Culture Plates Research Articles

Isolation and identification of intestinal cellulolytic bacteria from red swamp crayfish (Procambarus clarkii)

Red swamp crayfish (Procambarus clarkii) like to eat aquatic plants. The intestinal microorganism plays an important role in cellulose degradation and utilization. Isolating bacteria which can degrade cellulose from the intestines of P. clarkii can provide a theoretical basis for the development of probiotics in forage for P. clarkii. Three selective media including carboxymethyl-cellulose, microcrystalline cellulose, and cellobiose were used in plate cultures for 48h and then dyed with Congo red. It was found that 5 strains produced hydrolytic rings on 3 culture media at 48h. Based on the results of 16S rRNA molecular analysis, strains C, E, G, H, and M were identified as Citrobacter sp., Staphylococcus sp., Acinetobacter johnsonii, Shewanella sp., and Aeromonas caviae, respectively. Specifically, Staphylococcus sp. exhibited the strongest capacity for the degradation of cellulose. Shewanella sp. showed the strongest degradation capacity for cellobiose. Acinetobacter johnsonii and Shewanella sp. can degrade cellulose and are expected to be used as probiotic feed for P. clarkii. This study can provide a theoretical basis for the healthy culture of P. clarkii.

Toxicity and cytokine release from human dental pulp stem cells after exposure to universal dental adhesives cured by single peak and polywave LEDs

Objectivesto assess the impact of universal adhesives, cured with single-peak and polywave LEDs, on the metabolic activity and cytokine release of human dental pulp stem cells (hDPSCs). In addition, analyze the degree of conversion (DC) of the adhesives cured with the different LEDs. MethodsDiscs (5 mm diameter, 1 mm thick) were prepared using three universal adhesives: Single Bond Universal (SBU, 3 M ESPE), Optibond Universal (OBU, Kerr), and Zipbond Universal (ZBU, SDI). These discs were cured for 40 s using a single-peak (DeepCure, 3 M ESPE) or a polywave light-emmiting diode (LED) curing unit (Valo Grand, Ultradent). After 24 h, the specimens were placed in 24-well culture plates, each containing 1 mL of culture medium for 24 h. hDPSCs (1.8 ×104) were seeded in 96-well plates and allowed to grow for 24 h. Subsequently, the cells were exposed to the extracts (culture medium containing eluates from the adhesive discs) for an additional 24 h. Cells not exposed to the extracts were used as a control group. The mitochondrial metabolism was assessed using the MTT assay and the cytokine release evaluated through MAGPIX. The degree of conversion of the adhesives was analyzed using FTIR (n = 5). The results were analyzed by ANOVA two-way and Tukey’s test. ResultsOBU and ZBU eluates caused a statistically significant reduction in mitochondrial metabolism, regardless of the LED used, indicating their cytotoxicity. In contrast, SBU did not significantly affect the MTT results, resembling the control group. A higher release of cytokines IL-1, IL-6, IL-10, and TNF-α were found in association to ZBU. SBU, on the other hand, increased the release of IL-8. OBU did not influenced the cytokine release. SBU presented the higher DC, while OBU and ZBU had similar DC, lower than SBU. SignificanceIn conclusion, universal adhesives exhibit toxicity towards hDPSCs, but the extent of toxicity varies depending on the adhesive material. ZBU was associated with increased cytokine release, particularly pro-inflammatory mediators, from hDPSCs. The different LEDs did not influenced the cytotoxicity of the evaluated adhesives.

Conducting practical classes of phase II MBBS students (CBME batch) during COVID 19 pandemic: Experience of a paraclinical resident

ABSTRACT The Medical Council of India, now the National Medical council changed the teaching pattern of the Under Graduate Medical Course (MBBS) after 22 years. The new revised curriculum and teaching pattern is known as Competency Based Medical Education (CBME). CBME pattern started from the 2019 MBBS batch. In the second year of MBBS, they have to learn para clinical subjects, Pathology, Microbiology, and Pharmacology. Teaching Practical classes to MBBS students for the first year PG residents is a new experience for them, as they just start their paraclinical specialty journey, and it is even more difficult for them when they are teaching the CBME batch. To take one practical class MBBS, PG residents must start the preparation before at least one week. In that one week, we read the topic thoroughly with concepts. They must prepare the PowerPoint presentation followed by a microteaching presentation to the departmental faculties and have to do the correction to the presentation. PG residents make many arrangements like smear slides for staining, culture plates, and biochemical reactions to demonstrate to MBBS students but when only 4–5 students were attending the physical demonstration classes, all the preparation look useless.

