Analysis Of Activation Research Articles

Comparison of the Antioxidant Potency of Four Triterpenes of Centella asiatica against Oxidative Stress.

We comparatively evaluated the antioxidant properties of key triterpenes from Centella asiatica, including asiatic acid (AA), asiaticoside, madecassic acid, and madecassoside, in several cell types, including skin fibroblasts, macrophages, hepatocytes, and endothelial cells, under conditions promoting oxidative stress. AA conferred the highest viability on Hs68 cells exposed to ultraviolet B (UVB) irradiation. Triterpene pretreatment attenuated the UVB-induced generation of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as the UVB-induced depletion of glutathione (GSH) in skin fibroblasts. AA most potently inhibited UVB-induced MMP generation, resulting in increased intracellular collagen levels. Pretreatment with triterpenes, particularly AA, significantly improved cell viability and attenuated TBHP-induced levels of ROS, alanine aminotransferase, and aspartate aminotransferase in HepG2 cells. Triterpenes attenuated ROS levels and reduced MDA and GSH expression in EA.hy926 cells. In RAW264.7 macrophages, production of nitric oxide, tumor necrosis factor-α, and interleukin-6 (indicators of LPS-induced oxidative damage) was significantly reduced by treatment with any of the triterpenes. Statistical analyses of triterpene biological activities using principal component analysis and hierarchical clustering revealed that AA exerted the greatest overall influence and showed remarkable activity in Hs68 and HepG2 cells.

Parametric Analysis of Donor Activation for Glycosylation Reactions.

The chemical synthesis of complex oligosaccharides relies on efficient and highly reproducible glycosylation reactions. The outcome of a glycosylation is contingent upon several environmental factors, such as temperature, acidity, the presence of residual moisture, as well as the steric, electronic, and conformational aspects of the reactants. Each glycosylation proceeds rapidly and with a high yield within a rather narrow temperature range. For better control over glycosylations and to ensure fast and reliable reactions, a systematic analysis of 18 glycosyl donors revealed the effect of reagent concentration, water content, protecting groups, and structure of the glycosyl donors on the activation temperature. With these insights, we parametrize the first step of the glycosylation reaction to be executed reliably and efficiently.

Time Delay in Motor Vehicle Accident Arrival: A Critical Analysis of Trauma Team Activation.

Introduction This research aims to investigate the role of time since trauma (TST) in refining trauma team activation (TTA) criteria within a level I trauma center. We analyze the association between TST and post-emergency department (ED) disposition, proposing new insights for the enhancement of TTA criteria. Methods A retrospective analysis was conducted on a dataset comprising 3,693 patients presenting to a level I trauma center following motor vehicle accidents (MVAs) from 2016 to 2021. Data from a trauma registry, encompassing time of injury, time of ED arrival, TTA status, and post-ED disposition, were utilized. TST was calculated as the difference between the time of injury and the time of ED arrival. Patients that received TTA, full or partial, were categorized based on TST (less than one hour, one to two hours, and two or more hours). Statistical analyses, including chi-square tests, were performed using the Statistical Analysis System (SAS) (version 3.8, SAS Institute Inc., Cary, NC). Results Of the 1,261 patients meeting the criteria, 98.3% received TTA, with decreasing TTA rates observed with increasing TST (p = 0.0076). A significant association was found between TST and post-ED disposition for patients who received TTA (p = 0.0007). Compared to the other TST groups, a higher proportion of patients with a TST of two or more hours were admitted,sent to the intensive care unit (ICU), and sent to the operating room (OR). Conclusion The study indicates a statistically significant relationship between TST and TTA rates, challenging our assumptions about the decreased need for TTA over time. While a longer TST was associated with a lower percentageof TTA, patients with a TST of two or more hours demonstrated increased rates of admission, ICU utilization, and surgical interventions. This suggests that TTA criteria may benefit from refinement to include patients with longer TST. Acknowledging study limitations, such as a small sample size and retrospective design, this research contributes valuable insights into potential considerations for optimizing trauma care protocols.

