Decreased mRNA Levels Research Articles

Activating transcription factor 3 mediates apoptosis and cell cycle arrest in TP53-mutated anaplastic thyroid cancer cells

BackgroundIt is believed that loss of p53 function plays a crucial role in the progression of well to poorly differentiated thyroid cancers including anaplastic thyroid carcinoma (ATC). Given the poor prognosis of ATC due to its strong therapeutic resistance, there is a need to establish new therapeutic targets to extend the survival of ATC patients. Activating transcription factor 3 (ATF3) can inhibit the oncogenic activity of mutant p53 and, as a result, contribute to tumor suppression in several TP53-mutated cancers. Herein, we demonstrate that the ectopic overexpression of ATF3 leads to the suppression of oncogenic mutant p53 activity in chemo-resistant 8305 C thyroid cancer cells harboring R273C p53 gene mutation.MethodsThe biological behavior of 8305 C cells was assessed pre- and post-transfection with pCMV6-ATF3 plasmid using MTT assay, fluorescent microscopy, cell cycle, and annexin V/PI flow cytometric analysis. The effect of ectopic ATF3 overexpression on the cellular level of p53 was examined by western blotting assay. The mRNA expression levels of TP53, TAp63, ΔNp63, and SHARP1 were evaluated in ectopic ATF3-expressing cells compared to controls.ResultsThe overexpression of ATF3 in 8305 C thyroid cancer cells significantly decreased cell viability and induced apoptosis and cell cycle arrest in vitro. The immunoblotting of p53 protein revealed that ATF3 overexpression significantly increased the level of mutant p53 in 8305C cells compared to mock-transfected control cells. Additionally, elevated mRNA levels of TAp63 and SHARP1 and a decreased mRNA level of ΔNp63 were observed in PCMV6-AC-ATF3-transfected 8305 C cells with significant differences compared to the mock and untreated cells.ConclusionIn light of our findings, it is evident that therapeutic strategies aimed at increasing ATF3 expression or enhancing the interaction between ATF3 and mutant p53 can be a promising approach for the treatment of p53-mutated metastatic thyroid cancer.

Effects of H9N2 avian influenza virus infection on metabolite content and gene expression in chick DF1 cells

After viral infection, the virus relies on the host cell's complex metabolic and biosynthetic machinery for replication. However, the impact of avian influenza virus (AIV) on metabolites and gene expression in poultry cells remains unclear. To investigate this, we infected chicken embryo fibroblasts DF1 cells with H9N2 AIV at an MOI of 3. Our aim was to explore how H9N2 AIV alters DF1 cells metabolic pathways to facilitate its replication. We employed metabolomics and transcriptomics techniques to analyze changes in metabolite content and gene expression. Metabolomics analysis revealed a significant increase in glutathione-related metabolites, including reduced glutathione (GSH), oxidized glutathione (GSSG) and total glutathione (T-GSH) upon H9N2 AIV infection in DF1 cells. Elisa results confirmed elevated levels of GSH, GSSG, and T-GSH consistent with metabolomics findings, noting a pronounced increase in GSSG compared to GSH. Transcriptomics showed significant alterations in genes involved in glutathione synthesis and metabolism post-H9N2 infection. However, adding the glutathione synthesis inhibitor BSO exogenously significantly promoted H9N2 replication in DF1 cells. This was accompanied by increased mRNA levels of pro-inflammatory cytokines (IL-1β, IFN-γ) and decreased mRNA levels of anti-inflammatory cytokines (TGF-β, IL-13). BSO also reduced catalase (CAT) gene expression and inhibited its activity, leading to higher reactive oxygen species (ROS) and malondialdehyde (MDA) level in DF1 cells. qPCR results indicated decreased mRNA levels of Nrf2, NQO1, and HO-1 with BSO, ultimately increasing oxidative stress in DF1 cells. Therefore, the above results indicated that H9N2 AIV infection in DF1 cells activated the glutathione metabolic pathway to enhance the cell's self-defense mechanism against H9N2 replication. However, when GSH synthesis is inhibited within the cells, it leads to an elevated oxidative stress level, thereby promoting H9N2 replication within the cells through Nrf2/HO-1 pathway. This study provides a theoretical basis for future rational utilization of the glutathione metabolic pathway to prevent viral replication.

