Statistical optimization of environmental factors to produce the cytotoxic enniatins H, I and MK1688 against human multidrug resistance cancer cell lines.

The environmental conditions were optimized to produce the enniatin H, I, and MK1688 by Fusarium strain on cereal grain exhibiting anti-carcinogenic potential against MES-SA (human uterine sarcoma cell line), HCT15 (human colorectal carcinoma cancer cell line), and their multidrug resistance sublines. From the statistical optimization by response surface methodology, the optimal condition of independent variables affecting the response variables were 20.85 °C (temperature), 46.85% (w/w, initial moisture content), and 18.42 days (growth time) for ENN H; 23.31 °C, 44.15% (w/w) and 17.23 days for ENN I; 23.08 °C, 43.97% (w/w) and 17.06 days for ENN MK1688. In case of cytotoxic effects, ENNs significantly suppressed growth of cancer cell lines without multidrug resistance, and ENN I inhibited growth of cancer cell lines most strongly. These data will provide valuable point to produce the cyclic hexadepsipeptide exhibiting anti-carcinogenic potential from Fusarium strains.

Description and Characterization of the Odontella aurita OAOSH22, a Marine Diatom Rich in Eicosapentaenoic Acid and Fucoxanthin, Isolated from Osan Harbor, Korea.

Third-generation biomass production utilizing microalgae exhibits sustainable and environmentally friendly attributes, along with significant potential as a source of physiologically active compounds. However, the process of screening and localizing strains that are capable of producing high-value-added substances necessitates a significant amount of effort. In the present study, we have successfully isolated the indigenous marine diatom Odontella aurita OAOSH22 from the east coast of Korea. Afterwards, comprehensive analysis was conducted on its morphological, molecular, and biochemical characteristics. In addition, a series of experiments was conducted to analyze the effects of various environmental factors that should be considered during cultivation, such as water temperature, salinity, irradiance, and nutrients (particularly nitrate, silicate, phosphate, and iron). The morphological characteristics of the isolate were observed using optical and electron microscopes, and it exhibited features typical of O. aurita. Additionally, the molecular phylogenetic inference derived from the sequence of the small-subunit 18S rDNA confirmed the classification of the microalgal strain as O. aurita. This isolate has been confirmed to contain 7.1 mg g-1 dry cell weight (DCW) of fucoxanthin, a powerful antioxidant substance. In addition, this isolate contains 11.1 mg g-1 DCW of eicosapentaenoic acid (EPA), which is one of the nutritionally essential polyunsaturated fatty acids. Therefore, this indigenous isolate exhibits significant potential as a valuable source of bioactive substances for various bio-industrial applications.

Culture conditions in the IVF laboratory: state of the ART and possible new directions.

In the last four decades, the assisted reproductive technology (ART) field has witnessed advances, resulting in improving pregnancy rates and diminishing complications, in particular reduced incidence of multiple births. These improvements are secondary to advanced knowledge on embryonic physiology and metabolism, resulting in the ability to design new and improved culture conditions. Indeed, the incubator represents only a surrogate of the oviduct and uterus, and the culture conditions are only imitating the physiological environment of the female reproductive tract. In vivo, the embryo travels through a dynamic and changing environment from the oviduct to the uterus, while in vitro, the embryo is cultured in a static fashion. Importantly, while culture media play a critical role in optimising embryo development, a large host of additional factors are equally important. Additional potential variables, including but not limited to pH, temperature, osmolality, gas concentrations and light exposure need to be carefully controlled to prevent stress and permit optimal implantation potential. This manuscript will provide an overview of how different current culture conditions may affect oocyte and embryo viability with particular focus on human literature.

Evaluation and interpretation of cytogenetic test results based on biological relevance.

