UVRAG cooperates with cargo receptors to assemble the ER-phagy site.

ER-phagy is a selective autophagy process that targets specific regions of the endoplasmic reticulum (ER) for removal via lysosomal degradation. During cellular stress induced by starvation, cargo receptors concentrate at distinct ER-phagy sites (ERPHS) to recruit core autophagy proteins and initiate ER-phagy. However, the molecular mechanism responsible for ERPHS formation remains unclear. In our study, we discovered that the autophagy regulator UV radiation Resistance-Associated Gene (UVRAG) plays a crucial role in orchestrating the assembly of ERPHS. Upon starvation, UVRAG localizes to ERPHS and interacts with specific ER-phagy cargo receptors, such as FAM134B, ATL3, and RTN3L. UVRAG regulates the oligomerization of cargo receptors and facilitates the recruitment of Atg8 family proteins. Consequently, UVRAG promotes efficient ERPHS assembly and turnover of both ER sheets and tubules. Importantly, UVRAG-mediated ER-phagy contributes to the clearance of pathogenic proinsulin aggregates. Remarkably, the involvement of UVRAG in ER-phagy initiation is independent of its canonical function as a subunit of class III phosphatidylinositol 3-kinase complex II.

A genome-wide CRISPR/Cas9 knockout screen identifies TMEM239 as an important host factor in facilitating African swine fever virus entry into early endosomes.

African swine fever (ASF) is a highly contagious, fatal disease of pigs caused by African swine fever virus (ASFV). The complexity of ASFV and our limited understanding of its interactions with the host have constrained the development of ASFV vaccines and antiviral strategies. To identify host factors required for ASFV replication, we developed a genome-wide CRISPR knockout (GeCKO) screen that contains 186,510 specific single guide RNAs (sgRNAs) targeting 20,580 pig genes and used genotype II ASFV to perform the GeCKO screen in wild boar lung (WSL) cells. We found that knockout of transmembrane protein 239 (TMEM239) significantly reduced ASFV replication. Further studies showed that TMEM239 interacted with the early endosomal marker Rab5A, and that TMEM239 deletion affected the co-localization of viral capsid p72 and Rab5A shortly after viral infection. An ex vivo study showed that ASFV replication was significantly reduced in TMEM239-/- peripheral blood mononuclear cells from TMEM239 knockout piglets. Our study identifies a novel host factor required for ASFV replication by facilitating ASFV entry into early endosomes and provides insights for the development of ASF-resistant breeding.

Structural mechanism of human HCN1 hyperpolarization-activated channel inhibition by ivabradine.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a crucial role in regulating neuronal excitability. Despite growing evidence supporting the therapeutic potential of HCN1 inhibition in treating neurological disorders, the structural basis of channel inhibition by inhibitor has remained elusive. Here, we present the cryo-electron microscopy structure of human HCN1 channel in complex with inhibitor ivabradine, the drug on the market that acts on HCN channels. Combining electrophysiology, mutagenesis, and molecular dynamics simulations, our findings reveal that ivabradine binds to a previously unidentified pocket formed between the S4, S1, and HCN domain. Furthermore, through structure-based virtual screening, we identify two FDA-approved drugs that can inhibit the HCN1 channel by interacting with the ivabradine-binding site. Our results not only provide insights into the structural intricacies of ivabradine-mediated inhibition, but also offer a potential pharmacological framework for developing novel drugs targeting the HCN1 channel. The elucidation of these molecular interactions serves as a foundational step in advancing therapeutic strategies for modulating HCN1 activity, contributing to the broader landscape of drug discovery and development in this area.

Cryo-EM structure of human HCN3 channel and its regulation by cAMP.

HCN channels are important for regulating heart rhythm and nerve activity and have been studied as potential drug targets for treating depression, arrhythmia, nerve pain, and epilepsy. Despite possessing unique pharmacological properties, HCN channels share common characteristics in that they are activated by hyperpolarization and modulated by cAMP and other membrane lipids. However, the mechanisms of how these ligands bind and modulate HCN channels are unclear. In this study, we solved structures of full-length human HCN3 using cryo-EM and captured two different states, including a state without any ligand bound and a state with cAMP bound. Our structures reveal the novel binding sites for cholesteryl hemisuccinate in apo state and show how cholesteryl hemisuccinate and cAMP binding cause conformational changes in different states. These findings explain how these small modulators are sensed in mammals at the molecular level. The results of our study could help to design more potent and specific compounds to influence HCN channel activity and offer new therapeutic possibilities for diseases that lack effective treatment.

