Heat Shock Protein Research Articles

Microfocused Ultrasound With Visualization Induces Remodeling of Collagen and Elastin Within the Skin.

Microfocused ultrasound with real-time visualization (MFU-V) is often used for noninvasive skin lifting, by precisely targeting dermal and subcutaneous tissues to create thermal coagulation points (TCPs). These TCPs denature collagen and initiate a transient inflammatory response, ultimately attracting dermal fibroblasts and inducing efficient neocollagenesis and extracellular matrix (ECM) remodeling, yielding to MFU-V's desired skin-lifting effects. The current study investigates MFU-V's underlying mode of action based on the histological progression of TCPs in the skin, providing new insight into the technology's regenerative effect. Following standard triple-depth MFU-V treatment, invivo skin samples were assessed using histology and immunohistochemistry to evaluate TCPs, heat shock protein (HSP47), and elastin expression in fibroblasts. MFU-V treatment induced elongated, flame-like TCPs with denatured collagen at focal depths of 1.5, 3.0, and 4.5 mm within the skin-each corresponding to its respective transducer depth. Time-dependent progression of TCPs showed significantly increased scores of fibroblasts and mature collagen along with recruitment of HSP47-positive fibroblasts to TCP areas on Day 90. Collagen formation and later maturation were visualized. Newly synthesized elastin significantly increased in the TCP area on Day 90 compared to Day 14. This work provides histological evidence of stimulation and regeneration of newly synthesized elastin fibers after TCP induction. MFU-V-generated TCPs triggered the body's own healing cascade of collagen denaturation, transient inflammation, proliferation, and tissue remodeling, resulting in attraction of HSP47-positive fibroblasts to the TCP sites, and new collagen and elastin fiber regeneration by fibroblasts. Besides the well-described neocollagenesis, this study demonstrates that MFU-V treatment induces elastin neogenesis that may result not only in skin lifting but also in improved skin elasticity, providing an overall regenerative effect.

De novo strategy of organic semiconducting polymer brushes for NIR-II light-triggered carbon monoxide release to boost deep-tissue cancer phototheranostics.

The integration of photoacoustic imaging (PAI) and photothermal therapy (PTT) within the second near-infrared (NIR-II) window, offering a combination of high-resolution imaging and precise non-invasive thermal ablation, presents an attractive opportunity for cancer treatment. Despite the significant promise, the development of this noninvasive phototheranostic nanomedicines encounters challenges that stem from tumor thermotolerance and limited therapeutic efficacy. In this contribution, we designed an amphiphilic semiconducting polymer brush (SPB) featuring a thermosensitive carbon monoxide (CO) donor (TDF-CO) for NIR-II PAI-assisted gas-augmented deep-tissue tumor PTT. TDF-CO nanoparticles (NPs) exhibited a powerful photothermal conversion efficiency (43.1%) and the capacity to trigger CO release after NIR-II photoirradiation. Notably, the liberated CO not only acts on mitochondria, leading to mitochondrial dysfunction and promoting cellular apoptosis but also hinders the overexpression of heat shock proteins (HSPs), enhancing the tumor's thermosensitivity to PTT. This dual action accelerates cellular thermal ablation, achieving a gas-augmented synergistic therapeutic effect in cancer treatment. Intravenous administration of TDF-CO NPs in 4T1 tumor-bearing mice demonstrated bright PAI signals and remarkable tumor ablation under 1064nm laser irradiation, underscoring the potential of CO-mediated photothermal/gas synergistic therapy. We envision this tailor-made multifunctional NIR-II light-triggered SPB provides a feasible approach to amplify the performance of PTT for advancing future cancer phototheranostics.

Site-selectively Phosphorylated Hsp90 C-terminal Domain Variants Provide Access to Deciphering the Chaperone Code.

