Mouse Assay Research Articles

Oleuropein-driven reprogramming of the myeloid cell compartment to sensitise tumours to PD-1/PD-L1 blockade strategies

BackgroundPrevious studies have shown that functional systemic immunity is required for the efficacy of PD-1/PD-L1 blockade immunotherapies in cancer. Hence, systemic reprogramming of immunosuppressive dysfunctional myeloid cells could overcome resistance to cancer immunotherapy.MethodsReprogramming of tumour-associated myeloid cells with oleuropein was studied by quantitative differential proteomics, phenotypic and functional assays in mice and lung cancer patients. Combinations of oleuropein and two different delivery methods of anti-PD-1 antibodies were tested in colorectal cancer tumour models and in immunotherapy-resistant lung cancer models.ResultsOleuropein treatment reprogrammed monocytic and granulocytic myeloid-derived suppressor cells, and tumour-associated macrophages towards differentiation of immunostimulatory subsets. Oleuropein regulated major differentiation programmes associated to immune modulation in myeloid cells, which potentiated T cell responses and PD-1 blockade. PD-1 antibodies were delivered by two different strategies, either systemically or expressed within tumours using a self-amplifying RNA vector. Combination anti-PD-1 therapies with oleuropein increased tumour infiltration by immunostimulatory dendritic cells in draining lymph nodes, leading to systemic antitumour T cell responses. Potent therapeutic activities were achieved in colon cancer and lung cancer models resistant to immunotherapies, even leading to complete tumour regression.DiscussionOleuropein significantly improves the outcome of PD-1/PD-L1 blockade immunotherapy strategies by reprogramming myeloid cells.

Sweet taste receptors play roles in artificial sweetener-induced enhanced urine output in mice

Sweet taste receptors found in oral and extra oral tissues play important roles in the regulation of many physiological functions. Studies have shown that urine volume increases during the lifetime exposure to artificial sweeteners. However, the detailed molecular mechanism and the general effects of different artificial sweeteners exposure on urine volume remain unclear. In this study, we investigated the relationship between urinary excretion and the sweet taste receptor expression in mice after three artificial sweeteners exposure in a higher or lower concentration via animal behavioral studies, western blotting, and real-time quantitative PCR experiment in rodent model. Our results showed that high dose of acesulfame potassium and saccharin can significantly enhance the urine output and there was a positive correlation between K+ and urination volume. The acesulfame potassium administration assay of T1R3 knockout mice showed that artificial sweeteners may affect the urine output directly through the sweet taste signaling pathway. The expression of T1R3 encoding gene can be up-regulated specifically in bladder but not in kidney or other organs we tested. Through our study, the sweet taste receptors, distributing in many tissues as bladder, were indicated to function in the enhanced urine output. Different effects of long-term exposure to the three artificial sweeteners were shown and acesulfame potassium increased urine output even at a very low concentration.

Determination of pharmacological inhibition of ALDH2 by ethanol clearance in mice

ObjectivesRetinoic acid plays diverse physiological and pathophysiological roles in reproduction, immune function, energy metabolism and carcinogenesis. Because of the potential benefits of inhibiting retinoic acid biosynthesis in certain disease states, efforts are underway to develop inhibitors of retinoic acid biosynthesis via inhibition of the aldehyde dehydrogenase-1 A (ALDH1A) family of enzymes. However, many potential ALDH1A inhibitors also inhibit the related ALDH2 enzyme that plays a role in the metabolism of ethanol. Accurate in vitro assessment of ALDH2 inhibition is problematic, and to date, there are no published in vivo assays to determine inhibition of ALDH2 by candidate ALDH1A inhibitors. Study designTo address this, we developed a novel gas-chromatography-mass-spectrometry ethanol clearance assay in mice using orally administered ethanol and serial measurement of ethanol over time. We then used this assay to determine pharmacological inhibition of ALDH2 by candidate ALDH1A inhibitors. ResultsEthanol clearance in untreated male mice occurs within sixty minutes. Male mice treated with WIN 18,446, a known ALDH1A inhibitor that also inhibits ALDH2, demonstrated significant inhibition of ethanol clearance compared to untreated controls. Novel pyrazole and piperazine ALDH1A inhibitors were then tested with the piperazine inhibitor demonstrating ALDH2 inhibition via impaired ethanol clearance while the pyrazole inhibitor did not interfere with ethanol metabolism, suggesting a lack of ALDH2 inhibition. ConclusionsInhibition of ethanol clearance is a useful in vivo method of inferring pharmacologic inhibition of hepatic ALDH2. This assay may be useful in the development of novel ALDH1A specific inhibitors for a variety of therapeutic indications.

