Culture Density Research Articles

Efficient floral dip transformation method using Agrobacterium tumefaciens on Cosmos sulphureus Cav.

Yellow cosmos (Cosmos sulphureus Cav.) is a specific flowering plant and considered a suitable genetic engineering model. Agrobacterium-mediated plant transformation is commonly used for plant genetic engineering. Floral dip transformation is one of the plant genetic transformation methods, and it involves dipping flower buds into an Agrobacterium suspension. Studies on floral dip transformation of yellow cosmos have never been reported. Therefore, an efficient method in plant genetic engineering must be established. This study developed an effective and efficient floral dip transformation method for yellow cosmos.In this study, flower buds with sizes of 5–7 mm were used. Several parameters have been observed to optimize the floral dip method. These parameters included the optical density (OD600) of Agrobacterium culture, concentration of surfactant, and duration of flower bud dipping into the Agrobacterium suspension.The results showed that the floral dip method was most efficient when the flower buds were dipped into Agrobacterium suspension with OD600 = 0.8 and containing 5% sucrose and 0.1% Silwet L-77 for 30 s. This method enhanced the transformation efficiency at a rate of 12.78 ± 1.53%. The neomycin phosphotransferase II and green fluorescent protein genes with sizes of 550 and 736 bp, respectively, were confirmed by polymerase chain reaction. In addition, the transgenic plants were kanamycin resistant and fluorescent under ultraviolet light observation. This finding suggests that the proposed floral dip transformation provides new insights into efficient plant genetic engineering methods for yellow cosmos.

EFFECT OF LARVAL DENSITY AND DIET ON THE GROWTH AND SURVIVAL OF Perna perna MUSSELS IN RECIRCULATION SYSTEM (RAS).

This study evaluated the effect of initial larval stocking density and microalgae concentration in the diet on the growth and the number of competent larvae (number of live larvae at the end of the experiment retained on a sieve with mesh size of 210 µm) of Perna perna in a water recirculation system (RAS). The experiment was conducted using a completely randomized design and factorial scheme, with one factor being the food concentration at three levels (low: between 1.26 to 2.19, medium: between 2.36 to 3.94 and high: 5.03 to 7.29 x 104 cells mL⁻¹) and the second factor being the larval culture density, also at three levels (20, 80 and 200 larvae mL⁻¹), with 3 replications in each treatment. Larvae fed with the diet with low and high concentrations did not reach competence (210 µm) in 16 days. However, for larvae fed with the medium concentration (2.36 to 3.94 x 104 cells mL⁻¹), the average percentage of competent larvae, on day 16, was 21.8% ± 7.45, 27.4% ± 9.91 and 0.80% ± 0.26 at the densities 20, 80 and 200 larvae mL-1, respectively.

Study on the Microflora Structure in a Litopenaeus vannamei–Sinonovacula constricta Tandem-Culture Model Based on High-Throughput Sequencing under Different Culture Densities

In this study, we evaluated the intestinal contents of Pacific whiteleg shrimp (Litopenaeus vannamei), the visceral mass of razor clams (Sinonovacula constricta) and the water columns and the substrate sediments in different culture-density groups in a L. vannamei–S. constricta tandem-culture model by high-throughput sequencing of the 16S rRNA gene. The results show that the culture density affected the bacterial floral structure of the water columns, substrate sediment and razor-clam gut masses without making significant differences in the bacterial flora structure of the shrimp gut; the Shannon diversity indexes of the bacterial communities in the substrate sediment, shrimp gut and razor-clam gut masses were not significantly different among the density groups, and the Shannon diversity index of the bacterial communities in the water column was higher in the group with higher culture densities; at the phylum level, the dominant bacteria common to the shrimp guts, razor-clam visceral mass, water columns and substrate sediment were Proteobacteria and Bacteroidetes; Chloroflexi was the dominant bacterium specific to the substrate sediment; and Firmicutes was the dominant bacterium specific to the shrimp gut and razor-clam gut mass. We used national standards (GB 17378.4-2007, China) to evaluate the content of water-quality factors through the environmental factors and the genus-level correlation analysis of bacterial flora that follow: the dominant bacterium in the water column, uncultured_bacterium_f_Rhodobacteraceae, was negatively correlated with PO43−-P; the dominant bacteria in the substrate sediments, uncultured_bacterium_f_Anaerolineaceae and Woeseia, were significantly and negatively correlated with DO; and the dominant bacteria Lactococcus spp. in the razor-clam gut mass and the shrimp intestines were positively correlated with DO. These results show that culture density directly affects water-quality factors, which in turn affect the culture environment and the composition structure of the bacterial flora in a cultured organism.

