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Several Genome Wide linkage Studies on psoriasis performed to gain insight of genetic architecture of the disease. Caspase Recruitment Domain-containing family 14 (CARD14) also known as CARMA2 or BIMP2; cytogenic location: 17q25.3, is a scaffold protein that primarily controls the skin epidermis's nuclear factor kB (NF-kB) signaling pathway activity in skin epidermis, a master gene for inflammation, has been shown to be linked with rare, heritable form of psoriasis. CARD14 is predominantly expressed in keratinocytes and epithelial cells, but also in unidentified dermal cells. For better understanding of molecular processes involved in CARD14 underlying Indian psoriatic patients, we analyzed gene expression of 42 moderates to severe cases of plaque psoriasis and same number of controls using qPCR and its validation through Immunohistochemistry (IHC). This study identifies that the expression of CARD14 in dermal endothelial cells among patients with psoriasis and explores the potential functional consequences associated with an overactive CARD14 gene. Furthermore, the expression data from the western population was consistent with the results of the qPCR validation of the candidate gene. There is a significant correlation between Indian psoriasis vulgaris patients and CARD14 up-regulation, as evidenced by a roughly two-fold shift in lesional tissue expression. This provides insights into the pathways and genes linked to the pathogenesis of psoriasis. [ABSTRACT FROM AUTHOR]

The focal point of probiotic efficacy and a crucial factor influencing poultry cultivation lies in the level of intestinal inflammation. In conventional farming processes, the reduction of intestinal inflammation generally proves advantageous for poultry growth. This study investigated the impact of Bacillus amyloliquefaciens TL (B.A.-TL) on inflammatory factor expression at both tissue and cellular levels, alongside an exploration of main active secondary metabolites. The results demonstrated that broiler feeding with a basal diet containing 4 × 109 CFU/kg B.A.-TL markedly enhanced chicken growth performance, concomitant with a significant decrease in the expression of genes encoding inflammatory cytokines (e.g., CCL4, CCR5, XCL1, IL-1β, IL-6, IL-8, LITAF, and LYZ) in jejunum and ileum tissues. The extracellular polysaccharides of B.A.-TL (EPS-TL) exhibited notable suppression of elevated inflammatory cytokine expression induced by Escherichia coli O55 lipopolysaccharides (LPS) in chicken macrophage-like cells (HD11) and primary chicken embryonic small intestinal epithelial cells (PCIECs). Moreover, EPS-TL demonstrated inhibitory effect on NF-κB signaling pathway activation. These findings suggested that the metabolic product of B.A.-TL (i.e., EPS-TL) could partly mitigate the enhanced expression of inflammatory factors induced by LPS stimulation, indicating its potential as a key component contributing to the anti-inflammatory effects of B.A.-TL. [ABSTRACT FROM AUTHOR]

The deleterious impact of lead (Pb) pollution on human health is evident in both domestic and occupational settings, provoking an inflammatory response across multiple tissue, limited attention has been devoted to its adverse effects on colitis and the underlying mechanisms. Peiminine (PMI) has been recognized for its anti-inflammatory properties, yet its specific anti-inflammatory effects in lead-induced colitis models remain elusive. Through the establishment of both in vivo and in vitro lead exposure models, suggests that lead exposure can induce colitis and that PMI regulates lead exposure-induced colitis by inhibiting the NF-kB signaling pathway, and alleviates the ability of lead to apoptosis and inflammation levels in intestinal epithelial cells. Consequently, these results present a promising avenue for further exploration of the molecular mechanisms underlying lead-induced colitis, evaluation of the associated risks linked to lead exposure, and the development of therapeutic interventions for colitis resulting from lead exposure. [ABSTRACT FROM AUTHOR]

Abstract Several Genome Wide linkage Studies on psoriasis performed to gain insight of genetic architecture of the disease. Caspase Recruitment Domain-containing family 14 (CARD14) also known as CARMA2 or BIMP2; cytogenic location: 17q25.3, is a scaffold protein that primarily controls the skin epidermis’s nuclear factor kB (NF-kB) signaling pathway activity in skin epidermis, a master gene for inflammation, has been shown to be linked with rare, heritable form of psoriasis. CARD14 is predominantly expressed in keratinocytes and epithelial cells, but also in unidentified dermal cells. For better understanding of molecular processes involved in CARD14 underlying Indian psoriatic patients, we analyzed gene expression of 42 moderates to severe cases of plaque psoriasis and same number of controls using qPCR and its validation through Immunohistochemistry (IHC). This study identifies that the expression of CARD14 in dermal endothelial cells among patients with psoriasis and explores the potential functional consequences associated with an overactive CARD14 gene. Furthermore, the expression data from the western population was consistent with the results of the qPCR validation of the candidate gene. There is a significant correlation between Indian psoriasis vulgaris patients and CARD14 up-regulation, as evidenced by a roughly two-fold shift in lesional tissue expression. This provides insights into the pathways and genes linked to the pathogenesis of psoriasis.

Background: Chronic kidney disease (CKD) is the third-leading cause of premature mortality worldwide. It is characterized by rapid deterioration due to renal interstitial fibrosis (RIF) via excessive inflammatory infiltration. The aim of this study was to discover key immune-related genes (IRGs) to provide valuable insights and therapeutic targets for RIF in CKD. Materials and methods: We screened differentially expressed genes (DEGs) between RIF samples from CKD patients and healthy controls from a public database. Least absolute shrinkage and selection operator regression analysis and receiver operating characteristic curve analysis were applied to identify significant key biomarkers. The single-sample Gene Set Enrichment Analysis (ssGSEA) algorithm was used to analyze the infiltration of immune cells between the RIF and control samples. The correlation between biomarkers and immune cell composition was assessed. Results: A total of 928 DEGs between CKD and control samples from six microarray datasets were found, 17 overlapping immune-correlated DEGs were identified by integration with the ImmPort database, and six IRGs were finally identified in the model: apolipoprotein H (APOH), epidermal growth factor (EGF), lactotransferrin (LTF), lysozyme (LYZ), phospholipid transfer protein (PLTP), and secretory leukocyte peptidase inhibitor (SLPI). Two additional datasets and in vivo experiments indicated that the expression levels of APOH and EGF in the fibrosis group were significantly lower than those in the control group, while the expression levels of LTF, LYZ, PLTP, and SLPI were higher (all P < 0.05). These IRGs also showed a significant correlation with renal function impairment. Moreover, four upregulated IRGs were positively associated with various T cell populations, which were enriched in RIF tissues, whereas two downregulated IRGs had opposite results. Several signaling pathways, such as the "T cell receptor signaling pathway" and "positive regulation of NF-kB signaling pathway", were discovered to be associated not only with immune cell infiltration, but also with the expression levels of six IRGs. Conclusion: In summary, six IRGs were identified as key biomarkers for RIF, and exhibited a strong correlation with various T cells and with the NF-kB signaling pathway. All these IRGs and their signaling pathways may evolve as valuable therapeutic targets for RIF in CKD. [ABSTRACT FROM AUTHOR]