The evaluation and interpretation of cytogenetic test data are discussed from the perspective of biological relevance. The reliability of tests must be considered, before evaluation and interpretation. Statistical procedures are important for the evaluation of test data, but for human health risk assessment, biological relevance is essential. Cell culture conditions must be carefully considered. Cells must be healthy in the physiologically controlled culture medium. Osmolality, pH, and temperature are critical factors in keeping the culture medium physiologically normal and avoiding artifactual responses. Careful attention must be paid to the exposure of test chemicals to target cells, in both in vitro and in vivo tests. For in vivo tests, absorption, distribution, metabolism, and excretion are critical issues that affect the exposure of the target cells to the test chemical. The dose-response relationship and reproducibility are also critical factors in biological reliability. I also discuss why so many chemicals show positive results in in vitro cytogenetic assays.

Sodium-Ion-Free Fermentative Production of GABA with Levilactobacillus brevis CD0817.

Gamma-aminobutyric acid (GABA) has positive effects on many physiological processes. Lactic acid bacterial production of GABA is a future trend. This study aimed to produce a sodium-ion-free GABA fermentation process for Levilactobacillus brevis CD0817. In this fermentation, both the seed and fermentation media used L-glutamic acid instead of monosodium L-glutamate as the substrate. We optimized the key factors influencing GABA formation, adopting Erlenmeyer flask fermentation. The optimized values of the key factors of glucose, yeast extract, Tween 80, manganese ion, and fermentation temperature were 10 g/L, 35 g/L, 1.5 g/L, 0.2 mM, and 30 °C, respectively. Based on the optimized data, a sodium-ion-free GABA fermentation process was developed using a 10-L fermenter. During the fermentation, L-glutamic acid powder was continuously dissolved to supply substrate and to provide the acidic environment essential for GABA synthesis. The current bioprocess accumulated GABA at up to 331 ± 8.3 g/L after 48 h. The productivity of GABA was 6.9 g/L/h and the molar conversion rate of the substrate was 98.1%. These findings demonstrate that the proposed method is promising in the fermentative preparation of GABA by lactic acid bacteria.

[Suitability of Culture Broth and Conditions for Escherichia coli Growth and Gas Production as a Test for Food Additives in EC Broth].

The growth and gas production test for Escherichia coli in the microbiological examination of food additives is stipulated in the ninth edition of Japan's Specifications and Standards for Food Additives (JSFA) and described as a part of the "Confirmation Test for Escherichia coli" in "Microbial Limit Tests" in the same manuscript. The growth and gas production test for E. coli indicated that the positive or negative of "gas production and/or turbidity" in EC broth should be confirmed after incubating at 45.5±0.2℃ for 24±2 h. If both gas production and turbidity are negative, the culture is additionally incubated up to 48±2 h to determine E. coli contamination. The internationally referenced Bacteriological Analytical Manual of the U.S. FDA had revised the incubation temperature in tests for coliforms and E. coli from 45.5±0.2℃ to 44.5±0.2℃ in 2017. Therefore, we conducted research in anticipation of this temperature change being reflected in the microbiological examination of the JSFA. We used seven EC broth products and six food additives across eight products that are available in Japan in order to compare the growth and gas production at temperatures of 45.5±0.2℃ and 44.5±0.2℃ of E. coli NBRC 3972, which is designated as the test strain in JSFA. Both with/without food additives, the number of EC broth products in which medium turbidity and gas production by the strain were positive in three out of three tubes at all test times was greater at 44.5±0.2℃ than at 45.5±0.2℃. These results suggest that the growth and gas production test for E. coli could be more appropriately conducted by incubation at 44.5±0.2℃ in the "Confirmation Test for Escherichia coli" for E. coli in the JSFA in comparison to 45.5±0.2℃. Furthermore, there were differences in the growth and gas production of E. coli NBRC 3972 depending on the EC broth product used. Therefore, the importance of "Media growth promotion test" and "Method suitability test" in the ninth edition of the JSFA should be emphasized.

Construction and application of the genome-scale metabolic model of Streptomyces radiopugnans.

