Supplementation Of Culture Medium Research Articles

Perspective: Can early embryonic losses be reduced in lactating dairy cows?

Perspective: Can early embryonic losses be reduced in lactating dairy cows?

Peptide extract from spent yeast improves resistance of Saccharomyces cerevisiae to oxidative stress

Yeast cells face various stress factors during industrial fermentations, since they are exposed to harsh environmental conditions, which may impair biomolecules productivity and yield. In this work, the use of an antioxidant peptide extract obtained from industrial spent yeast was explored as supplement for Saccharomyces cerevisiae fermentation to prevent a common bottleneck: oxidative stress. For that, a recombinant yeast strain, producer of β-farnesene, was firstly incubated with 0.5 and 0.7 g/L peptide extract, in the presence and absence of hydrogen peroxide (an oxidative stress inducer), for 1–5 h, and then assayed for intracellular reactive oxygen species, and growth ability in agar spot assays. Results showed that under 2 mM H2O2, the peptide extract could improve cells growth and reduce reactive oxygen species production. Therefore, this antioxidant effect was further evaluated in shake-flasks and 2-L bioreactor batch fermentations. Peptide extract (0.7 g/L) was able to increase yeast resistance to the oxidative stress promoted by 2 mM H2O2, by reducing reactive oxygen species levels between 1.2- and 1.7-fold in bioreactor and between 1.2- and 3-fold in shake-flask fermentations. Moreover, improvements on yeast cell density of up to 1.5-fold and 2-fold, and on biomolecule concentration of up to 1.6-fold and 2.8-fold, in bioreactor and shake-flasks, respectively, were obtained. Thus, culture medium supplementation with antioxidant peptide extracted from industrial spent yeast is a promising strategy to improve fermentation performance while valuing biomass waste. This valorization can promote a sustainable and eco-friendly solution for the biotechnology industry by the implementation of a circular economy model.Key points• Peptide extract from spent yeast applied for the first time on yeast fermentation.• Antioxidant peptide extract enhanced S. cerevisiae oxidative stress resistance.• Fermentation performance under stress improved by peptide extract supplementation.

Strategies for controlling afucosylation in monoclonal antibodies during upstream manufacturing.

Core fucosylation is a highly prevalent and significant feature of N-glycosylation in therapeutic monoclonal antibodies produced by mammalian cells where its absence (afucosylation) plays a key role in treatment safety and efficacy. Notably, even slight changes in the level of afucosylation can have a considerable impact on the antibody-dependent cell-mediated cytotoxicity. Therefore, implementing control over afucosylation levels is important in upstream manufacturing to maintain consistent quality across batches of product, since standard downstream processing does not change afucosylation. In this review, the influences and strategies to control afucosylation are presented. In particular, there is emphasis on upstream manufacturing culture parameters and media supplementation, as these offer particular advantages as control strategies over alternative approaches such as cell line engineering and chemical inhibitors. The review discusses the relationship between the afucosylation influences and the underlying cellular metabolism to promote increased process understanding. Also, briefly highlighted is the value of empirical and mechanistic models in evaluating and designing control methods for core fucosylation.

WNT Co-Receptor LRP6 Is Critical for Zygotic Genome Activation and Embryonic Developmental Potential by Interacting with Oviductal Paracrine Ligand WNT2.

Mammalian preimplantation development depends on the interaction between embryonic autocrine and maternal paracrine signaling. Despite the robust independence of preimplantation embryos, oviductal factors are thought to be critical to pregnancy success. However, how oviductal factors regulate embryonic development and the underlying mechanism remain unknown. In the present study, focusing on WNT signaling, which has been reported to be essential for developmental reprogramming after fertilization, we analyzed the receptor-ligand repertoire of preimplantation embryonic WNT signaling, and identified that the WNT co-receptor LRP6 is necessary for early cleavage and has a prolonged effect on preimplantation development. LRP6 inhibition significantly impeded zygotic genome activation and disrupted relevant epigenetic reprogramming. Focusing on the potential oviductal WNT ligands, we found WNT2 as the candidate interacting with embryonic LRP6. More importantly, we found that WNT2 supplementation in culture medium significantly promoted zygotic genome activation (ZGA) and improved blastocyst formation and quality following in vitro fertilization (IVF). In addition, WNT2 supplementation significantly improved implantation rate and pregnancy outcomes following embryo transfer. Collectively, our findings not only provide novel insight into how maternal factors regulate preimplantation development through maternal-embryonic communication, but they also propose a promising strategy for improving current IVF systems.