GRWD1 Over-Expression Promotes Gastric Cancer Progression by Activating Notch Signaling Pathway via Up-Regulation of ADAM17.

Glutamate-rich WD repeat containing 1 (GRWD1) is over-expressed in a variety of malignant tumors and is considered to be a potential oncogene. However, its mechanism of action in gastric cancer (GC) is still unclear. Data analysis, Immunohistochemistry, and Western Blot (WB) were performed to verify the expression of GRWD1 in GC and para-cancerous tissues. The association between GRWD1 expression and tumor size, tissue differentiation, lymph node metastasis, TNM stage, and prognosis was analyzed according to the high and low expression levels of GRWD1. The relationship between GRWD1 and Notch pathway was verified by data analysis and WB. The effects of GRWD1 on the proliferation, migration, and invasion of GC cells were verified by cell proliferation, migration, and invasion assays. We confirmed that the high expression of GRWD1 promoted the proliferation of GC cells in vivo through the tumor formation assay in nude mice. The expression of GRWD1 was higher in GC tissues than in para-cancerous tissues, and its expression was positively correlated with tumor size, lymph node metastasis, and TNM stage, but negatively correlated with differentiation grade and prognosis. GRWD1 over-expression increased ADAM metallopeptidase domain 17 (ADAM17) expression and promoted Notch1 intracellular domain (NICD) release to promote GC cell proliferation, migration, and invasion in vitro. Results from animal studies have shown that high GRWD1 expression could promote GC cell proliferation in vivo by activating the Notch signaling pathway. GRWD1 promotes GC progression through ADAM17-dependent Notch signaling, and GRWD1 may be a novel tumor marker and therapeutic target.

Genotoxic and cytotoxic evaluation of venlafaxine in an acute and a subchronic assay in mouse.

The present research was made to determine the micronuclei and cytotoxic capacity of the antidepressant venlafaxine in an in vivo acute and subchronic assays in mouse. In the first study, we administered once 5, 50, and 250 mg/kg of the drug, and included a negative and a daunorubicin treated group. Observations were daily made during four days. The subchronic assay lasted 5 weeks with daily administration of venlafaxine (1, 5, and 10 mg/kg) plus a negative and an imipramine administered groups. Observations were made each week. In the first assay results showed no micronucleated polychromatic erythrocytes (MNPE) increase, except with the high dose at 72 h. The strongest cytotoxic effect was found with 250 mg/kg at 72 h (a 51% cytotoxic effect in comparison with the mean control level). In the subchronic assay no MNPE increase was found; however, with the highest dose a significant increase of micronucleated normochromatic erythrocytes was observed in the last three weeks (a mean of 51% respect to the mean control value). A cytotoxic effect with the two high doses in the last two weeks was observed (a polychromatic erythrocyte mean decrease of 52% respect to the mean control value). Results suggest caution with venlafaxine.

Antispasmodic and antidiarrheal effects of Juniperus oxycedrus L. on the jejunum in rodents.