Geosmin is one of the most common earthy-musty odor compounds, which is mainly produced by Streptomyces. Streptomyces radiopugnans was screened in radiation-polluted soil, which has the potential to overproduce geosmin. However, due to the complex cellular metabolism and regulation mechanism, the phenotypes of S. radiopugnans were hard to investigate. A genome-scale metabolic model of S. radiopugnans named iZDZ767 was constructed. Model iZDZ767 involved 1,411 reactions, 1,399 metabolites, and 767 genes; its gene coverage was 14.1%. Model iZDZ767 could grow on 23 carbon sources and five nitrogen sources, which achieved 82.1% and 83.3% prediction accuracy, respectively. For the essential gene prediction, the accuracy was 97.6%. According to the simulation of model iZDZ767, D-glucose and urea were the best for geosmin fermentation. The culture condition optimization experiments proved that with D-glucose as the carbon source and urea as the nitrogen source (4 g/L), geosmin production could reach 581.6 ng/L. Using the OptForce algorithm, 29 genes were identified as the targets of metabolic engineering modification. With the help of model iZDZ767, the phenotypes of S. radiopugnans could be well resolved. The key targets for geosmin overproduction could also be identified efficiently.

Metabolic engineering of Escherichia coli for biological production of 1, 3-Butanediol.

The production of 1,3-butanediol (1,3-BDO) from glucose was investigated using Escherichia coli as the host organism. A pathway was engineered by overexpressing genes phaA (acetyl-CoA acetyltransferase), phaB (acetoacetyl-CoA reductase), bld (CoA-acylating aldehyde dehydrogenase), and yqhD (alcohol dehydrogenase). The expression levels of these genes were optimized to improve 1,3-BDO production and pathways that compete with 1,3-BDO synthesis were disrupted. Culture conditions were also optimized, including the C: N ratio, aeration, induction time, temperature, and supplementation of amino acids, resulting in a strain that could produce 1,3-BDO at 257 mM in 36 h, with a yield of 0.51 mol/mol in a fed-batch bioreactor experiment. To the best of our knowledge, this is the highest titer of 1,3-BDO production ever reported using biological methods, and our findings provide a promising strategy for the development of microbial cell factories for the sustainable synthesis of other acetyl-CoA-derived chemicals.

Single-cell RNA sequencing reveals homogeneous transcriptome patterns and low variance in a suspension CHO-K1 and an adherent HEK293FT cell line in culture conditions.

Recombinant mammalian host cell lines, in particular CHO and HEK293 cells, are used for the industrial production of therapeutic proteins. Despite their well-known genomic instability, the control mechanisms that enable cells to respond to changes in the environmental conditions are not yet fully understood, nor do we have a good understanding of the factors that lead to phenotypic shifts in long-term cultures. A contributing factor could be inherent diversity in transcriptomes within a population. In this study, we used a full-length coverage single-cell RNA sequencing (scRNA-seq) approach to investigate and compare cell-to-cell variability and the impact of standardized and homogenous culture conditions on the diversity of individual cell transcriptomes, comparing suspension CHO-K1 and adherent HEK293FT cells. Our data showed a critical batch effect from the sequencing of four 96-well plates of CHO-K1 single cells stored for different periods of time, which was and may be therefore identified as a technical variable to consider in experimental planning. Besides, in an artificial and controlled culture environment such as used in routine cell culture technology, the gene expression pattern of a given population does not reveal any marker gene capable to disclose relevant cell population substructures, both for CHO-K1 cells and for HEK293FT cells. The variation observed is primarily driven by the cell cycle.

Development of a lactic acid bacteria strain that suppresses chronic inflammation and improves glucose and lipid metabolism.

Chronic inflammation caused by aging, obesity, and lifestyle disturbances can lead to the production of inflammatory cytokines and insulin resistance, reducing glucose and lipid metabolism. Lactic acid bacteria (LAB) have various bioactivities, and certain types of LAB have been reported to exhibit anti-inflammatory effects. We hypothesized that LAB strains, which can strongly induce the production of anti-inflammatory cytokines by immune cells in the intestinal tract, may improve glucose and lipid metabolism by suppressing chronic inflammation. We selected Lactiplantibacillus plantarum OLL2712 (OLL2712) from the LAB library owned by Meiji Co., Ltd. based on its ability to induce the production of interleukin-10 (IL-10), optimized the culture conditions of OLL2712 for industrial applications, and verified the efficacy of the strain in animal and clinical studies. The results showed that OLL2712 bacterial cells in the exponential phase had notably higher anti-inflammatory properties than the cells in the stationary phase and led to the inhibition of chronic inflammation and improvement of glucose and lipid metabolism in animal studies. Two randomized controlled trials consisting of healthy adults with elevated blood glucose levels or body mass indices (BMIs) also showed that the intake of OLL2712 suppressed the aggravation of chronic inflammation and improved glucose and lipid metabolism. This review identified a novel LAB strain that may contribute to diabetes and obesity prevention and demonstrated its clinical efficacy. In addition, the mechanism of action of this LAB strain through the intestinal immune system was partially elucidated, and the importance of optimizing the culture conditions of LAB was clarified.

