Tissue Culture Medium Research Articles

Oocytes Population and Development Competence of Bali Cattle Embryo In Vitro with Different Ovarian Reproductive Statuses

The present study aims to determine the potential of Bali cattle ovaries as sources of oocytes for in vitro embryo production based on different ovarian reproductive statuses. The ovaries were grouped into 4 categories: ovaries with no corpus luteum and dominant follicles (CL-DF-), those with corpus luteum and no dominant follicles (CL+DF-), those without corpus luteum but with dominant follicles (CL-DF+), and those with corpus luteum and dominant follicle (CL+DF+). The oocytes were collected via the slicing technique and grouped into 4 grades (a, b, c, and d). The oocyte’s maturation was performed using tissue culture medium 199 basic media. A drop sample (10–15 oocytes/drop) covered with mineral oil was then placed in a 5% CO2 incubator at a temperature of 38.5 ℃ for 24 h. Then, the samples were fertilized in 80 μL of fertilization medium with a final spermatozoa concentration of 1.5×106 spermatozoa/mL. After 5–6 h of in vitro fertilization, the oocytes were washed four times using the Charles Rosenkrans 1aa (CR1aa) medium. Then, the samples were cultured using the CR1aa as a base medium. The results showed no significant difference (p>0.05) for the 4 groups based on the oocyte population collected from one pair ovary as well as the number of oocytes that were suitable for maturation. However, group CL-DF+ showed a significant difference (p<0.05) in the rate of nuclear maturation (80.00±12.84), fertilization rate (80.00±4.72), and the ability of embryo development (60.19±22.45) when compared to group CL-DF-, CL+DF-, and CL+DF+. This study determines that the oocyte population of Bali cattle ovary pairs and oocytes quantity that are fit for maturation is not influenced by the reproductive status of the ovaries. However, the level of nuclear maturation, fertilization, and the ability of embryo development is higher in the ovaries without corpus luteum but with dominant follicles.

Surviving the host: Microbial metabolic genes required for growth of Pseudomonas aeruginosa in physiologically-relevant conditions.

Pseudomonas aeruginosa, like other pathogens, adapts to the limiting nutritional environment of the host by altering patterns of gene expression and utilizing alternative pathways required for survival. Understanding the genes essential for survival in the host gives insight into pathways that this organism requires during infection and has the potential to identify better ways to treat infections. Here, we used a saturated transposon insertion mutant pool of P. aeruginosa strain PAO1 and transposon insertion sequencing (Tn-Seq), to identify genes conditionally important for survival under conditions mimicking the environment of a nosocomial infection. Conditions tested included tissue culture medium with and without human serum, a murine abscess model, and a human skin organoid model. Genes known to be upregulated during infections, as well as those involved in nucleotide metabolism, and cobalamin (vitamin B12) biosynthesis, etc., were required for survival in vivo- and in host mimicking conditions, but not in nutrient rich lab medium, Mueller Hinton broth (MHB). Correspondingly, mutants in genes encoding proteins of nucleotide and cobalamin metabolism pathways were shown to have growth defects under physiologically-relevant media conditions, in vivo, and in vivo-like models, and were downregulated in expression under these conditions, when compared to MHB. This study provides evidence for the relevance of studying P. aeruginosa fitness in physiologically-relevant host mimicking conditions and identified metabolic pathways that represent potential novel targets for alternative therapies.

Asparagine Bioavailability Regulates the Translation of MYC oncogene in Lymphoid Malignancies

