Myeloid Differentiation Protein Research Articles

Myeloid cell nuclear differentiation antigen controls the pathogen-stimulated type I interferon cascade in human monocytes by transcriptional regulation of IRF7

Type I interferons (IFNs) are critical for anti-viral responses, and also drive autoimmunity when dysregulated. Upon viral sensing, monocytes elicit a sequential cascade of IFNβ and IFNα production involving feedback amplification, but how exactly this cascade is regulated in human cells is incompletely understood. Here we show that the PYHIN protein myeloid cell nuclear differentiation antigen (MNDA) is required for IFNα induction in monocytes. Unlike other PYHINs, this is not due to a pathogen sensing role, but rather MNDA regulated expression of IRF7, a transcription factor essential for IFNα induction. Mechanistically, MNDA is required for recruitment of STAT2 and RNA polymerase II to the IRF7 gene promoter, and in fact MNDA is itself recruited to the IRF7 promoter after type I IFN stimulation. These data implicate MNDA as a critical regulator of the type I IFN cascade in human myeloid cells and reveal a new role for human PYHINs in innate immune gene induction.

Sparstolonin B suppresses free fatty acid palmitate-induced chondrocyte inflammation and mitigates post-traumatic arthritis in obese mice.

Abnormal lipid metabolism, such as systemic increased free fatty acid, results in overproduction of pro‐inflammatory enzymes and cytokines, which is crucial in the development of obesity‐related osteoarthritis (OA). However, there are only a few drugs that target the lipotoxicity of OA. Recent researches have documented that the traditional Chinese medicine, Sparstolonin B (Ssn B), exerted anti‐inflammatory effects in various diseases, but not yet in OA. On the basis of this evidence, our works purposed to evaluate the effect of Ssn B on free fatty acid (FFA) palmitate (PA)‐stimulated human osteoarthritic chondrocytes and obesity‐associated mouse OA model. We found that Ssn B suppressed PA‐triggered inflammatory response and extracellular matrix catabolism in a concentration‐dependent approach. In vivo, Ssn B treatment inhibited cartilage degeneration and subchondral bone calcification caused by joint mechanical imbalance and alleviated metabolic inflammation in obesity. Mechanistically, co‐immunoprecipitine and molecular docking analysis showed that the formation of toll­like receptor 4 (TLR4)/myeloid differentiation protein‐2 (MD‐2) complex caused by PA was blocked by Ssn B. Subsequently, it leads to inactivation of PA‐caused myeloid differentiation factor 88 (MyD88)‐dependent nuclear factor‐kappaB (NF‐κB) cascade. Together, these findings demonstrated that Ssn B is a potential treatment agent for joint degenerative diseases in obese individuals.

Peripartal Rumen-Protected L-Carnitine Manipulates the Productive and Blood Metabolic Responses in High-Producing Holstein Dairy Cows.

This study aimed to monitor the effect of including rumen-protected L-carnitine (Carneon 20 Rumin-Pro, Kaesler Nutrition GmbH, Cuxhaven, Germany) in the transition diet on the productive and metabolic responses of multiparous high-producing Holstein dairy cows. Thirty-two multiparous cows were allocated in a completely randomized design to receive the same diet plus 60 g fat prill containing 85% palmitic acid (control, n = 16) or 100 g rumen-protected L-carnitine (RLC, n = 16); at 28 days before expected calving until 28 days in milk (DIM). Fat prill was included in the control diet to balance the palmitic acid content of both experimental diets. Milk production over the 28 DIM for the control and RLC groups was 46.5 and 47.7 kg, respectively. Milk fat content tended to increase upon rumen-protected L-carnitine inclusion (p = 0.1). Cows fed rumen-protected L-carnitine had higher fat- and energy-corrected milk compared with the control group. Pre- and post-partum administration of L-carnitine decreased both high- and low-density lipoprotein concentrations in peripheral blood of post-partum cows. The results of this study indicated that the concentration of triglycerides and beta-hydroxybutyrate was not significantly different between the groups, whereas the blood non-esterified fatty acid concentration was markedly decreased in cows supplemented with L-carnitine. Animals in the RLC group had a significant (p < 0.05) lower blood haptoglobin concentration at 7 and 14 DIM than the control. Animals in the RLC group had a lower concentration of blood enzymes than those of the control group. The mRNA abundance of Toll-like receptors 4, cluster of differentiation 14, and myeloid differential protein 2 did not significantly change upon the supplementation of L-carnitine in the transition diet. In summary, the dietary inclusion of RLC improved dairy cow's performance during the early lactation period. Greater production, at least in part, is driven by improved energy utilization efficiency and enhanced metabolic status in animals during the periparturient period.