Mass Cultivation of Microalgae: II. A Large Species Pulsing Blue Light Concept.

If mass cultivation of photoautotrophic microalgae is to gain momentum and find its place in the new "green future", exceptional optimizations to reduce production costs must be implemented. Issues related to illumination should therefore constitute the main focus, since it is the availability of photons in time and space that drives synthesis of biomass. Further, artificial illumination (e.g., LEDs) is needed to transport enough photons into dense algae cultures contained in large photobioreactors. In the present research project, we employed short-term O2 production and 7-day batch cultivation experiments to evaluate the potential to reduce illumination light energy by applying blue flashing light to cultures of large and small diatoms. Our results show that large diatom cells allow more light penetration for growth compared to smaller cells. PAR (400-700 nm) scans yielded twice as much biovolume-specific absorbance for small biovolume (avg. 7070 μm3) than for large biovolume (avg. 18,703 μm3) cells. The dry weight (DW) to biovolume ratio was 17% lower for large than small cells, resulting in a DW specific absorbance that was 1.75 times higher for small cells compared to large cells. Blue 100 Hz square flashing light yielded the same biovolume production as blue linear light in both the O2 production and batch experiments at the same maximum light intensities. We therefore suggest that, in the future, more focus should be placed on researching optical issues in photobioreactors, and that cell size and flashing blue light should be central in this.

A Rapid Method to Determine Growth‐Limiting Nutrient Concentrations for Yeast in a Microplate Spectrophotometer

Determining the minimal concentration of a substance-whether a compound used to inhibit cell growth or a growth-limiting nutrient-can be an arduous process. Carrying out the experiment in flasks or tubes and estimating cell density in a single-sample spectrophotometer has been a routine method of choice due to the ease of measurement and standardized conversions from optical density measurements to cell concentrations. However, when dealing with dozens or more samples, several challenges arise, including increased processing time, increased risk of contamination through repeated sampling, and an increased risk of confounding one sample for another. The protocol described here details a rapid method to estimate cell concentrations for such experiments using a microplate spectrophotometer. Using a microplate spectrophotometer to measure optical density of many cultures can be automated for high throughput and benefits from a reduced risk of contamination. Since one of the caveats of a microplate spectrophotometer is its low saturation limit, we further describe how to convert optical density readings of the microplate spectrophotometer into its single-sample spectrophotometric equivalent. To further illustrate the applicability of this protocol, we compare OD600 readings generated using a microplate spectrophotometer and a single-sample spectrophotometer for different nutrient starvations and show that the results are comparable. Overall, this method reduces the required resources, reduces the risk of contamination, and allows for faster processing of samples. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Generating an equation to convert microplate spectrophotometer readings to single-sample spectrophotometric values Basic Protocol 2: Evaluating growth-limiting nutrient concentrations using a microplate spectrophotometer.

Semiautomatic assessment of endothelial density and morphology in organ-cultured corneas - potential predictors for transplantation suitability and clinical outcome?

The quality of the endothelial cell layer is a major criterion for the approval of organ-cultured human donor-corneas for transplantation. We wanted to compare the predictive capacities of initial endothelial density and endothelium cell morphology for the approval of donor corneas for transplantation and for the clinical outcome after transplantation. The endothelial density and endothelium morphology in organ culture were examined by semiautomatic assessment of 1031 donor corneas. We performed a statistical analysis for correlations of donor-data and cultivation parameters regarding their predictive capacities for the final approval of donor corneas for transplantation and the clinical outcome of 202 transplanted patients. Corneal endothelium cell density proved to be the only parameter with a certain predictive capacity with regard to the final decision, whether donor corneas are suitable for transplantation - however, the correlation was low (area under the curve [AUC] = 0.655). Endothelial cell morphology lacked any predictive power (AUC = 0.597). The clinical outcome regarding visual acuity seemed to be largely independent from both corneal endothelial cell density and morphology. Sub-analyses on transplanted patients stratified for their diagnoses vindicated these findings. Higher endothelial density (above a cut-off level of 2000 cells/mm2), as well as better endothelial morphology do not seem to be critical for transplant-corneal functionality in organ culture and up to 2years after transplantation. Comparable long-term studies on graft survival are recommended to determine, whether the present endothelial density cut-off levels might be too stringent.