Non-essential amino acids are becoming increasingly important in cancer metabolism because of their roles beyond protein translation. These include providing precursors for macromolecule biosynthesis, serving as substrates for epigenetic modifications, regulating redox states, and modulating cell signaling. Acute lymphoblastic leukemia (ALL) is one such example where asparagine is shown to be conditionally essential for cancer growth and proliferation. Exploiting this metabolic vulnerability, L-Asparaginase (ASNase) has been used in the clinic to successfully treat ALL patients through depleting the circulating asparagine. Our investigation into the mechanism of resistance to ASNase in ALL cells yielded a striking correlation between asparagine bioavailability and c-MYC protein levels. c-MYC is a key transcription factor and has been shown to regulate the transcription of more than 10% of genes, including many metabolic genes, to meet the demands of tumor growth and progression. In addition, it has a well-documented role in normal hematopoiesis and hematological malignancies, including ALL, where high c-MYC levels have been shown to be a marker for leukemic initiation and growth. Using asparagine auxotrophic ALL cells that do not express asparagine synthetase (ASNS), the asparagine biosynthesis enzyme, we found that c-MYC protein levels were acutely depleted when asparagine was withdrawn from the tissue culture medium. However, c-MYC protein levels were sustained when ASNS cDNA was re-introduced into these cells. These results indicate the possibility of a reciprocal association between metabolism and a critical oncogene MYC. We also discovered that asparagine depletion inhibited the translation of c-MYC without changing its mRNA expression or altering the rate of protein degradation. Mechanistic Target of Rapamycin Complex 1 (mTORC1) and General Controlled Nonderepressible 2 (GCN2) are the two central molecular components involved in amino acid sensing and translation control. Asparagine starvation notably caused a decrease in global protein synthesis, mediated by suppression of mTORC1 and simultaneous activation of GCN2. Our studies using small molecule inhibitors and polysome profiling showed that repression of c-MYC mRNA translation is not due to the activation of the GCN2 pathway or a consequence of global translation suppression. In addition, both the '5' and '3' untranslated regions (UTRs) were not required for this inhibitory effect on c-MYC mRNA translation during asparagine depletion. Our results suggest that while asparagine depletion causes a defect in global translation at the initiation step, its effect on MYC mRNA translation is possibly due to elongation stalling. While ASNase can efficiently deplete circulating asparagine, its clinical applications are limited to ALL due to its low-level expression of ASNS. However, aspartate, a critical substrate for asparagine biosynthesis, can be pharmacologically targeted using electron transport chain (ETC) inhibitors. Our studies in ASNase-resistant ALL and lymphomas showed that while asparagine depletion alone did not affect c-MYC protein expression, co-treatment with ETC inhibitors (phenformin) showed a significant decrease in c-MYC protein levels. We extended this observation to test the synergy of ASNase and phenformin to treat an orthotopic MYC-driven B-cell lymphoma model. While either of the single treatments only modestly reduced the tumor burden, the double treatment showed the most significant reduction in the tumor burden, which correlated with a significant reduction of c-MYC protein expression. These results suggest that perturbing the asparagine biosynthesis pathway can be clinically employed to inhibit c-MYC protein expression and improve therapeutic outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

BabyBoom: 3-Dimensional Structure-Based Ligand and Protein Interaction Prediction by Molecular Docking.

Baby Boom (BBM) is a key transcription factor that triggers embryogenesis, enhances transformation and regeneration efficiencies, and regulates developmental pathways in plants. Triggering or activating BBM in non-model crops could overcome the bottlenecks in plant breeding. Understanding BBM's structure is critical for functional characterization and determination of interacting partners and/or ligands. The current in silico study aimed to study BBM's sequence and conservation across all plant proteomes, predict protein-protein and protein-ligand interactions, and perform molecular docking and molecular dynamics (MD) simulation to specifically determine the binding site amino acid residues. In addition, peptide sequences that interact with BBM have also been predicted, which provide avenues for altered functional interactions and the design of peptide mimetics that can be experimentally validated for their role in tissue culture or transformation media. This novel data could pave the way for the exploitation of BBM's potential as the master regulator of specialized plant processes such as apomixes, haploid embryogenesis, and CRISPR/Cas9 transgenic development.

Comparative Studies of Palmatine with Metformin and Glimepiride on the Modulation of Insulin Dependent Signaling Pathway In Vitro, In Vivo & Ex Vivo.