Dexmedetomidine mitigates myocardial ischemiareperfusion injury via regulation of HMGB1-TLR4-NF-κB signaling axis

Purpose: To study the effect of dexmedetomidine (Dex) on myocardial ischemia-reperfusion injury (MIRI), and the associated mechanism of action.Methods: Sixty Sprague-Dawley (SD) rats were assigned to sham, ischemia-reperfusion (I/R), Dex, and MD groups (methyllycaconitine prior to injection with Dex), with 15 rats in each group. Pathological changes in myocardial tissues were determined in all groups. Protein expression levels of HMGB1, TLR4, NF-κB and myeloid differentiation protein 88 (MyD88) in serum and myocardial tissues were assayed and compared.Results: Protein levels of HMGB1, TLR4, MyD88 and NF-κB were significantly higher in heart muscle I/R rats than those in sham group, but lower in heart muscle of rats in Dex group than in heart muscle of I/R rats (p &lt; 0.05). However, they were significantly up-regulated in MD group, relative to Dex group (p&lt; 0.05).Conclusion: Dex exerts a protective effect against ischemia/reperfusion-induced myocardial damage via HMGB1-TLR4-NF-κB signal axis via CAP, and thus, is a potential agent for the management of myocardial disease.

TiRNA signaling via stress-regulated vesicle transfer in the hematopoietic niche.

Extracellular vesicles (EVs) transfer complex biologic material between cells. However, the role of this process invivo is poorly defined. Here, we demonstrate that osteoblastic cells in the bone marrow (BM) niche elaborate extracellular vesicles that are taken up by hematopoietic progenitor cells invivo. Genotoxic or infectious stress rapidly increased stromal-derived extracellular vesicle transfer to granulocyte-monocyte progenitors. The extracellular vesicles contained processed tRNAs (tiRNAs) known to modulateprotein translation. 5'-ti-Pro-CGG-1 was preferentially abundant in osteoblast-derived extracellular vesicles and, when transferred to granulocyte-monocyte progenitors, increased protein translation, cell proliferation, and myeloid differentiation. Upregulating EV transfer improved hematopoietic recovery from genotoxic injury and survival from fungal sepsis. Therefore, EV-mediated tiRNA transfer provides astress-modulated signaling axis in the BM niche distinct from conventional cytokine-driven stress responses.

The inhibitory effect and mechanism of quetiapine on tumor progression in hepatocellular carcinoma in vivo.

Hepatocellular carcinoma (HCC) is the primary tumor of the liver and the fourth leading cause of cancer‐related death. Recently, several studies indicated the anti‐tumor potential of antipsychotic medicine. Quetiapine, an atypical antipsychotic, is used to treat schizophrenia, bipolar disorder, and major depressive disorder since 1997. However, whether quetiapine may show potential to suppress HCC progression and its underlying mechanism is persisting unclear. Quetiapine has been shown to induce apoptosis and inhibit invasion ability in HCC in vitro. Here, we established two different HCC (Hep3B, SK‐Hep1) bearing animals to identify the treatment efficacy of quetiapine. Tumor growth, signaling transduction, and normal tissue pathology after quetiapine treatment were validated by caliper, bioluminescence image, immunohistochemistry (IHC), and hematoxylin and eosin staining, respectively. Quetiapine suppressed HCC progression in a dose‐dependent manner. Extracellular signal‐regulated kinases (ERKs) and Nuclear factor‐κB (NF‐κB) mediated downstream proteins, such as myeloid leukemia cell differentiation protein (MCL‐1), cellular FLICE‐inhibitory protein (C‐FLIP), X‐linked inhibitor of apoptosis protein (XIAP), Cyclin‐D1, matrix metallopeptidase 9 (MMP‐9), vascular endothelial growth factor‐A (VEGF‐A) and indoleamine 2,3‐dioxygenase (IDO) which involved in proliferation, survival, angiogenesis, invasion and anti‐tumor immunity were all decreased by quetiapine. In addition, extrinsic/intrinsic caspase‐dependent and caspase‐independent pathways, including cleaved caspase‐3, −8, and − 9 were increased by quetiapine. In sum, the tumor inhibition that results from quetiapine may associate with ERK and NF‐κB inactivation.

