Culture Response Research Articles

Screening of cultivars for tissue culture response and establishment of genetic transformation in a high-yielding and disease-resistant cultivar of Theobroma cacao

A highly efficient transformation protocol is a prerequisite to developing genetically modified and genome-edited crops. A tissue culture system spanning culture initiation from floral material to conversion of embryos to plants has been tested and improved in Theobroma cacao. Nine cultivars were screened for their tissue culture response and susceptibility to Agrobacterium transfer-DNA delivery as measured through transient expression. These key factors were used to determine the genetic transformability of various cultivars. The high-yielding, disease-resistant cultivar INIAPG-038 was selected for stable transformation and the method was further optimized. Multiple transgenic events were produced using two vectors containing both yellow fluorescent protein and neomycin phosphotransferase II genes. A two-fold strategy to improve both T-DNA delivery and secondary somatic embryogenesis rates was conducted to improve overall transformation frequency. The use of Agrobacterium strain AGL1 and cotyledon tissue derived from secondary somatic embryos ranging in size between 4 to 10 mm resulted in the highest T-DNA delivery efficiency. Furthermore, the use of higher concentrations of basal salts and cupric sulfate in the medium increased the frequency of explants producing greater than ten embryos by five-fold and four-fold during secondary somatic embryogenesis, respectively. Consequently, an optimal combination of all these components resulted in a successful transformation of INIAPG-038 with 3.7% frequency at the T0 plant-level. Grafting transgenic scions with undeveloped roots to non-transgenic seedlings with healthy roots helped make plantlets survive and facilitated quick transplantation to the soil. The presented strategy can be applied to improve tissue culture response and transformation frequency in other Theobroma cacao cultivars.

Mesenchymal stem cell secretome decreases the inflammatory response in annulus fibrosus organ cultures.

Mesenchymal stem/stromal cell (MSC)-based therapies have been proposed for back pain and disc degeneration, despite limited knowledge on their mechanism of action. The impact of MSCs/their secretome on annulus fibrosus (AF) cells and tissue was analysed in bovine AF organ cultures (AF-OCs) exposed to upper-physiological cyclic tensile strain (CTS, 9 %, 1 Hz, 3 h/d) and interleukin (IL)-1β in a custom-made device. A 4 d treatment of the CTS + IL-1β-stimulated AF-OCs with MSC secretome downregulated the expression of inflammation markers [IL-6, IL-8, prostaglandin-endoperoxide synthase 2 (PTGS2)], complement system regulators [cluster of differentiation (CD)46, CD55, CD59] and matrix metalloproteinase 1 but also of tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2) and collagen type I. At the protein level, it was confirmed that IL-6, MMP-3 and collagen content was decreased in AF-OCs treated with the MSC secretome compared to the CTS + IL-1β stimulation alone. 9 d after treatment, a biomechanical peel-force test showed that the annular adhesive strength was significantly decreased by the MSC secretome treatment. Overall, MSC secretome had a stronger impact on AF tissue than MSCs in co-culture. The secretome contributed to a decrease in the inflammatory and catabolic status of AF cells activated by CTS + IL-1β and played a role in the regulation of the complement system. However, it also contributed to a decrease in collagen at the gene/protein level and in AF mechanical strength compared to the CTS + IL-1β stimulation alone. Therefore, the use of MSC secretome requires further investigation regarding its influence on disc matrix properties.

Rapid diagnosis and reduced workload for urinary tract infection using flowcytometry combined with direct antibiotic susceptibility testing.