177 So the Bone Flap Hit the Floor, Now What?

INTRODUCTION: Dropping the free bone flap on the operating room floor during a neurosurgical procedure is a rare and poorly documented accident. In fact, it has been reported in up to 3 out of 1000 operations involving craniotomies. The literature guiding its management is insufficient: to date, there is no protocol for sterilization of an accidentally contaminated flap in order to reposition it safely on the patient's skull. METHODS: Cadaveric human bone flaps were collected and intentionally dropped onto the floor of a neurosurgical operating room. Bacterial contamination from these flaps was quantified in colony forming units (CFU) counted on culture plates inoculated with the fragments. Identified species were included in a mixed preparation of microorganisms used to artificially contaminate fragments from 29 other individual flaps in a standardized manner. All 29 contaminated flaps were previously cut into five pieces and the following five sterilization protocols were completed on a fragment per flap each: rinsing with saline, mechanical debridement with sterile gauze, rinsing with antibiotics, rinsing with alcoholic chlorhexidine and flash sterilization by autoclave. Decontamination was measured by inoculation onto culture plates and CFU count. RESULTS: The observed contamination from a flap dropped on an operating room floor can be significant (up to 120 CFU cultured per flap). All protocols tested decreased bacterial load of the bone flaps to different degrees: saline by 95.7%, mechanical debridement by 97.6%, antibiotic bath by 99.5%, alcoholic chlorhexidine by 99.9% and flash sterilization by 100.0%. CONCLUSIONS: In the event of the accidental fall of a bone flap, decontamination by rinsing in an alcohol-chlorhexidine solution, which seems to represent the best combination of effectiveness versus the risks and the complexity of the method, could be considered.

Antibacterial Activity of the Vancomycin and Cefotaxime-Incorporated Total Etch Adhesive System - An In Vitro Study.

Marginal failure at the resin dentin interface promotes biofilm formation, which further leads to secondary caries and hypersensitivity. This likelihood also increases if residual bacteria are present following cavity preparation. In order to achieve a proper biological seal without jeopardizing bonding, efforts were made to functionalize the adhesive system with antibacterial activity. Aim and objectives: To appraise the antibacterial activity of a total-etch adhesive system against S. mutans with and without incorporation of antibiotics Vancomycin and Cefotaxime. A commercially available 5th-generation total-etch bonding agent (Te-Econorm) was used. S. mutans broth had been standardized and streaked over Muller-Hinton agar culture medium and round wells about 6 mm in diameter were made in the centre of the agar plates. Each experimental group comprised 10 samples, which include: Group 1 - 30µg Cefotaxime, Group 2- 30µg Cefotaxime + Bonding agent, Group 3- 30µg Vancomycin, Group 4- 30µg Vancomycin + Bonding agent, Group 5- Bonding agent, and Group 6- No material. Inoculated culture plates were examined for the zone of inhibition after incubation at 37° C for 24 hours. There was a significant difference in the mean diameter of zone of inhibition (p=0.000), with the maximum exhibited by Group 4, followed by Group 3 and Group 2. The least zone of inhibition was exhibited by Groups 1 and 5. The negative control showed no zone of inhibition. The combination of Vancomycin and bonding agent had superior antibacterial activity against S. mutans in comparison to cefotaxime and bonding agent.

Pseudomonas aeruginosa from a Tertiary Teaching Hospital: A Study of AmpC and Biofilm-producing Clinical Isolates

Abstract Introduction: Opportunistic Pseudomonas aeruginosa is one of the most prevalent bacteria with a broad spectrum of human-associated infections. The World Health Organization (WHO) claims that Pseudomonas aeruginosa infection is a typical healthcare-associated disease of critical or high concern. It is well known for being inherently resistant to many antibiotics and has the ability to produce biofilm. In the present study, we aimed to investigate P. aeruginosa with reference to biofilm and AmpC β-lactamases producing isolates in a tertiary care hospital. Materials and Methods: An observational cross-sectional study was performed at the Department of Microbiology, KIMS, KVV, Karad, and Krishna Hospital & Medical Research Center, Karad. (KH & MRC). A total of 180 isolates were subjected to this test to find out which produced the AmpC β -lactamase enzyme by the cefoxitin-cloxacillin double-disc synergy test method and biofilm production by tissue culture plate assay method. Results: Our study found a total of 97 isolates (53.89%) of AmpC production and biofilm production accounted for 151 (83.89%). Conclusion: AmpC- beta-lactamases are responsible for increasing the resistance of P. aeruginosa to several beta-lactam antibiotics and this study also showed that the clinical isolates of P. aeruginosa had a higher propensity to form biofilm and that there was a direct association between biofilm formation and antibiotic resistance.

The Biofilm Inhibition Properties of Glucosamine Gold Nanoparticles in Combination with Meropenem against Pseudomonas aeruginosa on the Endotracheal Tube: A Model of Biofilm-Related Ventilator-Associated Pneumonia.

