Changes In Expression Research Articles

Advances in the Regulation of Periostin for Osteoarthritic Cartilage Repair Applications

Emerging evidence indicates periostin (POSTN) is upregulated in patients with OA, and studies have shown that it can induce the activation of inflammatory cytokines and catabolic enzymes, making it a potential therapeutic target. Link N (LN) is a peptide fragment derived from the link protein and has been demonstrated as an anabolic-like factor and anti-catabolic and anti-inflammatory factors both in vitro and in vivo. This study aims to determine if LN can regulate POSTN expression and function in OA cartilage. Articular cartilage was recovered from donors undergoing total knee replacements to isolate chondrocytes and prepare osteochondral explants. Cells and explants were treated with POSTN and LN (1 and 100 μg) and measured for changes in POSTN expression and various matrix proteins, catabolic and proinflammatory factors, and signaling. To determine the effects of POSTN expression in vivo, a rabbit OA model was used. Immunoprecipitation and in silico modeling were used to determine peptide/POSTN interactions. Western blotting, PCR, and immunohistochemistry demonstrated that LN decreased POSTN expression both in vitro and in vivo. LN was also able to directly inhibit POSTN signaling in OA chondrocytes. In silico docking suggested the direct interaction of LN with POSTN at residues responsible for its oligomerization. Immunoprecipitation experiments confirmed the direct interaction of LN with POSTN and the destabilization of its oligomerization. This study demonstrates the ability of a peptide, LN, to suppress the overexpression and function of POSTN in OA cartilage.

Abstract A035: Infection of primary mammary fibroblasts with cytomegalovirus coordinately increases autotaxin-lysophosphatidate-inflammatory signaling, which could increase breast cancer metastasis

Abstract Background: Metastatic breast cancer is the most prevalent cause of mortality by cancer-related deaths in women. Our group showed that the 63% of Canadian breast cancer patients who were infected with CMV were 5.6-times more likely to develop Stage IV cancers with reduced relapse-free survival. Also, the metastatic phenotype of breast tumors and the number and sizes of lung metastases were increased in mice with latent infections. Fibroblasts are important sites of CMV infection. Cancer-associated fibroblasts (CAFs) are also major sites of autotaxin (ATX) secretion in breast tumors, whereas ATX production and CMV infection in breast cancer cells is negligible. ATX produces lysophosphatidate (LPA), which increases inflammatory cytokine production that stimulates further ATX secretion in a feedforward cycle. LPA promotes breast tumor growth and metastasis by signaling through six G protein-coupled receptors. Signaling is terminated by lipid phosphate phosphatases (LPPs)-1 and -3, which degrade LPA and are decreased in breast tumors. Hypothesis: Breast cancer metastasis could be increased by active CMV infection of CAFs through increased inflammation and LPA signaling. Results: Quiescent fibroblasts isolated and cultured from mammary tissue of non-tumor bearing mice (n>3) produced negligible quantities of ATX. LPA signaling was coordinately promoted in these primary fibroblasts infected for 48 h with 1 MOI of mouse CMV (n=3) with a 5-fold increase in mRNA for ATX (p=0.008), increased mRNAs for LPA2,3,4 receptors (p<0.05) and decreased mRNA expressions for LPP1 and LPP3 (p<0.05). An increase in the secretion of granulocyte monocyte-colony stimulating factor of 548 pg/ml (p=0.01) and Interleukin-6 by 8307 pg/ml (p=0.03) was observed post infection (n=5), thus creating a pro-inflammatory environment. Increased mRNA expression of fibroblast markers, including alpha-smooth muscle actin (p=0.005), fibroblast-associated protein (p=0.007), epidermal growth factor receptor (EGFR) (p=0.001) and transforming growth factor- ß receptor 1 (TGF-ßR1) (p=0.03) was also observed post-infection (n=3). The results were analyzed by student’s t-test. Assessment of these factors in breast cancer patients (n=1100) using the Tumor Immune Estimation Resource Analysis showed a positive correlation in the expression of EGFR and TGF-ßR1 with the LPA3,4 receptors (p<0.05). Conclusion: The changes in expression of ATX, LPA receptors and LPPs in CMV- infected fibroblasts are consistent with increased metastasis. Our results show that mCMV infection of primary mammary fibroblasts dramatically reprograms them to express CAF-like properties and promotes ATX-LPA-inflammatory signaling. CAFs are a major constituent of the tumor stroma. They promote tumor progression by secreting factors such as ATX, which increases metastasis, fibrosis and favors immunosuppression. Our results support the use of pharmacological agents to block signaling by the ATX-LPA-inflammatory cycle to treat breast and other cancers, especially for the majority of patients who are infected with CMV. Citation Format: Humayara Khan, Xiaoyun Tang, Frank Wuest, David N. Brindley, Denise G. Hemmings. Infection of primary mammary fibroblasts with cytomegalovirus coordinately increases autotaxin-lysophosphatidate-inflammatory signaling, which could increase breast cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A035.

