Percentage Of Dead Cells Research Articles

Evaluation of the effect of yellow laser (DPSS) light on human lymphocytes viability in vitro.

To evaluate the lymphocyte apoptotic impact of yellow laser light in vitro. The experimental study was conducted from November 2021 to April 2022 at the Postgraduate Medical Physics Laboratory, Mustansiriyah University, Baghdad, Iraq, and comprised blood samples from healthy volunteers. The samples were subjected to 1:1 dilution in isotonic phosphate buffered saline, and each sample was divided into two equal aliquots; one for irradiation, and other as control.The percentage of apoptotic lymphocyte was estimated before and after radiation exposure with low-level laser 589nm at 30J/cm², 50J/cm² and 70J/cm² energy intensities. Post-exposure evaluation was done immediately, and 1 and 2 hours after radiation on the viability of normal human cells. The vitality of the cells was determined using the trypan blue exclusion test. Data was analysed using SPSS 24. There were 26 healthy volunteers (16 male, 10 females) with an age range of (20-40) years. After the exposure, the percentage of lymphocytes that apoptose increased significantly when cells were irradiated at 70J/cm2 immediately compared to the controls (p≤ 0.05), but there was no significant differences with the 30J/cm² dose. After 1 and 2 hours, a significant reduction in the proportion of apoptotic lymphocytes was observed (p<0.05). Yellow low-level laser showed a noticeable protective effect on lymphocytes by decreasing the percentage of dead cells.

The Effects of Substrates and Sonication Methods on the Antioxidant Activity of Kefir Postbiotics

Sonoporation stimulates cell growth as it improves the permeability of the membrane and increases the uptake of impermeable molecules in the extracellular matrix. We evaluated the effects of substrates (whey, whole, and skim milk) and ultrasonic treatments (ultrasonication and thermosonication) on the antioxidant activity (AA) of water-soluble kefir postbiotics (WSKPs). The samples were evaluated in terms of antioxidant activity (ABTS, DPPH, FRAP, and ORAC), water-soluble protein content, proteolysis (SDS-PAGE profiles), and cell membrane permeability. The levels of AA in all WSKPs depended on the substrate and method of obtaining them. However, the WSKPs from whey had higher antioxidant activity with DPPH (11.11 mg TE/100 mL), ABTS (12.77 mg TE/100 mL), and FRAP (5.18 mg TE/100 mL). Also, the WSKPs from whey had the highest values for water-soluble protein (1.45–1.32 mg/mL) and proteolysis degree and the lowest percentage of dead cells (11.4–28%). These results suggest that the production of WSKPs from whey might add value to whey production. Furthermore, WSKPs have potential as a functional ingredient in the production of beverages or foods with antioxidant activity.

Mycobacterium intracellulare mediates macrophage pyroptosis by activating AIM2 and NLRP3 inflammasomes.

Clinically, the incidence of nontuberculous mycobacteria (NTM) lung disease is on the rise, and Mycobacterium intracellulare (M. intracellulare) has attracted much attention as a common opportunistic pathogen in clinical practice. So it is very important to study its immunopathogenic mechanism. In this study, the mechanism of M. intracellulare induced pyroptosis of macrophage was investigated. As shown in Fig.1, the secretion of IL-1β and IL-18 in J774A.1 cells increased with time after M. intracellulare infection and was affected by caspase-1 activation and K + efflux, while caspase-1 was significantly expressed in infected cells. Further from Fig.2, NLRP3,AIM2,ASC proteins were significantly expressed in J774A.1 cells after infection, indicating that the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammasome were involved in the infection process. In addition, when caspase-1 activity and K + efflux were inhibited, the expression of related proteins was significantly reduced. It indicates that the activation of NLRP3 and AIM2 is regulated by caspase-1 and K+. Figure3, the percentage of dead cells with cell membrane damage increases after infection and cleavage of GSDMD proteins occurs. In summary, infection of J774A.1 cells with M. intracellulare induces pyroptosis, and this process is mediated by caspase-1. Our study provides information for further understanding of the molecular mechanism of M. intracellulare infection.

(Invited) Carbon Nanostructures for Targeted Delivery and Health Monitoring in Plants