JAK and STAT5B mediate olfactory response of migratory locusts to their own volatiles

Janus kinase (JAK) and signal transducer and activator of transcription (STAT) signaling affect social aggregation, mood and psychiatric disorders, nociceptive and depressive behaviors. Olfactory dysfunction is one of the distinct symptoms of these behaviors, but function and mechanism of JAK and STAT in modulating olfaction remain largely unknown. Migratory locusts show olfactory preference for their own volatiles. We thus use this animal model to explore functions and mechanisms of JAK and STAT5B in mediating olfaction response to their own volatiles. Tissue distribution study shows that JAK and STAT5B express in antennae and brains, especially in antennal lobes and mushroom bodies in locust brains, and knockdown of these two genes by RNA interference (RNAi) in antennae and brains results in the loss of olfactory preference for locust volatiles, including chemical odorants indole and β-ionone. RNA-seq analysis reveals that JAK and STAT5B RNAi knockdown downregulates a functional class of transcripts in nucleoprotein complex, including heterogeneous nuclear ribonucleoprotein C (hnRNPC) and small nuclear ribonucleoprotein polypeptide F (SNRPF). HnRNPC and SNRPF mRNAs and proteins are also expressed in antennae and brains, and RNAi knockdown of these two genes reduces the percentage of locusts preferring volatiles, including chemical odorants indole and β-ionone. Furthermore, RNAi knockdown of dopamine receptor 1 (DopR1) results in the decrease of JAK mRNA level in antennae, and JAK/STAT5B, hnRNPC and SNRPF are required for dopamine receptor 1 (DopR1) to modulate olfactory preference for their own volatiles. This study confirms that JAK/STAT5B signaling modulates olfaction by affecting expression levels of hnRNPC and SNRPF, and this pathway is also required for DopR1 to modulate olfactory preference for their own volatiles. These findings highlight novel roles of JAK and STAT5B in modulating olfactory preference. This study provides novel insights into functional links among JAK/STAT5B signaling, RNA binding proteins and DopR1 underlying the modulation of olfactory behaviors.

A novel probiotic formula, BLLL, ameliorates chronic stress-induced depression-like behaviors in mice by reducing neuroinflammation and increasing neurotrophic factors.

Introduction: Probiotics have been recognized for their various biological activities, including antioxidant and anti-inflammatory properties. This study investigates the therapeutic effect of a novel probiotic formula, BLLL, consisting of Bifidobacterium breve, Lactobacillus plantarum, Lactobacillus paracasei, and Lactobacillus helveticus, on chronic stress-induced depression-like behaviors in mice. Methods: The BLLL formula or phosphate-buffered saline (PBS) was given orally at a dose of 2, 4, or 8 × 1010 CFU/kg once daily for 10days in mice treated with chronic unpredictable stress (CUS) treated or vehicle. Depression-like behaviors were assessed using the sucrose preference test (SPT), the forced swimming test (FST), and the tail suspension test (TST). The mRNA and/or protein expression of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), IL-4, IL-10, and chitinase-3-like protein 3 (CHI3L1, also known as Ym-1), as well as the concentration of nitrite, malondialdehyde (MDA), glutathione (GSH), and brain-derived neurotrophic factor (BDNF) in the hippocampus and medial prefrontal cortex were examined. Results: The BLLL formula treatment at a dose of 8 × 1010 CFU/kg, but not at a dose of 2 or 4 × 1010 CFU/kg, improved CUS-induced depression-like behaviors in mice, as shown by the decrease in immobility time in the TST and FST and the increase in sucrose intake in the SPT. Further analysis revealed that BLLL treatment suppressed the CUS-induced increase in IL-1β, IL-6, and TNF-α mRNA and protein levels, as well as the CUS-induced decrease in IL-4, IL-10, and Ym-1 mRNA and/or protein levels in the hippocampus and medial prefrontal cortex. In addition, treatment with the BLLL formula countered the CUS-induced increase in nitrite and MDA levels and the CUS-induced decrease in GSH content and BDNF concentration in the hippocampus and medial prefrontal cortex. Conclusion: These results demonstrate that the novel probiotic formula BLLL ameliorates chronic stress-induced depression-like behavior in mice by suppressing neuroinflammation and oxido-nitrosative stress in the brain.

Metal-Organic Framework: Fabrication of Nano Fluorescent Composite Materials and Treatment of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a common malignant tumor originating from liver cells, characterized by complex pathogenesis and limited treatment options such as surgery, chemotherapy, and transplantation. Cisplatin, an effective chemotherapeutic agent, disrupts cancer cell DNA but is hindered by side effects and the need for controlled sustained release to optimize efficacy. Metal-organic frameworks (MOFs) have emerged as promising nanocarriers for precise local drug delivery, reducing required doses and mitigating side effects of chemotherapeutic drugs, thus offering a potential avenue for hepatocellular carcinoma (HCC) treatment. In this research, a rectangular channel MOF (Rumgay H, Ferlay J, Martel C, Georges D, Ibrahim AS, Zheng R, Wei W, Lemmens VEPP, Soerjomataram I (2022) Global, regional and national burden of primary liver cancer by subtype. Eur J Cancer 161:108-118) carrier was synthesized using ligand L as the organic linker coordinated with Cu(II) and I(I). The MOF's structure and fluorescence properties were characterized. Additionally, to enhance substrate biocompatibility, composite carrier materials were prepared by incorporating polylactic acid (PLA) with 1, utilized for cisplatin loading. To evaluate the inhibitory effect of PLA-1@cisplatin on HCC, HepG-2 and Huh-7 HCC cell lines were treated with varying concentrations of the drug for 48h, and their cell viability was assessed. The results demonstrated a significant dose-dependent reduction in cell viability of both HepG-2 and Huh-7 cells. To explore the potential inhibitory mechanism of PLA-1@cisplatin on HCC, the mRNA levels of GADD45A and NACC1 in HepG-2 and Huh-7 cells post-treatment were measured. GADD45A expression, initially low in HCC cells, was significantly upregulated after drug treatment, while NACC1, typically highly expressed in HCC, showed a significant decrease in mRNA levels with increasing concentrations of PLA-1@cisplatin. These findings indicate that PLA-1@cisplatin effectively upregulates GADD45A expression and downregulates NACC1 expression. Overall, the developed cisplatin-loaded nanoparticle system holds promise for HCC treatment by reducing chemotherapy side effects and enhancing drug efficacy.

