Reporter Gene Research Articles

Swimming upregulates APOL3 through regulating macrophage polarization to inhibit glycolysis and the development of melanoma.

This study investigated the role of swimming exercise in regulating melanoma tumour growth and glycolysis in cancer cells, the specific mechanism involved was also studied. In our study, a murine melanoma tumour model was established to assess the impact of swimming on tumour growth. The mRNA and protein expressions were assessed using qRT-PCR, western blot, and IHC. The metabolic behavior of melanoma cells was examined through lactic acid level measurements and glucose consumption assessments. CCK-8 and colony formation assays were used to detect cell viability and proliferation. ELISA was employed to determine the levels of cytokines secreted by macrophages. The interaction between APOL3 and STAT3 was analyzed by dual luciferase reporter gene and ChIP assays. Our results demonstrated that swimming exercise suppressed melanoma growth in mice by suppressing glycolysis, which might be related to APOL3 upregulation. In addition, downregulation of APOL3 in melanoma was associated with poor prognosis, and APOL3 overexpression markedly suppressed melanoma cell proliferation by reducing glucose uptake and lactate production in vitro. Mechanistically, STAT3 directly down-regulated APOL3 transcription. Swimming upregulated APOL3 by inactivating the IL-6R-STAT3 signaling axis in melanoma cells by inhibiting the secretion of IL-6 by M2 macrophages. As expected, IL-6 secreted by M2 macrophages promoted glycolysis in melanoma cells by reducing APOL3 expression. In summary, swimming inactivated the IL-6R/STAT3 signaling axis in melanoma cells by inhibiting the secretion of IL-6 by M2 macrophages, which could suppress the growth of melanoma in the body by upregulating APOL3 to inhibit glycolysis.

Transcription Factor MAZ Potentiates the Upregulated NEIL3-mediated Aerobic Glycolysis, thereby Promoting Angiogenesis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is characterized by high vascularity and notable abnormality of blood vessels, where angiogenesis is a key process in tumorigenesis and metastasis. The main functions of Nei Like DNA Glycosylase 3 (NEIL3) include DNA alcoholization repair, immune response regulation, nervous system development and function, and DNA damage signal transduction. However, the underlying mechanism of high expression NEIL3 in the development and progression of HCC and whether the absence or silencing of NEIL3 inhibits the development of cancer remain unclear. Therefore, a deeper understanding of the mechanisms by which increased NEIL3 expression promotes cancer development is needed. Expression of NEIL3 and its upstream transcription factor MAZ in HCC tumor tissues was analyzed in bioinformatics efforts, while validation was done by qRT-PCR and western blot in HCC cell lines. The migration and tube formation capacity of HUVEC cells were analyzed by Transwell and tube formation assays. Glycolytic capacity was analyzed by extracellular acidification rate, glucose uptake, and lactate production levels. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter gene assays were utilized to investigate specific interactions between MAZ and NEIL3. NEIL3 and MAZ were substantially upregulated in HCC tissues and cells. NEIL3 was involved in modulating the glycolysis pathway, suppression of which reversed the stimulative impact of NEIL3 overexpression on migration and angiogenesis in HUVEC cells. MAZ bound to the promoter of NEIL3 to facilitate NEIL3 transcription. Silencing MAZ reduced NEIL3 expression and suppressed the glycolysis pathway, HUVEC cell migration, and angiogenesis. MAZ potentiated the upregulated NEIL3-mediated glycolysis pathway and HCC angiogenesis. This study provided a rationale for the MAZ/NEIL3/glycolysis pathway as a possible option for anti-angiogenesis therapy in HCC.

Long Non-coding RNA DLEU1 Promotes Progression of Osteoarthritis via miR-492/TLR8 Axis.

