Changes In Gene Transcription Research Articles

Age-related dysregulation of the retinal transcriptome in African turquoise killifish.

Age-related vision loss caused by retinal neurodegenerative pathologies is becoming more prevalent in our ageing society. To understand the physiological and molecular impact of ageing on retinal homeostasis, we used the short-lived African turquoise killifish, a model known to naturally develop central nervous system (CNS) ageing hallmarks and vision loss. Bulk and single-cell RNA-sequencing (scRNAseq) of three age groups (6-, 12-, and 18-week-old) identified transcriptional ageing fingerprints in the killifish retina, unveiling pathways also identified in the aged brain, including oxidative stress, gliosis, and inflammageing. These findings were comparable to observations in the ageing mouse retina. Additionally, transcriptional changes in genes related to retinal diseases, such as glaucoma and age-related macular degeneration, were observed. The cellular heterogeneity in the killifish retina was characterized, confirming the presence of all typical vertebrate retinal cell types. Data integration from age-matched samples between the bulk and scRNAseq experiments revealed a loss of cellular specificity in gene expression upon ageing, suggesting potential disruption in transcriptional homeostasis. Differential expression analysis within the identified cell types highlighted the role of glial/immune cells as important stress regulators during ageing. Our work emphasizes the value of the fast-ageing killifish in elucidating molecular signatures in age-associated retinal disease and vision decline. This study contributes to the understanding of how age-related changes in molecular pathways may impact CNS health, providing insights that may inform future therapeutic strategies for age-related pathologies.

Abstract PO3-26-04: Discovery of novel inhibitors of the epigenetic regulatory KAT6A histone acetyl transferase and their anti-tumor activity in breast cancer cells

Abstract KAT6A is a histone acetyltransferase (HAT) which directs chromatin structural rearrangement and subsequent changes in gene transcription through acetylation of histones, primarily specific lysine residues on histone H3. KAT6A is modified in cancer, including genomic amplification, overexpression, mutation, as well as genetic rearrangement resulting in the production of fusion proteins. Dysregulation of this MYST family member and its acetyl transferase activity is known to drive gene expression and tumorigenic progression in cancers. An inhibitor of KAT6A and related MYST family member KAT6B has entered phase 1 clinical trials with durable partial responses seen in a variety of tumor types, including estrogen-receptor positive (ER+) breast cancer previously treated with a CDK4/6 inhibitor (Sommerhalder, ASCO2023). Evidence of dose dependent pharmacodynamics (H3K23Ac inhibition) was observed in peripheral blood mononuclear cells and paired tumor biopsies. Isosterix has developed 2 distinct series of KAT6A selective inhibitors which engage in the active site of KAT6A in the acetyl CoA substrate binding pocket and inhibit histone acetylation activity. The kinetics of target enzyme binding and inhibition will be discussed for both series of inhibitors. These compounds have been characterized for potency and cellular activity in a variety of assays. Both series of compounds inhibit KAT6A-mediated histone acetylation with single digit nM potency in a biochemical assay as well as significant potency in the breast cancer tumor cell line ZR-75-1, inhibiting H3K23 acetylation. Using a thermal shift assay (CETSA) and mass spectrometry-based protein quantitation, direct target engagement of KAT6A within cells has been shown. These compounds reduce cell viability of a number of breast cancer cell lines with 10 days of compound administration confirming the dependence of these tumor cell lines on KAT6A acetyl transferase activity for proliferation and cell cycle progression. Compounds are orally bioavailable with excellent pharmacokinetic (PK) properties in mice including half-lives >5 hours with oral administration. Finally, Isosterix inhibitors demonstrated anti-tumor efficacy with oral administration in a ZR-75-1 mouse xenograft tumor model. Inhibitors against this novel epigenetic target demonstrate the potential for efficacy in ER+ breast cancer as well as other tumor indications through suppression of KAT6A-directed transcriptional induction and chromatin remodeling. Table. Compound Kat6A Biochemical IC50 (µM) ZR-75-1 H3K23 Acetylation EC50 (µM) Mouse PK (p.o.) Mouse PK (i.v.) Inhibitor class %F T½ (h) Cl (mL/min/kg) SL-IST-001 0.007 0.01 100 5.2 0.47 Series 1 SL-IST-002 0.007 0.005 100 6 0.48 Series 2 Citation Format: Leslie Holsinger, Roopa RaI, Rahul R. Nagawade, Benoy Pal, Samir Pawar, Pankaj Singh, Krishna Putta, Mahaboobi Jaleel, Tove Bostrom, Jakob Karen, Qianyang Huang, Alison Hannah. Discovery of novel inhibitors of the epigenetic regulatory KAT6A histone acetyl transferase and their anti-tumor activity in breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-26-04.

