Exposure of Toluene Diisocyanate Induces DUSP6 and p53 through Activation of TRPA1 Receptor

Toluene diisocyanate (TDI), a major intermediate agent used in the manufacturing industry, causes respiratory symptoms when exposed to the human body. In this study, we aimed to determine the molecular mechanism of TDI toxicity. To investigate the impact of TDI exposure on global gene expression, we performed transcriptomic analysis of human bronchial epithelial cells (BEAS-2B) after TDI treatment. Differentially expressed genes (DEGs) were sorted and used for clustering and network analysis. Among DEGs, dual-specificity phosphatase 6 (DUSP6) was one of the genes significantly changed by TDI exposure. To verify the expression level of DUSP6 and its effect on lung cells, the mRNA and protein levels of DUSP6 were analyzed. Our results showed that DUSP6 was dose-dependently upregulated by TDI treatment. Thereby, the phosphorylation of ERK1/2, one of the direct inhibitory targets of DUSP6, was decreased. TDI exposure also increased the mRNA level of p53 along with its protein and activity which trans-activates DUSP6. Since TRPA1 is known as a signal integrator activated by TDI, we analyzed the relevance of TRPA1 receptor in DUSP6 regulation. Our data revealed that up-regulation of DUSP6 mediated by TDI was blocked by a specific antagonist against TRPA1. TDI exposure attenuated the apoptotic response, which suggests that it promotes the survival of cancerous cells. In conclusion, our results suggest that TDI induces DUSP6 and p53, but attenuates ERK1/2 activity through TRPA1 receptor activation, leading to cytotoxicity.

Ontogeny of RORγt+ cells in the intestine of newborns and its role in the development of experimental necrotizing enterocolitis

Background Neonates possess an immature and plastic immune system, which is a major cause of some diseases in newborns. Necrotizing enterocolitis (NEC) is a severe and devastating intestinal disease that typically affects premature infants. However, the development of intestinal immune cells in neonates and their roles in the pathological process of NEC have not been elucidated. Results We examined the ontogeny of intestinal lamina propria lymphocytes in the early life of mice and found a high percentage of RORγt+ cells (containing inflammatory Th17 and ILC3 populations) during the first week of life. Importantly, the proportion of RORγt+ cells of intestinal lamina propria further increased in both NEC mice and patients tissue than the control. Furthermore, the application of GSK805, a specific antagonist of RORγt, inhibited IL-17A release and ameliorated NEC severity. Conclusions Our data reveal the high proportion of RORγt+ cells in newborn mice may directly contribute to the development of NEC.

Regulation of Life Extension Factor Klotho on Depressive-like Behaviors via Modulation of GluN2B Function in the Nucleus Accumbens

Klotho is a life extension factor that has an ability to regulate the function of GluN2B-containing N-methyl-D-aspartate receptors (NMDARs), whose dysfunction in the nucleus accumbens (NAc) underlies critical aspects of the pathophysiology of major depression. Here we study the functional relevance of klotho in the pathogenesis of depression. A chronic social defeat stress paradigm, where mice are either categorized as susceptible or unsusceptible group based on their performance in a social interaction test, was used in this study. We found that the expression of klotho was largely decreased in the NAc of susceptible mice when compared to control or unsusceptible group. Genetic knockdown of klotho in the NAc induced depressive-like behaviors in naive mice, while overexpression of klotho produced an antidepressive effect in normal mice and ameliorated the depressive-like behaviors in susceptible mice. Molecularly, knockdown of klotho in the NAc resulted in selective decreases of total and synaptic GluN2B expression that were identical to susceptible mice. Elevation of klotho in the NAc reversed the reductions of GluN2B expressions, as well as altered synaptic transmission and spine density in the NAc of susceptible mice. Furthermore, blockade of GluN2B with a specific antagonist abolished the beneficial effects of klotho elevation in susceptible mice. Collectively, we demonstrated that klotho in the NAc modulates depressive-like behaviors by regulating the function of GluN2B-containing NMDARs. These results reveal a novel role for klotho in the pathogenesis of depression, opening new insights into the molecular basis of major depression.

β-blocker eye drops affect ocular surface through β2 adrenoceptor of corneal limbal stem cells