To meet the demand for food and bioenergy of a rapidly growing global population requires novel and convergent approaches to enhance photosynthetic organism function. Nanotechnology is enabling the development of targeted and species independent tools for advancing plant bioengineering and precision agriculture.My research group has developed carbon nanotube-based sensors capable of real-time sensing of plant signaling molecules. These nanosensors enabled the creation of plant sentinels that communicate the health status of plants to electronic devices. Near-infrared (nIR) fluorescent single-walled carbon nanotubes (SWCNTs) were designed and interfaced with plant leaves to report hydrogen peroxide (H2O2), a key signaling molecule associated with the onset of plant stress. The sensor nIR fluorescence response (>900 nm) is quenched by H2O2 with selectivity against other stress-associated signaling molecules and within the plant physiological range (10–100 H2O2 μM). In vivo remote nIR imaging of H2O2 sensors enabled optical monitoring of plant health in response to stresses including UV-B light (−11%), high light (−6%), and a pathogen-related peptide (flg22) (−10%), but not mechanical leaf wounding (<3%). The sensor’s high biocompatibility was reflected on similar leaf cell death (<5%) and photosynthetic rates to controls without SWCNT. These optical nanosensors report early signs of stress and will improve our understanding of plant stress communication, provide novel tools for precision agriculture, and optimize the use of agrochemicals in the environment.We also developed targeted carbon-based nanomaterials as tools for precise chemical delivery (carbon dots, CDs) and gene delivery platforms (SWCNTs) to plant organelles (chloroplasts) and vasculature (phloem). A biorecognition approach of coating the nanomaterials with a rationally designed chloroplast targeting peptide improved the delivery of CDs with molecular baskets (TP-β-CD) for delivery of agrochemicals and of plasmid DNA coated SWCNT (TP-pATV1-SWCNT) from 47% to 70% and from 39% to 57% of chloroplasts in leaves, respectively. Plants treated with TP-β-CD (20 mg/L) and TP-pATV1-SWCNT (2 mg/L) had a low percentage of dead cells, 6% and 8%, respectively, similar to controls without nanoparticles, and no permanent cell and chloroplast membrane damage after 5 days of exposure. Orthogonally, the surfaces of carbon dot nanocarriers were functionalized with sucrose, enabling rapid and efficient foliar delivery into the plant phloem, a vascular tissue that transports sugars, signaling molecules, and agrochemicals through the whole plant. The chemical affinity of sucrose molecules to sugar membrane transporters on the phloem cells enhances the uptake of biocompatible carbon dots with β-cyclodextrin molecular baskets (suc-β-CD) that can carry a wide range of agrochemicals. The nanoparticle fluorescence emission properties allowed detection and monitoring of rapid translocation (<40 min) in the vasculature of wheat leaves by confocal and epifluorescence microscopy. The suc-β-CDs more than doubled the delivery of chemical cargoes into the leaf vascular tissue. The sucrose coating of nanoparticles approach enables unprecedented targeted delivery to roots with ≈70% of phloem-loaded nanoparticles delivered to roots.The use of plant biorecognition molecules mediated delivery provides an approach for guiding nanocarriers with their cargoes to plant organelles, cells and tissues and whole plants and engineering safer and efficient agrochemical and biomolecule delivery tools. Communication and actuation of plants mediated by nanosensors and targeted nanostructures can turn plants into technology and lead to a more precise and sustainable agriculture with reduced environmental impact.

The influence of hydroxyapatite coatings with different structure and crystallinity on osteogenesis stimulation

NiTi alloys are actively used in medicine as implants because of their mechanical properties. At the same time, however, the NiTi surface is not biologically active, which has a negative effect on osteogenesis. In addition, Ni is capable of causing various toxic reactions in the human body. It is therefore necessary to create a composite coating that is both bioactive and prevents the release of Ni ions. It has been shown that by changing the modes of plasma assisted RF sputtering of a calcium phosphate target, it is possible to obtain coatings with different contents of the amorphous phase. The coatings formed consist of hydroxyapatite and amorphous calcium phosphate. On the surface of a sample not treated with argon plasma, there are no phases containing calcium or phosphorus, although these elements are present on the surface. The different ways in which the CaP coatings were applied also affected their structure. It was found that the CaP coatings of samples M2 and M3 have mediocre corrosion resistance, dissolving quickly. This has a positive effect on the ability to osteogenesis, which was discovered as a result of in vitro tests. These tests have shown that the sample obtained at Idischarge = 30 A and Ubias = 30 V has the highest bioactivity and produces the lowest percentage of dead cells.

Antibacterial efficacy of silver nanoparticles, sodium hypochlorite, chlorhexidine, and hypochlorous acid on dentinal surfaces infected with Enterococcus faecalis.

The purpose of this study was to evaluate the antibacterial effect of silver nanoparticles (AgNPs) against Enterococcus faecalis and compare it with different irrigation solutions. This study was performed using 64 dentin blocks. E. faecalis suspension was dispensed to each sample and incubated under anaerobic conditions at 37°C throughout 21 days. After the inoculation period, the following solutions were added to each group and kept for 5 min: Group 1, 5.25% sodium hypochlorite (NaOCl); Group 2, 2.5% NaOCl; Group 3, 1% NaOCl; Group 4, 2% chlorhexidine (CHX); Group 5, 200 ppm hypochlorous acid (HOCl); and Group 6, AgNPs. The samples of positive control were treated with sterile saline. Biofilm viability assay was performed using the LIVE/DEAD BacLight Bacterial Viability Kit. Samples were examined using confocal laser scanning microscopy, respectively. There was no significant difference between the 5.25% NaOCl, 2.5% NaOCl, and 1%NaOCl groups (p > .05). However, these groups showed statistically higher antibacterial activity than the 2% CHX, 200 ppm HOCl, and AgNP groups. Also, 2% CHX showed greater percentage of dead cells compared with the AgNP and HOCl groups. While AgNPs group showed lower dead cell rate than all NaOCl groups and 2% CHX, it caused higher dead cells than 200 ppm HOCl group. The 200 ppm HOCl group showed the lowest percentage of dead cells (p < .05) Although the antibacterial effect of AgNPs is not as high as NaOCl and CHX, it has considerable bactericidal activity against E. faecalis and can be improved by further studies. RESEARCH HIGHLIGHTS: New antimicrobial approaches for root canal irrigation. Antimicrobial effect of silver nanoparticles against E. faecalis. Elimination of the biofilm layer for the success of endodontic treatment.