Abstract PR008: Cross-talk between metastatic cells and host systems: Neutrophil metabolic adaptation, immune profiling, and systemic metabolic shifts in tumor progression and cachexia

Abstract Oral squamous cell carcinoma (OSCC) is a prevalent type of head and neck cancer with a high mortality rate, partly due to late diagnosis often accompanied by metastasis. Cachexia, a complex syndrome marked by severe weight loss and muscle wasting, frequently accompanies advanced cancers including OSCC. Notably, 50-70% of OSCC patients exhibit cachexia at diagnosis, which persists post-treatment, hinting at a metastatic contribution to this syndrome. One significant challenge in studying cancer cachexia is the lack of well-characterized pre-clinical models that accurately reflect human disease. Traditional mouse models often involve subcutaneously transplanted cells, which fail to mimic the natural tumor-host interactions and metastatic progression seen in patients. These models do not typically exhibit metastasis and differ significantly from the genetic and molecular landscape of human cancers. To address these limitations, we developed a syngeneic mouse model by orthotopically transplanting MOC2 murine OSCC cells into the tongues of B6J mice, thereby closely mimicking the tumor microenvironment and metastatic behavior of human OSCC. By enhancing metastasis with a palm-enriched high-fat diet, we observed progressive tumor growth, metastasis, and body weight loss, independent of food intake reduction, indicating metabolic dysregulation rather than anorexia. Comprehensive analyses, including single-cell RNA sequencing (scRNAseq) of immune cells and transcriptomic and metabolomic profiling of tumor and host tissues, identified significant gene expression changes associated with metastasis and cachexia. Enhanced metastasis correlated with lipid metabolism changes in lung metastases, suggesting that metastatic cells exploit lipids for growth. Further, immune profiling revealed neutrophils' metabolic adaptation in metastasis, with implications for both tumor progression and cachexia. Targeting key metabolic and inflammatory pathways in tumor and immune cells holds potential for mitigating metastasis and cachexia. Early-stage serum metabolomics indicated systemic metabolic shifts, emphasizing the need to understand the interplay between tumor metastasis and host metabolism. These findings underscore the complex mechanisms underlying OSCC-induced cachexia, and the need for new targeted therapeutic strategies to disrupt this lethal synergy between cancer progression and metabolic deterioration of the host. Citation Format: Blanca Majem, Josefina Martin, Liudmila Shevkova, Claudia Bigas, Gloria Pascual, Salvador Aznar-Benitah. Cross-talk between metastatic cells and host systems: Neutrophil metabolic adaptation, immune profiling, and systemic metabolic shifts in tumor progression and cachexia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR008.

Association of NOX5 Expression with Sperm Activity and Motility in Pathospermic Infertile Men

Background: The newest NOX isoform, NOX5, has been found in mammalian spermatozoa. Many physiological and pathological situations in spermatozoa are mediated by reactive oxygen species (ROS). NOX5 is the main source of ROS in spermatozoa. Our purpose was to investigate the changes in NOX5 expression and the effect of NOX5 expression on sperm motility, chromatin integrity, and oxidative status in oligoasthenozoospermic compared to normozoospermic men. Methods: Semen samples were collected from 30 normozoospermic (NS) and 30 oligoasthenozoospermic (OAS) men. NOX5 protein expression in sperm samples was evaluated by immunohistochemistry and western blot. Oxidative stress status was evaluated by total antioxidant capacity (TAC), total oxidant capacity (TOC), and oxidative stress index (OSI) parameters. Chromatin integrity in spermatozoa was evaluated by toluidine blue staining. Results: NOX5 expression levels were significantly higher in OAS group than in NS group (p<0.001). In addition, chromatin integrity was significantly higher in the OAS group in comparison to NS group (p<0.001). TAC levels were higher in the NS group, but OSI and TOC levels were significantly higher in OAS group (p<0.001). It was found that NOX5 protein expression was positively correlated with oxidative stress and chromatin integrity and negatively correlated with motility (p<0.01). Conclusion: These results suggest that overexpression of NOX5 may be the source of excessive ROS production and oxidative stress injuries in oligoasthenozoospermic men. Considering that NOX5 expression is positively correlated with oxidative stress and chromatin integrity but negatively correlated with motility, it can be considered a biomarker to be used in assisted reproductive procedures.

