Changes In Expression Patterns Research Articles

Signal Transduction of Transient Receptor Potential TRPM8 Channels: Role of PIP5K, Gq-Proteins, and c-Jun.

Transient receptor potential melastatin-8 (TRPM8) is a cation channel that is activated by cold and "cooling agents" such as menthol and icilin, which induce a cold sensation. The stimulation of TRPM8 activates an intracellular signaling cascade that ultimately leads to a change in the gene expression pattern of the cells. Here, we investigate the TRPM8-induced signaling pathway that links TRPM8 channel activation to gene transcription. Using a pharmacological approach, we show that the inhibition of phosphatidylinositol 4-phosphate 5 kinase α (PIP5K), an enzyme essential for the biosynthesis of phosphatidylinositol 4,5-bisphosphate, attenuates TRPM8-induced gene transcription. Analyzing the link between TRPM8 and Gq proteins, we show that the pharmacological inhibition of the βγ subunits impairs TRPM8 signaling. In addition, genetic studies show that TRPM8 requires an activated Gα subunit for signaling. In the nucleus, the TRPM8-induced signaling cascade triggers the activation of the transcription factor AP-1, a complex consisting of a dimer of basic region leucine zipper (bZIP) transcription factors. Here, we identify the bZIP protein c-Jun as an essential component of AP-1 within the TRPM8-induced signaling cascade. In summary, with PIP5K, Gq subunits, and c-Jun, we identified key molecules in TRPM8-induced signaling from the plasma membrane to the nucleus.

Tumor-matched and unmatched cancer associated fibroblasts exhibit differential effect on proliferation and FMOD and MMP9 gene expression in head and neck squamous cell carcinoma cells when cocultured in spheroids

BackgroundCancer-associated fibroblasts (CAFs) are the major cellular component of the tumor microenvironment and are known to affect tumor growth and response to various treatments. This study was undertaken to investigate the crosstalk between tumor-matched or unmatched CAFs and head and neck squamous cell carcinoma (HNSCC) cells regarding tumor growth and treatment response.MethodsThree HNSCC cell lines (LK0412, LK0902 and LK0923), were cocultured in 2D or in 3D with their tumor-matched CAFs, site matched CAFs from other tumors or normal oral fibroblasts (NOFs). Cell proliferation was assessed as the amount of Ki67 positive cells/ spheroid area in formalin-fixed- paraffin-embedded 3D spheroids stained with Ki67 antibody. Viability after seven days of cisplatin treatment was measured with CellTiter-Glo 3D Viability Assay. The mRNA expression of CAF-associated markers (ACTA2, COL1A2, FAP, PDGFRα, PDGFRβ, PDPN, POSTN and S100A4) in CAFs before and after coculture with tumor cells as well as mRNA expression of CAF-induced genes (MMP1, MMP9 and FMOD) in tumor cells separated from CAFs after co-culture was measured with RT-qPCR. The expression of selected protein biomarkers was validated with immunohistochemistry based on previous mRNA expression results.ResultsThe proliferation of the LK0412 and LK0902 tumor spheroids varied significantly when cocultured with different CAFs and NOFs as shown by Ki-67 positive cells. RT‒qPCR analysis revealed different molecular profile of the analyzed HNSCC-derived CAFs concerning the expression of CAF-associated markers. The interaction between CAFs and HNSCC cells was more pronounced after coculture with unmatched CAFs as shown by changes in mRNA expression pattern of CAF-specific markers. Additionally, the unmatched CAFs significantly upregulated the mRNA expression of MMP1, MMP9 and FMOD in tumor cells compared to tumor-matched CAFs.ConclusionOur results indicate that tumor-matched CAFs are unique for each tumor and affect the proliferation and the gene/protein expression of tumor cells in a distinct manner. The interaction between tumor unmatched CAFs and HNSCC cells in the tumor spheroids is associated with significant changes in the mRNA expression of CAF-specific markers and significant increases in FMOD and MMP9 in tumor cells compared to when cocultured with tumor-matched CAFs. Taken together, our results show how important the selection of CAFs is to get a reliable in vitro model that mimics the patients’ tumor.

The Specific ROCK2 Inhibitor KD025 Alleviates Glycolysis through Modulating STAT3-, CSTA- and S1PR3-Linked Signaling in Human Trabecular Meshwork Cells.

