Supplementation Of Cultures Research Articles

Optimizing hexanoic acid biosynthesis in Saccharomyces cerevisiae for the de novo production of olivetolic acid

Medium chain fatty acids (MCFAs) are valuable platform compounds for the production of biotechnologically relevant chemicals such as biofuels and biochemicals. Two distinct pathways have been implemented in the yeast Saccharomyces cerevisiae for the biosynthetic production of MCFAs: (i) the mutant fatty acid biosynthesis (FAB) pathway in which the fatty acid synthase (FAS) complex is mutated and (ii) a heterologous multispecies-derived reverse β-oxidation (rBOX) pathway. Hexanoic acid has become of great interest as its acyl-CoA ester, hexanoyl-CoA, is required for the biosynthesis of olivetolic acid (OA), a cannabinoid precursor. Due to insufficient endogenous synthesis of hexanoyl-CoA, recombinant microbial systems to date require exogenous supplementation of cultures with hexanoate along with the overexpression of an acyl-CoA ligase to allow cannabinoid biosynthesis. Here, we engineer a recombinant S. cerevisiae strain which was metabolically optimized for the production of hexanoic acid via the FAB and rBOX pathways and we combine both pathways in a single strain to achieve titers of up to 120 mg L−1. Moreover, we demonstrate the biosynthesis of up to 15 mg L−1 OA from glucose using hexanoyl-CoA derived from the rBOX pathway.

Effect of dietary supplementation of yeast culture Saccharomyces cerevisiae in lactating female goats.

This study was designed to investigate the effects of adding a novel yeast culture, Saccharomyces cerevisiae refermented sorghum distiller's dried grains with solubles (SSDDGS), to the diets of lactating female goats on lactation performance and lamb growth performance. We divided 10 lactating Dazu black goats of similar age, weight, and offspring into two groups: one fed a pelleted diet with 50 g/day SSDDGS (ET), and the other without SSDDGS as a control (EC) for 7 weeks. We monitor the weight changes of each goat and collect blood and milk samples from experimental ewes at specific times for hormone and milk composition determination. We use ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) to detect metabolites in the serum of lactating ewes. Our results showed that SSDDGS supplementation significantly reduced female goats' average daily weight loss during weeks 2-4 of lactation and increased serum IGF-1 and prolactin levels at week 4 (p < 0.05). SSDDGS supplementation in early lactation significantly increased milk protein, lactose, and ash content (p < 0.05). UPLC-MS/MS analysis showed that SSDDGS changed the levels of 58 metabolites in the serum of lactating goats. These metabolites were mainly involved in the sohingolipid signaling pathway, and cysteine, methionine, and sphingolipid metabolism. In summary, Yeast culture SSDDGS reduced weight loss, enhanced milk quality, and modified metabolic profiles in early lactation goats, providing insight into the potential regulatory role and mechanism of yeast culture in lactation female goats.

Mouse liver sinusoidal endothelial cell responses to the glucocorticoid receptor agonist dexamethasone.

Liver sinusoidal endothelial cells (LSECs) which make up the fenestrated wall of the hepatic sinusoids, are active scavenger cells involved in blood waste clearance and liver immune functions. Dexamethasone is a synthetic glucocorticoid commonly used in the clinic and as cell culture supplement. However, the response is dependent on tissue, cell type, and cell state. The aim of this study was to investigate the effect of dexamethasone on primary mouse LSECs (C57BL/6J); their viability (live-dead, LDH release, caspase 3/7 assays), morphology (scanning electron microscopy), release of inflammatory markers (ELISA), and scavenging functions (endocytosis assays), and associated biological processes and pathways. We have characterized and catalogued the proteome of LSECs cultured for 1, 10, or 48h to elucidate time-dependent and dexamethasone-specific cell responses. More than 6,000 protein IDs were quantified using tandem mass tag technology and advanced mass spectrometry (synchronous precursor selection multi-notch MS3). Enrichment analysis showed a culture-induced upregulation of stress and inflammatory markers, and a significant shift in cell metabolism already at 10h, with enhancement of glycolysis and concomitant repression of oxidative phosphorylation. At 48h, changes in metabolic pathways were more pronounced with dexamethasone compared to time-matched controls. Dexamethasone repressed the activation of inflammatory pathways (IFN-gamma response, TNF-alpha signaling via NF-kB, Cell adhesion molecules), and culture-induced release of interleukin-6, VCAM-1, and ICAM-1, and improved cell viability partly through inhibition of apoptosis. The mouse LSECs did not proliferate in culture. Dexamethasone treated cells showed upregulation of xanthine dehydrogenase/oxidase (Xdh), and the transcription regulator Foxo1. The drug further delayed but did not block the culture-induced loss of LSEC fenestration. The LSEC capacity for endocytosis was significantly reduced at 48h, independent of dexamethasone, which correlated with diminished expression of several scavenger receptors and C-type lectins and altered expression of proteins in the endocytic machinery. The glucocorticoid receptor (NR3C1) was suppressed by dexamethasone at 48h, suggesting limited effect of the drug in prolonged LSEC culture. Conclusion: The study presents a detailed overview of biological processes and pathways affected by dexamethasone in mouse LSECs in vitro.

