Culture Conditions Research Articles

Effects of different trophic conditions on total fatty acids, amino acids, pigment and gene expression profiles in Euglena gracilis.

Euglena gracilis is a unique microalga that lacks a cell wall and is able to grow under different trophic culture conditions. In this study, cell growth, biomass production, and changes in the ultrastructure of E. gracilis cells cultivated photoautotrophically, mixotrophically, and under sequential-heterotrophy-photoinduction (SHP) were assessed. Mixotrophy induced the highest cell growth and biomass productivity (6.27 ± 0.59mg/L/d) in E. gracilis, while the highest content of fatty acids, 2.69 ± 0.04% of dry cell weight (DCW) and amino acids, 38.16 ± 0.08% of DCW was obtained under SHP condition. E. gracilis also accumulated significantly higher saturated fatty acids and lower unsaturated fatty acids when cultivated under SHP condition. Transcriptomic analysis showed that the expression of photosynthetic genes (PsbA, PsbC, F-type ATPase alpha and beta) was lower, carbohydrate and protein synthetic genes (glnA, alg14 and fba) were expressed higher in SHP-culture cells when compared to other groups. Different trophic conditions also induced changes in the cell ultrastructure, where paramylon and starch granules were more abundant in SHP-cultured cells. The findings generated in this study illustrated that aerobic SHP cultivation of E. gracilis possesses great potential in human and animal feed applications.

Growth potential, biochemical properties and nutrient removal efficiency of some freshwater microalgae and their consortia from wastewater

Impact of varying nitrate (NO3-N) and phosphate (PO4-P) concentrations and sewage water (SW) on the growth, nutrient removal, lipid accumulation, enzymatic antioxidant activity and phytochemical contents of the microalgae Scenedesmus dimorphus, Coelastrella tenuitheca, Chroococcus turgidus and Parachlorella kessleri under monoculture and their consortia have been investigated. High growth rates were observed for all the four algae in both mono and mixed culture conditions at enhanced concentrations of N (1500 mg/L NO3-N) and P (40 mg/L PO4-P). The species Scenedesmus dimorphus outperformed other microalgae growing in SW in efficiently removing nitrogen. The algal consortia of mixed species was found to be more effective in phosphorus removal. The carbohydrate and protein contents were highest in Parachlorella kessleri, about 37% and 44%, respectively, in SW cultivation. The algal consortia demonstrated highest starch content (4%) in nitrogen deprived growth medium. Highest lipid production (43%) was observed in the SW culture. The species Coelastrella tenuitheca, Chroococcus turgidus and Scenedesmus dimorphus irrespective of the growth media indicated significant accumulation of phenol, flavonoid and tannin. The DPPH, catalase and ascorbic peroxidase assay showed pronounced antioxidant activity. Nutrient (N and P) enrichment exhibited enhanced antioxidant enzymatic activity and accumulation of cell storage products.

Gene-level alignment of single-cell trajectories

AbstractSingle-cell data analysis can infer dynamic changes in cell populations, for example across time, space or in response to perturbation, thus deriving pseudotime trajectories. Current approaches comparing trajectories often use dynamic programming but are limited by assumptions such as the existence of a definitive match. Here we describe Genes2Genes, a Bayesian information-theoretic dynamic programming framework for aligning single-cell trajectories. It is able to capture sequential matches and mismatches of individual genes between a reference and query trajectory, highlighting distinct clusters of alignment patterns. Across both real world and simulated datasets, it accurately inferred alignments and demonstrated its utility in disease cell-state trajectory analysis. In a proof-of-concept application, Genes2Genes revealed that T cells differentiated in vitro match an immature in vivo state while lacking expression of genes associated with TNF signaling. This demonstrates that precise trajectory alignment can pinpoint divergence from the in vivo system, thus guiding the optimization of in vitro culture conditions.