Functional bowel disorders (FBD) have a major potential to degrade the standards of public life. Juniperus oxycedrus L. (J. oxycedrus) (Cupressaceae) has been described as a plant used in traditional medicine as an antidiarrheal medication. The present study is the first to obtain information on the antispasmodic and antidiarrheic effects of J. oxycedrus aqueous extract through in vitro and in vivo studies. An aqueous extract of J. oxycedrus (AEJO) was extracted by decoctioning air-dried aerial sections of the plant. Antispasmodic activity was tested in an isolated jejunum segment of rats exposed to cumulative doses of drogue extract. The antidiarrheic activity was tested using diarrhea caused by castor oil, a transit study of the small intestine, and castor oil-induced enteropooling assays in mice. In the jejunum of rats, the AEJO (0.1, 0.3 and 1 mg/ml) diminished the maximum tone induced by low K+ (25 mM), while it exhibited a weak inhibitory effect on high K+ (75 mM) with an IC50=0.49 ± 0.01 mg/ml and IC50=2.65 ± 0.16 mg/ml, respectively. In the contractions induced by CCh (10-6 M), AEJO diminished the maximum tone, similar to that induced by low K+ (25 mM). with an IC50=0.45 ± 0.02 mg/ml. The inhibitory effect of AEJO on low K+ induced contractions was significantly diminished in the presence of glibenclamide (GB) (0.3 µM) and 4-aminopyrimidine (4-AP) (100 µM), with IC50 values of 1.84 ± 0.09 mg/ml. and 1.63 ± 0.16 mg/ml, respectively). The demonstrated inhibitory effect was similar to that produced by a non-competitive antagonist acting on cholinergic receptors and calcium channels. In castor oil-induced diarrhea in mice, AEJO (100, 200, and 400 mg/kg) caused an extension of the latency time, a reduced defecation frequency, and a decrease in the amount of wet feces compared to the untreated group (distilled water). Moreover, it showed a significant anti-motility effect and reduced the amount of fluid accumulated in the intestinal lumen at all tested doses. These findings support the conventional use of Juniperus oxycedrus L. as a remedy for gastrointestinal diseases.

The Role MicroRNA-135a in Suppressing Tumor Growth in Kidney Cancer Through the Regulation of Phosphoprotein Phosphatase2A and the Activation of the AKT and ERK1/2 Signaling Pathways.

Background: Kidney cancer is a frequently occurring malignant tumor in the urinary system, with rising morbidity and mortality rates in recent times. Developing new biomarkers and therapeutic targets is essential to improve the prognosis of patients affected by kidney cancer. In recent years, miRNAs' role in tumorigenesis and development has received growing attention. miRNAs constitute a group of small non-coding RNA molecules that regulate gene expression, affecting various biological processes, including cell proliferation, differentiation, and apoptosis. Of the many miRNAs, miR-135a plays a pivotal role in several cancers. Nevertheless, the precise mechanisms and functions concerning miR-135a in renal cancer remain incompletely understood. Therefore, this study aims to analyze the effects of miR-135a on renal cancer replication and migration and its possible mechanisms, and to provide new strategies for the diagnosis and treatment of renal cancer. Methods: Renal cell lines (ACHN, A498) with stable hyperexpression of miR-135a and reduced expression of miR-135a were constructed by lentivirus packaging. The changes of replication, clone formation and migration ability of overexpressed miR-135a and overexpressed miR-135a in ACHN and A498 renal cell lines were detected. The possible mechanism of miR-135a affecting the replication of kidney cancer was analyzed by target gene prediction, double luciferase test, Western blotting and subcutaneous tumorigenicity assay in nude mice. Results: Hyperexpression of miR-135a can inhibit kidney cancer replication, whereas miR-135a knockdown potentially enhances replication. However, neither hyperexpression nor knockdown of miR-135a affects the migration ability of kidney cancer cells. The protein expression of PP2A-B56-γ, PP2A-Cα and PP2A-Cβ in renal cell line decreased after hyperexpression of miR-135a, while the protein expression of PP2A-B56-γ, PP2A-Cα and PP2A-Cβ increased after knockdown of miR-135a. In addition, the protein expression of p-Akt and p-ERK1/2 proteins in kidney cancer cells after hyperexpression of miR-135a were down-regulated, while the protein expression of p-Akt and p-ERK1/2 were up-regulated in kidney cancer cells after knockdown of miR-135a. In subcutaneous tumor formation experiments in nude mice, tumor size within nude mice in the miR-135a group was significantly smaller than in the control group. Conclusion: MiR-135a could suppress the replication of kidney cancer by modulating PP2A and AKT, ERK1/2 signaling pathways.