In Vitro Investigation of the Antimicrobial Potential and Cell Viability of Different Dosages of Ozone Therapy

ABSTRACT The aim of the study was to evaluate and compare, in vitro, the cytotoxicity of different applied ozone dosages in keratinocyte (NOK) and fibroblast (L929) cell lines, as well as the antimicrobial potential for Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). NOK and L929 cells and strains of gram-positive bacteria S. aureus and gram-negative bacteria E. coli were divided into seven experimental groups: ozone at doses from 0, 0.005, 0.01, 0.02, 0.03, 0.04 and 0.05 mg/L. The effect of ozone on cell viability/cytotoxicity was determined using the alamarBlue® assay, the Live/Dead® assay and the morphometric analysis of the percentage of live and dead cells. The results show that applied ozone dosages of 0.005, 0.01 and 0.02 mg/L showed the highest viability values of NOK and L929 cells, as well as a higher density and percentage of live cells in the 72-hour experimental. However, no difference was observed in the percentage of dead cells between the experimental groups. Furthermore, ozone promoted a reduction in CFUs at all tested dosages. With the results obtained in the present study, it is possible to confirm the restorative potential of ozone therapy in a dose-dependent response, as well as its antimicrobial potential.

Zinc oxide nanoparticles preserve the quality and fertility potential of rooster sperm during the cryopreservation process.

Sperm cryopreservation is one of the main methods for preserving rooster sperm for artificial insemination (AI) in commercial flocks. Yet, rooster sperm is extremely susceptible to reactive oxygen species (ROS) produced during the freezing process. Oxidative stress could be prevented by using nanoparticles containing antioxidants. The present study was conducted to investigate the effect of zinc oxide nanoparticles (ZnONP) in rooster semen freezing extender on quality parameters and fertility potential. For this aim, semen samples were collected and diluted in Lake extenders as follows: control: Lake without ZnONP, ZnO100: Lake with 100-μg zinc oxide (ZnO), ZnONP50: Lake with 50-μg ZnONP, ZnONP100: Lake with 100-μg ZnONP and ZnONP200: Lake with 200-μg ZnONP. After freezing and thawing, sperm motility, viability, membrane integrity, morphology, mitochondrial activity, acrosome integrity, DNA fragmentation, lipid peroxidation and ROS, as well as fertility and hatchability were assessed. According to the current results, higher rates of motility, membrane integrity, mitochondrial activity, acrosome integrity and live cells were detected in the ZnO100, ZnONP50 and ZnONP100 groups compared to other groups (p ≤ .05). Yet, the percentage of dead cells, DNA fragmentation, lipid peroxidation and ROS levels were lower in the mentioned groups (p ≤ .05). Furthermore, a higher percentage of fertility was observed in the ZnO100 and ZnONP100 groups than in the control group (p ≤ .05). In conclusion, the use of 100-μg ZnO and 50- to 100-μg ZnONP represents a valuable and safe additive material that could be used to improve the quality and fertility potential of rooster sperm under cryopreservation conditions.

Abstract 7563: Efficacy of JPI-547, novel dual PARP inhibitor, in endometrial cancer cell line

Abstract Objectives: PARP inhibitor is effective in treating DNA repair deficiency endometrial cancer. Endometrial cancer (EC) is one of the most common gynecological cancers in developed countries and its incidence is increasing, and the prognosis of patients with early stage and low-risk EC is very good, but the prognosis of progressive and recurrent EC is poor. Novel PARP inhibitor JPI-547 is a PARP and tankyrase dual inhibitor, and in cancer cells, tankyrase inhibitor plays an important role in DNA repair, telomere maintenance, and cell survival. This experiment was conducted to determine whether the reactivity of the PTEN mutant EEC was increased by using the Novel PARP inhibitor. Method: Human endometrioid endometrial cancer cell lines, Hec-1A, Hec-1B and Ishikawa were obtained. Cell viability assay for the drugs Olaparib and JPI-547 were assessed. Colonies consisting of at least 50 cells were counted. The number of dead cells was counted in five different areas of the hemocytometer, and the experiment was performed three times. The percentage of dead cells was analyzed compared to the untreated group. We confirmed the efficacy by siRNA transfection and immunofluorescence. Result: When treated with Olaparib, there was no change in cell viability in the rest of the cells except for Ishikawa cells, which are PTEN mutations. It decreased by 35.4% in Ishikawa cells, whereas when treated with JPI-547, all cell lines Hec-1A, Hec-1B, and Ishikawa cell viability decreased by 28%, 34.3%, and 48.7%, respectively. PTEN knock-down reduced cell visibility by 28% when treated with olaparib, whereas when treated with JPI-547, Hec-1A (siCont) and Hec-1A (siPTEN) decreased by 26% and 42%, respectively, in all cells regardless of whether PTEN knock-down is present. When treated with Olaparib, dead cells were identified only in PTEN knock-down cells, increasing by 22%. When treated with JPI-547, dead cells increased by 25.5% and 33% in all cells of Hec-1A (siCont) and Hec-1A (siPTEN), respectively. It was confirmed that JPI-547 can reduce cell viability regardless of the formation of Rad51 according to PTEN. Conclusion: JPI-547, a novel PARP inhibitor, showed good efficacy in PTEN mutation and PTEN-wild-type EEC cell lines than in conventional PARP inhibitor. The effect of reducing survival of ECC cell line with JPI-547 was confirmed with or without PTEN mutation. Further evaluation and in vivo experiments are needed to prove the efficacy of JPI-547 in EC. Citation Format: Shin-Wha Lee, So-Hyun Nam, Su-Bin Park. Efficacy of JPI-547, novel dual PARP inhibitor, in endometrial cancer cell line [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 7563.