High specificity of engineered T cells with third generation CAR (CD28-4-1BB-CD3-ζ) based on biotin-bound monomeric streptavidin for potential tumor immunotherapy

IntroductionImmunotherapy has revolutionized cancer treatment, and Chimeric Antigen Receptor T cell therapy (CAR-T) is a groundbreaking approach. Traditional second-generation CAR-T therapies have achieved remarkable success in hematological malignancies, but there is still room for improvement, particularly in developing new targeting strategies. To address this limitation, engineering T cells with multi-target universal CARs (UniCARs) based on monomeric streptavidin has emerged as a versatile approach in the field of anti-tumor immunotherapy. However, no studies have been conducted on the importance of the intracellular signaling domains of such CARs and their impact on efficiency and specificityMethodHere, we developed second-generation and third-generation UniCARs based on an extracellular domain comprising an affinity-enhanced monomeric streptavidin, in addition to CD28 and 4-1BB co-stimulatory intracellular domains. These UniCAR structures rely on a biotinylated intermediary, such as an antibody, for recognizing target antigens. In co-culture assays, we performed a functional comparison between the third-generation UniCAR construct and two second-generation UniCAR variants, each incorporating either the CD28 or 4-1BB as co-stimulatory domainResultsWe observed that components in culture media could inhibit the binding of biotinylated antibodies to monomeric streptavidin-CARs, potentially compromising their efficacy. Furthermore, third-generation UniCAR-T cells showed robust cytolytic activity against cancer cell lines upon exposure to specific biotinylated antibodies like anti-CD19 and anti-CD20, underscoring their capability for multi-targeting. Importantly, when assessing engineered UniCAR-T cell activation upon encountering their target cells, third-generation UniCAR-T cells exhibited significantly enhanced specificity compared to second-generation CAR-T cellsDiscussionFirst, optimizing culture conditions would be essential before deploying UniCAR-T cells clinically. Moreover, we propose that third-generation UniCAR-T cells are excellent candidates for preclinical research due to their high specificity and multi-target anti-tumor cytotoxicity

Refining microbial community metabolic models derived from metagenomics using reference-based taxonomic profiling.

Characterization of microbial community metabolic output is crucial to understanding their functions. Construction of genome-scale metabolic models from metagenome-assembled genomes (MAG) has enabled prediction of metabolite production by microbial communities, yet little is known about their accuracy. Here, we examined the performance of two approaches for metabolite prediction from metagenomes, one that is MAG-guided and another that is taxonomic reference-guided. We applied both on shotgun metagenomics data from human and environmental samples, and validated findings in the human samples using untargeted metabolomics. We found that in human samples, where taxonomic profiling is optimized and reference genomes are readily available, when number of input taxa was normalized, the reference-guided approach predicted more metabolites than the MAG-guided approach. The two approaches showed significant overlap but each identified metabolites not predicted in the other. Pathway enrichment analyses identified significant differences in inferences derived from data based on the approach, highlighting the need for caution in interpretation. In environmental samples, when the number of input taxa was normalized, the reference-guided approach predicted more metabolites than the MAG-guided approach for total metabolites in both sample types and non-redundant metabolites in seawater samples. Nonetheless, as was observed for the human samples, the approaches overlapped substantially but also predicted metabolites not observed in the other. Our findings report on utility of a complementary input to genome-scale metabolic model construction that is less computationally intensive forgoing MAG assembly and refinement, and that can be applied on shallow shotgun sequencing where MAGs cannot be generated.IMPORTANCELittle is known about the accuracy of genome-scale metabolic models (GEMs) of microbial communities despite their influence on inferring community metabolic outputs and culture conditions. The performance of GEMs for metabolite prediction from metagenomes was assessed by applying two approaches on shotgun metagenomics data from human and environmental samples, and validating findings in the human samples using untargeted metabolomics. The performance of the approach was found to be dependent on sample type, but collectively, the reference-guided approach predicted more metabolites than the MAG-guided approach. Despite the differences, the predictions from the approaches overlapped substantially but each identified metabolites not predicted in the other. We found significant differences in biological inferences based on the approach, with some examples of uniquely enriched pathways in one group being invalidated when using the alternative approach, highlighting the need for caution in interpretation of GEMs.

