Detection Of Viability Research Articles

Abstract PO4-28-04: Curcumin inhibits breast cancer cell proliferation by regulating ciRS-7/miR-7-5p/CKS2 axis

Abstract Background Breast cancer (BC) is the most common cancer in women worldwide. Curcumin is a polyphenolic turmeric-derived compound that has anti-proliferative and anti-tumor properties in different cancer types by acting on multiple molecules. Circular RNAs (circRNAs) are non-coding, single-stranded, covalently closed RNA molecules that act as regulators of the microRNA (miRNA) activity. Recent studies show that circRNAs are potential contributors to the onset and progression of many cancer types. CircRNA ciRS-7 acts as an oncogene and accelerates tumor progression by competitively suppressing miR-7-5p in BC. However, whether curcumin can regulate ciRS-7 to inhibit BC progression is still unclear. Method Breast Cancer cell lines (MCF-7 and T47D) and normal epithelial cell line (MCF-10A) were cultivated and treated with Curcumin (5μM, 10 μM, 20 μM) or DMSO as a control. Also, the cell lines were transfected with ciRS-7 siRNA, miR-7-5p mimic, and their non-targeted controls. The cell viability was detected by Cell Viability Detection Kit-8 (CVDK-8) using an ELISA plate reader. To detect cell migration, scratch assay was performed after 24hs of transfection and a phase contrast microscope was used to acquire images. The effect on apoptosis was detected by Annexin V-FITC Apoptosis Detection Kit using flow cytometry. Potential target genes of miR-7-5p were identified by searching for the overlapping genes in miRNet and miRTarBase v8 with the overexpressed genes in BC patient tissue samples in the TCGA database. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of ciRS-7, miR-7-5p, and the selected genes. qRT-PCR experiments were performed in duplicate and the 2−ΔΔCt method was used for relative quantitation analysis. Results Curcumin treatment, depending on the increased doses, decreased cell proliferation through inducing apoptosis in MCF-7 and T47D cancer cells. Curcumin's anti-proliferative effect was shown to be quite restricted on normal MCF-10A cells, as compared to MCF-7 and T47D cancer cells. In MCF-7 and T47D cells treated with curcumin, cell migration was dramatically inhibited. Curcumin has very little influence on the migration of MCF-10A cells. In both cancer cell group that was transfected with ciRS-7 siRNA and miR-7-5p mimic, it was observed that apoptosis was increased, proliferation was suppressed, and migration was decreased compared to its control groups. Moreover, the expression of ciRS-7 was found to be significantly decreased in the curcumin-treated group, while miR-7-5p was shown to be significantly higher. CKS2 gene one of the possible target genes of miR-7-5p that was identified by using in silico approaches, was also downregulated in curcumin-treated BC cell lines compared to its control group. ciRS-7 and CKS2 expression levels were found to be downregulated, whereas miR-7-5p expression level was found to be elevated in MCF-7 and T47D cells that were transfected with ciRS-7 siRNA. Additionally, CKS2 gene expression was found to be downregulated in miR-7-5p mimic transfected cells. Conclusion Curcumin is derived from the turmeric plant (Curcuma longa) and has been used since 3000 B.C. as a food additive. Nowadays, curcumin is one of the most essential anti-cancer substances that was examined. However, investigations on the influence of curcumin on the circRNA-miRNA-mRNA axis are scarce. Curcumin has been demonstrated to inhibit proliferation and induce apoptosis of BC cells via the ciRS-7/miR-7-5p/CKS2 axis in the present study. Keywords Breast Cancer, Curcumin, ciRS-7, miR-7-5p, CKS2 Citation Format: Asmaa Abuaisha, Murat Kaya, Ilknur Suer, Selman Emiroglu, Fahrunnisa Abanoz, Mustafa Tukenmez, Neslihan Cabıoğlu, Mahmut Muslumanoglu, Kivanc Cefle, Sukru Palanduz, Sukru Ozturk. Curcumin inhibits breast cancer cell proliferation by regulating ciRS-7/miR-7-5p/CKS2 axis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-04.

Liver Imaging Reporting and Data System Contrast-Enhanced US Nonradiation Treatment Response Assessment Version 2024.

