High-Performance NIR-II Fluorescent Type I/II Photosensitizer Enabling Augmented Mild Photothermal Therapy of Tumors by Disrupting Heat Shock Proteins.

NIR-II fluorescent photosensitizers as phototheranostic agents hold considerable promise in the application of mild photothermal therapy (MPTT) for tumors, as the reactive oxygen species generated during photodynamic therapy can effectively disrupt heat shock proteins. Nevertheless, the exclusive utilization of these photosensitizers to significantly augment the MPTT efficacy has rarely been substantiated, primarily due to their insufficient photodynamic performance. Herein, we present the utilization of high-performance NIR-II fluorescent type I/II photosensitizer (AS21:4) as a simple but effective nanoplatform derived from molecule AS2 to enhance the MPTT efficacy of tumors without any additional therapeutic components. By taking advantage of heavy atom effect, AS21:4 as a type I/II photosensitizer demonstrates superior efficacy in producing 1O2 (ΦΔ = 12.4%) and O2 •- among currently available NIR-II fluorescent photosensitizers with absorption exceeding 800 nm. In vitro and in vivo findings demonstrate that the 1O2 and O2 •- generated from AS21:4 induce a substantial reduction in the expression of HSP90, thereby improving the MPTT efficacy. The remarkable phototheranostic performance, substantial tumor accumulation, and prolonged tumor retention of AS21:4, establish it as a simple but superior phototheranostic agent for NIR-II fluorescence imaging-guided MPTT of tumors. This article is protected by copyright. All rights reserved.

Effects of Microtopography on Soil Microbial Community Structure and Abundance in Permafrost Peatlands.

Soil microorganisms play crucial roles in the stability of the global carbon pool, particularly in permafrost peatlands that are highly sensitive to climate change. Microtopography is a unique characteristic of peatland ecosystems, but how microtopography affects the microbial community structures and their functions in the soil is only partially known. We characterized the bacterial and fungal community compositions by amplicon sequencing and their abundances via quantitative PCR at different soil depths in three microtopographical positions (hummocks, flats, and hollows) in permafrost peatland of the Greater Xing'an Mountains in China. The results showed that the soil of hummocks displayed a higher microbial diversity compared to hollows. Microtopography exerted a strong influence on bacterial community structure, while both microtopography and soil depth greatly impacted the fungal community structure with variable effects on fungal functional guilds. Soil water content, dissolved organic carbon, total phosphorus, and total nitrogen levels of the soil mostly affected the bacterial and fungal communities. Microtopography generated variations in the soil water content, which was the main driver of the spatial distribution of microbial abundances. This information stressed that the hummock-flat-hollow microtopography of permafrost peatlands creates heterogeneity in soil physicochemical properties and hydrological conditions, thereby influencing soil microbial communities at a microhabitat scale. Our results imply that changes to the water table induced by climate warming inducing permafrost degradation will impact the composition of soil microbes in peatlands and their related biogeochemical functions, eventually providing feedback loops into the global climate system.

Variations in soil microbial community structure and extracellular enzymatic activities along a forest-wetland ecotone in high-latitude permafrost regions.

Permafrost degradation by global warming is expected to alter the hydrological processes, which results in changes in vegetation species composition and gives rise to community succession. Ecotones are sensitive transition areas between ecosystem boundaries, attract particular interest due to their ecological importance and prompt responses to the environmental variables. However, the characteristics of soil microbial communities and extracellular enzymes along the forest-wetland ecotone in high-latitude permafrost region remain poorly understood. In this study, we evaluated the variations of soil bacterial and fungal community structures and soil extracellular enzymatic activities of 0-10 cm and 10-20 cm soil layers in five different wetland types along environmental gradients, including Larix gmelinii swamp (LY), Betula platyphylla swamp (BH), Alnus sibirica var. hirsute swamp (MCY), thicket swamp (GC), and tussock swamp (CC). The relative abundances of some dominant bacterial (Actinobacteria and Verrucomicrobia) and fungal (Ascomycota and Basidiomycota) phyla differed significantly among different wetlands, while bacterial and fungal alpha diversity was not strongly affected by soil depth. PCoA results showed that vegetation type, rather than soil depth explained more variation of soil microbial community structure. ß-glucosidase and ß-N-acetylglucosaminidase activities were significantly lower in GC and CC than in LY, BH, and MCY, while acid phosphatase activity was significantly higher in BH and GC than LY and CC. Altogether, the data suggest that soil moisture content (SMC) was the most important environmental factor contributing to the bacterial and fungal communities, while extracellular enzymatic activities were closely related to soil total organic carbon (TOC), nitrate nitrogen (NO3--N) and total phosphorus (TP).