Mesenchymal Stem Cell-Derived Exosomes as a Neuroregeneration Treatment for Alzheimer’s Disease

Background: Alzheimer’s disease (AD) is the most prevalent kind of dementia and is a long-term degenerative disease. Pathologically, it is defined by the development of extracellular amyloid-β plaques and intracellular neurofibrillary tangles made up of hyperphosphorylated tau protein. This causes neuronal death, particularly in the hippocampus and cortex. Mesenchymal stem cell (MSC)-derived exosomes have been identified as possibly therapeutic and have promise for Alzheimer’s disease due to their regenerative characteristics. Methods: A systematic retrieval of information was performed on PubMed. A total of 60 articles were found in a search on mesenchymal stem cells, exosomes, and Alzheimer’s disease. A total of 16 ongoing clinical trials were searched and added from clinicaltrials.gov. We added 23 supporting articles to help provide information for certain sections. In total, we included 99 articles in this manuscript: 50 are review articles, 13 are preclinical studies, 16 are clinical studies, 16 are ongoing clinical trials, and 4 are observational studies. Appropriate studies were isolated, and important information from each of them was understood and entered into a database from which the information was used in this article. The clinical trials on mesenchymal stem cell exosomes for Alzheimer’s disease were searched on clinicaltrials.gov. Results: Several experimental investigations have shown that MSC-Exo improves cognitive impairment in rats. In this review paper, we summarized existing understanding regarding the molecular and cellular pathways behind MSC-Exo-based cognitive function restoration, with a focus on MSC-Exo’s therapeutic potential in the treatment of Alzheimer’s disease. Conclusion: AD is a significant health issue in our culture and is linked to several important neuropathological characteristics. Exosomes generated from stem cells, such as mesenchymal stem cells (MSCs) or neural stem cells (NSCs), have been examined more and more in a variety of AD models, indicating that they may be viable therapeutic agents for the treatment of diverse disorders. Exosome yields may be increased, and their therapeutic efficacy can be improved using a range of tailored techniques and culture conditions. It is necessary to provide standardized guidelines for exosome manufacture to carry out excellent preclinical and clinical research.

EphA4 Targeting Peptide-Conjugated Extracellular Vesicles Rejuvenates Adult Neural Stem Cells and Exerts Therapeutic Benefits in Aging Rats.

Aging and various neurodegenerative diseases cause significant reduction in adult neurogenesis and simultaneous increase in quiescent neural stem cells (NSCs), which impact the brain's regenerative capabilities. To deal with this challenging issue, current treatments involve stem cell transplants or prevention of neurodegeneration; however, the efficacy or success of this process remains limited. Therefore, extensive and focused investigation is highly demanding to overcome this challenging task. Here, we have designed an efficient peptide-based EphA4 receptor-targeted ligand through an in silico approach. Further, this strategy involves chemical conjugation of the peptide with adipose tissue stem cell-derived EV (Exo-pep-11). Interestingly, our newly designed engineered EV, Exo-pep-11, targets NSC through EphA4 receptors, which offers promising therapeutic advantages by stimulating NSC proliferation and subsequent differentiation. Our result demonstrates that NSC successfully internalized Exo-pep-11 in both in vitro culture conditions as well as in the in vivo aging rats. We found that the uptake of Exo-pep-11 decreased by ∼2.3-fold when NSC was treated with EphA4 antibody before Exo-pep-11 incubation, which confirms the receptor-specific uptake of Exo-pep-11. Exo-pep-11 treatment also increases NSC proliferation by ∼1.9-fold and also shows ∼1.6- and ∼2.4-fold increase in expressions of Nestin and ID1, respectively. Exo-pep-11 also has the potential to increase neurogenesis in aging rats, which is confirmed by ∼1.6- and ∼1.5-fold increases in expressions of TH and Tuj1, respectively, in rat olfactory bulb. Overall, our findings highlight the potential role of Exo-pep-11 for prospective applications in combating age-related declines in NSC activity and neurogenesis.

