Equivalent Cell Density Research Articles

Improvement of fracture toughness based on auxetic patterns fabricated by metallic extrusion in 3D printing

This study aims to evaluate the effect of auxetic patterns on the fracture toughness of 3D printed metallic specimens manufactured by metallic extrusion of 17-4PH stainless steel. When it is pulled in the horizontal direction, the auxetic material expands in the vertical axis. This occurs since it has a negative Poisson's ratio. In this work auxetic cell structures have been employed in fracture mechanics characterization of Single Edge Notch Bending SENB specimens in order to improve their fracture toughness dynamically during strain.SENB specimens were designed with different cell patterns for fracture toughness assessment. (i) Auxetic pattern at 0° parallel to the notch direction, (ii) at 90° perpendicular to the notch direction and (iii) a conventional honeycomb pattern is defined with equivalent cell density on the specimen surface. Three-point bending tests have been carried out to evaluate the fracture toughness of each specimen. The mechanical performance ratio of specimens was evaluated by dividing the absorbed energy till fracture over the specimen weight. The ratios were also compared to a solid specimen ratio taken as a reference in this study.The results show that all specimens with cell patterns absorb more strain energy than the solid specimen does. Although the solid specimen is the stiffest, both cell patterns (auxetic and honeycomb) have higher performance indices considering the important weight reduction. For around 35 % of reduction in the maximum measured load, the auxetic pattern at 0° can absorb up to 88% more energy compared to the solid specimen. This proves its capability of delaying the crack initiation and enhancing the fracture toughness.

In vivo transcriptome analysis provides insights into host-dependent expression of virulence factors by Yersinia entomophaga MH96, during infection of Galleria mellonella.

The function of microbes can be inferred from knowledge of genes specifically expressed in natural environments. Here, we report the in vivo transcriptome of the entomopathogenic bacterium Yersinia entomophaga MH96, captured during initial, septicemic, and pre-cadaveric stages of intrahemocoelic infection in Galleria mellonella. A total of 1285 genes were significantly upregulated by MH96 during infection; 829 genes responded to in vivo conditions during at least one stage of infection, 289 responded during two stages of infection, and 167 transcripts responded throughout all three stages of infection compared to in vitro conditions at equivalent cell densities. Genes upregulated during the earliest infection stage included components of the insecticidal toxin complex Yen-TC (chi1, chi2, and yenC1), genes for rearrangement hotspot element containing protein yenC3, cytolethal distending toxin cdtAB, and vegetative insecticidal toxin vip2. Genes more highly expressed throughout the infection cycle included the putative heat-stable enterotoxin yenT and three adhesins (usher-chaperone fimbria, filamentous hemagglutinin, and an AidA-like secreted adhesin). Clustering and functional enrichment of gene expression data also revealed expression of genes encoding type III and VI secretion system-associated effectors. Together these data provide insight into the pathobiology of MH96 and serve as an important resource supporting efforts to identify novel insecticidal agents.

Pall Xpansion® Bioreactor Supports Progenitor Cell Growth to >1 Million cells/cm2 and Proper Cell Differentiation

Background & Aim With the recent increase in early phase cell therapy clinical trials, there is a need for a manufacturing platform which can be implemented in research labs and easily scaled up to expedite process development studies, pre-clinical testing, and large-scale expansion of adherent human cells. Although various platforms such as traditional flatware and stirred tank bioreactors with microcarriers exist and have been well characterized, traditional flatware often does not allow for tight control of the cellular environment and is not scalable. Pall's Xpansion single-use bioreactor offers a tightly-controlled, scalable manufacturing platform for cell therapy applications, allowing for expedited process development, pre-clinical testing and large scale, high-density expansion of progenitor cells while maintaining their differentiation capacity. Methods, Results & Conclusion We have previously demonstrated efficient expansion of both epithelial cells (Vero, HEK293), human mesenchymal stem/stromal cells (hMSC), and progenitor cells in the Xpansion single-use bioreactor. Here we extend the progenitor cell findings by successfully expanding a proprietary progenitor cell to extremely high cell densities (> 1 million cells/cm2) reproducibly for 8 batches. In summary, all batches resulted in 1) cell expansion to > 1 million cells/cm2 and equivalent cell densities to the traditional flatware process control; 2) equivalent differentiation to a more mature cell fate as indicated by expression of key cell surface markers; and 3) self-assembly into characteristic three-dimensional structures.