Diversity and Distribution Characteristics of Soil Microbes across Forest-Peatland Ecotones in the Permafrost Regions.

Permafrost peatlands are a huge carbon pool that is uniquely sensitive to global warming. However, despite the importance of peatlands in global carbon sequestration and biogeochemical cycles, few studies have characterized the distribution characteristics and drivers of soil microbial community structure in forest-peatland ecotones. Here, we investigated the vertical distribution patterns of soil microbial communities in three typical peatlands along an environmental gradient using Illumina high-throughput sequencing. Our findings indicated that bacterial richness and diversity decreased with increasing soil depth in coniferous swamp (LT) and thicket swamp (HT), whereas the opposite trend was observed in a tussock swamp (NT). Additionally, these parameters decreased at 0-20 and 20-40 cm and increased at 40-60 cm along the environmental gradient (LT to NT). Principal coordinate analysis (PCoA) indicated that the soil microbial community structure was more significantly affected by peatland type than soil depth. Actinomycetota, Proteobacteria, Firmicutes, Chloroflexota, Acidobacteriota, and Bacteroidota were the predominant bacterial phyla across all soil samples. Moreover, there were no significant differences in the functional pathways between the three peatlands at each depth, except for amino acid metabolism, membrane transport, cell motility, and signal transduction. Redundancy analysis (RDA) revealed that pH and soil water content were the primary environmental factors influencing the bacterial community structure. Therefore, this study is crucial to accurately forecast potential changes in peatland ecosystems and improve our understanding of the role of peat microbes as carbon pumps in the process of permafrost degradation.

Response of Carbon Emissions and the Bacterial Community to Freeze-Thaw Cycles in a Permafrost-Affected Forest-Wetland Ecotone in Northeast China.

Climate warming can affect freeze-thaw cycle (FTCs) patterns in northern high-latitude regions and may affect permafrost carbon emissions. The response of carbon release and microbial communities to FTCs has not been well characterized. Here, we conducted laboratory incubation experiments to investigate the relationships among carbon emissions, bacterial community, and soil variables in a permafrost-affected forest-wetland ecotone in Northeast China. The emission rates of CO2 and CH4 increased during the FTCs. FTC amplitude, FTC frequency, and patch type had significant effects on carbon emissions. FTCs increased the contents of soil DOC, NH4+-N, and NO3--N but reduced bacterial alpha diversity. CO2 emissions were mainly affected by bacterial alpha diversity and composition, and the inorganic nitrogen content was the important factor affecting CH4 emissions. Our findings indicated that FTCs could significantly regulate CO2 and CH4 emissions by reducing bacterial community diversity and increasing the concentration of available soil substrates. Our findings shed new light on the microorganism-substrate mechanisms regulating the response patterns of the soil carbon cycle to FTCs in permafrost regions.

Vertical distribution of bacterial community diversity in the Greater Khingan Mountain permafrost region.