Biofilm-related infections play a significant role in the development and persistence of ventilator-associated pneumonia. Pseudomonas aeruginosa (P. aeruginosa) frequently causes biofilm-related infections associated with ventilator tubing. Glucosamine gold nanoparticles (AuNPs) may exhibit antibiofilm properties; however, more studies, including combinatorial therapy with antibiotics, are needed to explore their potential applications in clinical settings. This study aims to investigate the biofilm inhibition properties of glucosamine AuNPs in combination with meropenem against P. aeruginosa ATCC 9027 on the endotracheal tube. A biofilm inhibition assay of glucosamine AuNPs at 0.02 mg/mL, both singly and in combination with meropenem at 1 mg/mL, was carried out against P. aeruginosa ATCC 9027 on an endotracheal tube using the tissue culture plate method. Scanning electron microscopy was performed for visualization. Glucosamine AuNPs at 0.02 mg/mL combined with meropenem at 1 mg/mL showed greater biofilm inhibition (72%) on the endotracheal tube than glucosamine nanoparticles at 0.02 mg/mL alone (26%) (p = 0.001). The scanning electron microscopic visualization revealed that the untreated P. aeruginosa biofilm was denser than the glucosamine nanoparticles-treated biofilm, whether combined with meropenem or using glucosamine nanoparticles alone. The combination of glucosamine AuNPs and meropenem may have the synergistic effect of inhibiting biofilm production of P. aeruginosa on the endotracheal tubes of patients with mechanical ventilation. Conducting additional experiments to explore the impact of combining glucosamine-coated gold nanoparticles (AuNPs) with meropenem on the inhibition of biofilm production by clinical P. aeruginosa isolates would be beneficial.

Effect of diesel exhaust particles on RANK/RANKL expression in in vivo and in vitro models of middle ear inflammation

ObjectiveIncreasing evidence suggests a link between middle ear inflammation and the development of diesel exhaust particles (DEPs). Chronic middle ear inflammation can lead to bone damage and remodeling. This study aimed to explore the impact of DEPs on the expression of interleukin (IL)-6 and RANKL under conditions of middle ear inflammation. MethodsDEPs were collected by burning fuel in a diesel engine at the Gwangju Institute of Science and Technology. Human middle ear epithelial cells were cultured to 70–80% confluence in culture plates and then treated with DEPs at concentrations of 0, 5, 10, 20, 40, and 80 μg/mL for 24 h. Cell viability was assessed manually. B6.SJL mice, aged 9 weeks, were exposed to DEPs at a concentration of 200 μg/m3 for 1 h daily over a period of 28 days. The expression levels of IL-6, tumor necrosis factor α, RANKL, and RANK were evaluated using hematoxylin and eosin staining and western blot analysis of the harvested middle ear samples. ResultsThe viability of human middle ear epithelial cells was found to decrease in a dose-dependent manner after 24 h. The mRNA expression level of IL-6 exhibited the most significant increase at the 48-h mark. In contrast, the mRNA expression levels of RANKL and RANK showed a marked increase as early as 6 h post-exposure, with both genes subsequently displaying a time-dependent decrease. Histological analysis revealed that the middle ear mucosa was thicker in the group exposed to DEPs compared to the control group. Additionally, the protein expression levels of IL-6 and RANKL were elevated in the DEP-exposed group relative to the normal control group. ConclusionsWe confirmed the expression of osteoclast-related proteins in the mouse middle ear. These results imply that air pollutants might affect RANKL/RANK signaling, which is associated with bone remodeling.

Analysis of the Mycotoxin Levels and Expression Pattern of SWN Genes at Different Time Points in the Fungus Slafractonia leguminicola.

The fungal plant pathogen Slafractonia leguminicola produces two mycotoxins that affect animals: slaframine, which causes slobbers, and swainsonine, which causes locoism. Slafractonia leguminicola contains the swainsonine-associated orthologous gene clusters, "SWN", which include a multifunctional swnK gene (NRPS-PKS hybrid), swnH1 and swnH2 (nonheme iron dioxygenase genes), swnN and swnR (reductase genes), and swnT (transmembrane transporter). In addition to these genes, two paralogs of swnK, swnK1 (paralog1) and swnk2 (paralog2), are found in S. leguminicola. cDNAs from total mRNA were isolated from the S. leguminicola mycelia grown in the culture plates as well as from leaves inoculated with the fungal mycelia at different time points, and expression pattern of the SWN genes were analyzed using RT-qPCR. The concentrations of swainsonine and slaframine production from this fungus at different time points were also examined using liquid chromatography-mass spectrometry. The timing of gene expression was similar in cultured fungus and inoculated leaves and agreed with our proposed biosynthetic pathway. Substantially more swainsonine was produced than slaframine during time course studies.