ANTI-INFLAMMATORY EFFECTS OF PRECONDITIONED BONE MARROW MSC-DERIVED SECRETOME ON DEGENERATED HUMAN NUCLEUS PULPOSUS CELLS IN VITRO

Invertebral disc degeneration (IDD) is a degenerative disease involving a variety of musculoskeletal and spinal disorders such as lower back pain (LBP). Secretome derived from mesenchymal stem cells (MSCs) have exerted beneficial effect on tissue regeneration. In this study, the goal was to investigate the paracrine and the anti-inflammatory effects of secretome from interleukin IL1β preconditioned Bone Marrow MSCs (BMSCs) on human nucleus pulposus cells (hNPCs) in a 3D in vitro model.Secretome was collected from BMSCs (BMSCs-sec) after preconditioning with 10 ng/mL IL1β. hNPCs were isolated from surgical specimens, culture expanded in vitro, encapsulated in alginate beads and treated with: growth medium; IL1β 10 ng/mL; IL1β 10 ng/mL for 24 hours and then BMSCs-sec. We examined: i) cell proliferation and viability (flow cytometry), ii) nitrite production (Griess assay) and ROS quantification (Immunofluorescence) iii) glycosaminoglycan (GAG) amount (DMBB) and iv) gene expression levels of extracellular matrix (ECM) components and inflammatory mediators (qPCR). One-way ANOVA analysis was used to compare the groups under exam and data were expressed as mean ± S.D.In vitro tests showed an enhancement of hNPCs proliferation after treatment with BMSCs-sec (p ≤ 0.05) compared to IL1β group. After 24 hours, the percentage of dead cells was higher in IL1β treated hNPCs compared to control group and decreased significantly in combined IL1β and BMSCs-sec sample group (p ≤ 0.01). Nitrite and ROS production were significantly mitigated and GAGs content was improved by preconditioned BMSCs-sec (p ≤ 0.05). Furthermore, gene expression levels were modulated by BMSCs-sec treatment compared to controls.Our results supported the potential use of BMSCs' secretome as a cell-free strategy for IDD, overcoming the side effects of cell-therapy. Moreover, secretome derived from IL1β preconditioned BMSCs was able to reduce hNPCs death, attenuate ECM degradation and oxidative stress counteracting IDD progression.Acknowledgements: Financial support was received from the “iPSpine” and “RESPINE” Horizon 2020 projects.

Improvement of osteoblast adhesion, viability, and mineralization by restoring the cell cytoskeleton after bisphosphonate discontinuation in vitro.

Bisphosphonates are prescribed to treat excessive bone resorption in patients with osteoporosis. However, its use is associated with potential adverse effects such as medication-related osteonecrosis of the jaw, prompting the introduction of the drug holiday concept in patients prior to dentoalveolar surgery. Furthermore, bisphosphonate discontinuation has been studied in vivo, in humans, and in animal models. However, it is not known whether this approach could affect bone cells in vitro. Therefore, the objective of this study was to investigate the potential effects of bisphosphonate discontinuation on pre-osteoblast and osteoblast activities in vitro. Pre-osteoblasts (MC3T3) and osteoblasts were treated with bisphosphonate (alendronate) at concentrations of 1, 5, and 10 µM. Alendronate was then withdrawn at different time points. The negative control consisted of untreated cells (0 µM), while the positive control consisted of cells incubated with alendronate throughout the experiment. Cell viability, cell adhesion, cell cytoskeleton, mineralization, and gene expressions were investigated. Pre-osteoblasts and osteoblasts showed a decrease in cell viability after treatment with 5-10 μM alendronate for 4 days or longer. Two days of alendronate discontinuation significantly increased cell viability compared with the positive control. However, these levels did not reach those of the negative control. Bone nodule formation was reduced by alendronate. Discontinuation of alendronate regained bone nodule formation. Longer periods of discontinuation were more effective in restoring nodule formation than shorter periods. Addition of alendronate resulted in an increase in the percentage of dead cells, which, in turn, decreased when alendronate was discontinued. Alendronate affected the cell cytoskeleton by disassembling actin stress fibers. Cell adhesion and cell morphological parameters were also affected by alendronate. Discontinuation of alendronate restored cell adhesion and these parameters. Overall, the highest improvement after alendronate discontinuation was seen at 10 µM. However, alendronate treatment and discontinuation did not affect osteoblast gene expression. Discontinuation of alendronate helps to reverse the negative effects of the drug on cell viability, cell adhesion, and mineralization by restoring the cell cytoskeleton. Our data suggest the benefits of drug holiday and/or intermittent strategies for alendronate administration at the cellular level.

