely studied. Because of its importance in regulating cell signaling, precise and accurate measurements of protein phosphorylation across wide dynamic ranges are crucial to understanding how signaling pathways function. Although immunological assays are commonly used to detect phosphoproteins, their lack of sensitivity, specificity, and selectivity often make them unreliable for quantitative measurements of complex biological samples. Recent advances in Mass Spectrometry (MS)-based targeted proteomics have made it a more useful approach than immunoassays for studying the dynamics of protein phosphorylation. Selected reaction monitoring (SRM)—also known as multiple reaction monitoring (MRM)—and parallel reaction monitoring (PRM) can quantify relative and absolute abundances of protein phosphorylation in multiplexed fashions targeting specific pathways. In addition, the refinement of these tools by enrichment and fractionation strategies has improved measurement of phosphorylation of low-abundance proteins. The quantitative data generated are particularly useful for building and parameterizing mathematical models of complex phospho-signaling pathways. Potentially, these models can provide a framework for linking analytical measurements of clinical samples to better diagnosis and treatment of disease.

mathematical modeling↗

MEK5 suppresses osteoblastic differentiation

Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and is activated by its upstream kinase, MAPK kinase 5 (MEK5), which is a member of the MEK family. Although the role of MEK5 has been investigated in several fields, little is known about its role in osteoblastic differentiation. In this study, we have demonstrated the role of MEK5 in osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells and bone marrow stromal ST2 cells. We found that treatment with BIX02189, an inhibitor of MEK5, increased alkaline phosphatase (ALP) activity and the gene expression of ALP, osteocalcin (OCN) and osterix, as well as it enhanced the calcification of the extracellular matrix. Moreover, osteoblastic cell proliferation decreased at a concentration of greater than 0.5 μM. In addition, knockdown of MEK5 using siRNA induced an increase in ALP activity and in the gene expression of ALP, OCN, and osterix. In contrast, overexpression of wild-type MEK5 decreased ALP activity and attenuated osteoblastic differentiation markers including ALP, OCN and osterix, but promoted cell proliferation. In summary, our results indicated that MEK5 suppressed the osteoblastic differentiation, but promoted osteoblastic cell proliferation. These results implied that MEK5 may play a pivotal role in cell signaling to modulate the differentiation and proliferation of osteoblasts. Thus, inhibition of MEK5 signaling in osteoblasts may be of potential use in the treatment of osteoporosis. - Highlights: • MEK5 inhibitor BIX02189 suppresses proliferation of osteoblasts. • MEK5 knockdown and MEK5 inhibitor promote differentiation of osteoblasts. • MEK5 overexpression inhibits differentiation of osteoblasts.

60 APPLIED LIFE SCIENCES↗

Deubiquitinase USP33 is negatively regulated by β-TrCP through ubiquitin-dependent proteolysis

Ubiquitin-mediated proteolysis regulates cellular levels of various proteins, and therefore plays important roles in controlling cell signaling and disease progression. The Skp1-Cul1-F-box ubiquitin ligase β-TrCP is recognized as an important negative regulator for numerous key signaling proteins. Recently, the deubiquitinases (DUBs) have turned out to be essential to regulate signaling pathways related to human diseases. However, whether β-TrCP is able to regulate the deubiquitinase family members remains largely unexplored. Here, we found that β-TrCP downregulated cellular levels of endogenous USP33. We also revealed that β-TrCP interacted with USP33 independently of the classic binding motif for β-TrCP, and mediated USP33 degradation via the ubiquitin proteasome pathway. Furthermore, we found that the WD40 motif of β-TrCP and 201–400 amino acid motif of USP33 are required for the interaction between β-TrCP and USP33. Consequently, β-TrCP attenuated USP33-mediated inhibition of cell proliferation and cell invasion. Taken together, our study clarified that the E3 ligase β-TrCP regulates cellular USP33 levels by the ubiquitin-proteasomal proteolysis. - Highlights: •β-TrCP downregulates protein levels of USP33. •β-TrCP promotes USP33 ubiquitination and degradation. • The effect of β-TrCP on USP33 is independent of DSG motif. • β-TrCP attenuates USP33-mediated suppression of cell proliferation and invasion.