Antagonistic activities of mycoparasitic Pythium species against Fusarium oxysporum f. sp. lycopersici and Botrytis cinerea on tomatoes

In this study, antagonistic effects of Pythium acanthophoron, P. lycopersicum, P. oligandrum and P. paroecandrum against Fusarium oxysporum f. sp. lycopersici and Botrytis cinerea were investigated by in vitro and in vivo trials. In vitro mycoparasitic activities of Pythium species were determined by dual culture, inverted plate culture and agar diffusion tests. As a result of dual culture tests, suppressive effects of all mycoparasites were over 70% against mycelial growth of the pathogens. Inverted plate tests showed that antagonistic effects of mycoparasites regarding their volatile compounds were rather low. In the agar diffusion test, all mycoparasites showed antibiosis effect, however P. lycopersicum had the highest suppressive effect on both pathogens. In pot trials, mycoparasites were effective to protect tomato seedlings when pathogens were seperately inoculated, and suppressed the symptoms. When two pathogens were inoculated together, P. paroecandrum was ineffective against B. cinerea, but decreased the severity of wilt symptoms, while other mycoparasites totally inhibited both diseases. Chromatographic analyses made by using leaf samples taken 12, 24, 48 and 72 hours after pathogen inoculation showed meaningful increase on chlorogenic acid, caffeic acid and epicatechine, in the samples taken 48 hours after inoculation. Analyses after the inoculations of tomatoes with the mycoparasites and/or pathogens showed that mycoparasites also caused increase in the amounts of phenolics. This indicated that the mycoparasites could be effective to induce defense mechanisms of tomato plants against pathogens. Among them, P. oligandrum can be mentioned as the most effective mycoparasite regarding the induction of phenolics.

In vitro ANALYSIS OF BIOFILM FORMATION AND ANTIBIOTIC RESISTANCE OF UROPATHOGENIC Escherichia coli

Urinary tract infections are an exceedingly common worldwide problem, caused mostly by Gram-negative bacteria, especially Escherichia coli. Microbial biofilms are considered a serious public health problem. The potential of uropathogenic E. coli (UPEC) to produce biofilm was explored in the present study. The Congo red agar, tube- and tissue culture plate methods were used to evaluate the formation of biofilm by E. coli isolates. Of the 155 isolates, 101, 106 and 90 isolates were positive for these three methods, respectively. Subsequently, the sensitivity of E. coli isolates to antimicrobial agents (amikacin, cefepime, cefixime, cefotaxime, cefpodoxime, ceftazidime, ceftriaxone, ciprofloxacin, nitrofurantoin, gentamycin, nalidixic acid, ofloxacin and pefloxacin) was tested. There was no difference in the rate of biofilm detection between Congo red agar method and tube method. The antibiotic sensitivity test revealed that the biofilm producing isolates were multi-drug resistant. The study emphasized the necessity for developing alternative therapeutic approaches to overcome multi drug resistance arising from biofilm formation of UPEC.

Abstract 4248: Patient-derived MTAP-deleted bladder cancer organoid model: A unique platform for drug development