The Inhibitory Effect of KerraTM, KSTM, and MinozaTM on Human Papillomavirus Infection and Cervical Cancer.

Background and Objectives: Cervical cancer is one of the most common types of frequently found cancers in Thailand. One of the causative agents is the infection of the high-risk human papillomavirus (HPV) type 16 and 18. Traditional medicines are rich sources of bioactive compounds which are a valuable source for the development of novel cancer therapies. In this study, the therapeutic effects of 3 traditional medicines, KerraTM, KSTM, and MinozaTM, were studied on HeLa and CaSki cells. Materials and Methods: The effects of KerraTM, KSTM, and MinozaTM on cancer cells were evaluated through cytotoxicity and cell death assays. The infection assay using HPV-16 pseudovirus was also carried out. Results: All traditional medicines efficiently suppressed cell growths of HeLa and CaSki, with KerraTM being the most potent anticancer agent followed by KSTM and MinozaTM. KerraTM at 158 µg/mL and 261 µg/mL significantly increases the percentage inhibition of the HPV-16 pseudovirus infection in a pre-attachment step in a dose-dependent manner, while KSTM at 261 µg/mL efficiently inhibited viral infection in both pre-attachment and adsorption steps. However, KerraTM, KSTM, and MinozaTM at subtoxic concentrations could not reduce the viral E6 mRNA expressions of HPV-16 and HPV-18. Cell death assay by acridine orange/ethidium bromide showed that KerraTM increased population of dead cells in dose-dependent manner in both CaSki and HeLa. The percentage of secondary necrosis in KerraTM-treated CaSki was higher than that of HeLa cells, while the percentage of late apoptotic cells in HeLa was higher than that of CaSki, indicating that HeLa was more susceptible to KerraTM than CaSki. For KSTM and MinozaTM, these extracts at 250 µg/mL promoted autophagy over cell death. At 500 µg/mL, the percentage of dead cells in KerraTM was higher than that of KSTM and MinozaTM. Conclusions: KerraTM is a potent traditional medicine for promoting cancer cell death. KerraTM is possibly useful in the prevention and treatment of cervical cancer. Further investigation will be carried out to gain a better understanding of the biochemical mechanism and the pharmacological activity underlying this effect.

The Use of Next Generation Flow in Multiple Myeloma Patients: A Preliminary Real-Life Multicenter MRD Harmonization Experience

Introduction: Minimal residual disease (MRD) detection represents a sensitive tool to appropriately measure response in MM. The major concern about MRD detection in real-world setting is the reproducibility of results among different laboratories possibly due to methodological discrepancies. Therefore, a harmonized approach, according to criteria of Next generation Flow (NGF)/ Next generation sequencing (NGS) established by IMWG (Kumar S et al. Lancet Oncol 2016), is warranted. The aim of this project was to create an “Italian MM MRD network” using standardized NGF and NGS-MRD approach; here we report preliminary results of the NGF part. Methods: The “NGF harmonization project” includes 7 Italian Laboratories willing to commit to EuroFlow protocols after an initial MRD survey: Brescia (L1), Catania (L2), Padova (L3), Roma 1 (L4), Roma 2 (L5), S. Giovanni Rotondo (L6) and Torino (L7). Standardization of all flow cytometers settings was performed by implementation of the EuroFlow Standard Operating Protocol (SOP) for instrument setup and compensation for BD FASCCanto II, BD FACSLyric, BC Navios and BC DxFlex flow cytometers (www.euroflow.org). Bone marrow (BM) aspirates were collected from newly diagnosed MM patients treated with 4 cycles of Dara-VTD (Daratumumab, bortezomib, thalidomide, dexamethasone) induction followed by autologous stem cell transplantation (ASCT) in complete-response/very-good-partial-response at day 100 (+/- 15 days) after ASCT. Anonymized samples were split equally and shipped at room temperature to the participati ng laboratories and simultaneously analyzed. Analyses were carried out following NGF methodology (Flores-Montero J et al. Leukemia 2017) by using a two-tube 8-color antibody panel for monoclonal plasma cells identification (Tube 1:CD27, CD138, CD38, CD56, CD45, CD19, CD117, CD81 and Tube 2: CD27, CD138, CD38, CD56, CD45, CD19, cytoplasmic κ and λ light-chain). We aimed to acquire ≥ 3,000,000 events per tube for a sensitivity of at least 1 x 10 -5; a sample was considered MRD positive when ≥ 20 monoclonal plasma cells were detected. Intraclass Correlation Coefficient (ICC) was performed to evaluate degree of correlation and agreement between measurements; standard deviation (SD) and Coefficient of variation (CV) to measure variability of the dataset. Results. In stage 1 of the study, before starting MRD evaluation on freshsamples, 4 laboratories (L1, L2, L3, L7) performed a blinded analysis of the same 5 anonymous flow-cytometry files from a retrospective data repository of MM patients with/without MRD, to evaluate the inter-operator variability: 100% of the participants were concordant that samples #1, #2 and #4 were MRD positive. Sample #3 was considered MRD positive by 75% of participants whereas 75% considered sample #5 as MRD negative (ICC=0.91, 95% CI 0.69-0.98, p&amp;lt;0.001). In stage 2, a total of 7 samples have been distributed and processed after 48 hours from specimen collection, due to different shipping time in all laboratories. The inter-laboratory correlation study showed a concordance of 100 % for the sample #1 and sample #3 considered as MRD positive, 100% of the participants considered sample #5 and sample #6 as MRD negative, sample #2 was considered MRD positive by 71% of participants whereas sample #4 was considered MRD negative by 86 % of participants. Sample #7 was considered as not evaluable from all participants due to a high percentage of dead cells. (ICC=0.61, 95% CI 0.31-0.91, p&amp;lt;0.001) (Fig. 1). The highest difference in term of antigen expression of monoclonal plasma cells was recorded for CD45 and CD27. We obtained a median of 7 x 10 -6 of Limit of detection (LOD, range 6 x 10 -5 - 2 x 10 -6) and 1 x 10 -5 Limit of Quantification (LOQ, range 1 x 10 -4- 5 x 10 -6) for all samples. A centralized revision (in L7 lab) of all FCS files is ongoing to confirm our preliminary results; moreover, the study will be corroborated increasing the number of fresh samples. Conclusion: our preliminary results demonstrate the importance of a harmonized NGF-MRD assessment to improve the accuracy and comparability of MM-MRD testing among different laboratories. Major discrepancies have been found in fresh samples vs the retrospective dataset suggesting an impact of the transportation time and sample processing on the concordance of our results.