To investigate the biological significance of Rho-associated coiled-coil-containing protein kinase (ROCK) 2 in the human trabecular meshwork (HTM), changes in both metabolic phenotype and gene expression patterns against a specific ROCK2 inhibitor KD025 were assessed in planar-cultured HTM cells. A seahorse real-time ATP rate assay revealed that administration of KD025 significantly suppressed glycolytic ATP production rate and increased mitochondrial ATP production rate in HTM cells. RNA sequencing analysis revealed that 380 down-regulated and 602 up-regulated differentially expressed genes (DEGs) were identified in HTM cells treated with KD025 compared with those that were untreated. Gene ontology analysis revealed that DEGs were more frequently related to the plasma membrane, extracellular components and integral cellular components among cellular components, and related to signaling receptor binding and activity and protein heterodimerization activity among molecular functions. Ingenuity Pathway Analysis (IPA) revealed that the detected DEGs were associated with basic cellular biological and physiological properties, including cellular movement, development, growth, proliferation, signaling and interaction, all of which are associated with cellular metabolism. Furthermore, the upstream regulator analysis and causal network analysis estimated IL-6, STAT3, CSTA and S1PR3 as possible regulators. Current findings herein indicate that ROCK2 mediates the IL-6/STAT3-, CSTA- and S1PR3-linked signaling related to basic biological activities such as glycolysis in HTM cells.

Proteomic profiling of peanut hairy root culture unveils distinctive adaptive responses induced by elicitor treatment across diverse time intervals

Plants employ a diverse array of responses to counteract and defend against stressors. Utilizing quantitative proteomic analysis allows for the exploration of global proteomic dynamics in cells and tissues at specific time points. In order to comprehend the intricate mechanisms underlying plant responses, we conducted a proteomic investigation of peanut hairy root cultures induced with a combination of chitosan (CHT), methyl jasmonate (MeJA), and cyclodextrin (CD) at multiple time points. The study identified 292, 317, and 327 differentially expressed proteins (DEPs) in hairy root tissues at 24-, 48-, and 72 h intervals post-elicitation, respectively. Principal component analysis (PCA) distinctly clustered each timepoint of treatment and the control group. Gene ontology (GO) enrichment analysis highlighted terms such as "responding to biotic stimuli" and "defense response" during the early response, with the upregulation of Heat shock cognate 70 kDa protein, Wound-induced protein 1, and Disease resistance-like protein. The 72 h time point specifically showed an upregulation of superoxide dismutase (SOD) and auxin response factor, indicating a delayed response and illustrating how hairy roots adapt to stress in later stages. Furthermore, GO enrichment at later stages implicated alterations in protein production, involving both synthesis and degradation processes. Proteolytic enzymes, including those in the ubiquitin/proteasome pathway, were identified as facilitating protein degradation. The detection of small molecular weight (MW) peptides in the culture medium of elicited hairy roots suggested their origin from the proteolytic cleavage of precursor proteins. The GO enrichment of culture medium peptides pointed towards involvement in signaling pathways. The suggested signaling cascade acts as a potential link that connects the recognition of stress to the initiation of appropriate response to external stressors. This approach delves into dynamic changes in protein expression and secretion patterns over various intervals, providing insights into the plant's adaptive response to external stress.

Analysis of common differential gene expression in synovial cells of osteoarthritis and rheumatoid arthritis.

To elucidate potential molecular mechanisms differentiating osteoarthritis (OA) and rheumatoid arthritis (RA) through a bioinformatics analysis of differentially expressed genes (DEGs) in patient synovial cells, aiming to provide new insights for clinical treatment strategies. Gene expression datasets GSE1919, GSE82107, and GSE77298 were downloaded from the Gene Expression Omnibus (GEO) database to serve as the training groups, with GSE55235 being used as the validation dataset. The OA and RA data from the GSE1919 dataset were merged with the standardized data from GSE82107 and GSE77298, followed by batch effect removal to obtain the merged datasets of differential expressed genes (DEGs) for OA and RA. Intersection analysis was conducted on the DEGs between the two conditions to identify commonly upregulated and downregulated DEGs. Enrichment analysis was then performed on these common co-expressed DEGs, and a protein-protein interaction (PPI) network was constructed to identify hub genes. These hub genes were further analyzed using the GENEMANIA online platform and subjected to enrichment analysis. Subsequent validation analysis was conducted using the GSE55235 dataset. The analysis of differentially expressed genes in the synovial cells from patients with Osteoarthritis (OA) and Rheumatoid Arthritis (RA), compared to a control group (individuals without OA or RA), revealed significant changes in gene expression patterns. Specifically, the genes APOD, FASN, and SCD were observed to have lower expression levels in the synovial cells of both OA and RA patients, indicating downregulation within the pathological context of these diseases. In contrast, the SDC1 gene was found to be upregulated, displaying higher expression levels in the synovial cells of OA and RA patients compared to normal controls.Additionally, a noteworthy observation was the downregulation of the transcription factor PPARG in the synovial cells of patients with OA and RA. The decrease in expression levels of PPARG further validates the alteration in lipid metabolism and inflammatory processes associated with the pathogenesis of OA and RA. These findings underscore the significance of these genes and the transcription factor not only as biomarkers for differential diagnosis between OA and RA but also as potential targets for therapeutic interventions aimed at modulating their expression to counteract disease progression. The outcomes of this investigation reveal the existence of potentially shared molecular mechanisms within Osteoarthritis (OA) and Rheumatoid Arthritis (RA). The identification of APOD, FASN, SDC1, TNFSF11 as key target genes, along with their downstream transcription factor PPARG, highlights common potential factors implicated in both diseases. A deeper examination and exploration of these findings could pave the way for new candidate targets and directions in therapeutic research aimed at treating both OA and RA. This study underscores the significance of leveraging bioinformatics approaches to unravel complex disease mechanisms, offering a promising avenue for the development of more effective and targeted treatments.

