24-h Culture Research Articles

The NAD+ precursor nicotinamide riboside protects against postovulatory aging in vitro.

Postovulatory aging (POA) of oocytes is clinically significant as it mirrors the degeneration observed in maternally aged oocytes, leading to substantial impairments in oocyte quality and the success rates of artificial reproductive technology (ART). The molecular alterations associated with POA, such as the degeneration of the first polar body, an increase in perivitelline space, reactive oxygen species (ROS) accumulation, energy depletion, and chromosomal and DNA damage, underscore the urgency of finding interventions to mitigate these effects. This study aims to identify whether nicotinamide riboside (NR) can prevent POA during the process of in vitro culture and raise the success rates of ART. Taking advantage of an in vitro postovulatory oocyte aging model, we examined the morphological integrity and NAD+ levels of ovulated mouse MII oocytes after 24h of culturing. Following in vitro fertilization, we assessed the embryonic developmental potential of oocytes affected by POA. Using immunofluorescence and confocal microscopy, we measured the levels of ROS, mitochondrial function, and γH2AX. We also evaluated spindle assembly and chromosome alignment. Additionally, we detected the distribution of cortical granules to assess the metabolic and quality changes in POA oocytes with the supplementation of NR. To further our analysis, quantitative real-time PCR was conducted to measure the mRNA expression levels of antioxidant enzymes Sod1 and Gpx1 in the oocytes. With 200μM NR supplementation during in vitro culture for 24h, the oocytes from POA demonstrated reduced signs of aging-related decline in oocyte quality, including reduced ROS accumulation, improved mitochondrial function, and corrected mis-localization of cortical granules. This improvement in oocyte quality is likely due to the inhibition of oxidative stress via the NAD+/SIRT1 signaling pathway, which also helped to restore normal spindle assembly and chromosome alignment, as well as reduce the elevated levels of γH2AX, thereby potentially enhancing the embryonic development potential. Current research provides evidence that NR is an efficient and safe natural component that prevents the process of POA and is thus a potential ideal antiaging drug for raising the success rates of ART in clinical practice.

Hypoxia-Induced Inflammation in In Vitro Model of Human Blood-Brain Barrier: Modulatory Effects of the Olfactory Ensheathing Cell-Conditioned Medium.

Hypoxia compromises the integrity of the blood-brain barrier (BBB) and increases its permeability, thereby inducing inflammation. Olfactory ensheathing cells (OECs) garnered considerable interest due to their neuroregenerative and anti-inflammatory properties. Here, we aimed to investigate the potential modulatory effects of OEC-conditioned medium (OEC-CM) on the response of human brain microvascular endothelial cells (HBMECs), constituting the BBB, when exposed to hypoxia. HBMECs were utilized to establish the in vitro BBB model. OECs were isolated from mouse olfactory bulbs, and OEC-CM was collected after 48h of culture. The effect of OEC-CM treatment on the HBMEC viability was evaluated under both normoxic and hypoxic conditions at 6h, 24h, and 30h. Western blot and immunostaining techniques were employed to assess NF-κB/phospho-NF-κB expression. HIF-1α, VEGF-A, and cPLA2 mRNA expression levels were quantified using digital PCR. ELISA assays were performed to measure PGE2, VEGF-A, IL-8 secretion, and cPLA2 specific activity. The in vitro formation of HBMEC capillary-like structures was examined using a three-dimensional matrix system. OEC-CM attenuated pro-inflammatory responses and mitigated the HIF-1α/VEGFA signaling pathway activation in HBMECs under hypoxic condition. Hypoxia-induced damage of the BBB can be mitigated by novel therapeutic strategies harnessing OEC potential.

Effects of Platycodon grandiflorus on doxorubicin resistance and epithelial-mesenchymal transition of breast cancer cells via the p38 mitogen-activated protein kinase pathway.

With the increase of chemotherapy frequency for breast cancer, the drug resistance rate of patients is rising, accompanied by cell invasion and metastasis, thus causing mortality. We aimed to explore the mechanism by which Platycodon grandiflorus affects breast cancer cells in terms of the doxorubicin (Dox) resistance and epithelial-mesenchymal transition (EMT) via the p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway. MCF-7/R cell lines with resistance to Dox were established. After 24h of culture with DMEM (blank group), they were divided into Platycodon grandiflorus, Platycodon grandiflorus + Ophiopogon japonicus, Platycodon grandiflorus + Curcumae Rhizoma, Platycodon grandiflorus + Curcumae Rhizoma + U46619 groups. Flow cytometry, colony formation assay, as well as Transwell assay were performed to examine the cells for apoptosis, proliferation, and invasion, respectively. Western blotting was performed to measure the phosphorylated (p)-p38 MAPK-to-p38 MAPK ratio together with N-cadherin, vimentin, β-catenin, and E-cadherin protein expressions. Compared with the blank group, the half maximal inhibitory concentration (IC50), number of cell colonies, number of invading cells and expressions of proteins related to EMT (i.e. N-cadherin, vimentin, and β-catenin) significantly reduced, but increases in apoptosis rate, p-p38 MAPK/p38 MAPK ratio and E-cadherin protein expression were observed in different groups (P < 0.05). Compared with the Platycodon grandiflorus + Curcumae Rhizoma group, the Platycodon grandiflorus + Curcumae Rhizoma + U46619 group had significantly decreased IC50, cell colony count, invading cell count and β-catenin, N-cadherin, and vimentin expressions, in addition to elevated E-cadherin protein expression, apoptosis rate, and p-p38 MAPK/p38 MAPK ratio (P < 0.05). Platycodon grandiflorus can reverse the resistance of breast cancer cells to Dox and regulate their biological activities by activating the p38 MAPK signaling pathway.