Long non-coding RNAs (LncRNAs) are generally reported to participate in the development of Osteoarthritis (OA) by acting as competing endogenous RNAs (ceRNAs). However, the molecular mechanism is largely unknown. This study aimed to investigate the possible mechanisms contributing to osteoarthritis (OA). Four gene expression profiles from patients with OA were downloaded from a public database and integrated to screen important RNAs associated with OA. Differentially expressed (DE) lncRNAs, microRNAs (miRNAs), and mRNAs were filtered, and a ceRNA network was constructed. An in vitro OA model was established by treating chondrocytes with IL-1β. The expression levels of MMP-13, COL2A1, aggrecan, and RUNX2 were detected by qRT-PCR and western blot. Cell proliferation ability was detected by CCK-8 assay. Flow cytometry was used for apoptosis assay. A dual luciferase reporter gene was used to confirm the relationship between DLEU1, miR-492, and TLR8. An OA-related ceRNA network, including 11 pathways, 3 miRNAs, 7 lncRNAs, and 16 mRNAs, was constructed. DLEU1 and TLR8 were upregulated, and miR-492 was downregulated in IL-1β-induced chondrocytes. Overexpression of DLEU1 suppressed viability and promoted apoptosis and extracellular matrix (ECM) degradation in IL-1β induced chondrocytes. Luciferase reporter assay validated the regulatory relations among DLEU1, miR-492, and TLR8. Further study revealed that the effects of DLEU1 on chondrocytes could be reversed by miR-492. DLEU1 may be responsible for the viability, apoptosis, and ECM degradation in OA via miR-492/TLR8 axis.

Naringin promotes osteogenic potential in bone marrow-derived mesenchymal stem cells via mediation of miR-26a/Ski axis

BackgroundOsteonecrosis of the femoral head (ONFH) is a common orthopedic disease, which seriously affects the quality of life of patients. Naringin has protective effect on ONFH. In present study, we aimed to investigate the mechanism of Naringin regulating miR-26a in ONFH. MethodsTwo sequencing datasets (GSE89587 for micro-RNA, GSE123568 for mRNA) related to ONFH were obtained from the GEO database for bioinformatics analysis. Bone marrow-derived mesenchymal stem cells (BMSCs) were treated with adipogenic medium (AM) or osteogenic medium (OM), and then treated with 10 μM, 100 μM or 1000 μM Naringin. Gene and protein levels were detected by RT-qPCR and Western blotting. ALP activity and alizarin red staining (ARS) were applied to investigate the osteogenic differentiation of BMSCs. Oil red O staining was performed to test adipogenic differentiation. The content of triglycerides (TG) in BMSCs was detected by TG detection kit. Double luciferase reporter gene measured the interaction between miR-26a and Ski. ResultsBioinfomatic analyses indicated a significant downregulation of miR-26a and a significant upregulation of Ski in the peripheral blood of patients with ONFH. Naringin significantly promoted the osteogenic differentiation, and increased the ALP activity and ARS. Meanwhile, it decreased the adipogenic differentiation and inhibited TG levels. In addition, miR-26a was downregulated and Ski was increased in AM-treated BMSCs, while miR-26a was upregulated and Ski was decreased in OM-treated BMSCs. Furthermore, miR-26a promoted the osteogenic differentiation and suppressed the adipogenic differentiation in BMSCs. Moreover, Naringin enhanced osteogenic potential in BMSCs was reversed by knockdown of miR-26a or overexpression of Ski. ConclusionNaringin could promote osteogenic differentiation of BMSCs via mediation of miR-26a/Ski axis. Thereby, Naringin might be a new agent for ONFH treatment.

Diagnostic significance of serum hsa_circ_0000745 and hsa_circ_0001459 in ischemic stroke and its role in the prognosis of interventional therapy

ABSTRACT Objective We aimed to identify hsa_circ_0000745 and hsa_circ_0001459 expression, value as biomarkers in ischemic stroke (IS), and functions in BV2 cells. Methods RNA sequencing datasets in the GEO database were retrieved. The expression of circulating hsa_circ_0000745 and hsa_circ_0001459 was validated by RT-qPCR. The predictive values of hsa_circ_0000745 and hsa_circ_0001459 in the diagnosis and outcome of acute IS were evaluated using receiver operator characteristic curve analysis. BV2 cells were treated with lipopolysaccharide, followed by hsa_circ_0000745 or hsa_circ_0001459 downregulation and subsequent migration and apoptosis assay. The downstream miR-1287-5p was detected using the luciferase reporter gene assay. Results Hsa_circ_0000745 or hsa_circ_0001459 were upregulated in acute IS. Hsa_circ_0000745 or/and hsa_circ_0001459 differentiated between healthy control subjects and patients with IS, resulting in areas under curve (AUC) of 0.85 and 0.83, respectively. Hsa_circ_0000745 or hsa_circ_0001459 was positively correlated with serum pro-inflammatory cytokines and the NIHSS (P<0.001). Longitudinal and ROC analyses of hsa_circ_0001459 and hsa_circ_0000745 expression levels revealed the 90-day-outcome-predicting potential after stroke. Hsa_circ_0001459 and hsa_circ_0000745 promoted the apoptosis and inhibited the migration of LPS-induced BV2 cells. Hsa_circ_0001459 and hsa_circ_0000745 commonly sponged miR-1287-5p. Conclusions Hsa_circ_0001459 and hsa_circ_0000745 showed upregulations in IS and might have clinical utility as a diagnostic and outcome-predicting marker.

