Induction Of Genes Research Articles

Nicotinamide riboside targets mitochondrial unfolded protein response to reduce alcohol-induced damage in Kupffer cells.

The pathogenesis of alcohol-related liver disease (ALD) is closely linked to mitochondrial dysfunction and impaired cellular energy metabolism. In this study, we explored how ethanol triggers inflammation, oxidative stress, and mitochondrial dysfunction in Kupffer cells, i.e.hepatic resident macrophages, primarily focusing on the mitochondrial unfolded protein response (UPRmt) using immortalized mouse Kupffer cells (ImKCs) and mouse primary KCs. The UPRmt is a cellular defense mechanism activated in response to the perturbation of mitochondrial proteostasis to maintain mitochondrial integrity and function by upregulating the expression of mitochondrial chaperones and proteases. We also determined whether nicotinamide riboside (NR), a NAD+ precursor, could mitigate ethanol-triggered cellular damage. When ImKCs were exposed to 80 mm ethanol for 72 h, they displayed inflammation, oxidative stress, and impaired mitochondrial function with decreased mitochondrial content and deformed mitochondrial crista structure. NR, however, counteracted the effects of ethanol. Furthermore, ethanol increased mRNA and protein levels of UPRmt genes, such as mitochondrial chaperones and proteases, which were attenuated by NR. Notably, the ethanol-induced shift in the entry of activating transcription factor 5 (ATF5), a putative transcriptional regulator of UPRmt, to the nucleus from the mitochondria was abolished by NR. The induction of UPRmt genes by ethanol was significantly repressed when Atf5 was knocked down, indicating the role of ATF5 in the induction of UPRmt genes in ImKCs exposed to ethanol. We also confirmed the induction of UPRmt gene expression in mouse and human livers exposed to alcohol. Our findings demonstrate the ability of NR to alleviate ethanol-induced oxidative stress, inflammation, and mitochondrial dysfunction, partly by modulating the ATF5-dependent UPRmt pathway in ImKCs, suggesting its potential for ALD therapy. © 2024 The Pathological Society of Great Britain and Ireland.

Altered hypoxia- and redox-related transcriptional signatures in mitochondrial-DNA-depleted PC-3 cells

Rho 0 (ρ0) cells are widely used as a tool to investigate how the absence of respiring mitochondria affects a variety of physiological and pathological processes. Prominently, ρ0 cells have been used to study the role of mitochondrial reactive oxygen species (ROS) production and/or mitochondrial respiration in the stabilization of the hypoxia-inducible factor (HIF) in hypoxia. In this study, we cultured ρ0 and WT PC-3 cells in 5% O2 (physioxia) and Plasmax medium for 2 weeks prior to transcriptomic and functional analyses. RNA-seq showed that ρ0 PC-3 cells exhibit impaired induction of HIF-regulated genes when exposed to hypoxia, compared to wild-type (WT) cells. Surprisingly, when comparing the transcriptomes of ρ0 and WT cells in physioxia (5% O2), we found a strong presence of HIF-related gene signatures in ρ0 cells compared to WT. Among the HIF targets found to be upregulated in ρ0 cells are CA9, EGLN3, EPAS1, HK2, ENO2, and SLC2A1. Moreover, several Nrf2 targets were upregulated in ρ0 cells, including NQO1, HMOX1, GPX2, and SLC7A11, which is in line with ρ0 cells showing a significantly higher H2O2 efflux rate than WT cells. Given the alterations in HIF-dependent and Nrf2-dependent gene expression and basal ROS production observed in ρ0 PC-3 cells, we conclude that caution should be taken when interpreting the results from experiments that focus on ROS production and HIF signaling using ρ0 cells as a model.