60 APPLIED LIFE SCIENCES↗

Comparative Molecular Analysis of Cancer Behavior Cultured In Vitro, In Vivo, and Ex Vivo

Current pre-clinical models of cancer fail to recapitulate the cancer cell behavior in primary tumors primarily because of the lack of a deeper understanding of the effects that the microenvironment has on cancer cell phenotype. Transcriptomic profiling of 4T1 murine mammary carcinoma cells from 2D and 3D cultures, subcutaneous or orthotopic allografts (from immunocompetent or immunodeficient mice), as well as ex vivo tumoroids, revealed differences in molecular signatures including altered expression of genes involved in cell cycle progression, cell signaling and extracellular matrix remodeling. The 3D culture platforms had more in vivo-like transcriptional profiles than 2D cultures. In vivo tumors had more cells undergoing epithelial-to-mesenchymal transition (EMT) while in vitro cultures had cells residing primarily in an epithelial or mesenchymal state. Ex vivo tumoroids incorporated aspects of in vivo and in vitro culturing, retaining higher abundance of cells undergoing EMT while shifting cancer cell fate towards a more mesenchymal state. Cellular heterogeneity surveyed by scRNA-seq revealed that ex vivo tumoroids, while rapidly expanding cancer and fibroblast populations, lose a significant proportion of immune components. This study emphasizes the need to improve in vitro culture systems and preserve syngeneic-like tumor composition by maintaining similar EMT heterogeneity as well as inclusion of stromal subpopulations.

59 BASIC BIOLOGICAL SCIENCES↗

AMPK and vacuole-associated Atg14p orchestrate μ-lipophagy for energy production and long-term survival under glucose starvation

Dietary restriction increases the longevity of many organisms, but the cell signaling and organellar mechanisms underlying this capability are unclear. We demonstrate that to permit long-term survival in response to sudden glucose depletion, yeast cells activate lipid-droplet (LD) consumption through micro-lipophagy (µ-lipophagy), in which fat is metabolized as an alternative energy source. AMP-activated protein kinase (AMPK) activation triggered this pathway, which required Atg14p. More gradual glucose starvation, amino acid deprivation or rapamycin did not trigger µ-lipophagy and failed to provide the needed substitute energy source for long-term survival. During acute glucose restriction, activated AMPK was stabilized from degradation and interacted with Atg14p. This prompted Atg14p redistribution from ER exit sites onto liquid-ordered vacuole membrane domains, initiating µ-lipophagy. Our findings that activated AMPK and Atg14p are required to orchestrate µ-lipophagy for energy production in starved cells is relevant for studies on aging and evolutionary survival strategies of different organisms.

59 BASIC BIOLOGICAL SCIENCES↗

Differential RNA regulation by staphylococcal enterotoxins A and B in murine macrophages

Staphylococcal enterotoxin A (SEA) is significantly better than enterotoxin B (SEB) in activating tumor necrosis factor (TNF) secretion by B6MP102 cells. Both toxins bound to B6MP102 cells; however, SEB competed less effectively with SEA than SEA competed with SEB. This suggested that receptors unique to SEA were present on B6MP102 cells. Signal transduction occurred in response to both toxins. Within 30 s after addition, SEA and SEB significantly increased the F-actin concentration in B6MP102 cells. However, only SEA induced increased TNF mRNA levels. B6MP102 cells incubated with interferon-gamma and SEB secreted TNF. However, enhanced mRNA expression was delayed and the concentration of TNF secreted was less than that of B6MP102 cells stimulated with SEA. Although these data suggest that receptors unique to SEA are present on B6MP102 cells, they also indicate that staphylococcal enterotoxins differentially regulate TNF at the RNA level, perhaps because of differences in binding to the plasma membrane.