Molecular Basis of Artemisinin Derivatives Inhibition of Myeloid Differentiation Protein 2 by Combined in Silico and Experimental Study.

Artemisinin (also known as Qinghaosu), an active component of the Qinghao extract, is widely used as antimalarial drug. Previous studies reveal that artemisinin and its derivatives also have effective anti-inflammatory and immunomodulatory properties, but the direct molecular target remains unknown. Recently, several reports mentioned that myeloid differentiation factor 2 (MD-2, also known as lymphocyte antigen 96) may be the endogenous target of artemisinin in the inhibition of lipopolysaccharide signaling. However, the exact interaction between artemisinin and MD-2 is still not fully understood. Here, experimental and computational methods were employed to elucidate the relationship between the artemisinin and its inhibition mechanism. Experimental results showed that artemether exhibit higher anti-inflammatory activity performance than artemisinin and artesunate. Molecular docking results showed that artemisinin, artesunate, and artemether had similar binding poses, and all complexes remained stable throughout the whole molecular dynamics simulations, whereas the binding of artemisinin and its derivatives to MD-2 decreased the TLR4(Toll-Like Receptor 4)/MD-2 stability. Moreover, artemether exhibited lower binding energy as compared to artemisinin and artesunate, which is in good agreement with the experimental results. Leu61, Leu78, and Ile117 are indeed key residues that contribute to the binding free energy. Binding free energy analysis further confirmed that hydrophobic interactions were critical to maintain the binding mode of artemisinin and its derivatives with MD-2.

Myeloid differentiation protein-2 has a protective role in house dust mite-mediated asthmatic characteristics with the proinflammatory regulation of airway epithelial cells and dendritic cells.

Myeloid differentiation protein-2 (MD-2) is a lipopolysaccharide-binding protein involved in lipopolysaccharide signalling via Toll-like receptor 4 (TLR4). TLR4 plays an essential role in HDM-mediated allergic airway inflammation. Moreover, MD-2 is structurally similar to Der f 2, a major allergen from house dust mite (HDM). We aimed to clarify the role of MD-2 in the pathogenesis of HDM-mediated allergic airway inflammation. Wild-type (WT), TLR4 knockout and MD-2knockout mice were subjected to intranasal instillation of HDM extract, and asthmatic features were evaluated. We also evaluated gene sets regulated by MD-2 in HDM-treated airway epithelial cells and examined the function of dendritic cells from lymph nodes and from lungs. Aggravated allergic airway inflammation with increased airway hyperresponsiveness was observed in MD-2 knockout mice compared with WT and TLR4 knockout mice. Global gene expression analysis revealed an MD-2 regulated proinflammatory response and reconstituted TLR4 signalling in airway epithelial cells. The ability of dendritic cells to evoke an allergic immune response was enhanced in MD-2 knockout mice. MD-2 plays a protective role in HDM-induced airway allergy with the proinflammatory regulation of airway epithelial cells and dendritic cells. MD-2may serve as a therapeutic target in the treatment of asthma.

Increased Toll-like Receptor-MyD88-NFκB-Proinflammatory neuroimmune signaling in the orbitofrontal cortex of humans with alcohol use disorder.

BackgroundMany brain disorders, including alcohol use disorder (AUD), are associated with induction of multiple proinflammatory genes. One aspect of proinflammatory signaling is progressive increases in expression across cells and induction of other innate immune genes. High‐mobility group box 1 (HMGB1) heteromers contribute to amplification by potentiating multiple proinflammatory responses, including Toll‐like receptors (TLRs). TLR signaling recruits coupling proteins linked to nuclear transcription factors that induce proinflammatory cytokines and chemokines and their respective receptors. We tested the hypothesis that AUD induction of TLR expression increases levels of proinflammatory genes and cellular signaling cascades in association with neurodegeneration in the orbitofrontal cortex (OFC).MethodsPostmortem human OFC tissue samples (n = 10) from males diagnosed with AUD were compared to age‐matched moderate drinking controls (CON). Neuroimmune signaling molecules were assessed using immunohistochemistry for protein and reverse transcription polymerase chain reaction for messenger RNA (mRNA).ResultsIn the AUD OFC, we report induction of the endogenous TLR agonist HMGB1 as well as all TLRs assessed (i.e., TLR2‐TLR9) except TLR1. This was accompanied by increased expression of the TLR adaptor protein myeloid differentiation primary response 88 (MyD88), activation of the proinflammatory nuclear transcription factor nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NFκB), and downstream induction of proinflammatory cytokines, chemokines, and their corresponding receptors. Several of these proinflammatory signaling markers are expressed in glia and neurons. The induction of HMGB1‐TLR‐MyD88‐NFκB proinflammatory signaling pathways correlates with neurodegeneration (i.e., Fluoro‐Jade B), lifetime alcohol consumption, and age of drinking onset.ConclusionThese data implicate the induction of HMGB1‐TLR‐MyD88‐NFκB cascades through coordinated glial and neuronal signaling as contributors to the neurodegeneration seen in the postmortem human OFC of individuals with AUD.