CzcR-dependent reduction of catalase gene expression and induction of catalase activity in Pseudomonas aeruginosa during zinc excess

BackgroundPseudomonas aeruginosa is able to survive, grow, and cause severe infections at different sites throughout the human body owing to its ability to sense diverse signals and precisely modulate target gene expression using its abundant signaling systems. Release of zinc (Zn) and hydrogen peroxide (H2O2) within the phagocyte are two major host strategies to defend against bacterial infections. It was previously shown that the response regulator CzcR controls global gene expression including catalase genes during Zn excess, but regulatory mechanisms of catalase gene expression and the role of CzcR in H2O2 tolerance remain unclear.ResultsIn the study, comparative transcriptome analysis comprehensively described the CzcR-dependent and -independent gene regulatory pattern in P. aeruginosa during Zn excess, which revealed the counteractive co-regulation of two key H2O2-detoxifying catalase genes katA and katB through CzcR-dependent and -independent pathways in response to Zn excess. Protein-DNA interaction assay demonstrated that CzcR negatively regulates the expression of catalase genes katA and katB by directly binding to their promoters. While interestingly, we further showed that CzcR positively regulates H2O2 tolerance by inducing the catalase activity during Zn excess.ConclusionThis study reported the opposite functions of CzcR in negatively regulating the expression of catalase genes katA and katB but in positively regulating the activity of catalase and H2O2 tolerance during Zn excess in P. aeruginosa.

Cxcl10 is protective during mouse-adapted SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, remains endemic worldwide. Circulating levels of the chemokine CXCL10 are strongly positively associated with poor outcome; however, its precise role in SARS-CoV-2 pathogenesis and its suitability as a therapeutic target have remained undefined. Here, we challenged mice genetically deficient in Cxcl10 with a mouse-adapted strain of SARS-CoV-2. Infected male, but not female, Cxcl10-/- mice displayed increased mortality compared to wild type controls. Histopathological damage, inflammatory gene induction and virus load in the lungs of male mice were not broadly influenced by Cxcl10 deficiency. However, accumulation of B and T lymphocytes in the lung parenchyma of infected mice was reduced in the absence of Cxcl10. Thus, during acute SARS-CoV-2 infection, Cxcl10 regulates lymphocyte infiltration in lung and confers protection against mortality. Our preclinical model results do not support targeting CXCL10 therapeutically in severe COVID-19.

Osteocytic oxygen sensing: Distinct impacts of VHL and HIF-2alpha on bone integrity

Skeletal fracture resistance emerges from multiple components of bone structure like microarchitecture, matrix mineralization, and organization. These characteristics are engendered via mechanisms like the hypoxia-inducible factors (HIF) pathway, involving two paralogs, HIF-1α and HIF-2α. Under normoxia, HIF-α is targeted for degradation via von-Hippel Lindau (VHL); hypoxia enables HIF-α stabilization and induction of target genes. We previously showed that osteocytic Vhl deletion or expression of degradation-resistant HIF-2α cDR female mice each produced high bone mass, whereas degradation-resistant osteocytic HIF-1α produced no overt phenotype. We report within that Vhl cKO increased bone strength, while HIF-2α cDR displayed markedly reduced bone strength below Cre-negative controls. This suggests that VHL and HIF-2α drive distinct responses that promote disparate effects on bone strength. Both Vhl deletion or HIF-2α accumulation generated two discrete bone morphologies: an outer lamellar cortex and a woven, poorly mineralized endocortex that imparted dramatically different functional outcomes. Our studies reveal novel influence of osteocytic HIF-2α signaling on collagen matrix organization, mineralization, and bone strength.

The endocannabinoid anandamide mediates anti-inflammatory effects through activation of NR4A nuclear receptors.

Endocannabinoids are lipid mediators, which elicit complex biological effects that extend beyond the central nervous system. Tissue concentrations of endocannabinoids increase in atherosclerosis, and for the endocannabinoid N-arachidonoyl-ethanolamine (anandamide, AEA), this has been linked to an anti-inflammatory function. In this study, we set out to determine the anti-inflammatory mechanism of action of AEA, specifically focusing on vascular smooth muscle cells. RNA-sequencing, RT-qPCR, LC-MS/MS, NanoBit, ChIP, microscale thermophoresis, NMR structural footprinting, Gal4 reporter gene assays and loss of function approaches in cell and ex vivo organ culture were used. AEA pretreatment attenuated the cytokine-mediated induction of inflammatory gene expression such as CCL2. This effect was also observed in preparations obtained from cannabinoid receptor knockout mice and after pertussis toxin treatment. The anti-inflammatory effect of AEA required preincubation, suggesting an effect through gene induction. AEA increased the expression of the nuclear receptors NR4A1 and NR4A2. Knockdown and knockout of these receptors blocked the AEA-mediated anti-inflammatory effect in cell culture and aortic organ culture, respectively. Conversely, NR4A agonists (CsnB, C-DIM12) attenuated inflammatory gene expression. AEA binds to NR4A, and mutations in NR4A attenuated this effect. The interaction of AEA with NR4A caused recruitment of the nuclear corepressor NCoR1 to the CCL2 promoter, resulting in gene suppression. By binding to NR4A, AEA elicits an anti-inflammatory response in vascular smooth muscle cells. NR4A-binding by AEA analogues may represent novel anti-inflammatory agents.