Optimized Rhodobacter sphaeroides for the Production of Antioxidants and the Pigments with Antioxidant Activity.

The photosynthetic bacterium, Rhodobacter sphaeroides, is a bacterium that can grow in a variety of environments and produces substances with antioxidant effects. In this study, we investigated the culture conditions to increase the production of antioxidants in R. sphaeroides, which can grow under both aerobic and anaerobic conditions. After incubation in the exponential phase and stationary phase under both conditions, a 2,2-diphenyl-1-picrylhydrazyl assay was used to confirm the antioxidant effect. Although the highest antioxidant effect was shown in the stationary phase under aerobic conditions, the antioxidant effect of each cell was found to be greater when cultured under anaerobic conditions. The antioxidant activity of R. sphaeroides was increased by sonication. In addition, the contents of carotenoids and bacteriochlorophyll, which are pigments with antioxidant effects, produced by R. sphaeroides were measured. We confirmed that the content of carotenoids was higher in anaerobic conditions than in aerobic conditions. However, when measuring the content of the bacterium, we found that there was more content in aerobic conditions. Therefore, we confirm that when grown in anaerobic conditions, the antioxidant effect of R. sphaeroides is higher, which suggests that this antioxidant effect comes from the effect of carotenoid.

Optimal culture conditions of Piriformospora indica for volatile compound release to promote effective plant growth.

Piriformospora indica, which is an endophytic fungus that grows on various media in the absence of a host, emits plant growth promoting volatile organic compounds (VOCs). Kaefer medium (KF) has been shown to be the most suitable medium for P. indica growth; however, different media may differentially affect fungal metabolism which may in turn influence the VOC profiles of P. indica. To date, how the VOCs emitted from P. indica cultured on different media affect plant growth has not been well characterized. Here, we show that poor nutrient medium (PNM) promoted the growth of P. indica more effectively than potato dextrose agar (PDA) or KF medium. By contrast, plant total biomass and root fresh weight were increased 1.8-fold and 2.1-fold, when co-cultivated with P. indica cultured on PDA medium in comparison with KF or PNM medium, respectively. Furthermore, sucrose in the plant culture medium downregulated the fold-induction ratio of the plant growth promoted by P. indica VOCs.

Fucoxanthin Production of Microalgae under Different Culture Factors: A Systematic Review.

Fucoxanthin is one of the light-harvesting pigments in brown microalgae, which is increasingly gaining attention due to its numerous health-promoting properties. Currently, the production of microalgal fucoxanthin is not yet feasible from an economic perspective. However, the cultivation of microalgae at favourable conditions holds great potential to increase the viability of this fucoxanthin source. Hence, this study aimed to review the fucoxanthin production of microalgae under different conditions systematically. A literature search was performed using the Web of Science, Scopus and PubMed databases. A total of 188 articles were downloaded and 28 articles were selected for the current review by two independent authors. Microalgae appeared to be a more reliable fucoxanthin source compared to macroalgae. Overall, a consensus fucoxanthin production condition was obtained and proposed: light intensity ranging from 10 to 100 µmol/m2/s could achieve a higher fucoxanthin content. However, the optimal light condition in producing fucoxanthin is species-specific. The current review serves as an antecedent by offering insights into the fucoxanthin-producing microalgae response to different culture factors via a systematic analysis. With the current findings and recommendations, the feasibility of producing fucoxanthin commercially could be enhanced and possibly achieve practical and sustainable fucoxanthin production.