In Vitro Migration of Porcine Adipose-Derived Stem Cells in Platelet-Rich Plasma

The proliferation and migration of porcine adipose-derived stem cells (ASCs) have only begun to be studied in detail in vitro. The primary aim of these studies was to analyse in vitro ASC migration in varying concentrations of growth factors derived from the platelet-rich fraction of centrifuged plasma, the so-called platelet-rich plasma (PRP). ASCs were hypothesized to migrate at a faster velocity with increasing PRP concentrations. Whole blood was collected with a sodium citrate anticoagulant and subjected to two centrifugations. The first slower spin was to remove red blood cells to collect plasma, and the second faster spin was to collect the fraction with a high platelet concentration. Platelet concentration increased 3.5-fold compared to plasma, within the prescribed therapeutic range. For long-term culture of ASCs, DMEM supplemented with no more than 20% was necessary to maintain cell viability. ASC proliferation rate in PRP-supplemented media was comparable to that in fetal bovine serum (FBS), a standard cell culture media supplement. In addition, the velocity of ASC migration increased in cultures supplemented with 10%, 20% or 30% PRP. Generally, PRP was determined to be a media supplement with similar effects as FBS, potentially making it a suitable substitute for in vitro expansion of ASC populations. These results are likely partly explained by similarities in growth factor concentrations and their effects. Further characterization of PRP will be necessary to tease out the specific growth factor(s) responsible for the increase in swine ASC migration.

Optimal culture medium selection and supplementation for recombinant thaumatin II production by Komagataella phaffii

Sweet protein thaumatin, a promising alternative to table sugar, can overcome several drawbacks imposed by other sugar alternatives in the market today. Recombinant technology involving Komagataella phaffii (previously known as Pichia pastoris) has the potential to facilitate a uniform and large-scale production of this protein. To ensure maximal yield from the bioprocess, this study performed a screening to identify the media composition that exhibits the highest secretion yield of recombinant thaumatin II and favourable growth of the host organism. To achieve this, the most recommended media formulations for K. phaffii namely, BSM, FM22, MGY, BMGY, MBSM and d’Anjou media were investigated. It was identified from this study that the highest yields of recombinant thaumatin II were achieved from BMGY, FM22 and BSM media, irrespective of not exhibiting the highest cell growth. Despite exhibiting a lower cell density than FM22, the BMGY medium resulted in 62.79 mg/L of recombinant thaumatin II, which is 1–2 times higher than the former. This observation facilitated an understanding that the optimal medium for cell growth is not necessarily optimal for recombinant protein production. Moreover, a comparison between baffled and non-baffled flasks demonstrated that the former leads to a higher secretion yield, thereby indicating the importance of sufficient oxygen transfer into the medium. Furthermore, casamino acids and vitamins were supplemented to the media, where the latter contributed to an increased yield of thaumatin. As such, the highest protein yield of 68.60 mg/L was achieved for BMGY supplemented with vitamins.

Insulin-like growth factor-1 (IGF-1) selectively modulates the metabolic and lipid profile of bovine embryos according to their kinetics of development

Supplementation of culture media with IGF-1 during in vitro culture of embryos has had controversial results over the years. In the present study, we show that differences previously observed in response to IGF addition might be related to intrinsic heterogeneity of the embryos. In other words, the effects exerted by IGF-1 are dependent on the characteristics of the embryos and their ability to modulate metabolism and overcome stressful conditions, such as the ones found in a non-optimized in vitro culture system. To test this hypothesis, in vitro produced bovine embryos with distinct morphokinetics (fast- and slow-cleavage) were submitted to treatment with IGF-1 and then evaluated for embryo production rates, total cell number, gene expression and lipid profile. Our results show that remarkable differences were found when fast and slow embryos treated with IGF-1 were compared. Fast embryos respond by upregulating genes related to mitochondrial function, stress response, and lipid metabolism, whereas slow embryos presented lower mitochondrial efficiency and lipid accumulation. We conclude that indeed the treatment with IGF-1 selectively affects embryonic metabolism according to early morphokinetics phenotypes, and this information is relevant for decision-making in the design of more appropriate in vitro culture systems.