The ubiquitous molecular chaperone Heat shock protein 90 (Hsp90) is pivotal in many cellular processes through folding of client proteins under stressed and normal conditions. Despite intensive research on its function as a chaperone, the influence of posttranslational modifications on Hsp90 (the 'chaperone code'), and its interactions with co-chaperones and client proteins, still remains to be elucidated. The C-terminal domain (CTD) of Hsp90 is essential for formation of the active homodimer state of Hsp90 and contains recognition sites for co-chaperones and client proteins. Here we used expressed protein selenoester ligation to introduce site-selective phosphorylations in the Hsp90 CTD, while preserving the native amino acid sequence. The two phosphorylations do not affect the overall secondary structure, but in combination, slightly decrease the thermal stability of the CTD. The Hsp90 CTD functions as a chaperone in decreasing aggregation of model client proteins, but the C-terminal phosphorylations do not significantly alter the anti-aggregation activity for these clients. The optimization of expressed protein selenoester ligation to carry out several steps in one pot provides an efficient route to access site-specifically modified Hsp90 CTD variants, allowing the generation of Hsp90 variants with site-specific PTMs to decipher the chaperone code.

Porcine reproductive and respiratory syndrome virus degrades TANK-binding kinase 1 via chaperon-mediated autophagy to suppress type I interferon production and facilitate viral proliferation.

Porcine reproductive and respiratory syndrome virus (PRRSV) has led to significant economic losses in the global swine industry. Type I interferon (IFN-I) plays a crucial role in the host's resistance to PRRSV infection. Despite extensive research showing that PRRSV employs multiple strategies to antagonise IFN-I induction, the underlying mechanisms remain to be fully elucidated. In this study, we have discovered that PRRSV inhibits the production of IFN-I by degrading TANK-binding kinase 1 (TBK1) through chaperon-mediated autophagy (CMA). From a mechanistic standpoint, PRRSV nonstructural protein 2 (Nsp2) increases the interaction between the heat shock protein member 8 (HSPA8) and TBK1. This interaction leads to the translocation of TBK1 into lysosomes for degradation, mediated by lysosomal-associated membrane protein 2A (LAMP2A). As a result, the downstream activation of IFN regulatory factor 3 (IRF3) and the production of IFN-I are hindered. Together, these results reveal a new mechanism by which PRRSV suppresses host innate immunity and contribute to the development of new antiviral strategies against the virus.

Design, synthesis and biological studies of new isoxazole compounds as potent Hsp90 inhibitors.

Heat shock protein 90 (Hsp90), a molecular chaperone, contributes to the preservation of folding, structure, stability, and function proteins. In this study, novel compounds comprising isoxazole structure were designed, synthesized and their potential ability as Hsp90 inhibitors was validated through docking studies. The active site-based compounds were prepared through a multi-step synthesis process and their chemical structures were characterized employing FT-IR, NMR, and mass spectrometry analysis. Cytotoxic and Hsp90 inhibition activities of synthesized compounds were assessed by MTT assay and ELISA kit, respectively. Based on the obtained results, compound 5 exhibited the highest cytotoxicity (IC50; 14 µM) against cancer cells and reduced Hsp90 expression from 5.54 ng/mL in untreated (normal cells) to 1.56 ng/mL in cancer cells. Moreover, molecular dynamics (MD) simulation results indicated its high affinity to target protein and approved its excellent stability which is essential for exerting an inhibitory effect on cancer cell proliferation.

Prenatal Acoustic Signals Influence Nestling Heat Shock Protein Response to Heat and Heterophil-to-Lymphocyte Ratio in a Desert Bird

Heat shock proteins (HSPs) are essential to cellular protection against heat stress. However, the causes of inter-individual variation in HSP regulation remain unclear. This study aimed to test the impact of early-life conditions on the HSP response to heat in zebra finches. In this arid-adapted bird, incubating parents emit “heat-calls” at high temperatures, which adaptively alter offspring’s phenotypes. Embryos were exposed to heat-calls or control-calls, and at 13 days post-hatch nestlings were separated into two different experiments to test responses to either chronic nest temperature (“in-nest” experiment) or an acute “heat-challenge”. Blood samples were collected to measure levels of heat shock cognate 70, heat shock protein 90α, corticosterone and the heterophil-to-lymphocyte (H/L) ratio. In the in-nest experiment, both HSPs were upregulated in response to increasing nest temperatures only in control-calls nestlings (HSC70: p = 0.010, HSP90α: p = 0.050), which also had a marginally higher H/L ratio overall than heat-call birds (p = 0.066). These results point to a higher heat sensitivity in control-call nestlings. Furthermore, comparing across experiments, only the H/L ratio differed, being higher in heat-challenged than in in-nest nestlings (p = 0.009). Overall, this study shows for the first time that a prenatal acoustic signal of heat affects the nestling HSP response to postnatal temperature.