(1) Insulin resistance, a symptom of type 2 diabetes mellitus (T2DM), is caused by the inactivation of the insulin signaling pathway, which includes IRS-PI3K-IRS-1-PKC-AKT2 and GLUT4. Metformin (biguanide) and glimepiride (sulfonylurea) are both drugs that are derivatives of urea, and they are widely used as first-line drugs for the treatment of type 2 diabetes mellitus. Palmatine has been previously reported to possess antidiabetic and antioxidant properties. (2) The current study compared palmatine to metformin and glimepiride in a type 2 diabetes model for ADME and insulin resistance via the PI3K/Akt/GLUT4 signaling pathway: in vitro, in vivo, ex vivo, and in silico molecular docking. (3) Methods: Differentiated L6 skeletal muscle cells and soleus muscle tissue were incubated in standard tissue culture media supplemented with high insulin and high glucose as a cellular model of insulin resistance, whilst streptozotocin (STZ)-induced Sprague Dawley rats were used as the diabetic model. The cells/tissue/animals were treated with palmatine, while glimepiride and metformin were used as standard drugs. The differential gene expression of PI3K, IRS-1, PKC-α, AKT2, and GLUT4 was evaluated using qPCR. (4) Results: The results revealed that the genes IRS-PI3K-IRS-1-PKC-AKT2 were significantly down-regulated, whilst PKC-α was upregulated significantly in both insulin-resistant cells and tissue animals. Interestingly, palmatine-treated cells/tissue/animals were able to reverse these effects. (5) Conclusions: Palmatine appears to have rejuvenated the impaired insulin signaling pathway through upregulation of the gene expression of IRS-1, PI3K, AKT2, and GLUT4 and downregulation of PKC-expression, according to in vitro, in vivo, and ex vivo studies.

Potato (Solanum tuberosum L.) Plantlet Regeneration in Ammonium Nitrate Free Stock Solution-1 of Murashige &amp; Skoog (MS, 1962) Plant Tissue Culture Medium

Potato (Solanum tuberosum L.) plantlet regeneration potentiality was studied in ammonium nitrate (NH4NO3) free tissue culture medium. Four different treatments were designed from the compositions of the stock solution-01 of plant tissue culture medium. Those were -Treatment-1 (Stock solution -01 as recommended by Murashige &amp; Skoog (1962), Treatment-2 (MS stock solution-01 without having NH4NO3), Treatment-3 (MS stock solution-01without NH4NO3 but other component had double concentration), Treatment-4 (Readymade MS powder, Duchefa Biocheme, The Netherland). Shoot length, shoot diameter, node number and leaf number per plantlet were highest in Treatment-4 at 14, 21 and 28 days after inoculation (DAI). Shoot regeneration parameters were statistically similar with the treatment-3 and the check treatment-1. But the check treatment-1 showed better result in root number and root length (cm) as compared to treatment-3 and treatment-4. The treatment-2 showed lowest result in each of the said parameter. The stock solution-01 which was formulated without ammonium nitrate and has double dose of other ingredient has the potentiality for potato plantlet regeneration, but it was not as suitable as Readymade MS powder (Duchafa, The Netherland).

Rational design of a Nivolumab-based ANTI-PD-1 single chain variable fragment that blocks the interaction between PD-1 expressed on T-CELLS and PD-L1 ON CHO cells

Antibodies that block the interaction between PD-1 expressing T-cells and cancer cells expressing PD-L1 play a central role in contemporary immunotherapy regimes [1-3]. We previously reported the isolation of a single chain variable fragment (scFv) of the monoclonal anti-PD-1 antibody Nivolumab, that binds to purified PD-1 and blocked its interaction with PD-L1 [4]. This anti-PD-1 scFv did not, however, function in a cell-based assay designed to detect the disruption of the PD-1/PD-L1 interaction, a result likely due to its poor solubility in tissue culture media. Herein we report that following a series of structure-based rational design analyses, including Aggreescan3D, we have isolated a variant of the anti-PD-1 scFv having significantly improved solubility in tissue culture medium. Moreover, this soluble anti-PD-1 scFv variant disrupted the interaction between PD-1 expressed on Jurkat Cells and PD-L1 expressed on CHO cells. These findings are discussed in terms of the related observation that the residues mutated to form the anti-PD-1 variant are conserved in many other scFvs; thus, the properties of a range of scFvs will likely be enhanced by similar mutations of the conserved residues.

Effect of Plant Preservative MixtureTM on Endophytic Bacteria Eradication from In Vitro-Grown Apple Shoots.