The American College of Radiology Liver Imaging Reporting and Data System (LI-RADS) standardizes the imaging technique, reporting lexicon, disease categorization, and management for patients with or at risk for hepatocellular carcinoma (HCC). LI-RADS encompasses HCC surveillance with US; HCC diagnosis with CT, MRI, or contrast-enhanced US (CEUS); and treatment response assessment (TRA) with CT or MRI. LI-RADS was recently expanded to include CEUS TRA after nonradiation locoregional therapy or surgical resection. This report provides an overview of LI-RADS CEUS Nonradiation TRA v2024, including a lexicon of imaging findings, techniques, and imaging criteria for posttreatment tumor viability assessment. LI-RADS CEUS Nonradiation TRA v2024 takes into consideration differences in the CEUS appearance of viable tumor and posttreatment changes within and in close proximity to a treated lesion. Due to the high sensitivity of CEUS to vascular flow, posttreatment reactive changes commonly manifest as areas of abnormal perilesional enhancement without washout, especially in the first 3 months after treatment. To improve the accuracy of CEUS for nonradiation TRA, different diagnostic criteria are used to evaluate tumor viability within and outside of the treated lesion margin. Broader criteria for intralesional enhancement increase sensitivity for tumor viability detection. Stricter criteria for perilesional enhancement limit miscategorization of posttreatment reactive changes as viable tumor. Finally, the TRA algorithm reconciles intralesional and perilesional tumor viability assessment and assigns a single LI-RADS treatment response (LR-TR) category: LR-TR nonviable, LR-TR equivocal, or LR-TR viable.

How precisely can x-ray predict the viability of wild flower plant seeds?

The detection of seed viability is an important step in seed production, as well as in conservation and restoration practice. Due to random natural events, the quality and viability of seeds of wild flowering species vary substantially, and hence a quick and reliable method for seed viability assessment is desirable. X-rays provide information about the internal structures of a seed and therefore show promise for detection of viability and even germination capacity. Seeds of 207 accessions of 176 wild flowering plant species were x-rayed and the viability results were compared with combined germination-TZ test results. Of special interest was whether there are certain plant families for which x-ray is an appropriate method for viability detection, considering correlations with seed internal morphology, seed mass and/or shape. The comparison revealed a strong correspondence between viability determination by combined germination-TZ tests and by x-ray analysis. According to taxonomy and seed type, two main groups could be distinguished, that differed significantly in viability detection by x-ray and combined germination-TZ test. Whereas the evaluation of little/non-endospermic seeds gave approximately identical results, there was greater discrepancy for of endospermic seeds. Seeds of different sizes and shapes were evaluated similarly with both methods. Especially for little/non-endospermic seeds, x-ray can provide a useful and quick tool for viability detection, whereas for endospermic seeds, further research is needed. For the commercial seed industry, viability detection via x-ray should be the first step before further vigour testing is performed.

Chitosan/nanoclusters membrane-based sensors with antibacterial properties for rapid detection of bacterial viability and food preservation

In this work, copper nanoclusters (CuNCs) with tiny particle sizes were successfully obtained by a template-assisted synthesis approach. The interfacial interaction energies of cysteine (Cys) and CuNCs were simulated and calculated by molecular dynamics using the Materials Studio program package, and the anchoring sites of Cys on CuNCs surface in aqueous solution were investigated in detail by adsorption modeling. Chitosan (CS), a natural polymer, was used as a confining agent to enhance the excellent peroxidase (POD)-like activity of CuNCs. Therefore, the composite can catalyze the decomposition of hydrogen peroxide (H2O2) to generate hydroxyl radical (·OH) and possess higher antibacterial activity. A broad-spectrum antibacterial system based on CS@CuNCs was established for E. coli and S. aureus, avoiding the harmful effects of high concentration of H2O2 on organisms. In addition, a cascade reaction with glucose oxidase (GOx) was established for the specific consumption of glucose by bacteria, and a method for the visual colorimetric detection of bacterial viability within 30 min was established. Finally, based on the polymer properties of CS, a cling wrap with antibacterial properties was also prepared for food preservation. In summary, this study provides innovative proposals for the application of CS/nanocluster composite in food packaging, food safety, and even bioengineering.

Astaxanthin suppresses the malignant behaviors of nasopharyngeal carcinoma cells by blocking PI3K/AKT and NF-κB pathways via miR-29a-3p

BackgroundAs a natural extraction, astaxanthin is gaining increasing attention because of its safety and anti-tumor properties. It has been reported to participate in the progression of various types of cancer such as gastric cancer and ovarian cancer. Nevertheless, the role of astaxanthin in nasopharyngeal carcinoma (NPC) has not been investigated.ObjectThe study aimed to explore the anticancer mechanism of astaxanthin in regulating NPC cell proliferation, cell cycle progression, apoptosis, migration, and invasion.MethodsHuman NPC cells (C666-1) were treated with different concentrations of astaxanthin (0, 1, 10, 20 mg/mL) followed by detection of cell viability. Then, C666-1 cell proliferation, apoptosis, cell cycle progression, invasion, and migration in response to 10 mg/mL astaxanthin, LY294002 (PI3K/AKT inhibitor) or parthenolide (PTL; NF-κB inhibitor) treatment were measured using cell counting kit-8 assay, colony forming assay, flow cytometry analyses, Transwell assay, and wound healing assay, respectively. Western blotting was performed to quantify protein levels of factors involved in PI3K/AKT and NF-κB signaling pathways, cell cycle phase markers (Cyclin D1, p21) and apoptotic markers (Bcl-2 and Bax).ResultsC666-1 cell proliferation, invasion, and migration were significantly suppressed by astaxanthin while cell apoptosis and cell cycle arrest at G1 phase were effectively enhanced in the context of 10 mg/mL astaxanthin. Protein levels of p-AKT, p-P65 and p-IκB levels were suppressed by astaxanthin treatment. After LY294002 or PTL treatment, the suppressive impact of astaxanthin on C666-1 cell process was strengthened, accompanied by the more obvious decrease in cell activity and cell colony number, more enhanced cell apoptosis and G1 phase arrest, and further inhibited cell migration and invasion. Moreover, the inhibitory effect of astaxanthin on Cyclin D1 and Bcl-2 protein levels as well as the promoting impact of astaxanthin on p21 and Bax were also amplified in combination with LY294002 or PTL treatment.ConclusionsAstaxanthin significantly suppresses NPC cell proliferation, cell cycle arrest, migration, invasion while promoting cell apoptosis by inactivating PI3K/AKT and NF-κB pathways. The study first reveals the anticancer role of astaxanthin in NPC, providing a potential candidate for NPC treatment.