The Potential Role of RP105 in Regulation of Inflammation and Osteoclastogenesis During Inflammatory Diseases.

Inflammatory diseases have a negative impact on bone homeostasis via exacerbated local and systemic inflammation. Bone resorbing osteoclasts are mainly derived from hematopoietic precursors and bone marrow monocytes. Induced osteoclastogenesis during inflammation, autoimmunity, metabolic diseases, and cancers is associated with bone loss and osteoporosis. Proinflammatory cytokines, pathogen-associated molecular patterns, or endogenous pathogenic factors induce osteoclastogenic differentiation by binding to the Toll-like receptor (TLR) family expressed on surface of osteoclast precursors. As a non-canonical member of the TLRs, radioprotective 105 kDa (RP105 or CD180) and its ligand, myeloid differentiation protein 1 (MD1), are involved in several bone metabolic disorders. Reports from literature had demonstrated RP105 as an important activator of B cells, bone marrow monocytes, and macrophages, which regulates inflammatory cytokines release from immune cells. Reports from literature had shown the association between RP105 and other TLRs, and the downstream signaling mechanisms of RP105 with different “signaling-competent” partners in immune cells during different disease conditions. This review is focused to summarize: (1) the role of RP105 on immune cells’ function and inflammation regulation (2) the potential regulatory roles of RP105 in different disease-mediated osteoclast activation and the underlying mechanisms, and (3) the different “signaling-competent” partners of RP105 that regulates osteoclastogenesis.

MD2 blockage prevents the migration and invasion of hepatocellular carcinoma cells via inhibition of the EGFR signaling pathway.

The toll-like receptor (TLR) is an emerging signaling pathway in tumor invasion and metastasis. The activation of TLRs requires specific accessory proteins, such as the small secreted glycoprotein myeloid differentiation protein 2 (MD2), which contributes to ligand responsiveness. However, the role of MD2 in tumorigenesis and metastasis has rarely been reported. This study aimed to investigate the effects and underlying mechanisms of MD2 on the proliferation, migration, and invasion of hepatocellular carcinoma (HCC). Cell counting kit 8 (CCK8), cell colony formation, wound healing, and transwell assays were conducted to determine cell viability, proliferation, migration, and invasion, respectively. Quantitative real-time PCR (qRT-PCR) was performed to assess the expression of MD2 in HCC cell lines and human normal liver cell lines as well as the silencing efficiency of MD2 blockage. Western blot and qRT-PCR assays were performed to detect the protein and mRNA expression levels of epithelial mesenchymal transformation (EMT) markers and epidermal growth factor receptor (EGFR) signaling molecules. MD2 was highly expressed in HCC tissues and cell lines. High expression of MD2 was associated with poor prognosis of HCC patients. In addition, MD2 silencing slightly inhibited the proliferation of HepG2 and HCCLM3, and significantly suppressed cell migration and invasion. Furthermore, MD2 blockage could distinctly prevent the EMT process by increasing the protein and mRNA levels of E-cadherin and Occludin, and decreasing the levels of Vimentin, N-cadherin, and Snail. Finally, the phosphorylation level of EGFR as well as its downstream molecular Src, Akt, I-κBα, and p65 were downregulated in HCC cells with MD2 silencing. Our findings suggest that high expression of MD2 may affect the EMT, migration, and invasion via modulation of the EGFR pathway in HCC cells.