Bromuconazole exposure induces cardiac dysfunction by upregulating the expression LEF1

With the wide application of bromuconazole (BRO), a kind of triazole fungicide, the environmental problems caused by BRO have been paid more and more attention. In this study, adult male zebrafish were exposed to environmental related concentration and the maximum non-lethal concentration for zebrafish larvae (0,50 ng/L and 7.5 mg/L) for 7 days, respectively. Zebrafish exposed to BRO exhibited a significant reduction in body length and an increase in fatness index, indicating adverse physiological changes. Notably, the exposed zebrafish showed enlarged heart ventricular volumes and thinner heart walls. Transcriptome analysis of heart samples showed that BRO exposure mainly affected pathways related to cardiac energy metabolism. In addition, the amount of ATP in the heart tissue was correspondingly reduced, and the expression levels of genes related to controlling ion balance and myosin synthesis in the heart were also altered. The study extended its findings to the rat cardiomyocytes (H9C2), where similar cardiotoxic effects including changes in transcription of genes related to energy metabolism and heart function were also observed, suggesting a potential universal mechanism of BRO-induced cardiotoxicity. In a doxorubicin (DOX) induced larval zebrafish heart failure model, the expression of lymphoid enhancer-binding factor 1(LEF1), a key gene in the Wnt/β-catenin signaling pathway, was significantly increased in larval zebrafish and adult fish heart tissues and cardiomyocytes, suggesting that LEF1 might play an important role in BRO-induced cardiotoxicity. Taken together, BRO exposure could interfere with cardiac function and metabolic capacity by abnormal activation the expression of LEF1. The study emphasized the urgent need for monitoring and regulating BRO due to its harmful effects on the hearts of aquatic organisms.

Transcriptional and ultrastructural changes of macrophages after african swine fever virus infection

African swine fever (ASF) is a highly impactful infectious disease in the swine industry, leading to substantial economic losses globally. The causative agent, African swine fever virus (ASFV), possesses intricate pathogenesis, warranting further exploration. In this study, we investigated the impact of ASFV infection on host gene transcription and organelle changes through macrophage transcriptome sequencing and ultrastructural transmission electron microscopy observation. According to the results of the transcriptome sequencing, ASFV infection led to significant alterations in the gene expression pattern of porcine bone marrow derived macrophages (BMDMs), with 2404 genes showing upregulation and 1579 genes downregulation. Cytokines, and chemokines were significant changes in the expression of BMDMs; there was significant activation of pattern recognition receptors such as Toll-like receptors and Nod-like receptors. According to the observation of the ultrastructure, mitochondrial damage and mitochondrial autophagy were widely present in ASFV-infected cells. The reduced number of macrophage pseudopodia suggested that virus-induced structural changes may compromise pathogen recognition, phagocytosis, and signal communication in macrophages. Additionally, the decreased size and inhibited acidification of secondary lysosomes in macrophages implied suppressed phagocytosis. Overall, ASFV infection resulted in significant changes in the expression of cytokines and chemokines, accompanied by the activation of NLR and TLR signaling pathways. We reported for the first time that ASFV infection led to a reduction in pseudopodia numbers and a decrease in the size and acidification of secondary lysosomes.

Abstract 3217: Discovery of novel inhibitors of the epigenetic regulator Kat6a histone acetyl transferase and characterization of their anti-tumor activity

Abstract KAT6A and its paralog KAT6B are histone acetyltransferases (HATs) which direct chromatin structural rearrangement and subsequent changes in gene transcription through acetylation of histones, primarily specific lysine residues on histone H3. KAT6A is modified in cancer, including genomic amplification, overexpression, mutation, as well as genetic rearrangement, resulting in the production of fusion proteins. Dysregulation of this MYST family member and its acetyl transferase activity is known to drive gene expression and tumorigenic progression in cancers. An inhibitor of KAT6A and KAT6B entered phase 1 clinical trials (NCT04606446). With continuous daily dosing, durable partial responses were seen in estrogen-receptor positive (ER+) breast cancer patients who had progressed on a prior endocrine therapy and CDK4/6 inhibitor. In that study, evidence of dose dependent pharmacodynamics (H3K23Ac inhibition) was observed in peripheral blood mononuclear cells and paired tumor biopsies (Sommerhalder, ASCO2023). Isosterix has developed a series of structurally diverse small molecule KAT6A-selective inhibitors which engage at the active site in the acetyl CoA substrate binding pocket. Lead compounds are selective for KAT6A and have significant potency and cellular activity in a variety of assays; they inhibit KAT6A-mediated histone acetylation in a biochemical assay as well as pharmacodynamic dose-dependent inhibition of histone acetylation in the breast cancer tumor cell line ZR-75-1. These compounds reduce cell viability of ZR-75-1 in 10 days with continuous compound administration, confirming the dependence of this cell line on KAT6A acetyl transferase activity for proliferation and cell cycle progression. Lead inhibitors are orally bioavailable with excellent pharmacokinetic properties in rodents. They demonstrate dose-dependent anti-tumor efficacy with minimal weight loss with continuous daily dosing in mouse xenograft tumor models. Inhibitors against this novel epigenetic target demonstrate the potential for efficacy in not only ER+ breast cancer but also other tumor indications which exhibit transcriptional induction dependent on KAT6A-directed chromatin remodeling. Selection of a lead candidate is planned for early 2024 with IND enabling studies to follow. Citation Format: Leslie J. Holsinger, Roopa Rai, Rahul R. Nagawade, Benoy K. Pal, Samir G. Pawar, Krishna Anand Putta, Somashekar S.C., Mahaboobi Jaleel, Jianhong Wang, Alison L. Hannah. Discovery of novel inhibitors of the epigenetic regulator Kat6a histone acetyl transferase and characterization of their anti-tumor activity [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 3217.

