The Effects of Light Spectrum and Intensity, Seeding Density, and Fertilization on Biomass, Morphology, and Resource Use Efficiency in Three Species of Brassicaceae Microgreens.

Light is a critical component of indoor plant cultivation, as different wavelengths can influence both the physiology and morphology of plants. Furthermore, fertilization and seeding density can also potentially interact with the light recipe to affect production outcomes. However, maximizing production is an ongoing research topic, and it is often divested from resource use efficiencies. In this study, three species of microgreens-kohlrabi; mustard; and radish-were grown under five light recipes; with and without fertilizer; and at two seeding densities. We found that the different light recipes had significant effects on biomass accumulation. More specifically, we found that Far-Red light was significantly positively associated with biomass accumulation, as well as improvements in height, leaf area, and leaf weight. We also found a less strong but positive correlation with increasing amounts of Green light and biomass. Red light was negatively associated with biomass accumulation, and Blue light showed a concave downward response. We found that fertilizer improved biomass by a factor of 1.60 across species and that using a high seeding density was 37% more spatially productive. Overall, we found that it was primarily the main effects that explained microgreen production variation, and there were very few instances of significant interactions between light recipe, fertilization, and seeding density. To contextualize the cost of producing these microgreens, we also measured resource use efficiencies and found that the cheaper 24-volt LEDs at a high seeding density with fertilizer were the most efficient production environment for biomass. Therefore, this study has shown that, even with a short growing period of only four days, there was a significant influence of light recipe, fertilization, and seeding density that can change morphology, biomass accumulation, and resource input costs.

Non-uniformity in in vitro drug-induced cytotoxicity as evidenced by differences in IC50 values - implications and way forward.

Cell lines have proven indispensable for in vitro experiments and their utility as experimental models range from understanding the fundamental cell functioning to drug discovery. One of the most common utility of cell lines is for in vitro drug testing. Drug testing involves determining the cytotoxic effects of the drugs and such a measurement is expressed as the IC50 values of drugs. Although determination of IC50 values of drugs on cell lines is one of the most common in vitro experimental approaches, a significant amount of variations can be observed in the results obtained from such studies. Although the variations in the IC50 values of a drug on different cells lines can and should vary, the non-uniformity of such results reported from different studies using a particular drug on a specific cell line is a matter of concern. We present the IC50 values of 5 most commonly used drugs 5-fluorouracil, bleomycin, cisplatin, doxorubicin and methotrexate obtained from several in vitro cell line-based studies. Some of the factors which contribute to the non-uniformity of the IC50 values for a particular drug from different studies are discussed as three types of factors, the biological, non-biological and human factors. Also, ways in which such variations can be reduced to obtain universally common, reliable results are presented.

Effect of inoculation density of bone marrow mesenchymal stem cells cultured on calcium phosphate cement scaffold on osteogenic differentiation.

BACKGROUND: Calcium phosphate cements (CPCs) are biocompatible materials that have been evaluated as scaffolds in bone tissue engineering. At present, the stem cell density of inoculation on CPC scaffold varies. OBJECTIVE: The aim of this study is to analyze the effect of seeding densities on cell growth and osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) on a calcium phosphate cements (CPCs) scaffold. METHODS: BMMSCs derived from minipigs were seeded onto a CPC scaffold at three densities [1 million/mL (1M), 5 million/mL (5M) and 25 million/mL 25M)], and cultured for osteogenic induction for 1, 4 and 8 days. RESULTS: Well adhered and extended BMMSCs on the CPC scaffold showed significantly different proliferation rates within each seeding density group at different time points (P < 0.05). The number of live cells per unit area in 1M, 5M and 25M increased by 3.5, 3.9 and 2.5 folds respectively. The expression of ALP peaked at 4 days post inoculation with the fold-change being 2.6 and 2.8 times higher in 5M and 25M respectively as compared to 1M. The expression levels of OC, Coll-1 and Runx-2 peaked at 8 days post inoculation. CONCLUSIONS: An optimal seeding density may be more conducive for cell proliferation, differentiation, and extracellular matrix synthesis on scaffolds. We suggest the optimal seeding density should be 5 million/mL.