Structure-based virtual screening towards the discovery of novel thrombin inhibitors with Anti-HCC activities.

Hepatic carcinoma (HCC) is one of the most lethal malignant tumors in the world, and new treatment regimens for this disease are urgently needed. Studies have shown that thrombin stimulates tumor progression by forming fibrin and activating platelets. Dabigatran etexilate, a thrombin inhibitor, can inhibit the activity of thrombin and prevent the proliferation and metastasis of HCC in cells and nude mice. The present study was designed to find thrombin inhibitors with novel skeletons, and further confirm the correlation between thrombin inhibition and HCC prevention to identify potential anti-HCC drug leads. The potential thrombin inhibitors were firstly screened in the Topscience Database, and 20 potential active molecules were found by molecular docking. The effect of these molecules on thrombin inhibition, coagulation and tumor proliferation were evaluated, and the definite activity of ZXX-4 was identified. Further in vivo assays in nude mice showed that ZXX-4 inhibited tumor proliferation in nude mice, reduced tumor metastasis, and enhanced the clinical efficacy of first-line drug sorafenib for the treatment of HCC. ZXX-4 can be further explored as an anti-tumor lead compound with a novel skeleton, and inhibition of thrombin can serve as a potential treatment strategy for HCC.

THBS2 promotes gastric cancer progression and stemness via the Notch signaling pathway.

Thrombospondin-2 (THBS2), a secreted extracellular matrix protein, plays a crucial role in various biological processes including angiogenesis, tissue remodeling, and inflammation. Our study focuses on its function in human gastric cancer (GC). Through bioinformatics and tumor tissue analysis, we compared THBS2 expression in GC tissues and adjacent tissues, and predicted regulatory upstream and downstream molecules. The direct regulatory effect of miR-29b-3p on THBS2 was evaluated through dual-luciferase reporter assays, showing that miR-29b-3p targets the 3'-UTR of THBS2 mRNA, reducing its expression in GC cells. The influence of THBS2 on tumorigenesis and stemness was examined on protein expression levels via Western blot. In vivo, THBS2's role was investigated through xenograft and metastasis assays in nude mice, demonstrating that downregulation of THBS2 impairs GC tumorigenesis and liver metastasis. Our study identified THBS2 as a highly expressed prognostic factor in GC patients. Functionally, THBS2 promotes GC progression through the Notch signaling pathway by regulating Notch3, NEY1, and HES1 proteins, and sustains cancer stem cell-like characteristics by Notch3, including the expression of CD44, Nanog, OCT4, and SOX2. In sum, our study reveals that THBS2 promotes GC progression and stemness, modulated negatively by miR-29b-3p. This suggests potential therapeutic targets within the THBS2/Notch signaling axis for combating gastric cancer.

Bioinformatical analysis and experimental validation of endoplasmic reticulum stress-related biomarker genes in type 2 diabetes mellitus.