Medium supplementation with human, but not fetal calf serum facilitates endocytosis of PLGA nanoparticles by human primary B-lymphocytes via complement opsonization

The “biological identity” of nanoparticles (NPs) is governed by a shell consisting of various biomolecules that is formed upon exposure to biological media, the so-called biomolecule corona. Consequently, supplementation of cell culture media with e.g. different sera is likely to affect interactions between cells and NPs ex-vivo, especially endocytosis. We aimed to investigate the differential impact of human and fetal-bovine serum on the endocytosis of poly (lactic-co-glycolic acid) NPs by human peripheral blood mononuclear cells via flow cytometry. Furthermore, we employed different methods to inhibit endocytosis, providing mechanistic insights. The resulting biomolecule corona was characterized via denaturing gel electrophoresis. We found profound differences between human and fetal bovine serum regarding the endocytosis of fluorescently labeled PLGA nanoparticles by different classes of human leukocytes. Uptake by B-lymphocytes was particularly sensitive. We further present evidence, that these effects are mediated by a biomolecule corona. We demonstrate to our knowledge for the first time that the complement is an important contributor to the endocytosis of non-surface-engineered PLGA-nanoparticles prepared via emulsion solvent evaporation by human immune cells. Our data demonstrates that results obtained with xenogeneic culture supplements such as fetal bovine serum may have to be interpreted with caution.

Enhancement of Developmental Competence of Immature Cattle Oocytes with Leukemia inhibitory Factor as a Culture Media Supplement

Background: Leukemia Inhibitory Factor (LIF) plays an essential role in oocyte maturation and during early embryonic development. The present study aims to improve the in vitro cattle embryo production by supplementing culture media with LIF. Methods: Fresh ovaries and oviducts were collected from an abattoir in 0.9% saline (30-35°C) supplemented with antibiotics. Total 542 cumulus-oocyte complexes were aspirated from ovaries and cultured in maturation media in 5% CO2 incubator at 38.5°C with maximum humidity after 5-6 times washing. After 24 h matured oocytes were co-incubated with in vitro capacitated sperms in FBO medium. After 15-18 h cumulus cells were stripped off and presumptive zygotes were cultured in mCR2aa medium. After 40 to 42 h, cleavage was observed and embryos were cultured for 7-9 days. Culture media used to replace with fresh media after every 24 h. LIF was supplemented with 15, 30, 45 ng/ml. Result: Supplementation of LIF in culture media increased maturation rate, cleavage rate significantly (P less than 0.05). LIF Supplementation @ 30 ng/ml during culture increased blastocyst development (in control group 7.0±1.3 and 7.1±1.2, 12.9±0.4, 10.2±1.5 in 15 ng/ml, 30 ng/ml and 45 ng/ml respectively) significantly.

Supplementation of culture medium with quercetin improves mouse blastocyst quality and increases the expression of HIF-1α protein.