Endophytic contaminants are a common problem for the in vitro propagation of woody plants and have significant economic repercussions for the conservation of plant genetic resources and commercial micropropagation. In this study, first, the microbial contamination that appeared around the base of in vitro-grown apple shoots was identified as Bacillus megaterium. Then, plant preservative mixture (PPMTM) was used as a bactericidal agent in plant tissue culture. Its efficacy for eradicating endophytic B. megaterium in in vitro cultures of apple was tested. In vitro-contaminated shoots were grown in tissue culture medium supplemented with 0.2% v/v PPMTM for 12 weeks and then transferred to medium without any PPMTM and cultured for 24 weeks. This study showed that PPMTM is an effective agent for controlling the growth of B. megaterium. Our results highlight the species-specific response of apple shoots to PPMTM. PPMTM was effective in controlling endogenous microbial contaminations from apple varieties ‘Golden Delicious’, ‘Landsberger Renette’, ‘Suislepper’, and ‘Aport krovavo-krasnyi’; meanwhile, in ‘KG 7’ and ‘Gold Rush’, all the plants grown in the absence of PPMTM were still bacterially contaminated, even though they were pre-treated for 12 weeks in PPMTM-supplemented medium. These results therefore suggest the essentiality of further testing of extended incubation of PPMTM in these cultivars that had outbreaks of bacterial contamination.

Aerosol Survival, Disinfection and Formalin Inactivation of Nipah Virus.

Nipah virus is a relatively newly discovered emerging virus on the WHO list of priority pathogens which has the potential to cause outbreaks with high fatality rates. Whilst progress is being made in the development of animal models for evaluating vaccines and therapies, some of the more fundamental data on Nipah virus are lacking. We performed studies to generate novel information on the aerosol survival of Nipah virus and to look at the efficacy of two common disinfectants. We also performed studies to evaluate the inactivation of Nipah virus by using neutral buffered formalin. Nipah virus was relatively stable in a small particle (1–5 µm) aerosol in the dark, with it having a decay rate of 1.46%min−1. Sodium hypochlorite (at 10%) and ethanol (at 80%) reduced the titre of Nipah virus to undetectable levels. Nipah virus that was in tissue culture medium was also inactivated after 24 h in the presence of 10% formalin.

P12.06: Effect of Supplementing Taurine to the Developed Lowtemperature Islet Preservation Solution

Introduction: Pancreatic islet transplantation has recently emerged as one of the most promising therapeutic approaches for improving glycemic control in type 1 diabetes patients. However, one of the problems with islet transplantation is that it is impossible to culture the isolated islet for extended periods to while recipients are selected, tested and prepared for surgery. To address this problem, we developed the islet preservation solution, and to improve the function of the preservation solution, we added taurine and conducted a preservation experiment. Taurine is antioxidant activity and it is not a classical free radical scavenger. Therefore, its mechanism remains unclear but it controls osmotic pressure. Our hypothesis is that taurine supplementation can improve islet recovery and enhance islet function after low temperature preservation. Methods: CMRL 1066 medium (Corning) was used as a control group, and a comparison group was prepared some compounds OPTI(Optipharm presevation) solution (with taurine) and OPTI-T solution (without taurine). To confirm the preservation effect of these solutions, we isolated islet from adult Yucatan pig’s pancreas using standard enzymatic digestion (Nordmark) followed by Ficoll purification, these islets were preserved in a humidified tissue culture incubator with preservation solutions and preserved in 4℃ for up to 4 weeks. We compared islet survival in groups of islets. To islet viability and islet functionality, we utilized Glucose Stimulated Insulin Secretion (GSIS) for measuring insulin release, calculated stimulation index (SI) and AO/PI viability staining for during the preservation period. Results: Highest islet recovery was preserved in the group of pig islets cultured with Taurine as compared to the group of islets hypothermically preserved in standard tissue culture media over the 4 week preservation period (p<0.001 using ANOVA). The porcine islet viability/cell number ratio in the OPTI solution maintained a high survival rate for up to 2 weeks (94%). In contrast, porcine islet in OPTI-T solution and CMRL 1066 supplemented media significantly reduced viability after 1 weeks. OPTI solution (with taurine) was significantly higher than OPTI-T solution (without taurine) and CMRL 1066 during 4 weeks after preservation. Glucose stimulation insulin secretion test was performed for functional analysis. As a result, a level of porcine insulin was higher during 2 weeks in the OPTI solution than OPTI-T solution and CMRL 1066. But after 3 weeks preservation, insulin level in high glucose, significantly decreased. Therefore, the viability and functionality of islets were improved by adding taurine to the developed preservation solution. Conclusion: Taurine supplementation has been shown to highest islet recovery, viability and insulin secretion level for 2 weeks. Based on this data, we have performed 2 weeks cold storage of isolated islets for current clinical islet transplantation. Ministry of trade industry and energy, KEIT 20011276, NTIS 1415172664.