Comparison of Silver, Zinc Oxide, and Chitosan-mediated Nanoparticle synthesis and their antifungal activity against Oral Candidiasis

Aim: To analyze the effectiveness of silver, zinc oxide, and chitosan nanoparticles against oral candidiasis. Materials and Methods: Agar-well diffusion method: After the synthesis of AgNP, ZnONP, and ChNP In the agar plate, wells are cut, and 25 l, 50 l, and 100 l concentrations of the solution are placed. Reading was taken after 24 hours. The zone of inhibition was noted. Time-kill curve assay: RBA broth was prepared and sterilized, and 6 mL was added to all five test tubes. Candidia suspension was added to all five test tubes in the range of 5×105 CFU/ml. The first three tubes contain silver NP, zinc-oxide Np, and chitosan Np with three different concentrations; the fourth tube is considered the growth control, and the fifth tube is the standard (fluconazole). The incubation is done under suitable conditions for varied time intervals (1h, 2h, 3h, 4h, 5h). Then the percentage of dead cells is calculated at a wavelength of 540nm at regular time intervals. Results and Conclusion: From the agar well diffusion method, Ag Np showed 13mm , zinc-oxide Np 16 mm, and chitosan Np 17 mm zones of inhibition. From the time-kill assay, Chitosan Np at 50 µg/ml showed better bactericidal activity. Chitosan nanoparticles hold great potential as a novel approach for the management of candidiasis, offering improved drug delivery, enhanced antifungal activity, and the possibility of overcoming drug resistance.

Effect of different manual puncture methods on donkey embryo before vitrification

The application of embryo recovery and transfer technology in the donkey industry is far lower than that of horses and cattle. Sometimes the recovered embryos could not be transferred in time, which required embryo cryopreservation. The embryo cryopreservation technology is more conducive to the preservation and transportation of recovered embryos with excellent genetic traits. However, this technique for donkey embryos is not efficient and needs further optimization. The objective of this study was to evaluate the effect of different manual puncture methods on the viability and pregnancy rates of vitrified-thawed donkey embryos. A total of 117 donkey embryos were recovered on day 7–8 post-ovulation and were divided into four groups by random assortment. There were 28 embryos without puncture or cryopreservation (Control). 58 embryos were manually punctured using a 29G needle (VG, n = 29) or microneedle with a sharp tip of <10 μm (VM, n = 29), then vitrified in 15% EG + 15% DMSO + 0.5 M sucrose. Another 31 embryos were punctured with a microneedle and vitrified with 10% EG + 10% DMSO +0.5 M sucrose +2 mol/L proline (VMP). Both fresh embryos and vitrified-thawed embryos were incubated for 3 h (38.5 °C, 5% CO2 in air) before embryo transfer. The results showed that the embryo recovery rates on day 7.5 and 8 were higher than day 7 (P < 0.05). After incubation, dead cells were assessed and the percentages of dead cells in VM and VMP were lower than that in VG (P < 0.05), although both were higher than those in Control (P < 0.05). The pregnancy rates after 23 days post transfer were assessed and the results showed that the pregnancy rate in VG (8.0%) was lower than that in Control (41.7%), VM (24.0%) and VMP (29.6%) (P < 0.05). No pregnancies resulted from the 10 embryos with diameters ≤650 μm in VG, which lower than either VM (33.3%) or VMP (38.9%) (P < 0.05). While, there was no difference in pregnancy rates among all vitrification groups when embryos were >650 μm in diameter (P > 0.05). In conclusion, the embryo recovery rate on day 7 after ovulation was relatively low, and it was more appropriate to extend it to day 8. Microneedle puncture could reduce embryo damage and achieve a higher pregnancy rate compared with 29G needles. Proline has the potential to improve donkey embryo cryopreservation.