Regarding the low number of embryos that reach the blastocyst stage when cultured in vitro, this study aimed to evaluate the effects of quercetin on pre-implantation mouse (Mus musculus) embryos obtained using in vitro fertilization, especially during the passage from morula to blastocyst. Furthermore, we studied whether quercetin also affected the expression of hypoxia-inducible factor 1α (HIF-1α). The culture medium for the embryos was supplemented with quercetin, for long or short periods of time, and then the development potential, total cell number, apoptosis rates and expression of HIF-1α were studied to determine the effect of quercetin. Embryos failed to develop when cultured for long periods of time with quercetin, implying the possible toxic effects of this, alternatively antioxidant, compound. However, a short culture from morula to blastocyst significantly improved the development potential of in vitro produced embryos, increasing the final total cell number and reducing the apoptosis rate, observing similar results to those embryos cultured in low-oxygen concentrations or developed in utero. Furthermore, in embryos treated with quercetin for 2 or 4 h we found an increase in HIF-1α compared with untreated embryos. This work could imply a way to use quercetin in fertility clinics to improve the production of healthy blastocysts and, consequently, increase the success rates in assisted reproduction techniques.

Immunomodulatory effects of graded levels of docosahexaenoic acid (DHA) in common carp (Cyprinus carpio) – In vitro and in vivo approaches

The D-series resolvins formed from docosahexaenoic acid (DHA) work as anti-inflammatory mediators indicating the role of this long-chain polyunsaturated fatty acid in the immune system of mammals. However, such information is still limited in fish. The current study was conducted to evaluate the immunomodulatory effects of graded levels of DHA in common carp Cyprinus carpio by in vitro and in vivo approaches. In the in vitro experiment, the head kidney leukocytes isolated from common carp (body weight = 120.3 ± 12.4 g) were exposed to DHA at 0, 15, and 75 μM (corresponding to DHA0, DHA15, and DHA75) for 1 h; the cells were then immediately exposed to lipopolysaccharides (LPS) at a dose of 10 μg/ml for 4 or 24 h to stimulate the pro-inflammatory responses. The expression of several target genes involved in the inflammatory response (tlr4, nfkb, il-1, il-6, pge2, pla2, nf-kbi, il-10, and tgf-β1) and cytoprotection (hsp70, gpx1, and prdx3) was then assessed by RT-qPCR. Results showed that the pro-inflammatory response induced by LPS was confirmed through the upregulations of il-1 and il-6 expressions in the DHA0 group after 4 h of LPS exposure. The downregulations of il-6 in DHA15 and DHA75 cells after 4 h of LPS exposure compared to DHA0 indicated that the free DHA supplementation in the cell culture medium induced an anti-inflammatory response. Decreases of il-10 and nf-kbi expression were also observed in DHA-treated groups and the highest expression of hsp70 in DHA75 cells. In the in vivo experiment, common carp juveniles (21.7 ± 0.9 g) were fed to apparent satiation with a diet supplemented with DHA at 0, 6, and 20 g/kg for 8 weeks. After the feeding trial, the fish were challenged with Aeromonas veronii at 2.1 × 107 CFU/ml and the fish mortality was then recorded during 14 days. At the end of the feeding trial and the second day of bacterial infection, fish blood samples were collected for haematological parameters while liver and head kidney were used for assaying different immune variables. Results showed that the DHA supplementation in fish diet did not influence the fish growth and other husbandry parameters. The lowest value of fish mortality was recorded in DHA20-fed fish. The positive effects of DHA-supplemented diets were also found in myeloperoxidase (MPO), glutathione (GSH), and catalase (CAT) activities. In conclusion, the results suggest that DHA is able to modulate the inflammatory response in the immune celsl at a dose of 75 μM/mL and to boost disease resistance in common carp fed on a diet supplemented with DHA at 20 g/kg of feed.

Serine metabolism contributes to cell survival by regulating extracellular pH and providing an energy source in Saccharomyces cerevisiae.

Changes in extracellular pH affect the homeostasis and survival of unicellular organisms. Supplementation of culture media with amino acids can extend the lifespan of budding yeast, Saccharomyces cerevisiae, by alleviating the decrease in pH. However, the optimal amino acids to use to achieve this end, and the underlying mechanisms involved, remain unclear. Here, we describe the specific role of serine metabolism in the regulation of pH in a medium. The addition of serine to synthetic minimal medium suppressed acidification, and at higher doses increased the pH. CHA1, which encodes a catabolic serine hydratase that degrades serine into ammonium and pyruvate, is essential for serine-mediated alleviation of acidification. Moreover, serine metabolism supports extra growth after glucose depletion. Therefore, medium supplementation with serine can play a prominent role in the batch culture of budding yeast, controlling extracellular pH through catabolism into ammonium and acting as an energy source after glucose exhaustion.

