The B-RafV600E inhibitor dabrafenib selectively inhibits RIP3 and alleviates acetaminophen-induced liver injury

Receptor-interacting protein (RIP)3 is a critical regulator of necroptosis and has been demonstrated to be associated with various diseases, suggesting that its inhibitors are promising in the clinic. However, there have been few RIP3 inhibitors reported as yet. B-Raf^V600E inhibitors are an important anticancer drug class for metastatic melanoma therapy. In this study, we found that 6 B-Raf inhibitors could inhibit RIP3 enzymatic activity in vitro. Among them, dabrafenib showed the most potent inhibition on RIP3, which was achieved by its ATP-competitive binding to the enzyme. Dabrafenib displayed highly selective inhibition on RIP3 over RIP1, RIP2 and RIP5. Moreover, only dabrafenib rescued cells from RIP3-mediated necroptosis induced by the necroptosis-induced combinations, that is, tumor necrosis factor (TNF)α, TNF-related apoptosis-inducing ligand or Fas ligand plus Smac mimetic and the caspase inhibitor z-VAD. Dabrafenib decreased the RIP3-mediated Ser358 phosphorylation of mixed lineage kinase domain-like protein (MLKL) and disrupted the interaction between RIP3 and MLKL. Notably, RIP3 inhibition of dabrafenib appeared to be independent of its B-Raf inhibition. Dabrafenib was further revealed to prevent acetaminophen-induced necrosis in normal human hepatocytes, which is considered to be mediated by RIP3. In acetaminophen-overdosed mouse models, dabrafenib was found to apparently ease the acetaminophen-caused liver damage. The results indicate that the anticancer B-Raf^V600E inhibitor dabrafenib is a RIP3 inhibitor, which could serve as a sharp tool for probing the RIP3 biology and as a potential preventive or therapeutic agent for RIP3-involved necroptosis-related diseases such as acetaminophen-induced liver damage.Necroptosis, also known as programmed necrosis, is a kind of programmed cell death that occurs at conditions that result in blocking the execution of apoptosis.^{1, 2} The protein kinase receptor-interacting protein (RIP)3 is a serine/threonine protein kinase that has recently been demonstrated to be the critical regulator that switches cells from apoptosis to necroptosis.^{3, 4, 5, 6} The death receptor ligands, such as tumor necrosis factor (TNF)α, Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL), are classical inducers of apoptosis or necroptosis. By binding to their respective receptors, they lead to activation of functional caspase-8, which results in apoptosis by activating the effector caspases such as caspase-3 but inactivating the necroptic kinases such as RIP3. When caspase-8 is absent or inhibited by caspase inhibitors such as z-VAD, those death receptor ligands cause necroptosis, which can be augmented by Smac mimetic that promotes degradation of inhibitor of apoptosis proteins.^{3, 4, 5, 6}RIP3 is widely involved in physiological processes and pathological states.⁶ RIP3 deficiency not only rescues the lethality of caspase-8^−/− and FADD^−/− mice⁷ and restores normal proliferation of their T cells,⁶ but also protects hepatocytes from ethanol-induced injury and steatosis,⁸ rescues caspase-8 or FADD deficiency-induced massive inflammation in epithelium,⁹ prevents cerulean-induced acute necrotizing pancreatitis,^{3, 4} inhibits photoreceptor and cone cell death^{10, 11} and alleviates macrophage necrosis in advanced atherosclerosis lesions.¹² Acetaminophen is an extensively used analgesic and antipyretic. When taken in overdose, its most frequent toxicity is hepatotoxicity including fatal centrilobular hepatic necrosis.^{13, 14} Acetaminophen overdose is the most common cause of acute liver failure in the United States and the United Kingdom.¹⁵ It also causes 11.86% of acute liver failure in China.¹⁶ Enhanced levels of high-mobility group box-1 and necrosis keratin-18 marked occurrence of hepatic necrosis.¹⁴ Necrosis has been considered as the predominant mode of cell death in this case, for which RIP3 has been shown to be responsible.¹⁷ In addition, RIP3 might also be associated with carcinogenesis and tumor drug resistance to chemotherapeutics.^{18, 19} These lines of evidence suggest potential extensive uses of small-molecule RIP3 inhibitors in medical prevention or therapy.However, few RIP3 inhibitors have been reported²⁰ and no small-molecule RIP3 inhibitors have been investigated for the potential medical uses. One possible cause is that there lacks a proper RIP3 kinase assay for screening for its inhibitors at molecular levels, which should be highly sensitive, free of radioisotopes, and high throughput. We thus established a non-radioactive luminescent RIP3 kinase assay in this study. By using this assay, we found that 6 B-Raf inhibitors inhibited the RIP3 enzymatic activity in vitro. But only dabrafenib could rescue cells from RIP3-mediated necroptosis induced by TNFα, TRAIL or Fas ligand plus Smac mimetic and the caspase inhibitor z-VAD. Dabrafenib directly and ATP-competitively bound to RIP3 protein and caused highly selective inhibition on RIP3 over RIP1, RIP2 and RIP5. Dabrafenib was demonstrated to ease acetaminophen-induced necrosis in normal human hepatocytes and to prevent acetaminophen-induced liver injury in mice. Our study raises a possibility that the medical indications of the B-Raf^V600E inhibitor dabrafenib might be extended from cancers to RIP3-involved diseases.     