Microgravity and Human Body: Unraveling the Potential Role of Heat-Shock Proteins in Spaceflight and Future Space Missions

In recent years, the increasing number of long-duration space missions has prompted the scientific community to undertake a more comprehensive examination of the impact of microgravity on the human body during spaceflight. This review aims to assess the current knowledge regarding the consequences of exposure to an extreme environment, like microgravity, on the human body, focusing on the role of heat-shock proteins (HSPs). Previous studies have demonstrated that long-term exposure to microgravity during spaceflight can cause various changes in the human body, such as muscle atrophy, changes in muscle fiber composition, cardiovascular function, bone density, and even immune system functions. It has been postulated that heat-shock proteins (HSPs) may play a role in mitigating the harmful effects of microgravity-induced stress. According to past studies, heat-shock proteins (HSPs) are upregulated under simulated microgravity conditions. This upregulation assists in the maintenance of the proper folding and function of other proteins during stressful conditions, thereby safeguarding the physiological systems of organisms from the detrimental effects of microgravity. HSPs could also be used as biomarkers to assess the level of cellular stress in tissues and cells exposed to microgravity. Therefore, modulation of HSPs by drugs and genetic or environmental techniques could prove to be a potential therapeutic strategy to reduce the negative physiological consequences of long-duration spaceflight in astronauts.

Robust reference gene selection in Norway spruce: essential for real-time quantitative PCR across different tissue, stress and developmental conditions

Accurate gene expression analysis in Norway spruce (Picea abies) under diverse stress conditions requires the identification of stable reference genes for normalization. Notably, the literature lacks reports on suitable reference genes in Norway spruce. Here, we aimed to address this gap by identifying suitable reference genes for quantitative real-time PCR in Norway spruce across various stress conditions (drought, heat, pathogen infection) in seedlings, tissues (needle, phloem, root), and developmental stages (seedlings, mature trees). We evaluated the stability of 15 candidate reference genes and assessed their expression stability using five statistical algorithms (ΔCt, geNorm, NormFinder, BestKeeper, and RefFinder). Our results highlight ubiquitin-protein ligase (SP1), conserved oligomeric Golgi complex (COG7), and tubby-like F-box protein (TULP6) as the most stable reference genes, while succinate dehydrogenase (SDH5) and heat shock protein 90 (HSP90) were the least stable under various experimental conditions. COG7 and TULP6 are novel candidate reference genes reported for the first time. The expression stability of the identified reference genes was further validated using dehydrin-like protein 5 (PaDhn5) under drought conditions in Norway spruce. Pairwise variation analysis suggests that two reference genes were sufficient to normalize gene expression across all sample sets. This study provides a comprehensive analysis of reference gene stability under different experimental conditions and a catalog of genes for each condition, facilitating future functional genomic research in Norway spruce and related conifers.

HSP90 Complex From OLP Lesion Induces T-Cell Polarization via Activation of Dendritic Cells.

To investigate the effects of the heat shock protein 90 (HSP90) complex from oral lichen planus (OLP) lesion tissues on dendritic cell (DC) activation and the polarization of naïve T cells. Expression of HSP90 in OLP lesions and healthy control (HC) mucosa was evaluated by single-cell RNA sequence, IHC, qRT-PCR, and immunoblotting. HSP90 complex was extracted by immunoprecipitation from oral mucosa as the agonist of DCs. Expression of IFN-α and MHC was detected by flow cytometry. After cocultured with pre-stimulated DCs, polarization of naïve T cells was investigated by cytokine analysis. HSP90 was significantly higher in the lamina propria of OLP lesion and closely related to lymphocyte infiltration. HSP90 complex of OLP lesion activated DCs via TLR9 and increased their IFN-α secretion and MHC II expression. Pre-stimulated DCs increased the proportion of Th17 cells. HSP90 complex isolated from OLP lesion activated TLR9/IFN-α of DCs and further promoted the polarization of naïve T cells toward Th17 immunity.