Endoplasmic reticulum stress (ERS) is a prominent etiological factor in the pathogenesis of diabetes. Nevertheless, the mechanisms through which ERS contributes to the development of diabetes remain elusive. Transcriptional expression profiles from the Gene Expression Omnibus (GEO) datasets were analyzed and compared to obtain the differentially expressed genes (DEGs) in T2DM. Following the intersection with ERS associated genes, the ERS related T2DM DEGs were identified. Receiver operating characteristic (ROC) and Least Absolute Shrinkage and Selection Operator (LASSO) analysis were performed to screen out the ERS related biomarker genes and validate their diagnostic values. Gene expression level was detected by qPCR and Elisa assays in diabetic mice and patient serum samples. By analyzing the transcriptional expression profiles of the GEO datasets, 49 T2DM-related DEGs were screened out in diabetic islets. RTN1, CLGN, PCSK1, IAPP, ILF2, IMPA1, CCDC47, and PTGES3 were identified as ERS-related DEGs in T2DM, which were revealed to be involved in protein folding, membrane composition, and metabolism regulation. ROC and LASSO analysis further screened out CLGN, ILF2, and IMPA1 as biomarker genes with high value and reliability for diagnostic purposes. These three genes were then demonstrated to be targeted by the transcription factors and miRNAs, including CEBPA, CEBPB, miR-197-5p, miR-6133, and others. Among these miRNAs, the expression of miR-197-5p, miR-320c, miR-1296-3P and miR-6133 was down-regulated, while that of miR-4462, miR-4476-5P and miR-7851-3P was up-regulated in diabetic samples. Small molecular drugs, including D002994, D001564, and others, were predicted to target these genes potentially. qPCR and Elisa analysis both validated the same expression alteration trend of the ERS-related biomarker genes in diabetic mice and T2DM patients. These findings will offer innovative perspectives for clinical diagnosis and treatment strategies for T2DM.

4-Phenyl-thiazole-based dual inhibitors of fatty acid amide hydrolase and soluble epoxide hydrolase do not alleviate orofacial inflammatory pain in female rats

Pain arising from trigeminal systems such as headache is common, debilitating, and current treatments (e.g., sumatriptan) are limited. New treatments that target novel mechanisms of action may be required to innovate both short- and long-term pain therapy. Fatty acid amide hydrolase and soluble epoxide hydrolase are two pain-related enzymes that regulate pain and inflammation via independent pathways. We have previously demonstrated that simultaneous inhibition of these enzymes using a novel dual inhibitor alleviates acute inflammatory pain in the hindpaw and does not depress wheel running in rats. Here, we expanded on these findings and performed structure-activity relationships of our lead compound, the 4-phenyl-thiazole-based dual inhibitor SW-17, to generate 18 analogs and tested them for their inhibition at both enzymes. Conversion of the sulfonamide group to a tertiary amine led to a general decrease in the potency for the sEH enzyme, while this change was well-tolerated at the FAAH enzyme yielding several strong inhibitors. Six selected inhibitors were evaluated in mouse and rat sEH inhibition assays and results showed a species difference, i.e. 4-phenyl-thiazole-based analogs are significantly less or not active in mouse sEH compared to human and rat enzymes. The most potent inhibitor, SW-17, was evaluated in a plasma stability assay in human and rat plasma and showed moderate stability. However, SW-17 did not alleviate orofacial inflammatory pain in female rats compared to the traditional anti-migraine agent sumatriptan. Although modification of 4-phenyl-thiazole-based dual inhibitor SW-17 changes potencies at both FAAH and sEH, these approaches may not produce antinociception against trigeminal pain. Key Words: polypharmacology, formalin, inflammation, enzyme inhibition, structure-activity relationship studies

Isolation and identification of a duck-derived Japanese encephalitis virus and evaluation of its virulence in mice

Birds including domestic and wild birds, as the amplifying or reservoir hosts of JEV, were sensitive to JEV infection and could develop a sufficiently high viremia to infect mosquitoes. However, most of JEV positive reports in birds were based on molecular detection, with few viruses isolated from clinical cases. In this study, one JEV strain, designated duck/2022-SD-1, was first isolated and identified from blood samples of ducks in 2022 in Shandong province of China. The JEV duck/2022-SD-1 strain was classified into genotype I cluster and shared 96.5 to 99.5 % nucleotide sequence identity with other GI JEV strains. Biological characteristics revealed that duck/2022-SD-1 possessed similar replication ability to a virulent strain Beijing/2020–1. Based on the amino acid identity comparison of E protein, amino acid sites responsible for JEV virulence were conserved between duck/2022-SD-1 and other virulence strains. Through virulence assays in mice, we further determined that duck/2022-SD-1 was a highly virulent JEV strain with highly neuroinvasive in mice, which is similar to the virulence of another virulent strain Beijing/2020–1. Thus, the potential threat of JEV strains originating from domestic birds should be brought to people's attention.

