Dead Cells Research Articles

Detection of antimicrobial-induced survival/dead bacteria via mEos4b photoconversion: a preliminary study

The escalating prevalence of hospital-acquired infections poses a critical challenge for healthcare systems worldwide. Effective management requires rapid identification of pathogens and their antibiotic resistance profiles. In this study, we utilized the photoconvertible mEos4b protein, which transitions from green to red fluorescence upon blue light exposure, to distinguish live from dead bacteria. The mEos4b gene was cloned into a prokaryotic vector and expressed inEscherichia coliBL21. The Minimum Inhibitory Concentration (MIC) of the transgenic bacteria was determined for five antibiotics, followed by a post-antibiotic effect assessment over a two-hour exposure period. The optimal photoconversion time for mEos4b was established as 90 s, and confocal microscopy was used to visualize live (green) and dead (red) cells post-exposure. The mEos4b-TR system proved highly specific, accurately distinguishing live and dead bacteria without producing false positives, even in control groups, which is a common issue in commercial live-dead kits. By relying on cellular metabolic activity rather than dyes, this system minimizes nonspecific interactions and contamination, making it more reliable than traditional methods prone to false readings. These results highlight the potential of the mEos4b-TR system as a superior alternative for rapid, precise bacterial viability assessments, particularly in determining antibiotic susceptibility. This preliminary study demonstrates the system's differentiation of viable and non-viable cells, suggesting its potential application in future studies involving novel antibacterial agents to refine antibiotic sensitivity testing.

Integration of the bulk transcriptome and single-cell transcriptome reveals efferocytosis features in lung adenocarcinoma prognosis and immunotherapy by combining deep learning

BackgroundEfferocytosis (ER) refers to the process of phagocytic clearance of programmed dead cells, and studies have shown that it is closely related to tumor immune escape.MethodsThis study was based on a comprehensive analysis of TCGA, GEO and CTRP databases. ER-related genes were collected from previous literature, univariate Cox regression was performed and consistent clustering was performed to categorize lung adenocarcinoma (LUAD) patients into two subgroups. Lasso regression and multivariate Cox regression analyses were used to construct ER-related prognostic features, and multiple immune infiltration algorithms were used to assess the correlation between the extracellular burial-related risk score (ERGRS) and tumor microenvironment (TME). And the key gene HAVCR1 was identified by deep learning, etc. Finally, pan-cancer analysis of the key genes was performed and in vitro experiments were conducted to verify the promotional effect of HAVCR1 on LUAD progression.ResultsA total of 33 ER-related genes associated with the prognosis of LUAD were identified, and the prognostic signature of ERGRS was successfully constructed to predict the overall survival (OS) and treatment response of LUAD patients. The high-risk group was highly enriched in some oncogenic pathways, while the low-ERGRS group was highly enriched in some immune-related pathways. In addition, the high ERGRS group had higher TMB, TNB and TIDE scores and lower immune scores. The low-risk group had better immunotherapeutic response and less likelihood of immune escape. Drug sensitivity analysis revealed that BRD-K92856060, monensin and hexaminolevulinate may be potential therapeutic agents for the high-risk group. And ERGRS was validated in several cohorts. In addition, HAVCR1 is one of the key genes, and knockdown of HAVCR1 in vitro significantly reduced the proliferation, migration and invasion ability of lung adenocarcinoma cells.ConclusionOur study developed a novel prognostic signature of efferocytosis-related genes. This prognostic signature accurately predicted survival prognosis as well as treatment outcome in LUAD patients and explored the role of HAVCR1 in lung adenocarcinoma progression.

Practical application of PMA-qPCR assay for determination of viable cells of inter-species biofilm of Candida albicans-Staphylococcus aureus

Abstract Determining the number of viable cells by calculating colony-forming units is time-consuming. The evaluation of mixed biofilms consisting of different species is particularly problematic. Therefore, the aim of this study was to optimize a molecular method—propidium monoazide quantitative polymerase chain reaction (PMA-qPCR) - for accurate and consistent differentiation between living and dead cells. In the practical experimental example, the number of genome copies representing living cells was determined in a mixed biofilm of Candida albicans-Staphylococcus aureus inhibited by photodynamic inactivation. Optimal conditions such as PMA concentration and the duration of light exposure, the optimization of DNA isolation from the mixed biofilm and standardization of PMA-qPCR parameters were tested prior to the main experiment. The genome copy number was calculated based on the known amount of gDNA in the qPCR reaction and the genome size of the respective microorganism. The results showed that PDI in the presence of 1 mM methylene blue decreased the total genome copy number from 1.65x108 to 3.19x107, and from 4.39x107 to 1.91x107 for S. aureus and C. albicans (p < 0.01), respectively. The main disadvantage is overestimation of the number of living cells represented by genome copy numbers. Such cells are unable to reproduce and grow (no vitality), and are continuously dying. On the other hand, PMA-qPCR determines the copy numbers of all microbial species, including a mix of eukaryotic yeasts and prokaryotic bacteria in a biofilm in one step, which is a great advantage.