An Overview of Leishmania In Vitro Cultivation and Implications for Antileishmanial Screenings against Promastigotes

The quest for new drug candidates targeting neglected parasitic diseases has become increasingly urgent over the past decades. Advancements in formulating and optimizing drug delivery systems begin with basic research, including direct assays to evaluate the activity of molecules against parasitic stages maintained in laboratories; i.e., promastigotes. In the context of leishmaniasis, an endemic disease worldwide, the cultivation of Leishmania parasites can vary significantly across different laboratories. Factors such as culture media composition, pH, supplementation, and temperature can lead to varied drug responses in in vitro activity assays. This study aims to compile the parameters used in Leishmania spp. promastigotes cultivation protocols described in scientific articles published in indexed journals over the past ten years. The data reveal a lack of uniformity among Leishmania culture protocols, suggesting a potential bottleneck in comparing the leishmanicidal potential of in vitro drug candidates reported by different research groups. This condition is crucial to consider, because viability/inhibition assays should begin with fully-grown, healthy promastigote cultures capable of homogeneous division, thereby producing more reproducible results.

Dietary supplementation of distiller's grains yeast cultures improves performance and immunity by altering the intestinal flora of broilers.

Distiller's grains are a by-product of liquor production with a higher yield than liquor. Developing and utilizing distiller's grains well could alleviate the problem of scarce feed resources. Our present experiment was conducted with 6000 yellow-feathered broilers to study the effects of adding distiller's grains yeast cultures (DGYC) to the diet on growth performance and immunity of broilers. The broilers were divided into five groups, receiving different DGYC concentrations during two stages. Growth performance, intestinal microorganisms and immune organ development were measured. The results showed that groups B and D, supplemented with medium and high concentrations of DGYC, respectively, had significantly improved growth performance compared to the control group (P < 0.05). Group D also showed higher immune organ index (P < 0.01), increased serum total protein, high-density lipoprotein and immunoglobulin levels (P < 0.05) and lower levels of low-density lipoprotein, triglycerides, interleukin 1β and tumor necrosis factor α (P < 0.05). Hematoxylin and eosin staining confirmed improved immune organ development in group D (P < 0.05). Furthermore, in high-concentration group D, levels of short-chain fatty acids (SCFA; acetic, propionic and butyric acids) in cecal chyme were significantly increased (P < 0.05). The richness (Chao1) and diversity (Faith-pd) index of cecal microbiota were significantly higher in group D compared to the control group (P < 0.05). The microbial composition in group D differed from the control and medium-concentration group B. Seven bacteria (Clostridia-UCG-014, UCG-009, DTU089, UCG-010, Campylobacter, Harryflintia, Shuttleworthia) showed significant differences (P < 0.05). After DGYC feeding, DTU089 decreased, while other SCFA-producing bacteria increased (P < 0.05). Subsequently, KEGG function and corresponding signal pathway predictions were performed on bacteria with significant differences. Group D exhibited a higher enrichment of immune function pathways (P < 0.01) and showed significant changes in four immune signaling pathways according to the signal pathway heatmap. Our data suggest that high concentrations of DGYC can be applied as a feed additive for broilers that promotes growth, improves intestinal health and enhances certain immunity. © 2024 Society of Chemical Industry.

Serum-Free Medium Supplemented with Haematococcus pluvialis Extracts for the Growth of Human MRC-5 Fibroblasts.

Experiments are increasingly performed in vitro; therefore, cell culture technology is essential for scientific progress. Fetal bovine serum (FBS) is a key cell culture supplement providing growth factors, amino acids, and hormones. However, FBS is not readily available on the market, has contamination risks, and has ethical concerns. This study aimed to investigate Haematococcus pluvialis extracts (HE) as a potential substitute for FBS. Therefore, we assessed the effects of HE on cell maintenance, growth, and cycle progression in human lung fibroblasts (MRC-5). Cell progression and monosaccharide, fatty acid, and free amino acid compositions were analyzed using cell cycle analysis, bio-liquid chromatography, gas chromatography, and high-performance liquid chromatography, respectively. The results of nutritional profiles showed that the extracts contained essential amino acids required for synthesizing non-essential amino acids and other metabolic intermediates. Furthermore, most of the components present in HE were consistent with those found in FBS. HE enhanced cell viability and regulated cell cycle phases. Additionally, the interaction between growth factor cocktails and HE significantly improved cell viability, promoted cell cycle progression, and activated key cell cycle regulators, such as cyclin A and cyclin-dependent kinases 1 (CDK1). Our findings suggest that HE have considerable potential to substitute FBS in MRC-5 cell cultures and have functional and ethical advantages.