Deletion of the endothelial glycocalyx component endomucin leads to impaired glomerular structure and function.

Endomucin (EMCN), an endothelial-specific glycocalyx component, was found to be highly expressed by the endothelium of the renal glomerulus. We reported an anti-inflammatory role of EMCN and its involvement in the regulation of vascular endothelial growth factor (VEGF) activity through modulating VEGF receptor 2 (VEGFR2) endocytosis. The goal of this study is to investigate the phenotypic and functional effects of EMCN deficiency using the first global EMCN knockout mouse model. Global EMCN knockout mice were generated by crossing EMCN-floxed mice with ROSA26-Cre mice. Flow cytometry was employed to analyze infiltrating myeloid cells in the kidneys. The ultrastructure of the glomerular filtration barrier was examined by transmission electron microscopy, while urinary albumin, creatinine, and total protein levels were analyzed from freshly collected urine samples. Expression and localization of EMCN, EGFP, CD45, CD31, CD34, podocin, albumin, and α-smooth muscle actin were examined by immunohistochemistry. Mice were weighed regularly, and their systemic blood pressure was measured using a non-invasive tail-cuff system. Glomerular endothelial cells and podocytes were isolated by fluorescence-activated cell sorting for RNA-seq. Transcriptional profiles were analyzed to identify differentially expressed genes in both endothelium and podocytes, followed by gene ontology analysis of up- and down-regulated genes. Protein levels of EMCN, albumin, and podocin were quantified by Western blot. EMCN -/- mice were viable with no gross anatomical defects in kidneys. The EMCN -/- mice exhibited increased infiltration of CD45 + cells, with an increased proportion of Ly6G high Ly6C high myeloid cells and higher VCAM-1 expression. EMCN -/- mice displayed albuminuria with increased albumin in the Bowman's space compared to the EMCN +/+ littermates. Glomeruli in EMCN -/- mice revealed fused and effaced podocyte foot processes and disorganized endothelial fenestrations. We found no significant difference in blood pressure between EMCN knockout mice and their wild-type littermates. RNA-seq of glomerular endothelial cells revealed downregulation of cell-cell adhesion and MAPK/ERK pathways, along with glycocalyx and extracellular matrix remodeling. In podocytes, we observed reduced VEGF signaling and alterations in cytoskeletal organization. Notably, there was a significant decrease in both mRNA and protein levels of podocin, a key component of the slit diaphragm. Our study demonstrates a critical role of the endothelial marker EMCN in supporting normal glomerular filtration barrier structure and function by maintaining glomerular endothelial tight junction and homeostasis and podocyte function through endothelial-podocyte crosstalk.

The alleviation of difenoconazole-induced kidney injury in common carp (Cyprinus carpio) by silybin: Involvement of inflammation, oxidative stress, and apoptosis

Triazole fungicides, such as difenoconazole (DFZ), are frequently used to control fungus in crops that pollute water. The common carp (Cyprinus carpio) (hereafter referred to as “carp”) is an excellent bio-indicator of water quality. The seeds of the silymarin plant contain a flavonolignan called silybin (SYB), which is used to treat liver disease. To explore SYB's involvement in DFZ-triggered kidney damage in carps, an H&E assay was conducted, and ROS level was also examined. The results demonstrated that SYB alleviated DFZ-induced destruction of kidney tissue structure in carps, as well as alleviating the elevation of kidney ROS level in carps. RT-qPCR and Western blot were used to detect inflammation-, oxidative stress- and apoptosis-related factors at mRNA level and protein level. The experimental findings indicated that relative to the DFZ group, SYB + DFZ co-treatment reduced inflammation-related mRNA level of il-6, il-1β and tnf-α, elevated mRNA level of il-10. It also reduced protein expression levels of NF-κB and iNOS. In addition, SYB + DFZ co-treatment reduced DFZ-induced increase in the oxidative stress-related mRNA indicators sod and cat, and decreased the protein expression levels of Nrf2 and NQO1. SYB reduced the DFZ-induced increase in pro-apoptotic gene Bax mRNA and protein expression levels and the DFZ-induced decrease in anti-apoptotic gene Bcl-2 mRNA and protein expression levels. In summary, SYB potentially mitigates DFZ-induced kidney damage in carp by addressing inflammation, oxidative stress, and apoptosis. Our results establish a theoretical foundation for the clinical advancement of freshwater carp feeds.