Abstract Introduction: Approximately 30% of advanced bladder cancer (BCa) tumors exhibit MTAP gene deletion, which has been associated with luminal subtype BCa, poor response to chemotherapy, and shorter progression to metastatic disease (De Souza 2023). MTAP deletion has been proposed as a potential biomarker for response to pemetrexed and an emerging class of PRMT5 and MAT2A inhibitors in development for MTAP-deleted tumors. Here, we describe the first patient-derived 3D model of BCa with MTAP deletion and its translational potential as a drug sensitivity platform. Methods: A patient (pt) with metastatic BCa harboring MTAP deletion underwent a pelvic lymph node biopsy. A fresh tumor sample was processed under collagenase/dispase solution for 30 minutes and filtered through a 70-um cell strainer. Cells were cultures in low attachment plates with MammoCult™ Basal Medium (Stem Cell Technologies). After 5 passages and 3D spheroid formation, immunostaining for cytokeratin (CK) 5 and 6, GATA3, CK20, and Ki67 staining were performed to confirm the urothelial origin. The MTAP-del was confirmed by a western blot of the organoid specimen and compared to other BLCa cell lines (UMUC, RT4, J82, and 5637). The viability assays were performed using the CellTiter-Glo® 3D Cell kit. Next-generation sequencing (NGS) of primary BCa tumor revealed deletion of MTAP, CDKN2A, CDKN2B, and mutations in p53, TERT, and KDM6A. Results: The patient organoid sample was positive for the luminal CK5/6 markers and negative for GATA3. Ki67 and CK20 were primarily expressed in the nucleus of the same sample. The organoid retained morphologic and immunohistochemical features of the patient’s primary bladder tumor, showing the same squamous differentiation and similar Ki-67 proliferative index. The organoid displayed no evidence of the MTAP protein by western blot (similar findings with the MTAP-del cell lines UMUC and RT4). The organoid showed sensitivity to pemetrexed [pemetrexed IC50=0.12 uM (other MTAP-del BLCa IC50: 0.1-0.23 uM)] and resistance to gemcitabine and cisplatin [Gem IC50=55 nM (other BLCa IC50: 1-29 nM), Cis IC50=90.5 uM (other BLCa IC50: 2-17 uM). Conclusions: We describe the first patient-derived MTAP-deleted BCa organoid recapitulating the features of the primary tumor. The organoid displayed sensitivity to pemetrexed as described with MTAP-deleted BCa. The organoid represents a unique platform for testing novel therapeutic strategies for MTAP-deleted BCa. Citation Format: Maximilian Schwermann, Benedito Carneiro, Shaolei Lu, Andre De Souza, Matthew Hadfield, Anthony Mega, Galina Lagos, Sari Khaleel, Dragan Golijanin, Sheldon Holder, Praveen Srinivasan, William MacDonald, Andrew George, Lanlan Zhou, Leiqing Zhang, Wafik El-Deiry. Patient-derived MTAP-deleted bladder cancer organoid model: A unique platform for drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4248.

Abstract 2073: Human breast patient derived organoids: Development and characterization of a new tissue mimetic model

Abstract Worldwide, breast cancer (BC), is the most frequently diagnosed cancer among women. In the US, 1 out of 8 women may expect to be diagnosed with BC In their lifetime. Current models for studying BC tumor biology and BC drug discovery include 2D cultured cell lines and patient derived xenografts (PDX). As 2D cultured cells do not represent the 3D complexity of human tissue, and PDX models are time consuming and cost prohibitive, better tissue mimetic models are needed. Organoids are self-assembled 3D cultured cellular units that may mimic the corresponding human tissue structurally and functionally (1). Patient derived organoids (PDO) are developed from tissue of specific patients and may resemble the source tissue in molecular features, including recapitulation of patient specific responses to therapies (2). Here, we report development and characterization of novel breast PDO lines from fresh (≤24h post-surgery) and cryopreserved (≤7 days post-surgery) tissue. Breast tumor and normal, tumor adjacent (NTA) tissue were sourced from consented patients. Breast PDO were generated following modifications to a published protocol (3). Briefly, tissue samples were minced, digested with collagenase, and filtered. The filtrate was resuspended in Matrigel (Corning) and small drops were added on cell culture plates. Both tumor and NTA PDO were cultured using the same media and organoids were monitored using brightfield microscopy (Olympus CK40) and live cell imaging (Muvicyte, Perkin Elmer). PDO lines were analyzed for expression and distribution of important breast-specific biomarkers using confocal microscopy (ImagExpress, Molecular Devices). PDO lines displayed hormone receptors (ER, PR), cell surface (EpCAM, CD49f), cytoskeletal (cytokeratin 14, 19, E-cadherin, vimentin) and cell proliferation (Ki67) markers. Transcriptome profiles were analyzed using vendor services (Azenta). PDO were also tested for their susceptibility to breast cancer drugs, e.g., Tamoxifen, chlorambucil and Docetaxel. We have successfully generated one NTA ER+/PR+/HER2− PDO line and one tumor ER+/PR+/HER2− PDO line from different donors. Our breast PDO lines would be highly useful in understanding tumor biology, BC progression, signal transduction, and drug discovery/testing. Future efforts would include process scaling up and development of BC specific assays.

Abstract 92: Assessment of an in vitro potency assay for evaluation of immune cell-mediated cytotoxicity using the Omni Pro 12 automated imaging platform