We evaluated if flowcytometry, using Sysmex UF-5000, could improve diagnosis of urinary tract infections by rapid identification of culture negative and contaminated samples prior to culture plating, thus reducing culture plating workload and response time. We also evaluated if it is possible to reduce the response time for antibiotic susceptibility profiles using the bacteria information flag on Sysmex UF-5000 to differentiate between Gram positive and negative bacteria, followed by direct Antibiotic Susceptibility Testing (dAST) on the positive urine samples. One thousand urine samples were analyzed for bacteria, white blood cells and squamous cells by flowcytometry before culture plating. Results from flowcytometric analysis at different cut-off values were compared to results of culture plating. We evaluated dAST on 100 urine samples that were analyzed as positive by flowcytometry, containing either Gram positive or Gram negative bacteria. Using a cut-off value with bacterial count ≥100.000/mL and WBCs ≥10/μL, flowcytometry predicted 42,1% of samples with non-significant growth. We found that most contaminated samples contain few squamous cells. For 52/56 positive samples containing Gram negative bacteria dAST was identical to routine testing. Overall, there was concordance in 555/560 tested antibiotic combinations. Flowcytometry offers advantages for diagnosis of urinary tract infections. Screening for negative urine samples on the day of arrival reduces culture plating and workload, and results in shorter response time for the negative samples. The bacteria information flag predicts positive samples containing Gram negative bacteria for dAST with high accuracy, thus Antibiotic Susceptibility Profile can be reported the day after arrival. For the positive samples containing Gram negative bacteria the concordance was very good between dAST and Antibiotic Susceptibility Testing in routine. For positive samples containing Gram positive bacteria the results were not convincing. We did not find any correlation between epithelial cells and contamination.

Abstract LB220: Cellular Differentiation and Growth Control in the Human Colonic epithelium: Comparing Clinical Trial Outcomes to Responses in Human Colonoid Culture

Abstract Introduction: As part of our effort to understand the role of trace elements in colon polyp chemoprevention, we have conducted both colonoid culture (ex vivo) and human subject (in vivo) studies using Aquamin®— a calcium-, magnesium-, trace elements-rich, multi-mineral product derived from red marine algae as an intervention. Calcium alone was used as a comparator. Methods: Thirty subjects at-risk for colorectal cancer were enrolled and randomized to receive Aquamin® or calcium alone (each providing 800-mg calcium per day) or placebo. Colon biopsies were obtained before and after 90 days of intervention. We employed histologically normal colonic tissue from the same subjects (at baseline), to propagate in colonoid culture. Once established and expanded, we used the same interventions for a period of two weeks and compared the response (at 1.5mM) to the outcome of the studies conducted in human colonic biopsies after 90 days of intervention. Specifically, colonoid tissue and biopsies were subjected to analysis for markers of growth and differentiation by quantitative immunohistochemistry and tandem mass tag mass spectrometry-based proteomics. Results: The normal tissue colonoids demonstrated a high degree of differentiation as indicated by gross and microscopic appearance, and cytokeratin 20 (CK20) expression without either intervention. Only modest increases were seen in the CK20 expression with either calcium alone or Aquamin®. Similarly, CK20 expression was higher in baseline biopsies and there was no increase in the expression with calcium but a 5% increase seen with Aquamin® after 90 days of intervention. On proteomic screen, CK20 expression was increased by 40% in colonoid tissue in response to both interventions at 1.5mM, while CK20 expression increased by 10% and 21% with Calcium and Aquamin® in colon biopsies. When the colonoids were assessed for Ki67 (growth marker) expression, it was decreased by 17.2% and 18.4% with calcium alone and Aquamin® respectively as compared to the control. Analyzing colon biopsies after 90 days of intervention revealed that Aquamin® reduced the level of Ki67 expression by 20%, while no change was seen with calcium alone. Differential proteomic expression of proliferating cell nuclear antigen was decreased by 32% and 39% by calcium and Aquamin® at 1.5mM in colonoid tissue, whereas Ki67 protein was decreased by 4% and 22% with calcium and Aquamin®, respectively in colon biopsies. Conclusion: Colon tissue maintained in colonoid culture, thus, provides a good surrogate for human tissue collected in a clinical trial. It can be used to assess personalized responses or can provide a quick snapshot of the response in a relatively short time. Differential proteomic expression appears to be more sensitive than quantitative immunohistochemistry. A combination of calcium and additional trace elements proved to be more effective than calcium alone in altering differentiation and growth markers in either setting. Citation Format: Muhammad N. Aslam, Shannon McClintock, Mohamed Ali H Jawad-Makki, Karsten Knuver, Daniyal M. Nadeem, Haris Ahmad, Durga Attili, Danielle (Kim) Turgeon, James Varani. Cellular Differentiation and Growth Control in the Human Colonic epithelium: Comparing Clinical Trial Outcomes to Responses in Human Colonoid Culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB220.

