Application of amphiregulin in IVM culture of immature human oocytes and pre-insemination culture for COCs in IVF cycles.

Background: Amphiregulin (AR) is a growth factor that resembles the epidermal growth factor (EGF) and serves various functions in different cells. However, no systematic studies or reports on the role of AR in human oocytes have currently been performed or reported. This study aimed to explore the role of AR in human immature oocytes during in vitro maturation (IVM) and in vitro fertilization (IVF) in achieving better embryonic development and to provide a basis for the development of a pre-insemination culture medium specific for cumulus oocyte complexes (COCs). Methods: First, we examined the concentration of AR in the follicular fluid (FF) of patients who underwent routine IVF and explored the correlation between AR levels and oocyte maturation and subsequent embryonic development. Second, AR was added to the IVM medium to culture immature oocytes and investigate whether AR could improve the effects of IVM. Finally, we pioneered the use of a fertilization medium supplemented with AR for the pre-insemination culture of COCs to explore whether the involvement of AR can promote the maturation and fertilization of IVF oocytes, as well as subsequent embryonic development. Results: A total of 609 FF samples were examined, and a positive correlation between AR levels and blastocyst formation was observed. In our IVM study, the development potential and IVM rate of immature oocytes, as well as the fertilization rate of IVM oocytes in the AR-added groups, were ameliorated significantly compared to the control group (All P < 0.05). Only the IVM-50 group had a significantly higher blastocyst formation rate than the control group (P < 0.05). In the final IVF study, the maturation, fertilization, high-quality embryo, blastocyst formation, and high-quality blastocyst rates of the AR-added group were significantly higher than those of the control group (All P < 0.05). Conclusion: AR levels in the FF positively correlated with blastocyst formation, and AR involvement in pre-insemination cultures of COCs can effectively improve laboratory outcomes in IVF. Furthermore, AR can directly promote the in vitro maturation and developmental potential of human immature oocytes at an optimal concentration of 50 ng/ml.

Endophytic Aspergillus fumigatiaffinis: Novel paclitaxel production and optimization insights.

This study investigated the potential of endophytic fungi to produce paclitaxel (Taxol®), a potent anticancer compound widely employed in chemotherapy. This research aimed to identify, confirm, and characterize endophytic fungi capable of paclitaxel (PTX) production and assess their paclitaxel yield. Additionally, it aimed to investigate factors influencing paclitaxel production. A total of 100 endophytic fungal isolates were collected and identified from the roots of Artemisia judaica. Aspergillus fumigatiaffinis exhibited the highest PTX production (26.373 µg L-1) among the isolated endophytic fungi. The strain was identified as A. fumigatiaffinis (Accession No. PP235788.1). Molecular identification confirmed its novelty, representing the first report of PTX production by A. fumigatiaffinis, an endophyte of Artemisia judaica. Optimization through full factorial design of experiments (DOE) and response surface methodology (RSM) significantly enhanced PTX production to 110.23 µg L-1 from 1 g of dry weight of the fungal culture under optimal conditions of pH 8.0, 150 µg L-1 becozyme supplementation, and 18 days of fermentation in potato dextrose broth. The presence of paclitaxel was confirmed using thin layer chromatography, high performance liquid chromatography, and gas chromatography-mass spectrometry. These findings maximize the role of endophytic fungus to produce a secondary metabolite that might be able to replace the chemically produced PTX and gives an opportunity to provide a sustainable source of PTX eco-friendly at high concentrations. KEY POINTS: • Endophytic fungi, like A. fumigatiaffinis, show promise for eco-friendly paclitaxel production • Optimization strategies boost paclitaxel yield significantly, reaching 110.23 µg L -1 • Molecular identification confirms novelty, offering a sustainable PTX source.

Polyphosphate Plays a Significant Role in the Maturation of Spores in Myxococcus xanthus.