Study on Anti-Inflammatory Activity of Niloticin by Targeting MD-2 Based on Computer-Aided Drug Design and Biolayer Interferometry

Niloticin is an active compound from Cortex phellodendri, but its antiinflammatory activity has not yet been explored. The aim of the present study was to assess the drug potential of niloticin and to study the MD-2-targeting mechanism of its anti-inflammatory activity. Niloticin’s drug potential was analyzed using the Traditional Chinese Medicine Systems Pharmacology Database. Molecular docking and biolayer interferometry technology were used to explore the anti-inflammatory mechanism of niloticin by targeting myeloid differentiation protein 2 (MD-2), which mediates a series of Toll-Like Receptor (TLR) 4-dependent inflammatory responses. The cytokines involved in the LPSTLR4/ MD-2-NF-κB pathway were evaluated by ELISA, RT-PCR, and western blot. The results showed that niloticin has drug potential and could bind to MD- 2. Niloticin had no impact on cell viability. Niloticin could significantly decrease the levels of NO, IL-6, TNF-a, and IL-1β (P&lt;0.01) induced by LPS. IL-1β, IL-6, iNOS, TNF-a, and COX-2 mRNA expression levels were decreased by niloticin (all P&lt;0.01). Compared with the control group, TLR4, p65, MyD88, p-p65, and iNOS expression levels induced by LPS were suppressed by niloticin (all P&lt;0.01). In conclusion, niloticin is a potential MD-2 antagonist. It might interact with MD-2 to play an anti-inflammatory role by suppressing the activation of the LPS-TLR4/MD-2-NF-κB signaling pathway.

Proceedings of the 2020 Classic Examples in Toxicologic Pathology XXVII.

The histopathology slide seminar "Classic Examples in Toxicologic Pathology XXVII" was held on February 21 and 22, 2020, at the Department of Pathology at the University of Veterinary Medicine in Hannover, Germany, with joint organization by the European Society of Toxicologic Pathology. The goal of this annual seminar is to present and discuss classical and actual cases of toxicologic pathology. This article summarizes the presentations given during the seminar, including images of representative lesions. Ten actual and classical cases of toxicologic pathology, mostly induced by a test article, were presented. These included small intestine pathology and transcriptomics induced by a γ-secretase modulator, liver findings in nonhuman primates induced by gene therapy, drug-induced neutropenia in dogs, device-induced growth plate lesions, polycystic lesions in CAR/PXR double knockout mice, inner ear lesions in transgenic mice, findings in Beagle dogs induced by an inhibitor of the myeloid leukemia cell differentiation protein MCL1, findings induced by a monovalent fibroblast growth factor receptor 1 antagonist, kidney lesions induced by a mammalian target of rapamycin inhibitor in combination therapy, and findings in mutation-specific drugs.

Kaempferia parviflora extract inhibits TNF-α-induced release of MCP-1 in ovarian cancer cells through the suppression of NF-κB signaling

Ovarian clear cell carcinoma (OCCC) is an uncommon subtype of epithelial cell ovarian cancers (EOCs) that has poor response to conventional platinum-based therapy. Therefore, finding new potential therapeutic agents is required. Since inflammatory cytokine, tumor necrosis factor alpha (TNF-α), is strongly expressed in EOCs and associated with the level of tumor grade, disruption of this inflammation pathway may provide another potential target for OCCC treatment. We previously reported that Kaempferia parviflora (KP) extract decreased cell proliferation and induced apoptosis. However, the effects of KP on OCCC, especially the aspects related to inflammatory cytokines, have not been elucidated. Our current study demonstrated the effects of KP extract on cytokine production in TNF-α-induced OCCC TOV-21G cell line. This study showed that KP extract inhibited interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) production at both transcription and translation levels via the suppression of nuclear factor-kappa B (NF-κB) signal transduction. In contrast, KP extract increased the expression of inhibitor kappa B (IκB) protein which may delay NF-κB translocation into the nucleus upon TNF-α activation. Moreover, the suppression of cytokines released from KP treated-TOV-21G reduced the migration of monocyte cell (THP-1). KP extract also exhibited the inhibition of IL-6 and MCP-1 production from THP-1 activated by lipopolysaccharides (LPS). Cells treated with KP extract exhibited a decrease in extracellular signal-regulated kinases (ERK1/2) and protein kinase B (AKT) phosphorylation and induced myeloid leukemia cell differentiation protein Mcl-1 (MCL-1) expression. Suppression of inflammatory cytokine and chemokine production and inhibition of tumor-associated macrophage (TAM) migration support the possibility of using KP for OCCC treatment.