Angiogenesis in Free-Standing Two-Vasculature-Embedded Scaffold Extruded by Two-Core Laminar Flow Device.

Rapid construction of pre-vascular structure is highly desired for engineered thick tissue. However, angiogenesis in free-standing scaffold has been rarely reported because of limitation in growth factor (GF) supply into the scaffold. This study, for the 1st time, investigated angiogenic sprouting in free-standing two-vasculature-embedded scaffold with three different culture conditions and additional GFs. A two-core laminar flow device continuously extruded one vascular channel with human umbilical vein endothelial cells (HUVECs) and a 3 mg/ml type-1 collagen, one hollow channel, and a shell layer with 2% w/v gelatin-alginate (70:30) composite. Under the GF flowing condition, angiogenic sprouting from the HUVEC vessel had started since day 1 and gradually grew toward the hollow channel on day 10. Due to the medium flowing, the HUVECs showed elongated spindle-like morphology homogeneously. Their viability has been over 80% up to day 10. This approach could apply to vascular investigation, and drug discovery further, not only to the engineered thick tissue.

New Insights Into Microbial Induced Calcium Carbonate Precipitation Using Saccharomyces cerevisiae.

Saccharomyces cerevisiae plays an important role in the mineralization of many metal ions, but it is unclear whether this fungus is involved in the mineralization of calcium carbonate. In this study, S. cerevisiae was cultured under various conditions to explore its ability to perform microbially induced calcium carbonate precipitation (MICP). Organic acids, yeast extract, and low-carbon conditions were the factors influencing the biomineralization of calcium carbonate caused by S. cerevisiae, and biomolecules secreted by the fungus under different conditions could change the morphology, size, and crystal form of the biosynthesized mineral. In addition, transcriptome analysis showed that the oxidation of organic acids enhanced the respiration process of yeast. This implied that S. cerevisiae played a role in the formation of calcium carbonate through the mechanism of creating an alkaline environment by the respiratory metabolism of organic acids, which could provide sufficient dissolved inorganic carbon for calcium carbonate formation. These results provide new insights into the role of S. cerevisiae in biomineralization and extend the potential applications of this fungus in the future.

Optimization of Culture Conditions for the Generation of Canine CD20-CAR-T Cells for Adoptive Immunotherapy.

BACKGROUND/AIM: Chimeric antigen receptor (CAR) T cell therapy targeting CD20 has the potential to become a promising novel treatment for canine B cell lymphoid malignancy. However, the optimal approach for producing potent CAR-T cells with favorable phenotype for dogs remains unknown. In this study, we assessed several culture conditions and their effects on the phenotypic characteristics of CD20-CAR-T cells. MATERIALS AND METHODS: Canine CAR-T cells were generated by incubating with several mitogens in the presence or absence of Akt inhibitor. Gene transduction efficiency and phenotypic characteristics were determined by flow cytometry. RESULTS: Comparison of several kinds of mitogens revealed that stimulation with phytohemagglutinin has high transduction efficacy, whereas stimulation with concanavalin A was superior in memory T cell formation. Akt inhibition at the initial stage of CAR-T production tended to enhance transduction efficiency and memory T cell formation. CONCLUSION: This study provides a significant insight into the understanding of the ex vivo expansion of canine T cells in adoptive immunotherapy.

Role of temperature, nutrition, oxygen, osmolality, and bacterial strain in inducing a viable but non-culturable state in Campylobacter jejuni.

Campylobacter jejuni is a globally important foodborne pathogen that can exist environmentally in a viable but non-culturable (VBNC) state, leading to missed detection of VBNC cells in food and false results in epidemiological surveillance. To establish a method for its resuscitation from the VBNC state and enable better detection, the mechanisms by which C. jejuni are induced into the VBNC state should be understood in detail. However, experimental induction of the VBNC state can be rather time consuming. Therefore, in this study, we investigated the effects of temperature, nutrition, oxygen, and osmolality, in an attempt to achieve a shorter induction time for VBNC state in C. jejuni. Culture at 4 °C under aerobic conditions in nutrient-rich Mueller-Hinton broth was the most effective condition for inducing the VBNC state in the C. jejuni strain JCM 2013. However, in C. jejuni strains 81-176 and B17, this condition induced the VBNC state slower than in the JCM 2013 strain, suggesting that the bacterial strain characteristics also affected the induction of VBNC state. These findings provide novel insights into the effects of various conditions on inducing the VBNC state in C. jejuni.