ω3-PUFA alleviates neuroinflammation by upregulating miR-107 targeting PIEZO1/NFκB p65

BackgroundMiR-107 is reduced in sepsis and associated with inflammation regulation. Dietary supplementation with polyunsaturated fatty acids (ω3-PUFA) can increase the expression of miR-107; this study investigated whether the ω3-PUFA can effectively inhibit neuroinflammation and improve cognitive function by regulating miR-107 in the brain. MethodsThe LPS-induced mouse model of neuroinflammation and the BV2 cell inflammatory model were used to evaluate the effects of ω3-PUFA on miR-107 expression and inflammation. Intraventricular injection of Agomir and Antagomir was used to modulate miR-107 expression. HE and Nissl staining for analyzing hippocampal neuronal damage, immunofluorescence analysis for glial activation, RT-qPCR, and Western blot were conducted to examine miR-107 expression and inflammation signalling. ResultsThe result shows that LPS successfully induced the mouse neuroinflammation model and BV2 cell inflammation model. Supplementation of ω3-PUFA effectively reduced the secretion of pro-inflammatory factors TNFα, IL1β, and IL6 induced by LPS, improved cognitive function impairment, and increased miR-107 expression in the brain. Overexpression of miR-107 in the brain inhibited the nuclear factor κB (NFκB) pro-inflammatory signalling pathway by targeting PIEZO1, thus suppressing microglial and astrocyte activation and reducing the release of inflammatory mediators, which alleviated neuroinflammatory damage and improved cognitive function in mice. miR-107, as an intron of PANK1, PANK1 is subject to PPAR α Adjust. ω3-PUFA can activate PPARα, but ω3-PUFA upregulates brain miR-107, and PPARα/PANK1-related pathways may not be synchronized, and further research is needed to confirm the specific mechanism by which ω3-PUFA upregulates miR-107. ConclusionThe miR-107/PIEZO1/NFκB p65 pathway represents a novel mechanism underlying the improvement of neuroinflammation by ω3-PUFA.

Geometric Co-Ordinate Feature Point Estimation (Gfpe): A Novel Approach to Analyse Of Kinematic Characteristics of Yoga Asanas Based on Anybody Simulation Software

The kinematic characteristics of yoga asanas is crucial for comprehending the biomechanical demands and benefits associated with these postures. Utilizing AnyBody simulation software, researchers delve into the intricate movements and alignments involved in various yoga poses. Through advanced biomechanical modeling, AnyBody allows for the analysis of joint angles, muscle activations, and forces exerted on the body during asana practice. This paper presented an analysis of the kinematic characteristics of yoga asanas using AnyBody Simulation in conjunction with Geometric Co-ordinate Feature Point Estimation (GFPE). By integrating advanced biomechanical modelling capabilities of AnyBody with GFPE’s precision in identifying geometric features, this study aims to elucidate the intricate movements and alignments inherent in various yoga poses. Through comprehensive simulations, joint angles, muscle activations, and forces exerted on the body during asana practice are meticulously analyzed. The examination of body posture with GFPE in the downward-facing dog pose, the average joint angles of the shoulder, elbow, and wrist were found to be 130°, 150°, and 160°, respectively. Additionally, muscle activation levels were measured, with the triceps brachii exhibiting an average activation of 70% of maximum voluntary contraction during the pose.

Typical active jamming performance analysis of triangular FMCW

Typical active jamming performance analysis of triangular FMCW

Current status of interspecies somatic cell nuclear transfer and meta‐analysis of the effects of phylogenetic distance on embryonic and fetal development

Abstract Interspecies somatic cell nuclear transfer (iSCNT) has the capacity to rescue endangered species, ethically produce human embryonic stem cells, and allow for more specific analysis of pre‐implantation embryos. However, several obstacles negatively affect iSCNT efficiency. Over 100 iSCNT studies have been published between 1999 and the spring of 2023. This review details the possible benefits of iSCNT, describes the hypothesized issues that prevented the development of some embryos and/or live offspring from developing; and analyzes the cleavage, blastocyst, pregnancy, live birth, and iSCNT efficiency rates based on published data. Embryos produced by iSCNT may help with the rescue of endangered species and the production of human embryonic stem cells with the use of oocytes from domestic species, as well as in‐depth analyses of embryonic genome activation (EGA) and mRNA transcripts. But the combination of the cells of two species in a single embryo presents several challenges: lack of EGA synchronisation, presence of mitochondrial DNA (mtDNA) from both species in the embryo, requirement of cell and embryo culture optimization for each species combination, lack of embryo transfer data, and an extremely low overall efficiency in terms of producing live offspring. Additional research on the aforementioned topics is warranted to increase iSCNT efficiency.