Abstract 2452: Metformin administration in hypoglycemia compromises cancer cell viability in glioblastoma initiating cells

Abstract Cancer cells show higher energy and biomolecules requirements than healthy cells, making the targeting of cancer metabolic dysfunctions an appealing therapeutic approach. Unfortunately, tackling the tumor metabolism is a daunting task, due to the ability of cancer cells to re-organize their metabolism switching between glycolysis and oxidative phosphorylation. Cancer metabolic plasticity and re-programming is partially supported by altered ionic channels expression and activity, our laboratory has described Chloride Intracellular Channel 1 (CLIC1) as a marker of malignancy in tumor cells and a possible therapeutic target. CLIC1 is a metamorphic protein able to switch between cytoplasmic and transmembrane forms (tmCLIC1), the latter characterized by chloride conductance. TmCLIC1 was found to be overexpressed in several solid tumors where supports cancer proliferation and growth, while it is mainly absent in healthy cells. Recently, it has been demonstrated that tmCLIC1 activity is affected by biguanide compounds, in particular metformin. Metformin was recently repurposed as an anticancer drug with unclear mechanisms. We demonstrated that in hypoglycemia, metformin displays increased toxic effect on cancer cells, inducing apoptosis. Metformin inhibits Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A), and hypoglycemia upregulates PP2A subunit B56δ targeting the Glycogen Synthase Kinase 3 Beta (GSK3b). Metformin treatment in combination with hypoglycemia leads to de-phosphorylation of GSK3b protein resulting in degradation of induced myeloid leukemia cell differentiation protein (MCL-1), a pro-survival protein, and cell death (Elgendy et al., Cancer Cell 2019). In primary glioblastoma cell lines enriched in tumor-initiating-cells (GSCs), metformin has proven to be effective in reducing cell growth without affecting viability (Gritti et al., Oncotarget 2014). Here, we are going to show studies trying to establish whether CLIC1 is a component of the established PP2A-mediated signaling network. Our work aims to clarify metformin molecular mechanism of action and to assess a new potential therapeutic approach for solid tumors in which tmCLIC1 is found to be overexpressed. Citation Format: Francesca Cianci, Riccardo Cazzoli, Ivan Verduci, Saverio Minucci, Michele Mazzanti. Metformin administration in hypoglycemia compromises cancer cell viability in glioblastoma initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2452.

MYD88, NFKB1, and IL6 transcripts overexpression are associated with poor outcomes and short survival in neonatal sepsis

Toll-like receptor (TLR) family signature has been implicated in sepsis etiopathology. We aimed to evaluate the genetic profile of TLR pathway-related key genes; the myeloid differentiation protein 88 (MYD88), IL1 receptor-associated kinase 1 (IRAK1), the nuclear factor kappa-B1 (NFKB1), and interleukin 6 (IL6) in the blood of neonates with sepsis at the time of admission and post-treatment for the available paired-samples. This case–control study included 124 infants with sepsis admitted to the neonatal intensive care unit and 17 controls. The relative gene expressions were quantified by TaqMan Real-Time qPCR and correlated to the clinic-laboratory data. MYD88, NFKB1, and IL6 relative expressions were significantly higher in sepsis cases than controls. Higher levels of MYD88 and IL6 were found in male neonates and contributed to the sex-based separation of the cases by the principal component analysis. ROC analysis revealed MYD88 and NFKB1 transcripts to be good biomarkers for sepsis. Furthermore, patients with high circulatory MYD88 levels were associated with poor survival, as revealed by Kaplan–Meier curves analysis. MYD88, NFKB1, and IL6 transcripts showed association with different poor-outcome manifestations. Clustering analysis split the patient cohort into three distinct groups according to their transcriptomic signature and CRP levels. In conclusion, the study TLR pathway-related transcripts have a gender-specific signature, diagnostic, and prognostic clinical utility in neonatal sepsis.

Total saponins from Rhizoma Panacis Majoris inhibit proliferation, induce cell cycle arrest and apoptosis and influence MAPK signalling pathways on the colorectal cancer cell.