Abstract 4686: Identification of ART3 as a novel synthetic lethal partner of the BRCA1 mutation in breast cancer

Abstract Breast cancer (BC), the most prevalent malignancy in women, ranks fourth in cancer mortality as of 2022. Mutations in the BRCA genes significantly elevate BC risk and are associated with markedly reduced survival compared to non-carriers. These genes are crucial in homologous recombination, a mechanism essential for error-free DNA repair. Despite advancements, treatment options for BRCA mutation-driven cancers remain limited. Synthetic lethality (SL) has emerged as a promising strategy, with poly(ADP-ribose) polymerase inhibitors (PARPi) being the first developed drugs based on this principle. However, PARPi's effectiveness is limited, as nearly 40% of BRCA-mutated BCs are unresponsive, and resistance often develops in initial responders. Therefore, novel mutation-specific targeted therapies are urgently needed. In this study, we employed the novel computational method 'Mining Synthetic Lethals (MiSL)' to analyze TCGA pan-cancer primary tumor data, identifying ART3 as a potential SL partner of the BRCA1 germline mutation. Our approach focused on the intersection of genetic interactions and cancer vulnerabilities, providing a unique insight into SL relationships. siRNA-mediated silencing of ART3 in BRCA1-mutated cells consistently led to decreased cell viability, apoptosis induction, and G2/M cell cycle arrest. Following stable transfection with an inducible ART3 shRNA construct, we confirmed significant reduction in cell viability in BRCA1-deficient cells through colony formation assays. RNA sequencing of ART3 knockdown cells revealed transcriptional changes in genes involved in cell cycle progression, apoptosis, and growth and proliferation pathways compared with control cells. Immunofluorescent staining revealed increased DNA damage foci with decreased DNA repair in ART3 knockdown BRCA1-deficient cells. In conclusion, our study identifies ART3 as a novel SL partner of the BRCA1 mutation in BC. This discovery paves the way for developing new therapeutic strategies for BRCA1-mutated breast cancers and novel chemoprevention approaches for individuals carrying germline BRCA1 mutations. Future research will focus on further elucidating the molecular mechanisms underlying this SL interaction and exploring its therapeutic potential in clinical settings. Citation Format: Hannah Neiger, Xinyu Pei, Anh Nguyen, Wenjia Wang, Emily L. Siegler, James Zhou, Kristie Lau, Alexandra Tan, Yihui Shi. Identification of ART3 as a novel synthetic lethal partner of the BRCA1 mutation in breast cancer [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 4686.

Abstract 1681: Characterizing signaling and localization of Daple-FLT3 gene fusion in leukemia cells

Abstract Receptor tyrosine kinases (RTKs) are cell surface receptors that regulate cell survival, cell proliferation, and cell migration upon ligand binding. Genetic mutations leading to hyperactivation of RTKs are often seen in cancer and allow signaling pathways to be constitutively active. In some hematological malignanices, the coiled-coil domain (CCD) of Daple, a cell signaling scaffold protein, has been found in gene fusions with the RTKs FLT3 and PDGFRB, and to the intracellular kinase JAK2. It is hypothesized that this gene fusion product causes the tyrosine kinase domain of FLT3 to become activated and mislocalizes in the cell, leading to rampant cell signaling of either the MAPK, AKT, and STAT5 signal transduction cascades. Using fluorescently tagged Daple-FLT3, it was shown that the gene fusion product is subcellularly localizes to the peri-centrosome space across all leukemia cell lines tested. Ectopic expression demonstrated activation of the kinase domain and subsequent increases in phosphorylation of STAT5α and AKT, but no significant increase in the phosphorylation of MAPK was observed. Further investigation is needed to identify direct substrates of the Daple-FLT3 gene fusion and transcriptional changes. Identifying the direct kinase substrates and insights gained through understanding the gene fusion localization, affects on cell signaling, and changes in gene expression, may unveil novel therapeutic strategies into targeting this protein in cancer. Citation Format: Michael Acquazzino, Jason Ear. Characterizing signaling and localization of Daple-FLT3 gene fusion in leukemia 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 1681.