Cancer cell seeding density as a mechanism of chemotherapy resistance: a novel cancer cell density index based on IC50-Seeding Density Slope (ISDS) to assess chemosensitivity.

Although the 50% inhibitory concentration (IC50) is a commonly used measurement of chemosensitivity in cancer cells, it has been known to vary with the density of the treated cells (in that more densely seeded cells are more resistant to chemotherapeutic agents). Indeed, density-dependent chemoresistance may be a significant independent mechanism of therapy resistance. We examine the nature of cell density-dependent chemoresistance and explore possible underlying mechanisms. CellTiter-Glo assays and ethidium homodimer staining revealed that response to chemotherapy is density-dependent in all cancer cell lines tested. Our results prompted us to develop a novel cancer cell seeding density index of chemosensitivity, the ISDS (IC50-Seeding Density Slope), which we propose can serve as an improved method of analyzing how cancer cells respond to chemotherapeutic treatment compared to the widely-used IC50. Furthermore, western blot analysis suggests that levels of autophagy and apoptotic markers are modulated by cancer cell density. Cell viability experiments using the autophagy inhibitor chloroquine showed that chloroquine's efficacy was reduced at higher cell densities and that chloroquine and cisplatin exhibited synergy at both higher and lower cell densities in TOV-21G cells. We discuss alternative mechanisms of density-dependent chemoresistance and in vivo/clinical applications, including challenges of adjuvant chemotherapy and minimal residual disease. Taken together, our findings show that cell density is a significant contributor in shaping cancer chemosensitivity, that the ISDS (aka the Ujwal Punyamurtula/Wafik El-Deiry or Ujwal-WAF Index) can be used to effectively assess cell viability and that this phenomenon of density-dependent chemoresistance may be leveraged for a variety of biologic and cancer therapeutic applications.

Micropyramid-patterned, oxygen-permeable bottomed dish for high density culture of pancreatic islets.

The need for maintaining cell-spheroid viability and function within high-density cultures is unmet for various clinical and experimental applications, including cell therapies. One immediate application is for transplantation of pancreatic islets, a clinically recognized treatment option to cure type 1 diabetes; islets are isolated from a donor for subsequent culture prior to transplantation. However, high seeding conditions cause unsolicited fusion of multiple spheroids, thereby limiting oxygen diffusion to induce hypoxic cell death. Here we introduce a culture dish incorporating a micropyramid-patterned surface to prevent the unsolicited fusion and oxygen-permeable bottom for optimal oxygen environment. A 400µm-thick, oxygen-permeable polydimethylsiloxane sheet topped with micropyramid pattern of 400µm-base and 200µm-height was fabricated to apply to the 24-well plate format. The micropyramid pattern separated the individual pancreatic islets to prevent the fusion of multiple islets. This platform supported the high oxygen demand of islets at high seeding density at 260 islet equivalents cm-2, a 2-3-fold higher seeding density compared to the conventional islet culture used in a preparation for the clinical islet transplantations, demonstrating improved islet morphology, metabolism and function in a 4 d-culture. Transplantation of these islets into immunodeficient diabetic mice exhibited significantly improved engraftment to achieve euglycemia compared to islets cultured in the conventional culture wells. Collectively, this simple design modification allows for high-density cultures of three-dimensional cell spheroids to improve the viability and function for an array of investigational and clinical replacement tissues.

[Research progress of different cell seeding densities and cell ratios in cartilage tissue engineering].

Objective: To review the research progress of different cell seeding densities and cell ratios in cartilage tissue engineering. Methods: The literature about tissue engineered cartilage constructed with three-dimensional scaffold was extensively reviewed, and the seeding densities and ratios of most commonly used seed cells were summarized. Results: Articular chondrocytes (ACHs) and bone marrow mesenchymal stem cells (BMSCs) are the most commonly used seed cells, and they can induce hyaline cartilage formation in vitro and in vivo. Cell seeding density and cell ratio both play important roles in cartilage formation. Tissue engineered cartilage with good quality can be produced when the cell seeding density of ACHs or BMSCs reaches or exceeds that in normal articular cartilage. Under the same culture conditions, the ability of pure BMSCs to build hyaline cartilage is weeker than that of pure ACHs or co-culture of both. Conclusion: Due to the effect of scaffold materials, growth factors, and cell passages, optimal cell seeding density and cell ratio need further study.