Macauba (Acrocomia aculeata) pulp oil reduces fat accumulation and enhances the lifespan of Caenorhabditis elegans at low temperatures via fat-1- and fat-7-dependent pathway.

Macauba (Acrocomia aculeata) is a Brazilian palm tree whose oil in the pulp is rich in oleic acid and carotenoids. However, its physiological function remains unknown. This study aimed to investigate the effects of macauba pulp oil (MPO) on the metabolic link between lipid metabolism and lifespan using Caenorhabditis elegans (C. elegans). C. elegans were treated with 5.0mg/mL of MPO for analyzing triglyceride and glycerol accumulation, fatty acid profile, gene expression of lipid and oxidative metabolism proteins under cold (4°C) stress conditions, and lifespan analysis under stress conditions such as cold (4°C), heat (37°C), and oxidative (paraquat) stress. MPO significantly suppressed fat accumulation and increased glycerol (a lipolysis index) and the lifespan of C. elegans at low temperature (4°C). This was accompanied by decreased mRNA levels of the genes involved in lipogenesis (spb-1 and pod-2) and increased levels of the genes involved in fatty acid β-oxidation (acs-2 and nhr-49) and fat mobilization genes (hosl-1 and aak-2). Additionally, MPO treatment modulated fatty acid pools in C. elegans at low temperatures in that MPO treatment decreased saturated fatty acid levels and shifted the fatty acid profile to long-chain fatty acids. Moreover, the effect of MPO on fat accumulation at low temperatures was abolished in fat-7 mutants, whereas both fat-1 and fat-7 contribute, at least in part, to MPO-elevated survival of C. elegans under cold conditions. PRACTICAL APPLICATION: The results obtained in the present study may contribute to the understanding of the health benefits of consuming macauba pulp oil and consequently stimulate economic growth and the industrial application of this new type of oil, which may result in the creation of new jobs and increased value of small producers.

Nutriomic Effects of Precision Lipids on Murine Hepatic Triacylglycerol Alterations Induced by High-Fat Diets.

This study aims to investigate if a mixture of functional lipids (FLs), containing conjugated linoleic acid (CLA), tocopherols (TPs), and phytosterols (PSs), prevents some lipid alterations induced by high-fat (HF) diets, without adverse effects. Male CF1 mice (n = 6/group) were fed (4 weeks) with control (C), HF, or HF + FL diets. FL prevented the overweight induced by the HF diet and reduced the adipose tissue (AT) weight, associated with lower energy efficiency. After the intervention period, the serum triacylglycerol (TAG) levels in both HF diets underwent a decrease associated with an enhanced LPL activity (mainly in muscle). The beneficial effect of the FL mixture on body weight gain and AT weight might be attributed to the decreased lipogenesis, denoted by the lower mRNA levels of SREBP1-c and ACC in AT, as well as by an exacerbated lipid catabolism, reflected by increased mRNA levels of PPARα, ATGL, HSL, and UCP2 in AT. Liver TAG levels were reduced in the HF + FL group due to an elevated lipid oxidation associated with a higher CPT-1 activity and mRNA levels of PPARα and CPT-1a. Moreover, genes linked to fatty acid biosynthesis (SREBP1-c and ACC) showed decreased mRNA levels in both HF diets, this finding being more pronounced in the HF + FL group. The administration of an FL mixture (CLA + TP + PS) prevented some lipid alterations induced by a HF diet, avoiding frequent deleterious effects of CLA in mice through the modulation of gene expression related to the regulation of lipid metabolism.

IRE1/JNK Is the Leading UPR Pathway in 6-OHDA-Induced Degeneration of Differentiated SH-SY5Y Cells.

Parkinson's disease (PD) is a neurodegenerative disorder which affects dopaminergic neurons of the midbrain. Accumulation of α-synuclein or exposure to neurotoxins like 6-hydroxydopamine (6-OHDA) induces endoplasmic reticulum (ER) stress along with the unfolded protein response (UPR), which executes apoptosis via activation of PERK/CHOP or IRE1/JNK signaling. The present study aimed to determine which of these pathways is a major contributor to neurodegeneration in an 6-OHDA-induced in vitro model of PD. For this purpose, we have applied pharmacological PERK and JNK inhibitors (AMG44 and JNK V) in differentiated SH-SY5Y cells exposed to 6-OHDA. Inhibition of PERK and JNK significantly decreased genotoxicity and improved mitochondrial respiration, but only JNK inhibition significantly increased cell viability. Gene expression analysis revealed that the effect of JNK inhibition was dependent on a decrease in MAPK10 and XBP1 mRNA levels, whereas inhibition of either PERK or JNK significantly reduced the expression of DDIT3 mRNA. Western blot has shown that JNK inhibition strongly induced the XBP1s protein, and inhibition of each pathway attenuated the phosphorylation of eIF2α and JNK, as well as the expression of CHOP. Collectively, our data suggests that targeting the IRE1/JNK pathway of the UPR is a more effective option for PD treatment as it simultaneously affects more than one pro-apoptotic pathway.