A novel, simple and rapid assay to measure citrate level in bacterial culture for analysis of citrate consumption by bacteria

Citrate is essential for Krebs cycle in eukaryotes and serves as a carbon source for some bacteria to survive/grow. Available methods for measuring citrate level rely mainly on enzymatic reactions and/or sophisticated instrumentations. This study aimed to develop a novel, simple and rapid assay to quantify citrate in bacterial culture for analysis of citrate consumption. The assay was initially tested with 0.1–25.6 mM citrate in deionized (dI) water or complete M9 medium without/with 0.25 M HCl. Wavelength scan revealed maximum absorption of citrate at λ210 nm with linear calibration curve (R2 = 0.9997–0.9999) when HCl was added. Among negatively charged chemicals tested, only oxalate interfered with the assay. After 24-h inoculation of Klebsiella pneumoniae (the known citrate-utilizing bacterium) in (20 mM) citrate-containing complete M9 medium, the remaining citrate significantly decreased (by 22.20±7.13 % consumption). However, a mild decrease was also observed in the sample without bacterial inoculation, suggesting that some components of the complete medium interfered with the assay. It was clearly evidenced that M9 salt, CaCl2 and/or MgSO4 were responsible for such interference. Finally, citrate at low concentrations (0.1–6.4 mM) in M9 medium with CaCl2 and/or MgSO4 provided the linear calibration curve (R2 = 0.9451–0.9986). At 5 mM, citrate consumption by K. pneumoniae after 24-h culture was 46.88±0.47 %. In summary, we have successfully developed a novel, simple and rapid method for measuring citrate level in bacterial culture. It will be very useful for measuring citrate consumption by typical and atypical citrate-utilizing bacteria for classification, mechanistic and pathophysiologic studies.

Low molecular weight acids differentially impact Fusarium verticillioides transcription

Fusarium verticillioides is both an endophyte and pathogen of maize. During growth on maize, the fungus often synthesizes the mycotoxins fumonisins, which have been linked to a variety of diseases, including cancer in some animals. How F. verticillioides responds to other fungi, such as Fusarium proliferatum, Aspergillus flavus, Aspergillus niger, and Penicillium oxalicum, that coinfect maize, has potential to impact mycotoxin synthesis and disease. We hypothesize that low molecular weight acids produced by these fungi play a role in communication between the fungi in planta/nature. To address this hypothesis, we exposed 48-h maize kernel cultures of F. verticillioides to oxalic acid, citric acid, fusaric acid, or kojic acid and then compared transcriptomes after 30 min and 6 h. Transcription of some genes were affected by multiple chemicals and others were affected by only one chemical. The most significant positive response was observed after exposure to fusaric acid which resulted in >2-fold upregulation of 225 genes, including genes involved in fusaric acid synthesis. Exposure of cultures to the other three chemicals increased expression of only 3–15 genes. The predicted function and frequent co-localization of three sets of genes support a role in protecting the fungus from the chemical or a role in catabolism. These unique transcriptional responses support our hypothesis that these chemicals can act as signaling molecules. Studies with gene deletion mutants will further indicate if the initial transcriptional response to the chemicals benefit F. verticillioides.

Endometriotic tissue fragments are viable after cryopreservation in an ex vivo tissue model recapitulating the fibrotic microenvironment.

Is it possible to establish an ex vivo endometriosis model using cryopreserved endometriotic tissue fragments? Cryopreserved endometriotic tissue fragments remain viable after thawing and during at least 3days of culture and can therefore be used to establish an ex vivo endometriosis model to efficiently test potential therapeutic agents. Endometriosis is the most prevalent benign gynecologic disease with an enormous societal burden; however, curative therapies are still lacking. To efficiently test potential new therapies, an ex vivo model based on previously cryopreserved endometriotic tissue that recapitulates the different endometriosis subtypes and their microenvironment is highly desirable. Endometriotic tissue fragments of three different subtypes were obtained from 28 patients by surgical resection. After cryopreservation and thawing, viability and metabolic activity of these tissue fragments were assessed. Viability was compared with fresh fragments from 11 patients directly after surgical removal. Experimental intervention studies were performed in cryopreserved and thawed tissue fragments from two patients to confirm the usability of these tissues for ex vivo intervention studies. Endometriotic tissue fragments (n = 45) were cryopreserved according to three different protocols. After thawing, fragments were cultured for 24 h. A resazurin-based assay was performed to assess the metabolic activity of the tissue fragments. In addition, cell type-specific viability was analyzed by VivaFix, Hoechst 33342, and α-smooth muscle actin immunofluorescence staining and confocal microscopy. The presence of endometriosis was histologically confirmed based on hematoxylin-eosin staining. Cryopreserved and thawed tissue fragments were treated for 72 h with pirfenidone or metformin and COL1A1 and CEMIP gene expressions were assessed using RT-PCR and RT-qPCR, either in the whole tissue fragments or in myofibroblasts isolated by laser capture microdissection. Metabolic activity of endometriotic tissue fragments obtained from peritoneal (PER), ovarian (OMA), and deep (DE) endometriotic lesions was well preserved after cryopreservation in a dimethyl sulfoxide-based medium and was comparable with fresh tissue fragments. Relative metabolic activity compared to fresh tissue was 70% (CI: 92-47%) in PER, 43% (CI: 53-15%) in OMA and 94% (CI: 186-3%) in DE lesions. In fragments from PE lesions 92% (CI: 87-96%), from OMA lesions 95% (CI: 91-98%), and from DE lesions 88% (CI: 78-98%) of cells were viable after cryopreservation and thawing followed by a 24-h culture period. Differences in gene expression of fibrotic markers COL1A1 and CEMIP after 72-h treatment with pirfenidone or metformin could be detected in whole tissue fragments and in isolated myofibroblasts, indicating that cryopreserved and thawed endometriotic tissue fragments are suitable for testing anti-fibrotic interventions. N/A. Viability and metabolic activity of the endometriotic tissue fragments may have been partially compromised by damage sustained during the surgical procedure, contributing to inter-sample variance. The storage of viable endometriotic tissue fragments for later usage in an ex vivo model creates the possibility to efficiently test potential new therapeutic strategies and facilitates the exchange of viable endometriotic tissue between different research laboratories. This study was not financially supported by external funding. The authors declare no competing interest. N/A.