Soil microorganisms are crucial contributors to the function of permafrost ecosystems, as well as the regulation of biogeochemical cycles. However, little is known about the distribution patterns and drivers of high-latitude permafrost microbial communities subject to climate change and human activities. In this study, the vertical distribution patterns of soil bacterial communities in the Greater Khingan Mountain permafrost region were systematically analyzed via Illumina Miseq high-throughput sequencing. Bacterial diversity in the active layer was significantly higher than in the permafrost layer. Principal coordinate analysis (PCoA) indicated that the bacterial community structure in the active layer and the permafrost layer was completely separated. Permutational multivariate analysis of variance (PERMANOVA) detected statistically significant differentiation across the different depths. The relative abundance of the dominant phyla Chloroflexi (17.92%-52.79%) and Actinobacteria (6.34%-34.52%) was significantly higher in the permafrost layer than in the active layer, whereas that of Acidobacteria (4.98%-38.82%) exhibited the opposite trend, and the abundance of Proteobacteria (2.49%-22.51%) generally decreased with depth. More importantly, the abundance of bacteria linked to human infectious diseases was significantly higher in the permafrost layer according to Tax4Fun prediction analysis. Redundancy analysis (RDA) showed that ammonium nitrogen (NH4 +-N), total organic carbon (TOC), and total phosphorus (TP) were major factors affecting the bacterial community composition. Collectively, our findings provide insights into the soil bacterial vertical distribution patterns and major environmental drivers in high-latitude permafrost regions, which is key to grasping the response of cold region ecosystem processes to global climate changes.

Nitrite-dependent anaerobic methane oxidation bacteria and potential in permafrost region of Daxing'an Mountains.

Nitrite-dependent anaerobic methane oxidation (n-damo) acts as a crucial link between biogeochemical carbon and nitrogen cycles. Nevertheless, very few studies have characterized n-damo microorganisms in high-latitude permafrost regions. Therefore, this study investigated the vertical distribution and diversity of n-damo bacterial communities in soil from three forest types in the permafrost regions of the Daxing'an Mountains. A total of 11 and 8 operational taxonomic units (OTUs) of n-damo 16S rRNA and pmoA genes were observed, respectively. Remarkable spatial variations in n-damo bacteria community richness, diversity, and structure were observed at different soil depths. Moreover, the abundances of n-damo bacteria (16S rRNA and pmoA genes) varied between 1.55 × 104 to 1.47 × 105 and 1.31 × 103 to 3.11 × 104 copies g-1 dry soil (ds), as demonstrated by quantitative PCR analyses. 13CH4 stable isotope tracer assays indicated that the potential n-damo rates varied from 0 to 1.26 nmol g-1 day-1, with the middle layers (20-40 cm and 40-60 cm) exhibiting significantly higher values than the upper (0-20 cm) and deeper layers (80-100 cm) in all three forest types. Redundancy analyses (RDA) indicated that total organic carbon (TOC), nitrate (NO3--N), and nitrite (NO2--N) were key modulators of the distribution of n-damo bacterial communities. This study thus demonstrated the widespread occurrence of n-damo bacteria in cold and high-latitude regions of forest ecosystems and provided important insights into the global distribution of these bacteria. KEY POINTS: • This study detected n-damo bacteria in soil samples obtained from the permafrost region of three forest types in the Daxing'an Mountains. • The community composition of n-damo bacteria was mainly affected by soil depth and not forest type. • The abundances of n-damo bacteria first increased and then decreased at higher soil depths.

Soil organic carbon stabilization in permafrost peatlands.

In permafrost peatlands, the degradation of permafrost soil can raise soil temperature and alter moisture conditions, which increases the rate of loss of soil organic carbon (SOC). Here we selected three typical permafrost types that have very different active layer thicknesses but with soil originating from the same vegetation and which exist under comparable climatic conditions in the Da Xing'an mountain range: continuous permafrost, island permafrost, and island melting permafrost. To quantify the relative importance of control elements on SOC stabilization in these different permafrost types, we used correlation analysis to assess the relationship between organic carbon, physical and chemical properties and microorganisms, and explored the contribution of these factors to the accumulation of organic carbon. This study shows that the interaction between clay or silt, iron oxides and microorganisms have an important influence on the stability of organic carbon in permafrost peatlands.