Mucoadhesive Carrier-Mediated Oral Co-delivery of Bcl2 Inhibitors Improves Gastric Cancer Treatment.

Gastric cancer treatment is challenging due to the lack of early-stage diagnostic technology and targeted delivery systems. Currently, the available treatments for gastric cancer are surgery, chemotherapy, immunotherapy, and radiation. These strategies are either invasive or require systemic delivery, exerting toxicities within healthy tissues. By creation of a targeted delivery system to the stomach, gastric cancer can be treated in the early stages. Such an approach reduces the negative effects on the rest of the body by minimizing systemic absorbance and random localization. With this in mind, we developed a mucoadhesive vehicle composed of β-Glucan And Docosahexaenoic Acid (GADA) for controlled drug/gene delivery. In the current study, we investigated the therapeutic effect of codelivery Bcl2 inhibitors navitoclax (NAVI) and siRNA (Bcl2) via oral using GADA. The therapeutic efficacy of the GADA-mediated oral NAVI/siRNA was investigated in a gastric cancer mouse model. Higher Bcl2 inhibition efficacy was observed in Western blotting and TUNEL assay in mice treated with GADA/NAVI/siRNA compared to free NAVI, siRNA, and NAVI/siRNA. Histology (H&E) and immunohistochemistry (Ki67, TUNEL, and BCl2) analyses confirmed a significant reduction of the tumor region. Interaction between GADA and mucus resulted in retention for over 6 h and thereby sustained local payload release. The developed oral carrier GADA is an emerging vehicle that has promising potential in oral delivery of both small and large molecules, and their mucoadhesive property results in improved therapeutic efficacy with minimal side effects compared to conventional treatment. This study opens a new window for the effective delivery of oral medicine for the treatment of gastric cancer and other gastrointestinal diseases.

A novel montmorillonite clay-cetylpyridinium chloride material for reducing PFAS leachability and bioavailability from soils

Soils contaminated by per- and polyfluoroalkyl substances (PFAS) present a significant threat to both ecological and human health. Extensive research efforts are currently underway to develop effective strategies for immobilizing these chemicals in soils. In this study, calcium montmorillonite was modified with cetylpyridinium chloride (CPC-CM) to enhance its electrostatic and hydrophobic interactions with PFAS. CPC-CM exhibited high adsorption for perfluorooctanoate acid (PFOA), perfluorooctane sulfonate (PFOS) and 8:2 fluorotelomer sulfonic acids (8:2 FTSA) across initial concentrations of 50–1000 μg/L, outperforming both the parent CM and L-carnitine modified CM. Soil leaching tests demonstrated the superior immobilization capabilities of the CPC-CM, maintaining an average PFAS leaching rate below 7% after 120-day incubation. In the context of human exposure scenarios, the in vitro bioaccessibility and in vivo bioavailability of PFAS in soils were measured by gastrointestinal extraction and mouse assay. CPC-CM treatment effectively reduced the bioaccessibility (by up to 84%) and bioavailability (by up to 76%) of PFAS in soils. Furthermore, the safety and efficacy of CPC-CM were evaluated using enteric microorganisms of mice. CPC-CM treatment mitigated PFAS-induced changes in the abundance of Bacteroidetes and Firmicutes, thereby reducing PFAS-induced health risks for humans. Overall, CPC-CM synthesized in this study demonstrated superior adsorption performance and application safety, offering a highly promising approach for remediating PFAS-contaminated soil.