A microfluidic chip platform based on Pt nanozyme and magnetized phage composite probes for dual-mode detecting and imaging pathogenic bacteria viability

The detection of pathogen viability is critically important to evaluate its infectivity. In the study, an integrated microfluidic chip based on dual-mode analytical strategy was developed to rapidly realize detection of bacteria activity (with Salmonella typhimurium, S.T, as a model analyte). Firstly, the composite probes, including deactivated phage modified magnetic beads and nano Pt-antimicrobial peptide (AMP) which can specifically recognize Gram-negative bacteria as nanozyme were prepared. When the composite probes are introduced into the chip together with target bacteria, after enrichment, oscillating and magnetic separation, they will conjugate with S.T and produce a magnetic sandwich complex. The complex can catalyze tetramethylbenzidine (TMB)-H2O2 to produce visible colorimetric signals which is correspondent to the total S.T content. Simultaneously, PtNPs in the complex can produce hydroxyl radical oxidation (∙OH) by decomposing H2O2. Under the synergistic action of ∙OH and AMP, the captured live S.T can be lysed to release ATP and emit bioluminescence signals which corresponds to the live S.T concentration. Therefore, the chip can simultaneously detect and image S.T at different viability in one test. The dual-mode assay demonstrated high sensitivity (≤33 CFU/mL), high specificity (identifying strain), signal amplification (5 folds) and short time (≤40min). The chip array can detect four samples in one test and exhibited advantages of high-integration, -sensitivity, -specificity and miniaturization, which are suitable to rapidly detect and image pathogen's viability in trace level. The replacement of phage probes can detect other bacteria. It has a wide prospect in pathogens screening.

AFM-based nanomechanics and machine learning for rapid and non-destructive detection of bacterial viability

AFM-based nanomechanics and machine learning for rapid and non-destructive detection of bacterial viability

Turmeric Inhibits MDA-MB-231 Cancer Cell Proliferation, Altering miR-638-5p and Its Potential Targets.

Recent research suggests curcumin extracted from the turmeric plant may inhibit the proliferation of cancer cells by controlling the expression of microRNAs (miRNAs). The effect of phenolic curcumin on miR-638-5p and potential target gene expressions in the triple negative breast cancer (TNBC) cell line MDA-MB-231 was investigated in this study. GSE154255 and GSE40525 datasets were downloaded and analyzed using GEO2R to identify dysregulated miRNAs in TNBC. To find differently expressed genes in breast cancer (BRCA), The Cancer Genome Atlas Program data was examined. Utilizing in silico tools, KEGG, GO, and other enrichment analyses were performed. The databases miRNet, miRTarBase v8.0, and TarBase v.8 were used for miRNA and mRNA matching. Real-time quantitative reverse transcription polymerase chain reaction was used to examine the levels of miRNA and its targets in miRNA mimic transfected/curcumin-treated MDA-MB-231 cultures and controls. The cell viability detection kit-8 method was used to assess cell viability, and the scratch assay was used to conduct migration assessment. Bioinformatics analysis showed that miR-638-5p was significantly reduced in TNBC patients. Experimental results showed that miR-638-5p was upregulated in MDA-MB-231 treated with curcumin, while the potential target genes of miR-638-5p, CFL1, SIX4, MAZ, and CDH1 were downregulated. Mimic miR-638-5p transfection inhibited MDA-MB-231 cell proliferation and reduced migration and expression of CFL1, SIX4, and MAZ genes was decreased in mimic miR-638-5p transfected cells. These findings suggest that curcumin exerts its anticancer effects on MDA-MB-231 cells by modulating the expression of miR-638-5p and its possible target genes.