Preliminary Study of &lt;i&gt;Pleurotus ostreatus&lt;/i&gt; (Jacq.) P. Kumm. Spent Mushroom Compost as Nutrient Supplement on the Shoot Induction of Fig Tissue Culture

Spent mushroom compost is identified as waste substrate. As the mushroom agriculture industry is growing rapidly, the production also increases and contributes to large amounts of spent mushroom compost annually. The conversion of spent mushroom compost as waste to wealth is a great concept that can give value to agricultural industry and solid waste management of mushroom cultivation. The use of SMC as nurient supplement may reduce the production cost for in vitro culture of fig plant. This research is to study the use of Pleurotus ostreatus (Jacq.) P. Kumm. spent mushroom compost (SMC) as a nutrient supplement of the media in shoot induction of fig plant. Nodal segment of fig’s stem was cultured in MS media supplemented with different concentrations of SMC. With presence of SMC, 10% SMC have the highest shoot proliferation (2.167 ± 1.169) and length of shoot (0.154 ± 0.060 cm). The study revealed that in presence of 10% of SMC could produce new shoots at a frequency which was comparable to the control (p&lt;0.05; n=3; Tukey’s multiple range test). Among the different media combinations of plant growth regulators with SMC, 15 μM Kinetin with 10% SMC is the most effective treatment which gave maximum number of shoot proliferation (3.00 ± 1.27). 30 μM IAA with 10% SMC media have the highest leaves regenerated which were 1.33 ± 0.84. In conclusion, the SMC added in media for plant tissue culture had positive effect on shoot induction thus highlighted the potential of waste substrate of SMC to act as nutrient supplement for plant tissue culture.

Creation of a High-Fidelity, Low-Cost, Intraosseous Line Placement Task Trainer via 3D Printing.

The description of procedural task trainers includes their use as a training tool to hone technical skills through repetition and rehearsal of procedures in a safe environment before ultimately performing the procedure on a patient. Many procedural task trainers available to date suffer from several drawbacks, including unrealistic anatomy and the tendency to develop user-created 'landmarks' after the trainer tissue undergoes repeated manipulations, potentially leading to inappropriate psychomotor skill development. To ameliorate these drawbacks, a process was created to produce a high-fidelity procedural task trainer, created from anatomy obtained from computed tomography (CT) scans, that utilize ubiquitous three-dimensional (3D) printing technology and off-the-shelf commodity supplies. This method includes creating a 3D printed tissue mold capturing the tissue structure surrounding the skeletal element of interest to encase the bony skeletal structure suspended within the tissue, which is also 3D printed. A tissue medium mixture, which approximates tissue in both high-fidelity geometry and tissue density, is then poured into a mold and allowed to set. After a task trainer has been used to practice a procedure, such as intraosseous line placement, the tissue media, molds, and bones are reclaimable and may be reused to create a fresh task trainer, free of puncture sites and manipulation defects, for use in subsequent training sessions.

Evaluating suitable low-cost agar substitutes, clarity, stability, and toxicity for resource-poor countries' tissue culture media

Over 20% of plant species assessed are threatened with extinction. Most of these plants have food security implications, especially in developing countries. Traditional seeds and cutting propagation techniques cannot counter the loss rate, and tissue culture provides a fast alternative to conventional propagation techniques. However, tissue culture has been considered too expensive for developing countries negatively impacted by food insecurity. A gelling agent is the costliest media component in plant tissue culture. This study aims to assess different gelling agents to find suitable ones with low cost and acceptable gelling properties for developing countries, especially in rural areas. Plantain explants were propagated on 16 starch-based substrates to evaluate their suitability as tissue culture gelling agents. This study compared the cost of various substrates and their gelling properties, such as clarity, toxicity, and texture, with agar as a reference gelling agent. Some substrates, such as xanthan, had good gelling properties, but their cost was too high (5.98 Euro L−1) to be considered low-cost. Other substrates, such as cassava starch, did not have suitable gelling properties; however, the cost was low (0.99 Euro L−1). Two of the substrates, mung bean and Isabgol, had suitable gelling properties and cost less than one euro. Therefore, smallholder banana and plantain farmers in resource-poor countries can undertake tissue culture operations with mung bean and Isabgol as gelling agents with minimum cost.