A novel three-layer module BoMYB1R1-BoMYB4b/BoMIEL1-BoDFR1 regulates anthocyanin accumulation in kale.

Anthocyanin is an important pigment responsible for plant coloration and beneficial to human health. Kale (Brassica oleracea var. acephala), a primary cool-season flowers and vegetables, is an ideal material to study anthocyanin biosynthesis and regulation mechanisms due to its anthocyanin-rich leaves. However, the underlying molecular mechanism of anthocyanin accumulation in kale remains poorly understood. Previously, we demonstrated that BoDFR1 is a key gene controlling anthocyanin biosynthesis in kale. Here, we discovered a 369-bp InDel variation in the BoDFR1 promoter between the two kale inbred lines with different pink coloration, which resulted in reduced transcriptional activity of the BoDFR1 gene in the light-pink line. With the 369-bp insertion as a bait, an R2R3-MYB repressor BoMYB4b was identified using the yeast one-hybrid screening. Knockdown of the BoMYB4b gene led to increased BoDFR1 expression and anthocyanin accumulation. An E3 ubiquitin ligase, BoMIEL1, was found to mediate the degradation of BoMYB4b, thereby promoting anthocyanin biosynthesis. Furthermore, the expression level of BoMYB4b was significantly reduced by light signals, which was attributed to the direct repression of the light-signaling factor BoMYB1R1 on the BoMYB4b promoter. Our study revealed that a novel regulatory module comprising BoMYB1R1, BoMIEL1, BoMYB4b, and BoDFR1 finely regulates anthocyanin accumulation in kale. The findings aim to establish a scientific foundation for genetic improvement of leaf color traits in kale, meanwhile, providing a reference for plant coloration studies.

Afucosylated anti-EBOV antibody MIL77-3 engages sGP to elicit NK cytotoxicity.

MIL77-3 is one component of antibody cocktail that is produced in our lab and represents an effective regimen for animals suffering from Zaire Ebolavirus (EBOV) infection. MIL77-3 is engineered to increase its affinity for the FcγRIIIa (CD16a) by deleting the fucose in the framework region. The potential effects of this modification on host immune responses, however, remain largely unknown. Herein, we demonstrated that MIL77-3 recognized secreted glycoproptein (sGP), produced by EBOV, and formed the immunocomplex to potently augment antibody-dependent cytotoxicity of human peripheral blood-derived natural killer cells (pNKs), including CD56dim and CD56bright subpopulations, in contrast to the counterparts (Mab114, rEBOV548, fucosylated MIL77-3). Intriguingly, this effect was not observed when NK92-CD16a cell line was utilized and restored by the addition of beads-coupled or membrane-anchored sGP in combination with MIL77-3. Furthermore, sGP bound to unrecognized receptors on T cells contaminated in pNKs rather than NK92-CD16a cells. Administration of beads-coupled sGP/MIL77-3 complex in mice elicited NK activation. Overall, this work reveals an immune-stimulating function of sGP/MIL77-3 complex by triggering cytotoxic activity of NK cells, highlighting the necessity to evaluate the potential impact of MIL77-3 on host immune reaction in clinical trials. IMPORTANCE: Zaire Ebolavirus (EBOV) is highly lethal and causes sporadic outbreaks. The passive administration of monoclonal antibodies (mAbs) represents a promising treatment regimen against EBOV. Mounting evidence has shown that the efficacy of a subset of therapeutic mAbs in vivo is intimately associated with its capacity to trigger NK activity, supporting glycomodification of Fc region of anti-EBOV mAbs as a putative strategy to enhance Fc-mediated immune effector function as well as protection in vivo. Our work here uncovers the potential harmful influence of this modification on host immune responses, especially for mAbs with cross-reactivity to secreted glycoproptein (sGP) (e.g., MIL77-3), and highlights it is necessary to evaluate the NK-stimulating activity of a fucosylated mAb engaged with sGP when a new candidate is developed.