P-370 The age-associated increase in ovarian stiffness compromises follicle development and induces changes in the follicles’ transcriptome, resulting in poor-quality oocytes

Abstract Study question Does the age-associated increase in ovarian stiffness trigger the decline in oocyte quantity and quality observed in women of advanced maternal age? Summary answer An encapsulated in vitro follicle culture mimicking ovarian biomechanics revealed that age-associated increase in stiffness impacts follicle growth, modulates follicles’ transcriptome and reduces oocyte quality. What is known already Female reproductive aging is characterized by a progressive decline in oocyte quality and quantity, resulting in sub-fertility. We recently discovered that with aging the mouse ovary assumes a pro-fibrotic milieu which induces ovarian stiffness. Moreover, human ovaries also become stiffer with aging; however, it is not known whether this age-associated increase in stiffness impacts ovarian function. The present study aims to examine whether stiffness is a novel mechanism that mediates the age-associated progressive decline in oocyte quality and how stiffness affects the transcriptome of growing follicles, resulting in poor-quality oocytes. Study design, size, duration We used an in vitro alginate-encapsulated follicle culture to mimic young and old environments in which follicles grow. Alginate hydrogel synthesis replicated the soft (0.5% - 1.79±0.08 kPa) and stiff (2% - 4.56±2.03 kPa) environments of young and aged ovaries, respectively. Mouse secondary follicles were cultured in 0.5 or 2% alginate and used for i) long-term culture (day 0-10) to evaluate follicle development and oocyte quality; ii) short-term culture (hour 0-24) to investigate follicles’ transcriptome. Participants/materials, setting, methods During the long-term culture, markers of follicle development were evaluated (n = 31 follicles), including: follicle size, estradiol production (ELISA), cell death (cleaved-caspase3,CC3; immunohistochemistry). At D10, oocyte quality was inspected on isolated oocytes. For the short-term culture experiment, 32 follicles were analyzed by RNAsequencing at 3h (baseline) and 24h. We compared 3h and 24h gene expression (DESeq2,R/Bioconductor) in 0.5% and 2%. Significant differences reported at padj&amp;lt;0.05 and log2FoldChange±2. Two mice strains (CB6F1;CD1) were used in all experiments. Main results and the role of chance Follicles cultured in stiff environment showed a significant reduction in follicle size compared to follicles cultured in soft environment (0.5% 226.9±17.4um, 2% 160.8±9.9um; p &amp;lt; 0.0001). These size differences suggest that granulosa cells are not proliferating in a stiff environment. Estradiol production was reduced in follicles cultured at 2% (0.5% 11.3±15.9ng/ml, 2% 0.3±0.5ng/ml, p = 0.296), while CC3 intensity was increased (CC3 staining intensity/area, 0.5% 20.4±5.8a.u., 2% 27.1±4.3a.u., p &amp;lt; 0.05), suggesting that the stiff environment impacts granulosa cell viability. Oocyte quality significantly declined in follicles cultured in 2%, with 68.9±16.8% of the oocytes degenerated, compared to 23.6±9.2% in 0.5% alginate. The use of two different mice strains yielded comparable results. Using RNAsequencing analysis, we found 1033 differentially expressed genes (DEGs) between 3h and 24h of culture in 2% alginate, mostly involved in apoptosis, inflammation and collagen processes, biological pathways typically associated with aging. The 64% of those DEGs were upregulated at 24h. Conversely, the 1295 DEGs (61% upregulated at 24h) detected comparing 3h and 24h in 0.5% alginate were relate to metabolism, cell cycle and development pathways, biological processes associated to the normal behavior of growing follicles. These differences in gene expression suggest an early-response in the transcriptome of follicles cultured in a stiff environment. Limitations, reasons for caution The stiffness of the alginate gels was based on whole ovary biomechanics; future investigations are needed to measure each follicle-stage-specific stiffness. The changes in follicles’ transcriptome in the stiff environment were evaluated only in short-term culture. This study utilizes in vitro approaches, in vivo modulation of stiffness is currently undergoing. Wider implications of the findings Increased ovarian stiffness is observed in conditions such as aging or ovaries exposed to cancer treatments. Studying how stiffness impacts oocyte quality will allow the development of suitable in vitro systems to support human folliculogenesis and in vivo approaches to modulate biomechanics of the ovary, preserving female fertility. Trial registration number not applicable

Evaluation of cellular viability in chitosan/L-arginine hydrogels.