Different concentrations of fetal bovine serum affect cytokine modulation in Lipopolysaccharide-activated apical papilla cells in vitro.

Fetal bovine serum (FBS) is the most used supplement in culture media; however, it may interfere with in vitro assays via effects on cell proliferation and cytokine production. The ideal FBS concentration for assays using apical papilla cells (APCs) remains unknown. Therefore, this study aimed to evaluate the effects of FBS on APC activation, cell viability/proliferation, and cytokine production. Human APCs were cultured, plated, and maintained in media containing increasing concentrations of FBS for 24 h, 48 h, 72 h, 7 days, and 14 days in the presence of Lipopolysaccharide (LPS - 1 µg/mL). At each time point, the cells were subjected to the MTT assay. The cytokines transforming growth factor (TGF)-β1, osteoprotegerin (OPG), and interleukin (IL)-6, along with the chemokine CCL2, were quantified using the enzyme-linked immunosorbent assay at the 24-h time-point. Statistical analysis was performed using two-way analysis of variance (ANOVA) followed by Tukey's post-hoc test (p<0.05). In general, APCs exhibited increasing metabolic activity in an FBS concentration-dependent fashion, regardless of the presence of LPS. In contrast, FBS interfered with the production of all the cytokines evaluated in this study, affecting the response induced by the presence of LPS. FBS increased APC metabolism in a concentration-dependent manner and differentially affected the production of TGF-β1, OPG, IL-6, and CCL2 by APCs in vitro.

In vitro silicon supplementation enhanced acclimatisation and growth of sugarcane (Saccharum officinarum) via improved antioxidant and nutrient acquisition patterns in saline soil.

Salinity affects crop growth by modulating cellular ionic concentrations and generation of reactive oxygen species. Application of silicon (Si) has proved beneficial in ameliorating salinity-triggered plant growth and yield retardations. Leaf roll explants of three sugarcane (Saccharum officinarum ) genotypes (HSF-240, CPF-246, CPF-250) were cultured in Murashige and Skoog (MS) medium supplemented with K2 SiO3 . In vitro regenerated plantlets were acclimatised and grown in natural saline soil. In absence of Si, cv. CPF-246 exhibited better salt tolerance as indicted by maximum chlorophyll a and chlorophyll b contents, rate of photosynthesis and root K+ uptake along with less cellular hydrogen peroxide content. Silicon restricted root Na+ uptake but assisted in K+ , Ca2+ , Mg2+ and Fe2+ accretion in roots and their translocation towards shoots. Cv. HSF-240 and cv. CPF-250 exhibited more increase in photosynthetic pigment content, stomatal conductance and photosynthetic rate after addition of 25 or 50mgL-1 Si than control group. Optimum phenolic content and antioxidant enzyme activity along with decreased lipid peroxidation and hydrogen peroxide content were recorded in all three sugarcane genotypes raised in presence of 25 or 50mgL-1 Si. These findings signify Si supplementation (50mgL-1 ) in tissue culture medium and plant adaptation in saline soil. Further in vitro studies involving Si-mediated gene expression modulations in sugarcane protoplasts shall assist in deciphering cross-talk between Si uptake and cellular responses. The application of Si can further be tested for other plant species to devise strategies for improved crop growth and utilisation of saline areas for crop cultivation.

Development of chondrocyte-laden alginate hydrogels with modulated microstructure and properties for cartilage regeneration.