Enhancement of heat tolerance by salt stress in Tetraselmis striata CTP4: impacts on HSP gene expression, pigments, and proximal composition

AbstractAs the world average temperature is on the rise and heat waves are becoming more prevalent, microalgal producers have been facing significant challenges regarding the time periods during which they are able to grow less thermotolerant microalgae in outdoor production facilities. Therefore, it is urgent to explore how microalgae cope with thermal stress and under which growth conditions tolerance to temperatures close to or higher than their maximum thermal threshold is induced. For this purpose, Tetraselmis striata CTP4, a euryhaline microalga known for its thermotolerance, was selected. Tetraselmis striata belongs to the Chlorodendrophyceae, a clade that branched off early from other "core chlorophyte" clades, usually comprised of microalgae able to colonise freshwater habitats. Here, we present compelling evidence that the ability of this microalga to withstand otherwise lethal thermal upshifts to 40 °C is induced by exposure to higher salinity (35 ppt). In contrast, this response is abrogated at lower salinities. Concomitantly, the expression of genes encoding HSP70 and HSP100, two heat shock proteins known to mediate thermotolerance and tolerance to other stresses (e.g., salt stress) in fungi, animals and plants, was enhanced when exposed to both heat stress and higher salinities. This suggests that cross-protective mechanisms against abiotic stress appeared early during the evolution of the core chlorophytes and of Archaeplastida in general. This knowledge can be used to select novel strains and growth conditions that promote thermotolerance in microalgae that are grown in outdoor industrial production facilities in environments where heat waves are expected.

Reversing Zearalenone Toxicity: The Role of Hydroxytyrosol in Zebrafish.

Zearalenone (ZEA) is a widely distributed mycotoxin that presents a substantial worldwide health risk to animals. Several natural compounds have shown promise in mitigating the detrimental impacts of ZEA. This study examined the detoxification potential of previously identified compounds by utilizing zebrafish embryos as a model organism. Hydroxytyrosol stands out among these natural compounds. Our findings indicate that hydroxytyrosol effectively mitigated mortality, hatching delay, and phenotypic abnormalities induced by ZEA in the assessed embryos. Furthermore, hydroxytyrosol restored the frequency and intensity of tail coiling (TC) while decreasing the expression of heat shock proteins (HSPs) in the zebrafish embryos. Extended incubation with hydroxytyrosol demonstrated protective effects on zebrafish growth and morphology, muscle birefringence, and touch-evoked escape behavior. Subsequent investigations indicated that hydroxytyrosol reversed the expression of proapoptotic targets (e.g., bax and caspase8) and cell cycle regulators (e.g., p21, gadd45a, and rbl2), thereby mitigating apoptosis and G2 cell cycle arrest induced by ZEA in zebrafish embryos. Additionally, hydroxytyrosol decreased staining for senescence associated-β-galactosidase (SA-β-Gal). Notably, p53/FoxO pathway plays an important role in detoxification mechanisms. Overall, these novel findings highlight the potential of hydroxytyrosol to reverse ZEA-induced toxicity in multiple aspects. The mitigating effect of hydroxytyrosol on ZEA toxicity may have been underestimated.

Role of Platelet-Activating Factor in the Pathogenesis of Chronic Spontaneous Urticaria

Chronic spontaneous urticaria (CSU) is a debilitating condition characterized by mast cell activation. Platelet-activating factor (PAF) is produced by various immune cells, including mast cells, basophils, lymphocytes, and eosinophils, which play crucial roles in CSU pathogenesis. It induces mast cell degranulation, increases vascular permeability, and promotes the chemotaxis of inflammatory cells. These effects result in the release of inflammatory mediators, the development of edema, and the persistence of inflammation, which are key features of CSU. Notably, elevated PAF levels have been linked to heightened disease activity and resistance to antihistamine treatment in CSU patients. Despite these findings, the precise role of PAF in CSU pathogenesis remains unclear. Rupatadine, an antihistamine, and heat shock protein 10, a natural anti-inflammatory peptide that selectively inhibits PAF-induced mast cell degranulation, have demonstrated anti-PAF activity. Furthermore, with the molecular structure of the PAF receptor now identified, several experimental PAF receptor antagonists have been synthesized. However, there remains a significant need for the development of therapeutic options targeting PAF in CSU management.