Colorectal cancer (CRC) ranks third in incidence and second in mortality among all types of cancer, and due to its insidious onset and lack of early symptoms, it is usually diagnosed at a later stage. Saponins, a class of compounds abundant in plants, have been reported to possess prominent anti-tumour properties. The use of ginsenoside Rg3 in the clinical setting was authorized by the National Medicinal Products Administration of China. In the present study, total saponins from Rhizoma Panacis Majoris (RPMTG) were prepared, and the pharmacological mechanisms underlying the anti-CRC effects of RPMTG were investigated. The effect of RPMTG on the proliferation, cell cycle progression and apoptosis of HCT116 and SW620 cells were detected by MTT, flow cytometry and western blotting assays, and it was demonstrated that RPMTG could inhibit the proliferation of HCT116 and SW620 cells with IC50 values of 315.8 and 355.1 µg/ml, respectively, induce cell cycle arrest in the S and G0/G1 phase, and trigger apoptosis by downregulating the expression of the anti-apoptotic proteins Bcl-2, Bcl-xL and induced myeloid leukaemia cell differentiation protein Mcl-1, and increasing the expression of the pro-apoptotic proteins Bax and Bad, cleaved caspased-3 and poly(ADP)-ribose polymerase. These findings suggested that RPMTG induced apoptosis through mitochondrial-related pathways. In addition, RPMTG also decreased the expression of phosphorylated (p)-extracellular signal-regulated kinase and increased p-c-Jun N-terminal kinase (p-JNK) and p-p38. Moreover, the effects of RPMTG on cell proliferation and apoptosis were partially reversed when the JNK and p38 mitogen-activated protein kinase (MAPK) pathways were inhibited, indicating that RPMTG triggered apoptosis mainly via regulating JNK and p38 MAPK signalling. Therefore, RPMTG may have potential as an anti-CRC agent, and further evaluations are needed.

The Akt pathway mediates the protective effects of myeloid differentiation protein 1 in pathological cardiac remodelling.

AimsMyeloid differentiation protein 1 (MD1) was shown to ameliorate pressure overload‐induced cardiac hypertrophy and fibrosis by negatively regulating the MEK–ERK1/2 and NF‐κB pathways. However, whether MD1 modulates cardiac function and whether the Akt pathway mediates the benefits of MD1 in pressure overload‐induced cardiac remodelling remain unclear.Methods and ResultsMale cardiac‐specific transgenic MD1 (MD1‐TG) mice, MD1‐knockout (KO) mice and wild‐type (WT) littermates aged 8–10 weeks were subjected to sham operation and aortic banding (AB) for 4 weeks. Then, left ventricular (LV) hypertrophy, fibrosis and function of the mice were assessed. When compared with WT‐AB mice, MD1‐TGs showed decreased cross‐sectional area (CSA) of cardiomyocytes (P < 0.001), mRNA expression of β‐myosin heavy chain (β‐MHC) (P < 0.02), ratios of heart weight/body weight and heart weight/tibia length (P < 0.04) and collagen volume fraction (P < 0.001). The LV end‐diastolic diameter was reduced, and LV ejection fraction and fractional shortening were improved in MD1‐TG‐AB mice than in WT‐AB mice (P < 0.05). In cultured H9C2 cells, adenovirus vector‐mediated MD1 overexpression decreased angiotensin II‐induced mRNA expression of brain natriuretic peptide (BNP) and β‐MHC and cell CSA (P < 0.002), whereas knockdown of MD1 by shRNA exhibited opposite effects (P < 0.04). Mechanistically, MD1 suppressed pathological cardiac remodelling at least partly by blocking Akt pathway. Akt inactivation by MK2206 largely offset the pro‐hypertrophic effects of MD1 deficiency in angiotensin II‐stimulated cardiomyocytes.ConclusionsThe Akt pathway mediates the protective effects of MD1 in pressure overload‐induced cardiac remodelling in mice. Targeting MD1 may provide therapeutic strategy for the treatment of pathological cardiac remodelling and heart failure.