Myxococcus xanthus synthesizes polyphosphates (polyPs) with polyphosphate kinase 1 (Ppk1) and degrades short- and long-chain polyPs with the exopolyphosphatases, Ppx1 and Ppx2, respectively. M. xanthus polyP:AMP phosphotransferase (Pap) generates ADP from AMP and polyPs. Pap expression is induced by an elevation in intracellular polyP concentration. M. xanthus synthesized polyPs during the stationary phase; the ppk1 mutant died earlier than the wild-type strain after the stationary phase. In addition, M. xanthus cells cultured in phosphate-starved medium, H2O2-supplemented medium, or amino acid-deficient medium increased the intracellular polyP levels by six- to ninefold after 6 h of incubation. However, the growth of ppk1 and ppx2 mutants in phosphate-starved medium and H2O2-supplemented medium was not significantly different from that of wild-type strain, nor was there a significant difference in fruiting body formation and sporulation in starvation condition. During development, no difference was observed in the adenylate energy charge (AEC) values in the wild-type, ppk1 mutant, and pap mutant strains until the second day of development. However, after day 3, the ppk1 and pap mutants had a lower ADP ratio and a higher AMP ratio compared to wild-type strain, and as a result, the AEC values of these mutants were lower than those of the wild-type strain. Spores of ppk1 and pap mutants in the nutrient medium germinated later than those of the wild-type strain. These results suggested that polyPs produced during development may play an important role in cellular energy homeostasis of the spores by being used to convert AMP to ADP via Pap.

Optimized Workflow from Gene to Product.

This chapter outlines the workflow using the ExpiSf™ Expression System designed for high-density infection of suspension ExpiSf9™ cells. The system utilizes a chemically defined, serum-free, protein-free, and animal origin free medium, making it suitable for recombinant protein expression experiments. The ExpiSf™ chemically defined medium allows efficient transfection and baculovirus production directly within the same culture medium. The ExpiSf™ Expression System Starter Kit provides all necessary components, including cells, culture medium, and reagents needed to infect one (1) liter of cell culture. The system's versatility and animal origin free nature make it a valuable tool for various protein expression studies and biotechnological applications.

Dynamic Energy Budget model for E. coli growth in carbon and nitrogen limitation conditions.

The simulations and predictions obtained from mathematical models of bioprocesses conducted by microorganisms are not overvalued. Mechanistic models are bringing a better process understanding and the possibility of simulating unmeasurable variables. The Dynamic Energy Budget (DEB) model is an energy balance that can be formulated for any living organism and can be classified as a structured model. In this study, the DEB model was used to describe E. coli growth in a batch reactor in carbon and nitrogen substrate limitation conditions. The DEB model provides a possibility to follow the changes in the microbes' cells including their elemental composition and content of some important cell ingredients in different growth phases in substrate limitation conditions which makes it more informative compared to Monod's model. The model can be used as an optimal choice between Monod-like models and flux-based approaches. KEY POINTS: • The DEB model can be used to catch changes in elemental composition of E. coli • Bacteria batch culture growth phases can be explained by the DEB model • The DEB model is more informative compared to Monod's based models.

Intestinal Epithelial Co-Culture Sensitivity to Pro-Inflammatory Stimuli and Polyphenols Is Medium-Independent.

The complexification of in vitro models requires the compatibility of cells with the same medium. Since immune cells are the most sensitive to growth conditions, growing intestinal epithelial cells in their usual medium seems to be necessary. This work was aimed at comparing the sensitivity of these epithelial cells to pro-inflammatory stimuli but also to dietary polyphenols in both DMEM and RPMI-1640 media. Co-cultures of Caco-2 and HT29-MTX cells were grown for 21 days in the two media before their stimulation with a cocktail of TNF-α (20 ng/mL), IL-1ß (1 ng/mL), and IFN-γ (10 ng/mL) or with LPS (10 ng/mL) from E. coli (O111:B4). The role of catechins (15 µM), a dietary polyphenol, was evaluated after its incubation with the cells before their stimulation for 6 h. The RPMI-1640 medium did not alter the intensity of the inflammatory response observed with the cytokines. By contrast, LPS failed to stimulate the co-culture in inserts regardless of the medium used. Lastly, catechins were unable to prevent the pro-inflammatory response observed with the cytokines in the two media. The preservation of the response of this model of intestinal epithelium in RPMI-1640 medium is promising when considering its complexification to evaluate the complex cellular crosstalk leading to intestinal homeostasis.