Bridging the gap: advancing cancer cell culture to reveal key metabolic targets

Metabolic rewiring is a defining characteristic of cancer cells, driving their ability to proliferate. Leveraging these metabolic vulnerabilities for therapeutic purposes has a long and impactful history, with the advent of antimetabolites marking a significant breakthrough in cancer treatment. Despite this, only a few in vitro metabolic discoveries have been successfully translated into effective clinical therapies. This limited translatability is partially due to the use of simplistic in vitro models that do not accurately reflect the tumor microenvironment. This Review examines the effects of current cell culture practices on cancer cell metabolism and highlights recent advancements in establishing more physiologically relevant in vitro culture conditions and technologies, such as organoids. Applying these improvements may bridge the gap between in vitro and in vivo findings, facilitating the development of innovative metabolic therapies for cancer.

Modulation of Photosensitizing Responses in Cell Culture Environments by Different Medium Components

Many cell culture experiments are performed under light to evaluate the photodynamic or photosensitizing efficacy of various agents. In this study, the modulation of photosensitizing responses and phototoxicity under cell culture conditions by different medium components was investigated. The significant levels of reactive oxygen species (ROS) generated from DMEM, RPMI 1640, and MEM were observed under the irradiation of fluorescent light (FL) and white and blue LEDs, indicating that these media have their own photosensitizing properties; DMEM showed the most potent property. Phenol red-free DMEM (Pf-D) exhibited a stronger photosensitizing property than normal DMEM by 1.31 and 1.25 times under FL and blue LEDs, respectively; phenol red and riboflavin-free DMEM (PRbf-D) did not show any photosensitizing properties. The inhibitory effect on light transmission was more pronounced in DMEM than in RPMI, and the interference effect on green LED light was greatest at 57.8 and 27.4%, respectively; the effect disappeared in Pf-D. The media containing riboflavin induced strong phototoxicity in HaCaT keratinocytes by generating H2O2 under light irradiation, which was quenched by sodium pyruvate in the media. The presence of serum in the media was also reduced the phototoxicity; H2O2 levels in the media decreased serum content dependently. The phototoxicity of erythrosine B and protoporphyrin IX under FL was more sensitively pronounced in PRbf-D than in DMEM. The present results indicate that several medium components, including riboflavin, phenol red, sodium pyruvate, and serum, could modulate photosensitizing responses in a cell culture system by inducing photosensitizing activation and by interfering with irradiation efficacy and ROS generation.

Development and characterization of segment-specific enteroids from the pig small intestine in Matrigel and transwell inserts: insights into susceptibility to porcine epidemic diarrhea Virus

IntroductionThe critical early stages of infection and innate immune responses to porcine epidemic diarrhea virus (PEDV) at the intestinal epithelium remain underexplored due to the limitations of traditional cell culture and animal models. This study aims to establish a porcine enteroid culture model to investigate potential differences in susceptibility to infection across segments of the porcine small intestine (duodenum, jejunum, and ileum).MethodsIntestinal crypt cells from nursery pigs were cultured in Matrigel to differentiate into porcine enteroid monolayer cultures (PEMCs). Following characterization, PEMCs were enzymatically dissociated and subcultured on transwell inserts (PETCs) for apical surface exposure and infection studies. Characterization of region-specific PEMCs and PETCs included assessment of morphology, proliferation, viability, and cellular phenotyping via immunohistochemistry/immunocytochemistry and gene expression analysis. Subsequently, PETCs were inoculated with 105 TCID50 (50% tissue culture infectious dose)/mL of a high pathogenic PEDV non-S INDEL strain and incubated for 24 h. Infection outcomes were assessed by cytopathic effect, PEDV N protein expression (immunofluorescence assay, IFA), and PEDV N-gene detection (quantitative reverse transcription polymerase chain reaction, RT-qPCR).ResultsNo significant morphological and phenotypical differences were observed among PEMCs and PETCs across intestinal regions, resembling the porcine intestinal epithelium. Although PETCs established from different segments of the small intestine were susceptible to PEDV infection, jejunum-derived PETCs exhibited higher PEDV replication, confirmed by IFA and RT-qPCR.DiscussionThis segment-specific enteroid culture model provides a reliable platform for virological studies, offering a controlled environment that overcomes the limitations of in vivo and traditional cell culture methods. Standardizing culture conditions and characterizing the model are essential for advancing enteroid-based infection models.