Aqueous Ozone Exposure Inhibits Sporulation in the Cyclospora cayetanensis Surrogate Eimeria acervulina

Ozone is a potent disinfecting agent used to treat potable water and wastewater, effectively clearing protozoa such as Giardia and Cryptosporidium spp. It is unclear whether ozone treatment of water or fresh produce can reduce the spread of the emerging parasite Cyclospora cayetanensis, which causes cyclosporiasis in humans. Obtaining viable C. cayetanensis oocysts to evaluate inactivation methods is challenging because we lack the means to propagate them in vitro, because of delays in case reporting, and because health departments typically add inactivating fixatives to clinical specimens. Research in various surrogate organisms has sought to bolster understanding of the biology of C. cayetanensis. Among these surrogates is the poultry parasite Eimeria acervulina, a closely related and easily cultured parasite of economic significance. We used this surrogate to evaluate the consequences of ozone treatment, using the sporulation state as an indicator of infectious potential. Treating with ozonated water acidified with citric acid reduced sporulation ability in a dose-dependent manner; treatment with up to 4.93 mg/L initial concentration of ozone resulted in a 93% inactivation of sporulation by 7 days posttreatment. This developmental arrest was accompanied by transcriptional changes in genes involved in regulating the response to reactive oxygen species (ROS) in a time course that is consistent with the production of oxygen free radicals. This study shows that ozone is highly effective in preventing sporulation of E. acervulina, a model coccidian used as a surrogate for Cyclospora. Furthermore, ozone exposure induced molecular responses to general oxidative stress, documented with several well-characterized antioxidant enzymes.

Endogenous feline leukemia virus long terminal repeat integration site diversity is highly variable in related and unrelated domestic cats

Endogenous retroviruses (ERV) are indicators of vertebrate evolutionary history and play important roles as homeostatic regulators. ERV long terminal repeat (LTR) elements may act as cis-activating promoters or trans-activating enhancer elements modifying gene transcription distant from LTR insertion sites. We previously documented that endogenous feline leukemia virus (FeLV)-LTR copy number variation in individual cats tracks inversely with susceptibility to virulent FeLV disease. To evaluate FeLV-LTR insertion characteristics, we assessed enFeLV-LTR integration site diversity in 20 cats from three genetically distinct populations using a baited linker-mediated PCR approach. We documented 765 individual integration sites unequally represented among individuals. Only three LTR integration sites were shared among all individuals, while 412 sites were unique to a single individual. When primary fibroblast cultures were challenged with exogenous FeLV, we found significantly increased expression of both exogenous and endogenous FeLV orthologs, supporting previous findings of potential exFeLV-enFeLV interactions; however, viral challenge did not elicit transcriptional changes in genes associated with the vast majority of integration sites. This study assesses FeLV-LTR integration sites in individual animals, providing unique transposome genotypes. Further, we document substantial individual variation in LTR integration site locations, even in a highly inbred population, and provide a framework for understanding potential endogenous retroviral element position influence on host gene transcription.

D-Tetramethrin causes zebrafish hepatotoxicity by inducing oxidative stress and inhibiting cell proliferation

d-Tetramethrin is one of the main components of mosquito control products, and is widely used for the control of dengue fever and insecticide production. Due to its widespread use, d-tetramethrin is a ubiquitous environmental pollutant and poses potential risks to human health. However, the effects of d-tetramethrin on liver morphology and function are not clearly established. In this study, we used zebrafish as an animal model to analyze the acute and chronic effects of d-tetramethrin exposure on the liver. We exposed zebrafish larvae and adults to different concentrations of d-tetramethrin and examined the impact of d-tetramethrin on lipid and glycogen metabolism, cellular properties, oxidative stress, cell proliferation, and apoptosis in the liver. We also analyzed transcriptional changes in genes related to apoptosis, inflammation, and cell proliferation using qPCR. Zebrafish exposed to d-tetramethrin exhibited severe liver damage, as evidenced by the presence of vacuoles and nuclear distortion in liver cells. The liver area in zebrafish larvae of the treatment group was significantly smaller than that of the control group. Significant lipid accumulation and decreased glycogen levels were observed in the livers of both zebrafish larvae and adults exposed to d-tetramethrin. Furthermore, d-tetramethrin exposure induced apoptosis and inflammation in zebrafish embryos. Additionally, d-tetramethrin caused liver damage, metabolic dysfunction, and impaired liver function. These results suggest that d-tetramethrin induces liver toxicity in zebrafish, by inducing oxidative stress and inhibiting cell proliferation.

Stress-Related Gene Expression in Liver Tissues from Laying Hens Housed in Conventional Cage and Cage-Free Systems in the Tropics.

