Analysis Of Fluorescence Intensity Research Articles

Temporal fiber-optic fluorescence study of FAD-NADH interactions in phantoms for potential applications in rapid liquid optical biopsy procedures

Body fluids carry several biomarkers and genetic material that enable the detection of carcinogenesis and tumor metastasis. Owing to this, liquid biopsy is gaining importance and entering clinical practice as an adjunct modality to the current standard of practice in cancer detection. Liquid biopsy involving specimens such as urine is non-invasive and has biomarkers reflecting the tumor microenvironment. We investigate the normal and altered levels of cellular metabolic by-products in liquid phantoms and demonstrate their photon-induced excited-state interaction behaviour for potential diagnostic applications. Short-span temporal analysis of metabolic by-products FAD and NADH in liquid phantoms with fiber optic fluorescence spectroscopy is carried out. The mixed liquid phantom of FAD and NADH, showed excited state interactions that manifested as simultaneous inverse variation in the fluorescence emission from FAD and NADH in varying pH environments. Linear discriminant analysis of the time-varying integrated fluorescence intensity resulted in a 100% accuracy in classifying liquid samples between high and low metabolite concentrations. An extension of application of the experiment model for analysis of body fluids such as urine is also discussed.

Endothelin-A Receptor Activation Stimulates Th17 Cell Differentiation

Th17 cells are a major contributor to many pro-inflammatory disease conditions. Endothelin 1 (ET-1) through the ETA receptor promotes inflammation, however its role in T cell differentiation, especially T helper 17 (Th17) cells, is not fully understood. Importantly, ET-1 has been shown to be elevated in people with autoimmunity, diabetes, and salt-sensitive hypertension in which augmented Th17 cells are known to contribute to the pathogenesis. Therefore, we hypothesized that ETA receptor activation mediates Th17 cell differentiation. To test our hypothesis, we first treated C57BL/6J male mice (8–12-week-old, normal chow) with the ETA receptor antagonist (ABT-627, 10mg/kg/day) in the drinking water for five weeks. We measured Th17 cells in the colon and kidneys by flow cytometry. We found that ETA receptor blockade reduced the number of Th17 cells compared to that in vehicle control mice. Indeed, the absolute number of Th17 cells decreased from 461±61 Th17 cells/per two kidneys in vehicle control to 146±29 Th17 cells per two kidneys in the ABT-627 treated group (p=0.004), and from 8372±1454 Th17 cells to 3378±936 Th17 cells (p=0.028) in the whole colon. To determine whether this observation is a direct effect on IL17A induction, we evaluated ETA receptor antagonism under Th17 polarizing conditions in vitro. Naïve CD4+ T cells were isolated from the spleen and cultured under Th17 polarizing conditions (TGFβ and IL-6) in the presence or absence of ETA receptor antagonist (BQ-123, 1μM). We found that ETA receptor blockade directly attenuated Th17 differentiation in vitro. The frequency of IL17A-expressing cells within CD4+ T cells reduced from 11.37±0.68% in media control to 9.87±0.60% in BQ-123 treated cells (p=0.0006). Similarly, the relative mean fluorescence intensity (MFI) analysis revealed a reduction in IL17A production following ETA receptor blockade from 8937±871 relative units in media control to 7132±725 relative units in BQ-123 treated cells (p=0.0136). These data taken together support our hypothesis that the ETA receptor plays a key role in Th17 differentiation. Because of the critical role of Th17 cells in pathophysiology, our findings warrant further investigations for the use of ETA receptor antagonist in pro-inflammatory conditions. Tha Luong - U2C/TL1 Deep South KUH PRIME U2C DK133422 & TL1 DK139566 from the NIH/NIDDK Patrick Molina - F31HL151264 David & Jennifer Pollock - R01 DK134562. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Evaluation of volatile components from the tuber, fibrous roots, bud, stem and leaf tissues of Bletilla striata for its anti-colon cancer activity.