Transcriptional regulation of transcription factor genes WRI1 and LAFL during Brassica napus seed development.

The WRINKLED1 (WRI1) and LAFL [LEAFY COTYLEDON1 (LEC1), ABSCISIC ACID INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and LEC2] transcription factors play essential roles in governing seed development and oil biosynthesis. To gain a comprehensive understanding of the transcriptional regulation of WRI1 and LAFL, we conducted genome-wide association studies for the expression profiles of WRI1 and LAFL in developing seeds at 20 and 40 days after flowering (DAF) using 302 rapeseed (Brassica napus) accessions. We identified a total of 237 expression quantitative trait nucleotides (eQTNs) and 51 expression QTN-by-environment interactions (eQEIs) associated with WRI1 and LAFL. Around these eQTNs and eQEIs, we pinpointed 41 and 8 candidate genes with known transcriptional regulations or protein interactions with their expression traits, respectively. Based on RNA-seq and ATAC-seq data, we employed the XGBoost and Basenji models which predicted 15 candidate genes potentially regulating the expression of WRI1 and LAFL. We further validated the predictions via tissue expression profile, haplotype analysis, and expression correlation analysis, and verified the transcriptional activation activity of BnaC03.MYB56 (R2R3-MYB transcription factor 56) on the expression of BnaA09.LEC1 by dual-luciferase reporter and yeast one-hybrid assays. BnaA10.AGL15 (AGAMOUS-LIKE 15), BnaC04.VAL1 (VIVIPAROUS1/ABSCISIC ACID INSENSITIVE3-LIKE 1), BnaC03.MYB56, and BnaA10.MYB56 were co-expressed with WRI1 and LAFL at 20 DAF in M35, a key module for seed development and oil biosynthesis. We further validated the positive regulation of MYB56 on seed oil accumulation using Arabidopsis (Arabidopsis thaliana) mutants. This study not only delivers a framework for future eQEI identification but also offers insights into the developmental regulation of seed oil accumulation.

Dissociable contributions of the hippocampus and orbitofrontal cortex to representing task space in a social context.

The hippocampus (HC) and the orbitofrontal cortex (OFC) jointly encode a map-like representation of a task space to guide behavior. It remains unclear how the OFC and HC interact in encoding this map-like representation, though previous studies indicated that both regions have different functions. We acquired the functional magnetic resonance imaging data under a social navigation task in which participants interacted with characters in a two-dimensional "social space." We calculate the social relationships between the participants and characters and used a drift-diffusion model to capture the inner process of social interaction. Then we used multivoxel pattern analysis to explore the brain-behavior relationship. We found that (i) both the HC and the OFC showed higher activations during the selective trial than the narrative trial; (ii) the neural pattern of the right HC was associated with evidence accumulation during social interaction, and the pattern of the right lateral OFC was associated with the social relationship; (iii) the neural pattern of the HC can decode the participants choices, while the neural pattern of the OFC can decode the task information about trials. The study provided evidence for distinct roles of the HC and the OFC in encoding different information when representing social space.

The roles of cell wall polysaccharides in response to waterlogging stress in Brassica napus L. root.

BACKGROUND: Brassica napus L. (B. napus) is susceptible to waterlogging stress during different cultivation periods. Therefore, it is crucial to enhance the resistance to waterlogging stress to achieve a high and stable yield of B. napus. RESULTS: Here we observed significant differences in the responses of two B. napus varieties in root under waterlogging stress. The sensitive variety (23651) exhibited a more pronounced and rapid reduction in cell wall thickness and root integrity compared with the tolerant variety (Santana) under waterlogging stress. By module clustering analysis based on transcriptome data, we identified that cell wall polysaccharide metabolism responded to waterlogging stress in root. It was found that pectin content was significantly reduced in the sensitive variety compared with the tolerant variety. Furthermore, transcriptome analysis revealed that the expression of two homologous genes encoding polygalacturonase-inhibiting protein 2 (PGIP2), involved in polysaccharide metabolic pathways, was highly upregulated in root of the tolerant variety under waterlogging stress. BnaPGIP2s probably confer waterlogging resistance by inhibiting the activity of polygalacturonases (PGs), which in turn reduces the degradation of the pectin backbone polygalacturonic acid. CONCLUSIONS: Our findings demonstrate that cell wall polysaccharides in root plays a vital role in response to the waterlogging stress and provide a theoretical foundation for breeding waterlogging resistance in B. napus varieties.