Global egg production is mainly based on cage systems, which have been associated with negative effects on the welfare of birds. Stress factors in restrictive production systems can lead to changes in gene transcription and protein synthesis, ultimately impacting the quality of poultry products. The liver serves various metabolic functions, such as glycogen storage, and plays a crucial role in animals' adaptation to environmental changes. Consequently, both internal and external conditions can influence liver functions. The aim of this study was to evaluate the gene expression of AGP, CRP, NOX4, SOD1, CAT, GPX1, SREBF1, and FXR in the liver of laying hens under two different production systems. Liver tissues from Hy-Line Brown hens housed in conventional cage and cage-free egg production systems at 60 and 80 weeks of production were used. mRNA transcript levels were determined by qPCR using the relative quantification method and ACTB as the reference gene. AGP, SOD1, and SREBF1 gene expressions were significantly higher in the conventional cage group at the 60 weeks of production. Furthermore, the mRNA levels of transcripts related to oxidative stress and lipid metabolism were higher in the group of laying hens housed in conventional cages compared to those in cage-free systems. These results suggest differential gene expression of genes related to oxidative stress in liver tissues from hens housed in conventional cages compared to cage-free systems. The conditions of the egg production system can impact the gene expression of oxidative stress and lipid synthesis genes, potentially leading to changes in the metabolism and performance of hens, including egg quality.

The Interplay of TGF-β1 and Cholesterol Orchestrating Hepatocyte Cell Fate, EMT, and Signals for HSC Activation

Transforming growth factor-β1 (TGF-β1) plays important roles in chronic liver diseases, including metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD involves various biological processes including dysfunctional cholesterol metabolism and contributes to progression to metabolic dysfunction-associated steatohepatitis and hepatocellular carcinoma. However, the reciprocal regulation of TGF-β1 signaling and cholesterol metabolism in MASLD is yet unknown. Changes in transcription of genes associated with cholesterol metabolism were assessed by RNA sequencing of murine hepatocyte cell line (alpha mouse liver 12/AML12) and mouse primary hepatocytes treated with TGF-β1. Functional assays were performed on AML12 cells (untreated, TGF-β1 treated, or subjected to cholesterol enrichment [CE] or cholesterol depletion [CD]), and on mice injected with adenovirus-associated virus 8-control/TGF-β1. TGF-β1 inhibited messenger RNA expression of several cholesterol metabolism regulatory genes, including rate-limiting enzymes of cholesterol biosynthesis in AML12 cells, mouse primary hepatocytes, and adenovirus-associated virus-TGF-β1-treated mice. Total cholesterol levels and lipid droplet accumulation in AML12 cells and liver tissue also were reduced upon TGF-β1 treatment. Smad2/3 phosphorylation after 2 hours of TGF-β1 treatment persisted after CE or CD and was mildly increased after CD, whereas TGF-β1-mediated AKT phosphorylation (30min) was inhibited by CE. Furthermore, CE protected AML12 cells from several effects mediated by72hours of incubation with TGF-β1, including epithelial-mesenchymal transition, actin polymerization, and apoptosis. CD mimicked the outcome of long-term TGF-β1 administration, an effect that was blocked by an inhibitor of the type I TGF-β receptor. In addition, the supernatant of CE- or CD-treated AML12 cells inhibited or promoted, respectively, the activation of LX-2 hepatic stellate cells. TGF-β1 inhibits cholesterol metabolism whereas cholesterol attenuates TGF-β1 downstream effects in hepatocytes.

Transcriptome and metabolome analyses of Streptococcus gordonii DL1 under acidic conditions

ObjectivesStreptococcus gordonii is associated with the formation of biofilms, especially those that comprise dental plaque. Notably, S. gordonii DL1 causes infective endocarditis (IE). Colonization of this bacterium requires a mechanism that can tolerate a drop in environmental pH by producing acid via its own sugar metabolism. The ability to survive acidic environmental conditions might allow the bacterium to establish vegetative colonization even in the endocardium due to inflammation-induced lowering of pH, increasing the risk of IE. At present, the mechanism by which S. gordonii DL1 survives under acidic conditions is not thoroughly elucidated. The present study was thus conducted to elucidate the mechanism(s) by which S. gordonii DL1 survives under acidic conditions. MethodsWe analyzed dynamic changes in gene transcription and intracellular metabolites in S. gordonii DL1 exposed to acidic conditions, using transcriptome and metabolome analyses. ResultsTranscriptome analysis revealed upregulation of genes involved in heat shock response and glycolysis, and down regulation of genes involved in phosphotransferase systems and biosynthesis of amino acids. The most upregulated genes were a beta-strand repeat protein of unknown function (SGO_RS06325), followed by copper-translocating P-type ATPase (SGO_RS09470) and malic enzyme (SGO_RS01850). The latter two of these contribute to cytoplasmic alkalinization. S. gordonii mutant strains lacking each of these genes showed significantly reduced survival under acidic conditions. Metabolome analysis revealed that cytoplasmic levels of several amino acids were reduced. ConclusionsS. gordonii survives the acidic conditions by recovering the acidic cytoplasm using the various activities, which are regulated at the transcriptional level.