Body Scrub Salt Formulation from Cinnamomum burmannii (C. Ness & T. Ness) Blume Extract as an Antibacterial Against Staphylococcus aereus

Skincare is currently a major focus in the beauty industry with skin exfoliation being an important step. One product used to exfoliate the skin is a body scrub, which contains ingredients such as himalayan salt and cinnamon bark extract. Himalayan salt helps remove dead skin cells and moisturizes the skin, while cinnamon extract has antimicrobial properties that can protect the skin from bacteria. This study aims to formulate and test the effectiveness of body scrub with skin analyzer and antibacterial activity against Staphylococcus aureus. This research uses an experimental method with several stages, namely plant material collection, plant identification, powder making, extract making, preparation making, evaluation and stability, effectiveness test with skin analyzer and antibacterial activity against Staphylococcus aureus. The 10% body scrub has better effectiveness than 0%; 2.5% and 5% which obtained a percent increase in skin moisture (71.62%), a decrease in the number of blemishes on the skin (70.73%) and a decrease in pore size on the skin (76.38%) for 4 weeks of treatment. In testing antibacterial activity, the 10% concentration was also considered the optimal concentration because it had the greatest inhibition of 14.20 ± 0.59 mm. The results concluded that Himalayan salt and cinnamon bark extract can be formulated into a body scrub dosage form that meets the stability requirements and has the effectiveness in moisturizing, smoothing, and brightening the skin. The formula that has the most effective antibacterial activity is a body scrub containing extracts with a concentration of 10% against Staphylococcus aureus bacteria.

Rapid on-site detection of viable Vibrio parahaemolyticus in seafood using cis-diamminedichloroplatinum and colorimetric loop-mediated isothermal amplification (CDDP-LAMP).

Vibrio parahaemolyticus in seafood and marine environments poses significant health risks, causing gastroenteritis worldwide. Current detection methods fail to differentiate live from dead cells, leading to inaccuracies in food safety assessments. This study introduces a novel method combining cis-diamminedichloroplatinum (CDDP) with direct colorimetric loop-mediated isothermal amplification (LAMP) for rapid and accurate detection of viable V. parahaemolyticus cells in seafood samples. CDDP treatment at 37°C for 30min selectively inhibits DNA from dead cells, enhancing the specificity of the assay by ensuring only live cell DNA is amplified. The optimized CDDP-LAMP procedure detects alive V. parahaemolyticus within 1h, with results observable through a color change. The CDDP-LAMP assay demonstrates excellent specificity, identifying live V. parahaemolyticus cells while excluding dead cells and other bacteria. It shows a detection limit of 2.348CFU per reaction and successfully detects V. parahaemolyticus in seafood samples across different food matrices. This study is the first to combine CDDP with colorimetric LAMP for direct detecting viable bacteria in food, enhancing specificity by eliminating signals from dead cells. The CDDP-LAMP assay provides a rapid, accurate process for detecting viable V. parahaemolyticus cells, especially in resource-limited settings. It also gives a model for screening different bacterial pathogens, speeding up and improving foodborne illness risk assessments.

Enhanced activity of mixed-culture electroactive biofilms and sulfamethoxazole removal efficiency by adding N-acyl-homoserine lactones in bio-electrochemical system