Loss of two-pore channel 2 function in melanoma-derived tumours reduces tumour growth in vivo but greatly increases tumour-related toxicity in the organism

BackgroundMelanoma, a severe form of skin cancer, poses significant health risks due to its aggressive nature and potential for metastasis. The role of two-pore channel 2 (TPC2) in the development and progression of melanoma remains poorly understood. This study aims to investigate the impact of TPC2 knockout (KO) on melanoma-derived tumors, focusing on tumour growth and related toxicity in the organism.MethodsThe study utilized CHL-1 and B16 melanoma cell lines with TPC2 KO to assess the changes in proliferation dynamics. Methods included real-time monitoring of cell proliferation using the xCELLigence system, in vivo tumour growth assays in mice, histopathological analyses, inflammation marker assessment, and quantitative PCR (qPCR) for gene expression analysisResultsTPC2 KO was found to significantly alter the proliferation dynamics of CHL-1 and B16 melanoma cells. The in vivo studies demonstrated reduced tumor growth in TPC2 KO cell-derived tumors. However, a notable increase in tumor-related toxicity in affected organs, such as the liver and spleen, was observed, indicating a complex role of TPC2 in melanoma pathology.ConclusionsThe loss of TPC2 function in melanoma cells leads to reduced tumour growth but exacerbates tumour-related toxicity in the organism. These findings highlight the dual role of TPC2 in melanoma progression and its potential as a therapeutic target. Further research is needed to fully understand the mechanisms underlying these effects and to explore TPC2 as a treatment target in melanoma.Graphical

TRPA1 channel mediates methylglyoxal-induced mouse bladder dysfunction.

Introduction: The transient receptor potential ankyrin 1 channel (TRPA1) is expressed in urothelial cells and bladder nerve endings. Hyperglycemia in diabetic individuals induces accumulation of the highly reactive dicarbonyl compound methylglyoxal (MGO), which modulates TRPA1 activity. Long-term oral intake of MGO causes mouse bladder dysfunction. We hypothesized that TRPA1 takes part in the machinery that leads to MGO-induced bladder dysfunction. Therefore, we evaluated TRPA1 expression in the bladder and the effects of 1 h-intravesical infusion of the selective TRPA1 blocker HC-030031 (1nmol/min) on MGO-induced cystometric alterations. Methods: Five-week-old female C57BL/6 mice received 0.5% MGO in their drinking water for 12weeks, whereas control mice received tap water alone. Results: Compared to the control group, the protein levels and immunostaining for the MGO-derived hydroimidazolone isomer MG-H1 was increased in bladders of the MGO group, as observed in urothelium and detrusor smooth muscle. TRPA1 protein expression was significantly higher in bladder tissues of MGO compared to control group with TRPA1 immunostaining both lamina propria and urothelium, but not the detrusor smooth muscle. Void spot assays in conscious mice revealed an overactive bladder phenotype in MGO-treated mice characterized by increased number of voids and reduced volume per void. Filling cystometry in anaesthetized animals revealed an increased voiding frequency, reduced bladder capacity, and reduced voided volume in MGO compared to vehicle group, which were all reversed by HC-030031 infusion. Conclusion: TRPA1 activation is implicated in MGO-induced mouse overactive bladder. TRPA1 blockers may be useful to treat diabetic bladder dysfunction in individuals with high MGO levels.

Carcinogenic risk of food additive AF-2 banned in Japan: a case study on reassessment of genotoxicity