EFFECT OF FERTILIZATION ON GROWTH CHARACTERISTICS OF CYPRINUS CARPIO CULTURED IN RICE FIELDS IN IRAQ

This study was carried out to evaulate the effect of organic fertilization on the growth of common carp Cyprinus carpio cultured in rice fields and its effect on rice production as well as the possibilitd reducing the feed provided to fish. Four fields with an area of 100 m2 were cultured Yasmine rice represented four treatments. Common carp fish were cultured in it with a weight of 100 g/fish and a culture density of 100 fish/field. In the first treatment, organic manure was added without providing fish feed, second treatment, organic manure was added with fish feed, the third treatment without adding organic manure with fish feed, and the fourth treatment without adding organic manure and without providing fish feed. The results of the statistical analysis rate showed that T2 showed a significant increased(p≤0.05) in relative and specific growth in T2 compared to all treatments followed by T3. The results of the statistical analysis of the characteristics of the rice yield showed the number of dahlias, number of grains / dahlia, weight of 1000 grains and grain yield, showed a significant difference (p≤0.05) in T2 and T3 compared to T1and T4, while there were no significant differences(P>0.05) between treatments for the weight of 1000 grains trait. the study concluded that it is possible to apply the fish farming system in rice fields in Iraq by using organic fertilization (cow waste) and adding supplementary feed, as the T2 treatment gave the best growth for fish and rice.

Dynamic fry counting based on multi-object tracking and one-stage detection

Fry counting is an essential task in aquaculture that provides valuable information for feedstuff feeding, culture density adjustment, and fish economic benefit estimation. However, most current computer vision-based fry counting methods are designed for statically counting, with a fixed number of fry in a container. As a result, these methods are not suitable for dynamic scenarios, such as flowing water, where the effects of factors like water velocity and uneven lighting can cause errors in counting. To address this issue and improve accuracy, we propose a dynamic fry counting method capable of dealing with large-number fry counting. Specifically, we regard fry counting as a Multiple Object Tracking (MOT) problem based on Tracking-By-Detection (TBD) framework. Firstly, a dataset of streaming fry is constructed for training the detection model, and videos with varying numbers of fry are manually counted for counting tests. Secondly, due to the highly similar appearance of fry, Simple Online and Realtime Tracking (SORT) is combined with You Only Look Once version 5 Nano (YOLOv5-Nano) to perform fry tracking. To achieve stable tracking of fry movement, the SORT algorithm is improved based on multi-matching and trajectory recovery, reaching 82.6 % multi-object tracking accuracy. Finally, fry counting is realized based on the intersection of a counting line and moving trajectories. The results indicate that the proposed counting method achieves 96.4 % accuracy and can be implemented on CPU at 5–10 frames per second (fps) and GPU at 15–35 fps. In conclusion, the proposed method automatically counts the number of fry in videos with higher speed and accuracy, facilitating automated fishery farming.

Increasing cell culture density during a developmental window prevents fated rod precursors derailment toward hybrid rod-glia cells

In proliferating multipotent retinal progenitors, transcription factors dynamics set the fate of postmitotic daughter cells, but postmitotic cell fate plasticity driven by extrinsic factors remains controversial. Transcriptome analysis reveals the concurrent expression by postmitotic rod precursors of genes critical for the Müller glia cell fate, which are rarely generated from terminally-dividing progenitors as a pair with rod precursors. By combining gene expression and functional characterisation in single cultured rod precursors, we identified a time-restricted window where increasing cell culture density switches off the expression of genes critical for Müller glial cells. Intriguingly, rod precursors in low cell culture density maintain the expression of genes of rod and glial cell fate and develop a mixed rod/Muller glial cells electrophysiological fingerprint, revealing rods derailment toward a hybrid rod-glial phenotype. The notion of cell culture density as an extrinsic factor critical for preventing rod-fated cells diversion toward a hybrid cell state may explain the occurrence of hybrid rod/MG cells in the adult retina and provide a strategy to improve engraftment yield in regenerative approaches to retinal degenerative disease by stabilising the fate of grafted rod precursors.