Cryo-EM Structure and Biochemical Analysis of the Human Chemokine Receptor CCR8.

The C-C motif chemokine receptor 8 (CCR8) is a class A G-protein-coupled receptor that has emerged as a promising therapeutic target in cancer and autoimmune diseases. In the present study, we solved the cryo-electron microscopy (cryo-EM) structure of the human CCR8-Gi complex in the absence of a ligand at 2.58 Å. Structural analysis and comparison revealed that our apo CCR8 structure undergoes some conformational changes and is similar to that in the CCL1-CCR8 complex structure, indicating an active state. In addition, the key residues of CCR8 involved in the recognition of LMD-009, a potent nonpeptide agonist, were investigated by mutating CCR8 and testing the calcium flux induced by LMD-009-CCR8 interaction. Three mutants of CCR8, Y1133.32A, Y1724.64A, and E2867.39A, showed a dramatically decreased ability in mediating calcium mobilization, indicating their key interaction with LMD-009 and key roles in activation. These structural and biochemical analyses enrich molecular insights into the agonism and activation of CCR8 and will facilitate CCR8-targeted therapy.

Structural analysis of the human C5a-C5aR1 complex using cryo-electron microscopy.

The complement system is a complex network of proteins that plays a crucial role in the innate immune response. One important component of this system is the C5a-C5aR1 complex, which is critical in the recruitment and activation of immune cells. In-depth investigation of the activation mechanism as well as biased signaling of the C5a-C5aR1 system will facilitate the elucidation of C5a-mediated pathophysiology. In this study, we determined the structure of C5a-C5aR1-Gi complex at a high resolution of 3 Å using cryo-electron microscopy (Cryo-EM). Our results revealed the binding site of C5a, which consists of a polar recognition region on the extracellular side and an amphipathic pocket within the transmembrane domain. Furthermore, we found that C5a binding induces conformational changes of C5aR1, which subsequently leads to the activation of G protein signaling pathways. Notably, a key residue (M265) located on transmembrane helix 6 (TM6) was identified to play a crucial role in regulating the recruitment of ß-arrestin driven by C5a. This study provides more information about the structure and function of the human C5a-C5aR1 complex, which is essential for the proper functioning of the complement system. The findings of this study can also provide a foundation for the design of new pharmaceuticals targeting this receptor with bias or specificity.

Whole-transcriptome defines novel glucose metabolic subtypes in colorectal cancer.

Colorectal cancer (CRC) is the most prevalent malignancy of the digestive system. Glucose metabolism plays a crucial role in CRC development. However, the heterogeneity of glucose metabolic patterns in CRC is not well characterized. Here, we classified CRC into specific glucose metabolic subtypes and identified the key regulators. 2228 carbohydrate metabolism-related genes were screened out from the GeneCards database, 202 of them were identified as prognosis genes in the TCGA database. Based on the expression patterns of the 202 genes, three metabolic subtypes were obtained by the non-negative matrix factorization clustering method. The C1 subtype had the worst survival outcome and was characterized with higher immune cell infiltration and more activation in extracellular matrix pathways than the other two subtypes. The C2 subtype was the most prevalent in CRC and was characterized by low immune cell infiltration. The C3 subtype had the smallest number of individuals and had a better prognosis, with higher levels of NRF2 and TP53 pathway expression. Secreted frizzled-related protein 2 (SFRP2) and thrombospondin-2 (THBS2) were confirmed as biomarkers for the C1 subtype. Their expression levels were elevated in high glucose condition, while their knockdown inhibited migration and invasion of HCT 116 cells. The analysis of therapeutic potential found that the C1 subtype was more sensitive to immune and PI3K-Akt pathway inhibitors than the other subtypes. To sum up, this study revealed a novel glucose-related CRC subtype, characterized by SFRP2 and THBS2, with poor prognosis but possible therapeutic benefits from immune and targeted therapies.