Evaluation of chicken embryo extract and egg yolk extract as alternatives to basic cell culture medium supplement

BackgroundFetal calf serum (FCS), an existing cell culture supplement, is effective but has several drawbacks, including being expensive, requiring a lengthy process of production, and requiring a hard currency. With this in mind, we planned to evaluate chick embryo extract and egg yolk extracts in cell culture as alternatives to fetal calf serum (FCS).MethodsSpecific pathogen-free eggs were purchased from the National Veterinary Institute, Bishoftu, Ethiopia, and incubated in a humidified incubator at 37 °C for 11 days. Egg yolk extract (EYE) and chick embryo extract (CEE) were collected after the egg was opened with caution not to destroy the yolk sack or the chick embryo itself. Chick fibroblasts and Vero cells were cultured in minimum essential medium (MEM) supplemented with egg yolk extract or chick embryo extract at ratios of 0:10, 1:9, 2.5:7.5, and 5:5% fetal calf serum.ResultsFibroblast cell attachment was better in media supplemented with 5% CEE and 5% FCS. The confluency was also greater than 50% at this concentration. Vero cells cultured with 5% CEE and 5% FCS also exhibited very good cell attachment and a confluency of up to 70%. Viability and confluency were also observed at 5%:5% ratios of 50 and 70%, respectively.ConclusionThis investigation evaluated these two extracts as cell culture supplements and revealed promising results as alternatives to fetal calf serum. The limitation of this study is that it only used two cell types and additional cell lines, and different ratios should be tested. With the above findings, further research using different cell lines, ratios and conditions is warranted.

Thermodynamic Study on Biomimetic Legionella gormanii Bacterial Membranes.

The presented studies were aimed at determining the interactions in model membranes (Langmuir monolayers) created of phospholipids (PL) isolated from Legionella gormanii bacteria cultured with (PL + choline) or without (PL - choline) choline and to describe the impact of an antimicrobial peptide, human cathelicidin LL-37, on PL's monolayer behavior. The addition of choline to the growth medium influenced the mutual proportions of phospholipids extracted from L. gormanii. Four classes of phospholipids-phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL), and their mixtures-were used to register compression isotherms with or without the LL-37 peptide in the subphase. Based on them the excess area (Ae), excess (ΔGe), and total (ΔGm) Gibbs energy of mixing were determined. The thermodynamic analyses revealed that the PL - choline monolayer showed greater repulsive forces between molecules in comparison to the ideal system, while the PL + choline monolayer was characterized by greater attraction. The LL-37 peptide affected the strength of interactions between phospholipids' molecules and reduced the monolayers stability. Accordingly, the changes in interactions in the model membranes allowed us to determine the difference in their susceptibility to the LL-37 peptide depending on the choline supplementation of bacterial culture.

Integrated ultrasensitive metabolomics and single-cell transcriptomics identify crucial regulators of sheep oocyte maturation and early embryo development in vitro

IntroductionDevelopmental competence of oocytes matured in vitro is limited due to a lack of complete understanding of metabolism and metabolic gene expression during oocyte maturation and embryo development. Conventional metabolic analysis requires a large number of samples and is not efficiently applicable in oocytes and early embryos, thereby posing challenges in identifying key metabolites and regulating their in vitro culture system. ObjectivesTo enhance the developmental competence of sheep oocytes, this study aimed to identify and supplement essential metabolites that were deficient in the culture systems. MethodsThe metabolic characteristics of oocytes and embryos were determined using ultrasensitive metabolomics analysis on trace samples and single-cell RNA-seq. By conducting integrated analyses of metabolites in cells (oocytes and embryos) and their developmental microenvironment (follicular fluid, oviductal fluid, and in vitro culture systems), we identified key missing metabolites in the in vitro culture systems. In order to assess the impact of these key missing metabolites on oocyte development competence, we performed in vitro culture experiments. Furthermore, omics analyses were employed to elucidate the underlying mechanisms. ResultsOur findings demonstrated that betaine, carnitine and creatine were the key missing metabolites in vitro culture systems and supplementation of betaine and L-carnitine significantly improved the blastocyst formation rate (67.48% and 48.61%). Through in vitro culture experiments and omics analyses, we have discovered that L-carnitine had the potential to promote fatty acid oxidation, reduce lipid content and lipid peroxidation level, and regulate spindle morphological grade through fatty acid degradation pathway. Additionally, betaine may participate in methylation modification and osmotic pressure regulation, thereby potentially improving oocyte maturation and early embryo development in sheep. ConclusionTogether, these analyses identified key metabolites that promote ovine oocyte maturation and early embryo development, while also providing a new viewpoint to improve clinical applications such as oocyte maturation or embryo culture.