Abstract 170: Targeting colorectal cancer stem cells with the anticancer molecule thymoquinone

Abstract Introduction: 5-fluorouracil (5-Fu) remains the standard chemotherapy for metastatic colorectal cancer (CRC), but drug resistance and unpredictable cardiotoxicity limit its effectiveness. The high recurrence rates and the common resistance are thought to be due to a population of self-renewing cancer stem cells (CSCs). The black seed extract Thymoquinone (TQ) is a promising anticancer molecule known to inhibit cancer cell growth and progression in numerous cancer systems both in vitro and in vivo. This project aims to investigate the effect and mechanism of action of TQ on colon cancer stem/progenitor cells using two isogenic HCT116 colon cancer cell lines that differ in their 5-Fu drug sensitivity. Methods: Sphere-formation and propagation assays were used to assess the efficacy of TQ on targeting self-renewal capacity of colon CSCs enriched from the sensitive and resistant cell lines in 3D cultures over several generations in comparison to 2D monolayers. In addition, xenotransplantation experiments were used to assess for the tumor initiation of 2D vs. 3D cells. Results: Our results of TQ efficacy in 2D cell culture system showed that it reduced the viability of both cell lines. Importantly, our 3D results showed that TQ inhibits HCT116 colonosphere growth at 10-fold lower concentrations than those required to inhibit the growth of 2D monolayer cells. Interestingly, the injection of 100 spheres derived from HCT116 sensitive cell line and not the 2D equivalent cell density into NOD-SCID immunocompromised mice resulted in tumor development, suggesting that spheres are rich in cells with stem-like properties. Conclusion: In summary, our data suggests that TQ might be an effective treatment strategy and may prevent colorectal cancer recurrence by targeting CSCs. Citation Format: Farah R. Ballout, Maamoun Fatfat, Rana Abdel-Samad, Nadine Darwiche, Regine Schneider-Stock, Wassim Abou-Kheir, Hala-Gali Muhtasib. Targeting colorectal cancer stem cells with the anticancer molecule thymoquinone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 170.

Cardiosphere-Derived Cells Require Endoglin for Paracrine-Mediated Angiogenesis.

SummaryClinical trials of stem cell therapy to treat ischemic heart disease primarily use heterogeneous stem cell populations. Small benefits occur via paracrine mechanisms that include stimulating angiogenesis, and increased understanding of these mechanisms would help to improve patient outcomes. Cardiosphere-derived-cells (CDCs) are an example of these heterogeneous stem cell populations, cultured from cardiac tissue. CDCs express endoglin, a co-receptor that binds specific transforming growth factor β (TGFβ) family ligands, including bone morphogenetic protein 9 (BMP9). In endothelial cells endoglin regulates angiogenic responses, and we therefore hypothesized that endoglin is required to promote the paracrine pro-angiogenic properties of CDCs. Cre/LoxP technology was used to genetically manipulate endoglin expression in CDCs, and we found that the pro-angiogenic properties of the CDC secretome are endoglin dependent both in vitro and in vivo. Importantly, BMP9 pre-treatment of endoglin-depleted CDCs restores their pro-angiogenic paracrine properties. As BMP9 signaling is normally required to maintain endoglin expression, we propose that media containing BMP9 could be critical for therapeutic CDC preparation.

The Iron-Dependent Regulator Fur Controls Pheromone Signaling Systems and Luminescence in the Squid Symbiont Vibrio fischeri ES114

Bacteria often use pheromones to coordinate group behaviors in specific environments. While high cell density is required for pheromones to achieve stimulatory levels, environmental cues can also influence pheromone accumulation and signaling. For the squid symbiont Vibrio fischeri ES114, bioluminescence requires pheromone-mediated regulation, and this signaling is induced in the host to a greater extent than in culture, even at an equivalent cell density. Our goal is to better understand this environment-specific control over pheromone signaling and bioluminescence. Previous work with V. fischeri MJ1 showed that iron limitation induces luminescence, and we recently found that ES114 encounters a low-iron environment in its host. Here we show that ES114 induces luminescence at lower cell density and achieves brighter luminescence in low-iron media. This iron-dependent effect on luminescence required ferric uptake regulator (Fur), which we propose influences two pheromone signaling master regulators, LitR and LuxR. Genetic and bioinformatic analyses suggested that under low-iron conditions, Fur-mediated repression of litR is relieved, enabling more LitR to perform its established role as an activator of luxR. Interestingly, Fur may similarly control the LitR homolog SmcR of Vibrio vulnificus. These results reveal an intriguing regulatory link between low-iron conditions, which are often encountered in host tissues, and pheromone-dependent master regulators.