Tumour-associated macrophages and Schwann cells promote perineural invasion via paracrine loop in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is frequently accompanied by perineural invasion (PNI), which is associated with excruciating neuropathic pain and malignant progression. However, the relationship between PNI and tumour stromal cells has not been clarified. METHODS: The dorsal root ganglia or sciatic nerves nerve model was used to observe the paracrine interaction and the activation effect among Schwann cells, tumour-associated macrophages (TAMs), and pancreatic cancer cells in vitro. Next generation sequencing, enzyme-linked immunosorbent assay and chromatin immunoprecipitation were used to explore the specific paracrine signalling between TAMs and Schwann cells. RESULTS: We demonstrated that more macrophages were expressed around nerves that have been infiltrated by pancreatic cancer cells compared with normal nerves in murine and human PNI specimens. In addition, high expression of CD68 or GFAP is associated with an increased incidence of PNI and indicates a poor 5-year survival rate in patients with PDAC. Mechanistically, tumour-associated macrophages (TAMs) activate Schwann cells via the bFGF/PI3K/Akt/c-myc/GFAP pathway. Schwann cells secrete IL-33 to recruit macrophages into the perineural milieu and facilitate the M2 pro-tumourigenic polarisation of macrophages. CONCLUSIONS: Our study demonstrates that the bFGF/IL-33 positive feedback loop between Schwann cells and TAMs is essential in the process of PNI of PDAC. The bFGF/PI3K/Akt/c-myc/GFAP pathway would open potential avenues for targeted therapy of PDAC.

SCPL acyltransferases catalyze the metabolism of chlorogenic acid during purple coneflower seed germination.

The metabolism of massively accumulated chlorogenic acid is crucial for the successful germination of purple coneflower (Echinacea purpurea (L.) Menoch). A serine carboxypeptidase-like (SCPL) acyltransferase (chicoric acid synthase, CAS) utilizes chlorogenic acid to produce chicoric acid during germination. However, it seems that the generation of chicoric acid lags behind the decrease in chlorogenic acid, suggesting an earlier route of chlorogenic acid metabolism. We discovered another chlorogenic acid metabolic product, 3,5-dicaffeoylquinic acid, which is produced before chicoric acid, filling the lag phase. Then, we identified two additional typical clade IA SCPL acyltransferases, named chlorogenic acid condensing enzymes (CCEs), that catalyze the biosynthesis of 3,5-dicaffeoylquinic acid from chlorogenic acid with different kinetic characteristics. Chlorogenic acid inhibits radicle elongation in a dose-dependent manner, explaining the potential biological role of SCPL acyltransferases-mediated continuous chlorogenic acid metabolism during germination. Both CCE1 and CCE2 are highly conserved among Echinacea species, supporting the observed metabolism of chlorogenic acid to 3,5-dicaffeoylquinic acid in two Echinacea species without chicoric acid accumulation. The discovery of SCPL acyltransferase involved in the biosynthesis of 3,5-dicaffeoylquinic acid suggests convergent evolution. Our research clarifies the metabolism strategy of chlorogenic acid in Echinacea species and provides more insight into plant metabolism.

Preliminary analysis on the characteristics of light absorption coefficients in typical rivers of different river basins across China.