Marburg Virus Minigenome Assays.

This chapter describes minigenome systems for Marburg virus (MARV), which reconstitute the viral polymerase complex functions of gene expression and genome replication. Procedures covered herein include passage and seeding of cells, transfection, sample collection, and reporter gene assays.

Bacterial WYL domain transcriptional repressors sense single-stranded DNA to control gene expression.

Bacteria encode a wide array of immune systems to protect themselves against ubiquitous bacteriophages and foreign DNA elements. While these systems' molecular mechanisms are becoming increasingly well known, their regulation remains poorly understood. Here, we show that an immune system-associated transcriptional repressor of the wHTH-WYL-WCX family, CapW, directly binds single-stranded DNA to sense DNA damage and activate expression of its associated immune system. We show that CapW mediates increased expression of a reporter gene in response to DNA damage in a host cell. CapW directly binds single-stranded DNA by-products of DNA repair through its WYL domain, causing a conformational change that releases the protein from double-stranded DNA. In an Escherichiacoli CBASS system with an integrated capW gene, we find that CapW-mediated transcriptional activation is important for this system's ability to prevent induction of a λ prophage. Overall, our data reveal the molecular mechanisms of WYL-domain transcriptional repressors, and provide an example of how bacteria can balance the protective benefits of carrying anti-phage immune systems against the inherent risk of these systems' aberrant activation.

ERD14 regulation by the HY5- or HY5-MED2 module mediates the cold signal transduction of asparagus bean.

Cold stress affects the growth, development, and yield of asparagus bean (Vigna unguiculata subsp. sesquipedalis). Mediator (MED) complex subunits regulate the cold tolerance of asparagus bean, but the underlying regulatory mechanisms remain unclear. Here, VunMED2 positively responds to cold stress of asparagus beans. Under cold acclimation and freezing treatment, the survival rate, ROS scavenging activity, and expression levels of VunMED2 were increased in VunMED2 transgenic plants. Natural variation in the promoter of VunMED2 in two different cold-tolerant asparagus beans was observed. Under cold stress, the expression of the GUS reporter gene was higher in cold-tolerant plants than in cold-sensitive plants, and the expression of the GUS reporter gene was tissue-specific. VunHY5 positively influenced the expression of VunMED2 by binding to the E-box motif, and the transcriptional activation of the promoter was stronger in the cold-tolerant variety than in cold-sensitive plants. VunHY5 overexpression improved plant freezing resistance by increasing the antioxidant capacity and expression of dehydrin genes. VunHY5 and VunMED2 play a synergistic role in binding to the G-box/ABRE motif and transcriptionally activating the expression of VunERD14. VunERD14 complemented the med2 mutant, which could positively respond to plant freezing resistance by reducing membrane lipid peroxidation and improving the antioxidant capacity. Therefore, the VunHY5-VunERD14 module and the VunHY5-VunMED2-VunERD14 positive cascade effect are involved in the cold signal transduction in asparagus bean. Our findings have implications for the breeding of asparagus bean varieties with improved cold tolerance.

Structural and Functional Implications of Polyarginine Addition to Green Fluorescent Protein Expression in Saccharomyces cerevisiae INVSc1

Green Fluorescent Protein (EGFP) is widely used as a reporter gene, aiding in protein recovery and transduction studies. In this study, EGFP was tagged with eleven arginine residues (PolyR) and six histidine residues (His-tag) for purification. The aim was to enhance the synthesis of EGFP-PolyR in Saccharomyces cerevisiae and evaluate the effects of polyarginine modification on protein stability and expression levels. The expression of EGFP and EGFP-PolyR in S. cerevisiae was assessed through fluorescence measurements and protein levels. Structural analyses were conducted using in silico tools to investigate changes in beta strands and helices, which were validated through Western blots. Results showed that EGFP-PolyR maintained similar fluorescence levels to EGFP, but with notable structural changes. EGFP-PolyR's final beta strand terminates at Ala228, compared to Gly229 in EGFP, affecting the beta sheet's stability. Structural modifications also included altered helix lengths, with a longer helix 10 and shorter helix 9 in EGFP-PolyR. These alterations, along with shifts in helix-helix interactions, contribute to destabilization. Additionally, EGFP-PolyR exhibited unique gamma coils absent in EGFP, further differentiating its structure. The structural changes led to decreased protein expression and solubility, as indicated by Western blot analysis, with EGFP-PolyR showing significantly lower expression levels. The findings suggest that EGFP-PolyR is prone to aggregation and misfolding, characteristics often associated with aggregation-prone proteins.In conclusion, the polyarginine modification significantly impacts the structural integrity, stability, and solubility of EGFP. While fluorescence is retained, these changes hinder protein detectability and purification, highlighting the importance of considering structural alterations when modifying reporter proteins for experimental use.