D-glucaro-1,4-lactone improves Diethylnitrosamine induced hepatocellular carcinoma in rats via the uric acid-ROS pathway

Ethnopharmacological relevanceLiuwei dihuang pills is a famous Traditional Chinese Medicine with various anti-cancer properties. Over 50 pharmaceutical manufacturers produce Liuwei dihuang pills in China and an estimated millions of people around the world orally take it every day. D-glucaro-1,4-lactone (1,4-GL) was quantified to be about 12.0 mg/g in Liuwei dihuang pills and a primary bioactive component of it inhibiting the activity of β-glucuronidase in vivo. 1,4-GL can prevent and effectively inhibit various types of cancer. However, its exact mechanism of action remains unknown. The study would justify the traditional usage of Liuwei dihuang pills against cancers. Aim of the study1,4-GL, a bioactive ingredient derived from Liuwei dihuang pills, a famous Traditional Chinese Medicine, could delay the progression of diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in rats. The mechanism underpinning the effect, however, remains poorly understood. Materials and methodsHealthy and HCC rats were treated with or without 1,4-GL (40.0 mg/kg) and 1HNMR-based metabonomic analysis was employed. 10 metabolites in uric acid pathway were quantitatively determined by UPLC-MS/MS. The expression of xanthine dehydrogenase (XDH), SLC2A9 mRNA, and SLC2A9 protein was determined using RT-qPCR and Western Blot. The effect of 1,4-GL on HCC-LM3 cells was verified in vitro. The alterations of ROS activity, SLC2A9 and XDH gene levels were observed in NCTC-1469 cells induced by lipopolysaccharide (LPS) after 1,4-GL treatment. ResultsAfter the intervention of 1,4-GL, improved pathological morphology, liver lesions in HCC rats was observed with restored serum levels of AFP, AST, ALP, γ-GGT and Fisher's ratio. Hepatic metabonomics revealed that puring metabolism were significantly regulated by 1,4-GL in HCC rats. Uric acid, xanthine and hypoxanthine levels were quantified by UPLC-MS/MS and found to be nearly restored to control levels after 1,4-GL treatment in HCC rats. Changes in xanthine oxidase activity, XDH mRNA expression, and SLC2A9 mRNA and protein expression were also reversed. 1,4-GL treatment in LM3 HCC cells were consistent with the results in vivo. Furthermore, oxidative stress indicators such as T-SOD, GSH, CAT and MDA in serum and liver were improved after HCC rats treated with 1,4-GL. In vitro, 1,4-GL was observed to reduce lipopolysaccharide-induced ROS levels in NCTC-1469 cells with enhanced mRNA and protein expression of SLC2A9 and decreased mRNA level of XDH. ConclusionThe protective effects of 1,4-GL against DEN-induced HCC by reducing uric acid and ROS levels due to down-regulation of uric acid production and up-regulation of SLC2A9 expressions. 1,4-GL may represent a novel treatment that improves recovery from HCC by targeting uric acid-ROS pathway.

Dopamine receptors and key elements of the neurotrophins (BDNF, CDNF) expression patterns during critical periods of ontogenesis in the brain structures of mice with autism-like behavior (BTBR) or its absence (С57BL/6 J).

Analysis of the mechanisms underlying autism spectrum disorder (ASD) is an urgent task due to the ever-increasing prevalence of this condition. The study of critical periods of neuroontogenesis is of interest, since the manifestation of ASD is often associated with prenatal disorders of the brain development. One of the currently promising hypotheses postulates a connection between the pathogenesis of ASD and the dysfunction of neurotransmitters and neurotrophins. In this study, we investigated the expression of key dopamine receptors (Drd1, Drd2), brain-derived neurotrophic factor (Bdnf), its receptors (Ntrkb2, Ngfr) and the transcription factor Creb1 that mediates BDNF action, as well as cerebral dopamine neurotrophic factor (Cdnf) during the critical periods of embryogenesis (e14 and e18) and postnatal development (p14, p28, p60) in the hippocampus and frontal cortex of BTBR mice with autism-like behavior compared to the neurotypical C57BL/6 J strain. In BTBR embryos, on the 14th day of prenatal development, an increase in the expression of the Ngfr gene encoding the p75NTR receptor, which may lead to the activation of apoptosis, was found in the hippocampus and frontal cortex. A decrease in the expression of Cdnf, Bdnf and its receptor Ntrkb2, as well as dopamine receptors (Drd1, Drd2) was detected in BTBR mice in the postnatal period of ontogenesis mainly in the frontal cortex, while in the hippocampus of mature mice (p60), only a decrease in the Drd2 mRNA level was revealed. The obtained results suggest that the decrease in the expression levels of CDNF, BDNF-TrkB and dopamine receptors in the frontal cortex in the postnatal period can lead to significant changes in both the morphology of neurons and dopamine neurotransmission in cortical brain structures. At the same time, the increase in p75NTR receptor gene expression observed on the 14th day of embryogenesis, crucial for hippocampus and frontal cortex development, may have direct relevance to the manifestation of early autism.

