The leucine-rich repeat (LRR) domain of NLRP3 is required for NLRP3 inflammasome activation in macrophages.

The NLRP3 inflammasome is a critical component of innate immunity that defends the host from microbial infections. However, its aberrant activation contributes to the pathogenesis of several inflammatory diseases. Activation of the NLRP3 inflammasome induces the secretion of proinflammatory cytokines IL-1ß and IL-18 and pyroptotic cell death. NLRP3 contains a leucine-rich repeat (LRR) domain at its C terminus. Although posttranslational modifications in this LRR domain have been shown to regulate NLRP3 inflammasome activation, the role of the entire LRR domain in NLRP3 inflammasome activation remains controversial. Here, we generated mouse macrophages that express an endogenous NLRP3 mutant lacking the LRR domain. Deletion of the LRR domain diminished NLRP3 inflammasome activation in macrophages. Furthermore, using NLRP3-deficient macrophages that are reconstituted with NLRP3 mutants lacking the LRR domain, we found that deletion of the LRR domain inhibited NLRP3 inflammasome activation. Mechanistically, deletion of the LRR domain inhibited NLRP3 self-association, oligomerization, and interaction with the essential regulator NEK7. Our results demonstrate a critical role for the LRR domain in NLRP3 inflammasome activation.

Towards Lightweight Neural Networks for Garbage Object Detection.

In recent years, garbage classification has become a hot topic in China, and legislation on garbage classification has been proposed. Proper garbage classification and improving the recycling rate of garbage can protect the environment and save resources. In order to effectively achieve garbage classification, a lightweight garbage object detection model based on deep learning techniques was designed and developed in this study, which can locate and classify garbage objects in real-time using embedded devices. Focusing on the problems of low accuracy and poor real-time performances in garbage classification, we proposed a lightweight garbage object detection model, YOLOG (YOLO for garbage detection), which is based on accurate local receptive field dilation and can run on embedded devices at high speed and with high performance. YOLOG improves on YOLOv4 in three key ways, including the design of DCSPResNet with accurate local receptive field expansion based on dilated-deformable convolution, network structure simplification, and the use of new activation functions. We collected the domestic garbage image dataset, then trained and tested the model on it. Finally, in order to compare the performance difference between YOLOG and existing state-of-the-art algorithms, we conducted comparison experiments using a uniform data set training model. The experimental results showed that YOLOG achieved AP0.5 of 94.58% and computation of 6.05 Gflops, thus outperformed YOLOv3, YOLOv4, YOLOv4-Tiny, and YOLOv5s in terms of comprehensive performance indicators. The network proposed in this paper can detect domestic garbage accurately and rapidly, provide a foundation for future academic research and engineering applications.

The NLRP3 Inflammasome: An Overview of Mechanisms of Activation and Regulation.

The NLRP3 inflammasome is a critical component of the innate immune system that mediates caspase-1 activation and the secretion of proinflammatory cytokines IL-1ß/IL-18 in response to microbial infection and cellular damage. However, the aberrant activation of the NLRP3 inflammasome has been linked with several inflammatory disorders, which include cryopyrin-associated periodic syndromes, Alzheimer's disease, diabetes, and atherosclerosis. The NLRP3 inflammasome is activated by diverse stimuli, and multiple molecular and cellular events, including ionic flux, mitochondrial dysfunction, and the production of reactive oxygen species, and lysosomal damage have been shown to trigger its activation. How NLRP3 responds to those signaling events and initiates the assembly of the NLRP3 inflammasome is not fully understood. In this review, we summarize our current understanding of the mechanisms of NLRP3 inflammasome activation by multiple signaling events, and its regulation by post-translational modifications and interacting partners of NLRP3.

Therapeutic efficacy of a MMAE-based anti-DR5 drug conjugate Oba01 in preclinical models of pancreatic cancer.

Pancreatic cancer (PC) is among the most aggressive malignancies associated with a 5-year survival rate of <9%, and the treatment options remain limited. Antibody-drug conjugates (ADCs) are a new class of anticancer agents with superior efficacy and safety profiles. We studied the antitumor activity of Oba01 ADC and the mechanism underlying the targeting of death receptor 5 (DR5) in preclinical PC models. Our data revealed that DR5 was highly expressed on the plasma membrane of PC cells and Oba01 showed potent in vitro antitumor activity in a panel of human DR5-positive PC cell lines. DR5 was readily cleaved by lysosomal proteases after receptor-mediated internalization. Monomethyl auristatin E (MMAE) was then released into the cytosol to induce G2/M-phase growth arrest, cell death via apoptosis induction, and the bystander effect. Furthermore, Oba01 mediated cell death via antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. For improved potency, we investigated the synergetic effect of Oba01 in combination with approved drugs. Oba01 combined with gemcitabine showed better antiproliferative activity than either standalone treatment. In cell- and patient-derived xenografts, Oba01 showed excellent tumoricidal activity in mono- or combinational therapy. Thus, Oba01 may provide a novel biotherapeutic approach and a scientific basis for clinical trials in DR5-expressing patients with PC.

RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly.