The Effects of Smoking on Telomere Length, Induction of Oncogenic Stress, and Chronic Inflammatory Responses Leading to Aging.

Telomeres, potential biomarkers of aging, are known to shorten with continued cigarette smoke exposure. In order to further investigate this process and its impact on cellular stress and inflammation, we used an in vitro model with cigarette smoke extract (CSE) and observed the downregulation of telomere stabilizing TRF2 and POT1 genes after CSE treatment. hTERT is a subunit of telomerase and a well-known oncogenic marker, which is overexpressed in over 85% of cancers and may contribute to lung cancer development in smokers. We also observed an increase in hTERT and ISG15 expression levels after CSE treatment, as well as increased protein levels revealed by immunohistochemical staining in smokers' lung tissue samples compared to non-smokers. The effects of ISG15 overexpression were further studied by quantifying IFN-γ, an inflammatory protein induced by ISG15, which showed greater upregulation in smokers compared to non-smokers. Similar changes in gene expression patterns for TRF2, POT1, hTERT, and ISG15 were observed in blood and buccal swab samples from smokers compared to non-smokers. The results from this study provide insight into the mechanisms behind smoking causing telomere shortening and how this may contribute to the induction of inflammation and/or tumorigenesis, which may lead to comorbidities in smokers.

Particulate matter-induced gene expression patterns in human-derived cells based on 11 public gene expression datasets.

Exposure to particulate matter (PM) and house dust mite (HDM) can change the expression patterns of inflammation-, oxidative stress-, and cell death-related genes. We investigated the changes in gene expression patterns owing to PM exposure. This study examined the changes in gene expression patterns following PM exposure. We searched for differentially expressed genes (DEGs) following PM exposure using five cell line-based RNA-seq or microarray datasets and six human-derived datasets. The enrichment terms of the DEGs were assessed. DEG analysis yielded two gene sets. Thus, enrichment analysis was performed for each gene set, and the enrichment terms related to respiratory diseases were presented. The intersection of six human-derived datasets and two gene sets was obtained, and the expression patterns following PM exposure were observed. Two gene sets were obtained for cells treated with PM and their expression patterns were presented following verification in human-derived cells. Our findings suggest that exposure to PM2.5 and HDM may reveal changes in genes that are associated with diseases, such as allergies, highlighting the importance of mitigating PM2.5 and HDM exposure for disease prevention.

Synchronizing Drosophila larvae with the salivary gland reporter Sgs3-GFP for discovery of phenotypes in the late third instar stage.

The larval stage of the Drosophila melanogaster life cycle is characterized by rapid growth and nutrient storage that occur over three instar stages separated by molts. In the third instar, the steroid hormone ecdysone drives key developmental processes and behaviors that occur in a temporally-controlled sequence and prepare the animal to undergo metamorphosis. Accurately staging Drosophila larvae within the final third instar is critical due to the rapid developmental progress at this stage, but it is challenging because the rate of development varies widely across a population of animals even if eggs are laid within a short period of time. Moreover, many methods to stage third instar larvae are cumbersome, and inherent variability in the rate of development confounds some of these approaches. Here we demonstrate the usefulness of the Sgs3-GFP transgene, a fusion of the Salivary gland secretion 3 (Sgs3) and GFP proteins, for staging third instar larvae. Sgs3-GFP is expressed in the salivary glands in an ecdysone-dependent manner from the midpoint of the third instar, and its expression pattern changes reproducibly as larvae progress through the third instar. We show that Sgs3-GFP can easily be incorporated into experiments, that it allows collection of developmentally-equivalent individuals from a mixed population of larvae, and that its use enables precise assessment of changing levels of hormones, metabolites, and gene expression during the second half of the third instar.

