Dopamine (DA) plays a critical role in the brain, and the ability to directly measure dopaminergic activity is essential for understanding its physiological functions. We therefore developed red fluorescent GPCR-activation–based DA (GRAB DA ) sensors and optimized versions of green fluorescent GRAB DA sensors. In response to extracellular DA, both the red and green GRAB DA sensors exhibit a large increase in fluorescence, with subcellular resolution, subsecond kinetics, and nanomolar to submicromolar affinity. Moreover, the GRAB DA sensors resolve evoked DA release in mouse brain slices, detect evoked compartmental DA release from a single neuron in live flies, and report optogenetically elicited nigrostriatal DA release as well as mesoaccumbens dopaminergic activity during sexual behavior in freely behaving mice. Co-expressing red GRAB DA with either green GRAB DA or the calcium indicator GCaMP6s allows simultaneously tracking neuronal activity and dopaminergic signaling in distinct circuits in vivo .

Gastric cancer is the fourth most common cancer worldwide, with a high rate of death and low 5-year survival rate. To date, there is a lack of efficient therapeutic protocols for gastric cancer. Recent studies suggest that cancer stem cells (CSCs) are responsible for tumor initiation, invasion, metastasis, and resistance to anticancer therapies. Thus, therapies that target gastric CSCs are attractive. However, CSCs in human gastric adenocarcinoma (GAC) have not been described. Here, we identify CSCs in tumor tissues and peripheral blood from GAC patients. CSCs of human GAC (GCSCs) that are isolated from tumor tissues and peripheral blood of patients carried CD44 and CD54 surface markers, generated tumors that highly resemble the original human tumors when injected into immunodeficient mice, differentiated into gastric epithelial cells in vitro, and self-renewed in vivo and in vitro. Our findings suggest that effective therapeutic protocols must target GCSCs. The capture of GCSCs from the circulation of GAC patients also shows great potential for identification of a critical cell population potentially responsible for tumor metastasis, and provides an effective protocol for early diagnosis and longitudinal monitoring of gastric cancer.

RIPK2 mediates inflammatory signaling by the bacteria‐sensing receptors NOD1 and NOD2. Kinase inhibitors targeting RIPK2 are a proposed strategy to ameliorate NOD‐mediated pathologies. Here, we reveal that RIPK2 kinase activity is dispensable for NOD2 inflammatory signaling and show that RIPK2 inhibitors function instead by antagonizing XIAP‐binding and XIAP‐mediated ubiquitination of RIPK2. We map the XIAP binding site on RIPK2 to the loop between β2 and β3 of the N‐lobe of the kinase, which is in close proximity to the ATP‐binding pocket. Through characterization of a new series of ATP pocket‐binding RIPK2 inhibitors, we identify the molecular features that determine their inhibition of both the RIPK2‐XIAP interaction, and of cellular and in vivo NOD2 signaling. Our study exemplifies how targeting of the ATP‐binding pocket in RIPK2 can be exploited to interfere with the RIPK2‐XIAP interaction for modulation of NOD signaling.