The NLRP3 inflammasome, a critical component of the innate immune system, induces caspase-1 activation and interleukin (IL)-1ß maturation in response to microbial infection and cellular damage. However, aberrant activation of the NLRP3 inflammasome contributes to the pathogenesis of several inflammatory disorders, including cryopyrin-associated periodic syndromes, Alzheimer's disease, type 2 diabetes, and atherosclerosis. Here, we identify the receptor for activated protein C kinase 1 (RACK1) as a component of the NLRP3 complexes in macrophages. RACK1 interacts with NLRP3 and NEK7 but not ASC. Suppression of RACK1 expression abrogates caspase-1 activation and IL-1ß release in response to NLRP3- but not NLRC4- or AIM2-activating stimuli. This RACK1 function is independent of its ribosomal binding activity. Mechanistically, RACK1 promotes the active conformation of NLRP3 induced by activating stimuli and subsequent inflammasome assembly. These results demonstrate that RACK1 is a critical mediator for NLRP3 inflammasome activation.

One-step generation of mice carrying a conditional allele together with an HA-tag insertion for the delta opioid receptor.

G protein-coupled receptors (GPCRs) are important modulators of many physiological functions and excellent drug targets for many diseases. However, to study the functions of endogenous GPCRs is still a challenging task, partially due to the low expression level of GPCRs and the lack of highly potent and selective GPCR antibodies. Overexpression or knock-in of tagged GPCRs, or knockout of specific GPCRs in mice, are common strategies used to study the in vivo functions of these receptors. However, generating separate mice carrying tagged GPCRs or conditional alleles for GPCRs is labor intensive, and requires additional breeding costs. Here we report the generation of mice carrying an HA-tagged DOR (delta opioid receptor) flanked by LoxP sequences at the endogenous DOR locus using a single recombination step, aided by the TALEN system. These animals can be used directly to study the expression, localization, protein-protein interaction and signal transduction of endogenous DOR using anti-HA antibodies. By crossing with mice expressing tissue-specific Cre, these mice can also generate offspring with DOR knockout within specific tissues. These mice are powerful tools to study the in vivo functions of DOR. Furthermore, the gene modification strategy could also be used to study the functions of many other GPCRs.

Amidoalkylindoles as Potent and Selective Cannabinoid Type 2 Receptor Agonists with in Vivo Efficacy in a Mouse Model of Multiple Sclerosis.

Selective CB2 agonists represent an attractive therapeutic strategy for the treatment of a variety of diseases without psychiatric side effects mediated by the CB1 receptor. We carried out a rational optimization of a black market designer drug SDB-001 that led to the identification of potent and selective CB2 agonists. A 7-methoxy or 7-methylthio substitution at the 3-amidoalkylindoles resulted in potent CB2 antagonists (27 or 28, IC50 = 16-28 nM). Replacement of the amidoalkyls from 3-position to the 2-position of the indole ring dramatically increased the agonist selectivity on the CB2 over CB1 receptor. Particularly, compound 57 displayed a potent agonist activity on the CB2 receptor (EC50 = 114-142 nM) without observable agonist or antagonist activity on the CB1 receptor. Furthermore, 57 significantly alleviated the clinical symptoms and protected the murine central nervous system from immune damage in an experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis.

Kappa opioid receptor activation alleviates experimental autoimmune encephalomyelitis and promotes oligodendrocyte-mediated remyelination.

Multiple sclerosis (MS) is characterized by autoimmune damage to the central nervous system. All the current drugs for MS target the immune system. Although effective in reducing new lesions, they have limited effects in preventing the progression of disability. Promoting oligodendrocyte-mediated remyelination and recovery of neurons are the new directions of MS therapy. The endogenous opioid system, consisting of MOR, DOR, KOR and their ligands, has been suggested to participate in the pathogenesis of MS. However, the exact receptor and mechanism remain elusive. Here we show that genetic deletion of KOR exacerbates experimental autoimmune encephalomyelitis, whereas activating KOR with agonists alleviates the symptoms. KOR does not affect immune cell differentiation and function. Instead, it promotes oligodendrocyte differentiation and myelination both in vitro and in vivo. Our study suggests that targeting KOR might be an intriguing way to develop new MS therapies that may complement the existing immunosuppressive approaches.

On-line simultaneous deproteinization of biological samples and trace enrichment of three dipine series using a poly(N-isopropylacrylamide-co-ethyleneglycol dimethacrylate) monolith.

A porous poly(N-isopropylacrylamide-co-ethyleneglycol dimethacrylate) [poly(NIPAAm-co-EDMA)] monolithic column was prepared by in situ free-radical polymerization. The morphology of monolithic column and pressure drop across the columns were characterized. The results showed excellent permeability and high selectivity. Nifedipine, nitrendipine and nisoldipine were simultaneously selected to validate the extraction efficiency of the prepared monolith both in plasma and urine. The extracted nifedipine, nitrendipine and nisoldipine from plasma and urine samples have been on-line tested quantitatively by using the prepared monolith connected with RP-C18 column. The total analytical run time was 38 min. For all analytes, linear calibration curves were obtained over a range of 2-500 ng/mL with coefficient of correlation>0.997. Precision for inter- and intra-day assay showed acceptable results for quantitative assay with relative standard deviation (RSD) less than 12%. The accuracy and recovery was found to be in the range of 89-109% and 88-106%. The results indicated that the prepared monolith was feasible to be used as an on-line SPE sorbent material and the method was especially appropriate for multi-analytes monitoring in plasma and urine samples. Finally, the proposed method was successfully applied to simultaneously screen nifedipine, nitrendipine and nisoldipine in plasma.