Silencing of CDC42 inhibits contraction and growth-related functions in prostate stromal cells, which is mimicked by ML141

BackgroundProstate smooth muscle contraction and stromal growth may contribute to lower urinary tract symptoms suggestive of benign prostatic hyperplasia, but are incompletely understood. A role of the monomeric GTPase CDC42 for smooth muscle contraction and proliferation appears possible, but is unknown for the prostate. Here, we silenced CDC42 expression in prostate stromal cells (WPMY-1), and examined contractility, growth-related functions and responses to the presumed CDC42 inhibitor, ML141. MethodsWPMY-1 cells were transfected with scrambled or CDC42-specific siRNA, and characterized for GTPase activities, contraction, proliferation, colony formation, apoptosis, cell death and viability. Effects of ML141 were examined in cells with and without silencing. ResultsCDC42 silencing was confirmed by reduced mRNA and protein expression, and reduced CDC42 activity. Silencing impaired contraction (23–47 %), actin organization (25 %), proliferation (17–63 %), colony formation and viability (64–89 %), and increased the percentage of dead cells (2.6-fold). ML141 mimicked the phenotype of silencing in scrambled siRNA-transfected controls, and in non-transfected WPMY-1 cells, including inhibition of contraction, proliferation, colony formation and viability, breakdown of actin organization and increased cell death. In CDC42-silenced cells, ML141 still affected phalloiding organization, proliferation and cell death, with effect sizes resembling controls without silencing. ML141 inhibited RhoA activity in CDC42-silenced cells, but not in cells without silencing. ConclusionsCDC42 promotes contraction of prostate stromal cells, and drives stromal growth by CDC42-mediated proliferation and suppression of apoptosis-independent cell death. ML141 mimicks all effects of CDC42 silencing, but its specificity may be limited and depends on GTPase phenotypes of cells.

Mechanical and antibacterial properties of an experimental flowable composite containing Nb2O5 and NF_TiO2 nanoparticles.

This study developed an experimental flowable composite incorporated with niobium pentoxide (Nb2O5) combined or not with titanium dioxide co-doped with fluorine and nitrogen (NF_TiO2) and evaluated the mechanical and antibacterial properties. The experimental flowable composite (TEGDMA + BisGMA 1:1 + 60%wt – inorganic filler – borosilicate 0.7 μm) was formulated according to the type and concentration of Nb2O5 and NF_TiO2 (0.5, 1, 1.5 and 2 wt%) or NF_TiO2 + Nb2O5 (0.25, 0.5, 0.75 and 1 wt% - 1:1). The control groups were formed by the experimental composite without the incorporation of Nb2O5 and/or NF_TiO2 (GC-E) and by a commercial flowable composite (GC). The characterization of the surface of the composite and its particles was carried out using scanning electron microscopy (SEM) and energy dispersive x-rays (EDX). Specimens were manufactured and subjected to mechanical tests of flexural strength (FS) (n = 12), flexural modulus (FM) (n = 12), roughness (Ra) (n = 10), microhardness (n = 10), and contact angle (n = 10); and, to evaluate the antibacterial activity, they were submitted to tests of biofilm formation against S. mutans (CFU/mL) (n = 5), biofilm biomass by dry weight (n = 5) and confocal laser microscopy (%LIVE/DEAD) (n = 5). Data were submitted to one-way ANOVA and Tukey's post-hoc and, those that were not homoscedastic, but with normality, were submitted to Welch's ANOVA and Games-Howell's post-hoc. Dunnet's test was used to compare the controls with the other experimental groups (α = 5). The Nb2O5 particles had an average size of 32.4 μm and the nanoparticles (NPs) of NF_TiO2, 10 nm. EDX analysis identified isolated peaks of N, F, Ti, and Nb confirming the presence of these particles in the resin matrix. The 1.5% NF_TiO2 group had a higher FS and FM than the controls (p < 0.05). GC showed higher microhardness between groups (p < 0.05). There was no difference between the experimental groups regarding contact angle and roughness (p > 0.05), except for GC, which had the highest Ra values and the lowest contact angle between groups (p < 0.05). Composites containing 0.5%, 1%, 1.5%, and 2% Nb2O5, 1%, 1.5%, and 2% NF_TiO2 and 2% Nb2O5 + NF_TiO2 showed lower biofilm formation (p < 0.05), lower total biofilm biomass (p < 0.05), and a higher percentage of dead cells (44%, 52%, 52%, 79%, 42% 43%, 62%, 65%, respectively) than GC and GC-E (5% and 1%, respectively). It is concluded that the incorporation of 1.5% NF_TiO2 promoted a greater FS and FM among the experimental composites and that the addition of Nb2O5 particles (0.5%, 1%, 1.5%, and 2%), NF_TiO2 (1%, 1.5% and 2%) and the combination Nb2O5 + NF_TiO2 (2%) showed significant antibacterial effects.