Selenium Discrepancies in Fetal Bovine Serum: Impact on Cellular Selenoprotein Expression.

Selenium is an essential trace element in our diet, crucial for the composition of human selenoproteins, which include 25 genes such as glutathione peroxidases and thioredoxin reductases. The regulation of the selenoproteome primarily hinges on the bioavailability of selenium, either from dietary sources or cell culture media. This selenium-dependent control follows a specific hierarchy, with "housekeeping" selenoproteins maintaining constant expression while "stress-regulated" counterparts respond to selenium level fluctuations. This study investigates the variability in fetal bovine serum (FBS) selenium concentrations among commercial batches and its effects on the expression of specific stress-related cellular selenoproteins. Despite the limitations of our study, which exclusively used HEK293 cells and focused on a subset of selenoproteins, our findings highlight the substantial impact of serum selenium levels on selenoprotein expression, particularly for GPX1 and GPX4. The luciferase reporter assay emerged as a sensitive and precise method for evaluating selenium levels in cell culture environments. While not exhaustive, this analysis provides valuable insights into selenium-mediated selenoprotein regulation, emphasizing the importance of serum composition in cellular responses and offering guidance for researchers in the selenoprotein field.

Effects of the MCF-7 Exhausted Medium on hADSC Behaviour.

Stem cells possess the ability to differentiate into different lineages and the ability to self-renew, thus representing an excellent tool for regenerative medicine. They can be isolated from different tissues, including the adipose tissue. Adipose tissue and human adipose-derived stem cells (hADSCs) are privileged candidates for regenerative medicine procedures or other plastic reconstructive surgeries. The cellular environment is able to influence the fate of stem cells residing in the tissue. In a previous study, we exposed hADSCs to an exhausted medium of a breast cancer cell line (MCF-7) recovered at different days (4, 7, and 10 days). In the same paper, we inferred that the medium was able to influence the behaviour of stem cells. Considering these results, in the present study, we evaluated the expression of the major genes related to adipogenic and osteogenic differentiation. To confirm the gene expression data, oil red and alizarin red colorimetric assays were performed. Lastly, we evaluated the expression of miRNAs influencing the differentiation process and the proliferation rate, maintaining a proliferative state. The data obtained confirmed that cells exposed to the medium maintained a stem and proliferative state that could lead to a risky proliferative phenotype.

Anti-Persisters Activity of Lacticaseibacillus rhamnosus Culture Filtrates against Pseudomonas aeruginosa in Artificial Sputum Medium.

Persisters are antibiotic-tolerant bacteria, playing a role in the recalcitrance and relapse of many bacterial infections, including P. aeruginosa pulmonary infections in Cystic Fibrosis (CF) patients. Among novel antimicrobial strategies, the use of probiotics and their products is emerging as a particularly promising approach. The aim of this study was to evaluate the anti-persisters activity of culture filtrate supernatants of Lacticaseibacillus rhamnosus (LRM-CFS) against P. aeruginosa in artificial sputum medium (ASM), which resembles the CF lung environment. Planktonic persisters of two clinical strains of P. aeruginosa (PaCF1 and PaCF4) were obtained following two different procedures: (i) exposing stationary-phase cultures to cyanide m-chlorophenylhydrazone (CCCP) in LB medium; (ii) incubating stationary-phase cultures with high doses of tobramycin (128-fold MIC) in ASM. In addition, persisters from biofilm were obtained by exposing 48 h old biofilm of P. aeruginosa to 128 x MIC of ciprofloxacin. LRM-CFS at dilutions of 1:6 and 1:4 resulted in being bactericidal in ASM against both PaCF1 and PaCF4 persisters obtained after CCCP or tobramycin treatment. Moreover, LRM-CFS at dilution 1:4 caused a reduction of antibiotic-tolerant bacteria in the biofilm of both P. aeruginosa strains. Overall, LRM-CFS represents a promising adjuvant therapeutic strategy against P. aeruginosa recalcitrant infections in CF patients.

Methods for Cultivation of Bacteroides thetaiotaomicron and Analysis of Heme Metabolism by Mass Spectrometry and Spectroscopic Approaches.