Unveiling the regulatory mechanisms of nodules development and quality formation in Panax notoginseng using multi-omics and MALDI-MSI

IntroductionRenowned for its role in traditional Chinese medicine, Panax notoginseng exhibits healing properties including bidirectional regulatory effects on hematological system diseases. However, the presence of nodular structures near the top of the main root, known as nail heads, may impact the quality of the plant's valuable roots. ObjectivesIn this paper, we aim to systematically analyze nail heads to identify their potential correlation with P. notoginseng quality. Additionally, we will investigate the molecular mechanisms behind nail head development. MethodsMorphological characteristics and anatomical features were analyzed to determine the biological properties of nail heads. Active component analysis and MALDI mass spectrometry imaging (MALDI-MSI) were performed to determine the correlation between nail heads and P. notoginseng quality. Phytohormone quantitation, MALDI-MSI, RNA-seq, and Arabidopsis transformation were conducted to elucidate the mechanisms of nail head formation. Finally, protein-nucleic acid and protein–protein interactions were investigated to construct a transcriptional regulatory network of nodule development and quality formation. ResultsOur analyses have revealed that nail heads originate from an undeveloped lateral root. The content of ginsenosides was found to be positively associated with the amount of nail heads. Ginsenoside Rb1 specifically accumulated in the cortex of nail heads, while IAA, tZR and JAs also showed highest accumulation in the nodule. RNA-seq analysis identified PnIAA14 and PnCYP735A1 as inhibitors of lateral root development. PnMYB31 and PnMYB78 were found to form binary complexes with PnbHLH31 to synergistically regulate the expression of PnIAA14, PnCYP735A1, PnSS, and PnFPS. ConclusionOur study details the major biological properties of nodular structures in P. notoginseng and outlines their impact on the quality of the herb. It was also determined that PnMYB31- and PnMYB78-PnbHLH31 regulate phytohormones and ginsenosides accumulation, further affecting plant development and quality. This research provides insights for quality evaluation and clinical applications of P. notoginseng.

Robust autofocus method based on patterned active illumination and image cross-correlation analysis.

For the effectiveness of a computer-aided diagnosis system, the quality of whole-slide image (WSI) is the foundation, and a useful autofocus method is an important part of ensuring the quality of WSI. The existing autofocus methods need to balance focusing speed and focusing accuracy, and need to be optimized separately for different samples or scenes. In this paper, a robust autofocus method based on fiber bundle illumination and image normalization analysis is proposed. For various application scenes, it meets the requirements of autofocusing through active illumination, such as bright field imaging and fluorescence imaging. For different structures on samples, it ensures the autofocusing accuracy through image analysis. The experimental results imply that the autofocusing method in this paper can effectively track the change of the distance from the sample to the focal plane and significantly improve the WSI quality.

NrCAM-deficient mice exposed to chronic stress exhibit disrupted latent inhibition, a hallmark of schizophrenia.

The neuronal cell adhesion molecule (NrCAM) is widely expressed and has important physiological functions in the nervous system across the lifespan, from axonal growth and guidance to spine and synaptic pruning, to organization of proteins at the nodes of Ranvier. NrCAM lies at the core of a functional protein network where multiple targets (including NrCAM itself) have been associated with schizophrenia. Here we investigated the effects of chronic unpredictable stress on latent inhibition, a measure of selective attention and learning which shows alterations in schizophrenia, in NrCAM knockout (KO) mice and their wild-type littermate controls (WT). Under baseline experimental conditions both NrCAM KO and WT mice expressed robust latent inhibition (p = 0.001). However, following chronic unpredictable stress, WT mice (p = 0.002), but not NrCAM KO mice (F < 1), expressed latent inhibition. Analyses of neuronal activation (c-Fos positive counts) in key brain regions relevant to latent inhibition indicated four types of effects: a single hit by genotype in IL cortex (p = 0.0001), a single hit by stress in Acb-shell (p = 0.031), a dual hit stress x genotype in mOFC (p = 0.008), vOFC (p = 0.020), and Acb-core (p = 0.032), and no effect in PrL cortex (p > 0.141). These results indicating a pattern of differential effects of genotype and stress support a complex stress × genotype interaction model and a role for NrCAM in stress-induced pathological behaviors relevant to schizophrenia and other psychiatric disorders.