The intestinal barrier comprises a single layer of epithelial cells tightly joined to form a physical barrier. Disruption or compromise of the intestinal barrier can lead to the inadvertent activation of immune cells, potentially causing an increased risk of chronic inflammation in various tissues. Recent research has suggested that specific dietary components may influence the function of the intestinal barrier, potentially offering a means to prevent or mitigate inflammatory disorders. However, the precise mechanism underlying these effects remains unclear. Bovine colostrum (BC), the first milk from cows after calving, is a natural source of nutrients with immunomodulatory, anti-inflammatory, and gut-barrier fortifying properties. This novel study sought to investigate the transcriptome in BC-treated Zonulin transgenic mice (Ztm), characterized by dysbiotic microbiota, intestinal hyperpermeability, and mild hyperactivity, applying RNA sequencing. Seventy-five tissue samples from the duodenum, colon, and brain of Ztm and wild-type (WT) mice were dissected, processed, and RNA sequenced. The expression profiles were analyzed and integrated to identify differentially expressed genes (DEGs) and differentially expressed transcripts (DETs). These were then further examined using bioinformatics tools. RNA-seq analysis identified 1298 DEGs and 20,952 DETs in the paired (Ztm treatment vs. Ztm control) and reference (WT controls) groups. Of these, 733 DEGs and 10,476 DETs were upregulated, while 565 DEGs and 6097 DETs were downregulated. BC-treated Ztm female mice showed significant upregulation of cingulin (Cgn) and claudin 12 (Cldn12) duodenum and protein interactions, as well as molecular pathways and interactions pertaining to tight junctions, while BC-treated Ztm males displayed an upregulation of transcripts like occludin (Ocln) and Rho/Rac guanine nucleotide exchange factor 2 (Arhgf2) and cellular structures and interfaces, protein–protein interactions, and organization and response mechanisms. This comprehensive analysis reveals the influence of BC treatment on tight junctions (TJs) and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) signaling pathway gene expressions. The present study is the first to analyze intestinal and brain samples from BC-treated Ztm mice applying high-throughput RNA sequencing. This study revealed molecular interaction in intestinal barrier function and identified hub genes and their functional pathways and biological processes in response to BC treatment in Ztm mice. Further research is needed to validate these findings and explore their implications for dietary interventions aimed at improving intestinal barrier integrity and function. The MGH Institutional Animal Care and Use Committee authorized the animal study (2013N000013). [ABSTRACT FROM AUTHOR]

Pathogenic variants in the EDARADD gene result in autosomal recessive and autosomal dominant ectodermal dysplasia. This article reports on the fourth family in the world with ectodermal dysplasia 11A (ECTD11A) cause from a novel splicing variant in the EDARADD gene, identified by whole exome sequencing and confirmed by Sanger sequencing. The proband and his mother were heterozygous for the detected variant (NM_145861.4:c.161‐2A>T). The proband manifests unusual symptoms including hyperkeratotic plaques, slow‐growing hair, recurrent infection, and pectus excavatum. His mother presents hypohidrosis, extensive tooth decay, fragile nails, and sparse hair. Further studies on ECTD11A patients could be useful to characterizing the phenotype features more precisely. [ABSTRACT FROM AUTHOR]

Introduction. The recurrence of diffuse large B-cell cell lymphoma in the central nervous system in the vast majority of cases is a fatal manifestation of the disease. The study of the lymphoma mutational profile can improve the accuracy of the prognosis of relapse in the central nervous system and justify the selection of patients for preventive treatment. Aim. To evaluate the mutational profile of cases of diffuse large B-cell cell lymphoma with central nervous system damage in relapse based on the results of our own experiment on high-performance sequencing.Materials and methods. On the Illumina platform, full-exome sequencing of diagnostic samples of diffuse large B-cell cell lymphoma with relapses in the central nervous system was performed. A panel including more than 70 genes was analyzed.Results. Four main groups of genetic events can be distinguished in the group of studied samples, namely: combined mutations in the NF-kB (MYD88, NOTCH1, CD79B, CARD11) and JAK-STAT (PIM1, STAT6) signaling pathways, as well as aberrations in the main oncosuppressor TP53 and chromatin remodeling system genes (ARID1A, KMT2D, EP300, SMARCA4). A recurrent mutation c. 794T>C, p.L265P MYD88 was detected in the study group. Among other findings, mutations in the CIITA and CD58 genes should be noted, which are important in avoiding tumor cells from immune surveillance.Conclusion. Despite the apparent heterogeneity of the mutational profile of diffuse large B-cell cell lymphoma with relapses in the central nervous system, in most cases, tumor cells were characterized by genetic disorders leading to the production of a large number of pro-inflammatory cytokines by malignant lymphocytes, as well as aberrations that reduce immunogenicity and contribute to the avoidance of immune surveillance by the tumor.