Investigating bacteria-induced inflammatory responses using novel endometrial epithelial gland organoid models

IntroductionThe endometrium plays a crucial role in early human pregnancy, particularly in embryo implantation, survival, and growth. However, invasion and infection by pathogens can lead to endometritis, infertility, and poor reproductive outcomes. Understanding the mechanisms of endometritis and its impact on fertility remains limited. An infection model using patient-derived endometrial epithelial gland organoids (EEGOs) was established to advance in vitro studies on endometritis and related infertility.MethodsAn EEGOs infection model was constructed and characterized from human endometrium, treating the organoids with estrogen and progesterone to observe changes in the proliferative and secretory phases. The organoids were infected with E. coli, and the release of inflammatory cytokines in the supernatant was detected using ELISA. RNA-seq was employed to analyze the differences before and after E. coli treatment, and differential gene mRNA expression was validated using real-time quantitative PCR. Additionally, the effect of E2 in alleviating inflammation was assessed through markers of receptivity (PAEP, LIF, ITGβ), proliferation (Ki67), and barrier repair (ZO-1).ResultsThe constructed human EEGOs exhibited long-term expansion capability, genetic stability, and characteristic hormonal responses, strongly expressing epithelial markers (MUC1, E-Cadherin). After E. coli infection, the expression levels of inflammatory cytokines TNF-α, IL-8, and IFN-γ increased significantly (P < 0.05). RNA-seq indicated that the MAPK signaling pathway was activated post-infection, with increased expression levels of heat shock proteins and transcription factor mRNA. E2 treatment post-infection significantly decreased the mRNA expression of inflammatory genes IL-1β, IL8, IL6 and TNF-α compared to the E. coli infected group (P < 0.05). Additionally, the expression of genes related to receptivity, proliferation, and barrier repair was enhanced in the E2-treated organoids.ConclusionsOur findings demonstrate that patient-derived EEGOs are responsive to bacterial infection and are effective models for studying host-pathogen interactions in bacterial infections. These organoids revealed the anti-inflammatory potential of E2 in alleviating E. coli-induced inflammation, providing insights into the mechanisms of endometritis and its impact on infertility. The study supports the use of EEGOs as valuable tools for understanding endometrial health and developing targeted treatments.

Unlocking marine microbial treasures: new PBP2a-targeted antibiotics elicited by metals and enhanced by RSM-driven transcriptomics and chemoinformatics.

Elicitation through abiotic stress, including heavy metals, is a new natural product drug discovery technique. In this research, three compounds 1, 2, and 6, were achieved by triggering zinc and nickel on marine Sphingomonas sp. and Streptomyces sp., which were absent in normal culture. Compound 5 was obtained for the first time from marine bacteria. All compounds showed potent antibacterial activity against Staphylococcus aureus and bactericidal effect at 300µm, but 6 was more active. The potent compound 6 production was further enhanced through response surface methodology by optimizing the condition consisting of nickel 1mM ions, 20mg/L sucrose, 30mg/L salt and culture time 14days. Under these conditions, the SM-6 production was enhanced with a yield of 6.3mg/L, which was absent in the normal culture. Further transcriptome analysis of compound 6 unveiled its antibacterial activity on S. aureus by modulating heat shock protein genes, disrupting protein folding and synthesis, and perturbing cellular redox balance, leading to a comprehensive inhibition of normal bacterial growth. In addition, ADMET has shown that all compounds are safe for cardiac and hepatotoxicity. To determine the anti-bacterial mechanism, all compounds were docked with PBP2a and DNA gyrase enzyme, and TLR-4 protein for predicting vaccine construct, and the best docking score was achieved against PBP2a enzyme with the highest score of -10.2 for compound 6. In-silico cloning was carried out to ensure the expression of proteins generated and were cloned using S.aureus as a host. The simulation studies have shown that both SM-6-PBP2a and TLR-4-PBP2a complex are stable with the system. This study presents a new approach to anti-bacterial drug discovery from microorganisms through heavy metals triggering and enhancing the compound production through response surface methodology.