Bletilla striata (Thunb.) Rchb.f., a medicinal plant in the Orchidaceae family, is mainly found in East Asia and has extensive pharmacological activities. Plant's volatile components are important active ingredients with a wide range of physiological activities, and B. striata has a special odor and unique volatile components. Yet it has received little attention, hindering a full understanding of its phytochemical components. Employing the ultrasonic-assisted extraction method, the volatile components of B. striata's fibrous root, bud, aerial part and tuber were extracted, resulting in yields of 0.06%, 0.64%, 3.38% and 4.47%, respectively. A total of 78 compounds were identified from their chemical profiles using gas chromatography-mass spectrometry (GC-MS), including 45 components with the main compounds of linoleic acid (content accounting for 31.23%), n-hexadecanoic acid (13.53%), and octadecanoic acid (9.5%) from the tuber, 34 components with the main compounds of eicosane, 2-methyl- (28.42%), linoelaidic acid (10.43%), linoleic acid (4.53%), and n-hexadecanoic acid (6.91%) from the fibrous root, 38 components with the main compounds of pentadeca-6,9-dien-1-ol (9.29%), n-hexadecanoic acid (11%), eicosane,2-methyl- (23.43%), and linoleic acid (23.53%) from the bud, and 27 components with the main compounds of linoelaidic acid (5.97%), n-hexadecanoic acid (15.99%), and linolenic acid ethyl ester (18.9%) from the aerial part. Additionally, the growth inhibition activity against colon cancer HCT116 cells was evaluated using sulforhodamine B (SRB) assay and the thiazolyl blue tetrazolium bromide (MTT) assay, and the accumulation of reactive oxygen species (ROS) was determined using dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining and fluorescence intensity analysis. The volatile extracts exhibited significant growth inhibitory efficacy against HCT116 cells, with half-maximal inhibitory concentration (IC50) values of 3.65, 2.32, 2.42 and 3.89mg/mL in the SRB assay, and 3.55, 2.58, 3.12 and 4.80mg/mL in the MTT assay for the root, bud, aerial part, and tuber, respectively. Notably, treatment with the aerial part extract caused morphological changes in the cells and significantly raised the intracellular ROS level. In summary, the chemical profiles of the volatile components of B. striata were revealed for the first time, demonstrating a certain tissue specificity. Additionally, it demonstrated for the first time that these volatile extracts possess potent anti-colon cancer activity, highlighting the importance of these volatile components in B. striata's medicinal properties.

Abstract 6271: Novel insights into androgen receptor-variant 7 subnuclear localization and function in castration resistant prostate cancer

Abstract Prostate cancer (PC) stands as the most frequently diagnosed and second leading cause of cancer death among American men. PC mortality is predominantly attributed to castration-resistant prostate cancer (CRPC), often emerging within 2-3 years following the initiation of androgen deprivation therapy. This resistance primarily arises from sustained androgen receptor (AR) signaling, ultimately resulting in a compromised response to AR-directed therapies. AR splice variant expression is a key contributor to the persistent AR signaling in CRPC. AR-variant 7 (AR-V7) is the most clinically prevalent variant, expressed in about 75% of patients with CRPC and confers resistance to standard of care (SoC) treatments. AR-V7 differs from the full-length AR (AR-fl) because it lacks the ligand binding and hinge domains, rendering it resistant to SoC treatments. Currently, there are no selective inhibitors for AR-V7. Therefore, we sought to identify unique biological features of AR-V7, distinct from AR-fl, to exploit therapeutic options for targeting AR-V7. Previous studies have demonstrated high sequence homology, largely overlapping cistromes, and gene transactivation profiles between AR-V7 and AR-FL. Our published mechanistic studies revealed that AR-V7 utilizes a unique nuclear import pathway, not shared by AR-fl, allowing for constitutive import and activation of target genes in the nucleus. Herein, we report an additional novel subnuclear phenotype of AR-V7 in LNCAP95 and 22RV1 cells modeling CRPC. Staining with AR-V7- and AR-fl- specific antibodies revealed that unlike AR-fl, AR-V7 exhibited prominent nucleolar localization. To validate AR-V7 nucleolar localization, CRPC cells stained with fibrillarin (nucleolar marker), and subsequent fluorescence intensity analysis revealed that AR-V7 was predominantly localized in fibrillarin positive nucleoli, whereas AR-fl protein was solely localized in the nucleoplasm. This suggests a novel functional role of AR-V7 in the nucleus. To further confirm AR-V7 nucleolar localization, Actinomycin D (ActD) was used as a tool compound to disrupt nucleolar integrity. Treatment with ActD for 4 hours in CRPC cells significantly reduced the area of fibrillarin positive nucleoli and prompted AR-V7 re-distribution out of the nucleoli and into the nucleoplasm. Ongoing efforts to further investigate AR-V7 nucleolar function in CRPC cells include mutating the predicted nucleolar localization signal located in AR-V7 and identifying nucleolar-interacting proteins through proximity-labeling assays. In conclusion, we unveiled a novel nuclear compartmentalization of AR-V7 within the nucleolus, the primary hub for ribosomal biogenesis. These observations suggest that AR-V7 could regulate the high levels of protein synthesis occurring in CRPC cells, creating a window of a potential therapeutic opportunity for selective inhibition. Citation Format: Michelle K. Naidoo, Paraskevi Giannakakou. Novel insights into androgen receptor-variant 7 subnuclear localization and function in castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6271.