Osteogenic Differentiation Effect of Human Periodontal Ligament Stem-Cell Initial Cell Density on Autologous Cells and Human Bone Marrow Stromal Cells.

Stem cells have differentiation and regulation functions. Here, we discussed the impact of cell culture density on stem cell proliferation, osteoblastogenesis, and regulation. To discuss the effect of the initial culture density of human periodontal ligament stem cells (hPDLSCs) on the osteogenic differentiation of autologous cells, we found that the hPDLSC proliferation rate decreased with an increase in the initial plating density (0.5-8 × 104 cells/cm2) for the 48 h culture cycle. After hPDLSCs induced osteogenic differentiation for 14 days with different initial cell culture densities, the expression of osteoprotegerin (OPG) and runt-related transcription factor 2(RUNX2) and the OPG/ Receptor Activator of Nuclear Factor-κ B Ligand (RANKL) ratio were the highest in the hPDLSCs initially plated at a density of 2 × 104 cells/cm2, and the average cell calcium concentration was also the highest. To study hPDLSCs regulating the osteoblastic differentiation of other cells, we used 50 μg/mL of secreted exosomes derived from hPDLSCs cultured using different initial cell densities to induce human bone marrow stromal cell (hBMSC) osteogenesis. After 14 days, the results indicated that the gene expression of OPG, Osteocalcin(OCN,)RUNX2, and osterix and the OPG/RANKL ratio were the highest in the 2 × 104 cells/cm2 initial cell density group, and the average calcium concentration was also the highest. This provides a new idea for the clinical application of stem cell osteogenesis.

MP05-05 ANTIMICROBIAL AND ANTIBIOFILM PROPERTIES OF CHITOSAN: AN EX VIVO STUDY ON KIDNEY STONES

You have accessJournal of UrologyCME1 Apr 2023MP05-05 ANTIMICROBIAL AND ANTIBIOFILM PROPERTIES OF CHITOSAN: AN EX VIVO STUDY ON KIDNEY STONES Grace H. Holton, Daniel Massana Roquero, T. Jessie Ge, Kathleen E. Mach, Timothy J. Lee, Timothy C. Chang, and Joseph C. Liao Grace H. HoltonGrace H. Holton More articles by this author , Daniel Massana RoqueroDaniel Massana Roquero More articles by this author , T. Jessie GeT. Jessie Ge More articles by this author , Kathleen E. MachKathleen E. Mach More articles by this author , Timothy J. LeeTimothy J. Lee More articles by this author , Timothy C. ChangTimothy C. Chang More articles by this author , and Joseph C. LiaoJoseph C. Liao More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003216.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Up to 40% of kidney stones are colonized by bacterial biofilms. Stone fragmentation by ureteroscopic (URS) laser lithotripsy or percutaneous nephrolithotomy (PCNL) can disperse bacteria thereby increasing the risk of post-surgical infection, urosepsis, and stone recurrence. We hypothesized that the addition of chitosan, an established biopolymer with reported antimicrobial properties, may eradicate uropathogens and bacterial biofilms associated with kidney stones. METHODS: With IRB approval and patient consent, preoperative urine and intraoperative stone fragments were collected from patients undergoing URS and PCNL. In vitro biofilms and planktonic bacteria were assessed and quantitated by scanning electron microscopy. Colony forming units on agar plate culture and optical density were used to measure the bactericidal effects of chitosan on common uropathogens found in kidney stones. Stones were divided into a control group treated with saline for 90 minutes and an experimental group with a chitosan solution in saline. All samples were analyzed to determine bacterial presence, species, and biofilm integrity. RESULTS: In vitro culture demonstrated that as low as 0.0001% (w/v) chitosan inhibited growth in the planktonic form of the most common uropathogens found in stone formers (i.e. E. coli, Proteus mirabilis, Klebsiella pneumoniae and Staphylococcus aureus). Further, up to 90% of biofilms grown in vitro on a synthetic support or calcium oxalate stone were disrupted with 0.0005% (w/v) chitosan. Ex vivo experiments measuring the effect of chitosan on patient derived urine and stone fragments are underway to assess differences in urine and stone culture and if chitosan can effectively disrupt complex in vivo formed biofilms. CONCLUSIONS: Chitosan inhibits growth of both gram-negative and gram-positive uropathogens and disrupts their biofilms in vitro on kidney stones. Ex vivo experiments with patient derived stones will confirm the antimicrobial effects of chitosan on complex biofilms. Chitosan may be a promising additive to the saline irrigation to reduce post-surgical infections in kidney stone surgery. Source of Funding: NIH R21 DK131776 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e45 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Grace H. Holton More articles by this author Daniel Massana Roquero More articles by this author T. Jessie Ge More articles by this author Kathleen E. Mach More articles by this author Timothy J. Lee More articles by this author Timothy C. Chang More articles by this author Joseph C. Liao More articles by this author Expand All Advertisement PDF downloadLoading ...