BIOM-56. HEAT SHOCK PROTEINS PREDICT CLINICAL RESPONSE AND POTENTIATE LOCAL IMMUNOGENICITY AFTER LITT FOR GLIOBLASTOMA

Abstract Glioblastoma (GBM) remains one of the deadliest cancers despite advances in therapies. Laser interstitial thermal therapy (LITT) is a minimally invasive technique used to ablate intra-axial brain tumors. Non-ablative LITT-induced hyperthermia (33-43˚C) increases intra-tumoral mutational burden and neoantigen production, promoting neoantigen presentation via heat shock proteins (HSPs). HSPs are protein-folding chaperones that present novel intracellular antigens for immune recognition. We investigated HSPs as predictors of local immune response and clinical outcomes of GBM patients undergoing LITT. We conducted immune deconvolution of bulk RNA-sequencing data from The Cancer Genome Atlas to determine the correlation between 97 unique HSPs and tumor infiltration of CD8+, CD4+, B, macrophage, neutrophil, and dendritic cells. We evaluated the association between HSP expression and overall survival (OS). Pre-LITT biopsies from 9 patients (responders and non-responders) were analyzed via immunohistochemistry (IHC) to identify spatial HSP expression. Next-generation sequencing was utilized to validate findings and identify other predictive biomarkers. We identified two HSPs for further investigation: SACS and HSPA5. SACS was associated with improved survival based on TCGA data (14.9 vs. 11 months, HR:1.3, p<0.0001). HSPA5 was associated with worse outcomes (12.1 vs. 15.0 months, HR:0.73, p<0.0001). Immune deconvolution showed HSPA5 positively correlated with dendritic cell infiltration (r=0.50, p=6.1E-27) and SACS with CD8+ infiltration (r=0.31, p=6.8E-11). IHC of 9 patients demonstrated high SACS expression was associated with improved OS (p<0.05) after LITT. Multiplex IHC illustrated that immune cell infiltration was associated with therapeutic response post-LITT. Our ongoing immunohistochemical and genomic analyses aim to clarify the role of HSPs in enhancing immunogenicity in GBM. Further investigation into SACS and immune cell infiltration may reveal how hyperthermia via LITT improves response through neoantigen presentation.

Heat Shock Proteins expression in malaria and dengue vector.

The survival of mosquitoes under changing climatic conditions particularly temperature, is known to be supported by Heat Shock Proteins (HSPs). In view of climate change, it is imperative to know whether the mosquito vectors will be able to withstand the increased temperatures or perish. Therefore, the present study was undertaken on the expression of HSPs' gene in An. stephensi and Ae. aegypti by exposing them to temperatures ranging from 5 to 45°C for 15-180-minutes for once and continuously or with rest in between. We compared the temperature-tolerance of both the vectors in terms of expression of HSP83, HSP70, and HSP26 genes at varying degrees of temperature and duration. HSP70 and HSP26 were found distinctively expressed in both the vectors as compared to HSP83. With continuous exposure up to 180-minutes at 35°C and 40°C, HSP70 was found upregulated up to 35 and 47 folds in Ae. aegypti while in An. stephensi, the expression was only 1 fold. Between the genes, HSP70 was highly expressed at different temperatures followed by HSP26 and HSP83. The manifold up-regulation of HSP genes in Ae. aegypti than An. stephensi may be attributed to the robustness of Aedes vector in terms of temperature tolerance. This study has shown that Ae. aegypti and An. stephensi can withstand considerable temperature stress by expressing HSPs when exposed to variable temperature and duration. In view of changing climate, the study provides a clue that the vector of dengue and zika virus will be difficult to control.