Organic Carbon Mineralization and Bacterial Community of Active Layer Soils Response to Short-Term Warming in the Great Hing'an Mountains of Northeast China.

As a buffer layer for the energy and water exchange between atmosphere and permafrost, the active layer is sensitive to climate warming. Changes in the thermal state in active layer can alter soil organic carbon (SOC) dynamics. It is critical to identify the response of soil microbial communities to warming to better predict the regional carbon cycle under the background of global warming. Here, the active layer soils collected from a wetland-forest ecotone in the continuous permafrost region of Northeastern China were incubated at 5 and 15°C for 45 days. High-throughput sequencing of the 16S rRNA gene was used to examine the response of bacterial community structure to experimental warming. A total of 4148 OTUs were identified, which followed the order 15°C > 5°C > pre-incubated. Incubation temperature, soil layer and their interaction have significant effects on bacterial alpha diversity (Chao index). Bacterial communities under different temperature were clearly distinguished. Chloroflexi, Actinobacteria, Proteobacteria, and Acidobacteria accounted for more than 80% of the community abundance at the phylum level. Warming decreased the relative abundance of Chloroflexi and Acidobacteria, while Actinobacteria and Proteobacteria exhibited increasing trend. At family level, the abundance of norank_o__norank_c__AD3 and Ktedonobacteraceae decreased significantly with the increase of temperature, while Micrococcaccac increased. In addition, the amount of SOC mineralization were positively correlated with the relative abundances of most bacterial phyla and SOC content. SOC content was positively correlated with the relative abundance of most bacterial phyla. Results indicate that the SOC content was the primary explanatory variable and driver of microbial regulation for SOC mineralization. Our results provide a new perspective for understanding the microbial mechanisms that accelerates SOC decomposition under warming conditions in the forest-wetland ecotone of permafrost region.

Essential role for Cmtm7 in cell-surface phenotype, BCR signaling, survival and Igµ repertoire of splenic B-1a cells.

B-1a cells represent a distinct B cell population with unique phenotype, self-renewing capacity and restricted Igµ repertoire. They primarily locate in body cavity and also exist in spleen. The different subpopulations of B-1a cells are heavily affected by local environment. Our previous studies revealed that MARVEL-domain-containing membrane protein, CMTM7, was involved in B-1a cell development. Here, we focused its influence on peritoneal and splenic B-1a cells. Unlike peritoneal B-1a cells, we found that splenic Cmtm7-/- B-1a cells expressed higher level of CD5, CD80 and CD86 compared with WT counterparts. They also exhibited an enhanced tonic BCR signals in steady state. Though the cell viability was unaffected in vitro, Cmtm7 knockout markedly promoted splenic B-1a cell apoptosis in situ, which was likely associated with down-regulation of Il-5rα. With regard to Igµ repertoire, peritoneal and splenic Cmtm7-/- B-1a cells exhibit similar changes exemplified by the loss of VH11 and gain of VH12, whereas an increase in VH1 usage and skewed J segments from JH1 to JH2 and JH4 families could only be detected within splenic Cmtm7-/- B-1a cells. Overall, these data indicate that Cmtm7 functions differently in peritoneal and splenic B-1a cells and plays a more important role in splenic cells.

CMTM7 plays key roles in TLR-induced plasma cell differentiation and p38 activation in murine B-1 B cells.