Expression of fibroblast activation protein-α in human deep vein thrombosis

BackgroundFibroblast activation protein-α (FAP), a type-II transmembrane serine protease, is associated with wound healing, cancer-associated fibroblasts, and chronic fibrosing diseases. However, its expression in deep vein thrombosis (DVT) remains unclear. Therefore, this study investigated FAP expression and localization in DVT. MethodsWe performed pathological analyses of the aspirated thrombi of patients with DVT (n = 14), classifying thrombotic areas in terms of fresh, cellular lysis, and organizing reaction components. The organizing reaction included endothelialization and fibroblastic reaction. We immunohistochemically examined FAP-expressed areas and cells, and finally analyzed FAP expression in cultured dermal fibroblasts. ResultsAll the aspirated thrombi showed a heterogeneous mixture of at least two of the three thrombotic areas. Specifically, 83 % of aspirated thrombi showed fresh and organizing reaction components. Immunohistochemical expression of FAP was restricted to the organizing area. Double immunofluorescence staining showed that FAP in the thrombi was mainly expressed in vimentin-positive or α-smooth muscle actin-positive fibroblasts. Some CD163-positive macrophages expressed FAP. FAP mRNA and protein levels were higher in fibroblasts with low-proliferative activity cultured under 0.1 % fetal bovine serum (FBS) than that under 10 % FBS. Fibroblasts cultured in 10 % FBS showed a significant decrease in FAP mRNA levels following supplementation with hemin, but not with thrombin. ConclusionsThe heterogeneous composition of venous thrombi suggests a multistep thrombus formation process in human DVT. Further, fibroblasts or myofibroblasts may express FAP during the organizing process. FAP expression may be higher in fibroblasts with low proliferative activity.

The use of cell and larval assays to identify target genes for RNA interference-meditated control of the Australian sheep blowfly (Lucilia cuprina).

Flystrike, primarily caused by Lucilia cuprina, is a major health and welfare issue for sheep wool industries. Current chemical-based controls can have limited effectiveness due to the emergence of resistance in the parasite. RNA interference (RNAi), which uses double-stranded RNA (dsRNA) as a trigger molecule, has been successfully investigated for the development of innovative pest control strategies. Although RNAi offers great potential, the efficient identification, selection of target genes and delivery of dsRNA represent challenges to be overcome for the successful application of RNAi for control of L. cuprina. A primary L. cuprina (blowfly) embryo cell line (BFEC) was established and confirmed as being derived from L. cuprina eggs by PCR and amplicon sequencing. The BFECs were successfully transfected with plasmids and messenger RNA (mRNA) expressing fluorescent reporter proteins and dsRNA using lipid-based transfection reagents. The transfection of dsRNA into BEFC in this study suggested decreased mRNA levels of target gene expression, which suggested RNAi-mediated knockdown. Three of the dsRNAs identified in this study resulted in reductions of in target gene mRNA levels in BFEC and loss of biological fitness by L. cuprina larvae in a feeding bioassay. This study confirms that the novel BFEC cell line can be used to improve the efficacy of dsRNA-mediated screening to accelerate the identification of potential target genes in the development of RNAi mediated control approaches for L. cuprina. The research models established in this study are encouraging with respect to the use of RNAi as a blowfly control method, however further improvement and validation are required for field applicationsnot prefect, and could be ongoing developing. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

In vitro IL-15-activated human naïve CD8+ T cells down-modulate the CD8β chain and become CD8αα T cells.

Antigen-driven human effector-memory CD8+ T cells expressing low levels of the CD8β chain have been previously described. However, little is known on a possible antigen-independent trigger. We have examined the impact that IL-15 has on the expression of CD8β on purified human naïve CD8+ T cells after CFSE labeling and culture with IL-15. As expected, IL-15 induced naïve CD8+ T cells to proliferate and differentiate. Remarkably, the process was associated with a cell-cycle dependent down-modulation of CD8β from the cell surface, leading to the generation of CD8αβlow and CD8αβ- (i.e., CD8αα) T cells. In contrast, expression of the CD8α chain remained steady or even increased. Neither IL-2 nor IL-7 reproduced the effect of IL-15. Determination of mRNA levels for CD8α and CD8β isoforms by qPCR revealed that IL-15 promoted a significant decrease in mRNA levels of the CD8β M-4 isoform, while levels of the M-1/M-2 isoforms and of CD8α increased. Noteworthy, CD8+ T cell blasts obtained after culture of CD8+ T cells with IL-15 showed a cell-cycle dependent increase in the level of the tyrosine kinase Lck, when compared to CD8+ T cells at day 0. This study has shown for the first time that IL-15 generates CD8αα+αβlow and CD8αα+αβ- T cells containing high levels of Lck, suggesting that they may be endowed with unique functional features.