Quantification of the radiosensitization effect of high-Z nanoparticles on photon irradiated cells: combining Monte Carlo simulations and an analytical approach to the local effect model

In vitro experiments show significant reduction in the survival fraction of cells under irradiation treatments assisted with high-Z nanoparticles (NPs). In order to predict the radiosensitization effect of NPs, a modification of the local effect model (LEM), in which the energy deposition from NPs is assessed by Monte Carlo (MC) radiation transport codes, has been employed in the past. In this work, a combined framework that splits the consideration of the radiosensitization effect into two steps is proposed. The first step is the evaluation of the radial dose distribution (RDD) around a single NP ionized by a photon beam with given energy spectrum using MC simulation. Thereafter, an analytical approach based of the LEM and the calculated RDD is used for evaluation of the average dose and the average number of lethal lesions in a cell target due to a set of ionized NPs. The explicit expressions were derived for the case of a spherical cell target and the RDD describing by the power law function. RDDs around gold NPs (GNPs) of different radii were simulated using the MC technique and fitted by a power law function. The fitted RDD and the derived expressions were applied for calculation of the survival curves and relative biological effectiveness of a spherical MDA-MB-231 cell loaded with GNPs and irradiated with monoenergetic photons of 10–150 keV. The proposed framework provides a practical alternative to time-consuming MC simulations, enabling the assessment of the response of cell cultures to an irradiation treatment assisted with NPs for a wide variety of cell geometries, NP distributions and irradiation schemes.

TMOD-04. IMAGE-BASED DRUG RESPONSE PROFILING FROM PEDIATRIC TUMOR CELL SPHEROIDS USING PATIENT-BY-PATIENT DEEP TRANSFER LEARNING

Abstract Introduction Image-based phenotypic drug profiling is receiving increasing attention in drug discovery and precision medicine. Compared to classical end-point measurements quantifying drug response, image-based profiling enables both the quantification of drug response and characterization of disease entities and drug mechanisms of actions. In pediatric precision oncology, we aim to study drug response in patient-derived 3D spheroid tumor cell cultures and tackle the challenges of a lack of image-segmentation methods and limited patient-derived material. Methods We investigate deep transfer learning with patient-by-patient fine-tuning for cell-viability quantification. We fine-tune a convolutional neural network (pre-trained on ImageNet) with many cell-line-specific and few patient-specific assay controls. The method is validated using 3D cell cultures in 384-well microplates derived from cell lines with known drug sensitivities and tested with primary patient-derived samples. Network outputs at different drug concentrations are used for drug-sensitivity scoring; dense-layer activations are used in t-distributed stochastic neighbor embedding and clustering of drugs. Results Cell-line experiments confirm expected hits, such as effective treatment with BRAF inhibitors in a BRAF V600E mutated brain tumor model and NTRK inhibitors in a cell line harboring an NTRK-fusion, indicating the predictive power of deep learning to identify drug-hit candidates for individual patients. In patient-derived samples, clustering of drugs further confirms phenotypic similarity according to their mechanisms of actions. Combining drug scoring with phenotypic clustering may provide opportunities for complementary combination treatments. Conclusion Deep transfer learning with patient-by-patient fine-tuning is a promising, segmentation-free image-analysis approach for precision medicine and drug discovery based on 3D spheroid cell cultures.