The Zymoseptoria tritici effector Zt-11 contributes to aggressiveness in wheat.

Zymoseptoria tritici is an ascomycete fungus and the causal agent of Septoria tritici leaf blotch (STB) in wheat. Z. tritici secretes an array of effector proteins that are likely to facilitate host infection, colonisation and pycnidia production. In this study we demonstrate a role for Zt-11 as a Z. tritici effector during disease progression. Zt-11 is upregulated during the transition of the pathogen from the biotrophic to necrotrophic phase of wheat infection. Deletion of Zt-11 delayed disease development in wheat, reducing the number and size of pycnidia, as well as the number of macropycnidiospores produced by Z. tritici. This delayed disease development by the ΔZt-11 mutants was accompanied by a lower induction of PR genes in wheat, when compared to infection with wildtype Z. tritici. Overall, these data suggest that Zt-11 plays a role in Z. tritici aggressiveness and STB disease progression possibly via a salicylic acid associated pathway.

Similarities and distinctions in the activation of the Candida glabrata Pdr1 regulatory pathway by azole and non-azole drugs.

Candida glabrata has emerged as the second-leading cause of candidiasis due, in part, to its ability to acquire high-level resistance to azole drugs, a major class of antifungal that acts to block the biosynthesis of the fungal sterol ergosterol. The presence of azole drugs causes the induction of a variety of genes involved in controlling susceptibility to this drug class, including drug transporters and ergosterol biosynthetic genes such as ERG11. We found that the presence of azole drugs leads to an induction of genes encoding drug transporters and ERG11, while exposure of C. glabrata cells to antifungals of the phenothiazine class of drugs caused a much faster and larger induction of drug transporters but not ERG11. Coupled with further genetic analyses of the effects of azole and phenothiazine drugs, our data indicate that these compounds are sensed and responded to differentially in the yeast cell.

Gene expression profiling reveals host defense strategies for restricting Candida albicans invasion and gastritis to the limiting ridge of the murine stomach.

Candida albicans is a fungal constituent of the human gastrointestinal microbiota that can tolerate acidic environments like the stomach, where it can be associated with ulcers and chronic gastritis. In mice, C. albicans induces gastritis without concurrent intestinal inflammation, suggesting that the stomach is particularly prone to fungal infection. We previously showed that C. albicans invasion in the limiting ridge does not extend to or elicit an inflammatory response in the adjacent glandular region, indicating regionalized gastritis in the murine stomach. However, the molecular pathways involved in the host response to C. albicans specifically in the limiting ridge have not been investigated. Here, we found that gastric dysbiosis was associated with C. albicans limiting ridge colonization and gastritis. We isolated the limiting ridge and evaluated the expression of over 90 genes involved in mucosal responses. C. albicans infection triggered a type 3 immune response marked by elevated Il17a, Il17f, Il1b, Tnf, and Il36g, as well as an upregulation of Il12a, Il4, Il10, and l13. Chemokine gene induction (including Ccl2, Ccl3, Ccl4, Ccl1l, Cxcl1, Cxcl2, Cxcl9, and Cxcl10) coincided with an influx of neutrophils, monocytes/macrophages, and eosinophils. Hyphal invasion caused tissue damage, epithelial remodeling, and upregulation of genes linked to epithelium signaling and antimicrobial responses in the limiting ridge. Our findings support a need for continued exploration into the interactions between the immunological milieu, the host microbiota, and clinical interventions such as the use of antibiotics and immunotherapeutic agents and their collective impact on invasive candidiasis risk.