Alginate hydrogel is an attractive biomaterial for cell microencapsulation. The microarchitecture of hydrogels can regulate cellular functions. This study aims to investigate the applicability of sodium citrate buffer (SCB) as a culture medium supplement for modulating the microstructure of alginate microbeads to provide a favorable microenvironment for chondrogenic induction. The chondrocyte-laden microbeads, with and without TGF-β3 incorporation, were produced through an encapsulator. The obtained small-sized microbeads (~300 μm) were exposed to a treatment medium containing SCB, composed of varied concentrations of sodium citrate (1.10-1.57 mM), sodium chloride (3.00-4.29 mM), and ethylenediaminetetraacetic acid (0.60-0.86 mM) to partially degrade their crosslinked structure for 3 days, followed by culture in a normal medium until day 21. Scanning electron microscope micrographs demonstrated a loose hydrogel network with an enhanced pore size in the SCB-treated microbeads. Increasing the concentration of SCB in the treatment medium reduced the calcium content of the microbeads via a Na+ /Ca2+ exchange process and improved the water absorption of the microbeads, resulting in a higher swelling ratio. All the tested SCB concentrations were non-cytotoxic. Increases in aggrecan and type II collagen gene expression and their corresponding extracellular matrix accumulation, glycosaminoglycans, and type II collagen were vividly detected in the TGF-β3-containing microbeads with increasing SCB concentrations in the treatment medium. Our findings highlighted that the combination of SCB treatment and TGF-β3 incorporation in the chondrocyte-laden microbeads is a promising strategy for enhancing cartilage regeneration, which may contribute to a versatile application in cell delivery and tissue engineering.

Improving biorefinery sustainability and profitability by cultivating aquatic plants on ozonized distillery effluents

Industrial production of biogas offers a way to manage distillery leachate. The waste is usually subjected to anaerobic digestion for producing biogas. However, the effluent from anaerobic processes has high chemical oxygen demand (COD) and is harmful to the environment. An effective method of lowering COD is ozonation. Effluent from biogas plants after ozonation has the potential for use in breeding grounds for plants of the Lemnaceae family. Thus, they can provide a valuable additional source of biomass for the production of bioethanol. Lemna minor L. and Spirodela polyrhiza cultures were grown in media with the addition of 2.5% PFE, which had been treated by ozonation for between 6 and 50 min. Using ozonated effluent was an effective cultivation technique in all variants. The analyzed parameters were plant growth, chlorophyll index, fresh plant weight and photosynthetic traits (net photosynthesis, stomatal conductance, transpiration and concentration of intercellular CO2). The best growth of Lemna minor L. was observed in the media with PFE treated for 12 min. Similar effects were obtained for S. polyrhiza, with ozone treatment for 12 and 25 min. The results show the potential of using ozone-treated post-fermentation leachate as a supplement in culture media.

De novo testicular tissue generation from non-testicular cell lines, biologic and synthetic scaffolds: Current findings and future translational applications.

In recent decades, reproductive science has revolutionized the options for biological parenthood for the 20-50% of infertility cases affected by male factors. However, current solutions exclude those who are infertile due to absent testicular tissue. This includes anorchic 46, XY individuals due to trauma or congenital factors and transgender men with a 46, XX genotype. There is a clinical need for methods to restore testicular function independent of pre-existing testicular tissue. This mini-review analyzes studies that have applied non-testicular cell lines to generate germline and non-germline testicular parenchymal components. While only 46, XY cell lines have been evaluated in this context to date, the potential for future application of cell lines from 46, XX individuals is also included. Additionally, the role of varied culture methods, media supplementation, and biologic and synthetic scaffolds to further support testicular parenchyma generation are critiqued. De novo testicular tissue generation in this manner will require a focus on both cellular and environmental aspects of tissue engineering. Put together, these studies highlight the future potential for expanded clinical, reproductive, and endocrine management options for individuals who are currently excluded from aspects of biologic reproduction most consistent with their gender identity and reproductive preferences.

Natural Killer Cell Expansion and Cytotoxicity Differ Depending on the Culture Medium Used.