Traditional studies of cellular metabolism have relied on the use of radioisotopes. These have clear disadvantages associated with safety and waste generation. Furthermore, detection of the labeled species by scintillation counting provides only a quantification of its presence or absence. The use of stable isotopes, by contrast, allows the application of powerful, orthogonal spectroscopic approaches such as nuclear magnetic resonance spectroscopy (NMR) and various mass spectrometric methods. Using stable isotope labeling to study heme metabolism requires integrating methods for (a) generating the heme in labeled forms, (b) cultivating and quantifying the organism of choice in chemically defined media, to which labeled compounds can be added, (c) recovering cellular components and/or spent growth media, and (d) analyzing these materials for the labeled species using spectroscopic and mass spectrometric methods. These methods are summarized here in the context of Bacteroides thetaiotaomicron, a generally nonpathogenic anaerobe and heme auxotroph.

A Critical Role of Culture Medium Selection in Maximizing the Purity and Expansion of Natural Killer Cells.

Natural killer (NK) cells hold promise in cancer treatment due to their ability to spontaneously lyse cancer cells. For clinical use, high quantities of pure, functional NK cells are necessary. Combining adherence-based isolation with specialized media showed the unreliability of the isolation method, but demonstrated the superiority of the NK MACS® medium, particularly in suboptimal conditions. Neither human pooled serum, fetal calf serum (FCS), human platelet lysate, nor chemically defined serum replacement could substitute human AB serum. Interleukin (IL-)2, IL-15, IL-21, and combined CD2/NKp46 stimulation were assessed. IL-21 and CD2/NKp46 stimulation increased cytotoxicity, but reduced NK cell proliferation. IL-15 stimulation alone achieved the highest proliferation, but the more affordable IL-2 performed similarly. The RosetteSep™ human NK cell enrichment kit was effective for isolation, but the presence of peripheral blood mononuclear cells (PBMCs) in the culture enhanced NK cell proliferation, despite similar expression levels of CD16, NKp46, NKG2D, and ICAM-1. In line with this, purified NK cells cultured in NK MACS® medium with human AB serum and IL-2 demonstrated high cytotoxicity against primary glioblastoma stem cells.

Comparative genomic analysis and optimization of astaxanthin production of Rhodotorula paludigena TL35-5 and Rhodotorula sampaioana PL61-2.

Astaxanthin is a powerful antioxidant known to enhance skin, cardiovascular, eye, and brain health. In this study, the genome insights and astaxanthin production of two newly isolated astaxanthin-producing yeasts (TL35-5 and PL61-2) were evaluated and compared. Based on their phenotypic and genotypic characteristics, TL35-5 and PL61-2 were identified as basidiomycetous yeasts belonging to Rhodotorula paludigena and Rhodotorula sampaioana, respectively. To optimize astaxanthin production, the effects of cultural medium composition and cultivation conditions were examined. The optimal conditions for astaxanthin production in R. paludigena TL35-5 involved cultivation in AP medium containing 10 g/L glucose as the sole carbon source, supplemented with 1.92 g/L potassium nitrate, pH 6.5, and incubation at 20°C for 3 days with shaking at 200 rpm. For R. sampaioana PL61-2, the optimal medium composition for astaxanthin production consisted of AP medium with 40 g/L glucose, supplemented with 0.67 g/L urea, pH 7.5, and the fermentation was carried out at 20°C for 3 days with agitating at 200 rpm. Under their optimal conditions, R. paludigena TL35-5 and R. sampaioana PL61-2 gave the highest astaxanthin yields of 3.689 ± 0.031 and 4.680 ± 0.019 mg/L, respectively. The genome of TL35-5 was 20,982,417 bp in length, with a GC content of 64.20%. A total of 6,789 protein-encoding genes were predicted. Similarly, the genome of PL61-2 was 21,374,169 bp long, with a GC content of 64.88%. It contained 6,802 predicted protein-encoding genes. Furthermore, all essential genes involved in astaxanthin biosynthesis, including CrtE, CrtYB, CrtI, CrtS, and CrtR, were identified in both R. paludigena TL35-5 and R. sampaioana PL61-2, providing evidence for their ability to produce astaxanthin.