Abstract C007: Identifying the role of Interleukin-17 signaling in intestinal stem cells during pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC), the most aggressive type of pancreatic cancer, is predicted to become the second leading cause of cancer related deaths by 2030. Interleukin 17 (IL-17) signaling can promote tumor growth. In pancreatic cancer, IL-17 signaling in cancer cells is capable of triggering the formation of neutrophil extracellular traps (NETs) which is detrimental to the anti-tumor effect of immunotherapy. IL-17 signaling has been shown to have critical roles in maintaining gut homeostasis. Intestinal epithelial cells directly interact with the gut microbiome and are actively involved in infection control, injury repair, immune responses, and the production of antimicrobial peptides and cytokines, and need to be constantly replenished by intestinal stem cells (ISCs). Our lab’s study has previously shown that deletion of Interleukin 17 receptor A (IL-17RA) in the entire gut epithelium induces pro-tumorigenic IL-17 signaling due to the disruption of gut homeostasis, suggesting that immune responses induced by gut IL-17/IL-17RA signaling can affect the behavior of distant tumors. However, the mechanism of IL-17 signaling within specific gut epithelial compartments such as ISCs, enterocytes, secretory cells, etc. and their role in maintaining gut homeostasis and cancer risk are not well-studied until now. Thus, we aimed to identify the importance of IL-17 signaling in ISCs during pancreatic cancer. We hypothesized that disruption of IL-17/IL-17RA signaling within ISCs impacts tumor growth. Our preliminary data shows that IL-17 signaling in enterocytes maintains intestinal homeostasis and disruption of this axis triggers systemic increase in B cells that promotes tumor growth by increasing IL-17 levels in circulation. To identify the function of IL-17/IL-17RA signaling in ISCs, we utilized Leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5), a lineage marker for ISCs, to drive Cre enzyme tissue specific expression to specifically knockout Il17ra in gut ISCs - an inducible transgenic mouse model Il17ra fl/fl ;Lgr5- EGFP-CreERT2 (cKO). We used multiplex staining and imaging to validate the cell type specific loss of IL-17RA expression in tamoxifen treated cKO mice. We used implantable pancreatic tumor models and performed immunoprofiling using flow cytometry. We found that the pancreatic tumor growth was altered by the remote signal from gut ISCs in cKO mice. Furthermore, we performed RNAseq in both tumor and gut tissue which identified that the major changes in gene expression were associated with both innate and adaptive immune pathways, especially B cell activation. We conclude that the IL-17/IL-17RA signaling in gut ISCs changes intestinal immune responses locally that can affect distant tumor immunity. Citation Format: Haoyue Liu, Vidhi Chandra, Le Li, Olivereen Le Roux, Virginia Tahan, Florencia McAllister. Identifying the role of Interleukin-17 signaling in intestinal stem cells during pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C007.

Time-course RNA sequencing reveals high similarity in mRNAome between hepatic stellate cells activated by agalactosyl IgG and TGF-β1.

Previous studies have demonstrated the clinical relevance of aberrant serum immunoglobulin G (IgG) N-glycomic profiles in liver fibrosis and the pathogenic effects of agalactosyl IgG on activating hepatic stellate cells (HSCs). However, the dynamics of gene expression changes during HSC activation by agalactosyl IgG remain poorly understood. We performed RNA sequencing to analyze the mRNAome of human LX-2 HSCs at multiple time points after treatment with agalactosyl IgG and then compared these results with those obtained after normal IgG and transforming growth factor (TGF)-β1 treatments. Gene expression changes were significantly pronounced on day 5 and subsided by day 11 after HSC activation. A high degree of similarity in gene expression patterns between HSCs treated with agalactosyl IgG and TGF-β1 was observed, of which 1796 and 1785 differentially expressed genes (DEGs) were identified, respectively. Disease ontology analyses revealed that 114 and 105 DEGs in activated HSCs following agalactosyl IgG and TGF-β1 treatments, respectively, were linked to liver cirrhosis, hepatitis, fatty liver disease, hepatitis B, and alcoholic hepatitis, with CCL5 and FAS being the most commonly affected genes. DEGs associated with liver fibrosis or aforementioned liver diseases involved in gene annotation, physiological functions, and signaling pathways regarding secretion of cytokines and chemokines, expression of fibrosis-related growth factors and their receptors, modification of extracellular matrices, and regulation of cell viability in activated HSCs. In conclusion, this study characterized the dynamics of mRNAome and gene networks and identified the liver fibrosis-related DEGs during HSC activation by agalactosyl IgG and TGF-β1.