Exploring transient global transcriptional changes induced by ascorbic acid revealed via atKAS-seq profiling.

Transcription initiates the formation of single-stranded DNA (ssDNA) regions within the genome, delineating transcription bubbles, a highly dynamic genomic process. Kethoxal-assisted single-stranded DNA sequencing (KAS-seq) utilizing N3-kethoxal has emerged as a potent tool for mapping specific guanine positions in ssDNA on a genome-wide scale. However, the original KAS-seq method required the costly Accel-NGS Methyl-seq DNA library kit. This study introduces an optimized iteration of the KAS-seq technique, referred to as adapter-tagged KAS-seq (atKAS-seq), incorporating an adapter tagging strategy. This modification involves integrating sequencing adapters via complementary strand synthesis using random N9 tagging. Additionally, by harnessing the potential of ascorbic acid (ASC), recognized for inducing global epigenetic changes, we employed the atKAS-seq methodology to elucidate critical pathways influenced by short-term, high-dose ASC treatment. Our findings underscore that atKAS-seq enables rapid and precise analyses of transcription dynamics and enhancer activities concurrently. This method offers a streamlined, cost-efficient, and low-input approach, affirming its utility in probing intricate genomic regulatory mechanisms.

Metagenomics unveils the differences in the functions of microbial community of medium-temperature Daqu before and after maturation

Daqu is the saccharifying, fermenting, and aroma-producing agent used in Baijiu brewing, and its maturation is crucial for obtaining high-quality Daqu. Previous studies have explored the microbial community composition and diversity before and after maturation. However, little is known about the changes in the functions of microbial community. In this study, based on the analyses of enzyme activities and volatile compounds of medium-temperature Daqu before and after maturation, metagenomics was used to analyze the differences in the composition of microbial community and the potential functions, with the aim to explore the microorganisms involved in changes in enzyme activities and important volatiles. The results showed that the moisture (P≤0.05), starch content, liquefying activity, saccharifying activity (P≤0.05), and fermentative activity decreased, while the acidity and esterifying activity (P≤0.05) increased after Daqu maturation. In the meantime, the composition of volatile compounds changed significantly (P=0.001), with significant decreases in the contents of aromatic alcohols and esters as well as significant increases in the contents of pyrazines, ketones, and higher fatty alcohols. The relative abundances of Mucorales (34.8%-23.0%) and Eurotiales (34.3%-20.1%) decreased in matured Daqu, and functional predictions showed these changes decreased the gene abundances of α-amylase, α-glucosidase, alcohol dehydrogenase, and alcohol dehydrogenase (NADP+) (P > 0.05), resulting in lower levels of liquefying activity (P > 0.05), saccharifying activity (P≤0.05), fermentative activity (P > 0.05), as well as aromatic alcohols such as phenylethyl alcohol (P≤0.05). In addition, higher relative abundances of Saccharomycetales (2.9%-16.6%), Lactobacillales (14.9%-23.6%), and Bacillales (0.8%-3.8%) were observed after maturation, and they were conducive to improving the gene abundances of alcohol O-acetyltransferase, carboxylesterase, acetolactate decarboxylase, (R)-acetoin dehydrogenase, and (S)-acetoin dehydrogenase (P≤0.05), resulting in significantly higher levels of esterifying activity and pyrazines (P≤0.05). The microorganisms involved in the changes in enzyme activities and important volatiles before and after Daqu maturation were studied at the gene level in this work, which may facilitate further rational regulation for Daqu production.

Taste-Active Peptides from Triple-Enzymatically Hydrolyzed Straw Mushroom Proteins Enhance Salty Taste: An Elucidation of Their Effect on the T1R1/T1R3 Taste Receptor via Molecular Docking.