Transcriptional analysis of antibiotic resistance and virulence genes in multiresistant hospital-acquired MRSA

The staphylococcal chromosome cassette mec cannot solely explain the multiresistance phenotype or the relatively mild virulence profile of hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA). This study reports that several multiresistant HA-MRSA strains differently expressed genes that may support antibiotic resistance, modify the bacterial surface and influence the pathogenic process. Genes encoding efflux pumps (norA, arsB, emrB) and the macrolide resistance gene ermA were found to be commonly expressed by HA-MRSA strains, but not in the archetypal MRSA strain COL. At equivalent cell density, the agr system was considerably less activated in all MRSA strains (including COL) in comparison with a prototypic antibiotic-susceptible strain. These results are in contrast to those observed in recent community-acquired MRSA isolates and may partly explain how multiresistant HA-MRSA persist in the hospital setting.

Germ Line Gain of Function with SOS1 Mutation in Hereditary Gingival Fibromatosis

Mutation of human SOS1 is responsible for hereditary gingival fibromatosis type 1, a benign overgrowth condition of the gingiva. Here, we investigated molecular mechanisms responsible for the increased rate of cell proliferation in gingival fibroblasts caused by mutant SOS1 in vitro. Using ectopic expression of wild-type and mutant SOS1 constructs, we found that truncated SOS1 could localize to the plasma membrane, without growth factor stimuli, leading to sustained activation of Ras/MAPK signaling. Additionally, we observed an increase in the magnitude and duration of ERK signaling in hereditary gingival fibromatosis gingival fibroblasts that was associated with phosphorylation of retinoblastoma tumor suppressor protein and the up-regulation of cell cycle regulators, including cyclins C, D, and E and the E2F/DP transcription factors. These factors promote cell cycle progression from G(1) to S phase, and their up-regulation may underlie the increased gingival fibroblast proliferation observed. Selective depletion of wild-type and mutant SOS1 through small interfering RNA demonstrates the link between mutation of SOS1, ERK signaling, cell proliferation rate, and the expression levels of Egr-1 and proliferating cell nuclear antigen. These findings elucidate the mechanisms for gingival overgrowth mediated by SOS1 gene mutation in humans.

Relative effectiveness of kinetic analysis vs single point readings for classifying environmental samples based on community-level physiological profiles (CLPP)

The relative effectiveness of average-well-color-development-normalized single-point absorbance readings (AWCD) vs the kinetic parameters μm, λ, A, and integral (AREA) of the modified Gompertz equation fit to the color development curve resulting from reduction of a redox sensitive dye from microbial respiration of 95 separate sole carbon sources in microplate wells was compared for a dilution series of rhizosphere samples from hydroponically grown wheat and potato ranging in inoculum densities of 1×104−4×106 cells ml−1. Patterns generated with each parameter were analyzed using principal component analysis (PCA) and discriminant function analysis (DFA) to test relative resolving power. Samples of equivalent cell density (undiluted samples) were correctly classified by rhizosphere type for all parameters based on DFA analysis of the first five PC scores. Analysis of undiluted and 1:4 diluted samples resulted in misclassification of at least two of the wheat samples for all parameters except the AWCD normalized (0.50 abs. units) data, and analysis of undiluted, 1:4, and 1:16 diluted samples resulted in misclassification for all parameter types. Ordination of samples along the first principal component (PC) was correlated to inoculum density in analyses performed on all of the kinetic parameters, but no such influence was seen for AWCD-derived results. The carbon sources responsible for classification differed among the variable types with the exception of AREA and A, which were strongly correlated. These results indicate that the use of kinetic parameters for pattern analysis in CLPP may provide some additional information, but only if the influence of inoculum density is carefully considered.

Chaperone SecB from Escherichia coli Mediates Kinetic Partitioning via a Dynamic Equilibrium with Its Ligands

We have shown that the complexes between SecB, a chaperone from Escherichia coli, and two physiological ligands, galactose-binding protein and maltose-binding protein, are in rapid, dynamic equilibrium between the bound and free states. Binding to SecB is readily reversible, and each time the ligand is released it undergoes a kinetic partitioning between folding to its native state and re-binding to SecB. Binding requires that the polypeptide be devoid of tertiary structure; once the protein has folded, it is no longer a ligand. Conditions were established in which folding of the polypeptides was sufficiently slow so that at each cycle of dissociation rebinding was favored over folding and a kinetically stable complex between SecB and each polypeptide ligand was observed. Evidence that the ligand is continually released to the bulk solution and rebound was obtained by altering the conditions to increase the rate of folding of each ligand so that folding of the ligand was faster than reassociation with SecB thereby allowing the system to partition to free SecB and folded polypeptide ligand. We conclude that complexes between the chaperone SecB and ligands are in dynamic, rapid equilibrium with the free states. This mode of binding is simpler than that documented for chaperones that function to facilitate folding such as the Hsp70s and Hsp60s, where hydrolysis of ATP is coupled to the binding and release of ligands. This difference may reflect the fact that SecB does not mediate folding but is specialized to facilitate protein export. Without a requirement for exogenous energy it efficiently performs its sole duty: to keep proteins in a nonnative conformation and thus competent for export.