Effects of yeast culture supplementation on milk yield, rumen fermentation, metabolism, and bacterial composition in dairy goats.

The effects of yeast culture (YC) on dairy goat milk yield and potential effects of rumen microbial population changes on rumen fermentation are poorly understood. This study aimed to evaluate the effects of YC on milk yield and rumen fermentation in dairy goats and explore the potential microbial mechanisms. Forty Laoshan dairy goats with a weight of 51.23 ± 2.23 kg and daily milk yield of 1.41 ± 0.26 kg were randomly divided into 4 groups: control (no YC), YC1 (10 g/day per goat), YC2 (25 g/day per goat), and YC3 (40 g/day per goat). The pre-feeding period was 15 days, and the official period was 60 days. Laoshan dairy goats were milked twice daily, and the individual milk yield was recorded. On the last day of the official period, rumen fluid was collected to measure rumen fermentation, perform quantitative polymerase chain reaction (PCR), and detect metabolites. Compared to the control group, the YC group had greater milk yield; higher acetic acid, butyric acid, and total volatile fatty acid contents; and lower ammonia-N (NH3-N) content in the rumen (p < 0.05). YC increased the abundance of Clostridia_UCG-014 and Paraprevotella (p < 0.05). Differential metabolites L-leucine and aspartic acid were screened. This study revealed the microbial mechanisms linking the relative abundance of Paraprevotella and Clostridia_UCG-014 to L-leucine and aspartic acid utilization. These results describe the potential benefits of supplementing 10 g/day per goat YC in the diets of Laoshan dairy goats for improving the rumen environment and milk yield.

Immersive Anthropological Methodology towards Sustainable Ecotourism: A Case Study of Bali, Indonesia

Purpose: This research aims to investigate and implement an anthropological methodology for achieving sustainable eco-tourism in Bali, Indonesia. Research methods: This qualitative method used involves a thorough examination and comparison of diverse scholarly works, case studies, and relevant texts. The qualitative comparative analysis provides a robust framework for interpreting the complex interplay of ideas, perspectives, and contextual factors embedded in the literature building upon and expanding the understanding of the subject matter within the broader academic discourse. Results and discussion: The research underscores the critical issues of water supply, waste management, and infrastructure development in Bali. Sustainable solutions, including cultural inclusion, eco-friendly housing, and natural supplements, are proposed to alleviate these challenges. Implications: The results signify a crucial step towards fostering environmental conservation and responsible tourism practices in this popular destination.

Resveratrol Enhances Antioxidant and Anti-Apoptotic Capacities in Chicken Primordial Germ Cells through m6A Methylation: A Preliminary Investigation.

Avian primordial germ cells (PGCs) are essential in avian transgenic research, germplasm conservation, and disease resistance breeding. However, cultured PGCs are prone to fragmentation and apoptosis, regulated at transcriptional and translational levels, with N6-methyladenosine (m6A) being the most common mRNA modification. Resveratrol (RSV) is known for its antioxidant and anti-apoptotic properties, but its effects on PGCs and the underlying mechanisms are not well understood. This study shows that RSV supplementation in cultured PGCs improves cell morphology, significantly enhances total antioxidant capacity (p < 0.01), reduces malondialdehyde levels (p < 0.05), increases anti-apoptotic BCL2 expression, and decreases Caspase-9 expression (p < 0.05). Additionally, RSV upregulates the expression of m6A reader proteins YTHDF1 and YTHDF3 (p < 0.05). m6A methylation sequencing revealed changes in mRNA m6A levels after RSV treatment, identifying 6245 methylation sites, with 1223 unique to the control group and 798 unique to the RSV group. Combined analysis of m6A peaks and mRNA expression identified 65 mRNAs with significantly altered methylation and expression levels. Sixteen candidate genes were selected, and four were randomly chosen for RT-qPCR validation, showing results consistent with the transcriptome data. Notably, FAM129A and SFRP1 are closely related to apoptosis, indicating potential research value. Overall, our study reveals the protective effects and potential mechanisms of RSV on chicken PGCs, providing new insight into its use as a supplement in reproductive stem cell culture.