Proteomic characterization of the different stages of seed germination in Cupressus gigantea

Abstract Seed germination is a pivotal period of plant growth and development. This process can be divided into four major stages, swelling absorption, seed coat dehiscence, radicle emergence and radicle elongation. Cupressus gigantea, a tree native to Tibet, China, is characterized by its resistance to stresses such as cold, and drought, and has a high economic and ecological value. Nevertheless, given its unique geographic location, its seeds are difficult to germinate. Therefore, it is crucial to explore the mechanisms involved in seed germination in this species to improve the germination efficiency of its seeds, thereby protecting this high-quality resource. Here, our findings indicate that seed germination was enhanced when exposed to a 6-h/8-h light/dark photoperiod, coupled with a temperature of 20°C. Furthermore, the application of exogenous GA3 (1 mg/ml, about 2.9 mM) stimulated the germination of C. gigantea seeds. Subsequently, proteomics was used to detect changes in protein expression during the four stages of seed germination. We identified 34 differentially expressed proteins (DEPs), including 13 at the radicle pre-emergence stage, and 17 at the radicle elongation stage. These DEPs were classified into eight functional groups, cytoskeletal proteins, energy metabolism, membrane transport, stress response, molecular chaperones, amino acid metabolism, antioxidant system and ABA signalling pathway. Most of them were found to be closely associated with amino acid metabolism. Combined, these findings indicate that, along with temperature and light, exogenous GA3 can increase the germination efficiency of C. gigantea seeds. Our study also offers insights into the changes in protein expression patterns in C. gigantea seeds during germination.

Characterization of HCN channel subtypes at hypoglossal motoneurons throughout postnatal maturation in mice

The inspiratory phase of breathing includes a stiffening of the upper airway to decrease airway resistance and is mediated by excitatory motor output from hypoglossal motoneurons (XII MNs). This excitation decreases from wakefulness to sleep. This decrease in excitation is in large part due to increasing acetylcholine levels, which activate muscarinic acetylcholine receptors (mAChRs) that have a net inhibitory effect in adult preparations. By contrast, activation of mAChRs in neonatal rodent preparations has a net effect of potentiating inspiratory burst amplitude. Therefore, characterizing changes in muscarinic effects at XII MNs across postnatal maturation will help elucidate the mechanism underlying the shift from muscarinic excitation to inhibition. mAChRs modulate several effector ion channels including the hyperpolarization activated cyclic nucleotide gated (HCN) channel. HCN channels give rise to I h, a mixed cation current activated at hyperpolarized membrane potentials. I h additionally shows upregulation at XII MNs with postnatal maturation. Since there are four HCN subtypes (HCN1-4), the reported changes in I h may be caused by changes in HCN channel subtype levels. HCN1 and HCN2 are most prominent in adult XII MNs, whereas the expression of HCN1-4 in neonates is unknown. Thus, the purpose of this research is to characterize changes in HCN gene and channel expression levels at XII MNs across postnatal maturation. We hypothesis that both HCN1 and HCN2 subtypes will increase with postnatal maturation, and that HCN2 has higher levels than HCN1. To test this hypothesis, we used neuroanatomical techniques to evaluate changes in expression patterns of the four HCN proteins at the XII nuclei across postnatal maturation in CD-1 mice. We performed double-labeled immunofluorescence experiments against HCN1 and HCN2 and co-labeled with choline acetyltransferase (ChAT) on 20μm transverse brainstem slices across six postnatal age groups under identical conditions: P0-P1, P4-P6, P9-P10, P12-P14, P17-P19, and adult. Images of the XII nuclei were collected using a confocal microscope. ImageJ was used to determine the average XII MN HCN subtype intensity across postnatal maturation. Preliminary data suggest that HCN1 (n=2) channels undergo a decrease in expression at P17-19 before a final increase in adulthood (P0-1= 97-100%, P4-6= 92-100%, P9-10= 85-95%, P12-14= 77-97%, P17-19= 61-67%, adult= 71-82%). By contrast, preliminary data suggest that HCN2 (n=2) expression intensity remained relatively consistent with a decrease in labeling intensity at P4-6 (P0-1= 97-100%, P4-6=62-84%, P9-10= 45-95%, P12-14= 67-90%, P17-19= 50-93%, adult= 75-100%). These modest changes in labeling intensity of HCN channel subtypes may not contribute to the observed shift of mAChR modulation at XII MNs from excitation to inhibition with postnatal maturation. Biomedical Sciences, College of Graduate Studies; Department of Physiology, College of Graduate Studies, Midwestern University; NIH/NHLBI Funding: R15HL148870. 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.