Abstract The development of immunotherapies relies on the use of in vitro potency assays—which are key for understanding complex interactions between immune cells and cancer cells. Immune effector T-cells are a promising cancer therapy due to their innate cytotoxicity. In particular, CAR T-cell therapy uses genetically engineered T-cells that express a chimeric antigen receptor that binds to a specific antigen on tumor cells. Assessing the efficacy and potency of CAR T-cell therapies in vitro and at high throughputs is vital for the preclinical development of these promising therapies. Here, we describe an in vitro potency assay that uses an automated imaging platform to quantify immune cell-mediated cytotoxicity of cancer target cells by immune effector cells. A GFP fluorescent and HER-2 expressing lung cancer cell line, A549, was seeded into a 96-well cell culture plate. Then, HER-2 CAR T-cells were added at 24 hours post target cell seeding at various E:T ratios (1:10, 1:5, 1:2, 1:1, and 5:1). Fluorescent images of the target cells were captured every 6 hours by the Omni Pro 12, an automated, high-throughput, live-cell analysis platform designed for continuous multi-well imaging inside an incubator. Automated image analysis was performed using the Confluency Module in the Axion Portal to view attachment and proliferation and quantify cytolysis of fluorescent A549 target cells. Percent cytolysis of the target cells was calculated by comparing the green fluorescent confluency of treated wells to no treatment control wells. A549-GFP cells showed a dose-dependent decrease in confluency over time that correlated with increasing amounts of CAR T-cells. At 92 hours post HER-2 CAR T-cell addition, the 5:1 ET group demonstrated approximately 91.8% +/- 2.0% cytolysis of A549-GFP cells, while the 1:10 group demonstrated approximately 69.2% +/- 3.0% cytolysis. Future work will evaluate differences in CAR T-cell potency when co-cultured with target cells that express different levels of HER-2. Overall, the Omni Pro 12 platform enables continuous quantification of the potency and kinetics of immune cell-mediated cytolysis. Citation Format: Denise Sullivan, Lieke Stemkens, Inge Thijssen, Svenja Meiler, Danielle Califano, Stacie Chvatal, Daniel Millard. Assessment of an in vitro potency assay for evaluation of immune cell-mediated cytotoxicity using the Omni Pro 12 automated imaging platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 92.

Abstract 5770: Inhibition of KRAS G12D mutant with small molecules

Abstract Background: RAS mutations are occurring at a high rate in colorectal cancer, lung cancer, and pancreatic cancer. RAS is family of small GTPase proteins with important roles in cell growth, differentiation, proliferation, and survival. There are three types of RAS genes: HRAS, KRAS, and NRAS. The The RAS gene is the most commonly found oncogene in human cancer. KRAS exhibits a highest mutation rate compared to those of HRAS and NRAS in various cancer types. Methods: Colorectal cell line LIM1215, SW48 and NSCLC cell line NCI-H1975, NCI-H838 were used for MTT assays. Apoptosis assay was performed using the annexin V-FITC apoptosis detection kit (BioBud, Cat. LS-02-100). Colony forming assay was performed with 6-well culture plates by seeding 500 cells/well. The expression levels of proteins involved in Q2a-induced signaling pathway were determined by Western blot. In vivo study was performed with tumor xenografts. HT-29 cells were implanted into the right flank of 5-week-old BALB/c nude mice. Tumor size was determined by measuring the diameter (width, length) of the tumor every two days with a digital caliper. All group mice sacrificed at day 19. Results: Patients with cancer involving KRAS mutations are resistant to therapeutics that target epidermal growth factor receptor and show poor outcomes. In addition, effective treatments that target G12D in KRAS have not been developed. Thus, therapeutic agents targeting the G12D mutation in KRAS are needed. In this study, we identified the Q2a compound, a quinazoline-based derivative that specifically acts on the KRAS G12D mutation. This compound induced cell apoptosis by reducing phosphorylation of AKT in the PI3K pathway downstream of KRAS. Q2a compound specifically inhibited the growth of KRAS G12D mutant cells. In addition, cells with the KRAS G12D mutation were more sensitive to Q2a compound compared to the sensitivity of wild-type cells and those with other KRAS mutations, resulting in apoptosis. Treatment with the Q2a compound also decreased C-RAF phosphorylation in the MAPK pathway, which is downstream of KRAS. In KRAS G12D-mutated colorectal cancer xenografts, Q2a compound inhibited tumor production without significant liver toxicity. Conclusion: Q2a compounds show potential as therapeutic agents that target KRAS G12D mutations in colorectal and pancreatic cancers. Kyewords: Anti-Cancer Drug, KRAS G12D Mutant, apoptosis, colon cancerJi Yoon Lee, Dae-Hee Lee*Department of Marine Bio Food Science, Gangneung-Wonju national University Citation Format: Dae-Hee Lee, Ji Yoon Lee. Inhibition of KRAS G12D mutant with small molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5770.