Proteomics analysis of extracellular vesicles for biomarkers of autism spectrum disorder

BackgroundAutism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by symptoms that include social interaction deficits, language difficulties and restricted, repetitive behavior. Early intervention through medication and behavioral therapy can eliminate some ASD-related symptoms and significantly improve the life-quality of the affected individuals. Currently, the diagnosis of ASD is highly limited.MethodsTo investigate the feasibility of early diagnosis of ASD, we tested extracellular vesicles (EVs) proteins obtained from ASD cases. First, plasma EVs were isolated from healthy controls (HCs) and ASD individuals and were analyzed using proximity extension assay (PEA) technology to quantify 1,196 protein expression level. Second, machine learning analysis and bioinformatic approaches were applied to explore how a combination of EV proteins could serve as biomarkers for ASD diagnosis.ResultsNo significant differences in the EV morphology and EV size distribution between HCs and ASD were observed, but the EV number was slightly lower in ASD plasma. We identified the top five downregulated proteins in plasma EVs isolated from ASD individuals: WW domain-containing protein 2 (WWP2), Heat shock protein 27 (HSP27), C-type lectin domain family 1 member B (CLEC1B), Cluster of differentiation 40 (CD40), and folate receptor alpha (FRalpha). Machine learning analysis and correlation analysis support the idea that these five EV proteins can be potential biomarkers for ASD.ConclusionWe identified the top five downregulated proteins in ASD EVs and examined that a combination of EV proteins could serve as biomarkers for ASD diagnosis.

DDDR-41. A GENOME-WIDE CRISPR/CAS9 SCREEN IN GLIOBLASTOMA STEM CELLS UNVEILS SYNTHETIC LETHAL INTERACTIONS AND RESISTANCE MECHANISMS OF JIN001, A BLOOD-BRAIN BARRIER-PENETRANT HSP90 INHIBITOR

Abstract Glioblastomas (GBMs) are aggressive brain tumors characterized by extensive genetic heterogeneity and therapeutic resistance. Heat shock protein 90 (Hsp90) is a molecular chaperone that stabilizes and facilitates the proper folding of numerous proteins, including oncogenic drivers and is overexpressed in cancers. Hsp90 inhibitors can disrupt multiple oncogenic signaling pathways by degrading various client proteins essential for tumor growth and survival, making them attractive therapeutic candidates for GBMs. We have previously reported on the efficacy of Hsp90 inhibitors against gliomas. However, cancer cells develop resistance to Hsp90 inhibitors by upregulating compensatory chaperones (e.g., Hsp70), mutating client proteins, or activating alternative survival pathways, reducing the long-term efficacy of these agents. Combination approaches overcoming such resistance mechanisms are needed. JIN001, a novel Hsp90 inhibitor, demonstrates superior blood-brain barrier (BBB) penetration, and high potential for clinical translation. we performed a genome-wide CRISPR/Cas9 knockout screen using the Toronto Knockout Library v3 (TKOv3) with screen selection pressure from JIN001 in GBM stem cells (GSC272) to identify synthetic lethal interactions and resistance mechanisms associated with JIN001 treatment. Experiments were performed in triplicate with two time points, T22 and T37. Quality control analysis of the NGS data indicated excellent quality and consistency between different replicates. DrugZ analysis revealed a set of sensitive and resistant genes, informing potential combination therapies and further elucidating JIN001’s mechanism of action. Validation studies for the top synthetic lethal targets are underway to confirm the identified interactions and will be presented. This CRISPR-Cas9 screen offers valuable insights into JIN001’s therapeutic potential, reveals new combination strategies to overcome resistance to Hsp90 inhibitors and for use of these agents in other cancers, and paves the way for the development of more effective GBM combination treatment strategies.