Central responses to peripheral inflammation may include decreased expression of key apoptotic protease caspase-3 in brainstem

Microglia activation by proinflammatory stimuli, including lipopolysaccharide (LPS), is considered among the risk factors for neurodegeneration, but the LPS treatment may also have a neuroprotective effect, which leads to further analysis of the relationship between microglial activation and regulators of cell death. In the present work, a comparative study was carried out on proteins expression of marker for activated microglia Iba-1 and the apoptotic executor protease caspase-3 in the brainstem and prefrontal cortex of rats injected intraperitoneally with endotoxin at different doses and schedules. One day after LPS at a dose of 0.5 mg/kg, single, the Iba-1 and caspase-3 expression in both structures did not differ from control values. Endotoxin administration fourfold at the same dose over 7 days (once every 2 days) led one day after the last injection to a significant increase in the Iba-1 level in the brainstem, which was accompanied by a significant decrease in the expression of caspase-3. The same effects in this structure were observed 7 days after a single injection of LPS at a higher dose of 5 mg/kg. In a 7-day experiment, in contrast to the brainstem, no changes in caspase-3 expression were found in the frontal cortex, and an increase in Iba-1 expression was observed only after a single injection of LPS at a high dose. The detected decrease of caspase-3 level in the brain stem under neuroinflammatory conditions may reflect the development of neuroprotective processes, especially important for the structure responsible for such key body functions as respiration, blood pressure and heartbeat.

Mitochondrial dynamics and metabolic remodeling in xenograft of IPSC-derived human neural precursors

It is well recognized that the regulation of mitochondrial functions affects the differentiation and maturation of neurons. The study of these processes is of both fundamental and practical importance for regenerative neurobiology. Aim of the study: to characterize the mitochondrial fission changes and their relation to the activation of oxidative phosphorylation (metabolic shift) during maturation of human IPSC-derived neural precursors grafted into rat striatum. Wistar rats (n = 15) were unilaterally injected into the caudate nucleus with neural precursors derived from human IPSCs. Changes in localization and expression of neuronal differentiation markers: nestin, NeuN, neuronal enolase, as well as mitochondrial outer membrane protein, ATP synthase and mitochondrial fission protein Drp1 were assessed by immunostaining. Measurements were performed on graft cells 2 weeks, 3 and 6 months after surgery. Maturation of grafted neurons was associated with fluctuations morphometric parameters of the mitochondrial fraction and Drp1 levels. Increased mitochondrial fission was detected 3 months after transplantation, before an increase in ATP synthase staining by 6th month and a switch of transplanted cells to oxidative phosphorylation. The conducted experiment demonstrated a link between mitochondrial dynamics and changes in the metabolic profile and maturation of transplanted neurons. The regulation of mitochondrial dynamics may have future implications for developing methods to improve the integration of transplanted neurons into recepient brain structures.

Exploring the metabolic daylength measurement system: implications for photoperiodic growth.

Photoperiod is an environmental signal that varies predictably across the year. Therefore, the duration of sunlight available for photosynthesis and in turn the ability of plants to accumulate carbon resources also fluctuates across the year. To adapt to these variations in photoperiod, the metabolic daylength measurement (MDLM) system measures the photosynthetic period rather than the absolute photoperiod, translating it into seasonal gene expression changes linked to photoperiodic growth. In this Tansley Insight, we briefly summarize the current understanding of the MDLM system and highlight gaps in our knowledge. Given the system's critical role in seasonal growth, understanding the MDLM system is essential for enhancing plant adaptation to different photoperiods and optimizing agricultural production.