BackgroundCarcinogenic risk assessment studies have been repeatedly improved and are still being debated to find a goal. Evaluation might be changed if new approaches would be applied to some chemicals which means that new approaches may change the final assessment. In this paper, the risk assessment of a chemical, in particular the proper carcinogenicity, is examined using the long-banned food additive, 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide, AF-2, as a case study.ResultsFirst, Ames tests were carried out using strains TA1535, TA100, TA1538, and TA98 and their nitroreductase-deficient strains YG7127, YG7128, YG7129, and YG7130. The results showed that mutagenic activity was reduced by about 50% in the nitroreductase-deficient strains, indicating that part of the mutagenic activity shown in Ames test was due to bacterial metabolism. Second, in vivo genotoxicity tests were conducted, including the one that had not been developed in 1970’s. Both a micronucleus test and a gene mutation assay using transgenic mice were negative. Third, assuming it is a genotoxic carcinogen, the virtual safety dose of 550 μg/day was calculated from the TD50 in rats with a probability of 10−5.ConclusionAF-2 has been shown to be carcinogenic to rodents and has previously been indicated to be genotoxic in vitro. However, the present in vivo genotoxicity study, it was negative in the forestomach, a target organ for cancer, particularly in the gene mutation assay in transgenic mice. Considering the daily intake of AF-2 in the 1970s and its virtually safety dose, the carcinogenic risk of AF-2 could be considered acceptable.

Detection of Ivermectin Toxicity Using Some Biochemical and Immunological Assays in Mice

Detection of Ivermectin Toxicity Using Some Biochemical and Immunological Assays in Mice

Reproducible strategy for excisional skin-wound-healing studies in mice.

Wound healing is a complex physiological process involving various cell types and signaling pathways. The capability to observe the dynamics of wound repair offers valuable insights into the effects of genetic modifications, pharmaceutical interventions or other experimental manipulations on the skin-repair process. Here, we provide a comprehensive protocol for a full-thickness, excisional skin-wound-healing assay in mice, which can easily be performed by any scientist who has received an animal welfare course certificate and can be completed within ~3 h, depending on the number of animals. Crucially, we highlight the importance of considering key aspects of the assay that can dramatically contribute to the reliability and reproducibility of these experiments. We thoroughly discuss the experimental design, necessary preparations, wounding technique and analysis. In addition, we discuss the use of lineage-tracing techniques to monitor cell migration, differentiation and the contribution of different cell populations to the repair process. Overall, we explore key aspects of the skin-wound-healing assay, supplying a detailed procedure and guidelines essential for decreasing variability and obtaining reliable and reproducible results.

The Anti-Virulence Activities of the Antihypertensive Drug Propranolol in Light of Its Anti-Quorum Sensing Effects against Pseudomonas aeruginosa and Serratia marcescens.

The development of bacterial resistance is an increasing global concern that requires discovering new antibacterial agents and strategies. Bacterial quorum sensing (QS) systems play important roles in controlling bacterial virulence, and their targeting could lead to diminishing bacterial pathogenesis. In this context, targeting QS systems without significant influence on bacterial growth is assumed as a promising strategy to overcome resistance development. This study aimed at evaluating the anti-QS and anti-virulence activities of the β-adrenoreceptor antagonist propranolol at sub-minimal inhibitory concentrations (sub-MIC) against two Gram-negative bacterial models Pseudomonas aeruginosa and Serratia marcescens. The effect of propranolol on the expression of QS-encoding genes was evaluated. Additionally, the affinity of propranolol to QS receptors was virtually attested. The influence of propranolol at sub-MIC on biofilm formation, motility, and production of virulent factors was conducted. The outcomes of the propranolol combination with different antibiotics were assessed. Finally, the in vivo protection assay in mice was performed to assess propranolol's effect on lessening the bacterial pathogenesis. The current findings emphasized the significant ability of propranolol at sub-MIC to reduce the formation of biofilms, motility, and production of virulence factors. In addition, propranolol at sub-MIC decreased the capacity of tested bacteria to induce pathogenesis in mice. Furthermore, propranolol significantly downregulated the QS-encoding genes and showed significant affinity to QS receptors. Finally, propranolol at sub-MIC synergistically decreased the MICs of different antibiotics against tested bacteria. In conclusion, propranolol might serve as a plausible adjuvant therapy with antibiotics for the treatment of serious bacterial infections after further pharmacological and pharmaceutical studies.