Terminal differentiation of B cells into antibody-secreting cells is the foundation of humoral immune response. B-1 cells, which are different from B-2 cells, preferentially differentiate into plasma cells. CMTM7 is a MARVEL-domain-containing membrane protein predominantly expressed in B cells that plays an important role in B-1a cell development. The present study assessed CMTM7 function in response to antigen stimulation. Following immunization with T cell-dependent and T cell-independent antigens, Cmtm7-deficient mice exhibited decreased IgM but normal IgG responses in vivo. In vitro stimulation with LPSs induced Cmtm7-/- B-1 cell activation, whereas proliferation was marginally reduced. Notably, Cmtm7 deficiency markedly suppressed plasma cell differentiation in response to TLR agonists, accompanied by a decrease in IgM and IL-10 production. At the molecular level, loss of Cmtm7 repressed the downregulation of Pax5 and the upregulation of Xbp1, Irf4, and Prdm1. Furthermore, p38 phosphorylation was inhibited in Cmtm7-/- B-1 cells. Experiments using a p38 inhibitor revealed that p38 activation was essential for the terminal differentiation of B-1 cells, suggesting that Cmtm7 contributes to B-1 cell differentiation by maintaining p38 activation. Overall, the data reveal the crucial functions of CMTM7 in TLR-induced terminal differentiation and p38 activation in B-1 cells.

Emissions of CO2, CH4, and N2O Fluxes from Forest Soil in Permafrost Region of Daxing'an Mountains, Northeast China.

With global warming, the large amount of greenhouse gas emissions released by permafrost degradation is important in the global carbon and nitrogen cycle. To study the feedback effect of greenhouse gases on climate change in permafrost regions, emissions of CO2, CH4, and N2O were continuously measured by using the static chamber-gas chromatograph method, in three forest soil ecosystems (Larix gmelinii, Pinus sylvestris var. mongolica, and Betula platyphylla) of the Daxing'an Mountains, northeast China, from May 2016 to April 2018. Their dynamic characteristics, as well as the key environmental affecting factors, were also analyzed. The results showed that the flux variation ranges of CO2, CH4, and N2O were 7.92 ± 1.30~650.93 ± 28.12 mg·m-2·h-1, -57.71 ± 4.65~32.51 ± 13.03 ug·m-2·h-1, and -3.87 ± 1.35~31.1 ± 2.92 ug·m-2·h-1, respectively. The three greenhouse gas fluxes showed significant seasonal variations, and differences in soil CO2 and CH4 fluxes between different forest types were significant. The calculation fluxes indicated that the permafrost soil of the Daxing'an Mountains may be a potential source of CO2 and N2O, and a sink of CH4. Each greenhouse gas was controlled using different key environmental factors. Based on the analysis of Q10 values and global warming potential, the obtained results demonstrated that greenhouse gas emissions from forest soil ecosystems in the permafrost region of the Daxing'an Mountains, northeast China, promote the global greenhouse effect.

CMTM4 inhibits cell proliferation and migration via AKT, ERK1/2, and STAT3 pathway in colorectal cancer.

CMTM4 (CKLF-like MARVEL transmembrane domain containing 4), a potential tumor suppressor gene, is involved in several types of malignancies. It has been reported to be downregulated and exhibit anti-tumorigenic activities by regulating cell growth and cell cycle in clear cell renal cell carcinoma. It has also been identified as a tumor suppressor in hepatocellular carcinoma (HCC), and its negative expression is a risk factor for poor prognosis of HCC patients. In the present study, an integrated bioinformatics analysis based on The Cancer Genome Atlas (TCGA) database showed that CMTM4 was frequently reduced in colorectal cancer (CRC) and high expression of CMTM4 was associated with increased overall survival rates. Based on these findings, we adopted gain-of-function and lost-of-function strategies using SW480 and HT29 CRC cell lines which have relatively low and high endogenous CMTM4 levels, respectively. We observed impeded cell proliferation and migration upon overexpression of CMTM4 in SW480 cells, and the opposite effects were observed upon knockdown of CMTM4 in HT-29 cells. Cell signaling pathways essential for CRC progression were then examined, and the phosphorylation levels of AKT, ERK1/2, and STAT3 were found to be decreased by CMTM4 overexpression in SW480 cells and elevated by CMTM4 silencing in HT29 cells. Their inhibitors were used to validate that the three signaling pathways contributed to the inhibitory effects of CMTM4 on CRC cells. Taken together, our results suggest that CMTM4 plays a tumor suppressive role in CRC.