Adoptive cell therapy using umbilical cord blood (UCB)-derived allogeneic natural killer (NK) cells has shown encouraging results. However, because of the insufficient availability of NK cells and limited UCB volume, more effective culture methods are required. NK cell expansion and functionality are largely affected by the culture medium. While human serum is a major affecting component in culture media, the way it regulates NK cell functionality remains elusive. We elucidated the effects of different culture media and human serum supplementation on UCB NK cell expansion and functionality. UCB NK cells were cultured under stimulation with K562-OX40L-mbIL-18/21 feeder cells and IL-2 and IL-15 in serum-containing and serum-free culture media. The effects of the culture media and human serum supplementation on NK cell expansion and cytotoxicity were evaluated by analyzing the expansion rate, activating and inhibitory receptor levels, and the cytotoxicity of the UCB NK cells. The optimal medium for NK cell expansion was Dulbecco's modified Eagle's medium/Ham's F12 with supplements and that for cytotoxicity was AIM V supplemented with Immune Cell Serum Replacement. Shifting media is an advantageous strategy for obtaining several highly functional UCB NK cells. Live cell imaging and killing time measurement revealed that human serum enhanced NK cell proliferation but delayed target recognition, resulting in reduced cytotoxicity. Culture medium supplementation with human serum strongly affects UCB NK cell expansion and functionality. Thus, culture media should be carefully selected to ensure both NK cell quantity and quality for adoptive cell therapy.

Age-associated changes in miRNA profile of bovine follicular fluid.

This study shows that ageing affects miRNA profiles in follicular fluid, and an miRNA that is highly abundant in the follicular fluid of young cows supports the growth of oocytes derived from early antral follicles. We examined age-associated changes in miRNA profiles in the follicular fluid (FF) of cows. The role of miR-19b, which is abundant in the FF of young cows, in in vitro growth of early antral follicles (EAFs)-derived oocytes was assessed. FF was collected from the antral follicles of young (20-40 months) and aged (>120 months) cows. The miRNA profiles were similar between the FF of both age groups, whereas the abundance of some miRNAs differed between these samples. The miRNA profiles in granulosa cells (GCs) and the spent culture medium of oocyte-GC complexes (OGCs) derived from EAFs were distinct. Some miRNA groups overlapped among the GCs, culture media, and FFs. miR-19b was highly abundant in the FF of young cows, GCs, and culture medium. The supplementation of OGC culture medium with miR-19b increased the diameter, acetylation levels, and fertilisation ability of the oocytes. To assess whether miR-19b was functional in the GCs, a dual-luciferase assay, suppression of target protein, and RNA-sequencing of the GCs followed by functional annotation of the differentially expressed genes (DEGs) were conducted. Functional annotation of the DEGs suggested that miR-19b influences genes associated with FoxO signalling, endocytosis, and NR3C1 in GCs. These results suggest that in FFs, ageing affects the abundance of miRNAs that have important roles in oocyte development.

Neighbours in nodules: the interactions between Frankia sp. ACN10a and non-Frankia nodular endophytes of alder.

In the present study, we report the in vitro interactions between Frankia sp. ACN10a and non-Frankia nodular endophytes (NFNE) isolated from alder. The supernatant of NFNE grown in nitrogen-replete medium had neutral or negative effects on Frankia growth; none had a stimulatory effect. Inhibitory effects were observed for supernatants of some NFNE, notably Micromonospora, Pseudomonas, Serratia and Stenotrophomonas isolates. However, some NFNE-Frankia coculture supernatants could stimulate Frankia growth when used as a culture medium supplement. This was observed for supernatants of Frankia cocultured with Microvirga and Streptomyces isolates. In nitrogen-limited conditions, cocultures of Frankia with some NFNE, including some rhizobia and Cytobacillus, resulted in higher total biomass than Frankia-only cultures, suggesting cooperation, while other NFNE were strongly antagonistic. Microscopic observation of cocultures also revealed compromised Frankia membrane integrity, and some differentiation into stress resistance-associated morphotypes such as sporangia and reproductive torulose hyphae (RTH). Furthermore, the coculture of Frankia with Serratia sp. isolates resulted in higher concentrations of the auxinic plant hormone indole-3-acetic acid (IAA) and related indolic compounds in the culture supernatant. This study sheds new light on the breadth of microbial interactions that occur amongst bacteria that inhabit the understudied ecological niche of the alder nodule.