The objective of our study was to analyze and identify enzymatic peptides from straw mushrooms that can enhance salty taste with the aim of developing saltiness enhancement peptides to reduce salt intake and promote dietary health. We isolated taste-related peptides from the straw mushroom extract using ultrafiltration and identified them using UPLC-Q-TOF-MS/MS. The study found that the ultrafiltration fraction (500-2000 Da) of straw mushroom peptides had a saltiness enhancement effect, as revealed via subsequent E-tongue and sensory analyses. The ultrafiltration fractions (500-2000 Da) were found to contain 220 peptides, which were identified through UPLC-Q-TOF-MS/MS analysis. The interaction of these peptides with the T1R1/T1R3 receptor was also assessed. The investigation highlighted the significant involvement of Asp223, Gln243, Leu232, Asp251, and Pro254 in binding peptides from triple-enzymatically hydrolyzed straw mushrooms to T1R1/T1R3. Based on the binding energy and active site analysis, three peptides were selected for synthesis: DFNALPFK (-9.2 kcal/mol), YNEDNGIVK (-8.8 kcal/mol), and VPGGQEIKDR (-8.9 kcal/mol). Importantly, 3.2 mmol of VPGGQEIKDR increased the saltiness level of a 0.05% NaCl solution to that of a 0.15% NaCl solution. Additionally, the addition of 0.8 mmol of YNEDNGIVK to a 0.05% NaCl solution resulted in the same level of saltiness as a 0.1% NaCl solution.

Global sensitivity analysis of plasma instabilities via active subspaces

Active subspace analysis is a useful computational tool to identify and exploit the most important linear combinations in the space of a model’s input parameters. These directions depend inherently on a quantity of interest, which can be represented as a function from input parameters to model outputs. As the dynamics of many plasma models are driven by potentially uncertain parameter values, the utilization of active subspaces to perform global sensitivity analysis represents an important step in understanding how certain physical phenomena depend upon fluctuations in the values of these parameters. In the current paper, we construct and implement new computational methods to quantify the induced uncertainty within the growth rate generated by perturbations in a collisionless plasma modeled by the one-dimensional Vlasov–Poisson system near an unstable, spatially-homogeneous steady state in the linear regime.

Abstract 5354: High-resolution analysis of immune checkpoint activation utilizing a combined PD1/PD-L1 in situ proximity ligation assay (isPLA) and multiplex immunofluorescence (mIF) imaging approach

Abstract The immune checkpoint involving PD-1 and PD-L1 generates suppressive signals in T cells, which help to prevent autoimmunity by inducing a state of immune exhaustion. In the context of the tumor microenvironment (TME), however, cancer cells can manipulate these pathways to camouflage themselves from an immune attack. Blocking these checkpoints has thus emerged as a key immunotherapeutic tactic. Nonetheless, the success rate of immune checkpoint inhibitors (ICIs) has been mixed, even for patients who test positive for relevant diagnostic biomarkers. Patient stratification currently depends on immunohistochemical staining for checkpoint proteins like PD-L1, but these tests do not provide adequate stratification. A more comprehensive stratification, one that includes immune profiling of the TME plus an evaluation of immune checkpoint interactions, might provide better patient stratification and ICI responsiveness. Here we describe mIF that is augmented with a PD-1 and PD-L1 protein-protein interaction assay. Human formalin-fixed, paraffin-embedded (FFPE) tissue sections were subjected to standard histological processing and then incubated with primary antibodies against PD1 and PD-L1. Sections were then treated with oligonucleotide-modified secondary probes suitable for in situ Proximity Ligation Assay (isPLA), enabling the detection of PD1 and PD-L1 interactions. The PLA protocol utilized a fluorescent probe complementary to amplified nucleic acid to enhance the visibility of the protein interactions. The sample was then mounted in a closed microfluidic chamber and imaged on the CellScape™ platform for Precise Spatial Multiplexing. After imaging of the isPLA signal, the CellScape cyclic multiplex mIF staining approach was used to iteratively stain immune and structural markers on the tissue sample utilizing the VistaPlex™ Spatial Immune Profiling Assay Kit. This proof-of-concept study demonstrates that high-plex mIF can be augmented with an isPLA on human FFPE samples. The combined application of both methods allows visualization of PD1/PD-L1 protein-protein interactions and integrates this interaction within the spatial context of the surrounding cell populations. This approach allows for a more comprehensive insight into the interplay of different immune cell and non-immune cell populations during checkpoint activation processes in normal and neoplastic tissues. Citation Format: Arne Christians, Jannik Boog, Charles E. Jackson, Matthew H. Ingalls, J Spencer Schwarz, Sara Bodbin, Agata Zieba Wicher, Subham Basu, Oliver Braubach. High-resolution analysis of immune checkpoint activation utilizing a combined PD1/PD-L1 in situ proximity ligation assay (isPLA) and multiplex immunofluorescence (mIF) imaging approach [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 5354.