Mouse pharmacokinetics and metabolism of the phenylurea thiocarbamate NSC 161128

NSC 161128, a phenylurea thiocarbamate, displays activity against the NCI60 anti-cancer cell line panel and xenograft models. The metabolite N-methyl-N'-phenylurea (M10) has been detected in animal plasma; however, detection and quantification of other putative NSC 161128 metabolites have not been undertaken. The purpose of this study was to characterize the pharmacokinetics and metabolism of NSC 161128 in mice and under in vitro conditions. An LC-MS/MS assay was developed to evaluate stability and in vitro metabolism of NSC 161128 in liver microsomes and S9 fractions. Single-dose pharmacokinetic profiles for NSC 161128 and its metabolite M10 were obtained following intraperitoneal (I.P.) administration. A sensitive and specific positive ionization LC-MS/MS method for measuring NSC 161128 and its metabolites was developed. HPLC separation was achieved under gradient elution using an aqueous methanol mobile phase containing 0.05% formic acid and 0.05% ammonium hydroxide. The assay was linear over the range 1.0-1000ng/mL. NSC 161128 was stable in aqueous solution and tissue culture media, but not in plasma, where rapid degradation of NSC 161128 to the metabolite M10 was observed. Following I.P. administration of a 200mg/kg dose to male CD1 mice, the peak plasma concentration of NSC 161128 was 255ng/mL after 5min with a plasma half-life of 138min. Potential bioactivation of NSC 161128 was explored using mouse S9. An analytical LC-MS/MS method was successfully developed for the detection and quantification of NSC 161128 and its metabolites. These results increase the understanding of NSC 161128 pharmacokinetic and metabolic properties.

Developed applicability of a bacterial cellulose matrix as a gelling substitute for plant tissue culture media

Bacterial cellulose (BC) is a natural biodegradable, eco-friendly fiber, lying within the nanoscale range. It is reputable for its various physical and chemical qualities, like high hydrophilicity, immense crystallinity, ease of sterility, being toxin-free, and extremely pure. Adding to its wide applicability in different fields, this study evaluated the applicability of a developed gelling substitute for plant tissue culture media. The BC matrix was characterized under the acronym PLATIBACGEL (PLAnt TIssue Culture BActerial Cellulose GEL), formed by Komagataeibacter hansenii AS.5, preisolated from rotten apple waste. Scanning electron microscope, Fourier-transform infrared, X-ray diffractometer, and tensile strength analyses confirmed the formation of purified, porous, and heterogeneous densely packed multiple network polymers possessing cellulose properties. The water holding capacity (WHC) values of wet and dried BC membranes were 9179% and 226.9%, respectively, and the water absorption rate (WAR) of dry BC membranes was higher than that of wet membranes. Using BC as a tissue culture gelling agent, six genotypes from tomato and wheat seeds were cultured in vitro, for guaranteeing explant genetic diversity, over seven treatments. Treatment 5, included PLATIBACGEL as the main constituent, improved and sustained all in vitro seed germination, root penetration, and plant support. Likewise, repeated tomato micropropagation subcultures were successful. Results demonstrated applying PLATIBACGEL as a promising, reusable, cheap, and reliable alternative plant micropropagation media gelling agent. Wherefore, plant cellular developers and tissue-culturists can utilize bio-polymers like BC for better understanding plant cell response to different in vitro culturing conditions, with expected beneficial returns on gelling agents industry and markets as well.Graphical abstract

Effects of Aqueous and Ethanolic Extract of Rhazya stricta on Control of Date Contamination Under In Vitro Conditions