LDHB Mediates Histone Lactylation to Activate PD-L1 and Promote Ovarian Cancer Immune Escape

Background To investigate the effects of LDHB on lactylation of programmed cell death 1 ligand (PD-L1) and immune evasion of ovarian cancer. Methods Ovarian cancer cells were transfected with LDHB siRNA and cultured with primed T cells. Cell proliferation and viability were measured by cell counting kit 8 (CCK-8) and colony formation assay. The production of immune factors was detected by enzyme-linked immunosorbent assay (ELISA). The histone lactylation and activity of PD-L1 promoter were measured by chromatin immunoprecipitation (ChIP)-qPCR assay and luciferase reporter gene assay, respectively. Results Knockdown of LDHB notably inhibited the growth, glucose uptake, lactate production, and ATP production of ovarian cancer cells. Knockdown of LDHB enhanced the killing effects of T cells, led to increased production of immune activation factors IL-2, TNF-α, and IFN-γ, as well as elevated the levels of granzyme B and perforin. Mechanical study identified that LDHB regulated the H3K18 lactylation (H3K18la) modification on PD-L1 promoter region to promote its expression. Overexpression of PD-L1 abolished the immune activation effects that induced by siLDHB. Conclusion The LDHB modulated lactate production and the histone lactylation on PD-L1 promoter, which ultimately regulated its expression and participated in the immune evasion of ovarian cancer cells.

MiR-185-5p is Involved in Regulating the Abnormal Proliferation of Retinal Microvascular Endothelial Cells via Targeting CXCR4.

This study aimed to explore the expression profile of miR-185-5p in proliferative DR (PDR), and further evaluate its diagnostic value and possible mechanism of miR-185-5p in PDR. The level of miR-185-5p was detected by qRT-PCR. The ROC curve was established to estimate the diagnostic ability of miR-185-5p. Transwell experiment and cell counting kit-8 (CCK-8) assays were conducted to assess the effect of miR-185-5p on the migration and proliferation of human retinal endothelial cells (HRECs) induced by high glucose. Enzyme linked immunosorbent assay (ELISA) was used to detect the concentrations of inflammatory factors. The luciferase reporter gene experiment was used to prove the interaction between miR-185-5p and CXCR4. Compared to the control group, the expression of miR-185-5p was significantly up-regulated in both the type 2 diabetes mellitus (T2DM) group and the PDR groups, with higher levels in the PDR group than in the T2DM group. The ROC curve reveals that serum miR-185-5p can distinguish PDR patients from T2DM patients. MiR-185-5p levels in HRECs increased significantly after high glucose induction. High glucose induction also promoted the migration, proliferation and inflammation response of HRECs. However, when the intracellular miR-185-5p level was down-regulated by miR-185-5p inhibitor transfection, these effects were inhibited. The luciferase reporter gene assay showed that miR-185-5p directly targets CXCR4. The expression of miR-185-5p is out of balance in PDR and it may be involved in regulating the migration and proliferation of HRECs by regulating CXCR4.