Transcriptional Responses of Lacticaseibacillus rhamnosus to TNFα, IL-6, IL-8, and IL-10 Cytokines

The interaction between gut microbiota and the host immune system is a complex and understudied field, with cytokines like TNFα, IL-6, IL-8, and IL-10 playing pivotal roles. Commensal bacteria, including lactobacilli, respond to these cytokines through adaptive mechanisms that support their survival and function within the gut. While the influence of cytokines on pathogenic bacteria is well documented, their impact on commensal bacteria, particularly lactobacilli, remains underexplored. This study investigates the transcriptional responses of Lacticaseibacillus rhamnosus strains K32 and R19-3 to various cytokines using next-generation RNA sequencing (RNA-seq). Our findings reveal that cytokines, especially IL-8 and IL-10, significantly alter the L. rhamnosus transcriptome, affecting genes involved in carbohydrate metabolism, stress response, and transcriptional regulation. Notably, IL-8 and IL-10 induce a significant downregulation of genes related to the phosphotransferase system, suggesting a reduction in metabolic activity in response to inflammatory signals. This study unveils a previously unexplored aspect of L. rhamnosus adaptation, highlighting its intricate response to cytokine signals. By modulating gene expression, L. rhamnosus may mitigate the adverse effects of inflammation and promote gut health. These insights could inform the development of targeted probiotic therapies for inflammatory bowel disease (IBD) and other conditions with altered cytokine levels. Our results suggest that co-evolution between a host and gut microbiota enables bacteria to respond to specific cytokines through gene expression changes, revealing a unique and underexplored facet of the interaction between commensal bacteria and the host organism.

Biochemical characterization of the human ubiquitous glucose-6-phosphatase in neutrophil granulocytes.

Glucose-6-phosphatase-β (G6PC3) is a ubiquitous phosphatase present in the endoplasmic reticulum, which, unlike G6PC1, is not responsible for maintaining blood glucose level under starvation. Recently, G6PC3 has been shown to play an important role in neutrophil granulocytes, eliminating the toxic metabolite 1,5-anhydroglucitol-6-phosphate. The present study aimed to look for alternative substrates for the enzyme and outline the expression changes in the parts of this multicomponent system during neutrophil granulocyte differentiation. We determined the kinetic characteristics of recombinant human G6PC3 towards different sugar phosphates, and the transport of these compounds was also measured in rat liver microsomes. We found that all investigated sugar phosphates are substrates for G6PC3, although their microsomal transport is much slower than that of glucose-6-phosphate. Using the HL-60 promyelocytic leukemia cell line as an in vitro model system for myeloid differentiation, we found no significant differences in enzyme expression and phosphatase activity latency between undifferentiated and differentiated cells. Our results provide novel insights into the possible role of G6PC3 in the dephosphorylation of alternative sugar phosphates or their metabolites synthesized in the endoplasmic reticulum and confirm the potential feature of the enzyme in the promyelocytic stage as well. These findings contribute to our knowledge of intracellular carbohydrate metabolism of neutrophil granulocytes, which facilitates further research directions to better understand the underlying mechanisms of neutropenias.

HIF-1α knockdown attenuates phenotypic transformation and oxidative stress induced by high salt in human aortic vascular smooth muscle cells.

Increased dietary salt intake is a well-established risk factor for hypertension and related cardiovascular diseases, involving complex vascular remodeling processes. However, the specific role of hypoxia-inducible factor-1α (HIF-1α) in vascular pathophysiology under high-salt conditions remains poorly understood. This study investigates the role of HIF-1α in high-salt-induced vascular remodeling using human aortic vascular smooth muscle cells (HA-VSMCs) cultured in vitro. HA-VSMCs were divided into three groups: high-salt with HIF-1α knockdown (shHIF-1α + HS), negative control (shcontrol), and high-salt (HS). Cell viability, migration, gene expression, and protein levels were evaluated. High-salt conditions significantly increased mRNA expression of α-smooth muscle actin (α-SMA), smooth muscle protein 22 (SM22), angiotensin II type 1 receptor (AT1R), collagen I, and collagen III (p < 0.0001). HIF-1α knockdown partially attenuated these increases, particularly for α-SMA, SM22, and AT1R (p < 0.01). At the protein level, high-salt exposure markedly elevated expression of collagen III, HIF-1α, osteopontin (OPN), and angiotensin II (Ang II) (p < 0.0001). HIF-1α knockdown significantly reduced the high-salt-induced increases in collagen III and HIF-1α protein levels (p < 0.001) but had a limited effect on OPN and Ang II upregulation. Interestingly, SM22 protein expression was significantly decreased under high-salt conditions (p < 0.0001), an effect partially reversed by HIF-1α knockdown (p < 0.0001). These findings demonstrate that high-salt conditions induce complex changes in gene and protein expression in HA-VSMCs, with HIF-1α playing a crucial role in mediating many of these alterations. The study highlights the differential effects of HIF-1α on various markers of vascular remodeling and suggests that HIF-1α may be a potential therapeutic target for mitigating salt-induced vascular pathology. Further research is warranted to elucidate the mechanisms underlying the HIF-1α-dependent and -independent effects observed in this study.