Effects of UV-B and DMSO Treatments on the Production of Artemisinin in Low-Artemisinin Producing Artemisia annua Cell Cultures

Artemisinin is a sesquiterpene lactone derived from Artemisia annua L., and has a variety of biological properties, such as immunoregulatory and anticancer effects, and therapeutic applications. As a result of its naturally low production and compartmentalised synthesis, the irregular agricultural supply often leads to price fluctuations and reduction of the artemisinin inventory. In this study, elicitation efficiencies of ultraviolet B (UV-B) and dimethyl sulfoxide (DMSO) on a low-artemisinin producing (LAP) chemotype of the species A. annua were investigated. Exposure of cell suspension cultures to short-term UV-B radiation and DMSO treatment did not result in significant changes in artemisinin yield. The lack of stimulation could be associated with the growth condition such as the incubation duration after treatment, the physiological state of suspension-cultured cells, and the regulation of cellular metabolic homeostasis. Further molecular analysis with RT-PCR revealed the absence of mRNA transcripts of key genes ADS, DBR2, and ALDH1 which might affect artemisinin synthesis. This study demonstrated the complexity of stress-induced responses of cell suspension cultures in relation to metabolic processes which are important for artemisinin formation.

Maize transformation: history, progress, and perspectives.

Maize functional genomics research and genetic improvement strategies have been greatly accelerated and refined through the development and utilization of genetic transformation systems. Maize transformation is a composite technology based on decades' efforts in optimizing multiple factors involving microbiology and physical/biochemical DNA delivery, as well as cellular and molecular biology. This review provides a historical reflection on the development of maize transformation technology including the early failures and successful milestones. It also provides a current perspective on the understanding of tissue culture responses and their impact on plant regeneration, the pros and cons of different DNA delivery methods, the identification of a palette of selectable/screenable markers, and most recently the development of growth-stimulating or morphogenic genes to improve efficiencies and extend the range of transformable genotypes. Steady research progress in these interdependent components has been punctuated by benchmark reports celebrating the progress in maize transformation, which invariably relied on a large volume of supporting research that contributed to each step and to the current state of the art. The recent explosive use of CRISPR/Cas9-mediated genome editing has heightened the demand for higher transformation efficiencies, especially for important inbreds, to support increasingly sophisticated and complicated genomic modifications, in a manner that is widely accessible. These trends place an urgent demand on taking maize transformation to the next level, presaging a new generation of improvements on the horizon. Once realized, we anticipate a near-future where readily accessible, genotype-independent maize transformation, together with advanced genomics, genome editing, and accelerated breeding, will contribute to world agriculture and global food security.

Effects of coffee silverskin on growth performance, immune response, and disease resistance of Nile tilapia culture under biofloc system

The work herein was implemented to ascertain the possible consequences of coffee silverskin (CSS) on the growth, skin mucus, serum immunities, and disease resistance of Nile tilapia (Oreochromis niloticus) raised under biofloc system. Nile tilapia fingerlings (average weight 15.54 ± 0.21 g fish−1) were raised in 15 aquaria (150 L tank−1) at density of 20 fish per tank and fed five different diets: CSS1 (Control), CSS2 (10 g kg−1), CSS3 (20 g kg−1), CSS4 (40 g kg−1), and CSS5 (80 g kg−1) for 8 weeks. Growth rate and innate immune responses were calculated every four weeks, while the challenge test was conducted at the end of the experiment. The results determined that CSS dietary inclusion resulted in improved specific growth rate (SGR) and feed conversion ratio (FCR), compared to that of the control, except in fish fed the CSS5 diet. The optimum dose was recorded in fish fed CSS3. Similarly, fish fed CSS3 resulted in better innate immune responses than those of other incorporated diets, as well as the control. Notably, no significant differences in fish innate immune responses were detected between CSS5 and the control, or CSS2 and CSS4. Similarly, the CSS3 group presented the highest survival rates after the challenge test against Streptococcus agalactiae among all dietary inclusions. In conclusion, the dietary supplementation of 20 g kg−1 CSS (CSS3) may be used as an immunostimulant in tilapia cultured under biofloc system.