Salinity-driven changes in Salicornia cell wall nanomechanics and lignin composition

Widespread increases in soil salinization significantly reduce agricultural lands. Nevertheless, salt-tolerant plants, such as Salicornia europaea L., can play a crucial role in the reclamation of these lands. We selected S. europaea for this study due to its potential to mitigate soil salinization and its numerous applications, such as intercropping in agriculture, biocompounds production and bioenergy. This work was designed to determine whether different salinities induce significant biophysical, anatomical, lignin and gene transcript changes in S. europaea. Through atomic force microscopy (AFM), we revealed that salinity stimulated an increase in cell wall elasticity (CWE) as an important physiological mechanism of adaptation known as cell’s turgor conservation effect. Direct values of the cell wall stiffness subjected to salinity were obtained, with Young’s modulus (E) ranging from low and high salinity 0.52 to 0.03 MPa. The softening of the cell wall properly correlated with an increase in cell size, plant cells under strong salinity 1000 mM NaCl, swelled 5.4 times. The best salinity range found for its optimum growth was 200–400 mM NaCl. At higher salinities, we identified increases in the lignified xylem, large calcium oxalate crystals, and in transcript amounts of SeSOS1 and SeNHX1, which has a significant negative correlation with cell wall stiffness (−0.57 and −0.95, respectively). The high syringyl and guaiacyl ratio (S/G) in lignin for 0–400 mM NaCl may have influenced the rigidity and hydrophobicity of the cell walls. A positive S/G ratio and sugar yields are associated with higher bioethanol production, these values may also be useful for agriculture, biorefinery, biocompounds and plant-breeding applications. We conclude that salinity indeed influenced the cell wall traits of S. europaea. This halophyte is capable of markedly softening its cell wall as a way of adapting to the high level of salinity. Our presented insights and correlations not only provide a better understanding of cell wall remodelling but are also considered vital traits of adaptation strategies that this halophyte holds under salinity environment. These inputs can also be applied in future research aiming to produce biomass and biofuel or to improve the salinity tolerance during the cultivation of non-tolerant plants, crops and glycophytes, which is a significant challenge in agriculture, particularly in arid and coastal regions. The unique properties of the S. europaea cell walls could also inspire the development of materials with enhanced nanomechanical elasticity for resistance to osmotic stress.

Opioid-induced dysbiosis of maternal gut microbiota during gestation alters offspring gut microbiota and pain sensitivity

ABSTRACT There has been a rapid increase in neonates born with a history of prenatal opioid exposure. How prenatal opioid exposure affects pain sensitivity in offspring is of interest, as this may perpetuate the opioid epidemic. While few studies have reported hypersensitivity to thermal pain, potential mechanisms have not been described. This study posits that alterations in the gut microbiome may underly hypersensitivity to pain in prenatally methadone-exposed 3-week-old male offspring, which were generated using a mouse model of prenatal methadone exposure. Fecal samples collected from dams and their offspring were subjected to 16s rRNA sequencing. Thermal and mechanical pain were assessed using the tail flick and Von Frey assays. Transcriptomic changes in whole brain samples of opioid or saline-exposed offspring were investigated using RNA-sequencing, and midbrain sections from these animals were subjected to qPCR profiling of genes related to neuropathic and inflammatory pain pathways. Prenatal methadone exposure increased sensitivity to thermal and mechanical pain and elevated serum levels of IL-17a. Taxonomical analysis revealed that prenatal methadone exposure resulted in significant alterations in fecal gut microbiota composition, including depletion of Lactobacillus, Bifidobacterium, and Lachnospiracea sp and increased relative abundance of Akkermansia, Clostridium sensu stricto 1, and Lachnoclostridium. Supplementation of the probiotic VSL#3 in dams rescued hypersensitivity to thermal and mechanical pain in prenatally methadone-exposed offspring. Similarly, cross-fostering prenatally methadone-exposed offspring to control dams also attenuated hypersensitivity to thermal pain in opioid-exposed offspring. Modulation of the maternal and neonatal gut microbiome with probiotics resulted in transcriptional changes in genes related to neuropathic and immune-related signaling in whole brain and midbrain samples of prenatally methadone-exposed offspring. Together, our work provides compelling evidence of the gut-brain-axis in mediating pain sensitivity in prenatally opioid-exposed offspring.

High species homology potentiates quantitative inflammation profiling in zebrafish using immunofluorescence

Due to substantial homology between the human and zebrafish genome and a high level of conservation of the innate immune system across species, zebrafish larvae have become an invaluable research tool for studying inflammation and modelling inflammatory disease. However, further microscopy techniques need to be developed for better profiling of inflammation and in particular, integrated cytokine responses to different stimuli - approaches are currently largely limited to assessment of changes in cytokine gene transcription and in vivo visualisation using transgenics, which is limited in terms of the number of cytokines that may be assessed at once. In this study, after confirming substantial homology of human vs zebrafish cytokine amino acid sequences, immunofluorescence staining using antibodies directed at human cytokines was performed. Inflammatory cytokine signalling responses to experimental tailfin transection was assessed over 24 h (1 hpi (hours post injury), 2 hpi, 4 hpi, 24 hpi) in zebrafish larvae, with experimental end point at 120 h post fertilization (hpf). When immunofluorescence results were compared to responses observed in rodent and human literature, it is clear that the cytokines follow a similar response, albeit with a condensed total time course. Notably, tumor necrosis factor-α and monocyte chemoattractant protein-1 increased and remained elevated over the 24-h period. In contrast, interleukin-1β and interleukin-6 peaked at 4 hpi and 2 hpi respectively but had both returned to baseline levels by 24 hpi. Macrophage migration inhibitory factor was lowest at 1 hpi, potentially encouraging macrophage movement into the site of injury, followed by a sharp increase. This protocol provides valuable insight into inflammation over a time course and more so, provides an affordable and accessible method to comprehensively assess inflammation in zebrafish disease models.