ABSTRACT The addition of exogenous quorum sensing signaling molecules significantly enhanced the degradation efficiency of antibiotics, such as chloramphenicol in bio-electrochemical systems (BESs). However, the effects and mechanisms by which AHLs addition in BES facilitated the removal of sulfamethoxazole (SMX) remained inadequately explored. This study systematically compared the electrochemical performance and SMX removal efficiency in BES under two conditions: with and without the addition of N-acyl-homoserine lactones (AHLs) signaling molecules. In comparison to the control group, the AHL-treated group exhibited an increase in maximum output voltage from 340 to 489.67 mV, alongside a notable enhancement in SMX removal efficiency over 120 h ranging from 14.65% to 15.76%. Analyses of the live and dead cells and extracellular polymeric substances (EPS) composition revealed that following AHLs addition, both the ratio of live to dead cells and protein content within EPS increased by 12.66% and 74.37%, respectively. Furthermore, microbial community structure analysis indicated that after AHLs supplementation, there was a marked increase in the abundance of electroactive microorganisms as well as antibiotic-degrading and nitrogen-removing bacteria. Notably, Klebsiella – characterised by its electroactivity along with antibiotic degradation and nitrogen removal capabilities – exhibited a relative abundance reaching 56.84% in AHL, reflecting an increase of 28.31% compared to Blank; additionally, electroactive bacteria Dysgonomonas showed a relative abundance rise of 2.49%. Collectively, these findings suggested that enhancements in SMX removal efficiency upon AHLs addition were primarily driven by improvements in electrochemical performance coupled with alterations in microbial community structure.

Inside the Atacama Desert: uncovering the living microbiome of an extreme environment.

The Atacama Desert in Chile is one of the driest and most inhospitable places on Earth. To analyze the diversity and distribution of microbial communities in such an environment, one of the most important and challenging steps is DNA extraction. Using commercial environmental DNA extraction protocols, a mixture of living, dormant, and dead cells of microorganisms is extracted, but separation of the different DNA pools is almost impossible. To overcome this problem, we applied a novel method on soils across a west-east moisture transect in the Atacama Desert to distinguish between extracellular DNA (eDNA) and intracellular DNA (iDNA) at the cell extraction level. Here, we show that a large number of living and potentially active microorganisms, such as Acidimicrobiia, Geodermatophilaceae, Frankiales, and Burkholderiaceae, occur in the hyperarid areas. We observed viable microorganisms involved as pioneers in initial soil formation processes, such as carbon and nitrogen fixation, as well as mineral-weathering processes. In response to various environmental stressors, microbes coexist as generalists or specialists in the desert soil environment. Our results show that specialists compete in a limited range of niches, while generalists tolerate a wider range of environmental conditions. Use of the DNA separation approach can provide new insights into different roles within viable microbial communities, especially in low-biomass environments where RNA-based analyses often fail.IMPORTANCEThe novel e- and iDNA separation technique offers insights into the living community at the cell extraction level in the hyperarid Atacama Desert. This approach provides a new framework for analyzing the composition and structure of the potentially active part of the microbial communities as well as their specialization, ecological network and community assembly process. Our findings underscore the significance of utilizing alternative genomic techniques in low-biomass environments where traditional DNA- and RNA-based analyses may not be feasible. The results demonstrate the viability of the proposed study framework and show that specialized microorganisms are important in initial soil formation processes, including microbial-driven mineral weathering, as well as the fixation of carbon and nitrogen.

Abstract B026: Identification of small RNAs that function as multi-target therapeutics and as tools for novel target discovery in prostate cancer

Abstract Introduction: A major contributor to prostate cancer (PCa) mortality is the development of resistance to single agents targeting androgen receptor (AR) signaling. We have developed an unbiased shRNA-based negative selection screen to identify small RNAs (shRNAs and siRNAs) that inhibit PCa cell growth and survival (toxic sRNAs). Computational analyses of the most toxic sRNAs identified revealed that each can simultaneously target multiple known AR-coregulatory mRNAs. AR-coregulators modulate AR transcriptional response and are essential to AR signaling. We have proposed that the identified toxic sRNAs inhibit androgen signaling through seed-mediated targeting of androgen regulatory gene networks, resulting in inhibition of cell growth and viability. The goal of the current studies was to begin to analyze the broad therapeutic potential of these toxic sRNAs for PCa. We focused on analyzing i) their potential to thwart cell growth and/or viability when delivered to preclinical models of PCa, and ii) the targetome of the toxic sRNAs as a mean to uncover novel AR-coregulators that represent a targetable axis within androgen signaling. Methods: i) cationic lipid nanoparticles (LNPs), that efficiently deliver the toxic siRNAs to PCa cell lines, were used to deliver toxic siRNAs to organoids grown from PCa patient derived xenografts (PDX) models. 3D image analysis using the Ominer® software package was used to determine organoid count and size, the numbers of nuclei per organoid, and the fraction of dead cells to discriminate between cytotoxic and cytostatic effects. ii) to identify novel AR-coregulators, toxic siRNA direct targets were identified by sequencing of transcripts captured by biotinylated toxic siRNAs. Directly bound targets, were subjected to pathway enrichment and network analysis followed by identification of hub genes (Cytoscape). The effect of individually silencing potential AR-coregulators on the expression of androgen responsive genes was analyzed using Nanostring. Results and Conclusions: i) The effect of the toxic siRNAs on growth and viability of the PCa PDX-derived organoids will be presented and discussed as compelling preliminary data for therapeutic testing in more complex pre-clinical models (PCa PDXs). ii) Based on bioinformatic and downstream analysis of the direct targets of two toxic siRNAs, we selected 12 potentially novel AR- coregulators, with varied known functions including chromatin modifiers, transcriptional regulators, etc. Silencing of several of these potential AR-coregulators significantly affect expression of numerous AR responsive genes. Future experiments will focus on analyzing their interaction and functional association with the AR (i.e. effect of AR binding to chromatin). In conclusion, we have developed an assay that allowed the identification of toxic sRNAs for PCa cells and demonstrated their potential as therapeutics and as a tool for discovery of potential novel targets for PCa. Citation Format: Joshua M Corbin, Hamilton Young, Stefan Wilhelm, Adam S Asch, Chao Xu, Maria J Ruiz Echevarria. Identification of small RNAs that function as multi-target therapeutics and as tools for novel target discovery in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B026.