Maturation-Dependent Differences in the Re-innervation of the Denervated Dentate Gyrus by Sprouting Associational and Commissural Mossy Cell Axons in Organotypic Tissue Cultures of Entorhinal Cortex and Hippocampus.

Sprouting of surviving axons is one of the major reorganization mechanisms of the injured brain contributing to a partial restoration of function. Of note, sprouting is maturation as well as age-dependent and strong in juvenile brains, moderate in adult and weak in aged brains. We have established a model system of complex organotypic tissue cultures to study sprouting in the dentate gyrus following entorhinal denervation. Entorhinal denervation performed after 2 weeks postnatally resulted in a robust, rapid, and very extensive sprouting response of commissural/associational fibers, which could be visualized using calretinin as an axonal marker. In the present study, we analyzed the effect of maturation on this form of sprouting and compared cultures denervated at 2 weeks postnatally with cultures denervated at 4 weeks postnatally. Calretinin immunofluorescence labeling as well as time-lapse imaging of virally-labeled (AAV2-hSyn1-GFP) commissural axons was employed to study the sprouting response in aged cultures. Compared to the young cultures commissural/associational sprouting was attenuated and showed a pattern similar to the one following entorhinal denervation in adult animals in vivo. We conclude that a maturation-dependent attenuation of sprouting occurs also in vitro, which now offers the chance to study, understand and influence maturation-dependent differences in brain repair in these culture preparations.

Contemporary Film and the Empathy Controversy: Part 1

This article is written in two parts: Part 1 is a critique of Against Empathy: The Case for Rational Compassion by social psychologist Paul Bloom, and Part 2 is a reading of three U.S. films, produced between 2009 and 2012, that allow me to illustrate what is wrong with Bloom’s claim that we should endeavor to bypass empathy and cultivate more rationally effective forms of care. In Part 1, I introduce the project of both papers; however, my central aim is to mount several arguments against Bloom’s position. I argue that empathy is hard wired and therefore it is impossible to bypass, that Bloom’s own utilitarian ethics is significantly flawed, and that his central claim that empathy privileges those few we know best and who are closest to us is brought into question by the research that Bloom himself cites. However, the most important argument of Part 1—the argument that leads directly into my reading of filmic examples of empathy in Part 2—is that Bloom fails to understand how empathy is shaped by hegemonic ideological representations. This failure to consider in any meaningful way how culture and society shape affective response is a profound, but typical failure in the field of social psychology.

Effect of silver nitrate and growth regulators to enhance anther culture response in wheat (Triticum aestivum L.)

Application of chemical substances as stress pre-treatment factors may positively influence androgenetic responses in cereal and other crops. AgNO3 is an anti-ethylene compounds that played a significant role in combination with other chemicals for anther culture responses in cereal and other crop plants. For this study two local wheat cultivars viz. Kheri and Akbar were considered to evaluate the effect of AgNO3 and to optimize the suitable doses of plant growth regulators, amino acids and sucrose that supplemented in MS medium. Data were recorded on the basis of embryoids induction, regenerated green and albino plants. The results clearly stated that anther culture responses and its major outcomes on regeneration significantly increased with suitable dosages of chemicals. The most noteworthy increases embryo like structures and regenerated green plants accomplished by utilizing the combined effect of AgNO3 (50 mg/l) and as plant growth regulators IAA (1.0 mg/l) + kinetin (0.5 mg/l). Best embryo like structures (79.17%) and green plants (33.33%) were recorded in Kheri. The results clearly stated that reducing albinism and increasing embryos induction and green plants 50–75 mg/l silver nitrate along with optimum doses of IAA and kinetin showed very effective results in wheat.