Gastric cancer (GC), a common and high-mortality disease, still occupies an important position in current cancer research, and Helicobacter pylori (H. pylori) infection as its important risk factor has been a hot and challenging research area. Among the numerous pathogenic factors of H. pylori, the virulence protein CagA has been widely studied as the only bacterial-derived oncoprotein. It was found that CagA entering into gastric epithelial cells (GECs) can induce the dysregulation of multiple cellular pathways such as MAPK signaling pathway, PI3K/Akt signaling pathway, NF-kB signaling pathway, Wnt/b-catenin signaling pathway, JAK-STAT signaling pathway, Hippo signaling pathway through phosphorylation and nonphosphorylation. These disordered pathways cause pathological changes in morphology, adhesion, polarity, proliferation, movement, and other processes of GECs, which eventually promotes the occurrence of GC. With the deepening of H. pylori-related research, the research on CagA-induced abnormal signaling pathway has been updated and deepened to some extent, so the key signaling pathways activated by CagA are used as the main stem to sort out the pathogenesis of CagA in this paper, aiming to provide new strategies for the H. pylori infection and treatment of GC in the future. [ABSTRACT FROM AUTHOR]

To investigate the effect of combination of TNF-α and H2O2 induction on oxidative stress and inflammatory injury in rat cardiomyocytes and to constract a chronic inflammatory disease model that closely resembles oxidative stress and sustained inflammatory damage in cardiac myocytes, 200 µmol L-1 H2O2 for 1 h and 80 ng • mL-1 TNF-a for 12 h were used to set up oxidative injury model and inflammtatory injury model in H9c2 cells via CCK-8 assay, flow cytometry and enzyme-linked immunosorbent assay in this study. When H9c2 cells were treated with 200 µmol • L-1 H2O2 for 1 h followed by 80 ng • mL-1 TNF-α for 12 h in H9c2 cells, the level of oxidative stress indicator MDA increased, antioxidant enzyme SOD decreased, and inflammation-related protein expression and mRNA expression downstream of NF-kB signaling pathway was enhanced. The combination of 200 μmol • L-1 H2O2 for 1 h and 80 ng mL-1 TNF-a for 12 h induced oxidative stress and inflammatory injury in H9c2 cells, and the mechanism could be related to targeted regulation of NF-kB signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract Background There is increasing evidence that mesenchymal stem cells (MSCs) help shape the tumor microenvironment and promote tumor progression, and ion channels might play a critical role in this process. The objective of the present study was to explore the function and mechanism of MT-CAFs on progression of colon cancer. Methods Here, a gene chip was used for a general analysis of gene expression changes in MSC-transformed CAF cells (MT-CAFs). Bioinformatic tool and western blot screened out the ion channel protein TRPC3 with significantly increased expression, and identify the function through two-photon microscope. The progression of cancer was detected via MTS, transwell and Wound Healing. ELISA deected the secretion of inflammation factors. TRPC3/NF-KB axis was identified by western blot and immunofluorescence. Results TRPC3 can caused calcium influx, which further activated the NF-KB signaling pathway. Knockdown or inhibition of TRPC3 in MSCs significantly reduced the activation of NF-KB, and decreased the growth, migration, and invasion of MT-CAFs. After TRPC3 knockdown, the ability of MT- CAFs to promote tumor migration and invasion was impaired. Conversely, the upregulation of TRPC3 expression in MT-CAFs had the opposite effect. In vivo, TRPC3 expressed on MSCs also contributed to the tumorigenesis and progression of cancer cells. In addition, the Oncomine and GEPIA databases showed that TRPC3 expression is higher in colon cancer tissues compared with normal colon tissues, and was positively correlated with the expression of the CAF genes alpha-smooth muscle (α-SMA/ACTA2) and fibroblast activation protein Alpha. The disease-free survival of patients with positive TRPC3 expression in MSCs was significantly shorter than those with negative expression. Conclusions These results indicate that TRPC3 expressed on MT-CAFs plays a critical role in tumor progression via the NF-KB signaling pathway, and is correlated with poor prognosis in colon cancer patients. Therefore, TRPC3 may be a novel therapeutic target for the treatment of colon cancer.

This study explored the possible connection between the insulin resistance-targeting protein adipokine lipocalin-2 (LCN-2) and NF-κB signaling pathway in the inflammatory microenvironment in PCOS-IR model rats to determine the pharmacological mechanism of modified Cangfu Daotan decoction (MCDD) intervention for PCOS-IR. We used a high-fat diet (42 days) combined with letrozole (1 mg/kg/day, 42 days) to establish a PCOS-IR rat model. From the third week after modeling, the rats were given continuous administration of MCDD (high dose with 31.68 g/kg, medium dose with 15.84 g/kg, and low dose with 7.92 g/kg) for 28 days. Serum, ovarian tissue, liver, and adipose tissue were collected after the last gavage. Enzyme-linked immunosorbent assay, hematoxylin-eosin (HE) staining, Masson staining, qRT-PCR, and Western blot experiments were performed to detect various indicators. Our results showed that MCDD could reduce body weight and abdominal fat weight; restore normal estrous cycle and ovarian function; alleviate fatty liver; regulate HOMA-IR and OGTT index; reduce serum inflammatory factor levels, LCN-2 level, and gene expression; and regulate the insulin signal transduction and NF-κB pathways in PCOS-IR rats. Thus, MCDD may play a role in improving ovarian function in PCOS-IR rats by downregulating NF-κB/LCN-2 proteins and upregulating the gene expression of Insr/Irs-1/Glut4 in the insulin signaling pathway in the inflammatory environment.