Effect of metabolically divergent pig breeds and tissues on mesenchymal stem cell expression patterns during adipogenesis

BackgroundUnraveling the intricate and tightly regulated process of adipogenesis, involving coordinated activation of transcription factors and signaling pathways, is essential for addressing obesity and related metabolic disorders. The molecular pathways recruited by mesenchymal stem cells (MSCs) during adipogenesis are also dependent on the different sources of the cells and genetic backgrounds of donors, which contribute to the functional heterogeneity of the stem cells and consequently affect the developmental features and fate of the cells.MethodsIn this study, the alteration of transcripts during differentiation of synovial mesenchymal stem cells (SMSCs) derived from fibrous synovium (FS) and adipose synovial tissue (FP) of two pig breeds differing in growth performance (German Landrace (DL)) and fat deposition (Angeln Saddleback (AS)) was investigated. SMSCs from both tissues and breeds were stimulated to differentiate into adipocytes in vitro and sampled at four time points (day 1, day 4, day 7 and day 14) to obtain transcriptomic data.ResultsWe observed numerous signaling pathways related to the cell cycle, cell division, cell migration, or cell proliferation during early stages of adipogenesis. As the differentiation process progresses, cells begin to accumulate intracellular lipid droplets and changes in gene expression patterns in particular of adipocyte-specific markers occur. PI3K-Akt signaling and metabolic pathways changed most during adipogenesis, while p53 signaling and ferroptosis were affected late in adipogenesis. When comparing MSCs from FS and FP, only a limited number of differentially expressed genes (DEGs) and enriched signaling pathways were identified. Metabolic pathways, including fat, energy or amino acid metabolism, were highly enriched in the AS breed SMSCs compared to those of the DL breed, especially at day 7 of adipogenesis, suggesting retention of the characteristic metabolic features of their original source, demonstrating donor memory in culture. In contrast, the DL SMSCs were more enriched in immune signaling pathways.ConclusionsOur study has provided important insights into the dynamics of adipogenesis and revealed metabolic shifts in SMSCs associated with different cell sources and genetic backgrounds of donors. This emphasises the critical role of metabolic and genetic factors as important indications and criteria for donor stem cell selection.

Single-cell RNA sequencing reveals that an imbalance in monocyte subsets rather than changes in gene expression patterns is a feature of postmenopausal osteoporosis.

The role of monocytes in postmenopausal osteoporosis is widely recognized; however, the mechanisms underlying monocyte reprogramming remain unknown. In this study, single-cell RNA sequencing (scRNA-seq) was conducted on CD14+ bone marrow monocytes obtained from three postmenopausal women with normal bone mineral density (BMD) and three women with postmenopausal osteoporosis (PMOP). Monocle2 was used to classify the monocytes into 7 distinct clusters. The proportion of Cluster 1 significantly decreased in PMOP patients, while the proportion of Cluster 7 increased. Further analysis via GSEA, transcription factor activity analysis, and sc-metabolic analysis revealed significant differences between Clusters 1 and 7. Cluster 7 exhibited upregulated pathways associated with inflammation, immunity, and osteoclast differentiation, whereas Cluster 1 demonstrated the opposite results. Monocle2, TSCAN, VECTOR and scVelo data indicated that Cluster 1 represented the initial subset and that Cluster 7 represents one of the terminal subsets. BayesPrism and ssGSEA were employed to analyze the bulk transcriptome data obtained from the GEO database. The observed alterations in the proportions of Clusters 1 and 7 were validated and found to have diagnostic significance. CD16 serves as the marker gene for Cluster 7, thus leading to an increased proportion of CD16+ monocytes in women with PMOP. Flow cytometry was used to assess the consistency of outcomes with those of the bioinformatic analysis. Subsequently, an additional scRNA-seq analysis was conducted on bone marrow mononuclear cells obtained from three patients with PMOP and three postmenopausal women with normal BMD. The differential proportions of Cluster 1 and Cluster 7 were once again confirmed, with the pathological effect of Cluster 7 may attribute to cell-cell communication. The scRNA-seq findings suggest that an imbalance in monocyte subsets is a characteristic feature of PMOP. These findings elucidate the limitations of utilizing bulk transcriptome data for detecting alterations in monocytes, which may influence novel research inquiries.