Development of technology for culturing a cell line producing a single-domain antibody fused with the Fc fragment of human IgG1

Objectives. To develop an effective technology for the cultivation of Chinese hamster ovary (CHO) cells stably producing GamP2C5 antibody which is a component I of the GamCoviMab candidate drug for emergency prevention and therapy of infection caused by SARS-CoV-2 virus; to select optimal cultivation parameters and to scale this technology in production.Methods. The study was performed on CHO GamP2C5 (clone 78) cell culture, producing a single-domain antibody fused to the Fc fragment of human IgG1 GamP2C5. Different culture media and supplements were used. Cells were cultured in Erlenmeyer flasks, Biostat® RM 20 wave-mixed bioreactor, Ambr® 250 mini bioreactors, STR 200 stirred-tank bioreactor.Results. Using molecular-genetic and biotechnological methods, a stable clone producer of CHO GamP2C5 antibody, clone 78, was obtained. Then a technique was worked out for the cultivation of the obtained clone producer on different culture media. The most suitable cultivation regimes, culture media, and optimal supplements were selected. This technology was tested in laboratory conditions in a 10-L reactor, and then successfully scaled up for production at the MedGamal Branch of the Gamaleya National Research Center for Epidemiology and Microbiology.Conclusions. This study demonstrates the fundamental feasibility of developing and scaling up a culture technology, in order to produce a drug based on a modified single-domain antibody with virus neutralizing activity against different strains of SARS-CoV-2 virus.

O-005 The in vitro maturation rate of human oocytes is enhanced following uptake of extracellular vesicles derived from mature follicles

Abstract Study question Could follicular fluid derived extracellular vesicles (ffEVs) impact human oocyte in vitro maturation (IVM)? Summary answer Supplementation of IVM culture with ffEVs isolated from mature follicles enhanced oocyte maturation rates by &amp;gt; 20%, inducing changes in oocyte protein profile and organelle distribution. What is known already IVM involves the culture of immature germinal vesicle (GV) oocytes under set laboratory conditions to allow for their transition to mature metaphase II (MII) stage, which is confirmed by the extrusion of the first polar body. Efficient IVM could circumvent aggressive controlled ovarian stimulation (COS), reduce the cost and broaden the repertoire of infertility treatments. Animal studies suggest that extracellular vesicles (EVs), membranous nanosized vesicles containing different molecular content (e.g. nucleic acids, proteins) and present in the ovarian follicular fluid could facilitate oocyte maturation. The uptake of ffEVs by bovine, equine and feline oocytes, but not human, has been demonstrated. Study design, size, duration Women undergoing transvaginal oocyte retrieval after COS (n = 83) at University Hospital Zurich donated follicular fluid (n = 54 single follicles) and/or immature GV oocytes (n = 95). We aimed to: a) define differences in the protein cargo of ffEVs derived from follicles containing mature oocytes (MII-ffEVs, n = 10) versus (vs.) immature oocytes (GV-ffEVS, n = 5; metaphase I MI-ffEVs, n = 5), b) demonstrate the capacity of GV oocytes to uptake mature MII-ffEVs and c) determine the effect of MII-ffEVs supplementation on oocyte maturation. Participants/materials, setting, methods ffEVs were isolated by ultracentrifugation. The protein content of ffEVs was analysed by mass spectrometry. The uptake of fluorescently-labelled MII-ffEVs by GV oocytes (n = 15) was assessed by confocal microscopy. GVs were cultured for rescue-IVM in a timelapse incubator with MII-ffEVs (n = 45 GVs) or without (n = 40 GVs) and extrusion of polar body denoted maturation. The impact of MII-ffEVs supplementation on IVM-matured oocytes was assessed through single-cell proteomics and intracellular organelles appearance with transmission electron microscopy (TEM). Main results and the role of chance Successful isolation of ffEVs was confirmed by TEM and Western blotting analysis for characteristic protein markers of EVs (TSG101 and CD63). Mass spectrometry identified a total of 1340 proteins across ffEVs samples. Statistical analysis revealed differentially abundant proteins (DAPs) in ffEVs: 61 DAPs in the comparison MII-ffEVs vs. GV-ffEVs, 15 DAPs in MII-ffEVs vs. MI-ffEVs and 14 DAPs in MI-ffEVs vs GV-ffEVs (FDR&amp;lt;0.2). Among the 61 DAPs, 7 proteins in higher abundance in MII-ffEVs (&amp;gt;log2 fold change) were related to oocyte maturation, lipid accumulation, calcium binding and the coagulation cascade (e.g. FABP5, S100A9, F12, SERPINA10). Confocal microscopy confirmed the uptake of MII-ffEVs by GV oocytes after 24 hours of co-incubation. Supplementation of IVM culture with MII-ffEVs for 48 hours significantly increased the oocyte maturation rate compared to control by 22.8±9.4% (77.8% vs 55% mature oocytes respectively; P-value=0.0372). Single oocyte proteomics of IVM-matured oocytes revealed 59 DAPs (FDR 0.1) between ffEVs-supplemented and control groups. Among them, 37 DAPs were in higher abundance in ffEVs-supplemented mature oocytes (&amp;gt;log2 FC) including Hyaluronan Synthase 1 (HAS1) that is associated with oocyte maturation (6.55 fold increase). TEM revealed differences in oocyte organelle distribution and appearance, particularly of endoplasmic reticulum (RE) and RE-mitochondria complexes. Limitations, reasons for caution This study utilised immature oocytes from stimulated cycles, therefore these results should be interpreted within the context of rescue-IVM potential. Wider implications of the findings Our findings pave the way for further mechanistic studies on the role of ffEVs in supporting oocyte maturation and inspire the development of clinical IVM supplements as well as novel complementary regimes for COS based on ffEVs or their associated specific cargo. Trial registration number N/A