Purpose: To investigate the effect of phycocyanin on myocardial ischemia-reperfusion injury, and the possible mechanisms involved. Methods: Twenty-four Sprague-Dawley (SD) rats were randomly divided into Sham group (only threading without ligation), IRI group (myocardial ischemia-reperfusion injury group) and phycocyanin group (phycocyanin pretreatment + myocardial ischemia-reperfusion injury group). The heart was harvested and cardiomyocytes were isolated. Colorimetry was used to determine the contents of cardiomyocyte serum creatine phospho-MB (CK-MB), lactate dehydrogenase (LDH) and malondialdehyde (MDA), and the activities of total antioxidant capacity (T-AOC), catalase (CAT), glutathione (GSH), total superoxide dismutase (SOD) and other related oxidative stress indicators. Furthermore, apoptosis was evaluated using TUNEL staining. Protein levels of cardiac factor E2 related factor 2 (Nrf2), heme oxygenase-1 (HO-1), human NADPH dehydrogenase 1 (NQO1) and nuclear factor-κB (NF-κB) were evaluated by Western blot and immunohistochemistry. Results: Compared with the myocardial IRI group, the contents of CK-MB, LDH, MAD and ROS in the treated group were significantly decreased (p < 0.05), but the activities of SOD, GSH, SOD, CAT, and T-AOC in the myocardial tissues were significantly enhanced (p < 0.05). Moreover, the pathological changes in myocardial tissue were significantly reduced. In addition, the expression levels of Nrf2, HO-1 and NQO-1 were significantly up-regulated after phycocyanin pretreatment, while expression of NF-κB was significantly down-regulated (p < 0.05). Conclusion: Phycocyanin improves myocardial anti-oxidative stress via activation of Nrf2 signaling pathway, and also protects rats from myocardial ischemia-reperfusion injury by reducing inflammatory response via inhibition of NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Background: There is increasing evidence that mesenchymal stem cells (MSCs) help shape the tumor microenvironment and promote tumor progression, and ion channels might play a critical role in this process. The objective of the present study was to explore the function and mechanism of MT-CAFs on progression of colon cancer. Methods: Here, a gene chip was used for a general analysis of gene expression changes in MSC-transformed CAF cells (MT-CAFs). Bioinformatic tool and western blot screened out the ion channel protein TRPC3 with significantly increased expression, and identify the function through two-photon microscope. The progression of cancer was detected via MTS, transwell and Wound Healing. ELISA deected the secretion of inflammation factors. TRPC3/NF-KB axis was identified by western blot and immunofluorescence. Results: TRPC3 can caused calcium influx, which further activated the NF-KB signaling pathway. Knockdown or inhibition of TRPC3 in MSCs significantly reduced the activation of NF-KB, and decreased the growth, migration, and invasion of MT-CAFs. After TRPC3 knockdown, the ability of MT- CAFs to promote tumor migration and invasion was impaired. Conversely, the upregulation of TRPC3 expression in MT-CAFs had the opposite effect. In vivo, TRPC3 expressed on MSCs also contributed to the tumorigenesis and progression of cancer cells. In addition, the Oncomine and GEPIA databases showed that TRPC3 expression is higher in colon cancer tissues compared with normal colon tissues, and was positively correlated with the expression of the CAF genes alpha-smooth muscle (α-SMA/ACTA2) and fibroblast activation protein Alpha. The disease-free survival of patients with positive TRPC3 expression in MSCs was significantly shorter than those with negative expression. Conclusions: These results indicate that TRPC3 expressed on MT-CAFs plays a critical role in tumor progression via the NF-KB signaling pathway, and is correlated with poor prognosis in colon cancer patients. Therefore, TRPC3 may be a novel therapeutic target for the treatment of colon cancer. [ABSTRACT FROM AUTHOR]

Background & Aims: Eosinophils are the main inflammatory effector cells that damage gastrointestinal tissue in eosinophilic gastrointestinal diseases (EGIDs). Activation of the OX40 pathway aggravates allergic diseases, such as asthma, but it is not clear whether OX40 is expressed in eosinophils to regulate inflammation in EGIDs. In this study, we assessed the expression and effect of OX40 on eosinophils in WT and Ox40-/- eosinophilic gastroenteritis (EGE) mice. Methods: Eosinophil infiltration, ovalbumin (OVA)-specific Ig production, OX40 expression and inflammatory factor levels in the intestine and bone marrow (BM) were investigated to evaluate inflammation. Results: Weconfirmed thatOVA-challenged mice produced high levels of Ox40, Mbp, Ccl11, Il5, Il4, Il13, and Il6 mRNA and a low level of Ifng mRNA in the intestine. Increased eosinophils were observed in intestinal and lymph tissues, accompanied by significantly upregulatedOX40 and Type 2 cytokine production in eosinophils of EGEmice. Ox40 deficiencyamelioratedOVAinduced inflammation, eosinophil infiltration, and cytokine production in the intestine. Consistently, Ox40-/- eosinophils exhibited decreased proliferation and proinflammatory function. The stimulation of the agonistic anti-OX40 antibody, OX86, promoted the effect of OX40 on eosinophils. The present study also showed that Ox40 deficiency dampened the Traf2/6-related NF-kB signaling pathway in eosinophils. Conclusions: OX40 may play a critical role in the progress of OVA-induced EGE by promoting the maturation and function of eosinophils via the Traf2/6-related NF-kB signaling pathway. [ABSTRACT FROM AUTHOR]

Inflammatory reactions are part of a complex biological response that plays a vital role in the appearance of various stimuli resulting from tissue and cell damage, the invasion of pathogenic bacteria, and the formation of the subsequent adaptive immune response. The production of many triggers and mediators of inflammation, which are inducers of pro-inflammatory factors, is controlled by numerous differentiation programs, through which inflammation is resolved and tissue homeostasis is restored. However, prolonged inflammatory responses or dysregulation of pro-inflammatory mechanisms can lead to chronic inflammation. Modern advances in biotechnology have made it possible to characterize the anti-inflammatory activity of phlorotannins, polyphenolic compounds from brown seaweed, and the mechanisms by which they modulate the inflammatory response. The purpose of this review is to analyze and summarize the results of numerous experimental in vitro and in vivo studies, illustrating the regulatory mechanisms of these compounds, which have a wide range of biological effects on the body. The results of these studies and the need for further research are discussed. [ABSTRACT FROM AUTHOR]