#915 Role of VSIG4 in renal aging process

Abstract Background and Aims V-set Ig domain-containing 4 (VSIG4), known for its role in inflammation and fibrosis, is particularly interesting in the realm of chronic kidney diseases. However, its specific role in kidney aging, especially under the influence of diabetes, is not well understood. This study aims to elucidate the role of VSIG4 in the aging process of kidneys, particularly in the diabetes. Method This study used male db/db and db/m mice, beginning from the age of eight weeks and spanning 38 weeks. Urinary albumin and VSIG4 levels were assessed using 6-hour timed urine collections and ELISA. The intrarenal expression of VSIG4 and klotho were analyzed through qRT-PCR and Western blotting. Results Urinary albumin levels in db/db mice were significantly higher from 8 weeks onward compared to db/m mice. These levels increased progressively with age, peaking at 38 weeks. Similarly, urinary VSIG4 levels showed a significant initial increase in db/db mice, followed by a consistent rise with age. Interestingly, both urinary albumin and VSIG4 levels in db/m mice showed a sudden surge at 38 weeks. Notably, urinary VSIG4 levels paralleled these changes in albumin levels, with a correlation coefficient of 0.867 (p < 0.001). Intrarenal VSIG4 mRNA expression was markedly higher in diabetic mice and showed a significant increase at 16 and 38 weeks, with the 38-week levels being fivefold higher in db/m mice. The expression trends of VSIG4 protein were similar to its mRNA expression. The study also monitored intrarenal klotho expression to assess aging, which revealed a progressive decrease in both mRNA and protein levels with age. Conclusion This study highlights a significant upregulation of both urinary and intrarenal VSIG4 expressions in diabetic and non-diabetic mice. The strong association of urinary VSIG4 expression with albuminuria suggests its involvement in the renal aging process, potentially contributing to the pathogenesis of age-related kidney alterations.

Gut microbiota is necessary for pair-housing to protect against post-stroke depression in mice

The goal of this study is to investigate the role of microbiota-gut-brain axis involved in the protective effect of pair-housing on post-stroke depression (PSD). PSD model was induced by occluding the middle cerebral artery (MCAO) plus restraint stress for four weeks. At three days after MCAO, the mice were restrained 2 h per day. For pair-housing (PH), each mouse was pair housed with a healthy isosexual cohabitor for four weeks. While in the other PH group, their drinking water was replaced with antibiotic water. On day 35 to day 40, anxiety- and depression-like behaviors (sucrose consumption, open field test, forced swim test, and tail-suspension test) were conducted. Results showed pair-housed mice had better performance on anxiety- and depression-like behaviors than the PSD mice, and the richness and diversity of intestinal flora were also improved. However, drinking antibiotic water reversed the effects of pair-housing. Furthermore, pair-housing had an obvious improvement in gut barrier disorder and inflammation caused by PSD. Particularly, they showed significant decreases in CD8 lymphocytes and mRNA levels of pro-inflammatory cytokines (TNF-a, IL-1β and IL-6), while IL-10 mRNA was upregulated. In addition, pair-housing significantly reduced activated microglia and increased Nissl's body in the hippocampus of PSD mice. However, all these improvements were worse in the pair-housed mice administrated with antibiotic water. We conclude that pair-housing significantly improves PSD in association with enhanced functions of microbiota-gut-brain axis, and homeostasis of gut microbiota is indispensable for the protective effect of pair-housing on PSD.

Nicotinic acid attenuates amyloid β1-42-induced mitochondrial dysfunction and inhibits the mitochondrial pathway of apoptosis in differentiated SH-SY5Y cells

Alzheimer's disease (AD) is a chronic neurodegenerative disease characterized by progressive memory loss and behavioral disorders. The excessive accumulation of amyloid β (Aβ) and the formation of neurofibrillary tangles (NFTs) damage synaptic connections and the death of neurons. However, the underlying mechanisms of pathogenesis of AD remain unclear. Growing evidence indicates that impaired mitochondrial function may play a crucial role in the development of AD. In the current study, we investigated whether nicotinic acid (NA) could protect against amyloid β1-42-induced cytotoxicity in differentiated SH-SY5Y cells. Our results revealed the neuroprotective effects of NA on the differentiated SH-SY5Y cells treated with Aβ1-42. In detail, the 1-h pre-incubation with NA increased cell viability and lowered LDH levels. NA pre-incubation abolished Aβ1-42 treatment-associated alterations of mRNA levels of synaptic genes and enhanced the relative β3 Tubulin fluorescence intensity. NA eliminated the Aβ1-42-induced mitochondrial dysfunction by increasing the potential of mitochondrial membranes and maintaining a balance between the fusion and fission of mitochondria. Moreover, Aβ1-42 decreased mRNA levels of anti-apoptotic bcl2 and increased mRNA levels of pro-apoptotic: bim, bak, cytochrome c, and caspase 9. At the same time, the NA pre-treatment reduced Aβ1-42-dependent apoptotic death of differentiated SH-SY5Y cells.The above data suggest that NA presents a protective activity against Aβ1-42-induced cytotoxicity in differentiated SH-SY5Y cells by inhibiting the mitochondrial pathway of apoptosis and restoring the proper function of mitochondria.