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The monoamine neuromodulator dopamine (DA) plays a critical role in the brain, and the 21 ability to directly measure dopaminergic activity is essential for understanding its 22 physiological functions. We therefore developed the first red fluorescent GPCR-activation-23 based DA (GRABDA) sensors and optimized versions of green fluorescent GRABDA sensors 24 following our previous studies. In response to extracellular DA, both the red and green 25 GRABDA sensors have a large increase in fluorescence (ΔF/F0 values of 150% and 340%, 26 respectively), with subcellular resolution, subsecond kinetics, and nanomolar to 27 submicromolar affinity. Moreover, both the red and green GRABDA sensors readily resolve 28 evoked DA release in mouse brain slices, detect compartmental DA release in live flies with 29 single-cell resolution, and report optogenetically elicited nigrostriatal DA release as well as 30 mesoaccumbens dopaminergic activity during sexual behavior in freely behaving mice. 31 Importantly, co-expressing red GRABDA with either green GRABDA or the calcium indicator 32GCaMP6s provides a robust tool for simultaneously tracking neuronal activity and 33 dopaminergic signaling in distinct circuits in vivo. 35Dopamine (DA) is an essential monoamine neuromodulator produced primarily in the midbrain and 36 released throughout the central nervous system. A multitude of brain functions are regulated by DA, 37including motor control, motivation, learning and memory, and emotional control 1-9 . Consistent with 38these key physiological roles, altered DA signaling has been implicated in a variety of brain 39 disorders, including Parkinson's disease, addiction, schizophrenia, attention-deficit/hyperactivity 40 disorder, and posttraumatic stress disorder 10-20 . Thus, tools that can sense changes in DA 41 concentration with high spatiotemporal resolution, high specificity, and high sensitivity will greatly 42 facilitate our study of the diverse functions that the dopaminergic system plays under both 43 physiological and pathological conditions. 44Previous techniques for measuring DA dynamics, including microdialysis, electrochemical 45 probes, reporter cells, and gene expression-based assays, lack sufficient spatiotemporal resolution 46 and/or molecular specificity [21][22][23][24][25][26][27][28][29][30] . Recently, our group 31 and Patriarchi et al. 32 independently 47 developed two series of genetically encoded, G-protein-coupled receptor (GPCR)-based DA 48 sensors called GRABDA and dLight, respectively. Taking advantage of naturally occurring DA 49 receptors, these sensors convert a ligand-stabilized conformational change in the DA receptor into 50 an optical response via a conformation-sensitive fluorescent protein inserted in the receptor's third 51 intracellular loop. Our first-generation DA receptor-based sensors called GRABDA1m and 52 GRABDA1h were used to detect cell type-specific DA dynamics in several organisms, including 53 Drosophila, zebrafish, mice, and zebra finches 31,33-35 . Here, we employed semi-rational en...

miR-214-3p regulates ox-LDL-initiated autophagy in HUVECs by directly targeting the 3'UTR of ATG5 and may have a suitable role in the pathogenesis of atherosclerosis.

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Background: Pyroptosis is closely relevant to sepsis. However, the molecular mechanisms of pyroptosis in pneumonia-induced sepsis are still not fully understood. Thus, this study aimed to find the specific molecular pathways associated with pyroptosis and explore their relationship in pneumonia-induced sepsis.Methods: First, significant signaling pathways related to pneumonia-induced sepsis were screened by bioinformatics analysis based on GSE48080. The peripheral blood samples from patients with pneumoniainduced sepsis and healthy subjects were collected. Pneumonia-induced sepsis rat models were also established. Then, inflammatory response, pyroptosis, and regulatory T cells (Tregs)/T-helper 17 (Th17), Th1/Th2, and M1/M2 cell ratios in pneumonia-induced sepsis were evaluated.Results: IL-17 signaling pathway was significantly related to pneumonia-induced sepsis by bioinformatics analysis. Compared with healthy groups, the higher of Th17/Treg, Th1/Th2 and M1/M2 cell radios in the patients and sepsis rat model indicated that pneumonia-induced sepsis caused a severe inflammatory response. This result was confirmed by higher levels of pro-inflammatory factors (IL-6, TNF-α, IL-1β, and IL-18) and an inflammation indicator (LDH), as well as pyroptosis occurrence in sepsis. Additionally, the up-regulation of key molecules (HMGB1, RAGE, IL-17A, TRAF6 and NK-κB) in the IL-17 signaling pathway suggested the IL-17 pathway was activated. Moreover, the release of IL-1β and IL-18 and the levels of the molecules (NLRP3, NLRC4, Cleaved caspase-1, and Cleaved GSDMD) associated with caspase-1dependent pyroptosis were up-regulated in pneumonia-induced sepsis.Conclusions: As NK-κB activation can promote the development of caspase-1-dependent pyroptosis, these findings suggested that the activation of the IL-17 signaling pathway could promote pyroptosis in pneumonia-induced sepsis.