Abstract 3103: Alternative cell counting and viability detection using NexGreen/PI fluorescent stain on multiple low- and high-throughput image cytometry platforms

Abstract In the past decade, the increase in cell and gene therapy products such as biologics, antibodies, and CAR-T has significantly increased the need for precise, robust, and consistent cell counting and viability measurements. Simplified and automated workflows benefit from assays that measure sample characteristics independent of their buffer, media, or storage conditions. Reliable and robust results are thus required to maintain quality control of downstream processes. In this work, we demonstrate a novel fluorescence-based dye, NexGreen/PI, capable of live and dead cell detection comparable to AO/PI in multiple cell buffer/media conditions. Jurkat cells of different viabilities (low, intermediate, and high) were stained in parallel with either NexGreen/PI or AO/PI for 1-2 minutes and measured on multiple image cytometer platforms including low-throughput (Cellometer K2, Auto2000, and Spectrum) and high-throughput (Cellaca MX, Cellaca PLX, and Celigo) instruments. Both NexGreen/PI and AO/PI dyes are used as equal volumes added to cell samples (ex: 20 uL cells + 20 uL dye). Additionally, we tested if difficult to analyze cells in “spent” media (typically with a lower pH) or in PBS can be stained using NexGreen/PI compared to AO/PI. Results on the high-throughput platforms show that NexGreen/PI staining can discriminate between live and dead Jurkats, analogous to AO/PI in samples of low (10-25%), intermediate (40-60%), and high (80-95%) viabilities. Measured viability of all samples using NexGreen/PI was within 5% of the paired AO/PI-stained samples. Cell concentration results with either NexGreen/PI or AO/PI also show counts with less than 6% difference. Low-throughput instrument results are similar and show that viabilities of Jurkat cells match within 5% of each other independent of sample (low, intermediate, or high viability), while cell concentrations differ up to 6% when comparing NexGreen/PI and AO/PI. Cells in “spent” media or washed and resuspended in PBS can exhibit low AO signal intensity that can be difficult to detect in a robust fashion. NexGreen/PI-stained cells, however, showed an unaltered fluorescence signal in these “tricky” cell samples. In this work, we demonstrated the capability of NexGreen/PI to measure cell counts and viability using low- and high-throughput instruments, which enables fit-for-purpose for a wide range of samples. Thus, our experiments demonstrate that NexGreen/PI is scalable and can be of significant value for use in automation and high-throughput systems where multiple samples in different mediums can be precisely measured. Citation Format: Joydeep Mukherjee, Carolina Franco Nitta, Mackenzie Pierce, Sopaul Hem, Daniel Browe, Dmitry Kuksin, Leo Li-Ying Chan. Alternative cell counting and viability detection using NexGreen/PI fluorescent stain on multiple low- and high-throughput image cytometry platforms [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 3103.

Abstract 5978: Therapeutic targeting of MELK using a drug repurposing approach to combat TNBC cells

Abstract Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer linked to high chemo-resistant metastatic cases and limited therapeutic options due to the absence of conventional bioreceptors. Therefore, to treat TNBC, it is imperative to identify promising druggable biological targets. Experimental Procedures: An integrated bioinformatic analysis of microarray datasets was performed to identify significantly differentially expressed genes (DEGs) in TNBC. For selecting a potent therapeutic target, the function and significance of the upregulated DEGs in TNBC was investigated by an in silico study employing the UALCAN portal, String database, KM plotter, and Enrichr tools. This analysis included gene expression validation, insights into protein-protein interactions, survival analysis, and pathway mapping of the genes. To obtain potent drugs binding to the selected target MELK (maternal embryonic leucine zipper kinase), a drug repurposing approach was employed starting with virtual screening followed by molecular dynamic simulation (MDS) using GROMACS. Further, the anti-cancer activity of the top two drugs was validated by live-dead imaging, gene expression analyses, MTT-based cell viability and ROS detection assays. Results: Based on a stringent criterion for significant DEG identification (log2|FC|>1 & adjust P-value < 0.05), 25 overlapping upregulated genes were obtained from three selected datasets. Further, in silico analysis revealed that MELK is significantly upregulated across the four stages and subtypes of breast cancer, including TNBC. Higher MELK expression and its oncogenic interactions correlated with poor overall survival of TNBC patients. Evidence suggests that the existing inhibitor for MELK (OTSSP167) has shown poor specificity. Therefore, to block its activity, a library of 1293 FDA-approved drugs was screened against the kinase domain of MELK. MDS study and analysis using parameters like RMSD, RMSF and pair distance revealed that the top 10 drugs were strongly stabilized by H-bonds in the binding pocket of MELK, with minimal deviation and reduced fluctuations of MELK upon drug binding. Binding free energy calculation of drug-MELK interaction using the MM-PBSA method revealed that nine drugs possessed higher binding energy than the inhibitor. The top two drugs obtained from the study (originally kinase and reductase inhibitors respectively) were further validated for their in vitro anti-cancer efficacy in MDA-MB-231 and MDA-MB-468 cells. Treatment with these drugs exhibited (a) a dose-dependent decrease in proliferation, (b) significant cell death, (c) a marked increase in the cellular ROS levels, and (d) a decrease in the expression of MELK and its downstream targets in TNBC cell lines. Conclusion: The present study opens the avenue for using repurposed drugs as potential therapeutic molecules against MELK activity to combat TNBC progression. Citation Format: Arisha Arora, Shilpi Sarkar, Siddhartha S. Ghosh. Therapeutic targeting of MELK using a drug repurposing approach to combat TNBC cells [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 5978.