NASA Discipline Cell Biology↗

Planar Particle Imaging Doppler Velocimetry Developed

Two current techniques exist for the measurement of planar, three-component velocity fields. Both techniques require multiple views of the illumination plane in order to extract all three velocity components. Particle image velocimetry (PIV) is a high-resolution, high accuracy, planar velocimetry technique that provides valuable instantaneous velocity information in aeropropulsion test facilities. PIV can provide three-component flow-field measurements using a two-camera, stereo viewing configuration. Doppler global velocimetry (DGV) is another planar velocimetry technique that can provide three component flow-field measurements; however, it requires three detector systems that must be located at oblique angles from the measurement plane. The three-dimensional configurations of either technique require multiple (DGV) or at least large (stereo PIV) optical access ports in the facility in which the measurements are being conducted. Optical access is extremely limited in aeropropulsion test facilities. In many cases, only one optical access port is available. A hybrid measurement technique has been developed at the NASA Glenn Research Center, planar particle image and Doppler velocimetry (PPIDV), which combines elements from both the PIV and DGV techniques into a single detection system that can measure all three components of velocity across a planar region of a flow field through a single optical access port. In the standard PIV technique, a pulsed laser is used to illuminate the flow field at two closely spaced instances in time, which are recorded on a "frame-straddling" camera, yielding a pair of single-exposure image frames. The PIV camera is oriented perpendicular to the light sheet, and the processed PIV data yield the two-component velocity field in the plane of the light sheet. In the standard DGV technique, an injection-seeded Nd:YAG pulsed laser light sheet illuminates the seeded flow field, and three receiver systems are used to measure three components of velocity. The receiver systems are oriented at oblique angles to the light sheet in order to accurately resolve the three-component velocity. Each DGV receiver system contains two cameras, which share a common view of the illuminated flow through a beam-splitting cube. One camera views the illuminated flow directly (reference camera) and the second camera images the illuminated flow through an iodine vapor cell (signal camera). The laser frequency (wavelength) is adjusted so that the Doppler-shifted light from particles in the flow falls on an iodine absorption feature, see the following graph. The iodine vapor cell acts as a frequency-to-velocity filter by modulating the intensity of the transmitted light as a function of the flow velocity (Doppler shift). The ratio of the signal and reference images yields the component of the flow velocity along the bisector of the laser sheet propagation direction and the receiver system observation direction. The hybrid system employs a single-component DGV receiver system configured to simultaneously acquire PIV image data, as shown in the following diagram. The cameras used in the DGV receiver are replaced with PIV frame-straddling cameras, and the receiver system views the illuminated light sheet plane at 90 (as in the standard PIV configuration).

Wernet, Mark P.↗

Nascent histamine induces α-synuclein and caspase-3 on human cells

Highlights: • Nascent histamine alters cyclin expression pattern. • Nascent histamine increases expression of α-synuclein. • Nascent histamine activates caspase-3. - Abstract: Histamine (Hia) is the most multifunctional biogenic amine. It is synthetized by histidine decarboxylase (HDC) in a reduced set of mammalian cell types. Mast cells and histaminergic neurons store Hia in specialized organelles until the amine is extruded by exocytosis; however, other immune and cancer cells are able to produce but not store Hia. The intracellular effects of Hia are still not well characterized, in spite of its physiopathological relevance. Multiple functional relationships exist among Hia metabolism/signaling elements and those of other biogenic amines, including growth-related polyamines. Previously, we obtained the first insights for an inhibitory effect of newly synthetized Hia on both growth-related polyamine biosynthesis and cell cycle progression of non-fully differentiated mammalian cells. In this work, we describe progress in this line. HEK293 cells were transfected to express active and inactive versions of GFP-human HDC fusion proteins and, after cell sorting by flow cytometry, the relative expression of a large number of proteins associated with cell signaling were measured using an antibody microarray. Experimental results were analyzed in terms of protein–protein and functional interaction networks. Expression of active HDC induced a cell cycle arrest through the alteration of the levels of several proteins such as cyclin D1, cdk6, cdk7 and cyclin A. Regulation of α-synuclein and caspase-3 was also observed. The analyses provide new clues on the molecular mechanisms underlying the regulatory effects of intracellular newly synthetized Hia on cell proliferation/survival, cell trafficking and protein turnover. This information is especially interesting for emergent and orphan immune and neuroinflammatory diseases.