Apoptosis-induced exosomes from human exfoliated deciduous teeth enhance angiogenesis in human umbilical vein endothelial cells

Exosomes derived from the stem cells of human exfoliated deciduous teeth (SHED) hold promise for tissue regeneration. Apoptotic cells release a variety of extracellular vesicles that affect intercellular communication. This study aimed to investigate the angiogenic effects of SHED-derived exosomes modified via apoptosis induction on human umbilical vein endothelial cells (HUVECs). Apoptosis was induced in SHED via serum starvation for 3 weeks and confirmed by the upregulation of the apoptotic genes, caspase 3 and 9, and via annexin V staining. The apoptotic SHED-derived exosomes were isolated, characterized, and subjected to proteomic analysis. In vitro experiments were performed to assess the effects of apoptotic SHED exosomes on the proliferation, migration, and tube formation of HUVECs. The apoptosis-induced SHED showed increased cell viability and decreased numbers of dead cells compared with those of conventional cultures while retaining their identity as mesenchymal stem cells positive for CD44, CD73, and CD90. The apoptotic SHED-derived exosomes exhibited characteristic features, such as standard size, cup-shaped morphology, and positive staining, for exosomal markers CD9, CD63, and CD81. Proteins associated with apoptosis, programmed cell death, and cellular senescence were downregulated in the apoptotic SHED exosomes, whereas those associated with extracellular matrix organization were upregulated, indicating positive angiogenesis. HUVECs treated with apoptotic SHED exosomes exhibited significantly enhanced proliferation and migration compared with those treated with normal SHED exosomes. The mesh-like structures in the apoptotic SHED exosomes exhibited significantly increased signs of angiogenesis. The findings of this study provide new insights into the potential use of apoptotic SHED-derived exosomes in regenerative medicine.

Abstract 4118360: Lipocalin10 is Critical for Macrophage Efferocytosis to Repair Myocardial Ischemia/Reperfusion Injury