Mef2c regulates bone mass through Sost-dependent and -independent mechanisms

Mef2c is a transcription factor that mediates key cellular behaviors that promote endochondral ossification and bone formation. Previously, Mef2c has been shown to regulate Sost transcription via its osteocyte-specific enhancer, ECR5, and conditional deletions of Mef2cfl/fl with either Col1-Cre or Dmp1-Cre produced generalized high bone mass (HBM) consistent with Van Buchem Disease phenotypes. However, Sost−/−; Mef2cfl/fl; Dmp1-Cre mice produced a significantly higher bone mass phenotype that Sost−/− alone suggesting that Mef2c modulates bone mass through additional mechanisms, independent of Sost. To identify new Mef2c transcriptional targets important in bone metabolism, we profiled gene expression by single-cell RNA sequencing in subpopulations of cells isolated from Mef2cfl/fl; Dmp1-Cre and Mef2cfl/fl; Bglap-Cre femurs, both strains exhibiting similar high bone mass phenotypes. However, we found Mef2cfl/fl; Bglap-Cre to also display a growth plate defect characterized by an expansion of several osteoprogenitor subpopulations. Differential gene expression analysis identified a total of 96 up- and 2434 down- regulated genes in Mef2cfl/fl; Bglap-Cre and 176 up- and 1041 down- regulated genes in Mef2cfl/fl; Dmp1-Cre bone cell subpopulations compared to wildtype mice. Mef2c deletion affected the transcriptomes across several cell types including mesenchymal progenitors (MP), osteoprogenitors (OSP), osteoblast (OB), and osteocyte (OCY) subpopulations. Several energy metabolism genes such as Uqcrb, Ndufv2, Ndufs3, Ndufa13, Ndufb9, Ndufb5, Cox6a1, Cox5a, Atp5o, Atp5g2, Atp5b, Atp5 were significantly down regulated in Mef2c-deficient OBs and OCYs, in both strains. Binding motif analysis of promoter regions of differentially expressed genes identified Mef2c binding in Bone Sialoprotein (BSP/Ibsp), a gene known to cause increased trabecular BV/TV in the femurs of Ibsp−/− mice. Immunohistochemical analysis confirmed the absence of Ibsp protein in OBs and OCYs. These findings suggests that the HBM in Sost−/−; Mef2cfl/fl; Dmp1-Cre is caused by a multitude of transcriptional changes in genes that regulate bone formation, two of which are Sost and Ibsp.

Resident microbes shape the vaginal epithelial glycan landscape.

Epithelial cells are covered in carbohydrates (glycans). This glycan coat or "glycocalyx" interfaces directly with microbes, providing a protective barrier against potential pathogens. Bacterial vaginosis (BV) is a condition associated with adverse health outcomes in which bacteria reside in direct proximity to the vaginal epithelium. Some of these bacteria, including Gardnerella, produce glycosyl hydrolase enzymes. However, glycans of the human vaginal epithelial surface have not been studied in detail. Here, we elucidate key characteristics of the "normal" vaginal epithelial glycan landscape and analyze the impact of resident microbes on the surface glycocalyx. In human BV, glycocalyx staining was visibly diminished in electron micrographs compared to controls. Biochemical and mass spectrometric analysis showed that, compared to normal vaginal epithelial cells, BV cells were depleted of sialylated N- and O-glycans, with underlying galactose residues exposed on the surface. Treatment of primary epithelial cells from BV-negative women with recombinant Gardnerella sialidases generated BV-like glycan phenotypes. Exposure of cultured VK2 vaginal epithelial cells to recombinant Gardnerella sialidase led to desialylation of glycans and induction of pathways regulating cell death, differentiation, and inflammatory responses. These data provide evidence that vaginal epithelial cells exhibit an altered glycan landscape in BV and suggest that BV-associated glycosidic enzymes may lead to changes in epithelial gene transcription that promote cell turnover and regulate responses toward the resident microbiome.