Surface properties and Streptococcus mutans biofilm adhesion of ion-releasing resin-based composite materials

ObjectiveTo evaluate the adhesion of Streptococcus mutans (S. mutans) and related surface properties of ion-releasing resin-based composite (RBC) restorative materials. MethodsTwo ion-releasing RBCs, Activa (ACT) and Cention-N (CN), were compared to a conventional RBC (Z350) and a resin-modified glass ionomer cement (Fuji-II-LC). Ten disk-shaped specimens were fabricated for each material (n = 40). After standardized surface polishing procedure, the surface properties of the specimens were evaluated using surface roughness measurements by a profilometer and hydrophobicity using water contact angle measurements. To assess bacterial adhesion, the number of S. mutans bacteria was calculated from colony-forming units (CFU). Confocal laser scanning microscope analysis was done for qualitative & quantitative assessment. The data were analyzed using One-way ANOVA followed by Tukey's post-hoc test to compare the mean values of surface roughness, water contact angle and CFU values. To compare the mean dead cell percentage Kruskal-Wallis rank test and Conover test were used. A p-value of ≤ 0.05 was used to report the statistical significance. ResultsZ350 and ACT had the smoothest surfaces, followed by CN, and the roughest surface was seen in FUJI-II-LC. The lowest water contact angles were seen in CN, and Z350, and the highest were in ACT. S. mutans counts were the highest in ACT and the lowest in Z350 and CN. CN and Fuji-II-LC registered the highest percentage of dead bacterial cells, while the lowest were in ACT. SignificanceSurface properties did not significantly influence bacterial adhesion. More S. mutans bacteria accumulated on ACT than on the nanofilled composite and on CN. CN had antibacterial effects against Streptococcus mutans biofilms.

A novel glass ionomer cement with silver zeolite for restorative dentistry

ObjectiveTo develop an antimicrobial silver zeolite glass ionomer cement (SZ-GIC) and determine its biocompatibility, physical, adhesive and antibacterial properties. MethodsSilver nitrate and sodium zeolite were used to synthesize silver zeolite (SZ). SZ-GICs were prepared by incorporating SZ into GIC at 5% (SZ-GIC5), 2% (SZ-GIC2), or 1% (SZ-GIC1) by weight, respectively. The SZ-GICs were characterized by evaluating surface morphology, topography and elemental composition. SZ-GICs’ biocompatibility was assessed by evaluating cell cytotoxicity. Their physical properties were determined by testing setting time, compressive strength, flexural strength, water sorption and solubility. Their adhesive property was assessed by evaluating micro-tensile bond strength. Their antibacterial properties were assessed by evaluating biofilm growth kinetic, metabolic activity, viability and morphology. GIC was used as a control. ResultsSZ was a three-dimensional crystalline mineral. SZ-GICs (including SZ-GIC 5, 2 and 1) showed similar surface morphology and topography to GIC. SZ-GIC1 and GIC had no difference in cell cytotoxicity (p>0.05). SZ-GICs and GIC showed no difference in setting time (p>0.05). SZ-GICs had higher compressive and flexural strength than GIC (p<0.05). SZ-GIC2 and SZ-GIC1 showed lower water sorption and solubility than GIC (p<0.05). SZ-GICs had higher micro-tensile bond strength than GIC (p<0.05). Biofilms on SZ-GICs’ surfaces showed lower colony-forming units, decreased metabolic activities, higher percentages of dead cells and more ruptured bacterial cells compared with those on GIC. ConclusionSZ-GIC with silver zeolite at 1% by weight are as biocompatible as conventional GIC. The SZ-GICs have enhanced physical, adhesive and antibacterial properties than GIC. Clinical significanceA silver zeolite glass ionomer cement was developed. The SZ-GICs have great potential for caries prevention and management.

Abstract LB036: IRF8 regulates tumor cell sensitivity to intrinsic ferroptosis by repressing p53

Abstract Tumor cell resistance to ferroptosis, which is a recently discovered CTL induced cell death pathway, remains incompletely understood. We report here that interferon regulatory factor 8 (IRF8) functions as a regulator of tumor cell intrinsic ferroptosis. Genome-wide gene expression profiling identified ferroptosis pathway as an IRF8-regulated pathway in tumor cells. IRF8.KO tumor cells are resistant to intrinsic ferroptosis induction and also exhibit decreased ferroptosis in response to tumor specific CTL killing. Loss of IRF8 increases p53 express in tumor cells and knocking out Tp53 in IRF8.KO cells restored tumor cell sensitivity to intrinsic ferroptosis induction. We were able to show that treating tumor-bearing mice with IRF8-encoding plasmid NTC9385R-mIRF8 encapsulated in a DOTAP-cholesterol lipid nanoparticle significantly reduced tumor growth and increased the percentage of dead cells in the tumor. Our data therefore determine that IRF8 represses p53 expression to maintain tumor cell sensitivity to intrinsic ferroptosis. Citation Format: Dakota Poschel, Mercy Kehinde-Ige, John Klement, Dafeng Yang, Alyssa Merting, Natasha Savage, Huidong Shi, Kebin Liu. IRF8 regulates tumor cell sensitivity to intrinsic ferroptosis by repressing p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB036.