Abstract 1210: High-throughput time-resolved single-cell analysis of T-cell activation

Abstract How immune cells respond to stimuli is fundamental in developing adoptive cell therapies, vaccines, and other immunotherapies. T cells dynamically undergo activation, exhaustion, expansion, phenotype switching, and cell killing. Flow cytometry is the gold standard method for characterizing these at high throughput, typically by comparing T cell populations before and at different timepoints after treatment. This approach is limited to population-level analyses and overlooks any heterogeneity of individual T cell responses over time, which may contribute to variability in response. Here, we describe the development of a novel flow cytometry-based method (Time-lapse flowTM) to characterize immune cell responses over time at the single cell level. To measure the same cells repeatedly on a flow cytometer, we employed a new optical barcoding technology called laser particles (LPs) that provide individual cells with a unique spectral signature that can be tracked over the course of multiple measurements. Using a custom flow cytometer with fluorescence and LP detection, we performed a multi-day assay to capture how T cell marker expression changes over time after CD3/CD28 activation. Specifically, we used releasable antibodies to quantify PD1, CD69 and CD25 at each time-point and correlated these expression kinetics with cell function at the end of the assay by measuring cytokine secretion (IFNg, IL2, TNFa) after PMA/ionomycin stimulation. We found significant cellular heterogeneity in kinetic profiles of PD1, CD69 and CD25 expression, with different dominant ‘kinetic modes’ present for each marker. For example, while a large proportion of PD1 expression was transient (positive on day 1, but not on days 2 and 3), nearly all cells that express CD69 on day 1 retained their expression on day 2. We also found that T cells with late expression of PD1 had significantly more cytokine secretion compared to cells that were initially positive for PD1. These results were replicated with at least 3 replicate samples for multiple donors. Differences in kinetic phenotypes were not explained by conventional phenotyping with CD4, CD8, CD45RA and CCR7. To date, immune cell phenotyping with flow cytometry involves classifying cell types through their marker expression profiles at a single time-point. Here, we introduce a novel approach for acquiring time-resolved single-cell data with a flow cytometer and applied it to study T cell activation. We found novel kinetic phenotypes of T cells defined by their marker expression over time, which also correlated to their functional potential (cytokine secretion). These kinetic phenotypes could not be identified with conventional flow cytometry. Our approach could have significant implications for immunotherapy development, particularly in identifying the most suitable cell populations to use, understanding mechanisms of exhaustion and persistence, and elucidating the basis of donor variability. Citation Format: Sheldon J. Kwok, Sarah Forward, Emane Rose Assita, Trevor Brown, Marissa Fahlberg, Pratip Chattopadhyay. High-throughput time-resolved single-cell analysis of T-cell activation [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 1210.

Abstract 5274: Targeting retinoblastoma protein in tumor-associated macrophages suppresses ovarian cancer progression