Introduction: Efficient clearance of dead/dying cells by macrophages (MΦs) (termed efferocytosis) is critical for timely repairing the injured heart after ischemia/reperfusion (I/R). Prior work by us and others shows that Lipocalin 10 (Lcn10), a secreted protein of lipocalin family, can regulate MΦ polarization and is greatly downregulated in cardiac tissue of patients with heart failure. However, the potential role of Lcn10 in MΦ efferocytosis during cardiac I/R has never been investigated. Methods: Wild-type (WT) and Lcn10-knockout (KO) mice were subjected to 45 minutes of ischemia via the ligation of left anterior descending artery (LAD) followed by reperfusion. Cardiac function was measured by echocardiography. Flow cytometry was utilized to assess MΦ efferocytosis. Bone marrow-derived MΦs (BMDMs) were used to test efferocytosis in vitro and dissect mechanism. Dead cells were prepared by treating red-dye-labelled H9C2 cells with H 2 O 2 (1mM, 2h). Results: After co-culturing BMDMs with dead H9C2 cells for 2h, we observed a 25% reduction of efferocytosis in Lcn10-KO BMDMs, compared to WTs. Using Lcn10-KO mCherry mouse model (cardiomyocyte-specific overexpression of mCherry in Lcn10-KO background), we consistently found that cardiac MΦs in Lcn10-KO mice taken up fewer dead mCherry-myocytes at 4 days post-I/R surgery, leading to an increased accumulation of cardiac dead cells, higher serum levels of troponin I, greater myocardial fibrosis, and dramatically impaired cardiac function, compared to WT mCherry controls. In contrast, pre-treatment of BMDMs with recombinant Lcn10 protein (rLcn10) enhanced efferocytosis by 50%. Mechanistically, RNA-seq analysis revealed that many efferocytosis-related genes were dysregulated ( e.g., downregulation of Id 1, Id 3, Itga 8, Itga v, Cd 44, Cx3cr 1, Msr 1, and upregulation of Sirp α) in Lcn10-KO BMDMs, compared to WTs. Interestingly, most of these dysregulated genes are controlled by transcription factors Id1/3. Further studies showed that rLcn10 interacted with Cd44 and activated the expression of its downstream Id1/3, leading to enhanced MΦ efferocytosis. Importantly, rLcn10-mediated efferocytosis was greatly impaired by pre-treatment of MΦs with either anti-CD44 blocking antibody (KM201, 10µg/ml) or Id1/3 inhibitor (AGX51, 25µM). Conclusion: Our data indicate that Lcn10 is essential for MΦ efficient efferocytosis to repair I/R-induced cardiac damage though affecting the Cd44-Id1/3 signaling and its related MΦ surface receptors.

Efferocytosis: A new therapeutic target for stroke.

Efferocytosis refers to the process that phagocytes recognize and remove the apoptotic cells, which is essential for maintaining tissue homeostasis both in physiological and pathological conditions. Numerous studies have demonstrated that efferocytosis can prevent secondary necrosis and proinflammatory factor release, leading to the resolution of inflammation and tissue immunological tolerance in numerous diseases such as stroke. Stroke is a leading cause of death and morbidity for adults worldwide. Persistent inflammation triggered by the dead cells or cell debris is a major contributor to post-stroke brain damage. Effective efferocytosis might be an efficient strategy to minimize inflammation and restore brain homeostasis for neuronal regeneration and function recovery. In this review, we will discuss the phagocytes in the brain, the molecular mechanisms underlying efferocytosis, the role of efferocytosis in inflammation resolution, and the potential therapeutic applications targeting efferocytosis in stroke.

Prevalence and associated factors for isolated Malassezia species in patients with Dandruff in Mekelle City, Tigrai, Ethiopia

ObjectiveDandruff, a condition caused by lipophilic Malassezia fungi, is an excessive shed of dead skin cells from the scalp. Effective preventive and curative measures of the condition depend on knowledge and understanding of the prevalence of the condition, the common etiologic species, and the associated factors. This study aimed to investigate the prevalence, common etiologic species, and associated factors of Malassezia infection in Mekelle City, Ethiopia.MethodA facility-based cross-sectional study was conducted from February 2019 to June 2020 involving 217 participants who were visiting dermatology clinics to seek treatment for dandruff conditions. Information on the socio-demographic characteristics and hair care behaviors of the participants was obtained. Isolation and identification of Malassezia species from scalp scrapings using cultural and biochemical tests were carried out.ResultsOut of the 217 participants with dandruff, 111 (51.15%) were positive for Malassezia fungi. One hundred forty (140) Malassezia isolates were collected from the 111 positive participants. Further study of the isolates yielded three etiologic species: Malassezia globosa (67.15%), M. furfur (21.70%), and M. restricta (12.15%). Demographic characteristics, namely gender (AOR = 2.605; 95%CI: 1.427 − 4.757) and age (AOR = 2.667; 95%CI: 1.046 − 6.795), as well as hair care behaviors, namely use of hair oil (AOR = 2.964; 95%CI: 1.288 − 6.820), were associated with the presence of Malassezia species. However, the use of anti-dandruff shampoo (AOR = 2.782; 95%CI: 1.301 − 10.993) was negatively associated with the presence of Malassezia species among the participants with dandruff conditions. These findings open opportunities to devise effective prevention, management, and control measures for Malassezia-based dandruff conditions.