O-249 METAPHOR: METabolic imaging through AI-powered Phasor-based Hyperspectral analysis and Organelle recognition for the classification of human blastocysts

Abstract Study question Can we classify human blastocysts non-invasively using hyperspectral imaging? Summary answer HS imaging can significantly enhance our classification of human embryos and reveal numerous cellular features present in the embryo invisible to brightfield imaging. What is known already Non-invasive measurement of metabolism is a safe and powerful tool to reliably predict oocyte and embryo quality. We have previously shown that our METAPHOR technology, based on HS imaging coupled to artificial intelligence, was able to separate mouse oocyte categories according to maternal age with an average AUC of 96.2% and predict blastocyst formation (AUC 82.2%). Moreover, it was able to classify mouse embryos deprived from different nutrients with an average AUC of 93.7% showing clear superiority to grading provided by experienced embryologists (51%). Study design, size, duration A total of 74 human embryos donated for research at the blastocyst stage have been imaged to date. Embryos were warmed and imaged according to our established protocol and survival was statistically indistinguishable from control embryos. Participants/materials, setting, methods Embryos are imaged using multiphoton illumination near the infra-red. The HS detection covered the simultaneous measurement of the whole visible spectrum. Embryos are then allowed to implant in our proprietary 3D ex vivo platform. Implantation is monitored up until day 9 before immunostaining is performed to quantify presence and prevalence of lineage and implantation markers. Main results and the role of chance After imaging, embryos were placed on the 3D ex vivo implantation platform and let implant. Successful implantation (75.41%) was scored by observation of the characteristic deformation of collagen fibers within the matrix and staining for relevant cell lineages (OCT4, pMLC2). Interestingly, we observed that embryos with a higher trophectoderm (TE) grading tended to implant with a higher efficiency and penetrate deeper into the matrix than embryos with lower grading. HS images have unveiled a profound cellular diversity within embryos, elucidating distinctions between the Inner Cell Mass (ICM) and TE, as well as revealing different metabolic states defined by NADH dominance, FAD dominance, protoporphyrin levels, and apoptosis. HS raw images are processed using our HS-phasor approach which allows quick computing by converting the multidimensional HS data into comprehensive visual representation of bidimensional histograms and phasor plots. In summary, our analysis pipeline encompasses the algorithms to detect and segment the embryos, the computation of histogram and phasor-plot, and the classifier. AI allows us to classify embryos in distinct spectral spaces based on their metabolic features, paving the way to the establishment of a new grading system based on HS imaging. Limitations, reasons for caution The cohort size is still limited, and ongoing experiments will increase the dataset needed for more accurate prediction of embryo implantation. The implantation platform is an in vitro test that will require future clinical validation. Wider implications of the findings The safety and speed of METAPHOR warrants its incorporation at the IVF clinic, adding an additional layer of information to the current embryo selection methods. Moreover, the non-invasive measurement of the metabolic function opens new alleys of research such as the effect of media and culture supplements on embryo culture. Trial registration number Not applicable

Supplementation of yeast culture to low-fishmeal diets improves growth, intestinal health, and heat stress resistance in juvenile Chinese mitten crab (Eriocheir sinensis)