Legionella pneumophila widely exists in natural and artificial water environments, which enables it to infect people. L. pneumophila infection causes Legionnaires’ disease (LD), which is a significant but relatively uncommon respiratory infection. Approximately 90% of LD is caused by L. pneumophila serogroup 1 (Lp1). Meteorological conditions may affect the infectivity and virulence of Lp1, but the exact relationship between them is still unclear. In this study, we evaluated the virulence of Lp1 by screening of total 156 Lp1 strains isolated from cooling tower water in different regions of China by detecting their abilities to activate NF-kB signaling pathway in vitro. In addition, we screened the distribution of some selected virulence genes in these strains. The virulence, virulence gene distribution, and the meteorological factors were analyzed. We found that both the virulence and the distribution of virulence genes had a certain regional and meteorological correlation. Although the loss of several virulence genes showed significant effects on the virulence of Lp1 strains, the distribution of virulence genes had very limited effects on the virulence of Lp1. IMPORTANCE LD is likely to be underrecognized in many countries. Due to the widespread existence of L. pneumophila in natural and artificial water environments and to the lack of cross-protection against different strains, L. pneumophila is a potentially serious threat to human health. Therefore, effective monitoring of the virulence of L. pneumophila in the water environment is very important to prevent and control the prevalence of LD. Understanding the virulence of L. pneumophila can not only help us to predict the risk of possible outbreaks in advance but can also enable more targeted clinical treatment. This study highlights the importance of understanding the epidemiology and ecology of L. pneumophila isolated from public facilities in terms of public health and biology. Due to the potential for water sources to harbor and disseminate L. pneumophila and to the fact that geographical conditions influence the virulence of L. pneumophila, timely and accurate L. pneumophila virulence surveillance is urgently needed. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are a crucial type of non-coding RNAs involved in post-transcriptional regulation. The playing essential regulatory roles in the NF-κB signaling pathway and modulate the host immune response to diverse pathogens by targeting IκBα. However, the regulatory mechanism of miRNAs in relation with IκBα in Sebastes schlegelii remains unclear. In our study, we identified two copies of IkBα gene in black rockfish (Sebastes schlegelii), namely IkBα1 and IkBα2. Moreover, we have discovered that miRNA-530 can activate the NF-κB signaling pathway by inhibiting the expression of IκBα , thereby inducing the inflammatory response. This project comprehensively investigated the interactive regulatory roles of miRNA-530 in the NF-κB signaling pathway at both cellular and in vivo levels, while also elucidating the regulatory relationships between miRNA-530 and IκBα. In conclusion, our research confirmed that miRNA-530 can target the 3′UTR region of IκBα , resulting in a decrease in the expression of IκBα at the post-transcriptional level and inhibiting its translation. The findings contribute to the understanding of the regulatory network of non-coding RNA in teleosts and its subsequent regulation of the NF-κB signaling pathway by miRNAs. • According to binding sites and expression level, miRNA530 can target both IkBα1 and IkBα2. • miRNA-530 inhibits the expression of IkBα1 and IkBα2 at the mRNA level and protein level. • miRNA-530 promotes NF-κB pathway by targeting IκBα. [ABSTRACT FROM AUTHOR]

Objective To study the expressions of BMALl and CerbB-2 genes in nasopharyngeal carcinoma (NPC) and their related mechanisms. Methods Plasmid transfection. MTT, RT-PCR and Western blotting were used to detect the proliferation of NPC cells. the expressions of BMALl and CerbB-2 genes and proteins and the expression of NF-ıB signaling pathway. Results The MTT results showed that BMAL1 and CerbB-2 could affect the proliferation of NPC cells. RT-PCR results showed that BMALl gene was significantly down-regulated in NPC (CK:2.24±0.22, NPC:0.63±0.11, P

Inflammatory reactions are part of a complex biological response that plays a vital role in the appearance of various stimuli resulting from tissue and cell damage, the invasion of pathogenic bacteria, and the formation of the subsequent adaptive immune response. The production of many triggers and mediators of inflammation, which are inducers of pro-inflammatory factors, is controlled by numerous differentiation programs, through which inflammation is resolved and tissue homeostasis is restored. However, prolonged inflammatory responses or dysregulation of pro-inflammatory mechanisms can lead to chronic inflammation. Modern advances in biotechnology have made it possible to characterize the anti-inflammatory activity of phlorotannins, polyphenolic compounds from brown seaweed, and the mechanisms by which they modulate the inflammatory response. The purpose of this review is to analyze and summarize the results of numerous experimental in vitro and in vivo studies, illustrating the regulatory mechanisms of these compounds, which have a wide range of biological effects on the body. The results of these studies and the need for further research are discussed.

Objective To observe the effects and explore the mechanism of paeoniflorin (PF) on the inflammation and degeneration of LPS-induced human chondrocytes. Methods Discarded cartilage tissues from primary knee osteoarthritis (KO A) patients undergoing total knee arthroplasty at The Third Affiliated Hospital of Beijing University of Chinese Medicine from April 2019 to October 2019 were collected and digested into chondrocytes using pancreatin and collagenase. The effect of different concentrations of PF on the viability of chondrocytes was detected using the CCK-8 assay, and appropriate concentrations were selected for subsequent experiments. Chondrocytes were randomly divided into five groups，namely the blank group，the model group，and the low，medium，and high concentration PF intervention groups. Except for the blank group，chondrocytes in the other four groups were induced by LPS to simulate an arthritis environment. In the low，medium，and high concentration PF intervention groups? 10，20，and 50 jjlMoI/L PF were given respectively. The expressions of IL-1 p? IL-6，TNF-cc， MMP13 and ADAMTS5 in each group were detected by qPCR assay ； and the expression levels of NF-kB p65，IKB and IkK were detected by Western-blot. Results The results of CCK-8 assay showed that 10, 20，and 50 jjlMoI/L PF could significantly increase the level of proliferation and viability of human chondrocyte ； while excessively high concentration (200 jjlM) PF could inhibit chondrocyte viability. Further qPCR results showed that PF could significantly inhibit the expression levels of inflammatory factors IL-1 p? IL-6，TNF-a and cartilage degradation factors MMP13 and ADAMTS5 in human chondrocytes after LPS intervention, compared with the model group (尸 <0. 05) ; WB results showed that PF could significantly inhibit NF-kB p65 and IkK expresion and promotes IkB expression (P < 0, 05). Conclusion Paeoniflorin seems to inhibit LPS-induced human chondrocyte inflammation and delay cartilage degeneration by inhibiting the activation of NF-kB signaling pathway. [ABSTRACT FROM AUTHOR]

Objective The purpose of this study was to determine the protective effect of lipoic acid (ALA) on aged rat models and to explore its mechanism of action. Methods The rats were randomly divided into the following groups : the young rats control group (Con), the old rats group (Old), and the old rat + lipoic acid group (ALA). After adaptive feeding for 7 days, ALA was given for 12 weeks. ELISA analysis is used to analyze the bone metabolism indicators. Western blot analysis was performed to assess NF-KB, Caspase-3 and PARP protein expression. Results The result of this study indicate that ALA can significantly improve bone density and bone mass in aged rats. Elderly rats can significantly reduce the levels of TRACP-5b and [3-CTX after ALA administration (P < 0. 01), and significantly increase the levels of BLAP and OPG (P < 0. 01). Treatment with ALA can significantly reduce the expression of NF-KB, Caspase-3 and PARP in elderly rats, and inhibit NF-KB pathway conduct10n. Conclusion This study suggests that ALA can protect the bone mass by inhibiting NF-KB signaling pathway in aged rats. [ABSTRACT FROM AUTHOR]