CARACTERÍSTICAS MICROMORFOLÓGICAS DE SUSTRATOS ORGÁNICOS Y SU RELACIÓN CON RETENCIÓN DE AGUA Y CRECIMIENTO RADICAL

El estudio de la micromorfología permite comprender el funcionamiento hídrico y la respuesta fisiológica de los cultivos en un sustrato. En la investigación se evaluó el efecto de la micromorfología de mezclas de materiales orgánicos, sobre el contenido de agua y el crecimiento radical de crisantemo. La hipótesis fue que la descripción de la micromorfología de nuevas propuestas de sustratos permitiría inferir la contención, el movimiento del agua y en consecuencia el crecimiento radical. Materiales orgánicos se utilizaron como: aserrín (AP) y viruta de pino (VP), bagazo de henequén (BH), polvo de coco (PC) y un sustrato testigo (turba:Agrolita®). Siete mezclas se evaluaron plantando esquejes enraizados en contenedores con capacidad de 1250 mL. En las mezclas se determinó potencial hídrico (ψ), volumen radical (VR) y peso seco de raíces (PSR); en las hojas, el contenido relativo de agua (CRA) y el uso eficiente del agua (UEA). La micromorfología y la porosidad se caracterizaron a través de la descripción de secciones delgadas. Los resultados indicaron que la forma y distribución del tamaño de partículas, el grado de descomposición de la materia orgánica y el tipo y tamaño de poro, influyeron en la retención y el movimiento del agua en un sustrato. La mezcla de bagazo de henequén y aserrín en una relación 20:80, formó poros de empaquetamiento compuesto, de tamaños entre 50 y 500 μm y superó a la mezcla testigo en el uso eficiente del agua, sin diferencias significativas en potencial hídrico, contenido relativo de agua, volumen radical, ni peso seco de raíces. La combinación de polvo de coco y aserrín de pino formó poros de empaquetamiento compuesto y microporos <50 μm que dificultaron el movimiento del agua y afectaron el crecimiento de las raíces. En la mezcla 20PC:80VP el 80% de las partículas fueron >200 μm, con poros de empaquetamiento simples con 80% de tamaños de 200 a 500 μm que ocasionaron la percolación del agua y una disminución del (ψ) potencial hídrico.

Changes in photosynthesis, growth and biomass composition in outdoor Chlorella g120 culture during the metabolic shift from heterotrophic to phototrophic cultivation regime

Up to now, scarce information has been available regarding major photobiochemical changes that accompany the metabolic shift from heterotrophy to phototrophy in microalgae cells. In the present trials the trophic conversion from heterotrophic to phototrophic growth regime was studied in the microalga Chlorella vulgaris g120 grown in outdoor thin-layer cascades. The crucial question was whether Chlorella g120 can undergo the photoacclimation of its photosynthetic apparatus under natural irradiance. As for comparison the phototrophic strain Chlorella vulgaris R-117 was cultured in parallel.Various in-situ and ex-situ monitoring techniques namely oxygen production and chlorophyll fluorescence were tested to characterize physiological response of the Chlorella g120 culture during photoacclimation and correlate it with growth. We show that Chlorella g120 can undergo the metabolic shift from heterotrophic to phototrophic growth regime, but its conversion is. A rather high respiration rate was interpreted as a strategy to dissipate the unused light energy. Chlorella g120 was found as the strain characterized by a small-antenna size strain (low chlorophyll/cell ratio < 1%) which should be favorable for light utilization in dense cultures. The high carotenoid/chlorophyll ratio (= 0.63) may be of interest from biotechnological point of view for carotenoid (namely lutein) production.The build-up of dissolved oxygen (DO) concentration and high electron transport rate measured in-situ indicated high photosynthetic activity of outdoor microalgae cultures. These variables are usable on-line as reliable markers as they reflect the physiological status of microalgae culture. Highly productive cultures, like Chlorella R-117 have high photosynthesis which is inevitably accompanied by the build-up on high DO gradients. To maintain high biomass production in such strains we have to find an interplay among photosynthetic activity, gradients of DO concentration, biomass density and culture layer thickness that can secure high growth rate (and productivity).