Yeast culture (YC), a microecological product derived from yeast fermentation, can enhance animal growth, health, and resistance to heat stress. However, its impact on the economically significant crustacean Eriocheir sinensis has not been demonstrated. This study investigated the effects of low-fishmeal YC on the growth, intestinal health, and heat stress resistance of juvenile Chinese mitten crabs. Crabs (0.72 ± 0.01 g) were distributed across 42 tanks, and 40 crabs were randomly maintained per tank in six replicates. The crabs were fed a control diet (35% fish meal) or six YC concentrations (15% fish meal; 0, 0.8, 1.6, 2.4, 3.2, or 4.0 g/kg) for 56 days. After sample collection, the remaining crabs were subjected to a 72-h heat stress challenge at 32 °C. A moderate-yield YC diet significantly improved weight gain, specific growth rate, and digestive enzyme activities (α-amylase, trypsin, and lipase) compared to those of crabs fed a 15% fishmeal diet without YC. Notably, the diet with 3.2 g/kg YC supplementation was performed as well as crabs fed a 35% fishmeal diet. YC also enhanced intestinal morphology in the low-fishmeal diet group and reduced oxidative damage by increasing the expression of genes related to the peritrophic membrane and tight junction proteins. Furthermore, YC supplementation diversified the intestinal microbiota, significantly increased the abundance of Firmicutes and the genus Shewanella, increased acetate and butyrate levels and decreased the abundance of Proteobacteria. Correlation analysis revealed that weight gain and specific growth rate were associated with most digestive activity and intestinal health indicators. Following heat stress challenge, YC protected against oxidative stress and enhanced serum immunity at elevated temperatures. This study indicated that incorporating YC in low-fishmeal diets significantly increases juvenile crab growth, improves intestinal health, and mitigates oxidative stress induced by temperature elevation. The optimal YC concentration was determined to be 2.71 g/kg through regression analysis.

The Impact of Amotosalen Photochemical Pathogen Inactivation on Human Platelet Lysate

Background: Human Platelet Lysate (hPL) is a platelet-derived and growth factor-rich supplement for cell culture. It can be prepared from surplus platelet concentrates initially intended for transfusion. Amotosalen-based photochemical pathogen inactivation of platelet concentrates is used in a number of blood establishments worldwide to minimize the risk of pathogen transmission from donor to patient. Method: This pathogen inactivation method has not been formally validated for direct use on hPL. Here, we have studied the impact of pathogen inactivation on hPL and compared it to untreated hPL prepared from pathogen-inactivated platelet concentrates or control hPL. We used mass spectrometry, ELISA, and in vitro mesenchymal stem cell culture for determining residual amotosalen, final growth factor content, and cell doubling, respectively. Result: The data have shown amotosalen concentrations to be reduced a thousand-fold following pathogen inactivation, leaving trace quantities of photosensitizer molecules in the final hPL product. Some growth factors have been reported to be significantly more impacted in hPL that is directly pathogen-inactivated compared to both control conditions. This has no significant effect on the growth kinetics of adipose-derived mesenchymal stem cells. Conclusion: We have concluded direct amotosalen-based pathogen inactivation to have a measurable impact on certain growth factors in hPL, but this does not outweigh the likely benefits of reducing the odds of donor-to-patient pathogen transmission.

The role of fibroblast growth factor-2 in modulating the differentiation of periodontal ligament and alveolar bone-derived stem cells

ObjectiveThis study examined how range concentrations of Fibroblast Growth Factor-2 (FGF-2) influence the differentiation and activity of human-derived periodontal ligament (hPDLSCs) and alveolar bone-derived stem cells (haBMSCs). DesignhPDLSCs and haBMSCs were cultured with varying concentrations of FGF-2 (0, 1, 2.5, 5, 10, 20 ng/mL) and monitored for osteogenic differentiation through alkaline phosphatase (ALP) activity and quantification of gene expression (qRT-PCR) for osteogenesis markers. Additionally, alizarin red staining and a hydroxyproline colorimetric assay evaluated and quantified osteogenic matrix mineralization and collagen deposition. Statistical analyses were performed using one-way ANOVA or two-way ANOVA for multiple comparisons between groups. ResultsAt low FGF-2 concentrations, hPDLSCs differentiated toward an osteogenic lineage, whereas higher concentrations of FGF-2 inhibited osteogenesis and promoted fibroblastic differentiation. The effect of FGF-2 at the lowest concentration tested (1 ng/mL) led to significantly higher ALP activity than osteogenically induced positive controls at early time points and equivalent RUNX2 expression at early and later time points. FGF-2 supplementation of haBMSC cultures was sufficient, at all concentrations, to increase ALP activity at an earlier time point. Mineralization of haBMSC cultures increased significantly within 5–20 ng/mL FGF-2 concentrations under basal growth media conditions (α-minimal essential medium supplemented with 15 % fetal bovine serum and 1 % penicillin/streptomycin). ConclusionsFGF-2 has a dual capacity in promoting osteogenic and fibroblastic differentiation within hPDLSCs contingent upon the dosage and timing of administration, alongside supporting osteogenic differentiation in haBMSCs. These findings underscore the need for precision growth factors dosing when considering the design of biomaterials for periodontal regeneration.