Abstract Ovarian cancer is characterized by an immunosuppressive tumor microenvironment (TME) maintained by tumor-associated M2-like macrophages (TAMs) hindering anti-tumor responses and immunotherapy efficacy. An effective approach to target TAMs in ovarian cancer treatment is currently not available, but would be expected to release immunosuppressive pressure, enable a robust T cell response and improve immunotherapy outcomes. Retinoblastoma protein (Rb) is a well-known tumor suppressor and regulator of tumor cell proliferation. Accumulating evidence demonstrates its role in immune cells, in myeloid cells, in particular. However, these mechanisms remain poorly understood. Based on our previous data on Rb role in myeloid cell viability, we decided to test the effects of Rb modulation in macrophages. We used the small molecule, AP-3-84 compound, and newly developed analogs which bind the LxCxE motif of Rb and disrupt Rb interaction with its binding adaptor proteins. AP-3-84 induced cell death preferentially in macrophages, but not in T cells or tumor cells (without effects on cell proliferation). Gene and protein expression analysis revealed that Rb targeting induced major intracellular stress response programs and p53/mitochondria-related cell death pathways in TAMs. Moreover, we found that M2 type polarized macrophages expressed higher levels of Rb compared to M1 macrophages. In agreement with that, M2 type macrophages were significantly more sensitive to AP-3-84 treatment than M1 polarized cells. Next, we demonstrated that low dose therapeutic use of AP-3-84 in mice bearing ovarian cancer significantly re-shaped the immune composition of the TME by depleting TAMs and inducing remarkable T cell infiltration. Importantly, these AP-3-84 effects were immune-mediated, since identical AP-3-84 treatment in NSG immunodeficient mice had no effect on tumor growth. Similarly, anti-CD4/CD8 depletion also reverted AP-3-84 effects on cancer inhibition. An analysis of activation and differentiation markers in myeloid and T cell subsets in the TME showed a shift of the remaining macrophages towards an M1-like cell type accompanied by activation of T cells. Using ovalbumin-overexpressing ovarian cancer cell line, we documented the accumulation of antigen-specific T cells in the TME associated with a substantial delay of ovarian cancer upon AP-3-84 therapeutic Rb targeting. Ex vivo, we observed an analogous cell death induction by AP-3-84 treatment in TAMs from post-surgery ascites from ovarian cancer patients. Using available datasets, we further documented that an increase in Rb expression in the TME myeloid cells is associated with poorer prognosis. Overall, our data supports that therapeutic targeting of the Rb LxCxE motif is a promising approach for ovarian cancer treatment due to depletion of TAMs and re-shaping the ovarian cancer TME. Citation Format: Evgenii N. Tcyganov, Taekyoung Kwak, Xue Yang, Adi N. Poli, Colin Hart, Avishek Bhuniya, Joel Cassel, Andrew Kossenkov, Noam Auslander, Paridhima Sharma, Mark G. Cadungog, Stephanie Jean, Sudeshna Chatterjee-Paer, David Weiner, Laxminarasimha Donthireddy, Bryan Bristow, Rugang Zhang, Joseph M. Salvino, Luis J. Montaner. Targeting retinoblastoma protein in tumor-associated macrophages suppresses ovarian cancer progression [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 5274.

Abstract 4002: Investigating CAR-T cell efficacy and activation in the disseminated NALM6-luc human B-cell acute lymphoblastic leukemia model