Tag-free protein modification by lipoate ligase A: exploring substrate tolerance.

This study delves into the functional intricacies of lipoate ligase A (LplA), an enzyme showing great promise in bioconjugation due to its unique capacity for introducing azido groups into proteins without requiring a genetic tag. We aimed to enhance the understanding of LplA's functionality, particularly its substrate tolerance and the reliability of various analytical techniques. A pivotal aspect of our approach was incorporating azido groups into a range of proteins, followed by the addition of the fluorescent molecule Cy3 via click chemistry. Analysis of fluorescent intensity in the altered proteins indicated varying degrees of conjugation. Additionally, phenyl resin-based RP-HPLC facilitated effective separation of modified proteins, unmodified proteins, and remaining fluorescent tags post-separation. SASA analysis provided insights into conjugation trends, guiding the identification of proteins amenable to LplA's tag-free modification. Our findings demonstrate LplA's broad substrate tolerability for protein modification.

Minicells as an Escherichia coli mechanism for the accumulation and disposal of fluorescent cadmium sulphide nanoparticles

BackgroundBacterial biosynthesis of fluorescent nanoparticles or quantum dots (QDs) has emerged as a unique mechanism for heavy metal tolerance. However, the physiological pathways governing the removal of QDs from bacterial cells remains elusive. This study investigates the role of minicells, previously identified as a means of eliminating damaged proteins and enhancing bacterial resistance to stress. Building on our prior work, which unveiled the formation of minicells during cadmium QDs biosynthesis in Escherichia coli, we hypothesize that minicells serve as a mechanism for the accumulation and detoxification of QDs in bacterial cells.ResultsIntracellular biosynthesis of CdS QDs was performed in E. coli mutants ΔminC and ΔminCDE, known for their minicell-producing capabilities. Fluorescence microscopy analysis demonstrated that the generated minicells exhibited fluorescence emission, indicative of QD loading. Transmission electron microscopy (TEM) confirmed the presence of nanoparticles in minicells, while energy dispersive spectroscopy (EDS) revealed the coexistence of cadmium and sulfur. Cadmium quantification through flame atomic absorption spectrometry (FAAS) demonstrated that minicells accumulated a higher cadmium content compared to rod cells. Moreover, fluorescence intensity analysis suggested that minicells accumulated a greater quantity of fluorescent nanoparticles, underscoring their efficacy in QD removal. Biosynthesis dynamics in minicell-producing strains indicated that biosynthesized QDs maintained high fluorescence intensity even during prolonged biosynthesis times, suggesting continuous QD clearance in minicells.ConclusionsThese findings support a model wherein E. coli utilizes minicells for the accumulation and removal of nanoparticles, highlighting their physiological role in eliminating harmful elements and maintaining cellular fitness. Additionally, this biosynthesis system presents an opportunity for generating minicell-coated nanoparticles with enhanced biocompatibility for diverse applications.Graphical

Visualization of CO2-oil vanishing interface to determine minimum miscibility pressure using microfluidics

The determination of the minimum miscibility pressure (MMP) assumes paramount significance in evaluating CO2 enhanced-oil recovery strategies. We created a microfluidic method for determining the MMP of CO2 and oil samples by integrating the direct observation of the vanishing interface and the analysis of fluorescence intensity exhibited by oil samples. A microfluidic chip is designed with closed-end microchannels to mimic the blind-end fractures which contributes to reveal the complete picture of residual oil mobilization during CO2 miscible flooding. The maximum deviation of 3.83 % and 5.47 % unequivocally attested to the accuracy of our method by comparing measured MMPs with those reported in the literature and theoretically calculated in this study. The effects of alkane types, temperature and oil compositions on MMP was investigated. The MMPs for binary synthetic oils (C8 + C14) increased non-linearly with contents of tetradecane and increased significantly at C14 > 75 vol%. MMPs of nine n-alkanes, seven binary synthetic oils, four ternary synthetic oils and kerosene were provided reliably as the unprecedented new data. Such unique advantages as in-situ visualization, chip pattern customizability, user-independence, and the clear criterion render this research highly valuable for determining the MMP as well as elucidating the microscopic mechanisms underlying residual oil mobilization.

Detection of Vibrio parahaemolyticus Based on Magnetic and Upconversion Nanoparticles Combined with Aptamers.