Tumor reactive T cells from the cerebrospinal fluid of patients with leptomeningeal disease from melanoma.

2027 Background: Leptomeningeal disease (LMD) is a devastating complication from cancer, with a median survival of 6-8 weeks. The incidence of LMD is highest in melanoma (5-25%) carrying the worst prognosis. Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) showed complete and durable responses in patients with advanced metastatic melanoma. CSF-derived T cell populations in LMD shift to more exhausted phenotypes compared to extracranial and brain metastases. Ex vivo large T cell expansion can overcome this shortfall, with adoptive T cell transfer representing a novel therapeutic approach to treat melanoma LMD (M-LMD). The goal of this study is to optimize rapid CSF-derived tumor reactive T cell expansion in M-LMD, and evaluate the immune microenvironment in CSF compared to peripheral immunity and anti-tumoral response. Methods: CSF was collected from M-LMD patients (n=6) via lumbar puncture or Ommaya. Cells were plated following established TIL culture protocols to determine optimal expansion conditions i.e.: 1) 6000 IU/mL IL-2, 2) IL-2+OKT3, 3) IL-2+anti-4-1BB agonistic antibody, 4) IL-2+IL-7+IL-15+IL-21, 5) IL-2+anti-CD3/CD28 human dynabeads, &amp; 6) IL-2+anti-CD3/CD28/CD137 human dynabeads. After 4 weeks, T cells were propagated via rapid expansion protocol (REP) and phenotyped for CD4+ and CD8+ T cells with flow cytometry. T cells were co-cultured for 18-24 hours with HLA-matched melanoma cell lines to assess functionality, and IFN-y levels evaluated in supernatant. Ongoing studies are evaluating other biomarkers i.e., chemokines via Luminex. To examine the expanded TCR clonotypes, TCR beta seq was done in CSF-derived T cells at different times point of expansion and peripheral T cells. Results: Collected CSF yielded an average 8.12e4 viable cells for expansion (range 5e2-2.43e6). After initial culture in IL-2, 61.9% of samples showed increased cell yield with an average 101.68-fold expansion (range 2.35-1054). PreREP T cells were predominantly CD4+ (45.7%). REP was completed with a 100% success rate (mean 187.47-fold expansion). Post-REP flow cytometry analysis revealed similar results with further expansion of T cells. Additional culture supplements (Methods #2-6) produced similar expansion with a reduced T cell input requirement and demonstrated the potential to enrich for CD8+ T cells. M-LMD T cells were highly functional, producing substantial levels of IFN-y in response to HLA-matched melanoma cell lines. Anti-CD3/CD28 produced a larger expansion of T cells with higher levels of IFN-y. Preliminary TCR beta sequencing data revealed a distinct clonal distribution between peripheral and CSF-derived T cells. Conclusions: Therapies for LMD patients are desperately needed. Results demonstrate successful expansion of T cells ex vivo from CSF in M-LMD. Results raise potential to use autologous CSF-derived T cells as therapeutic strategy for patients with LMD.

Optimizing IVF Success: A Case Study of Melatonin-Assisted Frozen Embryo Transfer for Poor Oocyte Quality and Endometriosis.

The case study investigates the journey of a couple facing infertility. It intensifies the challenges, including poor oocyte quality and endometriosis. In spite of two failed in vitro fertilization cycles, the decision for ovum pickup (OPU) was made, followed by intra-cytoplasmic sperm injection (ICSI), embryo treatment with melatonin, and frozen embryo transfer (FET) to optimize the chances of a successful pregnancy. The couple opted for this approach. OPU yielded four poor-quality oocytes, prompting ICSI and melatonin treatment to enhance embryo quality. The embryos were exposed to culture supplementation with melatonin for 72 hours before being transferred to conventional media. After 5 days or 120 hours, the embryos developed into 3BB quality blastocysts, indicative of developmental stage and morphology. The blastocysts were then cryopreserved, and after 2 months, FET was conducted, resulting in the transfer of two embryos, which subsequently led to a positive pregnancy indication, as indicated by a β-hCG level of 233 mUI/ml measured 14 days post transfer. This approach highlights the effectiveness of melatonin supplementation in improving embryo quality and ultimately facilitating successful pregnancy in complex scenarios like endometriosis-related infertility.