Abstract 6775: 3D culture aggregation morphology depends on cell phenotype and gene expression profiles in lung cancer

Abstract Background: Three-dimensional (3D) cell culture allows for the reproduction of tissue structure and function in a state more akin to the in vivo environment compared to two-dimensional (2D) cell culture. It is established that 3D culture more faithfully replicates cell morphology, differentiation, proliferation, gene and protein expression, and responsiveness to stimulation, as well as drug metabolism. Focusing on the observation that lung cancer cell lines exhibit different clustering morphologies in 3D cell culture, we conducted a comparative study on the relationship between these clustering morphologies and the characteristics of the lung cancer cell lines. Methods: We cultured 30 lung cancer cell lines using 3D culture plate, and classified the types of cell clustering morphologies formed. We evaluated the molecular and cellular biology characteristics of all cell lines. Additionally, comprehensive gene expression analysis using RNA sequencing was performed on the 30 3D-cultured cell samples, and the characteristics of gene expression between different clustering types were compared. Results: The 3D culture aggregation morphology of 30 lung cancer cell lines were classified into three types (monolayer sheet, NOS, spheroid). Monolayer sheet-type cells exhibited higher proliferation, migration, and invasion capabilities compared to spheroid-type cells. In the transcriptomic bioinformatics analysis of the acquired gene expression data, GSEA analysis using Hallmark gene sets showed that the enrichment of epithelial-mesenchymal transition (EMT) and cancer metastasis-related genes in the monolayer type cells significantly compared to the spheroid type cells. Conversely, cell adhesion and intercellular binding-related gene sets were highly enriched in the spheroid type cells. Conclusion: 3D culture aggregation morphology represents phenotypic properties and transcriptomic profiles in non-small cell lung cancer cells. Citation Format: Naohiro Hayashi, Shimpei Tsudaka, Ken Suzawa, Tomoaki Higashihara, Yin Min Thu, Atsushi Matsuoka, Daisuke Mizuno, Ryo Yoshichika, Fumiaki Mukohara, Mao Yoshikawa, Kazuhiko Shien, Hiromasa Yamamoto, Shinichi Toyooka. 3D culture aggregation morphology depends on cell phenotype and gene expression profiles in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6775.

Abstract 2072: Human liver patient derived organoids: Development and characterization of a new tissue mimetic model

Abstract Liver is the largest solid organ and performs hundreds of vital functions including drug metabolism. Liver diseases, including viral infections, cancer and metabolic disorders account for about 2 million deaths annually, worldwide (1). Current models for studying liver biology and drug metabolism include 2D cultured primary cells/cell lines, which do not represent the metabolic and structural complexity of human tissue. In vivo models for liver diseases are time consuming, cost prohibitive and often ineffective. Therefore, better tissue mimetic liver models are needed. Organoids are self-assembled 3D cultured cellular units that may mimic the corresponding human tissue structurally and functionally (2). Patient derived organoids (PDO) are developed from tissue of specific patients and may resemble the source tissue in molecular features, including recapitulation of patient specific responses to therapies. Here, we report development and characterization of novel liver PDO lines from vendor-sourced cryopreserved tissue. We have developed biobanks of undifferentiated as well as differentiated liver PDO models.Human cadaver-sourced liver tissue were obtained with explicit donor consent. Liver PDO were generated following modification of a previously established protocol (3). Briefly, samples were minced, digested to single cells, and filtered. Specific number of cells were mixed with diluted Matrigel (Corning) and added as small drops on cell culture plates and grown in liver PDO specific growth media. Whenever appropriate, PDO were differentiated by culturing in differentiation-specific media in multi-well plates. In all cases, PDO were monitored using brightfield microscopy (Olympus CK40) and live cell imaging (Muvicyte, Perkin Elmer); and passaged every 6-8 days. Undifferentiated PDO were cryopreserved/thawed following in-house SOP to confirm viability. Liver-specific biomarker expression in the PDO lines were confirmed by confocal microscopy (ImageExpress, Molecular Devices). PDO lines were also assayed for ALT activity, albumin production and urea. PDO were cleared for microbial/viral contamination and confirmed as unique lines using single tandem repeat analyses. Transcriptome profiles were analyzed using vendor services. So far, we have successfully generated 3 liver PDO lines, both undifferentiated as well as differentiated. Our liver PDO lines would be highly useful in understanding liver biology and disease models; and will have significant usage in drug testing, including DMPK and ADME/Tox studies. Future efforts would include scaling up, development of liver specific assays and developing PDO from specific liver diseases, including hepatocellular carcinoma, MASLD and NASH.

Performance evaluation of Trichoderma reseei in tolerance and biodegradation of diuron herbicide in agar plate, liquid culture and solid-state fermentation.