Improving Cell Recovery: Freezing and Thawing Optimization of Induced Pluripotent Stem Cells.

Achieving good cell recovery after cryopreservation is an essential process when working with induced pluripotent stem cells (iPSC). Optimized freezing and thawing methods are required for good cell attachment and survival. In this review, we concentrate on these two aspects, freezing and thawing, but also discuss further factors influencing cell recovery such as cell storage and transport. Whenever a problem occurs during the thawing process of iPSC, it is initially not clear what it is caused by, because there are many factors involved that can contribute to insufficient cell recovery. Thawing problems can usually be solved more quickly when a certain order of steps to be taken is followed. Under optimized conditions, iPSC should be ready for further experiments approximately 4-7 days after thawing and seeding. However, if the freezing and thawing protocols are not optimized, this time can increase up to 2-3 weeks, complicating any further experiments. Here, we suggest optimization steps and troubleshooting options for the freezing, thawing, and seeding of iPSC on feeder-free, Matrigel™-coated, cell culture plates whenever iPSC cannot be recovered in sufficient quality. This review applies to two-dimensional (2D) monolayer cell culture and to iPSC, passaged, frozen, and thawed as cell aggregates (clumps). Furthermore, we discuss usually less well-described factors such as the cell growth phase before freezing and the prevention of osmotic shock during thawing.

Application of metabolic modeling for targeted optimization of high seeding density processes.

Process intensification by application of perfusion mode in pre-stage bioreactors and subsequent inoculation of cell cultures at high seeding densities (HSD) has the potential to meet the increasing requirements of future manufacturing demands. However, process development is currently restrained by a limited understanding of the cell's requirements under these process conditions. The goal of this study was to use extended metabolite analysis and metabolic modeling for targeted optimization of HSD cultivations. The metabolite analysis of HSD N-stage cultures revealed accumulation of inhibiting metabolites early in the process and flux balance analysis led to the assumption that reactive oxygen species (ROS) were contributing to the fast decrease in cell viability. Based on the metabolic analysis an optimized feeding strategy with lactate and cysteine supplementation was applied, resulting in an increase in antibody titer of up to 47%. Flux balance analysis was further used to elucidate the surprisingly strong synergistic effect of lactate and cysteine, indicating that increased lactate uptake led to reduced ROS formation under these conditions whilst additional cysteine actively reduced ROS via the glutathione pathway. The presented results finally demonstrate the benefit of modeling approaches for process intensification as well as the potential of HSD cultivations for biopharmaceutical manufacturing.

Development of a High Yielding Bioprocess for a Pre-fusion RSV Subunit Vaccine.

Respiratory syncytial virus (RSV) is a highly contagious virus causing severe infection in infants and the elderly. Various approaches are being used to develop an effective RSV vaccine. The RSV fusion (F) subunit, particularly the cleaved trimeric pre-fusion F, is one of the most promising vaccine candidates under development. The pre-fusion conformation elicits the majority of neutralizing antibodies during natural infection. However, this pre-fusion conformation is metastable and prone to conversion to a post-fusion conformation, thus hindering the potential of this construct as a vaccine antigen. The Vaccine Research Center (VRC) at the National Institutes of Health (NIH) designed a structurally stabilized pre-fusion F glycoprotein, DS-Cav1, that showed high immunogenicity and induced a neutralizing response in animal studies. To advance this candidate to clinical manufacturing, a production process that maintained product quality (i.e. a cleaved trimer with pre-fusion conformation) and delivered high protein expression levels was required. This report describes the development of the vaccine candidate including vector design and cell culture process development to meet these challenges. Co-transfection of individual plasmids to express DS-Cav1 and furin (for DS-Cav1 cleavage and activation) demonstrated a superior protein product expression and pre-fusion conformation compared to co-expression with a double gene vector. A top clone was selected based on these measurements. Protein expression levels were further increased by seeding density optimization and a biphasic hypothermia temperature downshift. The combined efforts led to a high-yield fed-batch production of approximately 1,500 mg/L (or up to 15,000 doses per liter) at harvest. The process was scaled up and demonstrated to be reproducible at 50 L-scale for toxicity and Phase I clinical trial use. Preliminary phase I data indicate the pre-fusion antigen has a promising efficacy (Crank et al., 2019).