Luciferase-mediated assay to detect the PAMP-triggered gene expression in transgenic Nicotiana benthamiana

ABSTRACT Luciferase is one of the bioluminescence-producing agents, which was widely used as a reporter enzyme for constructing bioassay systems to study gene expression with high accuracy and within a broad dynamic spectrum. Perception of pathogen associated molecular patterns (PAMPs) in plants often lead to significant transcriptional changes. The transcriptional changes of defense-related genes are often used as a marker to assay PAMP-triggered plant immune response. In this study, we showed that the marker gene CYP71D20 was rapidly activated in Nicotiana benthamiana upon treatment with the bacterial PAMP flg22 and the Phytophthora elicitin INF1. In addition, we generated transgenic N. benthamiana using the luciferase as a reporter gene to analyze CYP71D20 gene expression upon PAMP treatment. The transgenic line carrying the luciferase gene driven by CYP71D20 promoter was treated with the bacterial PAMP flg22 or Phytophthora elicitin INF1. Transcriptional activation of CYP71D20 was measured by monitoring the luciferase activity. The results showed that the LUC activity was increased after treatment with different PAMPs, indicating that the CYP71D20-promotor luciferase assay can be used to study the PAMP-triggered gene expression in N. benthamiana.

Functions of nucleolar complex associated 4 homolog in activated T cells

Nucleolar complex associated 4 homolog (NOC4L) is a key factor in ribosome biogenesis, and this study aims to investigate its roles in activated T cells from the perspective of translation regulation. Firstly, flow cytometry was employed to determine the expression levels of NOC4L in the CD4+ T cells under different conditions in the transgenic reporter mice expressing Noc4lmCherry. Subsequently, the expression of NOC4L along with cell proliferation was examined under Th1 and Th17 polarization conditions. Finally, in vitro experiments were conducted to identify the proteins interacting with NOC4L during the activation of Th1 and Th17 cells, on the basis of which the potential mechanisms of NOC4L were explored. The results showed that the expression level of NOC4L increased in activated CD4+ T cells, and the expression of NOC4L was closely associated with the proliferation and division of activated T cells. The in vitro experiments revealed interactions between NOC4L and proteins involved in ribosome assembly and cell proliferation during T cell activation. These findings lay a foundation for probing into the post-transcriptional regulation in helper T cells and hold profound significance for understanding the activation and regulatory mechanisms of T cells.

Features of Highly Homologous T-Cell Receptor Repertoire in the Immune Response to Mutations in Immunogenic Epitopes

CD8+ T-cell immunity, mediated through interactions between human leukocyte antigen (HLA) and the T-cell receptor (TCR), plays a pivotal role in conferring immune memory and protection against viral infections. The emergence of SARS-CoV-2 variants presents a significant challenge to the existing population immunity. While numerous SARS-CoV-2 mutations have been associated with immune evasion from CD8+ T cells, the molecular effects of most mutations on epitope-specific TCR recognition remain largely unexplored, particularly for epitope-specific repertoires characterized by common TCRs. In this study, we investigated an HLA-A*24-restricted NYN epitope (Spike448-456) that elicits broad and highly homologous CD8+ T cell responses in COVID-19 patients. Eleven naturally occurring mutations in the NYN epitope, all of which retained cell surface presentation by HLA, were tested against four transgenic Jurkat reporter cell lines. Our findings demonstrate that, with the exception of L452R and the combined mutation L452Q + Y453F, these mutations have minimal impact on the avidity of recognition by NYN peptide-specific TCRs. Additionally, we observed that a similar TCR responded differently to mutant epitopes and demonstrated cross-reactivity to the unrelated VYF epitope (ORF3a112-120). The results contradict the idea that immune responses with limited receptor diversity are insufficient to provide protection against emerging variants.

Conserved glucokinase regulation in zebrafish confirms therapeutic utility for pharmacologic modulation in diabetes

Glucokinase (GCK) is an essential enzyme for blood glucose homeostasis. Because of its importance in glucose metabolism, GCK is considered an attractive target for the development of antidiabetic drugs. However, a viable therapeutic agent has still to emerge, prompting efforts to improve understanding of the complex regulation and biological effects of GCK. Using the vertebrate organism zebrafish, an attractive model to study metabolic diseases and pharmacological responses, we dissected the complexities of gck regulation and unraveled effects of Gck modulation. We found that while gck expression in zebrafish islet cells is constitutive, gck expression in the liver is regulated by nutritional status, confirming similarity to the mammalian system. A combination of transgenic gck reporter lines and our diabetes model, the pdx1 mutant, allowed monitoring of gck expression under pathological conditions, revealing reduced gck expression and activity in the liver, which was unresponsive to nutrient stimulation, and decreased expression in the islet due to the reduced number of β-cells. Gck activation substantially ameliorated hyperglycemia in pdx1 mutants, without inducing oxidative stress responses in liver or islet. In-depth characterization of Gck activity and regulation at the cellular level in a whole-organism diabetes model clarifies its applicability as a drug target for therapies.

