Background Some chemokine receptors referred to as atypical chemokine receptors (ACKRs) are thought to non-signaling decoys because of their inability to activate typical G-protein signaling pathways. CXCR7, also known as ACKR3, binds to only two chemokines, SDF-1α and I-TAC, and recruits β-arrestins. SDF-1α also binds to its own conventional receptor, CXCR4, involving in homeostatic modulation such as development and immune surveillance as well as pathological conditions such as inflammation, ischemia, and cancers. Recently, CXCR7 is suggested as a key therapeutic target together with CXCR4 in such conditions. However, the molecular mechanisms underlying cellular responses and functional relation with CXCR7 and CXCR4 have not been elucidated, despite massive studies. Therefore, we aimed to reveal the molecular networks of CXCR7 and CXCR4 and compare their effects on cell migration. Methods Base on structural complementation assay using NanoBiT technology, we characterized the distinct mechanisms underlying β-arrestin2 recruitment by both CXCR4 and CXCR7. Crosslinking and immunoprecipitation were conducted to analyze complex formation of the receptors. Gene deletion using CRISPR and reconstitution of the receptors were applied to analysis of ligand-dependent ERK phosphorylation and cell migration. All experiments were performed in triplicate and repeated more than three times. Unpaired Student’s t-tests or ANOVA using PRISM5 software were employed for statistical analyses. Results Ligand binding to CXCR7 does not result in activation of typical signaling pathways via Gα subunits but activation of GRK2 via βγ subunits and receptor phosphorylation with subsequent β-arrestin2 recruitment. In contrast, CXCR4 induced Gαi activation and recruited β-arrestin2 through C-terminal phosphorylation by both GRK2 and GRK5. SDF-1α-stimulated ERK phosphorylation was facilitated by CXCR4, but not CXCR7. Heterodimerization of CXCR4 and CXCR7 was not confirmed in this study, while homodimerization of them was verified by crosslinking experiment and NanoBiT assay. Regarding chemotaxis, SDF-1α-stimulated cell migration was mediated by both CXCR4 and CXCR7. Conclusion This study demonstrates that SDF-1α-stimulated CXCR7 mediates β-arrestin2 recruitment via different molecular networking from that of CXCR4. CXCR7 may be neither a simple scavenger nor auxiliary receptor but plays an essential role in cell migration through cooperation with CXCR4.
To accelerate the reduction in tuberculosis (TB) incidence, it is necessary to optimize the use of innovative tools and approaches available within a local context. This study evaluated the use of an existing network of community health workers (CHW) for active case finding, in combination with mobile chest X-ray (CXR) screening events and the expansion of Xpert MTB/RIF testing eligibility, in order to reach people with TB who had been missed by the current system. A controlled intervention study was conducted from January 2018 to March 2019 in five intervention and four control districts of two low to medium TB burden cities in Viet Nam. CHWs screened and referred eligible persons for CXR to TB care facilities or mobile screening events in the community. The initial diagnostic test was Xpert MTB/RIF for persons with parenchymal abnormalities suggestive of TB on CXR or otherwise on smear microscopy. We analyzed the TB care cascade by calculating the yield and number needed to screen (NNS), estimated the impact on TB notifications and conducted a pre-/postintervention comparison of TB notification rates using controlled, interrupted time series (ITS) analyses. We screened 30,336 individuals in both cities to detect and treat 243 individuals with TB, 88.9% of whom completed treatment successfully. All forms of TB notifications rose by +18.3% (95% CI: +15.8%, +20.8%). The ITS detected a significant postintervention step-increase in the intervention area for all-form TB notification rates (IRR(β6) = 1.221 (95% CI: 1.011, 1.475); p = 0.038). The combined use of CHWs for active case findings and mobile CXR screening expanded the access to and uptake of Xpert MTB/RIF testing and resulted in a significant increase in TB notifications. This model could serve as a blueprint for expansion throughout Vietnam. Moreover, the results demonstrate the need to optimize the use of the best available tools and approaches in order to end TB.