Drosophila suzuki: Matsumura (Diptera: Drosophilidae): analysis of experimental conditions for its rearing and study in the laboratory

Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) is an invasive pest species that uses a wide variety of commercial fruit as a breeding and feeding resource. The damage to the fruit generated by D. suzukii´ oviposition causes considerable economic losses. Therefore, the maintenance of D. suzukii in the lab conditions constitutes a fundamental tool for research proposal. Two of the determining factors affecting the lab rearing conditions are the culture medium and density. In this study, we evaluated the impact of these factors on three components associated with fitness: development time, larval-adult viability and body size, comparing the effects of three culture media: cornmeal (polenta), mashed potato and plum when the flies were reared at three densities: 10, 30 and 50 larvae per 5ml of culture medium. Our results revealed that the flies reared the cornmeal culture medium and at lower densities provide the better conditions for the maintenance of D. suzukii under lab conditions.

Experimental and numerical investigation of the dynamic responses of longline aquacultural structures under waves

Mariculture is an important activity in the exploitation of marine resources, of which longline raft culture accounts for a large part of the production. The flexibility due to the structural characteristics and the front-to-back shielding effect between suspended culture units have great influence on the dynamic response of the structure under waves. There are relatively few systematic studies on the impacts of this flexibility and shielding effect. Laboratory experiments are carried out in our work to investigate the dynamic response of different forms of aquacultural structures under the action of regular and irregular waves, in which the structural flexibility is reasonably considered and modeled. Then based on the experimental results, a numerical model developed in our previous work is optimized to take the shielding effect into account. Through the improved numerical model, the influence of structural parameters and wave parameters on the mooring line tension is analyzed. The results indicate that the shielding effect reduces with the increase of wave frequency. The maximum mooring line tension generally decreases with the decrease of culture density, and the decreasing trend is gradually weakened. The increase of anchoring angle results in a smaller maximum mooring line tension in most cases due to the reducing anchoring system constrains. The maximum value of the mooring line tension under the action of regular and irregular waves generally appears around the relative structure length B/L=1.15∼1.35. The conclusions of the experiment and analysis can be used as a reference for the design of similar longline aquacultural structures to avoid structural failure.

Culture density influences the functional phenotype of human macrophages.