Trisomy 8 Remodels Chromatin and Activates Transcription of Runx1-Target Genes in Hematopoietic Stem Cell

Myelodysplastic syndrome (MDS) is a poor prognosis cancer that predominantly affects the elderly, arising from hematopoietic stem cell (HSC), resulting in bone marrow failure and predisposition to acute myelogenous leukemia (AML). It has long been known that numerical chromosome anomalies such as trisomy 8 are critical for the development of MDS. Trisomy 8 is known to be associated with relatively poor prognosis, and is a criterion for the diagnosis of MDS in patients. Systemic inflammation has been implicated in the selective advantage for clonal hematopoietic stem cells and the progression of MDS, such as trisomy 8 MDS associated with Behcet's disease. Next generation sequencing studies revealed that trisomy 8 MDS cells highly mutated RUNX1 transcriptional factor (TF) and ASXL1, an epigenetic modifier, both of which regulate HSC function and the differentiation, relative to MDS cells without trisomy 8, suggesting that those mutations may help trisomy 8 to drive the development of MDS. However, previous clinical studies failed to identify genes on the chromosome 8 commonly amplified among trisomy 8 MDS patients, which may account for the pathogenesis of MDS, the molecular mechanism of development of trisomy 8 MDS remains unclear. Thus, we hypothesized that trisomy 8 initiates the transformation by multiple malfunctions of epigenetic and transcriptional regulators in HSC other than a gene dosage effect due to the extra chromosome 8. In order to determine how trisomy 8 affects hematopoiesis, we established a new trisomy 8 model by introducing a human chromosome 8 to mimic trisomy 8 in mouse ES cells. We succeeded in generating trisomy 8 chimeric mice and confirmed the mice showing emergence of hematopoiesis in fetal liver but significantly decreased the chimerism of trisomy 8 cells than the control mice. Wecompetitively transplanted trisomy 8 fetal liver cells at E14.5 into lethally-irradiated wild-type primary recipient mice, and moreover serial transplanted hematopoietic stem and progenitor cells (HSPCs) which isolated from the primary recipient mice. We found that trisomy 8 reduced the self-renewal capacity of HSPC and impeded the differentiation, but it was not sufficient to develop myeloid malignancies in mice. To elucidate the molecular mechanism underlying the impaired hematopoiesis by trisomy 8, we performed RNA sequencing in HSCs isolated from the primary transplanted mice. Gene set enrichment analysis revealed that in comparison with the control HSCs, trisomy 8 HSCs had significantly positive enrichments in type I and type II interferon response genes, but also the canonical target genes of Runx1 and PU.1 TFs were positively enriched in trisomy 8 HSCs. In addition, ATAC-seq analysis revealed that trisomy 8 HSCs changed chromatin accessibility enriched with the binding motif of Runx TF, compared to control HSCs. Given the changes in chromatin accessibility in trisomy 8 HSC, we performed chromatin conformation capture sequencing (Hi-C) in HSPCs and found that trisomy 8 induced changes in chromatin structures such as topologically associating domain (TAD) and compartment A/B in the other chromosomes, which were partly associated with changes in transcription of genes. Since the Runx1 gene is critical for HSC integrity and differentiation, and loss-of-function mutations of RUNX1 were often found in human myeloid malignancies with trisomy 8, we attempted to determine whether Runx1 deletion cancel the impaired self-renewal capacity of trisomy 8 HSC. We found that Runx1 deletion partially canceled the impaired repopulating capacity of trisomy 8 cells in recipient mice, suggesting the selective advantage of loss of function mutation of the RUNX1 gene in trisomy 8 mosaic people and patients with trisomy 8 MDS. Furthermore, as Janus kinase (JAK) and TANK-binding kinase 1 (TBK1) are critical for interferon signalling and activation of their downstream target genes, we revealed that the inhibition of either JAK or TBK function ameliorated trisomy 8-induced chromatin remodelling in HSCs. Overall, trisomy 8 changes chromatin structure and transcription of genes on other chromosomes in HSC accompanied with the malfunction of Runx1 TF, at least in part, due to the activation of interferon signaling, and results in the impaired hematopoiesis. We demonstrated that trisomy 8 creates the basis for transformation of MDS via remodeling chromatin structures in HSC.

Effects of environmentally relevant concentrations of niclosamide on lipid metabolism and steroid hormone synthesis in adult female zebrafish

Niclosamide (NIC) is a commonly used molluscicide that reportedly disrupts the endocrine system and may lead to lipid metabolism disorders. However, few studies have investigated the mechanism by which NIC affects the endocrine system from the perspective of lipid metabolism. Adult female zebrafish were fed either a normal-fat diet (NFD) or a high-fat diet (HFD) and then exposed for 28 days to environmentally relevant concentrations of NIC. NIC accumulated most in the liver followed by the brain and then the gonads in both feeding conditions. Somatic index changes confirmed that HFD promotes fish growth, and NIC administration inhibits it. Lipid metabolites were decreased by NIC, as were levels of pregnenolone, androstenedione, estrogen, testosterone, and estradiol, suggesting that NIC impacted steroidogenesis. In addition, gene transcription changes related to the hypothalamic-pituitary-gonad-liver (HPGL) axis and altered ovarian histology strongly suggest that environmental relevant concentrations of NIC exposure may disrupt endocrine function. These findings highlighted that NIC exposure at environmentally relevant concentrations elicited endocrine-disruption effects may through impairing of steroid hormone synthesis.