Vibrio parahaemolyticus is a halophilic and heat-labile gram-negative bacterium and is the most prevalent foodborne bacterium in seafood. In order to develop a rapid and sensitive method for detecting the foodborne pathogenic bacterium Vibrio parahaemolyticus, an aptamer-modified magnetic nanoparticle and an aptamer-modified upconversion nanoparticle were synthesised and used as a capture probe and a signal probe, respectively. The aptamer-modified magnetic nanoparticle, V. parahaemolyticus cell, and aptamer-modified upconversion nanoparticle formed a sandwich-like complex, which was rapidly separated from a complex matrix using a magnetic force, and the bacterial concentration was determined by fluorescence intensity analysis. The results showed that the fluorescence intensity signal correlated positively with the concentration of V. parahaemolyticus in the range of 3.2 × 102 to 3.2 × 105 CFU/mL, with a linear equation of y = 296.40x - 217.67 and a correlation coefficient of R2 = 0.9610. The detection limit of the developed method was 4.4 CFU/mL. There was no cross-reactivity with other tested foodborne pathogens. This method is highly specific and sensitive for the detection of V. parahaemolyticus, and can achieve the qualitative detection of this bacterium in a complex matrix.

ADENOSINE INFLUENCES FOXP3 EXPRESSION OF T REGS VIA THE A2AR/CREB PATHWAY IN A MOUSE MODEL OF SEPSIS.

The adenosine concentration and forkhead box protein (Foxp3) expression in T regulatory cells (T regs ) are increased during sepsis. However, the mechanism by which adenosine induces Foxp3 expression is incompletely understood. A cecal ligation and puncture (CLP) model was constructed using C57BL/J mice. The plasma adenosine concentration and Foxp3 expression in splenic T regs were increased consistently for 15 days after sepsis onset. Analysis of the mean fluorescence intensity of Foxp3 and adenosine concentration in the same mice revealed a linear correlation. In the CLP model, adenosine 2a receptor (A2aR) blockade inhibited Foxp3 expression in T regs . In vitro activation of A2aR promoted Foxp3 expression in T regs and facilitated secretion of extracellular vesicles. Transcriptome sequencing revealed that A2aR blockade led to changes in cyclic adenosine monophosphate response element-binding protein (CREB) transcription in T regs in our sepsis model. Use of adenosine or A2aR agonists promoted CREB expression, CREB phosphorylation at S133, T reg expression of Foxp3, and enhanced inhibition of proliferation of cluster of differentiation (CD)4+ lymphocytes. A2aR blockade or inhibition of CREB expression inhibited Foxp3 expression in T regs . In the CLP model, use of CREB inhibitors could inhibit Foxp3 expression and reduce the bacterial load. In summary, adenosine in sepsis promotes CREB phosphorylation via A2aR which, in turn, upregulates Foxp3 expression in T regs .

A cyclic peptide-based PROTAC induces intracellular degradation of palmitoyltransferase and potently decreases PD-L1 expression in human cervical cancer cells.

Our previous research has found that degradation of palmitoyltransferase in tumor cells using a linear peptide PROTAC leads to a significant decrease in PD-L1 expression in tumors. However, this degradation is not a sustained and efficient process. Therefore, we designed a cyclic peptide PROTAC to achieve this efficient anti-PD-L1 effect. We designed and synthesized an improvement in linear peptide PROTAC targeting palmitoyltransferase DHHC3, and used disulfide bonds to stabilize the continuous N- and C-termini of the peptides to maintain their structure. Cellular and molecular biology techniques were used to test the effect of this cyclic peptide on PD-L1. In human cervical cancer cells, our cyclic peptide PROTAC can significantly downregulate palmitoyl transferase DHHC3 and PD-L1 expressions. This targeted degradation effect is enhanced with increasing doses and treatment duration, with a DC50 value much lower than that of linear peptides. Additionally, flow cytometry analysis of fluorescence intensity shows an increase in the amount of cyclic peptide entering the cell membrane with prolonged treatment time and higher concentrations. The Cellular Thermal Shift Assay (CETSA) method used in this study indicates effective binding between our novel cyclic peptide and DHHC3 protein, leading to a change in the thermal stability of the latter. The degradation of PD-L1 can be effectively blocked by the proteasome inhibitor MG132. Results from clone formation experiments illustrate that our cyclic peptide can enhance the proliferative inhibition effect of cisplatin on the C33A cell line. Furthermore, in the T cell-C33A co-culture system, cyclic peptides target the degradation of PD-L1, thereby blocking the interaction between PD-L1 and PD-1, and promoting the secretion of IFN-γ and TNF-α in the co-culture system supernatant. Our results demonstrate that a disulfide-bridged cyclic peptide PROTAC targeting palmitoyltransferase can provide a stable and improved anti-PD-L1 activity in human tumor cells.