The mammalian Ire1 inhibitor, 4µ8C, exhibits broad anti-Aspergillus activity in vitro and in a treatment model of fungal keratitis.

The fungal unfolded protein response consists of a two-component relay in which the ER-bound sensor, IreA, splices and activates the mRNA of the transcription factor, HacA. Previously, we demonstrated that hacA is essential for Aspergillus fumigatus virulence in a murine model of fungal keratitis (FK), suggesting the pathway could serve as a therapeutic target. Here we investigate the antifungal properties of known inhibitors of the mammalian Ire1 protein both in vitro and in a treatment model of FK. The antifungal activity of Ire1 inhibitors was tested against conidia of several A. fumigatus isolates by a broth microdilution assay and against fungal biofilm by XTT reduction. The influence of 4μ8C on hacA mRNA splicing in A. fumigatus was assessed through gel electrophoresis and qRT-PCR of UPR regulatory genes. The toxicity and antifungal profile of 4μ8C in the cornea was assessed by applying drops to uninfected or A. fumigatus-infected corneas 3 times daily starting 4 hours post-inoculation. Corneas were evaluated daily through slit-lamp imaging and optical coherence tomography, or at endpoint through histology or fungal burden quantification via colony forming units. Among six Ire1 inhibitors screened, the endonuclease inhibitor 4μ8C displayed the strongest antifungal profile with an apparent fungicidal action. The compound both blocked conidial germination and hyphal metabolism of A. fumigatus Af293 in the same concentration range that blocked hacA splicing and UPR gene induction (60-120 µM). Topical treatment of sham-inoculated corneas with 0.5 and 2.5 mM 4μ8C did not impact corneal clarity, but did transiently inhibit epithelialization of corneal ulcers. Relative to vehicle-treated Af293-infected corneas, treatment with 0.5 and 2.5 mM drug resulted in a 50% and >90% reduction in fungal load, respectively, the latter of which corresponded to an absence of clinical signs of infection or corneal pathology. The in vitro data suggest that 4μ8C displays antifungal activity against A. fumigatus through the specific inhibition of IreA. Topical application of the compound to the murine cornea can furthermore block the establishment of infection, suggesting this class of drugs can be developed as novel antifungals that improve visual outcomes in FK patients.

EPCO-04. H3F3A K27M MUTATION DRIVES A REPRESSIVE TRANSCRIPTOME VIA REDUCED SERINE 31 PHOSPHORYLATION BY MODULATING CHROMATIN ACCESSIBILITY, INDEPENDENT OF H3K27ME3 IN DIFFUSE MIDLINE GLIOMA

Abstract Heterozygous H3.3 K27M mutations are central to the pathogenesis of diffuse midline gliomas (DMG), causing a global reduction in H3K27 trimethylation (H3K27me3) and subsequent epigenetic dysregulation. H3.3 contains a unique Serine 31 (S31) phosphorylation site crucial for mitotic checkpoint regulation. In DMG cells, reduced mitotic S31 phosphorylation correlates with chromosomal instability and aneuploidy. Using a mouse model, we demonstrate that both H3.3K27M and H3.3S31A mutations promote high-grade glioma development, whereas wild-type H3.3 does not. Importantly, S31A, which maintains normal K27me, suggests that S31 phosphorylation loss alone is oncogenic. The H3.3K27M mutation disrupts epigenetic regulation by inhibiting the Polycomb Repressive Complex 2 (PRC2), leading to decreased H3K27me3 and unintended gene induction. Concurrently, K27M exerts a compensatory repressive effect on the transcriptome, mediated by reduced S31 phosphorylation, independent of PRC2 activity. Using CRISPR-engineered isogenic DMG cells and analyzing the transcriptome and chromatin accessibility with RNA-seq and ATAC-seq, we found that the K27M mutation produces a balanced gene expression profile, while PRC2 loss primarily leads to gene induction. Notably, cells with S31A mutations also maintain this balanced profile, linking the PRC2-independent effect of K27M to reduced S31 phosphorylation. Conversely, cells with wild-type K27 but S31A mutation show a repressive genomic state, evidenced by an up/down gene ratio of 0.75, owing to functional PRC2. We further show that the genes uniquely dysregulated by K27M or S31A, independent of PRC2, overlap significantly, with dysregulated genes displaying altered chromatin accessibility. This suggests that the effect of K27M and S31A mutations on gene regulation is primarily due to changes in chromatin state. This complex interplay points to targeting K27M-associated pathways, particularly S31 phosphorylation, as a promising therapeutic avenue in DMG, a malignancy with fewer effective treatments and dismal prognosis.