Integration of mRNA and miRNA Analysis Sheds New Light on the Muscle Response to Heat Stress in Spotted Sea Bass (Lateolabrax maculatus)

Temperature is a crucial environmental factor for fish. Elevated temperatures trigger various physiological and molecular responses designed to maintain internal environmental homeostasis and ensure the proper functioning of the organism. In this study, we measured biochemical parameters and performed mRNA–miRNA integrated transcriptomic analysis to characterize changes in gene expression profiles in the muscle tissue of spotted sea bass (Lateolabrax maculatus) under heat stress. The measurement of biochemical parameters revealed that the activities of nine biochemical enzymes (ALP, γ-GT, AST, GLU, CK, ALT, TG, LDH and TC) were significantly affected to varying degrees by elevated temperatures. A total of 1940 overlapping differentially expressed genes (DEGs) were identified among the five comparisons in the muscle tissue after heat stress. Protein–protein interaction (PPI) analysis of DEGs indicated that heat shock protein genes (HSPs) were deeply involved in the response to heat stress. In addition, we detected 462 differential alternative splicing (DAS) events and 618 DAS genes, which are closely associated with sarcomere assembly in muscle, highlighting the role of alternative splicing in thermal response regulation. Moreover, 32 differentially expressed miRNAs (DEMs) were identified in response to heat stress, and 599 DEGs were predicted as potential target genes of those DEMs, generating 846 DEG–DEM negative regulatory pairs potentially associated with thermal response. Function enrichment analysis of the target genes suggested that lipid metabolism-related pathways and genes were regulated by miRNAs. By analyzing PPIs of target genes, we identified 28 key negative regulatory pairs, including 13 miRNAs (such as lma-miR-122, lma-miR-200b-5p and novel-miR-444) and 15 target genes (such as hspa13, dnaja1, and dnajb1a). This study elucidates the molecular mechanisms of response to high-temperature stress and offers valuable information for the selection and breeding of heat-tolerant strains of spotted sea bass.

Comparative evaluation of the modulatory role of 1,25-dihydroxy-vitamin D3 on endoplasmic reticulum stress-induced effects in 2D and 3D cultures of the intestinal porcine epithelial cell line IPEC-J2

BackgroundThe use of conventional two-dimensional (2D) culture of the porcine intestinal epithelial cell (IEC) line IPEC-J2 in animal nutrition research has the disadvantage that IEC function is studied under unphysiological conditions, which limits the ability of transferring knowledge to the in vivo-situation. Thus, the aim of the present study was to establish a more convincing and meaningful three-dimensional (3D) culture of IPEC-J2 cells, which allows to study cell function in a more tissue-like environment, and to compare the effect of the endoplasmic reticulum (ER) stress inducer tunicamycin (TM) on ER stress indicators and the expression of tight junction proteins (TJP), inflammatory and apoptosis-related genes and the modulatory role of 1,25-dihydroxy-vitamin D3 (1,25D3) on these parameters in 2D and 3D cultures of IPEC-J2 cells.ResultsA published protocol for 3D culture of Caco-2 cells was successfully adopted to IPEC-J2 cells as evident from fully differentiated 3D IPEC-J2 spheroids showing the characteristic spherical architecture with a single layer of IPEC-J2 cells surrounding a central lumen. Treatment of 2D IPEC-J2 cells and 3D IPEC-J2 spheroids with TM for 24 h markedly increased mRNA and/or protein levels of the ER stress target genes, heat shock protein family A (Hsp70) member 5 (HSPA5) and DNA damage inducible transcript 3 (DDIT3), whereas co-treatment with TM and 1,25D3 did not mitigate TM-induced ER stress in IPEC-J2 cells in the 2D and the 3D cell culture. In contrast, TM-induced expression of pro-inflammatory [interleukin-6 (IL6), IL8] and pro-apoptotic genes [BCL2 associated X, apoptosis regulator (BAX), caspase 3 (CASP3), CASP8] and genes encoding TJP [TJP1, claudin 1 (CLDN1), CLDN3, occludin (OCLN), cadherin 1 (CDH1), junctional adhesion molecule 1 (JAM1)] was reduced by co-treatment with TM and 1,25D3 in 3D IPEC-J2 spheroids but not in the 2D cell culture.ConclusionsThe effect of 1,25D3 in the IPEC-J2 cell culture is dependent on the culture model applied. While 1,25D3 does not inhibit TM-induced expression of genes involved in inflammation, apoptosis and TJP in conventional 2D cultures of IPEC-J2 cells, TM-induced expression of these genes is abrogated by 1,25D3 in the more meaningful 3D IPEC-J2 cell culture model.