Exposure to microcystin-LR promotes astrocyte proliferation both in vitro and in vivo via Hippo signaling pathway

Microcystins (MCs) are toxic to the central nervous system of mammals. However, the direct toxicity of MCs on mammalian brain cells and the involved molecular mechanisms are not fully elucidated. Here, we incubated primary astrocytes, the major glial cell-type in the brain, with 0–12.5 μM concentrations of MC-LR for 48 h, and the impairment was evaluated. We found that MC-LR caused significant increases in the cell viability at the range of 0.05–1 μM concentrations with the highest density at 0.1 μM concentration. Treatment with 0.1 μM MC-LR induced YAP nuclear translocation and decreased the ratio of p-YAP to YAP. It also decreased mRNA levels of the upstream regulator (AMOT), and enhanced expressions of YAP interacted genes (Egfr, Tead1, and Ctgf) in primary astrocytes. Overexpression of AMOT significantly attenuated the increase of MC-LR-induced astrocyte proliferation and the expression of YAP downstream genes. These results indicate that Hippo signaling contributed to MC-LR-caused astrocyte proliferation. Further, reactive astrogliosis was observed in the mice brain after MC-LR exposure to environmentally relevant concentrations (20 or 100 μg/L) through drinking water for 16 weeks. Pathological observations revealed that 100 μg/L MC-LR exposure caused neuronal damages with characteristics of shrunken or vacuolation in the region of the cerebral cortex, striatum and cerebellum. These results were accompanied with increased oxidative stress and inflammatory response. Our data reveal the potential astrocytic mechanisms in MC-induced neurotoxicity and raise an alarm for neurodegenerative disease risk following daily exposure to MC-LR.

Galectin-receptor interaction: a key player in liver fibrosis induced by Schistosoma japonicum infection

BackgroundSchistosoma japonicum eggs lodge in the liver and induce a fibrotic granulomatous immune response in the liver of host. Galectin 3 (Gal-3) is a protein implicated in fibrosis in multiple organs. However, the pathology and molecular mechanisms promoting hepatic granuloma formation remain poorly understood.MethodsTo investigate the effect of blocking galectin-receptor interactions by α-lactose on liver immunopathology in mice with S. japonicum infection, C57BL/6 mice were infected with S. japonicum and alpha (α)-lactose was intraperitoneally injected to block the interactions of galectins and their receptors.ResultsCompared with S. japonicum-infected mice, there were significantly decreased Gal-3 mRNA and protein expression levels, decreased intensity of Gal-3 fluorescence in the liver, decreased serum ALT and AST levels, decreased egg numbers of S. japonicum in the liver section, attenuated hepatic and spleen pathology, and alleviated liver fibrosis accompanied with decreased protein expression levels of fibrosis markers [α-smooth muscle actin (α-SMA), collagen I, and collagen IV] in the liver of S. japonicum-infected mice blocked galectin-receptor interactions with hematoxylin-eosin staining, Masson’s trichrome staining, immunohistochemistry, or Western blot analysis. Compared with S. japonicum-infected mice, blocking galectin-receptor interactions led to increased eosinophil infiltration and higher eosinophil cationic protein (ECP) expression in the liver, accompanied by increased mRNA levels of eosinophil granule proteins [ECP and eosinophil peroxidase (EPO)], IL-5, CCL11, and CCR3 in the liver and decreased mRNA levels of Gal-3 and M2 macrophage cytokines (TGF-β, IL-10, and IL-4) in the liver and spleen by using quantitative real-time reverse transcription-polymerase chain reaction. In addition, there were increased Beclin1 protein expression and protein expression ratio of LC3B-II/LC3B-I and decreased p62 protein expression and protein expression ratios of phospho-mTOR/mTOR and phospho-AKT/AKT by Western blot; increased double-labeled F4/80+/LC3B+ cells by immunofluorescence staining; increased M1 macrophage polarization in the liver of S. japonicum-infected mice blocked galectin-receptor interactions by flow cytometric analysis and immunofluorescence staining.ConclusionsOur data found that blockage of galectin-receptor interactions downregulated Gal-3, which in turn led to reduced liver functional damage, elevated liver eosinophil recruitment, promoted macrophage autophagy through the Akt/mTOR signaling pathway, and alleviated liver pathology and fibrosis. Therefore, Gal-3 plays a pivotal role during S. japonicum infection and could be a target of pharmacologic potential for liver fibrosis induced by S. japonicum infection.Graphical