TMIC-82. CHARACTERIZATION OF GENETIC MARKERS AND BIOLOGICAL PROCESSES UNDERLYING THE TRANSITION OF GLIOBLASTOMA CELLS FROM A DIFFERENTIATED TO A STEM-CELL-LIKE STATE

Abstract BACKGROUND The main drivers of Glioblastoma (GBM) tumor growth are elevated angiogenesis and heightened characteristics of stemness. These driving factors make GBM an extremely invasive and aggressive tumor. While the GBM tumor microenvironment can be classified into several molecular subtypes, one common characteristic is the atypical induction of various signal transduction pathways including the receptor tyrosine kinases (RTKs), pro-angiogenic, and stem-cell-determining transcription factors, which all play important roles in GBM tumor formation, growth, and progression. OBJECTIVE To investigate the regulations of RTKs, pro-angiogenic, and stem-cell–determining transcription factors in two different tumor environments - monolayer cultures that induce a more differentiated state and neurosphere cultures that select for stem-like cells. METHODS Neurosphere- and monolayer-forming cell samples were cultured from four primary GBM cell lines including HK-336, HK-374, U87, and GS-154. RNA sequencing was performed at the Technology Center for Genomics and Bioinformatics at the University of California, Los Angeles. The transcriptomic data findings were validated by qPCR and western blot assays. RESULTS Our transcriptomic data revealed the significant upregulation of EGFR, PDGFR-α, VEGFA, and SOX2 in the neurosphere-forming HK-336, HK-374, U87, and GS-154 primary GBM cells. Consistent with the transcriptomic data, western blot, and qPCR assays demonstrated significantly higher expressions of these genes in the neurosphere-forming compared to the monolayer-forming GBM cells. Neurosphere-forming GBM cells possess stem-cell-like characteristics with the ability to self-renew and differentiate via the induction of various genes and disease progression pathways. CONCLUSIONS Neurosphere-forming GBM cells demonstrate upregulation of SOX2, EGFR, PDGFR-α, and VEGFA in RNA sequencing, western blot, and qPCR assays. Multipotent drug therapies designed to target multiple gene regulation pathways involving RTKs, pro-angiogenic, and stem-cell-determining transcription factors may be necessary to effectively treat GBM. Understanding the underlying mechanisms driving these aberrant cellular pathways could provide valuable insights for developing targeted therapies against this aggressive brain cancer.

AEBP1 is a negative regulator of skeletal muscle cell differentiation in oral squamous cell carcinoma

The tumor microenvironment plays a pivotal role in cancer development. We recently reported that in oral squamous cell carcinoma (OSCC), adipocyte enhancer-binding protein 1 (AEBP1) is abundantly expressed in cancer-associated fibroblasts (CAFs), leading to CAF activation and inhibition of CD8 + T cell infiltration. In the present study, we investigated whether AEBP1 contributes to the destruction and atrophy of muscle tissues in OSCC. By analyzing human skeletal muscle myoblasts (HSMMs), we found that AEBP1 is downregulated during muscle cell differentiation. Transcriptome analysis revealed that AEBP1 knockdown significantly upregulates myogenesis-related genes in HSMMs, and qRT-PCR and western blot analyses confirmed the induction of muscle-related genes, including MYOG, in HSMMs after AEBP1 knockdown. Conversely, ectopic expression of AEBP1 strongly suppressed myogenesis-related genes in HSMMs. Notably, indirect co-culture of HSMMs with OSCC cells led to AEBP1 upregulation and robust suppression of muscle-related genes in HSMMs. Treatment with TGF-β1 also upregulated AEBP1 and suppressed expression of muscle-related genes in HSMMs. Our findings suggest that AEBP1 is a negative regulator of skeletal muscle cell differentiation and that OSCC cells inhibit muscle cell differentiation, at least in part, by inducing AEBP1.