Cytosolic Ca 2+ levels ([Ca 2+ ] c ) change dynamically in response to inducers, repressors, and physiological conditions, and aberrant [Ca 2+ ] c concentration regulation is associated with cancer, heart failure, and diabetes. Therefore, [Ca 2+ ] c is considered as a good indicator of physiological and pathological cellular responses, and is a crucial biomarker for drug discovery. A genetically encoded calcium indicator (GECI) was recently developed to measure [Ca 2+ ] c in single cells and animal models. GECI have some advantages over chemically synthesized indicators, although they also have some drawbacks such as poor signal-to-noise ratio (SNR), low positive signal, delayed response, artifactual responses due to protein overexpression, and expensive detection equipment. Here, we developed an indicator based on interactions between Ca 2+ -loaded calmodulin and target proteins, and generated an innovative GECI sensor using split nano-luciferase (Nluc) fragments to detect changes in [Ca 2+ ] c . Stimulation-dependent luciferase activities were optimized by combining large and small subunits of Nluc binary technology (NanoBiT, LgBiT:SmBiT) fusion proteins and regulating the receptor expression levels. We constructed the binary [Ca 2+ ] c sensors using a multicistronic expression system in a single vector linked via the internal ribosome entry site (IRES), and examined the detection efficiencies. Promoter optimization studies indicated that promoter-dependent protein expression levels were crucial to optimize SNR and sensitivity. This novel [Ca 2+ ] c assay has high SNR and sensitivity, is easy to use, suitable for high-throughput assays, and may be useful to detect [Ca 2+ ] c in single cells and animal models.
BackgroundTraditional medicine (TM) still plays an important role in a number of health care systems around the world, especially across Asian and African countries. In Vietnam, however, little is known about preference for traditional medicine use. This study assessed the prevalence of use, preference, satisfaction, and willingness to pay for TM services amongst rural ethnic minority community.MethodsA cross-sectional survey in three provinces in the North and South of Vietnam.ResultsThe results showed a high level of satisfaction with TM services, with more than 90 % of respondents reporting improved health status given the use of TM. Indicators for preference of TM over modern medicine are a longer distance to health station; being in an ethnic minority; being female; and having had higher service satisfaction. Although we did not have a comparison group, the high level of satisfaction with TM services is likely the result of a project targeting community health workers and the public regarding TM education and access promotion. Indeed, the community health workers are credited with relaying the information about TM more than any other sources. This suggests the importance of community health workers and community health centers in the promotion of TM use.ConclusionsEthnic minority people prefer the use of traditional medicine services that supports the expansion of national programs and promotion of traditional medications.
Background C–C motif chemokine receptor 2 (CCR2), the main receptor for monocyte chemoattractant protein-1 (MCP-1), is expressed on immune cells, including monocytes, macrophages, and activated T cells, and mediates cell migration toward MCP-1 in inflammation-related diseases. The CCR2 gene encodes two isoforms: CCR2A and CCR2B. The CCR2B open reading frame is localized in a single exon, similar to other chemokine receptors, and CCR2A and CCR2B feature different amino acid sequences in their C-terminal intracellular loops due to alternative splicing. Most biochemical studies on CCR2-related cellular responses in the immune system have focused on CCR2B, with few reports focused on CCR2A. Understanding the functional properties of CCR2A in cellular responses may elucidate the roles played by MCP-1 and CCR2 in pathophysiological responses. Results CCR2 gene expression analysis in several cell types revealed that most adherent cells only expressed CCR2A, whereas CCR2B expression was dominant in monocytic cells. The C-terminal Helix 8 region of CCR2A contains few basic amino acids, which may be unfavorable for cell surface localization, as confirmed with the HiBiT assay. CCR2B contains many C-terminal Ser/Thr residues, similar to other chemokine receptors, which may be phosphorylated by G protein–coupled receptor kinases (GRKs) to promote β-arrestin recruitment and subsequent endocytosis. By contrast, CCR2A contains few C-terminal Ser/Thr residues, which are unlikely to be phosphorylated by GRKs. CCR2A localized on the cell surface is resistant to internalization, despite the interaction between Gβ and GRKs induced by ligand binding with CCR2A. CCR2A induced cellular responses at a relatively higher degree than CCR2B, although both receptors mediated signaling events through Gαq and Gαi. HeLa cells lacking CCR2A showed slowed growth compared with parent cells, regardless of MCP-1 stimulation, and their chemotactic activity toward MCP-1, in addition to basal motility, was significantly impaired. Conclusion MCP-1 and CCR2 may play pivotal roles in cancer progression by recruiting macrophages into cancer tissue. This study demonstrates that CCR2A but not CCR2B is expressed in solid cancer–derived cells. CCR2A is resistant to internalization by β-arrestin due to a distinct C-terminal region from CCR2B, which enhances MCP-1-stimulated responses, indicating that CCR2A may play essential roles in solid cancer progression.
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