Impact of ABCB1 and ABCG2 Transporter in Outcome of Gallbladder Cancer

BackgroundGallbladder cancer (GBC) is a biologically aggressive malignancy requiring appropriate biomarkers to improve its outcome. Role of ABC transporters (ABCB1 and ABCG2) has been linked to cancer aggressiveness, tumorigenesis and multidrug resistance. Herein, we studied the prognostic implication of ABCB1 and ABCG2 in GBC. MethodsFresh tissue (tumour & normal) samples collected from 54 patients who underwent R0 resection, were analysed for mRNA and protein expression of ABCB1 and ABCG2 by quantitative Real-Time PCR and western blotting respectively in this prospective study. The molecular findings were correlated with clinical-pathological parameters using χ2 and fisher exact test. The molecular changes in ABCB1 and ABCG2 was analysed for predicting overall survival (OS), disease free survival (DFS) and response to chemotherapy using Kaplan Meier log rank test and Cox regression multivariate analysis. ResultsThe mean age of the cohort was 50 ±13.2 with 26 (48.1%) patients had early stage gallbladder cancer (GBC). Overexpression of ABCB1 and ABCG2 was noted in 32/54(59%) and 40/54 (74%) cases respectively. The protein expression of ABCB1(P-glycoprotein) and ABCG2 (BCRP) was higher in 27/54 (50%) and 37/54 (59%) cases respectively. The mean OS and DFS was 20.7 ±11.5 and 19.3 ± 12.2 months at median follow up 24 months. The TNM stage, lymph node metastasis, and presence of gallstone were significant factors for predicting OS and DFS on multivariate analysis. Both ABCB1 and ABCG2 did not show any significant correlation with OS and DFS with similar incidence of late death and recurrence among over-expression and down-expression. Sub- group comparison suggests that change in expression pattern of ABCB1 and ABCG2 may not affect response to chemotherapy in GBC. ConclusionAltered expression of ABCB1 and ABCG2 may not be a useful prognostic marker for survival or response to chemotherapy in GBC. Presently, histo-pathological characteristics and associated gallstones are the important predictors for survival and recurrence in GBC.

Research Progress in Skin Aging and Immunity.

Skin aging is a complex process involving structural and functional changes and is characterized by a decrease in collagen content, reduced skin thickness, dryness, and the formation of wrinkles. This process is underpinned by multiple mechanisms including the free radical theory, inflammation theory, photoaging theory, and metabolic theory. The skin immune system, an indispensable part of the body's defense mechanism, comprises macrophages, lymphocytes, dendritic cells, and mast cells. These cells play a pivotal role in maintaining skin homeostasis and responding to injury or infection. As age advances, along with various internal and external environmental stimuli, skin immune cells may undergo senescence or accelerated aging, characterized by reduced cell division capability, increased mortality, changes in gene expression patterns and signaling pathways, and altered immune cell functions. These changes collectively impact the overall function of the immune system. This review summarizes the relationship between skin aging and immunity and explores the characteristics of skin aging, the composition and function of the skin immune system, the aging of immune cells, and the effects of these cells on immune function and skin aging. Immune dysfunction plays a significant role in skin aging, suggesting that immunoregulation may become one of the important strategies for the prevention and treatment of skin aging.

H3.1/3.2 regulate the initial progression of the gene expression program.

In mice, transcription from the zygotic genome is initiated at the mid-one-cell stage, and occurs promiscuously in many areas of the genome, including intergenic regions. Regulated transcription from selected genes is established during the two-cell stage. This dramatic change in the gene expression pattern marks the initiation of the gene expression program and is essential for early development. We investigated the involvement of the histone variants H3.1/3.2 in the regulation of changes in gene expression pattern during the two-cell stage. Immunocytochemistry analysis showed low nuclear deposition of H3.1/3.2 in the one-cell stage, followed by a rapid increase in the late two-cell stage. Where chromatin structure is normally closed between the one- and two-cell stages, it remained open until the late two-cell stage when H3.1/3.2 were knocked down by small interfering RNA. Hi-C analysis showed that the formation of the topologicallyassociating domain was disrupted in H3.1/3.2 knockdown (KD) embryos. Promiscuous transcription was also maintained in the late two-cell stage in H3.1/3.2 KD embryos. These results demonstrate that H3.1/3.2 are involved in the initial process of the gene expression program after fertilization, through the formation of a closed chromatin structure to execute regulated gene expression during the two-cell stage.

The effect of morphine administration on GluN3B NMDA receptor subunit mRNA expression in rat brain.