Using the transcriptome to evaluate the best reference genes for studying nutrition of the critically endangered Yangtze sturgeon (Acipenser dabryanus)

To better apply qRT-PCR in nutrition research, this study screened the reference genes (RGs) of the Yangtze sturgeon (Acipenser dabryanus) using the transcriptome. The results revealed that transcriptome sequencing of the liver, anterior gut (esophagus, cardia stomach, pyloric stomach), and posterior gut (pyloric caecum, duodenum, valvular gut, rectum) identified 179,199 unigenes with an N50 of 1747 bp and average length of 1127 bp. In the transcriptome, 19 unigenes with coefficients of variation <0.3, −0.5 < Log2FC <0.5 and FPKM >400 were screened. Excluding unannotated and repetitive unigenes, 9 potential reference genes were obtained, namely mitochondrial phosphoenolpyruvate carboxykinase (mPEPCK), Tc1 transporase (Tc1Tase), β-actin, elongation factor 2 kinase (EF2Kase), myeloid leukemia cell differentiation protein (Mcl1), histone H3.3 (hH3.3), elongation factor 1-alpha (EF1-α), thioredoxin interacting protein (txnip) and cofilin-2-like (CF2). In addition, 6 empirically commonly used RGs were also identified as potential candidates, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin (TUBB), β 2-microglobulin (B2M), succinate dehydrogenase complex, subunit A, flavoprotein variant (SDHA), hypoxanthine phosphoribosyltransferase 1 (HPRT1), and 18S rRNA. We designed the qRT-PCR primers of potential RGs and plotted a standard curve. The amplification efficiency of all genes is more than 91.5% (91.5–96.7%). Comprehensive analysis using Delta Cq, Bestkeeper, Normfinder and geNorm revealed that the expression stability of potential RGs is EF1-α > hH3.3 > CF2 > β-actin >18S rRNA >B2M > HPRT1 > GAPDH >mPEPCK >TUBB >Mcl1 > SDHA >EF2Kase > txnip >Tc1Tase in postprandial, EF1-α > β-actin >hH3.3 > CF2 > HPRT1 > B2M > mPEPCK >18S rRNA >SDHA >TUBB >GAPDH >Mcl1 > EF2Kase > txnip >Tc1Tase in fasting and refeeding, and β-actin >GAPDH >EF1-α > CF2 > hH3.3 > B2M >18S rRNA >HPRT1 > TUBB >Mcl1 > mPEPCK >EF2Kase > Tc1Tase > SDHA >Txnip during weaning. The weighted analysis of postprandial, fasting and refeeding, and weaning indicated that the stability of potential RGs is EF1-α > β-actin >hH3.3 > CF2 > B2M >18S rRNA >HPRT1 > GAPDH >mPEPCK >TUBB >Mcl1 > SDHA >EF2Kase > txnip >Tc1Tase. In summary, EF1-α and/or β-actin are recommended as RGs when qRT-PCR is used in the nutrition research of Yangtze sturgeon.

Nicotine and its metabolite cotinine target MD2 and inhibit TLR4 signaling

Nicotine is the principal alkaloid of tobacco often manufactured into cigarettes and belongs to a highly addictive class of drugs. Nicotine attenuates the neuroinflammation induced by microglial activation. However, the molecular target(s) underlying anti-inflammatory action of nicotine has not been fully understood. Considering the psychoactive substances morphine, cocaine, and methamphetamine act as xenobiotic-associated molecular patterns and can be specifically sensed by the innate immune receptor Toll-like receptor 4 (TLR4), here we sought to delineate whether nicotine and/or its metabolite cotinine may be recognized by the innate immune system via myeloid differentiation protein 2 (MD2), an accessory protein of TLR4 that is responsible for ligand recognition. MD2-intrinsic fluorescence titrations, surface plasmon resonance, and competitive displacement binding assays with curcumin (MD2 probe) demonstrated that both nicotine and cotinine targeted the lipopolysaccharide (LPS; TLR4 agonist) binding pocket of MD2 with similar affinities. The cellular thermal shift assay indicated that nicotine binding increased, while cotinine binding decreased, MD2 stability. These biophysical binding results were further supported by in silico simulations. In keeping with targeting MD2, both nicotine and cotinine inhibited LPS-induced production of nitric oxide and tumor necrosis factor alpha (TNF-α) and blocked microglial activation. Neither a pan nicotinic acetylcholine receptor (nAChR) inhibitor nor RNAi for nAChRs abolished the suppressive effect of nicotine- and cotinine-induced neuroinflammation. These data indicate that TLR4 inhibition by nicotine and cotinine at the concentrations tested in BV-2 cells is independent of classic neuronal nAChRs and validate that MD2 is a direct target of nicotine and cotinine in the inhibition of innate immunity.