Background: Contamination of tissue culture medium is a common problem that causes the loss of some or all cultures. Despite all measures taken in tissue culture laboratories to prevent culture contamination, bacterial and fungi contamination is still a significant problem under in vitro culture. Achieving a suitable method for controlling the contaminants in the in vitro culture medium will be a considerable development in the micropropagation of dates. Objectives: We aimed to investigate the effects of different aqueous and ethanolic extracts of Rhazya stricta on the disinfection of dates under in vitro culture. Methods: A factorial test was performed based on a completely randomized design with three replications. The study included three factors: Plant organs (leaves and roots), solvent type (ethanolic and aqueous), and extract concentration (0, 25, 50, and 75 mg/mL). Results: The lowest contamination (38.33%) was seen in the treatment with ethanolic root extract (50 ppm). In comparison, the control and the treatment containing aqueous leaf extract (with concentrations of 0 and 25 ppm) resulted in the highest contamination rate (100%) in tissue culture conditions. Conclusions: The alcoholic extract of medicinal plants can be used to disinfect the explants and the substrate.

Protection of Grouper Against Cryptocaryon irritans by Immunization With Tetrahymena thermophila and Protective Cross-Reactive Antigen Identification.

Vaccination is an effective method to prevent Cryptocaryon irritans infection. Although some vaccines have been developed, large-scale production of these vaccines is costly. Development of a heterogenous vaccine generated by low-cost antigens is an alternative method. In the present study, grouper immunized with Tetrahymena thermophila, a free-living ciliate that easily grows in inexpensive culture media at high density, showed protective immunity against C. irritans infection. Higher immobilization against C. irritans theronts was detected in T. thermophila–immunized grouper serum, which suggested the existence of a cross-reactive antibody in the serum. By immunoprecipitation and mass spectrometry analyses, tubulin was identified as a potential cross-reactive antigen between C. irritans and T. thermophila. Recombinant T. thermophila tubulin protein (rTt-tubulin) and its antibody were prepared, and immunofluorescence showed that both C. irritans and T. thermophila cilia were stained by the anti–rTt-tubulin antibody. Grouper immunized with rTt-tubulin showed a reduced infective rate after the C. irritans challenge. An enhanced level of C. irritans–binding immunoglobulin M (IgM) antibody was detected in serum from rTt-tubulin–immunized grouper. Moreover, specific antibodies were also found in the mucus and tissue culture medium from rTt-tubulin–immunized grouper. Overall, these findings suggested that vaccination with T. thermophila elicits cross-reactive protective immunity in grouper against C. irritans, and T. thermophila may be a potential heterologous antigen for vaccine development.

The Impact of Media Supplement on the Viability, Proliferation, and Differentiation Potential of Bone Marrow-Derived Mesenchymal Stem Cells

Background: Bone marrow-derived mesenchymal stem cells (bmMSCs) are one of the most promising therapeutic methods in modern medicine. These cells are grown in tissue culture media, commonly supplemented with fetal bovine serum (FBS), but such supplement carries a number of drawbacks, such as immunological reaction and composition variation. Another supplement known as insulin-transferrin-selenium could act as serum replacement and help enhance the experimental results and therapeutic value of MSCs. Objective: The objective was to investigate the effect of tissue culture supplement in regard to bone marrow MSCs viability, proliferation, and differentiation potential. Materials and Methods: Human bmMSCs were grown in tissue culture plates at seeding density of 5 × 103 cells/cm2. The cells were divided into two groups, 10% FBS supplement or insulin-transferrin-sodium (ITS) supplement. The viability of the cells was assessed with live/dead cells kit (ethidium homodimer-1 and calcein). Cell proliferation was assessed with MTS assay, while multilineage differentiation potential was assessed with human MSC functional identification kit. Results: Our results showed that the viability of MSCs was comparable between FBS and ITS supplement groups at 24 h and 48 h intervals. Both groups showed similar proliferation capacity after 48 h and 72 h of incubation with no significant statistical difference. The cells from both groups were able to differentiate into osteoblasts, adipocytes, and chondrocytes. Conclusion: Insulin-transferrin-selenium supplement could be used as an alternative to FBS in laboratory experiments and clinical applications (such as cell therapy) to avoid the drawbacks of FBS and enhance the outcome of these applications.