DDEL-18. OPTIMIZED METHOD TO QUANTIFY TTFIELDS-INDUCED PERMEABILIZATION OF CELL MEMBRANES

Abstract Tumor Treating Fields (TTFields) therapy is FDA-approved for glioblastoma. Using plate-based readouts of FITC-dextran fluorescent probes in U87 human glioblastoma cells, we previously reported increased uptake of FITC-dextrans (4-20kDa) post-TTFields exposure. However, this approach is unable to distinguish FITC-dextran uptake in live-vs-dead cells post-TTFields exposure. We demonstrate flow cytometry (FC) as a more robust and economical method to quantify the amount of FITC-dextrans entering the cells. We first identified the optimal [FITC-dextran] for the FC-based approach (titration 0-6.5mg/mL). Cells were then seeded onto coverslips and exposed to 200kHz TTFields, 1-3V/cm peak-to-peak (inovitro systemTM, Novocure), or no-TTFields (control), for 24-72h. Cells were harvested and stained for 1h using 4kDa or 20kDa FITC-dextrans. Cells were washed and analyzed by FC for mean±SD fluorescence intensity, and compared by unpaired t-test. Dead cells were excluded using propidium iodide staining. In the control vs. TTFields group, 4kDa FITC-dextrans had significantly increased uptake at 24h (4.9±0.3 vs. 8.0±1.7, p=0.01), 48h (6.5±0.2 vs. 8.3±1.0, p=0.01), and 72h (5.7±0.3 vs. 8.8±1.0, p=0.001). Likewise, in the TTFields group, the 20kDa FITC-dextrans had significantly increased uptake at 24h (5.6±0.3 vs. 7.5±0.9, p=0.006), 48h (4.8±0.3 vs. 6.6±0.8, p=0.006), and 72h (5.3±0.2 vs. 11.3±2.6, p=0.004). In the TTFields group, the 20kDa FITC-dextrans had significantly increased uptake between the 24h and 72h timepoints (p=0.03). The FC-based method utilized 0.36mg of FITC-dextrans/sample, much less compared to the 1-3mg recommended for plate-based methods. Another advantage of the FC-based method was exclusion of the dead cell population after TTFields exposure. We validated a FC-based-method that is economical and can reproducibly quantify the membrane permeabilization induced by TTFields. Compared to control conditions, TTFields increase membrane permeabilization of cells that remain alive after TTFields exposure, in a time-dependent manner for the 20kDa probe.

BIOM-64. EXPLOITING CELL-FREE DNA TO DISSECT MECHANISMS OF RESISTANCE TO TEMOZOLOMIDE IN GLIOBLASTOMA CELLS

Abstract Temozolomide (TMZ) is a cytotoxic alkylating agent that serves as a cornerstone in glioblastoma (GBM) treatment, yet is notorious for eliciting hypermutation in recurrent tumors that have developed drug resistance by downregulating mismatch repair (MMR). We reasoned that cell-free DNA (cfDNA), a non-invasive measure for detecting tumor-derived biomarkers, could be used to temporally examine the mechanism of resistance to TMZ. We therefore designed an in-vitro experiment using the patient-derived GBM43 cell line. Over five days, batches of cells were treated with either TMZ or vehicle. Cell counts and cfDNA were collected every 24 hours. We observed an associated increase in both dead cell counts and cfDNA yield in treated cells compared to untreated cells. In TMZ-treated samples, cfDNA showed a periodic fragmentation pattern consistent with internucleosomal cleavage and TMZ-induced apoptosis. Intriguingly, higher numbers of variants were found in the cfDNA of untreated samples when compared to treated cfDNA, possibly reflecting TMZ-induced selection of resistant subclones in treated samples. This was further supported by the finding that the average variant allele frequency (VAF) increased over time in TMZ-treated samples, consistent with purifying selection. Conversely, cfDNA from vehicle-treated samples demonstrated a decrease in the average VAF over time, consistent with clonal diversification. Although we did not observe an enrichment of the TMZ-associated mutational signature SBS11, we did find higher levels of defective MMR mutation signature SBS44 in treated samples, potentially indicating the selection of subclones with defective MMR mechanisms. Overall, our preliminary findings show that cfDNA can be used to resolve mechanisms of TMZ resistance over time. We are addressing open questions by performing longer treatment times, testing other patient-derived cell lines, and performing murine xenotransplantation experiments to test TMZ-associated hypermutation in vivo. Our work will contribute to our understanding of TMZ resistance, and importantly push the development of cfDNA-based biomarkers for measuring TMZ-sensitivity in patients.