CYTL1 inhibits tumor metastasis with decreasing STAT3 phosphorylation.

CYTL1 is a novel cytokine that was first identified in CD34+ hematopoietic cells. We previously prepared recombinant CYTL1 and verified that it chemoattracted human monocytes via the CCR2/ERK pathway. It has been reported that CYTL1 plays contradictory roles in neuroblastoma and lung cancer. We found that the expression level of CYTL1 was notably decreased and it was hypermethylated in various tumors, including breast and lung cancer, by bioinformatics analyses. After validating the expression of CYTL1 in lung cancer, we identified that CYTL1 exerted no obvious effect on tumor cell proliferation but inhibited their migration and invasion, and these effects were accompanied by decreasing STAT3 phosphorylation, using recombinant CYTL1 and CYTL1-overexpressing tumor cell lines. Furthermore, we constructed experimental and spontaneous metastasis models of breast cancer in BALB/c mice and found that CYTL1 significantly inhibited tumor metastasis in vivo. In summary, CYTL1 is a cytokine with tumor-suppressing characteristics that inhibits tumor metastasis and STAT3 phosphorylation in multiple types of tumors.

Cmtm7 knockout inhibits B-1a cell development at the transitional (TrB-1a) stage.

Innate-like B-1a cells are an important cell population for production of natural IgM and interleukin-10 (IL-10), and act as the first line against pathogens. We determined that CMTM7 is essential for B-1a cell development. Following Cmtm7 (CKLF-like MARVEL transmembrane domain-containing 7) knockout, B-1a cell numbers decreased markedly in all investigated tissues. Using a bone marrow and fetal liver adoptive transfer model and conditional knockout mice, we showed that the reduction of B-1a cells resulted from B-cell-intrinsic defects. Because of B-1a cell loss, Cmtm7-deficient mice produced less IgM and IL-10, and were more susceptible to microbial sepsis. Self-renewal and homeostasis of mature B-1a cells in Cmtm7-/- mice were not impaired, suggesting the effect of Cmtm7 on B-1a cell development. Further investigations demonstrated that the function of Cmtm7 in B-1a cell development occurred at the specific transitional B-1a (TrB-1a) stage. Cmtm7 deficiency resulted in a slow proliferation and high cell death rate of TrB-1a cells. Thus, Cmtm7 controls B-1a cell development at the transitional stage.

Publisher Correction: A new antagonist for CCR4 attenuates allergic lung inflammation in a mouse model of asthma.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Novel Adipokine, FAM19A5, Inhibits Neointima Formation After Injury Through Sphingosine-1-Phosphate Receptor 2.