Unraveling the complexities of antifungal susceptibility testing in Candida spp.: Insights from design of experiments

Our study delved into the intricate dynamics of antifungal susceptibility testing for Candida spp., employing a Design of Experiments approach. We systematically investigated the influence of pH, temperature, inoculum size, and glucose concentration on both growth patterns and inhibitory concentrations of Candida spp. Our findings underscore the nuanced interplay between these factors, revealing significant impacts on susceptibility outcomes. Notably, even minor adjustments in these parameters yielded substantial variations in growth and inhibitory concentrations, underscoring the critical importance of meticulous control over growth conditions in antifungal susceptibility testing protocols. Each Candida isolates exhibited unique susceptibility profiles, necessitating tailored culture conditions for accurate testing. Our study sheds light on the variability inherent in Candida spp. growth patterns and emphasizes the need for standardized protocols to ensure consistency across laboratories. By leveraging the design of experiments, our research provides a systematic framework for unraveling the complexities of antifungal susceptibility testing, offering valuable insights for optimizing testing protocols and informing clinical decision-making in antifungal treatment. These findings represent a significant step towards enhancing the efficacy and reliability of antifungal susceptibility testing in clinical practice.

Insights on aggregation-algae consortium based removal of sulfamethoxazole: Unraveling removal effect, enhanced method and toxicological evaluation

The escalating occurrence of the antibiotic Sulfamethoxazole (SMX) in the environment presents a significant global threat to ecological systems and human health. Despite the growing interest in using microalgae for antibiotic biodegradation, strategies to enhance SMX elimination remain underexplored. In this study, we isolated a novel aggregation-algae consortium (AAC) from a municipal wastewater treatment plant (WWTP) and examined its potential for SMX removal, optimized culture conditions, SMX metabolite fate and the physicochemical impact on microalgal cells. The findings revealed that the AAC demonstrated remarkable resistance to SMX, even at concentrations as high as 10 mg/L, and could degrade SMX via free radical reactions. Although ion repulsion limited the biodegradation of AAC, the addition of peptone and yeast extract resulted in a significant enhancement, increased by 16.71%, 39.12% and 46.77% of three SMX groups. Moreover, AAC exhibited exceptional adaptability in real wastewater, achieving removal of 87.05%, 97.39% and 20.80% for total dissolved nitrogen, total dissolved phosphorus and SMX, respectively. The decreased degradation toxicity of SMX following AAC treatment was further validated by ECOSAR software and in vitro tests using Caenorhabditis elegans. This study advanced our understanding of SMX biodegradation and provided a novel approach for treating wastewater contaminated with SMX.

Harnessing the antagonistic potential and unraveling the bioactive compounds of Streptomyces sp. isolate WAB2 to protect watermelon crop from Colletotrichum orbiculare