HC-HA/PTX3 from Human Amniotic Membrane Induced Differential Gene Expressions in DRG Neurons: Insights into the Modulation of Pain

Background: The biologics derived from human amniotic membranes (AMs) demonstrate potential pain-inhibitory effects in clinical settings. However, the molecular basis underlying this therapeutic effect remains elusive. HC-HA/PTX3 is a unique water-soluble regenerative matrix that is purified from human AMs. We examined whether HC-HA/PTX3 can modulate the gene networks and transcriptional signatures in the dorsal root ganglia (DRG) neurons transmitting peripheral sensory inputs to the spinal cord. Methods: We conducted bulk RNA-sequencing (RNA-seq) of mouse DRG neurons after treating them with HC-HA/PTX3 (15 µg/mL) for 10 min and 24 h in culture. Differential gene expression analysis was performed using the limma package, and Gene Ontology (GO) and protein–protein interaction (PPI) analyses were conducted to identify the networks of pain-related genes. Western blotting and in vitro calcium imaging were used to examine the protein levels and signaling of pro-opiomelanocortin (POMC) in DRG neurons. Results: Compared to the vehicle-treated group, 24 h treatment with HC-HA/PTX3 induced 2047 differentially expressed genes (DEGs), which were centered on the ATPase activity, receptor–ligand activity, and extracellular matrix pathways. Importantly, PPI analysis revealed that over 50 of these DEGs are closely related to pain and analgesia. Notably, HC-HA/PTX3 increased the expression and signaling pathway of POMC, which may affect opioid analgesia. Conclusions: HC-HA/PTX3 induced profound changes in the gene expression in DRG neurons, centered around various neurochemical mechanisms associated with pain modulation. Our findings suggest that HC-HA/PTX3 may be an important biological active component in human AMs that partly underlies its pain inhibitory effect, presenting a new strategy for pain treatment.

The H3K27me3 histone mark correlates with repression of colour and aroma development post-harvest in strawberry fruit.

Strawberry ripening is non-climacteric, and post-harvest fruit enter senescence and deteriorate rapidly. Chilled storage induces transcriptome wide changes in gene expression, including the down-regulation of aroma related genes. Histone marks are associated with transcriptional activation or repression; the H3K27me3 mark is mainly associated with repression of gene expression. Here genes associated with H3K27me3 were identified through ChIP-seq in ripe red strawberry fruit at harvest and after 5 days of chilled storage in the dark. The number of ChIP peaks increased with storage time, indicating an increased role for this mark in regulation of gene expression following chilled dark storage. Comparing ChIP-seq data to RNA-seq data from the same material identified 440 genes whose expression correlates with H3K27me3 repression. Abiotic stress genes, especially cold stress response genes, were down-regulated during storage. Increased association with the H3K27me3 mark indicates that they may be repressed via this epigenetic mark. Other functional groups included cell wall and carbohydrate metabolism. The association with the H3K27me3 mark of two transcription factors (FaHY5 and FaTRAB1) and FaADH, involved in ester biosynthesis, was validated by ChIP-PCR. These three genes are all down-regulated during storage and indicate a network of H3K27me3 gene repression affecting both anthocyanin and ester biosynthesis.

Single-nucleus transcriptomics reveals disease- and pathology-specific signatures in α-synucleinopathies.