EXTH-39. A BENCH-TOP MODEL FOR THE OPTIMIZATION OF ULTRASOUND PARAMETERS FOR SONODYNAMIC THERAPY OF GLIOBLASTOMA

Abstract BACKGROUND In-vitro studies have demonstrated that low frequency sound waves can interact with hemo-porphyrin molecule to induce the production of reactive oxygen species. This biophysical phenomenon can induce tumour cell death in glioblastoma cells in-vitro. The purpose of this study was to evaluate the production of reactive oxygen species and expression of apoptotic markers in glioblastoma cells in response to varying total treatment time and ultrasound power using a bench-top focused ultrasound (FUS) device. METHODS U251 cells were exposed to 5-Aminolevulenic Acid (5-ALA) at varying concentrations for up to 24 hours. The time point with peak proto-porphyrin IX (PPIX) fluorescence was established using fluorescence activated cell sorting (FACS). A transducer utilized at 0.91MHz was placed on an inverted stage with the focus targeted to the bottom of a standard 96-well cell culture plate. Pressure wave delivery was confirmed using a hydrophone. GBM cells were treated with 5-ALA alone, FUS alone, or 5-ALA + FUS and the cells were incubated for 24 hours prior to measuring ROS induction and cell death. ROS was measured using FACS with the CellROX Green Kit for Oxidative Stress Detection and Annexin-V was measured using the Dead Cell Stain Kit. RESULTS Peak fluorescence intensity for PPIX in the U251 cell line was found to be induced through incubation with 5-ALA for 24 hours with a concentration 200mg/mL. The fluorescence intensity of PPIX was found to be 313% greater in the treated group compared to the control group. Peak ROS was observed using a 0.91MHz transducer frequency for a total time of 120s with 6W average power. ROS was 61% and 66% greater in the SDT group compared to the FUS and 5-ALA group respectively. CONCLUSION Using this bench-top set up we successfully induced ROS and apoptosis in U251 cells. Establishing a reliable in-vitro model will allow us to further investigate the effects of other sonication parameters such as burst length.

Design and fabrication of PCF‐based terahertz sensor for breast cancer cell detection

AbstractBreast cancer is a type of cancer that is common in women worldwide, which emphasises its significance in identification with preventative treatment methods. The invented Photonic Crystal Fibre (PCF) exhibits outstanding performance in detecting Breast Cancer. The suggested model of the authors includes Hybrid layout within clad surface alongside Square Core. Introduced PCF detector exhibits max Relative Sensitivity (RS) of 96.82% as well 96.74% for breast cancer cell MCF‐7 as well MDA‐MB‐231 correspondingly. The authors also investigated the Confinement Loss of 1.642 × 10−10 dB/m, 2.461 × 10−10 dB/m with Effective Material Loss of 0.0473, 0.0565 cm−1 for the mentioned cells. Increased outcomes, customised therapy, plus quick action are made possible by swift identification in breast carcinoma. Timely malignancy detection reduces requirements to severe therapy by enabling simpler medicines. Additionally, making continuous illness detection easier, improving patient treatment. Furthermore, reliable evaluation contributes for investigating advancements that improve worldwide recognition as well as therapy alternatives. The introduced PCF Perhaps crucial in quick identification of these deadly cells as it has an extraordinary sensing ability. In conclusion, it has numerous possibilities in the healthcare sector.

Simvastatin Efficacy on Endodontic Biofilms: An In Vitro Study.

Mature biofilms were grown on dentin specimens and put in contact with the solutions to determine their antibiofilm activity. To assess residual antimicrobial activity, the dentin samples were immersed for 5 min in the solutions before microbial infection for 3 weeks. The study groups were: (1) 8% SIM, (2) 4% SIM, (3) 4% DC, (4) 2.5% sodium hypochlorite (NaOCl), and (5) 0.9% saline solution (SS). Cell viability was evaluated by means of the adenosine triphosphate (ATP) assay and flow cytometry (FC). The data collected were analyzed with the ANOVA test using Welch's correction followed by the Games-Howell test. The level of statistical significance was p < 0.05, and statistical analysis was performed using SPSS 23.0. All study groups reduced the biofilms significantly with respect to the control. The highest reduction percentage was obtained by 2.5% NaOCl, followed by SIM 8% and 4%, without statistically significant differences. In terms of residual activity, the 4% DC solution obtained a higher percentage of dead cells. Solutions of 4% and 8% SIM, and 4% Diclofenac, show antimicrobial and residual activity against multispecies endodontic biofilms.