Genetic analysis of rice seedling traits related to machine transplanting under different seeding densities.

BACKGROUND: Due to the diversity of rice varieties and cropping systems in China, the limitation of seeding density and seedling quality makes it hard to improve machine-transplanted efficiency. Previous studies have shown that indica and japonica varieties varied in machine transplanting efficiency and optimal seeding density. In this study, a RIL population derived from '9311' and 'Nipponbare' were performed to explore the seedling traits variations and the genetic mechanism under three seeding densities. RESULTS: The parents and RIL population exhibited similar trends as the seeding density increased, including seedling height and first leaf sheath length increases, shoot dry weight and root dry weight decreases. Among the 37 QTLs for six traits detected under the three seeding densities, 12 QTLs were detected in both three seeding densities. Five QTL hotspots identified clustered within genomic regions on chromosomes 1, 2, 4, 6 and 11. Specific QTLs such as qRDW1.1 and qFLSL5.1 were detected under low and high seeding densities, respectively. Detailed analysis the QTL regions identified under specific seeding densities revealed several candidate genes involved in phytohormones signals and abiotic stress responses. Whole-genome additive effects showed that '9311' contributed more loci enhancing trait performances than 'Nipponbare', indicating '9311' was more sensitive to the seeding density than 'Nipponbare'. The prevalence of negative epistasis effects indicated that the complementary two-locus homozygotes may not have marginal advantages over the means of the two parental genotypes. CONCLUSIONS: Our results revealed the differences between indica rice and japonica rice seedling traits in response to seeding density. Several QTL hotspots involved in different traits and specific QTLs (such as qRDW1.1 and qFLSL5.1) in diverse seeding densities had been detected. Genome-wide additive and two-locus epistasis suggested a dynamic of the genetic control underlying different seeding densities. It was concluded that novel QTLs, additive and epistasis effects under specific seeding density would provide adequate information for rice seedling improvement during machine transplanting.

Growth and Stem Cell Characteristics of Tendon-Derived Cells with Different Initial Seeding Densities: An In Vitro Study in Mouse Flexor Tendon Cells.

Tendon stem/progenitor cells (TSPCs) are considered promising seed cells for tendon regeneration. Previous studies reported that a low seeding density favors TSPC growth, whereas a high seeding density favors tenocyte growth. We aimed to distinguish TSPCs from tenocytes by seeding tendon-derived cells at a density gradient. In this study, tendon-derived cells were isolated from flexor digitorum profundus tendons of mice and seeded at the initial densities of 50, 500, 5,000, and 50,000/cm2. We found that distinct cell colonies were formed from cells with initial seeding densities of 50 and 500/cm2, but colonies were not discernible for cells seeded at 5,000 and 50,000/cm2. There was a positive correlation between cell proliferation rate and seeding density, but a negative correlation between cell senescence and seeding density. The cell proliferation rate decreased gradually during serial passages. All cells exhibited restricted differentiation potentials, and expressed stem cell markers and relatively high levels of tenogenic markers without notable differences among cells seeded at different densities. We concluded that a pure population of TSPCs could not be isolated from mouse digital flexor tendons through culturing cells at a density gradient. Cells seeded at low densities had very limited proliferative ability and did not show more prominent stem cell characteristics when compared with cells seeded at high densities.

Effects of cell phenotype and seeding density on the chondrogenic capacity of human osteoarthritic chondrocytes in type I collagen scaffolds.