Cellular retinoic acid binding proteins regulate germ cell proliferation and sex determination in zebrafish.

Cellular retinoic acid (RA) binding proteins (Crabps) solubilize intracellular RA and transport it to its nuclear receptors or cytoplasmic degradation enzymes. Despite their extreme conservation across chordates, genetic studies of Crabp function have revealed few essential functions. We have generated loss-of-function mutations in all four zebrafish Crabps and find essential roles for Crabp2s in gonad development and sex determination. Transgenic RA reporters show strong RA-responses in germ cells at the bipotential stage of gonad development. Double mutants lacking the functions of both Crabp2a and Crabp2b predominantly become male, which correlates with their smaller gonad size and reduced germ cell proliferation during gonad development at late larval and early juvenile stages. In contrast, mutants lacking the functions of both Crabp1a and Crabp1b have normal sex ratios. Exogenous RA treatments at bipotential gonad stages increase germ cell number, consistent with a direct role for RA in promoting germ cell proliferation. Our results suggest essential functions for Crabps in gonad development and sex determination.

Noninvasive Imaging of Transgene Expression in Neurons Using Chemical Exchange Saturation Transfer MRI.

Advances in gene therapy, especially for brain diseases, have created new imaging demands for noninvasive monitoring of gene expression. While reporter gene imaging using co-expression of fluorescent protein-encoding gene has been widely developed, these conventional methods face significant limitations in longitudinal invivo applications. Magnetic resonance imaging (MRI), specifically chemical exchange saturation transfer (CEST) MRI, provides a robust noninvasive alternative that offers unlimited depth penetration, reliable spatial resolution, and specificity toward particular molecules. In this study, we explore the potential of CEST-MRI for monitoring gene expression in neurons. We designed a CEST polypeptide reporter expressing 150 arginine residues and evaluated its expression in the living brain after viral vector delivery. A longitudinal study performed at one and 2 months postinjection showed that specific CEST signal was observable. In particular, the CEST contrast exhibited distinct peaks at 0.75 and 1.75 ppm, consistent with the expected hydroxyl and guanidyl protons resonance frequencies. Histological study confirmed the specific neuronal expression of the transgene evidenced by the fluorescence signal from the td-Tomato fluorophore fused to the polypeptide. The ability to image noninvasively a neuron-specific CEST-MRI reporter gene could offer valuable insights for further developments of gene therapy for neurological disorders.

Antibiotic tolerance due to restriction of cAMP-Crp regulation by mannitol, a non-glucose-family PTS carbon source.

Enzyme-IIA (EIIAGlc, Crr) of the phosphotransferase system (PTS) connects the uptake of glucose-family sugars to the cAMP-Crp regulatory cascade; phosphorylated EIIAGlc enhances cAMP-Crp activity, which then contributes to the antibiotic-mediated accumulation of reactive oxygen species (ROS) and cell death. Defects in PTS cause antibiotic and disinfectant tolerance. We report that mannitol, a carbon source whose uptake does not use EIIAGlc, reduces antibiotic-mediated killing of Escherichia coli without affecting antibiotic minimal inhibitory concentration. Thus, mannitol promotes antibiotic tolerance. The tolerance pathway was defined by the loss of ciprofloxacin lethality from the deletion of ptsI (first gene in PTS), mtlA (mannitol-specific Enzyme-II), cyaA (cAMP synthase), and crp (cAMP receptor protein) but not crr (EIIAGlc). A crp* mutant, which encodes a constitutively active Crp that bypasses the need for cAMP activation, also decreased mannitol-mediated antibiotic tolerance, as did exogenous cAMP. Thus, inhibition of antibiotic lethality by mannitol involves both PTS-mediated mannitol uptake and suppression of cAMP-Crp action, independent of EIIAGlc. Mannitol suppressed the downstream antibiotic-mediated transcription of genes involved in NADH production and cellular respiration, expression of a superoxide reporter gene (soxS), and accumulation of antibiotic-mediated ROS. Similar phenomena were observed with mannose and sorbitol, demonstrating that non-glucose PTS carbon sources can cause antibiotic tolerance by a novel path that reduces the ROS-promoting activity of cAMP-Crp. The work emphasizes that antibiotic tolerance, which contributes to disease relapse and the need for prolonged antibiotic treatment, can result from commonly consumed carbohydrates. This finding, plus mutations that interfere specifically with antibiotic lethality, makes tolerance a high probability event.IMPORTANCEBacterial tolerance constitutes a significant threat to anti-infective therapy and potentially to the use of disinfectants. Deficiency mutations that reduce glucose uptake, central carbon metabolism, and cellular respiration confer antibiotic/disinfectant tolerance by reducing the accumulation of reactive metabolites, such as reactive oxygen species. We identified novel environmental generators of tolerance by showing that non-glucose carbohydrates, such as mannitol, mannose, and sorbitol, generate tolerance to multiple antibiotic classes. Finding that these sugars inhibit a universal, stress-mediated death pathway emphasizes the potential danger of compounds that block the lethal response to severe stress. Immediate practical importance derives from mannitol being a popular food sweetener, a treatment for glaucoma, and a dehydrating agent for treating cerebral edema, including cases caused by bacterial infection: antibiotic tolerance could contra-indicate the use of mannitol and related carbohydrates during antibiotic treatment. Overall, the work shows that the presence of sugars must be considered during antimicrobial and perhaps disinfectant use.