Opioid addiction is critically dependent on the activation of N‑methyl‑D‑aspartate (NMDA) receptors, which are widely found in the mesocorticolimbic system. Meanwhile, opioid addiction may affect the expression level of NMDA receptor subunits. The existence of GluN3 subunits in the NMDA receptor's tetramer structure reduces the excitatory current of the receptor channel. We evaluated the changes in the mRNA expression pattern of the GluN3B subunit of the NMDA receptor in rat brains following acute and chronic exposure to morphine. Chronic, escalating intraperitoneal doses of morphine or saline were administered twice daily to male Wistar rats for six days. Two other groups were injected with a single acute dose of morphine or saline. The mRNA level of the GluN3B subunit of the NMDA receptor in the striatum, hippocampus, and nucleus accumbens (NAc) was measured by real‑time PCR. mRNA expression of the GluN3B subunit was considerably augmented (3.15 fold) in the NAc of animals chronically treated with morphine compared to the control group. The difference between rats that were chronically administered morphine and control rats was not statistically significant for other evaluated brain areas. In rats acutely treated with morphine, no significant differences were found for GluN3B subunit expression in the examined brain regions compared to the control group. It was concluded that chronic exposure to morphine notably increased the GluN3B subunit of the NMDA receptor in NAc. The extent of the impact of this finding on opioid addiction and its features requires further evaluation in future studies.

Inflammatory response in dairy cows caused by heat stress and biological mechanisms for maintaining homeostasis.

Climate change increases global temperatures, which is lethal to both livestock and humans. Heat stress is known as one of the various livestock stresses, and dairy cows react sensitively to high-temperature stress. We aimed to better understand the effects of heat stress on the health of dairy cows and observing biological changes. Individual cows were divided into normal (21-22 °C, 50-60% humidity) and high temperature (31-32 °C, 80-95% humidity), respectively, for 7-days. We performed metabolomic and transcriptome analyses of the blood and gut microbiomes of feces. In the high-temperature group, nine metabolites including linoleic acid and fructose were downregulated, and 154 upregulated and 72 downregulated DEGs (Differentially Expressed Genes) were identified, and eighteen microbes including Intestinimonas and Pseudoflavonifractor in genus level were significantly different from normal group. Linoleic acid and fructose have confirmed that associated with various stresses, and functional analysis of DEG and microorganisms showing significant differences confirmed that high-temperature stress is related to the inflammatory response, immune system, cellular energy mechanism, and microbial butyrate production. These biological changes were likely to withstand high-temperature stress. Immune and inflammatory responses are known to be induced by heat stress, which has been identified to maintain homeostasis through modulation at metabolome, transcriptome and microbiome levels. In these findings, heat stress condition can trigger alteration of immune system and cellular energy metabolism, which is shown as reduced metabolites, pathway enrichment and differential microbes. As results of this study did not include direct phenotypic data, we believe that additional validation is required in the future. In conclusion, high-temperature stress contributed to the reduction of metabolites, changes in gene expression patterns and composition of gut microbiota, which are thought to support dairy cows in withstanding high-temperature stress via modulating immune-related genes, and cellular energy metabolism to maintain homeostasis.

Kidney toxicity and transcriptome analyses of male ICR mice acutely exposed to the mushroom toxin α-amanitin

Amatoxins are responsible for most fatal mushroom poisoning cases, as it causes both hepatotoxicity and nephrotoxicity. However, studies on amatoxin nephrotoxicity are limited. Here, we investigated nephrotoxicity over 4 days and nephrotoxicity/hepatotoxicity over 14 days in mice. The organ weight ratio, serological indices, and tissue histology results indicated that a nephrotoxicity mouse model was established with two stages: (1) no apparent effects within 24 h; and (2) the appearance of adverse effects, with gradual worsening within 2–14 days. For each stage, the kidney transcriptome revealed patterns of differential mRNA expression and significant pathway changes, and Western blot analysis verified the expression of key proteins. Amanitin-induced nephrotoxicity was directly related to RNA polymerase II because mRNA levels decreased, RNA polymerase II-related pathways were significantly enriched at the transcription level, and RNA polymerase II protein was degraded in the early poisoning stage. In the late stage, nephrotoxicity was more severe than hepatotoxicity. This is likely associated with inflammation because inflammation-related pathways were significantly enriched and NF-κB activation was increased in the kidney.