Macrophages (MΦ) are commonly cultured in vitro as a model of their biology and functions in tissues. Recent evidence suggests MΦ to engage in quorum sensing, adapting their functions in response to cues about the proximity of neighboring cells. However, culture density is frequently overlooked in the standardization of culture protocols as well as the interpretation of results obtained in vitro. In this study, we investigated how the functional phenotype of MΦ was influenced by culture density. We assessed 10 core functions of human MΦ derived from the THP-1 cell line as well as primary monocyte-derived MΦ. THP-1 MΦ showed increasing phagocytic activity and proliferation with increasing density but decreasing lipid uptake, inflammasome activation, mitochondrial stress, and secretion of cytokines IL-10, IL-6, IL-1β, IL-8, and TNF-α. For THP-1 MΦ, the functional profile displayed a consistent trajectory with increasing density when exceeding a threshold (of 0.2 x 103 cells/mm2), as visualized by principal component analysis. Culture density was also found to affect monocyte-derived MΦ, with functional implications that were distinct from those observed in THP-1 MΦ, suggesting particular relevance of density effects for cell lines. With increasing density, monocyte-derived MΦ exhibited progressively increased phagocytosis, increased inflammasome activation, and decreased mitochondrial stress, whereas lipid uptake was unaffected. These different findings in THP-1 MΦ and monocyte-derived MΦ could be attributed to the colony-forming growth pattern of THP-1 MΦ. At the lowest density, the distance to the closest neighboring cells showed greater influence on THP-1 MΦ than monocyte-derived MΦ. In addition, functional differences between monocyte-derived MΦ from different donors could at least partly be attributed to differences in culture density. Our findings demonstrate the importance of culture density for MΦ function and demand for awareness of culture density when conducting and interpreting in vitro experiments.

Role of interleukin-6 and interleukin-10 in morphological and functional changes of the blood–brain barrier in hypertriglyceridemia

BackgroundHypertriglyceridemia is closely linked to atherosclerosis related inflammatory processes and blood–brain barrier (BBB) dysfunction. Using apolipoprotein B-100 (APOB-100) transgenic mice, an animal model of chronic hypertriglyceridemia, we analyzed BBB function and morphology in vitro and ex vivo. Our objective was to determine which BBB characteristics are produced mainly by interleukin (IL)-6, an atherosclerosis promoting cytokine, and whether these actions can be antagonized by IL-10, an anti-inflammatory cytokine.MethodsBrain endothelial and glial cell cultures and brain microvessels were isolated from wild type (WT) and APOB-100 transgenic mice and were treated with IL-6, IL-10 and their combination. First, IL-6 and IL-10 production was measured in WT and APOB-100 microvessels using qPCR. Then functional parameters of endothelial cell cultures were analyzed and immunocytochemistry for key BBB proteins was performed.ResultsIL-6 mRNA levels were higher in brain microvessels than in brain parenchyma of APOB-100 transgenic mice. Transendothelial electric resistance and P-glycoprotein activity were lower, and paracellular permeability was higher in cultured APOB-100 brain endothelial cells. These features were sensitive to both IL-6 and IL-10 treatments. A decreased P-glycoprotein immunostaining was measured in transgenic endothelial cells under control conditions and in WT cells after treating them with IL-6. This effect was antagonized by IL-10. Changes in immunostaining for tight junction proteins were observed after IL-6 exposure, which were in part antagonized by IL-10. In glial cell cultures an increase in aquaporin-4 immunolabeling in the transgenic group and an increase in microglia cell density in WT glia cultures was detected after IL-6 treatment, which was antagonized by IL-10. In isolated brain microvessels a decrease in P-glycoprotein immunolabeled area fraction was measured in APOB-100 microvessels under control conditions and in WT microvessels after every cytokine treatment. ZO-1 immunolabeling showed characteristics similar to that of P-glycoprotein. No change was seen in claudin-5 and occludin immunoreactive area fractions in microvessels. A decrease in aquaporin-4 immunoreactivity was measured in WT microvessels treated by IL-6, which was antagonized by IL-10.ConclusionIL-6 produced in microvessels contributes to BBB impairment observed in the APOB-100 mice. We showed that IL-10 partly antagonizes the effects of IL-6 at the BBB.