There is a lack of studies evaluating the toxicity of nitric oxide (NO) precursors in chitosan/L-arginine hydrogels and their topical administration. However, clarifying the characteristics of these elements is essential for their possible use in non-surgical techniques of tooth movement acceleration. Such characteristics include interaction with different cell types, metabolism and drug safety. This in vitro study aimed to assess the cytotoxicity of chitosan hydrogels on human HeLa cells using different concentrations of L-arginine. The hydrogels were synthesized in a materials engineering laboratory, with a controlled environment, using 4 different L-arginine concentrations of 0%, 10%, 15%, and 20%. Once the hydrogels were prepared, their physical and chemical properties were characterized, and viability analysis was performed using 2 different methods, including a 48-h assay with Artemia salina nauplii and a 24-h cell culture with human HeLa cells followed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation assay. Data analysis was performed using a Mann-Whitney U test to evaluate positive and negative controls in the cell culture, with a significance level of 0.01. A Wilcoxon paired test contrasted the 24-h compared to 48-h Artemia salina assays, with a Kruskal-Wallis and post hoc Dunn test used to compare groups using a significance level of 0.05. In the more viscous hydrogels, Artemia salina nauplii decreased drastically in 24 h, while the 15% and 20% hydrogels had no statistical differences from the negative control. The 10% and 20% hydrogels were statistically different from the negative control when comparing cell culture data. Our findings suggest that chitosan/L-arginine hydrogels could be used in humans without toxic effects. However, more trials and tests are needed to evaluate tooth movement rate during orthodontic treatment.

Early assessment of blood culture negativity as a potential support tool for antimicrobial stewardship

ObjectiveTo assess whether 48-h negative blood culture (BC) bottles are still negative at the classic 120-h incubation endpoint and whether 48 h might be the time to make antimicrobial therapy decisions. MethodsData from the first collected bottles from bloodstream infection (BSI) episodes of single patients were retrospectively analyzed. Probabilities of bottles being negative at the classic endpoint were calculated from 0 to 120 h of incubation. ResultsAmong BC-negative episodes (4018/4901 [82.0%]), most (2097/4018 (52.2%) occurred in medicine patients. At 48 h, probability was 100.0% (95% CI, 99.9–100.0) for all 4018 patients. Of these, 1244 (31.0%) patients remained on antibiotics until 120 h. Excluding 401 (32.2%) patients who received antibiotics for another (non-bloodstream) infection, 843 (67.8%) of 1244 patients could have merited early (48-h) discontinuation of antibiotics. Stopping treatment in these patients would have led to saving 5201 days of access (943 [18.1%] days), watch (3624 [69.7%] days), or reserve (634 [12.2%]) AWaRe groups’ antibiotics, which correspond to 65.6% (5201/7928) of days of administered antibiotics in all 1244 patients. ConclusionAs an early indicator of BC negativity, the 48-h endpoint could reliably support antimicrobial stewardship, but the clinical judgment remains imperative especially when BSI is highly suspected.

Embryonic growth and effect of embryonic age on quantitative and functional characteristics of duck primary hepatocytes

Primary hepatocytes (PH) have been widely used in metabolic and disease-resistance mechanism research. However, hepatocyte isolation (HI) remains challenging in ducks. This study aimed to explore embryonic growth and the effect of embryonic age (EA) on the quantitative and functional characteristics of PH in ducks. For embryonic growth, the size and weight of the embryo and liver were determined from 6 to 28 EA (E6-E28, similar below). As EA increased, the corresponding size and weight grew significantly. Specifically, embryonic length varied from 12.5 mm to 133.0 mm, and liver width varied from 2.0 mm to 26.2 mm. Embryonic weight ranged from 0.259 g to 53.58 g, and liver weight ranged from 0.007 g to 1.765 g. Liver index initially decreased and then increased with a ratio ranging from 1.06 to 3.29%. For quantitative and functional characteristics, they were determined from E6 to E22, as there were no obvious liver features before E6 and few cells obtained after E22. The number of cells isolated in liver increased from E6 to E16 and then sharply decreased from E16 to E22. The viability remained relatively stable from E6 to E10 and then decreased from E12 to E22. The comprehensive intensity of hepatic glycogen was stronger at E8 and E14. Albumin expression increased markedly from E6 to E18 by qPCR, and the overall albumin expression was stronger at E8 and E14 by immunofluorescence assay. Hepatocyte purity exceeded 90% except for E20 and E22. During culture, cell clusters appeared after 24-h culture, which were identified as nonhepatocytes. The growth curve showed an initial increase in cell quantity followed by a decrease, another increase, and then remaining stable. In conclusion, EA had a significant effect on the quantitative and functional characteristics of PH, and the suitable EA for HI were E8 and E14. Considering better operability and quantity, E14 was the optimal EA, laying a solid foundation for further hepatocyte purification, nutrient metabolism, and disease-resistance mechanism explorations in ducks.

Efficient derivation of embryonic stem cells and primordial germ cell-like cells in cattle.

The induction of the germ cell lineage from pluripotent stem cells (in vitro gametogenesis) will help understand the mechanisms underlying germ cell differentiation and provide an alternative source of gametes for reproduction. This technology is especially important for cattle, which are among the most important livestock species for milk and meat production. Here, we developed a new method for robust induction of primordial germ cell-like cells (PGCLCs) from newly established bovine embryonic stem (bES) cells. First, we refined the pluripotent culture conditions for pre-implantation embryos and ES cells. Inhibition of RHO increased the number of epiblast cells in the pre-implantation embryos and dramatically improved the efficiency of ES cell establishment. We then determined suitable culture conditions for PGCLC differentiation using bES cells harboring BLIMP1-tdTomato and TFAP2C-mNeonGreen (BTTN) reporter constructs. After a 24-h culture with bone morphogenetic protein 4 (BMP4), followed by three-dimensional culture with BMP4 and a chemical agonist and WNT signaling chemical antagonist, bES cells became positive for the reporters. A set of primordial germ cells (PGC) marker genes, including PRDM1/BLIMP1, TFAP2C, SOX17, and NANOS3, were expressed in BTTN-positive cells. These bovine PGCLCs (bPGCLCs) were isolated as KIT/CD117-positive and CD44-negative cell populations. We anticipate that this method for the efficient establishment of bES cells and induction of PGCLCs will be useful for stem cell-based reproductive technologies in cattle.