OBJECTIVE: Matrix-associated autologous chondrocyte implantation (MACI) achieves good clinical efficacy in young patients with focal cartilage injury; however, phenotypic de-differentiation of chondrocytes cultured in monolayer and the treatment of older OA patients are still challenges in the field of cartilage tissue engineering. This study aimed to assess the in vitro re-differentiation potential and in vivo chondrogenic capacity of human OA chondrocytes inoculated into collagen I scaffolds with different cellular phenotypes and seeding densities. METHODS: OA chondrocytes and articular chondrocyte-laden scaffolds were cultured over 2 weeks in in vitro. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and histological staining were used to detect the mRNA expression profiles and extracellular matrix secretion of chondrocyte-specific markers. OA chondrocyte-laden collagen I scaffolds with different cellular phenotypes, and seeding densities were implanted into SCID mice over 4 weeks to evaluate the chondrogenic capacity in vivo. RESULTS: Increased COL2a1, ACAN, COMP, SOX9, and BMP2 expression levels and decreased COL1a1, VCAN, MMP13, and ADAMTS5 amounts were observed in OA chondrocytes seeded in collagen I scaffolds; Implantation of phenotypically superior OA chondrocytes in collagen I scaffolds at high density could improve the chondrogenic capacity of human OA chondrocytes, as confirmed by RT-qPCR assessed gene expression patterns in vitro and histological evaluation in vivo. CONCLUSIONS: Freshly isolated chondrocytes from OA patients could be a source of replacement for articular chondrocytes being commonly used in MACI. Implantation of phenotypically superior OA chondrocytes in collagen I scaffolds at high density could be a promising tool for the treatment of elderly OA patients.

Effect of cell density on formation of three-dimensional cartilaginous constructs using fibrin & human osteoarthritic chondrocytes.

Background & objectives: Seeding density is one of the major parameters affecting the quality of tissue-engineered cartilage. The objective of this study was to evaluate different seeding densities of osteoarthritis chondrocytes (OACs) to obtain the highest quality cartilage. Methods: The OACs were expanded from passage 0 (P0) to P3, and cells in each passage were analyzed for gross morphology, growth rate, RNA expression and immunochemistry (IHC). The harvested OACs were assigned into two groups: low (1×10[7] cells/ml) and high (3×10[7] cells/ml) cell density. Three-dimensional (3D) constructs for each group were created using polymerised fibrin and cultured for 7, 14 and 21 days in vitro using chondrocyte growth medium. OAC constructs were analyzed with gross assessments and microscopic evaluation using standard histology, IHC and immunofluorescence staining, in addition to gene expression and biochemical analyses to evaluate tissue development. Results: Constructs with a high seeding density of 3×10[7] cells/ml were associated with better quality cartilage-like tissue than those seeded with 1×10[7] cells/ml based on overall tissue formation, cell association and extracellular matrix distribution. The chondrogenic properties of the constructs were further confirmed by the expression of genes encoding aggrecan core protein and collagen type II. Interpretation & conclusions: Our results confirmed that cell density was a significant factor affecting cell behaviour and aggregate production, and this was important for establishing good quality cartilage.

Impact of various culture conditions on ex vivo expansion of polyclonal T cells for adoptive immunotherapy.

Currently, adoptive immunotherapy is considered as one of the leading treatments in cancer. Successful adoptive immunotherapy depends on producing large numbers of desired T cells ex vivo for infusion. This requires an effective protocol for maximum functional T-cell expansion while keeping the time and costs to a minimum. Current T-cell expansion protocols are diverse in their methodology, and a universal protocol of expansion is wanting. Also, new findings regarding T-cell biology, signaling, and activation have reshaped the strategies of T-cell propagation over the years, introducing new ways to expand T cells. Here, we reviewed different conditions for blood-derived polyclonal T-cell expansion so as to elucidate the influential factors of T-cell expansion and their efficacy.

Bench-to-bedside optimization of mesenchymal stem cell isolation, processing, and expansion for in vivo administration.