ABSTRACT Watermelon (Citrullus lanatus Thumb) is a significant vegetable crop globally and is prone to anthracnose disease caused by Colletotrichum spp. The use of chemical fungicides for managing crop diseases has adverse impacts on the environment and human health. Therefore, it is important to use biocontrol agents for disease management. In this study, streptomycetes were assessed as a potential biocontrol agent against anthracnose pathogen, Colletotrichum orbiculare WEC2. The potent Streptomyces sp. isolate WAB2 was isolated from healthy watermelon plants and showed remarkable antifungal efficacy against C. orbiculare WEC2. Rigorous assays confirmed its performance against C. orbiculare WEC2. Bioactive compounds produced by Streptomyces sp. isolate WAB2 were identified using Gas Chromatography–Mass Spectrometry (GC–MS). An SEM study indicated the parasitic action of Streptomyces sp. isolate WAB2 against the pathogen. The formulated talc-based product of Streptomyces sp. isolate WAB2 was effective when applied as a seed treatment + foliar spray against C. orbiculare WEC2 under pot culture conditions. These treatments reduced the disease incidence (31.81%) and exhibited a higher disease reduction (52.56%), compared to the untreated control. Therefore, treatment with talc-based Streptomyces sp. isolate WAB2 is a sustainable solution for anthracnose disease management, and it facilitates eco-friendly crop protection practices against the disease in watermelon. RESEARCH HIGHLIGHTS Isolated streptomycete strains from healthy watermelon plants and screened for its efficacy against the pathogen, Colletotrichum orbiculare WEC2. Streptomyces sp. isolate WAB2 showed significant antifungal activity against the pathogen. Bio-active compounds produced by Streptomyces sp. isolate WAB2 was identified by Gas Chromatography–Mass Spectrometry (GC–MS). Using scanning electron microscopy (SEM), the parasitic activity of Streptomyces sp. isolate WAB2 against the pathogen was detected. Developed a talc-based formulation of Streptomyces sp. isolate WAB2 and tested it as a seed treatment plus foliar spray, effectively reducing the disease incidence.

Establishment and characterization of a novel cell line derived from spotted sea bass Lateolabrax maculatus and its susceptibilities to iridoviruses

The spotted sea bass (Lateolabrax maculatus), which holds significant commercial value, is extensively cultivated in China, Japan, and Southeast Asian countries. This study reports the establishment and characterization of a novel continuous cell line, designated as LMK cells, derived from the kidney tissue of spotted sea bass, The LMK cell line, primarily composed of epithelial-like cells, has been successfully subcultured in Medium 199 supplemented with 10 % fetal bovine serum (FBS) for over 60 passages. Mitochondrial genome sequencing and cytochrome c oxidase subunit I gene amplification verified the origin of the LMK cells from L. maculatus, distinguishing them from L. japonicus. Optimal culture conditions for LMK cells at passage 35 were determined to be 10 % FBS at 27 °C. Chromosomal analysis indicated that the cell line possesses 48 chromosomes. To assess viral susceptibility, LMK cells were exposed to the red sea bream iridovirus and largemouth bass ranavirus. Both iridoviruses induced cytopathic effects within 3 days post-infection and triggered immune-related gene responses. This study demonstrates that the LMK cell line is a valuable resource for molecular, virological, and immunological research in L.maculatus and other sea bass species.

Establishment and characterization of a novel human gallbladder cancer cell line, GBC-X1

In this study, we successfully established a novel gallbladder cancer cell line, designated as GBC-X1, derived from a primary tumor of a gallbladder cancer patient. By comprehensively analyzing the cell line’s phenotype, molecular characteristics, biomarkers, and histological characteristics, we confirmed that GBC-X1 serves as a valuable model for investigating the pathogenesis of gallbladder cancer and developing therapeutic agents. GBC-X1 has been continuously cultured for one year, with over 60 stable passages. Morphologically, GBC-X1 exhibits typical features of epithelial tumors. The population doubling time of GBC-X1 is 32 h. STR analysis validated a high consistency between GBC-X1 and the patient’s primary tumor. Karyotype analysis revealed an abnormal hypertetraploid karyotype for GBC-X1, characterized by representative karyotypes of 98, XXXX del (4) p (12) del (5) p (21) der (10). Under suspension culture conditions, GBC-X1 efficiently forms tumor balls, while subcutaneous inoculation of GBC-X1 cells into NXG mice leads to xenograft formation with a rate of 80%. Drug sensitivity testing demonstrated that GBC-X1 is resistant to oxaliplatin and sensitive to 5-FU, gemcitabine, and paclitaxel. Immunohistochemistry revealed positive expression of CK7, CK19, E-cadherin, MMP-2, CD44, SOX2, and TP53 in GBC-X1 cells, weak positive expression of Vimentin, and a Ki67 positive rate of 35%. Our research highlights GBC-X1 as a novel gallbladder cancer cell line and emphasizes its potential as an effective experimental model for investigating the pathogenesis of gallbladder cancer and drug development.