60 APPLIED LIFE SCIENCES↗

Synthetic Phage for Tissue Regeneration

Controlling structural organization and signaling motif display is of great importance to design the functional tissue regenerating materials. Synthetic phage, genetically engineered M13 bacteriophage has been recently introduced as novel tissue regeneration materials to display a high density of cell-signaling peptides on their major coat proteins for tissue regeneration purposes. Structural advantages of their long-rod shape and monodispersity can be taken together to construct nanofibrous scaffolds which support cell proliferation and differentiation as well as direct orientation of their growth in two or three dimensions. This review demonstrated how functional synthetic phage is designed and subsequently utilized for tissue regeneration that offers potential cell therapy.

59 BASIC BIOLOGICAL SCIENCES↗

Finding a partner in the ocean: molecular and evolutionary bases of the response to sexual cues in a planktonic diatom

Microalgae play a major role as primary producers in aquatic ecosystems. Cell signalling regulates their interactions with the environment and other organisms, yet this process in phytoplankton is poorly defined. Using the marine planktonic diatom Pseudo-nitzschia multistriata, we investigated the cell response to cues released during sexual reproduction, an event that demands strong regulatory mechanisms and impacts on population dynamics. We sequenced the genome of P. multistriata and performed phylogenomic and transcriptomic analyses, which allowed the definition of gene gains and losses, horizontal gene transfers, conservation and evolutionary rate of sex-related genes. We also identified a small number of conserved noncoding elements. Sexual reproduction impacted on cell cycle progression and induced an asymmetric response of the opposite mating types. G protein-coupled receptors and cyclic guanosine monophosphate (cGMP) are implicated in the response to sexual cues, which overall entails a modulation of cell cycle, meiosis-related and nutrient transporter genes, suggesting a fine control of nutrient uptake even under nutrient-replete conditions. The controllable life cycle and the genome sequence of P. multistriata allow the reconstruction of changes occurring in diatoms in a key phase of their life cycle, providing hints on the evolution and putative function of their genes and empowering studies on sexual reproduction

Plant Sciences↗

Neutron scattering to study membrane systems: from lipid vesicles to living cells.

The existence and role of lateral lipid organization in biological membranes has been studied and contested for more than 30 years. Lipid domains, or rafts, are hypothesized as scalable compartments in biological membranes, providing appropriate physical environments to their resident membrane proteins. This implies that lateral lipid organization is associated with a range of biological functions, such as protein co-localization, membrane trafficking, and cell signaling, to name just a few. Neutron scattering techniques have proven to be an excellent tool to investigate these structural features in model lipids, and more recently, in living cells. I will discuss our recent work using neutrons to probe the structure and mechanical properties in model lipid systems and our current efforts in using neutrons to probe the structure and organization of the bilayer in a living cell. These efforts in living cells have used genetic and biochemical strategies to generate a large neutron scattering contrast, making the membrane visible. I will present our results showing in vivo bilayer structure and discuss the outlook for this approach.

Nickels, Jonathan D.↗

Thermodynamics of signal transduction systems and fluctuation theorem in a signal cascade

Biochemical chain reactions are signal transduction cascades that can transmit biological information about the intracellular environment. In this study, we modelled a chain reaction as a code string for applying information theory. Herein, we assumed that cell signal transduction selects a strategy to maximize the transduced signal per signal event duration. To investigate the same, we calculated the information transmission capacity of the reaction chain by maximizing the average entropy production rate per reaction time, indicating the idea of the entropy coding method. Moreover, we defined a signal cascade trajectory. Subsequently, we found that the logarithm of the forward and reverse transition ratio per reaction time is equal to the entropy production rate, which derives the form of the fluctuation theorem in signal transduction. Our findings suggest the application of information entropy theory for analysing signal transduction.