Aim: In this study, we aimed at identifying the optimal conditions for isolation, processing and expansion of mesenchymal stem cells (MSCs). Methods: Porcine bone marrow was obtained from either small- or large-volume bone marrow aspirate (BMA). Next, three BMA processing methods were compared. Finally, the best condition was selected from various culture parameters, including basal media, supplementation and seeding density. Results: Our results demonstrate that a small-volume BMA and direct plating yields significantly higher concentration of MSCs. Basal media supplementation with 10% platelet lysate and seeding density of 1000 cells/cm2 can generate large numbers of multipotent MSCs with augmented function and low population doublings. Conclusion: This work provides guidance for preparation of robust MSCs for future clinical trials.

Increasing Cell Seeding Density Improves Elastin Expression and Mechanical Properties in Collagen Gel-Based Scaffolds Cellularized with Smooth Muscle Cells.

Vascular tissue engineering combines cells with scaffold materials in vitro aiming the development of physiologically relevant vascular models. For natural scaffolds such as collagen gels, where cells can be mixed with the material solution before gelation, cell seeding density is a key parameter that can affect extracellular matrix deposition and remodeling. Nonetheless, this parameter is often overlooked and densities sensitively lower than those of native tissues, are usually employed. Herein, the effect of seeding density on the maturation of tubular collagen gel-based scaffolds cellularized with smooth muscle cells is investigated. The compaction, the expression, and deposition of key vascular proteins and the resulting mechanical properties of the constructs are evaluated up to 1 week of maturation. Results show that increasing cell seeding density accelerates cell-mediated gel compaction, enhances elastin expression (more than sevenfold increase at the highest density, Day 7) and finally improves the overall mechanical properties of constructs. Of note, the tensile equilibrium elastic modulus, evaluated by stress-relaxation tests, reach values comparable to native arteries for the highest cell density, after a 7-day maturation. Altogether, these results show that higher cell seeding densities promote the rapid maturation of collagen gel-based vascular constructs toward structural and mechanical properties better mimicking native arteries.

Effect of Monocyte Seeding Density on Dendritic Cell Generation in an Automated Perfusion-Based Culture System.

Dendritic cells (DCs) are increasingly important for research and clinical use but obtaining sufficient numbers of dendritic cells is a growing challenge. We systemically investigated the effect of monocyte (MO) seeding density on the generation of monocyte-derived immature DCs (iDCs) in MicroDEN, a perfusion-based culture system, as well as 6-well plates. Cell surface markers and the ability of the iDCs to induce proliferation of allogeneic T cells were examined. The data shows a strong relationship between iDC phenotype, specifically CD80/83/86 expression, and T cell proliferation. MicroDEN generated iDCs proved better than well plate generated iDCs at inducing T cell proliferation within the 200k-600k MO/cm2 seeding density range studied. We attribute this to perfusion in MicroDEN which supplies fresh differentiation medium continuously to the differentiating MOs while concurrently removing depleted medium and toxic byproducts of cellular respiration. MicroDEN generated fewer iDCs on a normalized basis than the well plates at lower MO seeding densities but generated equivalent numbers of iDCs at 600k MO seeding density. These results demonstrate that MicroDEN is capable of generating greater numbers of iDCs with less manual work than standard well plate culture and the MicroDEN generated iDCs have greater ability to induce T cell proliferation.

Canola Root-Associated Microbiomes in the Canadian Prairies.