Culture Condition of Bone Marrow Stromal Cells Affects Quantity and Quality of the Extracellular Vesicles.

Extracellular vesicles (EVs) released by bone marrow stromal cells (BMSCs) have been shown to act as a transporter of bioactive molecules such as RNAs and proteins in the therapeutic actions of BMSCs in various diseases. Although EV therapy holds great promise to be a safer cell-free therapy overcoming issues related to cell therapy, manufacturing processes that offer scalable and reproducible EV production have not been established. Robust and scalable BMSC manufacturing methods have been shown to enhance EV production; however, the effects on EV quality remain less studied. Here, using human BMSCs isolated from nine healthy donors, we examined the effects of high-performance culture media that can rapidly expand BMSCs on EV production and quality in comparison with the conventional culture medium. We found significantly increased EV production from BMSCs cultured in the high-performance media without altering their multipotency and immunophenotypes. RNA sequencing revealed that RNA contents in EVs from high-performance media were significantly reduced with altered profiles of microRNA enriched in those related to cellular growth and proliferation in the pathway analysis. Given that pre-clinical studies at the laboratory scale often use the conventional medium, these findings could account for the discrepancy in outcomes between pre-clinical and clinical studies. Therefore, this study highlights the importance of selecting proper culture conditions for scalable and reproducible EV manufacturing.

Identification of a Cowden syndrome patient with a novel PTEN mutation and establishment of patient-derived induced pluripotent stem cells.

Cowden syndrome (CS) is an autosomal dominant inherited disorder characterized by multiple hamartomas in various organs such as the mucosa, skin, and gastrointestinal tract. Patients with CS are at high risk for breast and thyroid cancers. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor gene that negatively regulates the AKT pathway, and PTEN mutations are known to be the major causes of this syndrome. However, the pathogenesis of this syndrome has not been clarified. Here, we present a case of a Japanese woman with multiple oral polyps, breast cancer, and thyroid cancer who was clinically diagnosed with CS. We obtained DNA and RNA samples from the patient's peripheral blood mononuclear cells (PBMCs) and buccal mucosa tumor. Next-generation sequencing revealed novel germline mutations (c.1020delT and c.1026G > A) in exon 8 of PTEN. Sanger sequencing identified no PTEN transcript from the mutant allele. Furthermore, CS-specific induced pluripotent stem cells (CS-iPSCs) were established from PBMCs of the patient under feeder- and serum-free culture. Compared with healthy PBMCs and iPSCs, both of the CS-derived PBMCs and CS-iPSCs exhibited significantly reduced expression of the PTEN transcript. The transcriptional variant, PTENδ, was increased in CS-iPSCs, suggesting that it may be the cause of the disease.

Effective contamination control strategies facilitating axenic cultivation of Haematococcus pluvialis: Risks and challenges.

With industrialization, anthropogenic mishandlings have resulted in the discharge of abundant amount of CO2 into the atmosphere. This has triggered an unnatural warming that has dramatically increased the Earth's temperature in a short duration. This problem can be addressed by the biological conversion of CO2; several studies have been conducted using H. pluvialis culture that produces high value-added materials, such as astaxanthin and omega-3 fatty acids. However, although H. pluvialis has a high market value, the market size is quite small. Because H. pluvialis cells are susceptible to contamination due to its slow growth rate, hence large-scale culture of H. pluvialis without reliable contamination control strategies poses significant risks. This review comprehensively discusses the contamination that occurs during the culturing of H. pluvialis in various culture systems under different culture conditions. The review also discusses the strategies in controlling the biotic contaminants, such as bacteria and fungi.