Development of biocompatible superelastic materials and shape memory implants based on titanium nickelide for the creation of highly effective medical technologies

The paper presents fundamental and applied research in a new direction in science and technology, which has a world priority – medical materials and implants with shape memory. The main directions of the development of a new generation of more advanced biocompatible superelastic implantable materials and long-term shape memory implants based on titanium nickelide are formulated. The physical, biological principles of creating a new class of biocompatible superelastic materials based on titanium nickelide with a certain set of properties for various fields of medicine using technologies of induction melting, self-propagating high-temperature synthesis, and sintering are presented.Among the materials created, the development of alloys for long-term functioning in a living organism is of great importance: monolithic elastic, porous and ultra-thin porous-permeable materials, fabric and textile materials made of thin filaments based on titanium nickelide. The main medical and technical requirements for a new class of implants of the future have been identified. Criteria for the biomechanical compatibility of metallic materials under deformation conditions in aggressive tissue media have been determined. Conditions are formulated under which monolithic and porous materials exhibit properties that are optimal for implantation. A comprehensive study of the properties of cell incubators made of porous titanium nickelide was carried out, and the morphological characteristics and patterns of cell development in the porous structure of bulk biomaterials made of titanium nickelide were revealed.The main directions of creation of unique medical technologies based on the developed materials are characterized. The principles of application of biocompatible superelastic materials in various fields of medicine are described. Titanium nickelide-based materials and implants with shape memory implanted into the body are intended for a wide range of medical purposes in dentistry, traumatology, surgery, ophthalmology, vascular surgery, oncology and other fields of medicine as implants for osteosynthesis, endoprosthetics, replacement of tissue defects, creating anastomoses and support frames of organs.

Abstract 1965: Complete remission in advanced epithelial ovarian cancer is predicted by metabolic signatures measured in the media of patient’s primary culture 3D tumor explants: A metabolomic analysis

Abstract Ovarian cancer is the leading cause of gynecologic cancer death with over 21,000 new diagnosis and 13,000 deaths each year in the US. Treatment with platinum-based chemotherapy induces remission in up to 80% of patients yet the majority relapse and die from their disease within 5 years. We recently reported the analysis of patients with advanced ovarian cancers using plasma metabolomics to predict response and survival (D’Amora et al Gynecol Oncol, October 2021). As part of that study human tumor explants were submitted for the measurement of platinum resistance by ex vivo analysis of programmed cell death (EVA/PCD࣪). Media obtained from the non-drug exposed control cultures were examined by mass spectrometry for the quantification of 186 individual metabolites using the Biocrates’ p180 platform. Results obtained with the tissue culture media identify biochemical signatures that correlate with the achievement of complete pathologic remission. Among the most discrimination findings were the media concentrations of phosphatidyl choline (PC) aa c42:0 and the ratio of (PC) aa C40:2 divided by (PC) ae C42:1 suggesting either hyperactivity of lipid elongases or defective beta oxidation of very long chain fatty acids in drug refractory patients. When we compared the results of 8 patients who achieved pCR with 3 patients with residual disease the aa C42:0 concentration and the ratio aa C42:0/ae C42:1 provided an AUC= 0.958 (p. &amp;lt; 0.01) The findings suggest that peroxisomal dysfunction leads to the accumulation of very long chain fatty acid lipids. These species are known to induce endoplasmic reticulum and oxidative stress and are associated with the neurodegenerative diseases like Zellweger syndrome. We have reported similar findings in drug refractory breast cancer patients (da Silva et al, Oncotarget, August Aug 2018) suggesting common lipid metabolic changes in drug resistant adenocarcinomas. The results of these analyses could provide prognostic and predictive information for patients diagnosed with advanced epithelial ovarian cancers. Further studies correlating these findings with plasma metabolic signatures are underway as will be reported. Citation Format: Adam J. Nagourney, Paulo D'Amora, Ismael Dale Cotrim Guerreiro Silva, Steven S. Evans, Robert A. Nagourney. Complete remission in advanced epithelial ovarian cancer is predicted by metabolic signatures measured in the media of patient’s primary culture 3D tumor explants: A metabolomic analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1965.