Direct Uptake and Intracellular Dissolution of HgS Nanoparticles: Evidence from a Bacterial Biosensor Approach.

Mercury sulfide nanoparticles (HgSNPs), which occur widely in oxic and anoxic environments, can be microbially converted to highly toxic methylmercury or volatile elemental mercury, but it remains challenging to assess their bioavailability. In this study, an Escherichia coli-based whole-cell fluorescent biosensor was developed to explore the bioavailability and microbial activation process of HgSNPs. Results show that HgSNPs (3.17 ± 0.96 nm) trigger a sharp increase in fluorescence intensity of the biosensor, with signal responses almost equal to that of ionic Hg (Hg(II)) within 10 h, indicating high bioavailability of HgSNP. The intracellular total Hg (THg) of cells exposed to HgSNPs (200 μg L-1) was 3.52-8.59-folds higher than that of cells exposed to Hg(II) (200 μg L-1), suggesting that intracellular HgSNPs were only partially dissolved. Speciation analysis using size-exclusion chromatography (SEC)-inductively coupled plasma mass spectrometry (ICP-MS) revealed that the bacterial filtrate was not responsible for HgSNP dissolution, suggesting that HgSNPs entered cells in nanoparticle form. Combined with fluorescence intensity and intracellular THg analysis, the intracellular HgSNP dissolution ratio was estimated at 22-29%. Overall, our findings highlight the rapid internalization and high intracellular dissolution ratio of HgSNPs by E. coli, and intracellular THg combined with biosensors could provide innovative tools to explore the microbial uptake and dissolution of HgSNPs.

Antibacterial activity of the novel oxazolidinone contezolid (MRX-I) against Mycobacterium abscessus.

To evaluate contezolid (MRX-I) antibacterial activity against Mycobacterium abscessus in vitro and in vivo and to assess whether MRX-I treatment can prolong survival of infected zebrafish. MRX-I inhibitory activity against M. abscessus in vitro was assessed by injecting MRX-I into zebrafish infected with green fluorescent protein-labelled M. abscessus. Thereafter, infected zebrafish were treated with azithromycin (AZM), linezolid (LZD) or MRX-I then maximum tolerated concentrations (MTCs) of drugs were determined based on M. abscessus growth inhibition using one-way ANOVA. Linear trend analysis of CFU counts and fluorescence intensities (mean ± SE values) was performed to detect linear relationships between MRX-I, AZM and LZD concentrations and these parameters. MRX-I anti-M. abscessus minimum inhibitory concentration (MIC) and MTC were 16 μg/mL and 15.6 μg/mL, respectively. MRX-I MTC-treated zebrafish fluorescence intensities were significantly lower than respective LZD group intensities (whole-body: 439040 ± 3647 vs. 509184 ± 23064, p < 0.01); head: 74147 ± 2175 vs. 95996 ± 8054, p < 0.05). As MRX-I concentration was increased from 0.488 μg/mL to 15.6 μg/mL, zebrafish whole-body, head and heart fluorescence intensities decreased. Statistically insignificant differences between the MRX-I MTC group survival rate (78.33%) vs. corresponding rates of the 62.5 μg/mL-treated AZM MTC group (88.33%, p > 0.05) and the 15.6 μg/mL-treated LZD MTC group (76.67%, p > 0.05) were observed. MRX-I effectively inhibited M. abscessus growth and prolonged zebrafish survival when administered to M. abscessus-infected zebrafish, thus demonstrating that MRX-I holds promise as a clinical treatment for human M. abscessus infections.

Automated Analysis of Telomere Intensity from Quantitative Fluorescence In-Situ Hybridization using CellProfiler

Telomeres are noncoding DNA sequences that protect chromosome ends from degradation. Quantitative fluorescence in situ hybridization (Q-FISH) is the favored technique for measuring telomere length. It can be applied to individual chromosomes in metaphase cells. Much of the currently available software to analyze fluorescence signal intensity as a proxy for telomere length is only commercially available. This study sought to develop a pipeline for the automated analysis of telomere fluorescence intensities in metaphase Q-FISH using modules from the open-source, freely available CellProfiler software. Intensities telomere and centromere were stained with fluorophore-conjugated peptide nucleic acid probes then evaluated using our pipeline. Telomere and centromere intensities were measured in a total of 30 images, giving an average relative telomere intensity of 22.17 %. Overall, our study showed that the pipeline can automatically identify and measure fluorescence signals from telomeres and centromeres in approximately 36 sec/image. However, the pipeline requires high-quality metaphase chromosome images with minimal touching and overlapping of chromosomes. Nevertheless, CellProfiler provides a free and flexible platform for the automated analysis of Q-FISH images that can be easily optimized and shared between researchers