Detection of Mycoplasma hyopneumoniae viability using a PCR-based assay

Mycoplasma hyopneumoniae detection in clinical specimens is accomplished by PCR targeting bacterial DNA. However, the high stability of DNA and the lack of relationship between bacterial viability and DNA detection by PCR can lead to diagnostic interpretation issues. Bacterial messenger RNA is rapidly degraded after cell death, and consequently, assays targeting mRNA detection can be used for the exclusive detection of viable bacterial cells. Therefore, this study aimed at developing a PCR-based assay for the detection of M. hyopneumoniae mRNA and at validating its applicability to differentiate viable from inert bacteria. Development of the RNA-based PCR encompassed studies to determine its analytical sensitivity, specificity, and repeatability, as well as its diagnostic accuracy. Comparisons between DNA and mRNA detection for the same target gene were performed to evaluate the ability of the RNA-based PCR to detect exclusively viable M. hyopneumoniae after bacterial inactivation using various methods. The RNA-based PCR was also compared to the DNA-based PCR as a tool to monitor the growth of M. hyopneumoniae in vitro. Under the conditions of this study, the developed RNA-based PCR assay detected only viable or very recently inactivated M. hyopneumoniae, while the DNA-based PCR consistently detected cells irrespective of their viability status. Changes in growth activity over time were only observable via RNA-based PCR. This viability PCR assay could be directly applied to evaluate the clearance of M. hyopneumoniae or to determine the viability of the bacterium at late stages of eradication programs.

Tetrahydropalmatine inhibiting mitophagy through ULK1/FUNDC1 pathway to alleviate hypoxia/reoxygenation injury in H9c2 cells

This study explored the specific mechanism by which tetrahydropalmatine(THP) inhibited mitophagy through the UNC-51-like kinase 1(ULK1)/FUN14 domain containing 1(FUNDC1) pathway to reduce hypoxia/reoxygenation(H/R) injury in H9c2 cells. This study used H9c2 cells as the research object to construct a cardiomyocyte H/R injury model. First, a cell viability detection kit was used to detect cell viability, and a micro-method was used to detect lactate dehydrogenase(LDH) leakage to evaluate the protective effect of THP on H/R injury of H9c2 cells. In order to evaluate the protective effect of THP on mitochondria, the chemical fluorescence method was used to detect intracellular reactive oxygen species, intramitochondrial reactive oxygen species, mitochondrial membrane potential, and autophagosomes, and the luciferin method was used to detect intracellular adenosine 5'-triphosphate(ATP) content. Western blot was further used to detect the ratio of microtubule-associated protein 1 light chain 3(LC3) membrane type(LC3-Ⅱ) and slurry type(LC3-Ⅰ) and activated cleaved caspase-3 expression level. In addition, ULK1 expression level and its phosphorylation degree at Ser555 site, as well as the FUNDC1 expression level and its phosphorylation degree of Ser17 site were detected to explore its specific mechanism. The results showed that THP effectively reduced mitochondrial damage in H9c2 cells after H/R. THP protected mitochondria by reducing the level of reactive oxygen species in cells and mitochondria, increasing mitochondrial membrane potential, thereby increasing cellular ATP production, enhancing cellular activity, reducing cellular LDH leakage, and finally alleviating H/R damage in H9c2 cells. Further studies have found that THP could reduce the production of autophagosomes, reduce the LC3-Ⅱ/LC3-Ⅰ ratio, and lower the expression of the apoptosis-related protein, namely cleaved caspase-3, indicating that THP could reduce apoptosis by inhibiting autophagy. In-depth studies have found that THP could inhibit the activation of the ULK1/FUNDC1 pathway of mitophagy and the occurrence of mitophagy by reducing the phosphorylation degree of ULK1 at Ser555 and FUNDC1 at Ser17. The application of ULK1 agonist BL-918 reversely verified the effect of THP on reducing the phosphorylation of ULK1 and FUNDC1. In summary, THP inhibited mitophagy through the ULK1/FUNDC1 pathway to reduce H/R injury in H9c2 cells.

Screening of low-toxic zinc oxide nanomaterials and study the apoptosis mechanism of NSC-34 cells.