Canola is one of the most economically important crops in Canada, and the root and rhizosphere microbiomes of a canola plant likely impact its growth and nutrient uptake. The aim of this study was to determine whether canola has a core root microbiome (i.e., set of microbes that are consistently selected in the root environment), and whether this is distinct from the core microbiomes of other crops that are commonly grown in the Canadian Prairies, pea, and wheat. We also assessed whether selected agronomic treatments can modify the canola microbiome, and whether this was associated to enhanced yield. We used a field experiment with a randomized complete block design, which was repeated at three locations across the canola-growing zone of Canada. Roots and rhizosphere soil were harvested at the flowering stage of canola. We separately isolated total extractable DNA from plant roots and from adjacent rhizosphere soil, and constructed MiSeq amplicon libraries for each of 60 samples, targeting bacterial, and archaeal 16S rRNA genes and the fungal ITS region. We determined that the microbiome of the roots and rhizosphere of canola was consistently different from those of wheat and pea. These microbiomes comprise several putative plant-growth-promoting rhizobacteria, including Amycolatopsis sp., Serratia proteamaculans, Pedobacter sp., Arthrobacter sp., Stenotrophomonas sp., Fusarium merismoides, and Fusicolla sp., which correlated positively with canola yield. Crop species had a significant influence on bacterial and fungal assemblages, especially within the roots, while higher nutrient input or seeding density did not significantly alter the global composition of bacterial, fungal, or archaeal assemblages associated with canola roots. However, the relative abundance of Olpidium brassicae, a known pathogen of members of the Brassicaceae, was significantly reduced in the roots of canola planted at higher seeding density. Our results suggest that seeding density and plant nutrition management modified the abundance of other bacterial and fungal taxa forming the core microbiomes of canola that are expected to impact crop growth. This work helps us to understand the microbial assemblages associated with canola grown under common agronomic practices and indicates microorganisms that can potentially benefit or reduce the yield of canola.

Constant Isothiocyanate-Release Potentials across Biofumigant Seeding Rates.

Biofumigation is an integrated pest-management method involving the mulching of a glucosinolate-containing cover crop into a field in order to generate toxic isothiocyanates (ITCs), which are effective soil-borne-pest-control compounds. Variation in biofumigation efficacy demonstrates a need to better understand the factors affecting pest-control outcomes and develop best practices for choosing biofumigants, growth conditions, and mulching methods that allow the greatest potential isothiocyanate release. We measured the glucosinolate concentrations of six different commercial varieties of three biofumigant plant species: Brassica juncea (ISCI99, Vitasso, and Scala) Raphanus sativus (Diablo and Bento), and Sinapis alba (Ida Gold). The plants were grown in the range of commercially appropriate seeding rates and sampled at three growth stages (early development, mature, and 50% flowering). Within biofumigant species, the highest ITC-release potentials were achieved with B. juncea cv. ISCI99 and R. sativus cv. Bento. The highest ITC-release potential occurred at the 50% flowering growth stage across the species. The seeding rate had a minor impact on the ITC-release potential of R. sativus but had no significant effects on the ITC-release potentials of the B. juncea or S. alba cultivars.

Metabolic shift in density-dependent stem cell differentiation.

BACKGROUND: Vascular progenitor cells (VPCs) derived from embryonic stem cells (ESCs) are a valuable source for cell- and tissue-based therapeutic strategies. During the optimization of endothelial cell (EC) inductions from mouse ESCs using our staged and chemically-defined induction methods, we found that cell seeding density but not VEGF treatment between 10 ng/mL and 40 ng/mL was a significant variable directing ESCs into FLK1+ VPCs during stage 1 induction. Here, we examine potential contributions from cell-to-cell signaling or cellular metabolism in the production of VPCs from ESCs seeded at different cell densities. METHODS: Using 1D 1H-NMR spectroscopy, transcriptomic arrays, and flow cytometry, we observed that the density-dependent differentiation of ESCs into FLK1+ VPCs positively correlated with a shift in metabolism and cellular growth. RESULTS: Specifically, cell differentiation correlated with an earlier plateauing of exhaustive glycolysis, decreased lactate production, lower metabolite consumption, decreased cellular proliferation and an increase in cell size. In contrast, cells seeded at a lower density of 1,000 cells/cm2 exhibited increased rates of glycolysis, lactate secretion, metabolite utilization, and proliferation over the same induction period. Gene expression analysis indicated that high cell seeding density correlated with up-regulation of several genes including cell adhesion molecules of the notch family (NOTCH1 and NOTCH4) and cadherin family (CDH5) related to vascular development. CONCLUSIONS: These results confirm that a distinct metabolic phenotype correlates with cell differentiation of VPCs.