Enhancing the dispersibility of commercial pea protein ingredients using stirred media milling: Potential mechanisms of action

This study found that stirred media milling (SMM) showed great potential in enhancing the functional properties of commercial legume proteins. The effects of SMM on protein dispersibility and structure in 5% pea protein solutions were investigated. The milling increased pea protein dispersibility index from around 12 to 99%, over an 8-fold increase. During milling, the D[4,3] of protein particles decreased from around 122 μm to 320 nm and the magnitude of the zeta potential increased from 21.6 to 30.0 mV. Microscopy analysis revealed that the morphology of the protein particle changed from aggregated, irregular, and dense before milling to uniformly distributed and spherical after milling. And hydrophobic interactions were the primary intramolecular forces maintaining the structure of the water-dispersible protein particles. Intrinsic fluorescence intensity, surface hydrophobicity, and free sulfhydryl group analysis of the pea proteins increased after milling, and there was a net change in the secondary structure of the proteins from β-sheet and random coil to α-helix and β-turn. SDS-PAGE analysis revealed that insoluble protein aggregates transformed into water-dispersible ones after milling. These results suggested that the conformation and aggregate state of protein molecules were changed during milling. This study indicates that the stirred media milling is a simple and effective means of enhancing pea protein dispersibility, which could expand the application of plant protein in the food industry.

Time-Resolved Fluorescence Spectroscopy of Molecularly Imprinted Nanoprobes as an Ultralow Detection Nanosensing Tool for Protein Contaminants.

Currently, optical sensors based on molecularly imprinted polymers (MIPs) have been attracting significant interest. MIP sensing relies on the combination of the MIP's selective capability, which is conveyed to the polymeric material by a template-assisted synthesis, with optical techniques that offer exquisite sensitivity. In this work, we devised an MIP nanoparticle optical sensor for the ultralow detection of serum albumin through time-resolved fluorescence spectroscopy. The Fluo-nanoMIPs (&empty;~120 nm) were synthetized using fluorescein-O-methacrylate (0.1×, 1×, 10× mol:mol versus template) as an organic fluorescent reporter. The ability of 0.1× and 1×Fluo-nanoMIPs to bind albumin (15 fM-150 nM) was confirmed by fluorescence intensity analyses and isothermal titration calorimetry. The apparent dissociation constant (Kapp) was 30 pM. Conversely, the 10× fluorophore content did not enable monitoring binding. Then, the time-resolved fluorescence spectroscopy of the nanosensors was studied. The 1×Fluo-nanoMIPs showed a decrease in fluorescence lifetime upon binding to albumin (100 fM-150 nM), Kapp = 28 pM, linear dynamic range 3.0-83.5 pM, limit of detection (LOD) 1.26 pM. Selectivity was confirmed testing 1×Fluo-nanoMIPs against competitor proteins. Finally, as a proof of concept, the nanosensors demonstrated detection of the albumin (1.5 nM) spiked in wine samples, suggesting a possible scaling up of the method in monitoring allergens in wines.

Preparation of sodium-containing coacervates via high-voltage electrostatic field treatment: Saltiness perception of prefabricated chicken patties

Sodium-containing soy protein isolates (SPI)/apple pectin (AP) coacervate dispersion were prepared via high-voltage electrostatic field (HVEF) application to inhomogeneous spatial distribution of sodium as a strategy for reduction of salt content and enhancing the saltiness of prefabricated chicken patties. SPI/AP coacervates were prepared by HVEF processing at pH of 4.0, HVEF voltage of 32 kV, and ratio of SPI: AP to 5:1 which was optimized according to Artificial neural network (ANN)-coupled with genetic algorithm. Fluorescence intensity and FTIR analysis demonstrated that the HVEF treatment caused alteration of protein conformation, improved hydrogen bonding and electrostatic interactions between SPI and AP. At the NaCl concentration of 100 mM, HVEF treated sodium-containing SPI/AP coacervates achieved maximum phase separation as well as coacervate yield (85.23 ± 0.51%). HVEF treated sodium-containing SPI/AP coacervates displayed increased sodium fluorescence intensities, higher sodium concentration difference between coacervate phase and supernatant, and smaller particle sizes. Furthermore, HVEF treated coacervate dispersion showed higher sodium release in vitro during oral digestion. The saltiness of prefabricated chicken patties prepared using HVEF treated coacervates dispersion allowed a reduction in the sodium content of 43.59%. The increase of saltiness perception and sodium release in prefabricated chicken patties is attributed to enhanced water mobility in the matrix and distribution of inhomogeneous sodium.