α-synucleinopathies are progressive neurodegenerative disorders characterized by intracellular aggregation of α-synuclein, yet their molecular pathogenesis remains unknow. Here, we explore cell-specific changes in gene expression across different α-synucleinopathies. We perform single-nucleus RNA sequencing on nearly 300,000 nuclei from the prefrontal cortex of individuals with idiopathic Parkinson's disease (iPD, n = 20), Parkinson's disease caused by LRRK2 mutations (LRRK2-PD, n = 7), multiple system atrophy (MSA, n = 6) and healthy controls (n = 13). iPD and LRRK2-PD exhibit a largely overlapping cell type-specific signature, which is distinct from that of MSA, and includes an overall decrease of the transcriptional output in neurons. Notably, most of the differential expression signal in iPD and LRRK2-PD is concentrated in a specific deep cortical neuronal subtype expressing adrenoceptor alpha 2A. While most differentially expressed genes are highly cell type- and disease-specific, PDE10A is found consistently downregulated in most cortical neurons, and across all three diseases. Finally, exploiting the variable presence and/or severity of α-synuclein pathology in LRRK2-PD and iPD, we identify cell type-specific signatures associated with α-synuclein pathology, including a neuronal upregulation of the SNCA gene itself, encoding α-synuclein. Our findings provide novel insights into the cell-specific transcriptional landscape of the α-synucleinopathy spectrum.

Luminal epithelial cells integrate variable responses to aging into stereotypical changes that underlie breast cancer susceptibility.

Effects from aging in single cells are heterogenous, whereas at the organ- and tissue-levels aging phenotypes tend to appear as stereotypical changes. The mammary epithelium is a bilayer of two major phenotypically and functionally distinct cell lineages: luminal epithelial and myoepithelial cells. Mammary luminal epithelia exhibit substantial stereotypical changes with age that merit attention because these cells are the putative cells-of-origin for breast cancers. We hypothesize that effects from aging that impinge upon maintenance of lineage fidelity increase susceptibility to cancer initiation. We generated and analyzed transcriptomes from primary luminal epithelial and myoepithelial cells from younger <30 (y)ears old and older >55y women. In addition to age-dependent directional changes in gene expression, we observed increased transcriptional variance with age that contributed to genome-wide loss of lineage fidelity. Age-dependent variant responses were common to both lineages, whereas directional changes were almost exclusively detected in luminal epithelia and involved altered regulation of chromatin and genome organizers such as SATB1. Epithelial expression of gap junction protein GJB6 increased with age, and modulation of GJB6 expression in heterochronous co-cultures revealed that it provided a communication conduit from myoepithelial cells that drove directional change in luminal cells. Age-dependent luminal transcriptomes comprised a prominent signal that could be detected in bulk tissue during aging and transition into cancers. A machine learning classifier based on luminal-specific aging distinguished normal from cancer tissue and was highly predictive of breast cancer subtype. We speculate that luminal epithelia are the ultimate site of integration of the variant responses to aging in their surrounding tissue, and that their emergent phenotype both endows cells with the ability to become cancer-cells-of-origin and represents a biosensor that presages cancer susceptibility.

Establishment and Application of a New Radiation Biodosimetric Method Based on the Quantitative RPA-SHERLOCK Amplification Technology.

Biodosimetry is a key diagnostic tool for radiation exposure, risk assessment and treatment planning of acute radiation sickness. To effectively respond to a large-scale radiological incident, there is a need for the development of biodosimetric methods with fast, portable, and convenient operating advantages. We employed the recombinase polymerase amplification specific high-sensitivity enzymatic reporter unlocking (RPA-SHERLOCK) technology to establish a method for fast radiation dose assessment by measuring the expression level of radiation-inducible genes. Moreover, we proposed for the first time the principle of quantitative detection of curve slopes based on this method. Using this new method, changes in mRNA expression were confirmed in a number of radiation-sensitive genes (XPC, CDKN1A, and ATM) in human lymphocytes after irradiation. The standard curve of the dose-effect relationship was established, which can be used to quickly determine the exposed dose of the irradiated samples. Compared with traditional detection methods such as RT-qPCR, this method was found to be more convenient, fast and easy to operate. With the same amount of template input as RT-qPCR, the detection time of this method can be shortened to less than 20 min. The detection instrument required by this method is also more portable than a qPCR system.

Activation of Hippocampal Neuronal NADPH Oxidase NOX2 Promotes Depressive-Like Behaviour and Cognition Deficits in Chronic Restraint Stress Mouse Model.