Dinoflagellate cell density limits explored using Scrippsiella lachrymosa cultured in flow-through cages

Dinoflagellates constitute one of the most important groups of primary producers and micro-zooplankton on earth, common in both marine and freshwater environments. Despite their prominent position among phytoplankton, they are difficult to grow into dense cultures in the laboratory. This discrepancy between field and laboratory indicates serious limitations caused by the laboratory culturing conditions. A difficult to study but important factor is the constraints of enclosure in a limited volume of water. We conducted an experiment wherein the dinoflagellate Scrippsiella lachrymosa was grown in “flow cells” – 100 cm3 cylindrical cages constructed from plankton net, inserted in larger volumes of growth medium, allowing an exchange of medium without dilution of the culture. Cell numbers far exceeding the normal for culturing of this species and dinoflagellates in general were attained, even though the experiment was terminated before cultures reached stationary phase. A cell number ten times higher than under regular batch culturing was achieved (up to 340,000 cells mL−1). Pattern formation was distinct in cultures when cells were plentiful and water movements caused cell accumulation, not dispersion. High cell density concurrent with access to new growth medium promoted induction of the sexual cell cycle. The results indicate serious limitations to growth set by enclosure in a limited water volume in laboratory experiments; thus, maximum growth rates of dinoflagellates in favourable field conditions may be vastly underestimated. Cell accumulation behavior of dinoflagellates during the sexual life cycle may together with physical transport by larger forces in nature explain sudden bloom occurrences.

Lipophilic Statins Inhibit YAP Nuclear Localization, Coactivator Activity, and Migration in Response to Ligation of HLA Class I Molecules in Endothelial Cells: Role of YAP Multisite Phosphorylation.

Solid-organ transplant recipients exhibiting HLA donor-specific Abs are at risk for graft loss due to chronic Ab-mediated rejection. HLA Abs bind HLA molecules expressed on the surface of endothelial cells (ECs) and induce intracellular signaling pathways, including the activation of the transcriptional coactivator yes-associated protein (YAP). In this study, we examined the impact of lipid-lowering drugs of the statin family on YAP localization, multisite phosphorylation, and transcriptional activity in human ECs. Exposure of sparse cultures of ECs to cerivastatin or simvastatin induced striking relocalization of YAP from the nucleus to the cytoplasm and inhibited the expression of the YAP/TEA domain DNA-binding transcription factor-regulated genes connective tissue growth factor and cysteine-rich angiogenic inducer 61. In dense cultures of ECs, statins prevented YAP nuclear import and expression of connective tissue growth factor and cysteine-rich angiogenic inducer 61 stimulated by the mAb W6/32 that binds HLA class I. Exposure of ECs to either cerivastatin or simvastatin completely blocked the migration of ECs stimulated by ligation of HLA class I. Exogenously supplied mevalonic acid or geranylgeraniol reversed the inhibitory effects of statins on YAP localization either in low-density ECs or high-density ECs challenged with W6/32. Mechanistically, cerivastatin increased the phosphorylation of YAP at Ser127, blunted the assembly of actin stress fiber, and inhibited YAP phosphorylation at Tyr357 in ECs. Using mutant YAP, we substantiated that YAP phosphorylation at Tyr357 is critical for YAP activation. Collectively, our results indicate that statins restrain YAP activity in EC models, thus providing a plausible mechanism underlying their beneficial effects in solid-organ transplant recipients.

Effects of Different Culture Densities on the Acoustic Characteristics of Micropterus salmoide Feeding

The intensity and frequency of the acoustic signals generated by different behaviors of largemouth bass (Micropterus salmoides) have different characteristics. The acoustic signals generated during feeding can be used to analyze the characteristic patterns of their used behavior, which can provide a theoretical basis for applications such as automatic feeding based on acoustic signals. We used passive acoustics combined with video to study the feeding acoustic signals of largemouth bass in a recirculating water culture system (4, 8, 12, and 16 fish/m3). The result of the time–frequency and power spectrum analysis of the feeding sound signals showed that the short-time average amplitude of the feeding signal was well distinguished from the background noise, both the swallowing and the chewing sounds were positively correlated with the culture density, and the correlation between the swallowing sound and number of fish was stronger; at different culture densities, the short-time average zero-crossing in the swallowing phase of the largemouth bass suddenly dropped to about 500 and rose to about 1000 in the chewing process. Therefore, both the chewing and the swallowing sounds can be used as parameters to automatically identify the largemouth bass feeding process signal. The spectrum entropy in the feeding process of the largemouth bass was maintained at about 4–6 for different culture densities. In the power spectrum, the main feeding sounding frequencies of the largemouth bass at different farming densities and the distinguishable spectral range of the background noised ranged from 1 to 20 kHz, the main peak frequency of the feeding sound was within the range of 1.2 to 3.0 kHz, and the main power value of the power spectrum was positively correlated with farming density.