Invention and characterization of a systemically administered, attenuated and killed bacteria-based multiple immune receptor agonist for anti-tumor immunotherapy.

Activation of immune receptors, such as Toll-like (TLR), NOD-like (NLR) and Stimulator of Interferon Genes (STING) is critical for efficient innate and adaptive immunity. Gram-negative bacteria (G-NB) contain multiple TLR, NOD and STING agonists. Potential utility of G-NB for cancer immunotherapy is supported by observations of tumor regression in the setting of infection and Coley's Toxins. Coley reported that intravenous (i.v.) administration was likely most effective but produced uncontrollable toxicity. The discovery of TLRs and their agonists, particularly the potent TLR4 agonist lipopolysaccharide (LPS)-endotoxin, comprising ~75% of the outer membrane of G-NB, suggests that LPS may be both a critical active ingredient and responsible for dose-limiting i.v. toxicity of G-NB. This communication reports the production of killed, stabilized, intact bacteria products from non-pathogenic G-NB with ~96% reduction of LPS-endotoxin activity. One resulting product candidate, Decoy10, was resistant to standard methods of cell disruption and contained TLR2,4,8,9, NOD2 and STING agonist activity. Decoy10 also exhibited reduced i.v. toxicity in mice and rabbits, and a largely uncompromised ability to induce cytokine and chemokine secretion by human immune cells in vitro, all relative to unprocessed, parental bacterial cells. Decoy10 and a closely related product, Decoy20, produced single agent anti-tumor activity or combination-mediated durable regression of established subcutaneous, metastatic or orthotopic colorectal, hepatocellular (HCC), pancreatic, and non-Hodgkin's lymphoma (NHL) tumors in mice, with induction of both innate and adaptive immunological memory (syngeneic and human tumor xenograft models). Decoy bacteria combination-mediated regressions were observed with a low-dose, oral non-steroidal anti-inflammatory drug (NSAID), anti-PD-1 checkpoint therapy, low-dose cyclophosphamide (LDC), and/or a targeted antibody (rituximab). Efficient tumor eradication was associated with plasma expression of 15-23 cytokines and chemokines, broad induction of cytokine, chemokine, innate and adaptive immune pathway genes in tumors, cold to hot tumor inflammation signature transition, and required NK, CD4+ and CD8+ T cells, collectively demonstrating a role for both innate and adaptive immune activation in the anti-tumor immune response.

Characterization of A Bronchoscopically Induced Transgenic Lung Cancer Pig Model for Human Translatability.

There remains a need for animal models with human translatability in lung cancer (LC) research. Findings in pigs have high impact on humans due to similar anatomy and physiology. We present the characterization of a bronchoscopically-induced LC model in Oncopigs carrying inducible KRAS G12D and TP53 R167H mutations. Twelve Oncopigs underwent 29 injections via flexible bronchoscopy. Eighteen Adenovirus-Cre recombinase gene (AdCre) inductions were performed endobronchially (n=6) and transbronchially with a needle (n=12). Eleven control injections were performed without AdCre. Oncopigs underwent serial contrast-enhanced chest CT with clinical follow-up for 29 weeks. Following autopsy, lung and organ tissues underwent histopathology, immunohistochemistry, and RNA-sequencing with comparative analysis with The Cancer Genome Atlas (TCGA) human LC data. All 18 sites of AdCre injections had lung consolidations on CT imaging. Transbronchial injections led to histopathologic invasive cancer and/or carcinoma in situ (CIS) in 11/12 (91.7%), and invasive cancer (excluding CIS) in 8/12 (66.6%). Endobronchial inductions led to invasive cancer in 3/6 (50%). A soft tissue metastasis was observed in one Oncopig. Immunohistochemistry confirmed expression of Pan-CK+/epithelial cancer cells, with macrophages and T cells infiltration in the tumor microenvironment. Transcriptome comparison showed 54.3% overlap with human LC (TCGA), in contrast to 29.88% overlap of KRAS-mutant mouse LC with human LC. The transgenic and immunocompetent Oncopig model has a high rate of LC following bronchoscopic transbronchial induction. Overlap of the Oncopig LC transcriptome with human LC transcriptome was noted. This pig model is expected to have high clinical translatability to the human LC patient.