Effect of inoculant density, formulation, dispersion and soil nutrient amendment on the removal of carbofuran residues from contaminated soil

Our study was aimed at investigating some factors affecting the use of a carbofuran-degrading bacterium as an inoculant to remove carbofuran residues from contaminated soil. Soil samples inoculated with a liquid cell suspension of strain C28 to give an initial density ranging from 10 3 to 10 7 cfu g −1 (dry weight of soil) significantly improved pesticide removal. When the soil was inoculated with a granular formulation of strain C28 to give theoretical cell densities of 5 × 10 3, 5 × 10 4 and 5 × 10 5 cfu g −1 (dry wt of soil), a significant increase in the rate of carbofuran degradation, as compared to uninoculated samples, was seen only for the highest inoculum size. The rate of carbofuran degradation in non-inoculated samples was significantly reduced by chloroform fumigation, but inoculation of natural and chloroform-fumigated soil with equivalent cell densities of strain C28 in both liquid and granular formulation led to a significant increase in the rate of carbofuran breakdown. The effect of soil glucose amendment on pesticide degradation by strain C28, in a liquid or solid formulation, depended on the concentration of the sugar and on inoculant density. A low concentration of glucose significantly reduced the lag phase before pesticide degradation by a low density inoculant. A high concentration, however, led to a significant reduction of the degradation rate for all inoculum sizes. Mixing the siol after inoculation was essential to obtain rapid degradation of the pesticide. When theoretically-equivalent cell densities were introduced in 0.1 and 1 g of microgranules, no significant difference in the rate of pesticide degradation was observed in these experiments.

Equivalent cell density in three areas of neonatal rat cerebral cortex

Using the disector method, equivalent cell densities (number of cells per unit volume) were found in the frontal (motor), parietal (somatosensory) and occipital (visual) cortex of neonatal rat (1.2 × 10 6 cells/mm 3). As determined with GABA post-embedding immunocytochemistry on semithin sections, at least 11% of these cells expressed GABA. Because neonate frontal cortex is thicker than the parietal or the occipital cortex, the number of cells under an unit area of pial surface was greater in this cortex. This clearly differed from the adult pattern, where the greatest number of cells beneath an unit pial area is that of the somatosensory versus the frontal and the occipital cortex. It suggests that the attainment of the adult number of cells in a cortical column is achieved through differential cell death in the different areas and that the amount of thalamic input could be a crucial determinant of the adult cytoarchitecture.

PHYSIOLOGICAL MODIFICATIONS RELATED TO DENSITY INCREASE IN PERIPHYTIC ASSEMBLAGES 1

ABSTRACT Periphytic communities in running waters were examined as they developed on granite rocks, concrete balls and glass slides. At equivalent cell densities, no differences in pigment concentrations, species diversity or production levels were found among the different substrata examined. Development of the assemblage appeared to result from the elongation of short algal filaments which had initially settled on the surface. As these communities matured, a distinct canopy and understory developed. Cellular metabolisms were comparable among the communities. In the understory of the communities, even though the cellular content of chl a and b did not differ, chl c and carotenoid pigment concentrations were higher than those in the over‐story. Bicarbonate assimilation of Tabellaria fenestrata (Lyng.) Külz. and Eunotia pectinalisi var. pectinalis (O. F. Müll?) Rabh. was higher than that of the more abundant Tabellaria flocculosa (Roth.)Kütz. var. flocculosa IV (sensu Koppen) at both high and low cell densities. This probably reflects a seasonal succession of colonizing species. Glucose assimilation appeared to be mainly attributable to bacterial activity, and algal cells of the upper layer were less active than those of the bottom. The small amount of glucose that was incorporated by the algal cells was probably absorbed passively since its amount was in direct proportion to cell volumes.

Retinal crowding alters the morphology of alpha ganglion cells.