The present study evaluated the performance of the fungus Trichoderma reesei to tolerate and biodegrade the herbicide diuron in its agrochemical presentation in agar plates, liquid culture, and solid-state fermentation. The tolerance of T. reesei to diuron was characterized through a non-competitive inhibition model of the fungal radial growth on the PDA agar plate and growth in liquid culture with glucose and ammonium nitrate, showing a higher tolerance to diuron on the PDA agar plate (inhibition constant 98.63 mg L-1) than in liquid culture (inhibition constant 39.4 mg L-1). Diuron biodegradation by T. reesei was characterized through model inhibition by the substrate on agar plate and liquid culture. In liquid culture, the fungus biotransformed diuron into 3,4-dichloroaniline using the amide group from the diuron structure as a carbon and nitrogen source, yielding 0.154mg of biomass per mg of diuron. A mixture of barley straw and agrolite was used as the support and substrate for solid-state fermentation. The diuron removal percentage in solid-state fermentation was fitted by non-multiple linear regression to a parabolic surface response model and reached the higher removal (97.26%) with a specific aeration rate of 1.0 vkgm and inoculum of 2.6 × 108 spores g-1. The diuron removal in solid-state fermentation by sorption on barley straw and agrolite was discarded compared to the removal magnitude of the biosorption and biodegradation mechanisms of Trichoderma reesei. The findings in this work about the tolerance and capability of Trichoderma reesei to remove diuron in liquid and solid culture media demonstrate the potential of the fungus to be implemented in bioremediation technologies of herbicide-polluted sites.

Development of an ultrafast PCR to detect clinically relevant acquired vancomycin-resistance genes from cultured enterococci.

VRE are increasingly described worldwide. Screening of hospitalized patients at risk for VRE carriage is mandatory to control their dissemination. Here, we have developed the Bfast [VRE Panel] PCR kit, a rapid and reliable quantitative PCR assay for detection of vanA, vanB, vanD and vanM genes, from solid and liquid cultures adaptable to classical and ultrafast real-time PCR platforms. Validation was carried out on 133 well characterized bacterial strains, including 108 enterococci of which 64 were VRE. Analytical performances were determined on the CFX96 Touch (Bio-Rad) and Chronos Dx (BforCure), an ultrafast qPCR machine. Widely used culture plates and broths for enterococci selection/growth were tested. All targeted van alleles (A, B, D and M) were correctly detected without cross-reactivity with other van genes (C, E, G, L and N) and no interference with the different routinely used culture media. A specificity and sensitivity of 100% and 99.7%, respectively, were determined, with limits of detection ranging from 21 to 238 cfu/reaction depending on the targets. The Bfast [VRE Panel] PCR kit worked equally well on the CFX and Chronos Dx platforms, with differences in multiplexing capacities (five and four optical channels, respectively) and in turnaround time (45 and 16 minutes, respectively). The Bfast [VRE Panel] PCR kit is robust, easy to use, rapid and easily implementable in clinical microbiology laboratories for ultra-rapid confirmation of the four main acquired van genes. Its features, especially on Chronos Dx, seem to be unmatched compared to other tools for screening of VRE.

A novel approach to determine the critical survival threshold of cellular oxygen within spheroids via integrating live/dead cell imaging with oxygen modeling.

Defining the oxygen level that induces cell death within 3-D tissues is vital for understanding tissue hypoxia; however, obtaining accurate measurements has been technically challenging. In this study, we introduce a noninvasive, high-throughput methodology to quantify critical survival partial oxygen pressure (pO2) with high spatial resolution within spheroids by using a combination of controlled hypoxic conditions, semiautomated live/dead cell imaging, and computational oxygen modeling. The oxygen-permeable, micropyramid patterned culture plates created a precisely controlled oxygen condition around the individual spheroid. Live/dead cell imaging provided the geometric information of the live/dead boundary within spheroids. Finally, computational oxygen modeling calculated the pO2at the live/dead boundary within spheroids. As proof of concept, we determined the critical survival pO2 in two types of spheroids: isolated primary pancreatic islets and tumor-derived pseudoislets (2.43 ± 0.08 vs. 0.84 ± 0.04 mmHg), indicating higher hypoxia tolerance in pseudoislets due to their tumorigenic origin. We also applied this method for evaluating graft survival in cell transplantations for diabetes therapy, where hypoxia is a critical barrier to successful transplantation outcomes; thus, designing oxygenation strategies is required. Based on the elucidated critical survival pO2, 100% viability could be maintained in a typically sized primary islet under the tissue pO2 above 14.5 mmHg. This work presents a valuable tool that is potentially instrumental for fundamental hypoxia research. It offers insights into physiological responses to hypoxia among different cell types and may refine translational research in cell therapies.NEW & NOTEWORTHY Our study introduces an innovative combinatory approach for noninvasively determining the critical survival oxygen level of cells within small cell spheroids, which replicates a 3-D tissue environment, by seamlessly integrating three pivotal techniques: cell death induction under controlled oxygen conditions, semiautomated imaging that precisely identifies live/dead cells, and computational modeling of oxygen distribution. Notably, our method ensures high-throughput analysis applicable to various cell types, offering a versatile solution for researchers in diverse fields.