Creation of rice doubled haploids with low amylose content using in vitro anther culture.

In vitro androgenesis is a unique model for producing homozygous doubled haploid plants. The use of haploid biotechnology accelerates to obtain of doubled haploid plants, which is very important in rice breeding. The purpose of this work is to improve the production of doubled haploids in rice anther culture in vitro and selection of doubled haploid plants with valuable traits. The study the influence of nutrient media on the production of calli and plant regeneration processes in anther culture of 35 rice genotypes was revealed a significant influence of nutrient media on callus production. It was shown that the addition to culture medium phytohormones ratio with high level of cytokinin (5.0 mg/L BAP) and a low level of auxin (0.5 mg/L NAA), supplemented with amino acid composition promotes high production of green regenerated plants (68.75%) compared to albino plants (31.25%). As a result, doubled haploid lines of the glutinous variety Violetta were selected, which characterized by a low amylose content variation (from 1.86 to 2.80%). These doubled haploids are superior to the original variety in some yield traits and represent valuable breeding material.

Enrichment culture evaluation and characterization of Streptococcus agalactiae among pregnant women in Japan.

Introduction. Maternal screening tests and prophylactic antibiotics are important to prevent neonatal and infant group B streptococcal (GBS) infections.Hypothesis/Gap Statement. The performance of enrichment broth media for GBS screening that are available in Japan is unclear. Whole-genome data of GBS isolates from pregnant women in Japan is lacking.Aim. The aim of this study was to compare the protocol performance of six enrichment broths and two subculture agar plates, which were all available in Japan, for GBS detection. In addition, we showed whole-genome data of GBS isolates from pregnant women in Japan.Methodology. We collected 133 vaginal-rectal swabs from pregnant women visiting clinics and hospitals in Nagasaki Prefecture, Japan, and compared the protocol performance of 6 enrichment broths and 2 subculture agar plates. All GBS isolates collected in this study were subjected to whole-genome sequencing analysis.Results. We obtained 133 vaginal-rectal swabs from pregnant women at 35-37 weeks of gestation from 8 private clinics and 2 local municipal hospitals within Nagasaki Prefecture, Japan. The detection rate of the protocol involving the six enrichment broths and subsequent subcultures varied between 95.5 and 100 %, depending on the specific choice of enrichment broth. The GBS carriage rate among pregnant women in this region was 18.8 %. All 25 isolates derived from the swabs were susceptible to penicillin, whereas 48 and 36 % of the isolates demonstrated resistance to erythromycin and clindamycin, respectively. The distribution of serotypes was highly diverse, encompassing seven distinct serotypes among the isolates, with the predominant serotype being serotype V (n = 8). Serotype V isolates displayed a tendency towards increased resistance to erythromycin and clindamycin, with all resistant isolates containing the ermB gene.Conclusion. There was no difference in performance among the culture protocols evaluated in this study. GBS strains isolated from pregnant women appeared to have greater genomic diversity than GBS strains detected in neonates/infants with invasive GBS infections. To confirm this result, further studies with larger sample sizes are needed.

Using Mathematical Optimization Models to Improve the Efficiency of Duckweeds (Wolffia arrhiza and Lemna minor) Micropropagation.

The method of plant micropropagation is widely used to obtain genetically homogeneous and infection-free plants for the needs of various industries and agriculture. Optimization of plant growth and development conditions plays a key role in economically successful micropropagation. Computer technologies have provided researchers with new approaches for modeling and a better understanding of the role of the factors involved in plant growth in vitro. To develop new models for optimizing growth conditions, we used plants with a high speed of vegetative in vitro reproduction, such as duckweed (Wolffia arrhiza and Lemna minor). Using the development of the optimal modeling of the biological processes, we have obtained the prescriptions for an individually balanced culture medium that enabled us to obtain 1.5-2.0 times more duckweed biomass with a 1.5 times higher protein concentration in the dry mass. Thus, we have demonstrated that the method of optimization modeling of the biological processes based on solving multinomial tasks from the series of quadratic equations can be used for the optimization of trophic needs of plants, specifically for micropropagation of duckweeds in vitro.