Single-cell atlas of rainbow trout peripheral blood leukocytes and profiling of their early response to infectious pancreatic necrosis virus.

The recent development of single cell sequencing technologies has revolutionized the state-of-art of cell biology, allowing the simultaneous measurement of thousands of genes in single cells. This technology has been applied to study the transcriptome of single cells in homeostasis and also in response to pathogenic exposure, greatly increasing our knowledge of the immune response to infectious agents. Yet the number of these studies performed in aquacultured fish species is still very limited. Thus, in the current study, we have used the 10x Genomics single cell RNA sequencing technology to study the response of rainbow trout (Oncorhynchus mykiss) peripheral blood leukocytes (PBLs) to infectious pancreatic necrosis virus (IPNV), an important trout pathogen. The study allowed us to obtain a transcriptomic profile of 12 transcriptionally distinct leukocyte cell subpopulations that included four different subsets of B cells, T cells, monocytes, two populations of dendritic-like cells (DCs), hematopoietic progenitor cells, non-specific cytotoxic cells (NCC), neutrophils and thrombocytes. The transcriptional pattern of these leukocyte subpopulations was compared in PBL cultures that had been exposed in vitro to IPNV for 24h and mock-infected cultures. Our results revealed that monocytes and neutrophils showed the highest number of upregulated protein-coding genes in response to IPNV. Interestingly, IgM+IgD+ and IgT+ B cells also upregulated an important number of genes to the virus, but a much fainter response was observed in ccl4 + or plasma-like cells (irf4 + cells). A substantial number of protein-coding genes and genes coding for ribosomal proteins were also transcriptionally upregulated in response to IPNV in T cells and thrombocytes. Interestingly, although genes coding for ribosomal proteins were regulated in all affected PBL subpopulations, the number of such genes transcriptionally regulated was higher in IgM+IgD+ and IgT+ B cells. A further analysis dissected which of the regulated genes were common and which were specific to the different cell clusters, identifying eight genes that were transcriptionally upregulated in all the affected groups. The data provided constitutes a comprehensive transcriptional perspective of how the different leukocyte populations present in blood respond to an early viral encounter in fish.

Expression of Apoptosis and Autophagy Genes in HeLa and HEK 293 Cells under Conditions of Nutrient Deprivation

The goal of the study was a comparing the degree of development of autophagy in the human cervical carcinoma cells of HeLa-V and HeLa-R sublines and non-tumor human embryonic kidney cells HEK 293 under two types of starvation conditions – 24- and 48-h culture in serum-free DMEM medium and 4-h incubation in Earle’s minimal medium. The work assessed cell viability using MTT method and the expression of apoptosis (BCL2, BAX, CASP3) and autophagy (ULK1, BECN1, ATG5, ATG14, MAP1LC3B) genes using real-time PCR. Cultivation under serum starvation and Earl’s medium resulted in a significant decrease in the viability of HEK 293 cells, but had no influence on HeLa-V and HeLa-R cells. In the tumor cells of both lines, the expression of anti-apoptotic gene BCL2 increased, while in HEK 293 cells the BCL2/BAX ratio decreased and CASP3 gene was activated. In HeLa-V and HeLa-R cells, nutrient deprivation induced the stimulation of various combinations of genes ULK1, BECN1, ATG5 and ATG14 implicated in the initial stages of autophagy, but none of the treatments affected the expression of MAP1LC3B gene. In HEK 293 cells, serum starvation led to increase in expression level of BECN1, ATG5, ATG14 and MAP1LC3B genes. Thus, stimulation of autophagy in HeLa cells, especially HeLa-R, prevents the development of apoptotic processes, while in HEK 293 cells the processes of apoptosis and autophagy occur in parallel. Culture in the serum-free DMEM for 48 h appears to be most effective way to induce autophagy in tumor cell lines and, accordingly, the most suitable model for studying the role of autophagy in the development of their resistance to apoptotic pathway of death.

First report of a Herbaspirillum sp. causing leaf spots on Boston Fern (Nephrolepis exaltata) in Florida.