Abstract 5634: Tristetraprolin induces an antitumorigenic phenotype in triple negative breast cancer via a novel non-canonical mechanism

Abstract The ultimate cause of death for most triple negative breast cancer (TNBC) patients is metastatic disease, which has a five-year survival rate of 12.8%. This low survival rate is partly due to the lack of targeted treatments for metastatic TNBC, resulting from the heterogeneity of the disease. As such, further research is needed to identify novel molecular mechanisms that can be targeted to suppress TNBC malignancy. Tristetraprolin (TTP) is an RNA-binding protein that binds to AU-rich elements (AREs) in the 3’ UTRs of select mRNAs, including many that encode proteins involved in cancer-related processes, and targets these mRNAs for degradation. Loss of TTP in tumors correlates with increased disease severity and decreased survival, suggesting a role as a tumor suppressor in multiple cancers, including breast cancer. While previous research has shown that TTP can suppress proliferation in breast cancer, the mechanism remains weakly defined. To elucidate how TTP impacts tumorigenic phenotypes in advanced breast cancer, we stably transfected a FLAG-TTP-expressing construct into three aggressive and metastatic TNBC cell lines. We analyzed TTP-induced changes in gene expression patterns among these cell lines using RNA-sequencing and observed that TTP alters the expression levels of many genes. Gene Set Enrichment Analysis (GSEA) of our datasets revealed that TTP affects multiple pathways, including significant suppression of pathways that promote cell growth, various metabolic processes contributing to tumor growth, and the response to hypoxia. As these pathways highly associated with tumorigenesis, we hypothesized that TTP would attenuate multiple tumor phenotypes. Our subsequent functional analyses revealed that TTP potently suppressed four tumor phenotypes in these cell models: cell proliferation, stem cell frequency, migration, and invasiveness. We hypothesized that inhibition of proliferation was mediated by cell cycle alterations, however, flow cytometry analysis revealed no substantial cell cycle perturbations between TTP-expressing cells and controls. Further analysis of cell cycle regulators revealed minimal changes in expression of proteins encoded by known or suspected TTP target mRNAs. Actinomycin D assays surprisingly revealed that TTP had no effect on the decay kinetics of several known TTP targets, suggesting that TTP’s antitumorigenic properties observed in TNBC cells are independent of its RNA-destabilizing function. To confirm this finding, we constructed cell models expressing an RNA-binding defective mutant of TTP, C147R, which recapitulated suppression of the four tumorigenic phenotypes seen with the wild-type protein. Collectively, these findings reveal that expression of TTP can potently suppress several oncogenic phenotypes in TNBC cells via a mechanism independent of its canonical RNA-binding/destabilizing functions. Citation Format: Julia L. Rutherford, Megan B. Stemberger, Rohaan Mahmud, Christina R. Ross, Elizabeth J. White, Gerald M. Wilson. Tristetraprolin induces an antitumorigenic phenotype in triple negative breast cancer via a novel non-canonical mechanism [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 5634.

Abstract 5599: Role of HOX gene expression in differentiation of colon cancer stem cells

Abstract HOX genes encode a highly conserved set of transcription factors critical for stem cell (SC) function and embryonic development. Several HOX genes have also been implicated in a myriad of cancers, including colorectal cancer (CRC). However, how HOX genes play a role in SC regulation and contributes to cancer development is unclear. HOX expression is regulated by WNT, FGF, and retinoic acid (RA) signaling - pathways which also become dysregulated in cancer. Goal: To determine how regulation of HOX genes specifies differentiation of colonic SCs into specialized cell lineages and how dysregulation of HOX genes contributes to CRC development. Hypotheses: i) Each of the different cell types within human colonic epithelium has a distinct HOX gene expression signature; ii) All-trans retinoic acid (ATRA) induced differentiation of CRC cells changes HOX expression that correlates with specific HOX expression signatures in normal colon and CRC cells. Results: Immunostaining shows that specific HOX proteins (e.g., HOXA9 & HOXC9) are selectively expressed in ALDH-positive SCs and expression of these HOX genes is increased in CRC tissues compared to normal epithelium. Bioinformatics analysis predicted that these HOX genes (plus HOXA4, HOXA10, HOXC8) are regulated by RA signaling. Accordingly, we studied how ATRA influences HOX expression in CRC cells (HT29 & SW480). We found ATRA treatment: 1) Decreases proliferation and increases neuroendocrine cell (NEC) differentiation; and 2). Leads to specific changes in HOX gene expression patterns. Using NanoString profiling, we also discovered that LGR5+ SCs, ALDH+ SCs and GLP2R+ NECs have unique HOX signatures. By defining how these HOX gene expression changes in response to ATRA treatment in comparison to the HOX gene signatures of different colonic cell lineages will help us understand the role that HOX genes play in SC differentiation and how dysregulation of HOX gene expression contributes to CRC development. Citation Format: Brian T. Osmond, Caroline O. Facey, Lynn O. Opdenaker, Chi Zhang, Bruce M. Boman. Role of HOX gene expression in differentiation of colon cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5599.