Glycated Albumin Triggers an Inflammatory Response in the Human Airway Epithelium and Causes an Increase in Ciliary Beat Frequency.

The role of inflammation in airway epithelial cells and its regulation are important in several respiratory diseases. When disease is present, the barrier between the pulmonary circulation and the airway epithelium is damaged, allowing serum proteins to enter the airways. We identified that human glycated albumin (GA) is a molecule in human serum that triggers an inflammatory response in human airway epithelial cultures. We observed that single-donor human serum induced IL-8 secretion from primary human airway epithelial cells and from a cystic fibrosis airway cell line (CF1-16) in a dose-dependent manner. IL-8 secretion from airway epithelial cells was time dependent and rapidly increased in the first 4 h of incubation. Stimulation with GA promoted epithelial cells to secrete IL-8, and this increase was blocked by the anti-GA antibody. The IL-8 secretion induced by serum GA was 10–50-fold more potent than TNFα or LPS stimulation. GA also has a functional effect on airway epithelial cells in vitro, increasing ciliary beat frequency. Our results demonstrate that the serum molecule GA is pro-inflammatory and triggers host defense responses including increases in IL-8 secretion and ciliary beat frequency in the human airway epithelium. Although the binding site of GA has not yet been described, it is possible that GA could bind to the receptor for advanced glycated end products (RAGE), known to be expressed in the airway epithelium; however, further experiments are needed to identify the mechanism involved. We highlight a possible role for GA in airway inflammation.

Endogenous phytonutrient, phytochemical, and phytohormone levels modulate in-vitro callus induction and plant regeneration in finger millet (Eleusine coracana) genotypes

Finger millet (Eleusine coracana) is an orphan crop that has prominent nutritional traits such as high calcium. Biotechnological interventions in this crop suffer from constraints like genotypic dependency leading to inefficient regeneration and transformation systems. Biofortification requires improvements in the available plant tissue culture methods with major emphasis on underutilized crops like finger millet. Keeping this in view, the impact of inherent composition of phytonutrients (calcium, carbohydrates, proteins), phytochemicals (phenolics and flavonoids), and phytohormones (Abscisic Acid, ABA and Gibberellic Acid, GA3) on tissue culture responsiveness of four finger millet genotypes was studied. The study elucidated that the nutritional and phytochemical/hormone composition of seed/mature embryos as explants influence callus induction and regeneration on use of different phytohormones. There was significant (p < 0.05) callus induction at 1.5 mg/L 2, 4-D (2,4-Dichlorophenoxyacetic Acid) and 0.5 mg/L BAP (6-Benzylaminopurine) in all four genotypes. The high calcium variety GP-45 showed enhanced callusing and regeneration in comparison to other genotypes. Callus Induction Frequency and Shoot Regeneration Frequency exhibited significant positive correlation with calcium content and significant negative correlation with protein and flavonoid content for all the genotypes. The results demonstrate the importance of genotype selection based on inherent phytonutrient and phytohormone composition on tissue culture for crop improvement.

MƏDƏNİ QLOBALLAŞMA VƏ DÖVLƏTDAXİLİ MÜNAQİŞƏLİLİK

Nowadays, the impacts of globalization are also noticeable in culture. For this reason, in addition to economic, social, and political confrontations, the increasing conflicts of nationalism within the state because of cultural globalization have been investigated in the present article. The possible consequences of the intersection of globalizing forces and local culture have been researched. The types of reactions to cultural changes associated with globalization and the responses of local cultures to them are analyzed. Key words: cultural globalization, internal conflicts, local culture, democracy

Angiotensin II, ATP and high extracellular potassium induced intracellular calcium responses in primary rat brain endothelial cell cultures.