Towards characterization of cell culture conditions for reliable proteomic analysis: in vitro studies on A549, differentiated THP-1, and NR8383 cell lines.

Proteomic investigations result in high dimensional datasets, but integration or comparison of different studies is hampered by high variances due to different experimental setups. In addition, cell culture conditions can have a huge impact on the outcome. This study systematically investigates the impact of experimental parameters on the proteomic profiles of commonly used cell lines-A549, differentiated THP-1 macrophage-like cells, and NR8383-for toxicity studies. The work focuses on analyzing the influence at the proteome level of cell culture setup involving different vessels, cell passage numbers, and post-differentiation harvesting time, aiming to improve the reliability of proteomic analyses for hazard assessment. Mass-spectrometry-based proteomics was utilized for accurate protein quantification by means of a label-free approach. Our results showed that significant proteome variations occur when cells are cultivated under different setups. Further analysis of these variations revealed their association to specific cellular pathways related to protein misfolding, oxidative stress, and proteasome activity. Conversely, the influence of cell passage numbers on the proteome is minor, suggesting a reliable range for conducting reproducible biological replicates. Notable, substantial proteome alterations occur over-time post-differentiation of dTHP-1 cells, particularly impacting pathways crucial for macrophage function. This finding is key for the interpretation of experimental results. These results highlight the need for standardized culture conditions in proteomic-based evaluations of treatment effects to ensure reliable results, a prerequisite for achieving regulatory acceptance of proteomics data.

β-glucans, SAM, and GSH fluctuations in barley anther tissue culture conditions affect regenerants’ DNA methylation and GPRE

BackgroundMicrospore embryogenesis is a process that produces doubled haploids in tissue culture environments and is widely used in cereal plants. The efficient production of green regenerants requires stresses that could be sensed at the level of glycolysis, followed by the Krebs cycle and electron transfer chain. The latter can be affected by Cu(II) ion concentration in the induction media acting as cofactors of biochemical reactions, indirectly influencing the production of glutathione (GSH) and S-adenosyl-L-methionine (SAM) and thereby affecting epigenetic mechanisms involving DNA methylation (demethylation—DM, de novo methylation—DNM). The conclusions mentioned were acquired from research on triticale regenerants, but there is no similar research on barley. In this way, the study looks at how DNM, DM, Cu(II), SAM, GSH, and β-glucan affect the ability of green plant regeneration efficiency (GPRE).ResultsThe experiment involved spring barley regenerants obtained through anther culture. Nine variants (trials) of induction media were created by adding copper (CuSO4: 0.1; 5; 10 µM) and silver salts (AgNO3: 0; 10; 60 µM), with varying incubation times for the anthers (21, 28, and 35 days). Changes in DNA methylation were estimated using the DArTseqMet molecular marker method, which also detects cytosine methylation.Phenotype variability in β-glucans, SAM and GSH induced by the nutrient treatments was assessed using tentative assignments based on the Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy. The effectiveness of green plant regeneration ranged from 0.1 to 2.91 plants per 100 plated anthers. The level of demethylation ranged from 7.61 to 32.29, while de novo methylation reached values ranging from 6.83 to 32.27. The paper demonstrates that the samples from specific in vitro conditions (trials) formed tight groups linked to the factors contributing to the two main components responsible for 55.05% of the variance (to the first component DNM, DM, to the second component GSH, β-glucans, Cu(II), GPRE).ConclusionsWe can conclude that in vitro tissue culture conditions affect biochemical levels, DNA methylation changes, and GPRE. Increasing Cu(II) concentration in the IM impacts the metabolism and DNA methylation, elevating GPRE. Thus, changing Cu(II) concentration in the IM is fair to expect to boost GPRE.