With the increasing application of ZnO nanomaterials (ZnO-NMts) in the biomedical field, it is crucial to assess their potential risks to humans and the environment. Therefore, this study aimed to screen for ZnO-NMts with low toxicity and establish safe exposure limits, and investigate their mechanisms of action. The study synthesized 0D ZnO nanoparticles (ZnO NPs) and 3D ZnO nanoflowers (ZnO Nfs) with different morphologies using a hydrothermal approach for comparative research. The ZnO-NMts were characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Mouse brain neuronal cells (NSC-34) were incubated with ZnO NMts for 6, 12, and 24h, and the cell morphology was observed using TEM. The toxic effects of ZnO Nfs on NSC-34 cells were studied using CCK-8 cell viability detection, reactive oxygen species (ROS) measurement, caspase-3 activity detection, Annexin V-FITC/PI apoptosis assay, and mitochondrial membrane potential (Δφm) measurement. The results of the research showed that ZnO-NMts caused cytoplasmic vacuolization and nuclear pyknosis. After incubating cells with 12.5µgmL-1 ZnO-NMts for 12h, ZnO NRfs exhibited the least toxicity and ROS levels. Additionally, there was a significant increase in caspase-3 activity, depolarization of mitochondrial membrane potential (Δφm), and the highest rate of early apoptosis.This study successfully identified ZnO NRfs with the lowest toxicity and determined the safe exposure limit to be<12.5µgmL-1 (12h). These findings will contribute to the clinical use of ZnO NRfs with low toxicity and provide a foundation for further research on their potential applications in brain disease treatment.

Metabolic imaging of human embryos is predictive of ploidy status but is not associated with clinical pregnancy outcomes: a pilot trial.

Does fluorescence lifetime imaging microscopy (FLIM)-based metabolic imaging assessment of human blastocysts prior to frozen transfer correlate with pregnancy outcomes? FLIM failed to distinguish consistent patterns in mitochondrial metabolism between blastocysts leading to pregnancy compared to those that did not. FLIM measurements provide quantitative information on NAD(P)H and flavin adenine dinucleotide (FAD+) concentrations. The metabolism of embryos has long been linked to their viability, suggesting the potential utility of metabolic measurements to aid in selection. This was a pilot trial enrolling 121 IVF couples who consented to have their frozen blastocyst measured using non-invasive metabolic imaging. After being warmed, 105 couples' good-quality blastocysts underwent a 6-min scan in a controlled temperature and gas environment. FLIM-assessed blastocysts were then transferred without any intervention in management. Eight metabolic parameters were obtained from each blastocyst (4 for NAD(P)H and 4 for FAD): short and long fluorescence lifetime, fluorescence intensity, and fraction of the molecule engaged with enzyme. The redox ratio (intensity of NAD(P)H)/(intensity of FAD) was also calculated. FLIM data were combined with known metadata and analyzed to quantify the ability of metabolic imaging to differentiate embryos that resulted in pregnancy from embryos that did not. De-identified discarded aneuploid human embryos (n = 158) were also measured to quantify correlations with ploidy status and other factors. Statistical comparisons were performed using logistic regression and receiver operating characteristic (ROC) curves with 5-fold cross-validation averaged over 100 repeats with random sampling. AUC values were used to quantify the ability to distinguish between classes. No metabolic imaging parameters showed significant differences between good-quality blastocysts resulting in pregnancy versus those that did not. A logistic regression using metabolic data and metadata produced an ROC AUC of 0.58. In contrast, robust AUCs were obtained when classifying other factors such as comparison of Day 5 (n = 64) versus Day 6 (n = 41) blastocysts (AUC = 0.78), inner cell mass versus trophectoderm (n = 105: AUC = 0.88) and aneuploid (n = 158) versus euploid and positive pregnancy embryos (n = 108) (AUC = 0.82). The study protocol did not select which embryo to transfer and the cohort of 105 included blastocysts were all high quality. The study was also limited in number of participants and study sites. Increased power and performing the trial in more sites may have provided a stronger conclusion regarding the merits of the use of FLIM clinically. FLIM failed to distinguish consistent patterns in mitochondrial metabolism between good-quality blastocysts leading to pregnancy compared to those that did not. Blastocyst ploidy status was, however, highly distinguishable. In addition, embryo regions and embryo day were consistently revealed by FLIM. While metabolic imaging detects mitochondrial metabolic features in human blastocysts, this pilot trial indicates it does not have the potential to serve as an effective embryo viability detection tool. This may be because mitochondrial metabolism plays an alternative role post-implantation. This study was sponsored by Optiva Fertility, Inc. Boston IVF contributed to the clinical site and services. Becker Hickl, GmbH, provided the FLIM system on loan. T.S. was the founder and held stock in Optiva Fertility, Inc., and D.S. and E.S. had options with Optiva Fertility, Inc., during this study. The study was approved by WCG Connexus IRB (Study Number 1298156).