The meninges shield the nervous system from diverse, rather harmful stimuli and pathogens from the periphery. This tissue is composed of brain endothelial cells (BECs) that express diverse ion channels and chemical-transmitter receptors also expressed by neurons and glial cells to communicate with each other. However, information about the effects of ATP and angiotensin II on BECs is scarce, despite their essential roles in blood physiology. This work investigated in vitro if BECs from the meninges from rat forebrain respond to ATP, angiotensin II and high extracellular potassium, with intracellular calcium mobilizations and its second messenger-associated pathways. We found that in primary BEC cultures, both ATP and angiotensin II produced intracellular calcium responses linked to the activation of inositol trisphosphate receptors and ryanodine receptors, which led to calcium release from intracellular stores. We also used RT-PCR to explore what potassium channel subunits are expressed by primary BEC cultures and freshly isolated meningeal tissue, and which might be linked to the observed effects. We found that BECs mainly expressed the inward rectifier potassium channel subunits Kir1.1, Kir3.3, Kir 4.1 and Kir6.2. This study contributes to the understanding of the functions elicited by ATP and angiotensin II in BECs from rat meninges. SIGNIFICANCE OF THE STUDY: Brain endothelial cells (BECs) express diverse ion channels and membrane receptors, which they might use to communicate with neurons and glia. This work investigated in vitro, if BECs from the rat forebrain respond to angiotensin II and ATP with intracellular calcium mobilizations. We found that these cells did respond to said substances with intracellular calcium mobilizations linked to inositol trisphosphate and ryanodine receptor activation, which led to calcium release from intracellular stores. These findings are important because they might uncover routes of active communication between brain cells and endothelial cells.

Transcriptomic and isotopic data reveal central role of ammonium in facilitating the growth of the mixotrophic dinoflagellate, Dinophysis acuminata

Dinophysis spp. are mixotrophs that are dependent on specific prey, but are also potentially reliant on dissolved nutrients. The extent to which Dinophysis relies on exogenous N and the specific biochemical pathways important for supporting its autotrophic and heterotrophic growth are unknown. Here, the nutritional ecology of Dinophysis was explored using two approaches: 1) 15N tracer experiments were conducted to quantify the concentration-dependent uptake rates and associated kinetics of various N compounds (nitrate, ammonium, urea) of Dinophysis cultures and 2) the transcriptomic responses of Dinophysis cultures grown with multiple combinations of prey and nutrients were assessed via dinoflagellate spliced leader-based transcriptome profiling. Of the N compounds examined, ammonium had the highest Vmax and affinity coefficient, and lowest Ks for both pre-starved and pre-fed cultures, collectively demonstrating the preference of Dinophysis for this N source while little-to-no nitrate uptake was observed. During the transcriptome experiments, Dinophysis grown with nitrate and without prey had the largest number of genes with lower transcript abundances, did not increase abundance of transcripts associated with nitrate/nitrite uptake or reduction, and displayed no cellular growth, suggesting D. acuminata is not capable of growing on nitrate. When offered prey, the transcriptomic response of Dinophysis included the production of phagolysosomes, enzymes involved in protein and lipid catabolism, and N acquisition through amino acid degradation pathways. Compared with cultures only offered ammonium or prey, cultures offered both ammonium and prey had the largest number of genes with increased transcript abundances, the highest growth rate, and the unique activation of multiple pathways involved in cellular catabolism, further evidencing the ability of Dinophysis to grow optimally as a mixotroph. Collectively, this study evidences the key role ammonium plays in the mixotrophic growth of Dinophysis and reveals the precise biochemical pathways that facilitate its mixotrophic growth.

Hydrogel Scaffolds: Cell‐Laden Gradient Hydrogel Scaffolds for Neovascularization of Engineered Tissues (Adv. Healthcare Mater. 7/2021)

In articule number 2001706 by Georgia Papavasiliou and co-workers, vascular cell-laden gradient PEG hydrogels with five types of gradient combinations including immobilized cell adhesion peptide concentration (RGD), stiffness, and protease-sensitivity are designed to investigate their role on spatial variations in vascular sprouting responses in 3D culture.