Is the Excessive Use of Microblogs an Internet Addiction? Developing a Scale for Assessing the Excessive Use of Microblogs in Chinese College Students

More and more college students are using microblogs, with some excessive users demonstrating addiction-like symptoms. However, there is currently no published scale available for use in assessing excessive use of these microblogs, a significant impediment to advancing this area of research. We collected data from 3,047 college students in China and developed a Microblog Excessive Use Scale (MEUS) for Chinese college students, comparing it with criteria used for assessing Internet addiction. Our diagnostic scale featured three factors, two of which–“withdrawal and health problem” and “time management and performance”–are already included in Internet addiction assessment scales. The third factor, “social comfort,” does not appear in Internet addiction assessment scales. Our study found that females have significantly higher MEUS scores than males, and that total MEUS scores positively correlated with scores from “self-disclosure” and “real social interaction” scales. These findings differ from results obtained in previous investigations into Internet addiction. Our results indicate that some characteristics of the excessive use of microblogs are different to those of Internet addiction, suggesting that microblog overuse may not correspond exactly to the state of Internet addiction.     

Aryl hydrocarbon receptor activation by Lactobacillus reuteri tryptophan metabolism alleviates Escherichia coli-induced mastitis in mice

The intestinal microbiota has been associated with the occurrence and development of mastitis, which is one of the most serious diseases of lactating women and female animals, but the underlying mechanism has not yet been elucidated. Aryl hydrocarbon receptor (AhR) activation by microbiota tryptophan metabolism-derived ligands is involved in maintaining host homeostasis and resisting diseases. We investigated whether AhR activation by microbiota-metabolic ligands could influence mastitis development in mice. In this study, we found that AhR activation using Ficz ameliorated mastitis symptoms, which were related to limiting NF-κB activation and enhancing barrier function. Impaired AhR activation by disturbing the intestinal microbiota initiated mastitis, and processed Escherichia coli (E. coli)-induced mastitis in mice. Supplementation with dietary tryptophan attenuated the mastitis, but attenuation was inhibited by the intestinal microbiota abrogation, while administering tryptophan metabolites including IAld and indole but not IPA, rescued the tryptophan effects in dysbiotic mice. Supplementation with a Lactobacillus reuteri (L. reuteri) strain with the capacity to produce AhR ligands also improved E. coli-induced mastitis in an AhR-dependent manner. These findings provide evidence for novel therapeutic strategies for treating mastitis, and support the role of metabolites derived from the intestinal microbiota in improving distal disease.     

The Salicylic Acid Signaling Pathway Plays an Important Role in the Resistant Process of Brassica rapa L. ssp. pekinensis to Plasmodiophora brassicae Woronin

Journal of Plant Growth Regulation - Clubroot disease, caused by Plasmodiophora brassicae Woronin infection, leads to significant yield and economic losses in cruciferous vegetables. However, the...     