High-glutathione mesenchymal stem cells isolated using the FreSHtracer probe enhance cartilage regeneration in a rabbit chondral defect model

BackgroundMesenchymal stem cells (MSCs) are a promising cell source for cartilage regeneration. However, the function of MSC can vary according to cell culture conditions, donor age, and heterogeneity of the MSC population, resulting in unregulated MSC quality control. To overcome these limitations, we previously developed a fluorescent real-time thiol tracer (FreSHtracer) that monitors cellular levels of glutathione (GSH), which are known to be closely associated with stem cell function. In this study, we investigated whether using FreSHtracer could selectively separate high-functioning MSCs based on GSH levels and evaluated the chondrogenic potential of MSCs with high GSH levels to repair cartilage defects in vivo.MethodsFlow cytometry was conducted on FreSHtracer-loaded MSCs to select cells according to their GSH levels. To determine the function of FreSHtracer-isolated MSCs, mRNA expression, migration, and CFU assays were conducted. The MSCs underwent chondrogenic differentiation, followed by analysis of chondrogenic-related gene expression. For in vivo assessment, MSCs with different cellular GSH levels or cell culture densities were injected in a rabbit chondral defect model, followed by histological analysis of cartilage-regenerated defect sites.ResultsFreSHtracer successfully isolated MSCs according to GSH levels. MSCs with high cellular GSH levels showed enhanced MSC function, including stem cell marker mRNA expression, migration, CFU, and oxidant resistance. Regardless of the stem cell tissue source, FreSHtracer selectively isolated MSCs with high GSH levels and high functionality. The in vitro chondrogenic potential was the highest in pellets generated by MSCs with high GSH levels, with increased ECM formation and chondrogenic marker expression. Furthermore, the MSCs’ function was dependent on cell culture conditions, with relatively higher cell culture densities resulting in higher GSH levels. In vivo, improved cartilage repair was achieved by articular injection of MSCs with high levels of cellular GSH and MSCs cultured under high-density conditions, as confirmed by Collagen type 2 IHC, Safranin-O staining and O’Driscoll scores showing that more hyaline cartilage was formed on the defects.ConclusionFreSHtracer selectively isolates highly functional MSCs that have enhanced in vitro chondrogenesis and in vivo hyaline cartilage regeneration, which can ultimately overcome the current limitations of MSC therapy.Graphical

Effect of p38 inhibitor on the proliferation of chicken muscle stem cells and differentiation into muscle and fat

Inhibiting the p38 mitogen-activated protein kinase (MAPK) signaling pathway delays differentiation and increases proliferation of muscle stem cells in most species. Here, we aimed to investigate the effect of p38 inhibitor (p38i) treatment on the proliferation and differentiation of chicken muscle stem cells. Chicken muscle stem cells were collected from the muscle tissues of Hy-line Brown chicken embryos at embryonic day 18, then isolated by the preplating method. Cells were cultured for 4 days in growth medium supplemented with dimethyl sulfoxide or 1, 10, 20 μM of p38i, then subcultured for up to 4 passages. Differentiation was induced for 3 days with differentiation medium. Each treatment was replicated 3 times. The proliferation and mRNA expression of paired box 7 gene and myogenic factor 5 gene, as well as the mRNA expression of myogenic differentiation marker gene myogenin were significantly higher in p38i-treated cultures than in control (p<0.05), but immunofluorescence staining and mRNA expression of myosin heavy chain (MHC) were not significantly different between the two groups. Oil red O staining of accumulated lipid droplets in differentiated cell cultures revealed a higher lipid density in p38i-treated cultures than in control; however, the expression of the adipogenic marker gene peroxisome proliferator activated receptor gamma was not significantly different between the two groups. p38 inhibition in chicken muscle stem cells improves cell proliferation, but the effects on myogenic differentiation and lipid accumulation require additional analysis. Further studies are needed on the chicken p38-MAPK pathway to understand the muscle and fat development mechanism.