Nicotinamide adenosine dinucleotide phosphate oxidases (NOX) play important roles in mediating stress-induced depression. Three NOX isotypes are expressed mainly in the brain: NOX2, NOX3 and NOX4. In this study, the expression and cellular sources of these NOX isoforms was investigated in the context of stress-induced depression. Chronic restraint stress (CRS)-induced depressive-like behaviour and cognitive deficits were evaluated by tail suspension tests, forced swimming tests and the Morris water maze test. Hippocampal NOX expression was determined by immunofluorescence staining and western blotting. The hippocampal levels of the brain-derived neurotrophic factor (BDNF) mRNA were determined via quantitative real-time -polymerase chain reaction. Glucocorticoid levels in the hippocampus were measured using ELISA kits. In the mouse CRS model, a significant increase in NOX2 expression was observed in the hippocampus, whereas no significant changes in NOX3 and NOX4 expression were detected. Next, NOX2 expression was primarily localised to neurons (NeuN+) but not microglia (Iba-1+) or astrocytes (GFAP+). Treatment with gp91ds-tat, a specific NOX2 inhibitor, effectively mitigated the behavioural deficits induced by CRS. The decreased expression of the BDNF mRNA in the hippocampus of CRS mice was restored upon gp91ds-tat treatment. A positive correlation was identified between neuronal NOX2 expression and serum glucocorticoid levels. Our study indicated that neuronal NOX2 may be a critical mediator of depression-like behaviours and spatial cognitive deficits in mice subjected to CRS. Blockade of NOX2 signalling may be a promising therapeutic strategy for depression.

Time-dependent changes in genome-wide gene expression and post-transcriptional regulation across the post death process in silkworm.

Despite death marking the end of life, several gene expression and miRNA-mediated post-transcriptional regulation events may persist or be initiated. The silkworm (Bombyx mori) is a valuable model for exploring life processes, including death. In this study, we combined transcriptomics and miRNAomics analyses of young, old, and postmortem silkworms across the entire process after death to unravel the dynamics of gene expression and miRNA-mediated post-transcriptional regulation. In total, 171 genes exhibited sustained differential expression in postmortem silkworms compared to the pre-death state, which are primarily involved in nerve signaling, transport, and immune response. Postmortem time-specific genes were associated with cell cycle regulation, thermogenesis, immunity, and zinc ion homeostasis. We found that the down-regulated expression of 36 genes related to transcription, epigenetic modification, and homeostasis resulted in a significant shift in global gene expression patterns at 2h post-death. We also identified five mRNA-miRNA pairs (i.e., bmo-miR-2795-mhca, 2784-achi, 2762-oa1, 277-5p-creb, and 1000-tcb1) associated with stress hormone regulation, transcription activity, and signal transduction. The roles of these pairs were validated through in vivo experiments using miRNA mimics in silkworms. The findings provide valuable insights into the intricate mechanisms underlying the transcriptional and miRNA-mediated post-transcriptional regulation events in animals after death.

Prognostic Significance of PD-L1 Expression on Circulating Myeloid-Derived Suppressor Cells in NSCLC Patients Treated with Anti-PD-1/PD-L1 Checkpoint Inhibitors

Even though anti-PD-1/PD-L1 immune checkpoint inhibitors (ICIs) in non-small cell lung cancer (NSCLC) have improved survival, a high percentage of patients still do not respond to ICIs. Myeloid-derived suppressor cells (MDSCs) are circulating cells that express PD-L1 and can infiltrate and proliferate in the tumor microenvironment, inducing immunosuppression. By evaluating changes in PD-L1 expression of live peripheral blood MDSCs, we are able to define a new PD-L1 index, useful in predicting ICI escape in NSCLC patients before initiating anti-PD-1/PD-L1 immunotherapy. In this study, a cohort of 37 NSCLC patients was prospectively analyzed, obtaining independent PD-L1 indexes. In patients with a PD-L1 index &gt; 5.88, progressive disease occurred in 58.33% of patients [median progression-free survival (PFS) = 5.73 months; 95%CI = 2.67–20.53], showing significant differences when compared with patients with a PD-L1 index ≤ 5.88, in whom 7.69% progressed and median PFS was not reached (NR); p-value = 0.0042. Overall survival (OS) was significantly worse in patients with a high vs. low PD-L1 index (41.67% vs. 76.92%; median OS = 18.03 months, 95%CI = 6.77–25.23 vs. NR, 95%CI = 1.87-NR; p-value = 0.035). The PD-L1 index can be applied to stratify NSCLC patients according to their probability of response to ICIs at baseline. In addition to quantifying tumoral expression, this index could be used to compare nonresponse to treatment.