Plant Micropropagation and Temporary Immersion Systems.

Temporary immersion systems (TIS) have been widely recognized as a promising technology for micropropagation of various plant species. The TIS provides a suitable environment for culture and allows intermittent contact of the explant with the culture medium at different immersion frequencies and aeration of the culture in each cycle. The frequency or immersion is one of the most critical parameters for the efficiency of these systems. The design, media volume, and container capacity substantially improve cultivation efficiency. Different TIS have been developed and successfully applied to micropropagation in various in vitro systems, such as sprout proliferation, microcuttings, and somatic embryos. TIS increases multiplication and conversion rates to plants and a better response during the ex vitro acclimatization phase. This article covers the use of different immersion systems and their applications in plant biotechnology, particularly in plant tissue culture, as well as its use in the massive propagation of plants of agroeconomic interest.

Micropropagation of Agave angustifolia "Espadín" Used in the Production of Mezcal.

This chapter presents the methodological approach for the in vitro propagation of Agave angustifolia "espadin," the base material to produce mezcal. The protocol used in each stage of the crop is addressed in detail, considering the changes in the culture medium and the characteristics of the plant material at each stage. The importance of integrated management between the multiplication and growth phase, as part of the in vitro selection strategy, is mentioned.

In Vitro Propagation of Threatened Agave Species Using a Two-Stage System.

Agaves are plants with multiple possibilities of use and are naturally tolerant to low water availability conditions and high temperatures. This makes them species of great interest in the context of the necessary substitution of crops due to climate change. Unfortunately, the overexploitation of wild specimens has endangered many species of the genus that have not been domesticated or cultivated intensively. In vitro mass culture and propagation techniques have emerged as a very efficient option to produce agave plants that can be used without damage to the natural populations. A protocol is presented here for the in vitro micropropagation of agaves in a two-stage process. In the first step, clusters of slightly differentiated shoots are generated from stem segments cultivated on a semisolid medium added with cytokinin. In a second step, these shoot clusters are cultured in temporary immersion bioreactors where they grow and complete their differentiation, and then the shoots are rooted and transferred to soil. This protocol has been successfully applied to several threatened species of the Agave genus.

In Vitro Propagation of "Salsaparrilha" [Herreria salsaparrilha Mart. (Asparagaceae)]: An Antisyphilitic Medicinal Species.

An efficient procedure for in vitro propagation of Herreria salsaparrilha Martius was established from single-node explants (fourth and fifth nodes from apex to the base) derived from donor plants maintained under shading-house conditions. After surface sterilization, explants are inoculated in test tubes containing 15 mL of Murashige and Skoog (MS) medium without growth regulators. Cultures are maintained under 35 µmol m-2 s-1 irradiance, a 16/8-h light/dark light regime, at 26 ± 2 °C. The subcultures are carried out under the same conditions, adding 6-benzyladenine 1.0 mg/L and Phytagel® 2.8 g/L. Shoots are elongated and rooted by transferring individual shoots to half-strength MS medium without growth regulators. After 25-30 days, elongated rooted shoots are transferred to plastic pots containing 25-30 mL of sterile distilled water, covered with a transparent plastic bag, and kept under the same growth room conditions for 2 days. Plants are transferred to cups containing autoclaved and washed sand and kept in a shading house (50% light interception) for acclimatization. True-to-type adult plants were successfully recovered under ex vitro conditions.

Protocol for Generating Arabidopsis thaliana Cell Suspension Cultures Under Different Light Conditions.

Plant cell suspension cultures (PCSCs) are in vitro-cultured cells that can divide indefinitely in a sterile growth medium. These PCSCs can be derived from various plant tissues, such as the root, stem, leaves, or seeds, and are maintained in a suitable culture medium containing nutrients, vitamins, hormones, and other essential components necessary for their growth. PCSCs have extensive applications in biotechnology, particularly in producing pharmaceutical and chemical compounds. This chapter presents a protocol for generating cell lines from Arabidopsis thaliana root callus under different light conditions, which can be used to investigate the effects of light on plant cell growth and development. The protocol described in this chapter is a valuable tool for researchers interested in utilizing PCSCs in their studies.