Abstract Introduction: Chimeric Antigen Receptor T (CAR-T) cell therapy has emerged as a promising approach for treating hematological malignancies, particularly B-cell acute lymphoblastic leukemia (B-ALL). This study aimed to delve into the activation dynamics and therapeutic efficacy of CD19 CAR-T cells in a disseminated NALM6-luc human B-ALL murine model. Experimental design: NALM6-Luc-mCh-Puro cells were implanted intravenously in female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. Animals were treated with CD19 CAR-T cells alone and in combination with ibrutinib. The anti-tumor activity was assessed, and the persistence and activation of CAR-T cells in the blood were evaluated using flow cytometry. Additionally, ddPCR analysis was employed to quantify CAR copy numbers, providing insights into the T-cell persistence over time. Results: Treatment with CD19 CAR-T cells exhibited notable anti-cancer activity and that activity was enhanced in combination with ibrutinib, in line with other reports. The increased time to progression was noted as 69.2% for the CAR-T group and 138.4% for the combination of CAR-T and ibrutinib compared to control. Flow cytometry analysis showed that CAR-T cells persisted in the blood throughout the study. The mean CAR-T absolute cell count for both CAR-T and CAR-T plus ibrutinib treated groups increased from 2 cell/µL blood on Day 0 to 11 and 51 cells/µL of blood, respectively, by Day 39. The progressive increase of PD-1+ and LAG-3+ percentage cells correlates positively with tumor burden, indicating a dynamic link between biomarker upregulation and the presence of tumor cells in mice. ddPCR analysis revealed an initial CAR copy decrease in all groups, followed by gradual increase in tumor-bearing mice treated with CD19 CAR-T cells, with a dramatic increase observed by Day 39. Results from CAR-T treated and CAR-T plus ibrutinib treated groups were comparable, showing mean copy numbers per ng of DNA increasing from less than 1 on day 4 to 17.9 and 23.3, respectively, on day 39. Importantly, no changes were noted in non-tumor implanted mice, emphasizing the tumor dependency for CAR-T cell expansion. Conclusion: This study emphasizes the marked anti-cancer activity of CD19 CAR-T cells in the NALM6-Luc-mCh-Puro B-ALL murine model. Comprehensive analysis of CAR-T cell persistence and activation, including insights from ddPCR, provides a robust understanding of therapeutic response dynamics. The observed in vivo anti-cancer efficacy aligns with the persistence of CAR-T cells revealed by flow cytometry and the dynamic expansion captured through ddPCR analysis. Our results indicate ddPCR and flow cytometry as complementary and independent methods for assessing the systemic CAR-T cell persistence, contributing to optimizing therapy for B-cell acute lymphoblastic leukemia. Citation Format: Prashant Pandey, David Draper, Sheri Barnes, Yewei Xing, Derrik Germain, Lauren Kucharczyk, Roys Stacey. Investigating CAR-T cell efficacy and activation in the disseminated NALM6-luc human B-cell acute lymphoblastic leukemia model [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 4002.

Multimodal active subspace analysis for computing assessment oriented subspaces from neuroimaging data

Background:For successful biomarker discovery, it is essential to develop computational frameworks that summarize high-dimensional neuroimaging data in terms of involved sub-systems of the brain, while also revealing underlying heterogeneous functional and structural changes covarying with specific cognitive and biological traits. However, unsupervised decompositions do not inculcate clinical assessment information, while supervised approaches extract only individual feature importance, thereby impeding qualitative interpretation at the level of subspaces. New Method:We present a novel framework to extract robust multimodal brain subspaces associated with changes in a given cognitive or biological trait. Our approach involves active subspace learning on the gradients of a trained machine learning model followed by clustering to extract and summarize the most salient and consistent subspaces associated with the target variable. Results:Through a rigorous cross-validation procedure on an Alzheimer’s disease (AD) dataset, our framework successfully extracts multimodal subspaces specific to a given clinical assessment (e.g., memory and other cognitive skills), and also retains predictive performance in standard machine learning algorithms. We also show that the salient active subspace directions occur consistently across randomly sub-sampled repetitions of the analysis. Comparison with Existing Method(s):Compared to existing unsupervised decompositions based on principle component analysis, the subspace components in our framework retain higher predictive information. Conclusions:As an important step towards biomarker discovery, our framework not only uncovers AD-related brain regions in the associated brain subspaces, but also enables automated identification of multiple underlying structural and functional sub-systems of the brain that collectively characterize changes in memory and proficiency in cognitive skills related to brain disorders like AD.

Comparison of face-to-face and online flipped learning: academic achievement, epistemological and pedagogical beliefs

PurposeThe ultimate objective of this study is to compare the impact of face-to-face and online flipped learning on students' academic achievements and their perspectives on learning and teaching, offering valuable insights to the field.Design/methodology/approachThe study utilized a quasi-experimental research method that involves pre-test and post-test control groups.FindingsThe results indicated that face-to-face and online flipped learning positively impacted learning, with comparable contributions to academic achievement. However, we found that online flipped learning did not affect students' beliefs about learning and teaching, while face-to-face flipped learning positively influenced them.Originality/valueAs distance learning becomes increasingly important in our modern era, this research aims to explore the use of active learning methods, including discussion, writing, animation, drawing, association, analysis, knowledge measurement and games, in virtual learning environments, such as online flipped learning. The study seeks to enhance the existing literature on the impact of face-to-face and online flipped learning models on student success. Additionally, it aims to address a significant gap in the literature by determining the effect of these models on students' epistemological and pedagogical beliefs, which can impact their motivation, learning outcomes, academic achievements and decision-making processes.