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.

Mechanism of MiR-145a-3p/Runx2 pathway in dexamethasone impairment of MC3T3-E1 osteogenic capacity in mice.

In this experiment, we screened key miRNAs involved in the dexamethasone-induced decrease in osteogenic capacity of mouse precursor osteoblasts MC3T3-E1 over and investigated their specific regulatory mechanisms. In this experiment, cell counting kit assay was utilized to act on MC3T3-E1 cells at 0, 5μM, 10μM, 15μM concentrations of dexamethasone for 24h, 48h and 72h to observe the changes in cell viability in order to select the appropriate dexamethasone concentration. Apoptosis and reactive oxygen species were detected by flow cytometry. The transcription of osteogenesis-related genes (Runx2, ALP, OCN, OPN, OPG, COL1A1) and protein expression levels (Runx2, ALP, OCN, OPN) were detected by Western Blot and qRT-PCR to validate the changes in cellular osteogenesis. The differentially expressed miRNAs related to MC3T3-E1 osteogenic differentiation after dexamethasone action were screened out. The expression levels of selected target miRNAs were verified in the experimental group and the control group by qRT-PCR. The miRNA inhibitor was transfected to knock down miRNA in dexamethasone-induced MC3T3-E1 injury. Alkaline phosphatase staining and flow cytometry were performed to detect apoptosis and reactive oxygen species changes. transcript and protein expression levels of osteogenesis-related genes in mouse MC3T3-E1 were detected by qRT-PCR and Western blot experiments. By miRNA target gene prediction, luciferase reporter gene experiments, qRT-PCR and Western blot experiments were used to verify whether the selected target miRNAs targeted the target gene. First, it was determined that 10μM dexamethasone solution was effective in inducing a decrease in osteogenic function in mouse MC3T3-E1 by CCK8 experiments, which showed a significant decrease in alkaline phosphatase activity, a decrease in calcium nodules as shown by alizarin red staining, an increase in apoptosis and reactive oxygen species as detected by flow cytometry, as well as a decrease in the expression of osteogenesis-related genes and proteins. Five target miRNAs were identified: miR-706, miR-296-3p, miR-7011-5p, miR-145a-3p, and miR-149-3p. miR-145a-3p, which had the most pronounced and stable expression trend and was the most highly expressed miRNA, was chosen as the target of this experiment by qRT-PCR analysis. -145a-3p, as the subject of this experiment. Knockdown of miR-145a-3p in MC3T3-E1 cells after dexamethasone action significantly improved the expression of their impaired osteogenic indicators. It was shown that after knocking down the target miRNA, alkaline phosphatase staining was significantly increased compared with the dexamethasone-stimulated group and approached the level of the blank control group. Meanwhile, the expression of osteogenic function-related proteins and genes also increased in the dexamethasone-stimulated group after knocking down miR-145a-3p, and approached the level of the blank control group. A direct targeting relationship between miR-145a-3p and Runx2 was indeed confirmed by luciferase reporter gene assays, qRT-PCR and Western blot experiments. The results indicated that dexamethasone impaired the osteogenic differentiation ability of MC3T3-E1 cells by inducing the up-regulation of miR-145a-3p expression. MiR-145a-3p inhibited the osteogenic differentiation ability of MC3T3-E1 cells by targeting and suppressing the expression level of Runx2 protein. Inhibition of miR-145a-3p levels significantly improved the osteogenic differentiation ability of MC3T3-E1 cells.