It has been hypothesized that the dendritic field size of individual retinal ganglion cells is regulated early in development by interactions among neighboring cells (Wässle and Reiman, Proc. R. Soc. Lond. B. 200:441-461, 1978). An opportunity to test this hypothesis is provided by the consequences of prenatal enucleation that results in an increased density of ganglion cells in the remaining retina (Chalupa et al., Neuroscience 12:1139-1146, 1984). In the present study we compared dendritic field diameters of alpha ganglion cells in normal retinas to those of adult cats that were monocularly enucleated before birth (on embryonic days 42 and 51) and 6 days after birth. In each animal ganglion cells were labeled by central injections of horseradish peroxidase. The retinas were incubated according to a modified Hanker-Yates procedure and whole-mounted. All cells were sampled from the temporal retina along a corridor established by a line drawn through the optic disk and the area centralis. Alpha cells were differentiated into ON and OFF subtypes on the basis of their level of stratification in the inner plexiform layer (Peichl and Wässle, Proc. R. Soc. Lond. B. 212:139-156, 1981). Dendritic field and soma diameters were determined from complete cell drawings by calculating the mean of the two longest orthogonal diameters. Our main findings are: Early monocular enucleation does not disrupt the mosaics of ON and OFF alpha ganglion cells in the remaining retina of adult animals. Within the retinal corridor sampled, the density of alpha cells was substantially greater than normal in the remaining retina of the prenatal enucleates at all eccentricities except the far periphery. Except at the far periphery, the dendritic field diameters of the prenatally enucleated animals were significantly smaller than normal when compared at equivalent eccentricities. However, when compared at equivalent cell densities, dendritic field dimensions in the prenatally enucleated and normal animals were found to be similar. In the prenatal enucleates the somas of these neurons were also significantly smaller than normal. If the monocular enucleation was postnatal, however, the density and the dendritic field diameters of alpha cells did not differ appreciably from normal. These results indicate that the morphology of retinal ganglion cells can be influenced by increasing the density of developing ganglion cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Flow rate and bead size as critical parameters for immobilized-yeast reactors

The productivity of immobilized yeast cell reactors varies with a number of parameters, including flow, amount and growth rate of yeast, bead size and type of medium. Variation of these parameters has a pronounced effect on reaction rate. This paper presents typical ranges for these productivities and demonstrates the patterns of changes that take place when bead size, flow and reaction medium are varied. Saccharomyces cerevisiae cells were immobilized in calcium alginate beads for the production of ethanol. The productivity of immobilized yeast in a batch reactor (0.2 g ethanol/g yeast · h) was only two-thirds that of free cells suspended at an equivalent cell density (0.3 g ethanol/g yeast · h). Different flow rates and bead sizes were used to ‘optimize’ the productivity. The productivity of 3.34 mm beads at a flow rate of 8.8 litre h −1 (superficial velocity: 0.12 cm s −1 ) was 95% higher than that at 1.0 l h −1. Maximum productivities of 0.34, 0.27, 0.22 g/g yeast· h were obtained (at a flow rate of 8.8 l h −1 ) for 9.2% yeast-immobilized beads of 3.34, 4.45 and 5.65 mm in diameter, respectively.

Potassium transport in normal and transformed mouse 3T3 cells

The components of unidirectional K influx and efflux have been investigated in the 3T3 cell and the SV40 transformed 3T3 cell in expontntial and stationary growth phase. Over the cell densities used for transport experiments the 3T3 cell goes from exponential growth to density dependent inhibition of growth (4 X 10(4) to 4 X 10(5) cell cm-2) whereas the SV40 3T3 maintains exponential or near exponential growth (4 X 10(4) to 1 X 10(6) cell cm-2). In agreement with previous observations, volume per cell and mg protein per cell decrease with increasing cell density. Thus, transport measurements have been expressed on a per volume basis. Total unidirectional K influx and efflux in the 3T3 cell is approximately double that of the SV40 3T3 cell at all cell densities investigated. Both cell types have similar volumes initially and show similar decreases with increasing cell density. Thus, in this clone of the 3T3 cell SV40 transformation specifically decreases unidirectional K flux. The magnitude of the total K flux does not change substantially for either cell line during transition from sparse to dense cultures. However, the components of the K transport undergo distinct changes. Both cell lines possess a ouabain sensitive component of K influx, presumably representing the active inward K pump. Both also possess components of K influx and efflux sensitive to furosemide. The data suggest this component represents a one-for-one K exchange mechanism. The fraction of K influx mediated by the ouabain sensitive component is reduced to one half its value when exponential versus density inhibited 3T3 cells are compared (63% versus 31% of total influx). No comparable drop occurs in the SV40 3T3 cell at equivalent cell densities (64% versus 56% of total influx). Thus, the pump mediated component of K influx would appear to be correlated with growth. In contrast, the furosemide sensitive component represents approximately 20% of the total unidirectional K influx and efflux in both cell lines in sparse culture. At high cell densities, where growth inhibition occurs in the 3T3 cell but not the SV40 3T3, the furosemide sensitive component doubles in both cell lines. Thus, the apparent K-K exchange mechanism is density dependent rather than growth dependent.