Infrared laser–induced gene expression in single cells characterized by quantitative imaging in Physcomitrium patens

A spatiotemporal understanding of gene function requires the precise control of gene expression in each cell. Here, we use an infrared laser–evoked gene operator (IR-LEGO) system to induce gene expression at the single-cell level in the moss Physcomitrium patens by heating a living cell with an IR laser and thereby activating the heat shock response. We identify the laser irradiation conditions that provide higher inducibility with lower invasiveness by changing the laser power and irradiation duration. Furthermore, we quantitatively characterize the induction profile of the heat shock response using a heat-induced fluorescence reporter system after the IR laser irradiation of single cells under different conditions. Our data indicate that IR laser irradiation with long duration leads to higher inducibility according to increase in the laser power but not vice versa, and that the higher laser power even without conferring apparent damage to the cells decelerates and/or delayed gene induction. We define the temporal shift in expression as a function of onset and duration according to laser power and irradiation duration. This study contributes to the versatile application of IR-LEGO in plants and improves our understanding of heat shock-induced gene expression.

The long non-coding RNA lncRNA-DRNR enhances infectious bronchitis virus replication by targeting chicken JMJD6 and modulating interferon-stimulated genes expression via the JAK-STAT signalling pathway.

Infectious bronchitis virus (IBV) is the causative agent of infectious bronchitis (IB), a severe disease that primarily affects young chickens and poses a significant challenge to the global poultry industry. Understanding the complex interaction between the virus and its host is vital for developing innovative antiviral strategies. Long non-coding RNA (lncRNA) plays a crucial role in regulating host antiviral immune responses. Our previous studies have shown that IBV infection disrupts the stability of lncRNA in host cells, indicating a potential regulatory role for lncRNA in IBV pathogenesis. It is still not clear how lncRNA precisely modulates IBV replication. In this study, we observed down-regulation ofMSTRG.26120.58 (named lncRNA-DRNR) expression in various chicken cell lines upon IBV infection. We demonstrated that silencing lncRNA-DRNR using siRNA enhances intracellular replication of IBV. Through exploring genes encoding proteins upstream and downstream of lncRNA-DRNR within a 100kb range, we identified chJMJD6 (chicken JMJD6) as a potential target gene negatively regulated by lncRNA-DRNR expression levels. Furthermore, chJMJD6 inhibits STAT1 methylation, thereby affecting the induction of interferon-stimulated genes (ISGs) through the activation of the IFN-β-mediated JAK-STAT signalling pathway, ultimately promoting the intracellular replication of IBV. In summary, our findings reveal the critical role played by lncRNA-DRNR during IBV infection, providing novel insights into mechanisms underlying coronavirus-induced disruption in lncRNA stability.

EXECUTER1 and singlet oxygen signaling: A reassessment of nuclear activity.

Chloroplasts are recognized as environmental sensors, capable of translating environmental fluctuations into diverse signals to communicate with the nucleus. Among the reactive oxygen species produced in chloroplasts, singlet oxygen (1O2) has been extensively studied due to its dual roles, encompassing both damage and signaling activities, and the availability of conditional mutants overproducing 1O2 in chloroplasts. In particular, investigating the Arabidopsis (Arabidopsis thaliana) mutant known as fluorescent (flu) has led to the discovery of EXECUTER1 (EX1), a plastid 1O2 sensor residing in the grana margin of the thylakoid membrane. 1O2-triggered EX1 degradation is critical for the induction of 1O2-responsive nuclear genes (SOrNGs). However, a recent study showed that EX1 relocates from chloroplasts to the nucleus upon 1O2 release, where it interacts with WRKY18 and WRKY40 (WRKY18/40) transcription factors to regulate SOrNG expression. In this study, we challenge this assertion. Our confocal microscopy analysis and subcellular fractionation assays demonstrate that EX1 does not accumulate in the nucleus. While EX1 appears in nuclear fractions, subsequent thermolysin treatment assays indicate that it adheres to the outer nuclear region rather than localizing inside the nucleus. Furthermore, luciferase complementation imaging and yeast two-hybrid assays reveal that EX1 does not interact with nuclear WRKY18/40. Consequently, our study refines the current model of 1O2 signaling by ruling out the nuclear relocation of intact EX1 as a means of communication between the chloroplast and nucleus.