Abstract PD2-06: Implications of Heterogeneity in Breast Tumor Cell MHC-I Expression on Immunity and Therapeutic Resistance

Abstract Background Immune checkpoint inhibitors (ICIs) targeting the PD-1/L1 axis are approved in early-stage treatment for triple-negative breast cancer (TNBC), but only a fraction of patients benefit. Tumor expressed antigens bound to major histocompatibility complex-I (MHC-I) are required for CD8-mediated tumoricidal activity, and thus, response to anti-PD-1/L1 targeted ICI. However, many breast tumors downregulate, or heterogeneously express, MHC-I, making them less susceptible to ICIs. Tumor cells downregulating MHC-I may be effectively targeted by natural killer (NK) cells due to ‘missing self’ signals. However, this heterogeneity in MHC-I expression is poorly modeled in most preclinical studies, limiting our understanding of how to overcome ICI resistance in the context of heterogeneous MHC-I expression, as is often observed clinically. Objective We aimed to 1) quantitatively delineate how intratumoral heterogeneity in MHC class I expression affects immune responses and immunotherapy outcomes in mouse models and 2) determine whether targeting inhibitory signals on NK cells can overcome ICI resistance in MHC-I heterogenous TNBC. Methods We performed quantitative immunofluorescence for MHC-I, CD8, CD56, and pan-cytokeratin on breast cancer tumors from diverse subtypes (n=314) to obtain single-cell-resolution MHC-I expression and spatial information of tumor and immune cells. Fluorescence intensity and spatial analysis were processed to output individual tumor/stromal cell MHC-I expression and the composition of the local tumor microenvironment. To determine the functional effect of MHC-I heterogeneity in vivo, we generated a CRISPR-guided B2m knockout (B2m-null) in a murine orthotopic model (EMT6). We then combined MHC-I-proficient and MHC-I-deficient isogenic lines at various ratios to model how populations of MHC-I loss affected the immune microenvironment. We also assessed a second, intrinsically heterogenous (MHC-I expression) TNBC model E0771. To evaluate changes in the microenvironment, we used flow cytometry and an immune NanoString panel to evaluate gene expression patterns in tumor cells and infiltrating immune cells. Results TNBC patients had the highest MHC-I expression level across tumor cells, but also the highest variability and probability of demonstrating bimodal MHC-I expression (consisting of high and low/absent cells within a single tumor). ER+ tumors were unimodally low. Using spatial analysis, we identified that heterogenous MHC-I tumors had significantly higher levels of infiltrating NK cells(Paired T-Test: p=0.03). In murine models, even 10% or less of MHC-I-null (B2m-null) EMT6 cells in the tumor injection resulted in acquisition of ICI resistance. Interestingly, heterogeneity in expression of MHC-I resulted in a substantial infiltration by NKG2A+ NK cells compared to MHC-I high or -low tumors (Student T-Test: p=.002). Activation of these infiltrating NK cells via anti-NKG2A and anti-PD-L1 combination treatment restored complete responses in heterogeneous EMT6 tumors, and significantly extended survival in both E0771 (Mantel-Cox: p&amp;lt; 0.0001) and EMT6 models (Mantel-Cox: &amp;lt; 0.0001). Additionally, anti-NKG2A and anti-PDL1 combination treatment improved complete response in the heterogenous MHC-I EMT6 model to 30% and in the parental EMT-6 tumors to 70%. Conclusion Combined therapy with anti-NKG2A (targeting NK cells) and anti-PD-L1 (targeting CD8+ T cells) can restore immunotherapy responses and overcome resistance due to lack of MHC-I expression in tumor cell subpopulations. Citation Format: Brandie C. Taylor, Xiaopeng Sun, Justin Balko, Paula Gonzalez-Ericsson, Melinda Sanders. Implications of Heterogeneity in Breast Tumor Cell MHC-I Expression on Immunity and Therapeutic Resistance [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD2-06.

Toxicity of the Mycotoxin Deoxynivalenol on Early Cleavage of Mouse Embryos by Fluorescence Intensity Analysis.