Boston fern (Nephrolepis exaltata) samples were submitted by a nursery operation in Florida separately to the University of Florida Plant Diagnostic Center (UFPDC, Gainesville, FL) and to the North Carolina State University Plant and Pest Diagnostic Lab (NCSU PPDL, Raleigh, NC) in October 2021. Symptoms included tan spots on pinnules, some of which progressed into pinnule blight (Fig. S1). Bacterial streaming was noted from samples in both labs. Leaf spot margins were excised, macerated in sterile tap water, then streaked onto nutrient agar (NA) plates and incubated for 48 h at 27°C. Individual representative colonies that were opaque, creamy white, mucoid, and round with smooth margins were transferred and streaked onto additional NA plates. One strain from each lab (G21-1742, UFPDC and NC40101, NCSU PPDL) was selected for subsequent characterization. A suspension of each strain was adjusted to 108 CFU/mL and infiltrated into tobacco and tomato leaves, and confluent necrosis was observed 72 h after infiltration. The isolates were Gram-negative, oxidase-positive, HR-positive on tomato and tobacco, aerobic, not pectolytic, and nonfluorescent on King's Medium B. DNA was extracted from G21-1742 using Qiagen Stool kit (Qiagen cat#51604) and the 16S rRNA gene from strain G21-1742 was amplified using 16SrRNA universal primers UP1 (5'-TACGTGCCAGCAGCCGCGGTAATA-3') and UP2 (5'-AGTAAGGAGGGTATCCAACCGCA-3') (Kuppusamy et al. 2014). The amplicon was sequenced and submitted to NCBI (Genbank Accession No. OR004801). BLASTn analysis of 16S rRNA of G21-1742 resulted in 99.7% sequence identity to the type strain of Herbaspirillum huttiense subsp. huttiense ATCC 14670T (Genbank Accession NR_024698). The 16S rRNA sequence of NC40101 was identical to that of G21-1742. To determine if the G21-1742 strain was pathogenic, Boston fern plants were inoculated by suspending bacterial cells in tap water from a 24h culture grown on NA, adjusting the suspension to 108 CFU/mL and spraying the suspension on one three-week old frond from each of three healthy Boston fern plants. A second frond from each plant was sprayed with sterile tap water. Each treated frond was individually sealed in a clear plastic bag for 24h at approximately 25°C. Inoculated plants remained on the greenhouse bench after the plastic bags were removed. The inoculation experiment was repeated once. After 4 days, tan spots were observed on pinnules of inoculated plants that were identical to the original submitted samples, while no symptoms developed on water-inoculated plants. Bacterial strains were reisolated from symptomatic plants and were morphologically identical to G21-1742. The 16S rRNA sequence of the reisolated strain was identical to G21-1742. Additionally, we conducted MLSA analysis using 12 housekeeping genes (See Table S2 for housekeeping genes and accession numbers) from the fern strains and the corresponding housekeeping genes for the type strains of 13 Herbaspirillum species, which placed the fern strains most closely with H. huttiense (see Fig. S2). This is the first known report of a Herbaspirillum sp. on Boston fern, an important ornamental crop, that renders the plants aesthetically unsaleable. Previously, a Herbaspirillum sp. was reported in Florida to cause a leaf spot and blight on greenhouse grown tomato seedlings (Obradovic et al. 2007).

Magnesium in dairy cattle nutrition: A meta-analysis on magnesium absorption in dairy cattle and assessment of simple solubility tests to predict magnesium availability from supplemental sources

Supplemental Mg sources differ in bioavailability, and solubility is one of the determining factors. We explored whether and which in vitro solubility tests could reliably differentiate the quality of supplemental Mg sources. In experiment 1, we compared 3 chemical methods using an acetic acid solution (50 mL/L, termed vinegar test), a 1 M ammonium nitrate solution, and an artificial rumen buffer fluid without rumen microbiota. The Mg solubility results suggested the vinegar test was the best method due to its robustness, simplicity, and reproducibility. In experiment 2, we validated the reliability of the vinegar test using 4 MgO sources from experiment 1 and 12 new MgO sources plus a laboratory-grade MgO as a standard. Accordingly, we repeated the vinegar test with short (0.5 h) and long (3.0 h) incubation times on these sources and then conducted ruminal incubations in 24-h batch culture experiments. The repeated vinegar test resulted in similar results as in experiment 1. Linear regression across both experiments showed the soluble Mg content (g/kg) = 44.46 (±2.55) × pH - 142.9 (±14.9), root mean square error (RMSE) = 10.2, P slope <0.001, and concordance correlation coefficient (CCC) = 0.953. The predictable pH range was from 4 to 6. The equation cannot be applied to low-alkaline sources such as Mg sulfate, Mg acetate, or a group of MgO with exceptionally high alkaline properties showing a cluster of pH above 8.5. Solubility of the MgO sources in the vinegar test ranged from 5 to 35%, whereas the 24-h ruminal incubations led to more solubility (15-70%). Nevertheless, the differences among most MgO sources were parallel to the data from the in vitro rumen solubility. Next, we performed a meta-analysis of published studies (21 studies, 94 treatments) to assess the true Mg absorption in vivo and potential factors affecting Mg absorption in dairy cows. It appeared that on average dairy cows absorbed about 20% of the Mg intake (range 10-40%), regardless of their lactation status. We revealed a new strategy to predict Mg absorption relative to dietary K as follows: true Mg absorption (g/d) = 0.3395 (±0.025, P < 0.001) × Mg intake (g/d) - 1.9273 (±1.16, P = 0.11) when dietary K ≤20 g/kg DM, and 0.154 (±1.06, P = 0.05) + 0.209 (±0.026, P < 0.001) × Mg intake (g/d) when dietary K >20 g/kg DM (RMSE = 2.19). This strategy improved the accuracy of prediction as compared with the existing prediction (CCC = 0.922 vs. 0.845). Still, over- or underestimations inherent to individual studies were evident and might be related to unaccountable factors, especially the quality of supplemental Mg sources. In conclusion, the vinegar test is a useful tool to rank inorganic Mg sources with alkaline properties. Including in vitro solubility data in Mg nutrition research could help to refine the prediction of bioavailable Mg contents and increase precision in feed formulation.