Objective To observe the effect of resveratrol on the number of osteoclasts and F-actin ring area induced by titanium particles in mouse macrophage cell line RA W264. 7, and to explore its mechanism. Methods The mouse macrophage cell line RA W264. 7 was divided into Sham group, Vehicle group, Low-Res group, Mid-Res group, High-Res group; cells in the Sham group were added with minimal medium, the Vehicle group with titanium particles + minimal me dium, Low-Res group with titanium particles+ minimal medium + resveratrol ( 1 µmol/L), Mid-Res group with titanium particles + minimal medium+ resveratrol ( 10 µmol/L), and High-Res group with titanium particles + minimal medium + resveratrol ( 15 µmol/L), respectively. After 5 days of cell culture in each group, the number of osteoclasts in each group was detected by TRAP staining, and the area of F-actin ring of osteoclasts in each group was detected by phalloidin staining. Real-time PCR was used to detect TNF--α and IL-1β mRNA in each group, ELISA was used to detect TNF-a and IL-1β protein in each group, and Western blotting was used to detect p-p65 and p-IKB protein. Results In the Vehicle group, more osteoclasts with larger volume, wine red, and multiple nuclei were observed. After intervention with different concentrations of resveratrol, the number of osteoclasts decreased as the drug concentration increased; the number of osteo clasts in the Sham group, Vehicle group, Low-Res group, Mid-Res group, and High-Res group were 0, 145. 31 ± 7. 87, 129. 63 ± 4. 12, 85. 51 ± 3. 65, 69. 76 ± 5. 63, respectively; except between the Vehicle group and the Low-Res group, significant differences were found between the other groups (all P < 0. 05) . A small number of large F-actin rings was ob served in the Vehicle group; the F-actin rings in the Low-Res group and the Mid-Res group were slightly reduced as com pared with those of the Vehicle group, and the multi-nuclear cells in the High-Res group were significantly reduced; the F actin ring areas of the Sham group, Vehicle group, Low-Res group, Mid-Res group, High-Res group were 0, ( 3 642. 31 ± 987. 01), (3 265. 75 ± 871. 98), ( 2 497.45 ± 712. 61), and ( 1 741. 81 ± 716. 07) µm², with statistically significant difference between groups (all P < 0. 05) . The relative expression or expression levels of TNF-α mRNA, IL-1 ~ mRNA, TNF--α protein, IL-1~protein, p-p65 protein, and p-IKB protein in cells of the Low-Res group, Mid-Res group, and High Res group were lower than those of the Vehicle group, and the decreased degree was related to the resveratrol concentration. Conclusion Resveratrol has a concentration-dependent effect on the number of osteoclasts and F-actin ring area in duced by titanium particles in the RAW264. 7 mouse macrophage cell line, and the mechanism of the inhibition is related to the NF-KB signalling pathway. [ABSTRACT FROM AUTHOR]

Objective To observe the apoptosis, proliferation and expression of NF-K B signaling pathway-related pro teins in the skin squamous cell carcinoma ( cSCC) cell lines transfected with Survivin shRNA. Methods A431 cells with the highest relative expression of Survivin mRNA were divided into the sh-Survivin group and sh-NC group. Survivin shRNA was transfected into cells of the sh-Survivin group, and NC shRNA was transfected into cells of the sh-NC group. Cell cycle assay was used to detect apoptosis rate of each group at 48 h after transfection. At the 1st, 2nd, 3rd and 4th days of trans fection, the proliferation ability of each group was detected by MTS method (represented by OD value). After transfection for48 h, NF-KB inhibitory protein (IKBa), phosphorylated IKB kinasea/[3 (p-IKKa/[3) and phosphorylated p65 (p p65) were detected by Wes tern blotting. Results The apoptosis rates of the sh-Survivin group and sh-NC group were 19. 21 % ± 2. 54% and 4. 52% ± 0. 31 %, respectively, with statistically significant difference ( P < 0. 05). OD values of the sh-Survivin group were 0. 27 ± 0. 01, 0. 32 ± 0. 04, 0. 37 ± 0. 03, and 0. 54 ± 0. 08 on day 1, 2, 3, and 4, respective ly, and those of sh-NC group were 0. 27 ± 0. 02, 0. 38 ± 0. 03, 0. 67 ± 0. 05, and 0. 88 ± 0. 07, respectively. OD value of the sh-Survivin group on day 3 and 4 were significantly lower than those of the sh-NC group (all P < 0. 05). The relative expression of IKBa, p-IKKa/[3and p-p65 in the sh-Survivin group were 0. 61 ± 0. 11, 0. 13 ± 0. 06 and 0. 09 ± 0. 05, re spectively, and those in the sh-NC group were 0. 11 ± 0. 04, 0. 76 ± 0. 15 and 0. 81 ± 0. 19, respectively, with statistically significant difference between every two groups (all P < 0. 05) . Conclusion The apoptosis rate of cSCC cells transfected with Survivin shRNA increases, proliferation ability decreases, and NF-KB signaling pathway is inhibited; interfering Sur vivin may promote apoptosis and inhibit proliferation of cSCC cells by inhibiting NF-KB signaling pathway. [ABSTRACT FROM AUTHOR]

Recently, some researches show that intervertebral disc degeneration is significantly related to metabolic diseases, especially diabetes, but the specific mechanism has not been further studied. This study aims to the role of high glucose microenvironment and NF-kappa B signaling pathway in IVDD and to explore the mechanism of high glucose-induced degeneration of nucleus pulposus cells through NF-kappa B signaling pathway. It may provide a new strategy and method for preventing and treating diabetic-related low back pain. Nucleus pulposus cells was cultured in 5.5 mmol/L, 15 mmol/L, 30 mmol/L and 100 mmol/L glucose medium and the expression of MMP-3, MMP-13, Aggrecan and CollagenII in nucleus pulposus cells was detected by RT-PCR. Bay11-7082 was used to inhibit the activation of NF-kappa B signaling pathway and the expression of MMP-3, MMP-13, Aggrecan, CollagenII and NF-kappa B in nucleus pulposus cells was detected by RT-PCR and Western Blot. RT-PCR showed that at different concentration, the expression of Aggrecan and CollagenII decreased with the increasing of concentration, while the expression of MMP-3 and MMP-13 increased with the increasing of concentration. RT-PCR and Western Blot showed that after using Bay11-7082, the expression of Aggrecan and CollagenII increased and the expression of MMP-3 and MMP-13 decreased in the high glucose group. High glucose microenvironment is correlated with intervertebral disc degeneration. High glucose microenvironment induces intervertebral disc degeneration by mediating NF-kappa B signaling pathway. [ABSTRACT FROM AUTHOR]