Background Topical application of β-blocker eye drops induces damage to the ocular surface in clinical. However, the mechanism involved remains incompletely understood. The purpose of this study was to investigate the influence and mechanism of β-blocker eye drops on corneal epithelial wound healing. Methods Corneal epithelial wound healing models were constructed by epithelial scraping including in the limbal region and unceasingly received eye drops containing 5 mg/mL β-blocker levobunolol, β1-adrenoceptor (β1AR)-specific antagonist atenolol or β2-adrenoceptor (β2AR)-specific antagonist ICI 118, 551. For the migration assay, the murine corneal epithelial stem/progenitor cells (TKE2) were wounded and subsequently incubated with levobunolol, atenolol, or ICI 118, 551. The proliferation and colony formation abilities of TKE2 cells treated with levobunolol, atenolol, or ICI 118, 551 were investigated by CCK-8 kit and crystal violet staining. The differentiation marker Cytokeratin 3 (CK3), the stem cell markers-Cytokeratin 14 (CK14) and Cytokeratin 19 (CK19), and corneal epithelium regeneration-related signaling including in Ki67 and the phosphorylated epithelial growth factor receptor (pEGFR) and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) were assessed by immunofluorescence staining. Results Levobunolol and ICI 118, 551 impaired corneal wound healing, decreased the expressions of CK3, CK14, and CK19 after limbal region scraping in vivo and reduced the migration and proliferation of TKE2 in vitro, whereas atenolol had no significant effect. Moreover, levobunolol and ICI 118, 551 inhibited corneal wound healing by mediating the expression of Ki67, and the phosphorylation of EGFR and ERK1/2 in the limbal and regenerated corneal epithelium. Conclusion β-blocker eye drops impaired corneal wound healing by inhibiting the β2AR of limbal stem cells, which decreased corneal epithelial regeneration-related signaling. Therefore, a selective β1AR antagonist might be a good choice for glaucoma treatment to avoid ocular surface damage.

The Endothelin Receptor Antagonist Macitentan Inhibits Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) infection is an important cause of morbidity and mortality in immunocompromised patients and a major etiological factor for congenital birth defects in newborns. Ganciclovir and its pro-drug valganciclovir are the preferred drugs in use today for prophylaxis and treatment of viremic patients. Due to long treatment times, patients are at risk for developing viral resistance to ganciclovir and to other drugs with a similar mechanism of action. We earlier found that the endothelin receptor B (ETBR) is upregulated during HCMV infection and that it plays an important role in the life cycle of this virus. Here, we tested the hypothesis that ETBR blockade could be used in the treatment of HCMV infection. As HCMV infection is specific to humans, we tested our hypothesis in human cell types that are relevant for HCMV pathogenesis; i.e., endothelial cells, epithelial cells and fibroblasts. We infected these cells with HCMV and treated them with the ETBR specific antagonist BQ788 or ETR antagonists that are approved by the FDA for treatment of pulmonary hypertension; macitentan, its metabolite ACT-132577, bosentan and ambrisentan, and as an anti-viral control, we used ganciclovir or letermovir. At concentrations expected to be relevant in vivo, macitentan, ACT-132577 and BQ788 effectively inhibited productive infection of HCMV. Of importance, macitentan also inhibited productive infection of a ganciclovir-resistant HCMV isolate. Our results suggest that binding or signaling through ETBR is crucial for viral replication, and that selected ETBR blockers inhibit HCMV infection.

Co-treatment With Everolimus, an mTOR-Specific Antagonist, or Downregulation of ELK1 Enhances the Sensitivity of Pancreatic Cancer Cells to Genistein

Genistein is a natural isoflavone with pharmacological or potentially anti-tumor properties. However, the resistance of cancer cells to genistein remains a major obstacle. This study focused on the mechanism implicated in the resistance of pancreatic cancer (PC) cells to genistein and the mechanism of action. First, key molecules and signaling pathways related to genistein resistance in PC cells were explored using bioinformatics tools. DEP domain containing MTOR interacting protein (DEPTOR), a typical inhibitor of the mammalian target of rapamycin (mTOR) signaling, was predicted to be poorly expressed in the genistein-resistant PC cells. Thereafter, genistein-resistant PC cells (Panc-1 and PaCa) were constructed. Altered expression of DEPTOR was introduced in cells, and everolimus (ELM), an mTOR-specific antagonist, was administrated in cells as well to examine their roles in genistein resistance. The cell apoptosis was examined in vitro and in vivo in mouse xenograft tumors. The upstream regulator of DEPTOR was predicted via bioinformatic tools. The bioinformatic analyses showed that the PI3K/AKT/mTOR signaling pathway was activated in the setting of DEPTOR downregulation in genistein-resistant PC cells. DEPTOR overexpression reduced the 50% inhibiting concentration (IC50) of genistein in PC cells and suppressed mTOR phosphorylation, and it increased caspase-3 activity, LDH release and apoptosis in PC cells. ELM treatment enhanced the sensitivity of PC cells to genistein in vitro and it strengthened the tumor-eliminating role of genistein in mice. ETS transcription factor ELK1 (ELK1), a transcription factor that negatively regulated DEPTOR transcription, was suppressed by genistein. Upregulation of ELK1 suppressed DEPTOR transcription and reduced the genistein sensitivity of cells, and it also blocked the genistein-sensitizing roles of ELM in PC cells. In conclusion, this study demonstrated that ELK1 reduces DEPTOR transcription, leading to mTOR phosphorylation and the drug resistance of PC cells.