59 BASIC BIOLOGICAL SCIENCES↗

Gene expression and pathway analysis of human hepatocellular carcinoma cells treated with cadmium

Cadmium (Cd) is a toxic and carcinogenic metal naturally occurring in the Earth's crust. A common route of human exposure is via diet and cadmium accumulates in the liver. The effects of Cd exposure on gene expression in human hepatocellular carcinoma (HepG2) cells were examined in this study. HepG2 cells were acutely-treated with 0.1, 0.5, or 1.0 μM Cd for 24 h; or chronically-treated with 0.01, 0.05, or 0.1 μM Cd for three weeks and gene expression analysis was performed using Affymetrix GeneChip® Human Gene 1.0 ST Arrays. Acute and chronic exposures significantly altered the expression of 333 and 181 genes, respectively. The genes most upregulated by acute exposure included several metallothioneins. Downregulated genes included the monooxygenase CYP3A7, involved in drug and lipid metabolism. In contrast, CYP3A7 was upregulated by chronic Cd exposure, as was DNAJB9, an anti-apoptotic J protein. Genes downregulated following chronic exposure included the transcriptional regulator early growth response protein 1. Ingenuity Pathway Analysis revealed that the top networks altered by acute exposure were lipid metabolism, small molecule biosynthesis, cell morphology, organization, and development; while top networks altered by chronic exposure were organ morphology, cell cycle, cell signaling, and renal and urological diseases/cancer. Many of the dysregulated genes play important roles in cellular growth, proliferation, and apoptosis, and may be involved in carcinogenesis. In addition to gene expression changes, HepG2 cells treated with cadmium for 24 h indicated a reduction in global levels of histone methylation and acetylation that persisted 72 h post-treatment. - Highlights: • A common route of human exposure to the carcinogenic metal cadmium is via diet. • HepG2 cells were treated acutely or chronically with varying doses of cadmium. • Gene expression analysis was performed using Affymetrix Human Gene 1.0 Arrays. • Acute and chronic exposures altered the expression of 333 and 181 genes, respectively. • Acute cadmium exposure altered global levels of histone methylation and acetylation.

60 APPLIED LIFE SCIENCES↗

WE-EF-BRA-08: Cell Survival in Modulated Radiation Fields and Altered DNA-Repair at Field Edges

Purpose: Tissue damage prognoses in radiotherapy are based on clonogenic assays that provide dose dependent cell survival rates. However, recent work has shown that apart from dose, systemic reactions and cell-cell communication crucially influence the radiation response. These effects are probably a key in understanding treatment approaches such as microbeam radiation therapy (MRT). In this study we tried to quantify the effects on a cellular level in spatially modulated radiation fields. Methods: Pancreas carcinoma cells were cultured, plated and irradiated by spatially modulated radiation fields with an X-ray tube and at a synchrotron. During and after treatment cells were able to communicate via the intercellular medium. Afterwards we stained for DNA and DNA damage and imaged with a fluorescence microscope. Results: Intriguingly we found that DNA damage does not strictly increase with dose. Two cell entities appear that have either a high or a low amount of DNA lesions, indicating that DNA damage is also a cell stress reaction. Close to radiation boundaries damage-levels became alike; they were higher than expected at low and lower than expected at high doses. Neighbouring cells reacted similarly. 6 hours after exposure around 40% of the cells resembled in their reactions neighbouring cells more than randomly chosen cells that received the same dose. We also observed that close to radiation boundaries the radiation induced cell-cycle arrest disappeared and the size of DNA repair-centres increased. Conclusion: Cell communication plays an important role in the radiation response of tissues and may be both, protective and destructive. These effects may not only have the potential to affect conventional radiotherapy but may also be exploited to spare organs at risk by intelligently designing irradiation geometries. To that end intensive work is required to shed light on the still obscure processes in cell-signalling and radiation biology.

60 APPLIED LIFE SCIENCES↗