As a vital constituent of water's optical properties, the absorption coefficients influence the distribution of underwater light field, consequently impacting the structures and functional patterns of riverine ecosystems. In this study, the light absorption of non-algal particulates (ad(λ), m-1), phytoplankton (aph(λ), m-1) and CDOM (ag(λ), m-1) of 380 water samples collected from 133 rivers in eight external river basins across China from 2013 to 2023 were examined to determine the optical absorption characteristics. Results showed significant differences in ad(λ), aph(λ) and ag(λ) across different basins. ① The water bodies of eight basins can be categorized into 5 dominant types of absorption coefficients. ② In eastern China, ag(440) exhibited a northeast-high and southwest-low spatial distribution pattern. The Songliao River Basin had the highest ag(440) than other basins. The higher slope S of ag(λ) in rivers compared to lakes and reservoirs confirm river water primarily derive CDOM from external sources, distinguishing them from lakes and reservoirs. ③ The Huaihe and Haihe River Basins had higher ad(440) and aph(440) values, primarily due to lower terrain and human activities, leading to the accumulation of suspended particles and nutrients. And soil erosion from the Loess Plateau caused significant differences in ad(440) between the upper and middle reaches of the Yellow River Basin. These findings hold significant implications for understanding the optical characteristics of rivers in China.

Elevated serum IGFBP-1 levels correlate with cognitive deficits in treatment-resistant and chronic medicated schizophrenia patients.

BACKGROUND: Schizophrenia is a debilitating psychiatric disorder with diverse cognitive impairments. Insulin-like growth factor binding protein 1 (IGFBP-1), a ubiquitous negative regulator of IGF signaling, crosses the blood-brain barrier after peripheral synthesis. Given the crucial role of IGF signaling in cognitive function, we reasoned that altered serum IGFBP-1 concentrations might be associated with cognitive impairments in schizophrenia. To test this hypothesis, we examined the relationship between serum IGFBP-1 levels and cognitive performance in both medicated and treatment-resistant schizophrenia (TRS) patients. METHODS: Serum IGFBP-1 was measured in 31 TRS patients, 49 chronic medicated schizophrenia (CMS) patients, and 53 healthy controls. Clinical symptom severity was evaluated using the Positive and Negative Syndrome Scale (PANSS) and cognitive functions using the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS). RESULTS: Both TRS and CMS patients exhibited cognitive deficits compared to healthy controls (p < 0.05). Serum IGFBP-1 concentration differed significantly among groups (F=36.805, p < 0.001) and post hoc tests demonstrated significantly higher concentrations in both schizophrenia groups compared to controls (p < 0.001). Further, serum IGFBP-1 concentration was higher in the TRS group than the CMS group (p = 0.048). Correlation analysis identified a significant relationship between serum IGFBP-1 and attention in the TRS group (r = 0.411, p = 0.021), immediate memory in the CMS group (r = -0.417, p = 0.003), and RBANS total score in the CMS group (r = -0.368, p = 0.009). Multiple regression analysis adjusting for confounding factors revealed that serum IGFBP-1 was independently associated with attention in TRS patients (p = 0.016, 95 %CI. 0.002-0.015) and immediate memory in CMS patients (p = 0.022, 95 %CI-0.012 to -0.001). CONCLUSIONS: Elevated serum IGFBP-1 concentration may serve as a predictive biomarker for distinct cognitive deficits in TRS and CMS patients. Further investigations are warranted.

Chirped periodically poled lithium-niobate-on-insulator micro-waveguides for broadband second-harmonic generation.

Periodically poled lithium niobate (LN) waveguides based on quasi-phase matching schemes, benefiting from their high nonlinear coefficient (d33) and strong optical confinement, are widely employed for implementing efficient second-harmonic generation (SHG). Here, we report broadband SHG in z-cut chirped periodically poled lithium-niobate-on-insulator (CPPLNOI) ridge micro-waveguides. Nearly 90-nm-wide SHG at the telecom band is achieved, along with an averaged normalized efficiency of 7.5%/(W·cm2). We also demonstrate simultaneous generation of second as well as cascaded third and fourth harmonics under direct pumping of femtosecond pulses. This work would benefit applications for frequency conversion of a wideband coherent light source.

A FRET based ultrasensitive fluorescent aptasensor for 6'-sialyllactose detection.