Mutually Antagonistic Protein Pairs of Cancer

Switch-like behavior of tumorigenesis could be governed by antagonistic gene and protein pairs with mutual inhibition. Unlike extensive analysis of gene expression, search for protein level antagonistic pairs has been limited. Here, potential cancer type specific antagonist protein pairs with mutual inhibition were obtained from large scale datasets. Cancer samples or cancer types were compared to retrieve potential protein pairs with contrasting differential expression patterns. Analysis of two different protein expression datasets showed that a few proteins participate in most of the mutually antagonistic relationships. Some proteins with highly antagonistic profile were identified, which could not be attained from a differential expression or a correlation based analysis. The antagonistic protein pairs are sparsely connected by molecular interactions. Glioma, melanoma, and cervical cancer, are more frequently associated with antagonistic proteins than most of the other cancer types. Integrative analysis of mutually antagonist protein pairs contributes to our understanding of systems level changes of cancer.

Antagonism of Cerebral High Mobility Group Box 1 Ameliorates Dendritic Cell Dysfunction in Sepsis

Sepsis has emerged as a global health issue, and accounts for millions of deaths in intensive care units. Dysregulation of the immune response reportedly contributes to the pathogenesis and progression of this lethal condition, which involves both the dysfunction of immune cells and incompetent immunomodulatory mechanisms. High mobility group box 1 (HMGB1) is known as a later inflammatory mediator and is critically involved in the severity and prognosis of sepsis by inducing intractable inflammation and dysfunction of various immune cells. In the present study, we found that intracerebroventricular (ICV) injection of Box A, a specific antagonist of HMGB1, restored the dysregulated response of splenic dendritic cells (DCs) in septic mice by enhancing the expression of surface molecules, including CD80, CD86, and MHC-II, as well as improving DC priming of T lymphocytes. Cerebral HMGB1 was also confirmed to have potent inhibitory effects on DC functions when administrated by ICV injection in normal mice. The brain cholinergic system was found to mediate the immunomodulatory effects of central HMGB1, as it exhibited enhanced activity with persistent HMGB1 exposure. Furthermore, the inhibitory effects of cerebral HMGB1 on the response of peripheral DCs were also blocked by α7nAchR gene knockout. These findings provide novel insight into the relationship between cerebral HMGB1 and splenic DC dysfunction during sepsis, which is, at least in part, dependent on cholinergic system activity.

Mice Lacking GABAA Receptor δ Subunit Have Altered Pharmaco-EEG Responses to Multiple Drugs

In the brain, extrasynaptically expressed ionotropic, δ subunit-containing γ-aminobutyric acid A-type receptors (δ-GABAARs) have been implicated in drug effects at both neuronal and behavioral levels. These alterations are supposed to be caused via drug-induced modulation of receptor ionophores affecting chloride ion-mediated inhibitory tonic currents. Often, a transgenic mouse model genetically lacking the δ-GABAARs (δ-KO) has been used to study the roles of δ-GABAARs in brain functions, because a specific antagonist of the δ-GABAARs is still lacking. We have previously observed with these δ-KO mice that activation of δ-GABAARs is needed for morphine-induced conditioning of place preference, and others have suggested that δ-GABAARs act as targets selectively for low doses of ethanol. Furthermore, activation of these receptors via drug-mediated agonism induces a robust increase in the slow-wave frequency bands of electroencephalography (EEG). Here, we tested δ-KO mice (compared to littermate wild-type controls) for the pharmaco-EEG responses of a broad spectrum of pharmacologically different drug classes, including alcohol, opioids, stimulants, and psychedelics. Gaboxadol (THIP), a known superagonist of δ-GABAARs, was included as the positive control, and as expected, δ-KO mice produced a blunted pharmaco-EEG response to 6 mg/kg THIP. Pharmaco-EEGs showed notable differences between treatments but also differences between δ-KO mice and their wild-type littermates. Interestingly mephedrone (4-MMC, 5 mg/kg), an amphetamine-like stimulant, had reduced effects in the δ-KO mice. The responses to ethanol (1 g/kg), LSD (0.2 mg/kg), and morphine (20 mg/kg) were similar in δ-KO and wild-type mice. Since stimulants are not known to act on δ-GABAARs, our findings on pharmaco-EEG effects of 4-MMC suggest that δ-GABAARs are involved in the secondary indirect regulation of the brain rhythms after 4-MMC.

Polifunctional Liposomes with Trace Elements and Vitamins to Prevent SARS-Cov2 Infection

After many months of the SARS-CoV2 pandemic, in the absence of a vaccine or effective drug therapy, we offer a new generation supplement. The supplement is formulated with trace elements and vitamins of undisputed immunostimulating efficacy, included in the individual liposomes, which act as carriers and therefore we define multifunctional, to improve the absorption and bioavailability of the substances used. We focus the discussion on the role of Zinc as an effective immunostimulant and specific antagonist of the SARS-Cov2 virus.