BACKGROUND: Obesity plays crucial roles in the development of cardiovascular diseases. However, the mechanisms that link obesity and cardiovascular diseases remain elusive. Compelling evidence indicates that adipokines play an important role in obesity-related cardiovascular diseases. Here, we found a new adipokine-named family with sequence similarity 19, member A5 (FAM19A5), a protein with unknown function that was predicted to be distantly related to the CC-chemokine family. We aimed to test whether adipose-derived FAM19A5 regulates vascular pathology on injury. METHODS: DNA cloning, protein expression, purification, and N-terminal sequencing were applied to characterize FAM19A5. Adenovirus infection and siRNA transfection were performed to regulate FAM19A5 expression. Balloon and wire injury were performed in vivo on the rat carotid arteries and mouse femoral arteries, respectively. Bioinformatics analysis, radioactive ligand-receptor binding assays, receptor internalization, and calcium mobilization assays were used to identify the functional receptor for FAM19A5. RESULTS: We first characterized FAM19A5 as a secreted protein, and the first 43 N-terminal amino acids were the signal peptides. Both FAM19A5 mRNA and protein were abundantly expressed in the adipose tissue but were downregulated in obese mice. Overexpression of FAM19A5 markedly inhibited vascular smooth muscle cell proliferation and migration and neointima formation in the carotid arteries of balloon-injured rats. Accordingly, FAM19A5 silencing in adipocytes significantly promoted vascular smooth muscle cell activation. Adipose-specific FAM19A5 transgenic mice showed greater attenuation of neointima formation compared with wild-type littermates fed with or without Western-style diet. We further revealed that sphingosine-1-phosphate receptor 2 was the functional receptor for FAM19A5, with a dissociation constant (Kd) of 0.634 nmol/L. Inhibition of sphingosine-1-phosphate receptor 2 or its downstream G12/13-RhoA signaling circumvented the suppressive effects of FAM19A5 on vascular smooth muscle cell proliferation and migration. CONCLUSIONS: We revealed that a novel adipokine, FAM19A5, was capable of inhibiting postinjury neointima formation via sphingosine-1-phosphate receptor 2-G12/13-RhoA signaling. Downregulation of FAM19A5 during obesity may trigger cardiometabolic diseases.

Virtual Sorting Has a Distinctive Advantage in Identification of Anticorrelated Genes and Further Negative Regulators of Immune Cell Subpopulations.

Immune cells are highly plastic in both gene expression and cell phenotype. We have established a method of gene expressional plasticity and virtual sorting to evaluate immune cell subpopulations and their characteristic genes in human CD4+ T cells. In this study, we continued to investigate the informatics mechanism on the effectiveness of virtual sorting. We found that virtual sorting had an overall positive correlation to the Pearson correlation in the identification of positively correlated genes. However, owing to nonlinear biological anticorrelation, virtual sorting showed a distinctive advantage for anticorrelated genes, suggesting an important role in the identification of negative regulators. In addition, based on virtual sorting results, we identified two basic gene sets among highly plastic genes, i.e., highly plastic cell cycle-associated molecules and highly plastic immune and defense response-associated molecules. Genes within each set tended to be positively connected, but genes between two sets were often anticorrelated. Further analysis revealed preferential transcription factor binding motifs existed between highly plastic cell cycle-associated molecules and highly plastic immune and defense response-associated molecules. Our results strongly suggested predetermined regulation, which was called an immune cell internal phenotype, should exist and could be mined by virtual sorting analysis. This provided efficient functional clues to study immune cell phenotypes and their regulation. Moreover, the current substantial virtual sorting results in both CD4+ T cells and B cells provide a useful resource for big-data-driven experimental studies and knowledge discoveries.

A new antagonist for CCR4 attenuates allergic lung inflammation in a mouse model of asthma.

CCR4 is highly expressed on Th2 cells. CCR4 ligands include CCL22 and CCL17. Chemokine-like factor 1 can also mediate chemotaxis via CCR4. We designed and synthetized novel CCR4 antagonists, which were piperazinyl pyridine derivatives, for disrupting the interaction between three ligands and CCR4. We also determined whether these novel CCR4 antagonists could alleviate allergic asthma in a mouse. For identifying the potent compounds in vitro, we used chemotaxis inhibition and competition binding assays induced by CCL22, CCL17 and one of CKLF1's C-terminal peptides, C27. We found compound 8a which showed excellent potency in blocking the interaction of CCR4 and its three ligands. For studying the specificity of compounds, we chose chemotaxis inhibition assays with different receptors and ligands. We found compound 8a had excellent receptor specificity and exerted few influence on the interaction of other receptors and their ligands. In the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, compound 8a had no obvious cytotoxicity till the higher concentration (16 µM). For determining the potency of compounds in blocking the interaction of CCR4 in vivo, we used the ovalbumin induced allergic asthma model in mice. Our study demonstrated that CCR4 blockaded by compound 8a effectively attenuated airway hyperresponsiveness, airway eosinophilia and Th2 cytokines.