As a kind of human milk oligosaccharide, 6'-sialyllactose (6'-SL) plays an important role in promoting infant brain development and improving infant immunity. The content of 6'-SL in infant formula milk powder is thus one of the important nutritional indexes. Since the lacking of efficient and rapid detection methods for 6'-SL, it is of great significance to develop specific recognition elements and establish fast and sensitive detection methods for 6'-SL. Herein, using 6'-SL specific aptamer as the recognition element, catalytic hairpin assembly as the signal amplification technology and quantum dots as the signal label, a fluorescence biosensor based on fluorescence resonance energy transfer (FRET) was constructed for ultra-sensitive detection of 6'-SL. The detection limit of this FRET-based fluorescent biosensor is 0.3 nM, and it has some outstanding characteristics such as high signal-to-noise ratio, low time-consuming, simplicity and high efficiency in the actual sample detection. Therefore, it has broad application prospect in 6'-SL detection.

Graphene oxide-enhanced colorimetric detection of Mec A gene based on toehold-mediated strand displacement.

Mec A, as a representative gene mediating resistance to ß-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA), allows a new genetic analysis for the detection of MRSA. Here, a sensitive, prompt, and visual colorimetry is reported to detect the Mec A gene based on toehold-mediated strand displacement (TMSD) and the enrichment effect of graphene oxide (GO). The Mec A triggers to generate the profuse amount of signal units of single-stranded DNA (SG) composed of a long single-stranded base tail and a base head: the tail can be adsorbed and enriched on the surface of GO; the head can form a G quadruplex structure to exert catalytic function towards 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid). Therefore, through the enrichment effect of GO, the signal units SG reflects different degrees of signal amplification on different substrates (such as aqueous solution or filter membrane). This strategy demonstrates a broad linear working range from 100 pM to 1.5 nM (solution) and 1 pM to 1 nM (filter membrane), with a low detection limit of 39.53 pM (solution) and 333 fM (filter membrane). Analytical performance in real samples suggests that this developed colorimetry is endowed with immense potential for clinical detection applications.

ACKR1+ Endothelial Cells Mediate Leukocyte Infiltration and Synergize with SFRP2/ASPN+ Fibroblasts to Promote Skin Fibrosis in Systemic Sclerosis.

BACKGROUND: Skin fibrosis is the most typical pathological manifestation of systemic sclerosis (SSc) and localized scleroderma (LS) with unclear etiology and few effective treatments. Though excessive collagen secretion by fibroblasts is the primary cause of skin fibrosis, many lines of evidence suggested that vascular damage was the initiating event and various cell types along with fibroblasts worked together to contribute to the pathogenesis of skin fibrosis. OBJECTIVES: We sought to explore the relationships between vascular endothelial cell lesions and immune cell infiltration, along with the cell-cell interactions among various cell types within the fibrotic skin ecosystem. METHODS: Single-cell RNA-seq (10x Genomics) was performed on skin biopsies of 3 healthy donors and 7 SSc patients in Chinese. The additional 3 localized scleroderma patients' data from NCBI database (GSE160536) were integrated by Harmony. CellChat package (v1.5.0) was applied to analyze cell communication network. Transwell assay and subcutaneous bleomycin (BLM) injection in mice were used to explore the role of ACKR1 on immune cell infiltration. Milo single-cell western blot was applied to show the activation of fibroblast subclusters. RESULTS: A total of 62,295 cells were obtained and subpopulations of stromal and immune cells were identified. Interaction network analysis revealed that multiple chemokines secreted by macrophages, pericytes, and pro-inflammatory fibroblasts could bind with Duffy antigen/receptor for chemokines (ACKR1), which is highly expressed on ACKR1+ endothelial cells of lesion skin. Transwell assay revealed that over-expressed ACKR1 in HUVEC facilitated leukocyte infiltration under the treatment of IL8. The BLM mice showed enhanced ACKR1 expression, massive immune cell infiltration, and fibrosis in skin, which could be attenuated by ACKR1 inhibition. Furthermore, infiltrated macrophages with TGFB1 or PDGFB high production could activate SFRP2/ASPN+ fibroblasts to contribute to excessive accumulation of extracellular matrix (ECM), and the SOX4-ASPN axis plays an important role in the TGF-ß signaling cascade and the etiology of skin fibrosis. CONCLUSIONS: Our results reveal that highly expressed ACKR1 in endothelial cells of fibrotic skin tissue promotes immune cell infiltration, and SFRP2/ASPN+ fibroblasts synergize to exacerbate skin fibrosis.