Use of 2,3,5-triphenyl tetrazolium chloride for detection of Fusarium semitectum viability in soybean seeds

Use of 2,3,5-triphenyl tetrazolium chloride for detection of Fusarium semitectum viability in soybean seeds

H3K27 acetylation activated-PDLIM7 promotes castration-resistant prostate cancer progression by inducing O-Glycosylation of YAP1 protein

Castration-resistant prostate cancer (CRPC) is a fatal disease that evolves from prostate cancer due to drug resistance after long-term androgen deprivation therapy. In this study, we aimed to find novel molecular targets for treating CRPC. Through peptidome, we screened out polypeptides dysregulated in the serum of CRPC patients. According to RT-qPCR analysis and cell viability detection, we chose PDZ and LIM Domain 7 (PDLIM7) as the research object. As demonstrated by loss-of-function assays, silencing of PDLIM7 could suppress CRPC cell proliferation, migration, and angiogenesis. Moreover, PDLIM7 knockdown enhanced the sensitivity of CRPC cells to docetaxel treatment. Subsequently, we found that CBP/p300 increases the H3K27ac level in the PDLIM7 promoter to activate PDLIM7. Mechanism experiments such as IP and western blot revealed that PDLIM7 interacted with YAP1 to induce O-Glycosylation of YAP1 and thus stabilize YAP1 protein. Rescue assays demonstrated that PDLIM7 promoted the malignant processes of CRPC cells through YAP1. Finally, an animal study validated that PDLIM7 aggravated tumor growth. In conclusion, our findings highlighted the oncogenic role of PDLIM7 upregulated by CBP/p300-induced H3K27ac enhancement in CRPC by stabilizing YAP1.

Protective effect and regulatory mechanism of salidroside on skin inflammation induced by imiquimod in psoriasis mice

Salidroside (SAL) is a glucoside of tyrosol commonly existing in the roots of Rhodiola rosea. This study unveils the protective effect of SAL on skin inflammation in imiquimod (IMQ)-induced psoriasis. The mouse model of psoriasis was established by local application of IMQ, and SAL efficacy was evaluated through PASI scoring, H&E staining, and skin tissue pathology observation. The HaCaT cell model was established by interferon (IFN)-γ induction, followed by MTT assay detection of cell viability, detection of ROS, SOD, MDA, and CAT levels in skin tissues and cells using reagent kits, ELISA detection of inflammatory factors (TNF-α, IL-6, IL-1β), and qRT-PCR detection of psoriasis-related genes (S100a9, Cxcl1, Cxcl2) as well as miR-369-3p and SMAD2 expressions. The binding relationship between miR-369-3p and SMAD2 was validated using dual-luciferase reporter assay. SAL treatment reduced PASI scores and alleviated psoriasis symptoms of IMQ-induced mice, and also augmented the viability and subsided the oxidative stress and inflammation of IFN-γ-treated HaCaT cells. SAL treatment restrained miR-369-3p expression but elevated SMAD2 expression. Mechanistically, miR-369-3p targeted SMAD2 expression. miR-369-3p overexpression or SMAD2 inhibition partially offset the alleviating effect of SAL on psoriasis skin inflammation. In conclusion, SAL alleviates skin inflammation in IMQ-induced psoriasis mice via the miR-369-3p/SMAD2 axis.

Bovine serum albumin and folic acid-modified aurum nanoparticles loaded with paclitaxel and curcumin enhance radiotherapy sensitization for esophageal cancer

Background Nanocarrier systems have been used in the study of esophageal cancer (EC) and other diseases, with significant advantages in improving the non-targeted and nonspecific toxicity of traditional formulations. Some chemotherapeutic drugs and high atomic number nanomaterials have sensitization effects on ionizing radiation and can be used as chemoradiation sensitizers. Methods Aurum (Au) nanoparticles were modified by bovine serum albumin (BSA) and folic acid (FA), and were core-loaded with paclitaxel (PTX) and curcumin (CUR). The basic characteristics of FA-BSA-Au@PTX/CUR nanomedicines were evaluated by transmission electron microscopy, Fourier transform infrared spectroscopy, and Malvern Zetasizer. The encapsulation and release of drugs were monitored by ultraviolet–visible spectrophotometry (UV–Vis). The biological toxicity and radiotherapy sensitization effect of FA-BSA-Au@PTX/CUR were observed by cell viability, colony formation, cell apoptosis, cell cycle distribution, and γ-H2AX analysis experiments. Results The prepared nanomedicines showed good stability and spherical morphology. The results of cell uptake and cell viability detection revealed that FA-BSA-Au@PTX/CUR could specifically target EC cell KYSE150 and exert a certain inhibitory effect on proliferation, with no obvious toxicity on healthy cells Het-1A. In addition, the results of the colony formation experiment, cell apoptosis detection, cell cycle distribution, and γ-H2AX analysis showed that compared with X-rays alone, FA-BSA-Au@PTX/CUR combined with X-rays exhibited relatively stronger radiotherapy sensitization and anti-tumor activity. Conclusions FA-BSA-Au@PTX/CUR could target EC cancer cells and act as a safe and effective radiotherapy sensitizer to improve the radiotherapy efficacy of EC.