BACKGROUND: The relationship between nuclear factor {NF)-KB signaling pathway and intervertebral disc degeneration is a hotspot in the field of orthopedics. In-depth investigation on various signaling pathways in the intervertebral disc contributes to understanding the mechanism of intervertebral disc degeneration. OBJECTIVE: To investigate the regulation of the serum containing Yishen Huoxue Tongluo Recipe on the NF-KB signal pathway of human intervertebral disc nucleus pulposus cells under different hydrostatic pressures, attempting to explore the possible therapeutic mechanism and target of Yishen Huoxue Tongluo Recipe in the treatment of degenerative diseases of the intervertebral disc from the perspective of molecular biology. METHODS: Passage 3 Human intervertebral disc nucleus pulposus cells were divided into eight groups and were cultured in the drug-containing serum of Yishen Huoxue Tongluo Recipe. The cells were intervened for 2, 4, and 6 hours under different hydrostatic loading conditions {0.3, 1, and 3 MPa). The morphology and growth of nucleus pulposus cells were observed by an inverted phase contrast microscope. Ultrastructural changes of intervertebral disc nucleus pulposus cells were observed by a transmission electron microscopy. Cell counting kit-8 method was used to detect the proliferation activity of nucleus pulposus cells. Annexin V-FITC/Propidium Iodide apoptotic kit was used to double-stain nucleus pulposus cells to detect the cell apoptotic rate. Western blot method was used to detect the changes of NF-KB p65, Collagen II, ADAMTS-4, MMP-13 and Caspase-3 in nucleus pulposus cells. RESULTS AND CONCLUSION: {1) Under the same pressure and time, the morphology and growth of nucleus pulposus cells in the pressure+drug-containing serum groups were better than those in the normal pressure group and the simple pressure groups. Among them, the 0.3 and 1 MPa pressure+drug-containing serum groups had more intact cell morphology and better cell growth than the 3 MPa pressure+ drug-containing serum group. {2} The proliferative activity of nucleus pulposus cells was higher in the pressure+drug-containing serum group, and there was a significant difference between the 0.3 MPa pressure+drug-containing serum group and the 0.3 MPa simple pressure group (P < 0.05). (3) The apoptotic rate of nucleus pulposus cells in the pressure+drug-containing serum group was lower than that in the normal pressure group and simple pressure intervention group (P < 0.05). (4} The expression of Collagen II and Caspase-3 increased in the pressure+drug-containing serum group, while the expression of NF-kappa B p65, ADAMTS-4 and MMP-13 decreased in the pressure+drug-containing serum group (P < 0.05). To conclude, Yishen Huoxue Tong/uo Recipe can increase cell activity, reduce cell apoptosis and effectively delay the degeneration of nucleus pulposus cells. Its mechanism is likely to promote the expression of Collagen II and Caspase-3 through the NF-KB signaling pathway of nucleus pulposus cells of intervertebral disc, and inhibit the expression of NF-KB p65, ADAMTS-4 and MMP-13. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Shenyang Agricultural University is the property of Journal of Shenyang Agricultural University Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of Henan Agricultural Sciences is the property of Editorial Board of Journal of Henan Agricultural Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the third leading cause of cancer-related deaths worldwide. Despite recent advances in treatment options, therapeutic management of HCC remains a challenge, emphasizing the importance of exploring novel targets. MALT1 paracaspase is a druggable signaling molecule whose dysregulation has been linked to hematological and solid tumors. However, the role of MALT1 in HCC remains poorly understood, leaving its molecular functions and oncogenic implications unclear. Here we provide evidence that MALT1 expression is elevated in human HCC tumors and cell lines, and that correlates with tumor grade and differentiation state, respectively. Our results indicate that ectopic expression of MALT1 confers increased cell proliferation, 2D clonogenic growth, and 3D spheroid formation in well differentiated HCC cell lines with relatively low MALT1 levels. In contrast, stable silencing of endogenous MALT1 through RNA interference attenuates these aggressive cancer cell phenotypes, as well as migration, invasion, and tumor-forming ability, in poorly differentiated HCC cell lines with higher paracaspase expression. Consistently, we find that pharmacological inhibition of MALT1 proteolytic activity with MI-2 recapitulates MALT1 depletion phenotypes. Finally, we show that MALT1 expression is positively correlated with NF-kB activation in human HCC tissues and cell lines, suggesting that its tumor promoting functions may involve functional interaction with the NF-kB signaling pathway. This work unveils new insights into the molecular implications of MALT1 in hepatocarcinogenesis and places this paracaspase as a potential marker and druggable liability in HCC. [ABSTRACT FROM AUTHOR]