A microfabricated lab-on-chip with three-dimensional electrodes for microscopic observation of bioelectromagnetic effects of cells

Electromagnetic (EM) signals are widely used in electronic instruments and biomedical systems and might have affected the human bodies surrounded by them. However, the interaction mechanism of EM signals with biological structures is poorly understood. We propose a micro-fabricated low-frequency EM stimulation lab-on-chip with three-dimensional interdigital electrodes for observation of cell lines with microscope. The field strength between the electrodes at various frequencies is estimated through simulation. An electric field strength of 4.45Vrms/m is reached in the culture medium with a 10Vpp, 10 kHz input signal. According to the simulation results, the high end of the applicable frequency range of the testbench is 3 MHz. A prototype is fabricated using full-wafer microfabrication techniques. The impedance of the prototype between 20 Hz and 30 MHz is characterized. Moreover, human cell line HEK293T is cultured in the testbench for 24 h and observed using microscope to check the biocompatibility of the electrodes. The prototype is thus applicable to long-term microscopic observation of cell lines for study of EM effect on biological structures. The 24-h cell culturing experiment with and without EM stimulation with the proposed prototype shows that the cell growth is obviously affected by a 10 kHz EM signal.

Anti-proliferative, anti-migration, and anti-invasion activity of novel hesperidin glycosides in non-small cell lung cancer A549 cells.

Several attempts have been made to synthesize and investigate modified flavonoids to improve their potential anticancer efficacy. This study aimed to determine the in vitro anti-viability, anti-migration, and anti-invasive effects of two novel hesperidin glycosides, hesperidin glucoside (HG1) and hesperidin maltoside (HG2), compared to original hesperidin and diosmin. Inhibitory effects on normal (MRC5) and cancer (A549) cell viability of hesperidin glycosides were investigated by the trypan blue and MTS assays. A scratch assay determined the suppressive effects on cancer cell migration, and inhibition of cancer cell invasion was investigated through Matrigel™. The selectivity index (SI), a marker of cell toxicity, was also determined for A549 relative to MRC5 cells. The cell viability trypan blue and MTS assays showed similar results of the inhibition of A549 cancer cells; HG1 and HG2 had lower IC50 than original hesperidin and diosmin. The SI of HG1 and HG2 was > 2 after 72-h culture. Investigation of cell migration showed that HG1 and HG2 inhibited the ability of gap closure in a time- and dose-dependent manner. The infiltration of the Matrigel™-coated filter by A549 cells was suppressed in the presence of HG1 and HG2. This result implied that HG1 and HG2 could inhibit cancer cell invasion. Our results suggest the inhibition of cancer cell migration and invasion in a time- and concentration-related manner with a favorable toxic profile. Moreover, HG1 and HG2 appeared potentially better agents than the original hesperidin for future anticancer development.

3D printed hybrid scaffolds for bone regeneration using calcium methoxyethoxide as a calcium source.

Introduction: Hybrids consist of inorganic and organic co-networks that are indistinguishable above the nanoscale, which can lead to unprecedented combinations of properties, such as high toughness and controlled degradation. Methods: We present 3D printed bioactive hybrid scaffolds for bone regeneration, produced by incorporating calcium into our "Bouncy Bioglass", using calcium methoxyethoxide (CME) as the calcium precursor. SiO2-CaOCME/PTHF/PCL-diCOOH hybrid "inks" for additive manufacturing (Direct Ink Writing) were optimised for synergy of mechanical properties and open interconnected pore channels. Results and Discussion: Adding calcium improved printability. Changing calcium content (5, 10, 20, 30, and 40 mol.%) of the SiO2-CaOCME/PTHF/PCL-diCOOH hybrids affected printability and mechanical properties of the lattice-like scaffolds. Hybrids containing 30 mol.% calcium in the inorganic network (70S30CCME-CL) printed with 500µm channels and 100µm strut size achieved the highest strength (0.90 ± 0.23MPa) and modulus of toughness (0.22 ± 0.04MPa). These values were higher than Ca-free SiO2/PTHF/PCL-diCOOH hybrids (0.36 ± 0.14MPa strength and 0.06 ± 0.01MPa toughness modulus). Over a period of 90days of immersion in simulated body fluid (SBF), the 70S30CCME-CL hybrids also kept a stable strain to failure (~30 %) and formed hydroxycarbonate apatite within three days. The extracts released by the 70S30CCME-CL hybrids in growth medium did not cause cytotoxic effects on human bone marrow stromal cells over 24h of culture.

P-424 Effects of in vitro activation vs fragmentation on human ovarian tissue in culture