Detection of viability and clinical implications using positron emission tomography/magnetic resonance imaging in patients with acute myocardial infarction

Abstract Background PET/MRI has been used for almost 10 years to assess myocardial perfusion, viability, and inflammatory and infiltrative processes of heart. By combining the advantages of FDG-PET and LGE-MRI, it is already the modality of choic for myocardial viagility and inflammatory disease [1]. The purpose of this study is to evaluate the role of viability on PET/MRI to predict improvement if echocardiographic findings such as regional wall motion abnormality (RWMA) after successful percutaneous coronary intervention (PCI) for patients with acute myocardial infarction (AMI). Method A total of 154 patients with AMI who was performed cardiac PET/MRI within 7 days after PCI were enrolled. We performed TTE twice before and after PCI in 103 patients. We categorized these patients according to presence of viability on PET-MRI: group A (N=53, patients with viability on PET-MRI) and group B (N=50, patients without viability on PET-MRI) and compared. The primary end point is improvement of regional wall motion abnormality (RWMA) on TTE. We set the definition of viability in PET/MRI as FDG uptake is 50% or more and LGE transmurality is 50% or less [2]. Results Clinical and angiographic findings were similar between the two groups except peak high sensitivity Troponin-I(hs TnI). The value of peak hs TnI of Group A was significant lower than that of Group B(20.22±26.42 vs. 39.28±30.59, p=0.001). Among 53 patients in group A, 17 patients (32.1%) showed the result of slight improvement of RWMA on TTE and 13 patients (24.5%) showed result of disappeared RWMA on TTE. In contrast, among 50 patients in group B, 19 patients (38%) showed the result of slight improvement and only 4 patients (8%) showed result of disappeared RWMA on TTE. The proportion of patients who showed slight improvement of RWMA between two groups are not statistically significant (32.1% vs. 38%, p=0.529). But, the proportion of patients who showed disappeared RWMA between two groups are statistically significant (24.5% vs. 8%, p=0.024). The value of improvement of LVEF of Group A is significantly higher than that of Group B(6.89±8.26 vs. 3.86±6.55, p=0.043). Conclusions PET/MRI is useful tool to detect viability in patients with AMI. It seems to be predictable to improve cardiac function for the patients with viability on PET-MRI.Baseline characteristicsPrimary endpoint

Abstract 14203: Speckle Tracking Analysis During Dobutamine Stress Echocardiography in Patients Undergoing Percutaneous Coronary Intervention for Chronic Total Occlusion

Background: Dobutamine stress echocardiography (DSE) is often used as a non-invasive diagnostic modality for detection of myocardial ischemia and viability. A “biphasic response” in DSE can occur, in which contractility improves in dysfunctional segments with low dose stage and then becomes dysfunctional again at higher doses due to ischemia. We investigated if speckle tracking analysis during DSE can evaluate patients undergoing percutaneous coronary intervention (PCI) for chronic total occlusion (CTO). Methods: DSE was performed one day before CTO-PCI in consecutive 30 patients (18 female, age=71±11 years). Since 27 patients had 1 vessel, and 3 patients had 2 vessel CTO lesions, total of 33 regions were analyzed. Global (GLS) and regional longitudinal strain (RLS) were calculated as an averaged value of peak longitudinal strain in the apical 4-, 2-chamber, and long axis views at baseline echo, at low-dose, at high-dose DSE, and 6 months after CTO-PCI using available software (QLAB, Philips Medical Systems). Results: CTO-PCI was successfully done in all patients. GLS and RLS were feasible in all regions. Overall, GLS (-15.5±2.8 at baseline v.s. -17.3±3.8, p&lt;0.001) and left ventricular ejection fraction (LVEF) (56±6% v.s. 58±7%, p&lt;0.01) significantly improved 6 months after CTO-PCI. There was also significant improvement of RLS for the obstructed CTO regions (-13.8±3.6 v.s. -16.5±4.2, p&lt;0.005). Although in 24 patients (26 regions) with biphasic response during DSE (Figure), GLS (-15.7±2.5 v.s. -17.9±3.6, p&lt;0.01), LVEF (54±6% v.s. 56±7%, p&lt;0.05), and RLS (-13.8±3.5 v.s. -16.8±4.0, p&lt;0.01) improved significantly, GLS (-14.9±4.2 v.s. -15.8±5.0, p=n.s.), LVEF (59±3% v.s. 60±3%, p=n.s.), and RLS (-13.9±4.5 v.s. -15.5±5.7, p=n.s.) didn’t change in 6 patients (7 regions) without biphasic response. Conclusion: Speckle tracking analysis is feasible during DSE. CTO-PCI may improve both global and regional cardiac functions in patients with myocardial viability.