Deoxynivalenol is a mycotoxin, produced by Fusarium from contaminated corn, wheat, and other grains, that induces multiple effects in humans and animals, including cytotoxic, genotoxic, immunotoxic, and carcinogenic effects. Recent studies show that deoxynivalenol also affects the reproductive system of mammals, including oocyte quality. However, the effects of deoxynivalenol on early embryonic development have not been reported. In this study, fluorescence intensity analysis was used to show that deoxynivalenol disrupted the first cleavage of the zygote. The high deoxynivalenol dose disturbed the movement of the pronucleus after fertilization, while the low deoxynivalenol dose caused aberrant spindle morphology during the metaphase of the first cleavage. Further analysis showed that the reactive oxygen species level increased in the deoxynivalenol-exposed two-cell embryos, indicating oxidative stress. Moreover, deoxynivalenol caused DNA damage in the embryos, as positive γH2A.X signals were detected in the nucleus. These events led to the early apoptosis of mouse embryos, which was confirmed by autophagy. Taken together, our study provides evidence for the toxicity of deoxynivalenol during early embryonic development in the mouse model.

Single Prolonged Stress Decreases the Level of Adult Hippocampal Neurogenesis in C57BL/6, but Not in House Mice

Many people experience traumatic events during their lives, but not all of them develop severe mental pathologies, characterized by high levels of anxiety that persists for more than a month after psychological trauma, such as posttraumatic stress disorder (PTSD). We used a single prolonged stress protocol in order to model PTSD in long-inbred C57BL/6 and wild-derived (house) female mice. The susceptibility of mice to single prolonged stress was assessed by behavior phenotyping in the Open Field and Elevated Plus Maze, the level of neuroinflammation in the hippocampus was estimated by real-time PCR to TNFα, IL-1β, IL-6, IL-10, Iba1 and GFAP, as well as immunohistochemical analysis of microglial morphology and mean fluorescence intensity for GFAP+ cells. The level of neurogenesis was analyzed by real-time PCR to Ki67, Sox2 and DCX as well as immunohistochemistry to Ki67. We showed that long-inbread C57BL/6 mice are more susceptible to a single prolonged stress protocol compared to wild-derived (house) mice. Stressed C57BL/6 mice demonstrated elevated expression levels of proinflammatory cytokines TNFα, IL-1β, and IL-6 in the hippocampus, while in house mice no differences in cytokine expression were detected. Expression levels of Iba1 in the hippocampus did not change significantly after single prolonged stress, however GFAP expression increased substantially in stressed C57BL/6 mice. The number of Iba+ cells in the dentate gyrus also did not change after stress, but the morphology of Iba+ microglia in C57BL/6 animals allowed us to suggest that it was activated; house mice also had significantly more microglia than C57BL/6 animals. We suppose that decreased microglia levels in the hippocampus of C57BL/6 compared to house mice might be one of the reasons for their sensitivity to a single prolonged stress. Single prolonged stress reduced the number of Ki67+ proliferating cells in the dentate gyrus of the hippocampus but only in C57BL/6 mice, not in house mice, with the majority of cells detected in the dorsal (septal) hippocampus in both. The increase in the expression level of DCX might be a compensatory reaction to stress; however, it does not necessarily mean that these immature neurons will be functionally integrated, and this issue needs to be investigated further.

Adriamycin-loaded exosome with anti-CD20 aptamers selectively suppresses human CD20+ melanoma stem cells.

Targeting CD20+ melanoma cancer stem cells (CSCs) subset is essential for treating melanoma. Anti-CD20 aptamer-modified exosomes (ACEXO) loaded with Adriamycin could be a therapeutic strategy for targeting CSCs. Exosomes loaded with Adriamycin were modified with anti-CD20 aptamer and characterized by size and molecular markers using transmission electron microscope and dynamic light scattering. The uptake of ACEXO into CD20+ cells was checked, and its cytotoxicities in CD20+ melanoma cells, HEK 293T, and 3T3 cells were evaluated. At the same time, the in vivo distribution of ACEXO in the tumor-bearing mice model was determined. The particle size of the exosome is about 80-100nm. Western blot analysis showed that they expressed the characteristic exosome markers: CD9 and CD63. Quantitative analysis of the mean fluorescence intensity after 4h incubation showed that ACEXO significantly improved Adriamycin uptake. Notably, the ACEXO killed only CD20+ melanoma cells. In addition, they exhibited good biocompatibility with both 293T and 3T3 cells at all doses. After intravenous injection, exosome distribution data showed that ACEXO's accumulation in the tumor is higher than anti-CD20-modified exosomes (AEXO)'s at all time points, and the accumulation increased as time prolonged. Addition of ACEXO reduces the number of tumorspheres in A375 or WM266-4 cells compared to untreated controls or AEXO-treated group. More important, while treating melanoma tumor-bearing mice, ACEXO-treated group showed the lowest tumor weight without body weight loss. ACEXO loaded with Adriamycin could suppress tumor cell growth in vitro and in vivo, probably by targeting CD20+ melanoma CSCs.