Epigenomic studies in sorghum reveal differential enrichment of multiple histone marks at clade A PP2C genes in response to drought

Epigenetic regulation is essential for plant development and stress responses, as numerous stress-responsive genes are modulated epigenetically. However, most research has focused on individual histone marks. This study expands on previous work by examining the genome-wide profiles of seven histone marks (H3K9ac, H3K27ac, H3K4me3, H3K36me3, H3K27me3, H2A.Z, and H3K4me2) in sorghum leaves and roots under PEG-induced drought stress. Results revealed that five histone marks (excluding H3K36me3 and H3K27me3) were significantly associated with drought-responsive gene expression. The differential enrichment of these marks may enhance the induction of drought-responsive genes. Specifically, a subset of genes showed multiple histone marks' differential enrichment, with most clade A PP2C genes exhibiting enrichment for four marks after PEG treatment. This suggests a critical role for robust PP2C gene induction in sorghum’s drought response. Additionally, promoter cis-element analysis identified ERF family transcription factors as potential mediators of histone mark enrichment under drought conditions, providing new insights into the interaction between epigenetic modifications and transcriptional regulation in plant stress responses.

Liposome encapsulating pine bark extract in Nile tilapia: Targeting interrelated immune and antioxidant defense to combat coinfection with Aeromonas hydrophila and Enterococcus faecalis

Application of smart delivery systems for encapsulation of natural ingredients provides novel avenues and is being frequently developed. Thus, we aimed to highlight the effects of cyclosome liposomal pine bark extract (CL-PBE) on Nile tilapia growth, immunomodulation, antioxidant capacity and resistance against coinfection with Aeromonas hydrophila and Enterococcus faecalis and their associated virulence genes. The experiment was conducted on four fish groups receiving a control diet (control) along with three baseline meals supplemented with 200, 400 and 600 mg/kg diet of CL-PBE (CL-PBE 200, 400 and 600, respectively). At the end of the 12-weeks feeding trial, the tilapias were intraperitoneally challenged with virulent A. hydrophila strain and five days later, E. faecalis challenge was carried out. The results revealed that tilapias fed diets fortified with CL-PBE displayed significantly enhanced growth rate and feed conversion ratio in a dose-dependent manner. Moreover, we demonstrated that CL-PBE had potent antioxidant property presented by modulation of several markers of oxidative stress; substantial reductions in reactive oxygen species, hydrogen peroxide and malondialdehyde levels, an elevation in total antioxidant capacity and boosting glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) activities in fish serum and muscle tissues. This was also correlated with augmenting the expression of CAT, SOD, GSH-Px, Nrf2 and caspase-1 genes alongside reducing those of COX-2, HSP70 and iNOS genes in response to CL-PBE. Our data demonstrated that CL-PBE fortification counteracted the overly pronounced inflammatory response-mediated induction of IL-1β, TNF-α, MHCII and TLR2 genes at the transcriptional levels post coinfection together with promotion in MUC2 and IL-10 genes expression. Notably, our findings displayed optimal well-functioning fish immune system post dietary supplementation of CL-PBE for the protection against coinfection with A. hydrophila and E. faecalis. This was evident from the decline of their counts and hence encompassing the capacity to reduce cumulative mortality percentage in conjunction with interference with their virulence via the significant downregulatory effects of CL-PBE on E. faecalis esp and gelE and A. hydrophila act and fla virulence genes. Taken together, our study strongly suggested dietary inclusion of CL-PBE for Nile tilapias with superior growth performance and significant economic benefits coupled with potent stimulatory effects on antioxidant capacity and immune response expediting our detailed understanding of how coinfection with A. hydrophila and E. faecalis was controlled.