• We demonstrated that Syringaresinol can amelirate the IL-1β-stimulated ECM degradation and inflammation in mouse chondrocyte by inhibiting the NFκB pathways in vitro. In vivo, it can decrease the OARSI scores in DMM mouse models. Besides, these results mean that Syr might be a potential candidate for OA prevention and treatment. Osteoarthritis (OA) is a now regarded as a worldwide whole joint disease with synovial inflammation, cartilage degeneration, and subchondral sclerosis. Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used drugs for OA treatment which only relieve the symptoms and restrain the progression of OA. However, various severe adverse effects often occur in patients with long-term NSAIDs use, which heavily burdens the healthcare system and impacts the quality of life. Therefore, it is much imperative to identify alternative drugs with increased efficacy. Syringaresinol (Syr), a naturally occurring phytochemical which belonging to the lignan group of polyphenols, shows anti-tumor and anti-oxidant activities, which to benefit human health. Studies has shown Syr can regulate the inflammatory response by modulating the secretion and expression level of cytokines IL-6, IL-8, and tumor necrosis factor (TNF)-α. it also shows the inhibitory effect on NF-κB pathway in mouse cells. In the present study, we aimed to demonstrate the anti-inflammatory effects of Syr in OA. In vitro Syr treatment in IL-1β-activated mouse chondrocytes significantly restrained the expression of NO, PGE2, IL-6, TNF-α, INOS, COX-2 and MMP-13. Moreover, it considerably ameliorated the degradation of aggrecan and collagen II. Furthermore, the phosphorylation of the NF-kB signaling pathway was significantly suppressed by Syr. Moreover, in vivo , the cartilage degeneration was attenuated and the increased Osteoarthritis Research Society International (OARSI) scores were reversed in the DMM + Syr group, comprared to those in the DMM group. In sum, our study demonstrated that Syr can attenuate the inflammation in vitro and further verified its effect on OA in vivo. Thus, Syr might be a potent therapeautic alternative for OA treatment. [ABSTRACT FROM AUTHOR]

Background: Zinc finger protein A20 (tumor necrosis factor alpha-induced protein 3) functions as a potent negative feedback inhibitor of the nuclear factor-kB (NF-kB) signaling. It exerts these effects by interrupting the activation of IkB kinase beta (IKKß), the most critical kinase in upstream of NF-kB, and thereby controlling inflammatory homeostasis. We reported previously that electroacupuncture (EA) could effectively suppress IKKß activation. However, the mechanism underlying these effects was unclear. Therefore, the current study further explored the effects of EA on A20 expression in rat brain and investigated the possible mechanism of A20 in anti-neuroinflammation mediated by EA using transient middle cerebral artery occlusion (MCAO) rats. Methods: Rats were treated with EA at the "Baihui (GV20)," "Hegu (L14)," and "Taichong (Liv3)" acupoints once a day starting 2 h after focal cerebral ischemia. The spatiotemporal expression of A20, neurobehavioral scores, infarction volumes, cytokine levels, glial cell activation, and the NF-kB signaling were assessed at the indicated time points. A20 gene interference (overexpression and silencing) was used to investigate the role of A20 in mediating the neuroprotective effects of EA and in regulating the interaction between neuronal and glial cells by suppressing neuronal NF-kB signaling during cerebral ischemia/reperfusion-induced neuroinflammation. Results: EA treatment increased A20 expression with an earlier peak and longer lasting upregulation. The upregulated A20 protein was predominantly located in neurons in the cortical zone of the ischemia/reperfusion. Furthermore, neuronal A20 cell counts were positively correlated with neurobehavioral scores but negatively correlated with infarct volume, the accumulation of pro-inflammatory cytokines, and glial cell activation. Moreover, the effects of EA on improving the neurological outcome and suppressing neuroinflammation in the brain were reversed by A20 silencing. Finally, A20 silencing also suppressed the ability of EA to inhibit neuronal NF-kB signaling pathway. Conclusions: Ischemia/reperfusion cortical neurons in MCAO rats are the main cell types that express A20, and there is a correlation between A20 expression and the suppression of neuroinflammation and the resulting neuroprotective effects. EA upregulated neuronal A20 expression, which played an essential role in the antiinflammatory effects of EA by suppressing the neuronal NF-kB signaling pathway in the brains of MCAO rats. [ABSTRACT FROM AUTHOR]

This study aimed to identify the therapeutic ability of a novel toll-like receptor (TLR) 5 agonist, KMRC011, on ulcerative colitis induced by Citrobacter rodentium and dextran sulfate sodium in a C57BL/6N mouse model. Ulcerative colitis was induced in the mice by the oral administration of 1% dextran sulfate sodium in sterile drinking water for seven days ad libitum , followed by C. rodentium infection on the seventh day by intra-gastric administration (DSS-CT group). KMRC011 was administered intramuscularly at both 24 h and 15 min before (Treatment 1 group), and at both 15 min and 24 h after (Treatment 2 group) the C. rodentium infection. The length of the large intestine and histopathological counts were significantly greater and mucosal thickness was significantly thinner in the Treatment 1 group compared to the DSS-CT and Treatment 2 groups. Il-6 and Il-10 mRNA expression levels were upregulated, while Ifn-γ and Tnf-α mRNA expression levels were significantly downregulated in the Treatment 1 group, compared to the DSS-CT group. NF-κB p65 expression level was elevated due to ulcerative colitis in the DSS-CT group, but was significantly downregulated in the Treatment 1 group. Overall, KMRC011 showed protective effects against murine colitis by inhibiting NF-κB signaling.

This study explored the possible connection between the insulin resistance-targeting protein adipokine lipocalin-2 (LCN-2) and NF-κB signaling pathway in the inflammatory microenvironment in PCOS-IR model rats to determine the pharmacological mechanism of modified Cangfu Daotan decoction (MCDD) intervention for PCOS-IR. We used a high-fat diet (42 days) combined with letrozole (1 mg/kg/day, 42 days) to establish a PCOS-IR rat model. From the third week after modeling, the rats were given continuous administration of MCDD (high dose with 31.68 g/kg, medium dose with 15.84 g/kg, and low dose with 7.92 g/kg) for 28 days. Serum, ovarian tissue, liver, and adipose tissue were collected after the last gavage. Enzyme-linked immunosorbent assay, hematoxylin-eosin (HE) staining, Masson staining, qRT-PCR, and Western blot experiments were performed to detect various indicators. Our results showed that MCDD could reduce body weight and abdominal fat weight; restore normal estrous cycle and ovarian function; alleviate fatty liver; regulate HOMA-IR and OGTT index; reduce serum inflammatory factor levels, LCN-2 level, and gene expression; and regulate the insulin signal transduction and NF-κB pathways in PCOS-IR rats. Thus, MCDD may play a role in improving ovarian function in PCOS-IR rats by downregulating NF-κB/LCN-2 proteins and upregulating the gene expression of Insr/Irs-1/Glut4 in the insulin signaling pathway in the inflammatory environment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Zhang, Yang, Gu, Zhang, Kuang, Mai, Zheng, Yu, Lu, Zeng, Cao and Long.)