Dissecting the dynamics of cell death pathways in Hirschsprung’s disease: a comparative analysis of viable and non-viable cells under proinflammatory conditions

PurposeThe present study explores the dynamics of cell death in Hirschsprung’s disease (HSCR) and control (CO) groups under inflammatory stress conditions.MethodsUsing flow cytometry, we analyzed intestinal colonic organoid cultures derived from the ganglionic segment of the HSCR and CO groups. Our analysis focused on the quantification of RIPK1-independent and RIPK1-dependent apoptosis, as well as necroptosis in both viable and non-viable cells under acute and chronic inflammatory stress.ResultsOur findings indicate that HSCR cells are particularly vulnerable to inflammation during acute proinflammatory stress, as evidenced by an increase in dead cells (Zombie +). Under chronic conditions, adaptive changes are observed in both HSCR and CO groups, indicating survival mechanisms. These adaptations are uniquely altered in HSCR, suggesting an impaired response to chronic inflammation. HSCR cells show significantly decreased RIPK1-dependent apoptosis in acute scenarios compared to chronic ones, unlike the CO group, implying varied responses to different inflammatory stresses. In non-viable cells, considerable changes in RIPK1-dependent apoptosis under chronic conditions in HSCR indicate a heightened inflammatory response compared to CO.ConclusionThis research provides insights into cell death regulation in HSCR under inflammatory stress by using patient-derived organoids, underscoring the complexity of its inflammatory response.

Mitochondrial donation as a mechanism of participation by mesenchymal stromal cells in regenerative processes

Mesenchymal stromal cells (MSCs) are universal regulators of regenerative processes due to their ability to secrete regulatory molecules or replace dead cells through differentiation in the appropriate direction. Recently, another mechanism for the beneficial effects of MSCs on damaged tissue has been discovered, such as the transfer of mitochondria into its cells in response to stress signals. MSCs can transfer mitochondria through tunneling nanotubes that form a communication bridge between cells, through gap junctions, by release as part of extracellular vesicles or in free form, and as a result of complete or partial fusion with recipient cells. In damaged cells that received mitochondria from MSCs, impaired energy metabolism is restored and oxidative stress is reduced, which is accompanied by increased survival, and in some cases also increased proliferation or a change in differentiation status. The restoration of energy after the transfer of mitochondria from MSCs has a beneficial effect on the functional activity of recipient cells and suppresses inflammatory reactions. A significant contribution of the MSC mitochondrial donation to the therapeutic efficacy of MSCs has been repeatedly demonstrated in models of damage to various organs in experimental animals. This stimulates the search for methods to enhance the process of mitochondrial donation. However, it should be taken into account that MSCs are able to transfer mitochondria to malignant cells as well, thereby stimulating tumor growth and increasing its resistance to chemotherapy. These data make it necessary to evaluate the prospects for the use of MSCs in cell therapy with caution. On the other hand, they can serve as a basis for the search for new therapeutic targets in the treatment of oncological diseases.

SIRT3 mitigates high glucose-induced damage in retinal microvascular endothelial cells via OPA1-mediated mitochondrial dynamics

Oxidative stress in endothelial cells is pivotal in diabetic retinopathy (DR), with mitochondrial homeostasis being crucial to mitigate this stress. This study explored the roles of mitochondrial sirtuins (SIRTs) in high glucose (HG)-induced oxidative stress, related endothelial impairment, and mitochondrial homeostasis damage in rat retinal microvascular endothelial cells (RMECs). RMECs were cultured under HG or equivalent osmotic conditions. Cell viability was assessed using the Cell Counting Kit-8 assay, whereas cell death and survival were determined via calcein-AM/propidium iodide double staining. Reactive oxygen species (ROS) levels were measured using 2′,7′-dichlorofluorescein fluorescence. Expression of mitochondrial SIRTs3-5 and key mitochondrial homeostasis molecules was quantified by the quantitative real-time polymerase chain reaction and confirmed by western blotting. Mitochondrial morphology was evaluated using electron microscopy and the MitoTracker fluorescent probe. A SIRT3-overexpressing RMEC line was constructed to assess the role of SIRT3 in oxidative stress and mitochondrial dynamics. After 48 h of HG exposure, cell viability was significantly reduced, with a concomitant increase in cell death and ROS levels, alongside a marked decrease in SIRT3 expression and molecules associated with mitochondrial dynamics. SIRT3 overexpression reversed these effects, particularly increasing the mitochondrial fusion-related molecule, optic atrophy 1 (OPA1). However, the OPA1 inhibitor, MYLS22, blocked the protective effect of SIRT3, leading to more dead cells, a higher ROS level, and intensified mitochondrial fragmentation. These results suggest that SIRT3 is involved in HG-induced imbalanced mitochondrial dynamics of endothelial cells in DR, potentially through the OPA1 pathway.