A Micromanipulation-Actuated Large-Scale Screening to Identify Optimized Microphysiological Model Parameters in Skeletal Muscle Regeneration.

Hydrogel-based 3D cell cultures are extensively utilized to create biomimetic cellular microstructures. However, there is still lack of effective method for both evaluation of the complex interaction of cells with hydrogel and the functionality of the resulting micro-structures. This limitation impedes the further application of these microstructures as microphysiological models (microPMs) for the screening of potential culture condition combinations to enhance the skeletal muscle regeneration. This paper introduces a two-probe micromanipulation method for the large-scale assessment of viscoelasticity and contractile force (CF) of skeletal muscle microPMs, which are produced in high-throughput via microfluidic spinning and 96-well culture. The collected data demonstrate that viscoelasticity parameters (E* and tanδ) and CF both measured in a solution environment are indicative of the formation of cellular structures without hydrogel residue and the subsequent generation of myotubes, respectively. This study have developed screening criterias that integrate E*, tanδ, and CF to examine the effects of multifactorial interactions on muscle fiber repair under hypoxic conditions and within bioprinted bipennate muscle structures. This approach has improved the quality of hypoxic threshold evaluation and aligned cell growth in 3D. The proposed method is useful in exploring the role of different factors in muscle tissue regeneration with limited resources.

3D bioprinted respiratory disease model: Exploring the importance of culture conditions and controlled release in modelling infection

The burden of respiratory illnesses is large, having a significant impact on healthcare systems worldwide. As researchers work to better understand chronic diseases as well as newly emerging respiratory viruses, the need for improved respiratory models has become evident. While three-dimensional (3D) bioprinting has been illustrated as a feasible method to create complex cellularized constructs or respiratory models, it has yet to be determined whether including relevant biomechanical stimulus and/or including relevant growth factors has a significant impact on the response of respiratory tissue models to infection. In this study, an alginate/gelatin/collagen solution was synthesized and characterized in terms of rheology, printability, degradation, mechanical properties, and biocompatibility. The bioink which incorporated primary human pulmonary fibroblasts and THP-1 cells was bioprinted to form hierarchical 3D constructs, which were then seeded with primary human bronchial epithelial cells to form the respiratory tissue model. To explore the importance of growth factors and culture conditions in modelling infection, we strategically developed a hepatocyte-growth-factor-loaded nanoparticle system and incorporated them into the bioink for bioprinting the respiratory tissue model and then cultured the model under dynamic conditions in a breath-mimicking bioreactor. The effect of including growth factors and dynamic culture conditions was examined over a 28-day period, followed by the infection of these constructs with influenza A virus. It was determined that these constructs support infection, demonstrating a more clinically relevant infection pattern than 2D models. It was further determined that inclusion of hepatocyte growth factor aids in epithelial cell growth, while inclusion of biomechanical stimulus increases cellular metabolism and has a moderating effect on response to infection.

Improved detection of mycobacteria in CF and tissue samples grown in mycobacteria growth indicator tube incubated at 30°C compared to conventional growth conditions of liquid and solid media.

Our study shows that the addition of mycobacteria growth indicator tube (MGIT) liquid culture incubated at 30°C improves the detection of mycobacteria from CF and tissue samples. MGIT incubated at 30°C recovered significantly more mycobacterial isolates than MGIT incubated at 37°C and significantly more isolates than either Lowenstein Jensen or Stonebrink solid media incubated at either 36°C or 30°C. Of 202 mycobacterial isolates recovered from 1,549 specimens, 170 were recovered from MGIT incubated at 30°C, followed by MGIT incubated at 37°C with 124 isolates and solid media culture conditions that recovered between 95 and 107 mycobacterial isolates. All conventional culture conditions combined without MGIT incubated at 30°C recovered 166 isolates. MGIT incubated at 30°C recovered the highest number of isolates detected exclusively by a single culture condition and recovered mycobacterial isolates of highly relevant mycobacterial species, including Mycobacterium abscessus and Mycobacterium tuberculosis.