Co-production of Tet(X) and MCR-1, two resistance enzymes by a single plasmid

Tigecycline and colistin are few of ‘last-resort’ antibiotic defences used in anti-infection therapies against carbapenem-resistant bacterial pathogens. The successive emergence of plasmid-borne tet(X) tigecycline resistance mechanism and mobile colistin resistance (mcr) determinant, renders them clinically useless. Here, we report that co-carriage of tet(X6) and mcr-1 gives co-resistance to both classes of antibiotics by a single plasmid in Escherichia coli. Tet(X6), the new tigecycline resistance enzyme is functionally defined. Both Tet(X6) and MCR-1 robustly interfere accumulation of antibiotic-induced reactive oxygen species (ROS). Unlike that mcr-1 exerts fitness cost in E. coli, tet(X6) does not. In the tet(X6)-positive strain that co-harbors mcr-1, tigecycline resistance is independently of colistin resistance caused by MCR-1-mediated lipid A remodelling, and vice versa. In general consistency with that of MCR-1, Tet(X6) leads to the failure of tigecycline treatment in the infection model of G. mellonella. Taken together, the co-production of Tet(X) and MCR-1 appears as a major clinic/public health concern.     

SPC-Cre-ERT2 Transgenic Mouse for Temporal Gene Deletion in Alveolar Epithelial Cells

Although several Cre-loxP-based gene knockout mouse models have been generated for the study of gene function in alveolar epithelia in the lung, their applications are still limited. In this study, we developed a SPC-Cre-ER^T2 mouse model, in which a tamoxifen-inducible Cre recombinase (Cre-ER^T2) is under the control of the human surfactant protein C (SPC) promoter. The specificity and efficiency of Cre-ER^T2 activity was first evaluated by crossing SPC-Cre-ER^T2 mouse with ROSA26R mouse, a β-galactosidase reporter strain. We found that Cre-ER^T2 was expressed in 30.7% type II alveolar epithelial cells of SPC-Cre-ER^T2/ROSA26R mouse lung tissues in the presence of tamoxifen. We then tested the tamoxifen-inducible recombinase activity of Cre-ER^T2 in a mouse strain bearing TSC1 conditional knockout alleles (TSC1^fx/fx). TSC1 deletion was detected in the lungs of tamoxifen treated SPC-Cre-ER^T2/TSC1^fx/fx mice. Therefore this SPC-Cre-ER^T2 mouse model may be a valuable tool to investigate functions of genes in lung development, physiology and disease.     

A Mathematical Framework for Combining Decisions of Multiple Experts toward Accurate and Remote Diagnosis of Malaria Using Tele-Microscopy

We propose a methodology for digitally fusing diagnostic decisions made by multiple medical experts in order to improve accuracy of diagnosis. Toward this goal, we report an experimental study involving nine experts, where each one was given more than 8,000 digital microscopic images of individual human red blood cells and asked to identify malaria infected cells. The results of this experiment reveal that even highly trained medical experts are not always self-consistent in their diagnostic decisions and that there exists a fair level of disagreement among experts, even for binary decisions (i.e., infected vs. uninfected). To tackle this general medical diagnosis problem, we propose a probabilistic algorithm to fuse the decisions made by trained medical experts to robustly achieve higher levels of accuracy when compared to individual experts making such decisions. By modelling the decisions of experts as a three component mixture model and solving for the underlying parameters using the Expectation Maximisation algorithm, we demonstrate the efficacy of our approach which significantly improves the overall diagnostic accuracy of malaria infected cells. Additionally, we present a mathematical framework for performing ‘slide-level’ diagnosis by using individual ‘cell-level’ diagnosis data, shedding more light on the statistical rules that should govern the routine practice in examination of e.g., thin blood smear samples. This framework could be generalized for various other tele-pathology needs, and can be used by trained experts within an efficient tele-medicine platform.