Abstract Study question What is the added value of in vitro activation (IVA) protocol compared with fragmentation only in human ovarian tissue culture? Summary answer Although histological assessment shows that the IVA increases follicle survival and growth, IVA and fragmentation stimulate extensive and nearly identical transcriptomic changes in cultured tissues. What is known already Treatments based on activation of the PTEN/PI3K pathway in ovarian tissue in vitro have been administrated to refractory primary ovarian insufficiency (POI) patients and resulted in live births following auto-transplantation of the tissue. However, some studies have shown that mere fragmentation of the tissue produces comparative effects, questioning the added value of chemical stimulation protocol which could potentially activate oncogenic pathways. Study design, size, duration Thirty-three ovarian cortical biopsies were obtained from consenting women undergoing elective caesarean section at Karolinska University Hospital Huddinge. The samples were fragmented for culture studies. Half of the fragments were exposed to bpV (HOpic) +740Y-P (IVA group) during the first 24 h of culture while half were exposed to vehicle only (fragmentation only group). Subsequently, both groups were cultured for additional 6 days. Media change was performed every other day. Participants/materials, setting, methods Effects on follicles were evaluated by counting and scoring follicles before and after the 7-day culture via HE-stained serial sections. Follicle function was assessed by quantification of steroids by UPLC-MS/MS at the end of the culture. Transcriptomic effects were measured by RNA-sequencing (RNA-seq) of the tissue after the 24-h initial culture step. Selected differentially expressed genes (DEGs) were validated by qPCR and immunofluorescence in independent culture experiments. Main results and the role of chance Compared to fragmentation only group, significantly higher follicle survival rate, increased secondary follicle number and sizes were found in IVA group. No significant differences were detected in levels of steroids in culture media on day 7 between the two groups. In our RNA-seq data, when comparing the IVA group to fragmentation only group, only 110 DEGs were found with the relaxed cutoff of FDR&amp;lt;0.1. The signaling pathways affected by IVA involved follicle growth but also inflammation and DNA damage. However, we found that the gene expression was profoundly affected in both IVA and fragmentation only groups compared to the fresh tissue: in total 3,676 and 4,223 DEGs were found (FDR&amp;lt;0.001), respectively. The top enriched gene sets in both groups included several pathways that are known to modulate follicle growth, e.g. PI3K/AKT, MTORC1. We also found that classical steroidogenesis genes (e.g. CYP11A1, CYP17A1) were significantly downregulated in both groups after 24-h culture. Moreover, upregulation of genes related to glycolysis, a process that was shown to promote activation of primordial follicles, and its upstream regulator were shown in the RNA-seq data. This effect was validated by qPCR (ENO1, PKM, LDHA, p &amp;lt; 0.0001). Future validation will be performed using western blot and immunofluorescence. Limitations, reasons for caution The study was performed in an in vitro model where tissue was isolated from the regulation of hypothalamic-pituitary-ovarian (HPO) axis. And thus, further experiments with xeno-transplantation may be needed to explore the effect of IVA in vivo in the future. Wider implications of the findings The impact of 24-h culture on gene expression in ovarian tissue far exceeds the effect of IVA. Yet, follicle growth was stimulated by IVA, which may suggest effects on specific cell populations that are diluted in bulk transcriptomics. Cell type-specific impacts need further studies to conclude about effectiveness of IVA. Trial registration number Not applicable

P-090 The in vitro motility characteristics of human sperm collected by microfluidic device

Abstract Study question Do the in vitro motility characteristics of sperm collected using a microfluidic device (ZyMot) differ from those of sperm collected by density gradient centrifugation (DGC)? Summary answer ZyMot-collected sperm had longer-lasting motility than those collected by DGC, but the head beat-cross frequency was inferior due to the absence of washing in vitro. What is known already ZyMot (DxNow, Inc., USA) can sort highly motile sperm with low reactive oxygen species (ROS), and DNA damage, without centrifugation. In IUI cycles, although the total number of ZyMot collected sperm was lower than that of DGC, it was reported that the pregnancy rate of ZyMot cycles was higher than that of DGC cycles. Washing sperm with a medium is essential for in vitro capacitation; however, the protocol for microfluidic devices lacks this procedure. The aim of this study was to clarify the motility characteristics of ZyMot-collected sperm. We also examined how washing ZyMot-collected sperm affected motility. Study design, size, duration The specimens were provided by patients who underwent semen analysis between October 2022 and January 2023, with written consent. We used 12 samples with a semen volume of 2.5 ml or more, or a motile sperm concentration of 4 million/ml or more. The same donor samples were divided into three groups (DGC, ZyMot, and ZyMot with wash). Dunnett’s test was performed with the DGC group as the control group. Participants/materials, setting, methods In the DGC group, 2-layer DGC (400G, 20 min) was performed, followed by washing (400G, 5 min). In the ZyMot group, ZyMot was used according to the manufacturer’s protocol. In the ZyMot with wash group, ZyMot-collected sperm was centrifugally washed (300G, 5 min). All samples were cultured at 30 °C until motility analysis. Sperm motility was analyzed using a computer-assisted sperm analysis system (SMAS) after 0, 4, and 24 h of semen preparation. Main results and the role of chance There was no significant difference in the motility rate of the two ZyMot groups compared to that of the DGC group at 0 and 4h after sperm preparation. On the other hand, the motility of the ZyMot group tended to be higher (53.3%, p = 0.0787), and the motility of the ZyMot with wash group was significantly higher (85.4%, p &amp;lt; 0.001) than that of the DGC group (33.5%), after 24h of sperm preparation. The straight-line velocity, curvilinear velocity, and average speed of the ZyMot and ZyMot with wash group did not differ from those of the DGC group at 0 and 4h. All speed parameters of the ZyMot with wash group were significantly higher than those of the DGC group, after 24h of culturing. Regarding sperm head movement, there was no significant difference in the amplitude of lateral head displacement between the two ZyMot groups compared to that of the DGC group at 0 and 4h of culture. In contrast, the sperm head beat-cross frequency of the ZyMot group was lower than that of the DGC group (9.18±0.31 Hz vs 8.71±0.49 Hz, p = 0.0409). Both head movement parameters of the ZyMot with wash group were significantly higher than those of the DGC group. Limitations, reasons for caution We chose semen samples with a high number of motile sperm because we divided the sperm into three experimental groups. Therefore, it is unclear whether our findings are applicable to patients with poor semen parameters. Wider implications of the findings ZyMot may be advantageous for IUI cycles because ZyMot-collected sperm maintains sustainable motility for extended periods. Alternately, ZyMot may be unfavorable compared to DGC in conventional IVF cycles due to inferior sperm head movement, which is a marker of capacitation. However, this disadvantage can be overcome by washing. Trial registration number not applicable