Almonertinib and alflutinib show novel inhibition on rare EGFR S768I mutant cells.

PURPOSE: EGFR classical mutations respond well to EGFR tyrosine kinase inhibitors. However, it is uncertain whether currently available EGFR-TKIs are effective against rare EGFR mutations and compound mutations. Herein, the effectiveness of almonertinib and alflutinib, the third-generation tyrosine kinase inhibitors developed in China, on rare EGFR S768I mutations and compound mutations is identified. METHODS: In this study, using CRISPR method, four EGFR S768I mutation cell lines were constructed, and the sensitivity of EGFR to almonertinib and alflutinib was tested, with positive controls being the 1st (gefitinib), 2nd (afatinib), and 3rd (osimertinib) generation drugs. RESULTS: The present results indicate that almonertinib and alflutinib can effectively inhibit cell viability and proliferation in rare EGFR S768I mutations through the ERK or AKT pathways in a time-dependent manner, by blocking the cell cycle and inhibiting apoptosis. CONCLUSIONS: These findings suggest that almonertinib and alflutinib may be potential therapeutic options for non-small cell lung cancer patients with the EGFR S768I mutation.

A novel MICA/B-targeted chimeric antigen receptor augments the cytotoxicity of NK cells against tumor cells.

Chimeric antigen receptor (CAR)-modified immune cells have emerged as a promising approach for cancer treatment, but single-target CAR therapy in solid tumors is limited by immune escape caused by tumor antigen heterogeneity and shedding. Natural killer group 2D (NKG2D) is an activating receptor expressed in human NK cells, and its ligands, such as MICA and MICB (MICA/B), are widely expressed in malignant cells and typically absent from healthy tissue. NKG2D plays an important role in anti-tumor immunity, recognizing tumor cells and initiating an anti-tumor response. Therefore, NKG2D-based CAR is a promising CAR candidate. Nevertheless, the shedding of MICA/B hinders the therapeutic efficacy of NKG2D-CARs. Here, we designed a novel CAR by engineering an anti-MICA/B shedding antibody 1D5 into the CAR construct. The engineered NK cells exhibited significantly enhanced cytotoxicity against various MICA/B-expressing tumor cells and were not inhibited by NKG2D antibody or NKG2D-Fc fusion protein, indicating no interference with NKG2D-MICA/B binding. Therefore, the developed 1D5-CAR could be combined with NKG2D-CAR to further improve the obstacles caused by MICA/B shedding.

FNeXter: A Multi-Scale Feature Fusion Network Based on ConvNeXt and Transformer for Retinal OCT Fluid Segmentation.

The accurate segmentation and quantification of retinal fluid in Optical Coherence Tomography (OCT) images are crucial for the diagnosis and treatment of ophthalmic diseases such as age-related macular degeneration. However, the accurate segmentation of retinal fluid is challenging due to significant variations in the size, position, and shape of fluid, as well as their complex, curved boundaries. To address these challenges, we propose a novel multi-scale feature fusion attention network (FNeXter), based on ConvNeXt and Transformer, for OCT fluid segmentation. In FNeXter, we introduce a novel global multi-scale hybrid encoder module that integrates ConvNeXt, Transformer, and region-aware spatial attention. This module can capture long-range dependencies and non-local similarities while also focusing on local features. Moreover, this module possesses the spatial region-aware capabilities, enabling it to adaptively focus on the lesions regions. Additionally, we propose a novel self-adaptive multi-scale feature fusion attention module to enhance the skip connections between the encoder and the decoder. The inclusion of this module elevates the model's capacity to learn global features and multi-scale contextual information effectively. Finally, we conduct comprehensive experiments to evaluate the performance of the proposed FNeXter. Experimental results demonstrate that our proposed approach outperforms other state-of-the-art methods in the task of fluid segmentation.

Macrophage-derived exosomal HMGB3 regulates silica-induced pulmonary inflammation by promoting M1 macrophage polarization and recruitment.

BACKGROUND: Chronic inflammation and fibrosis are characteristics of silicosis, and the inflammatory mediators involved in silicosis have not been fully elucidated. Recently, macrophage-derived exosomes have been reported to be inflammatory modulators, but their role in silicosis has not been explored. The purpose of the present study was to investigate the role of macrophage-derived exosomal high mobility group box 3 (HMGB3) in silica-induced pulmonary inflammation. METHODS: The induction of the inflammatory response and the recruitment of monocytes/macrophages were evaluated by immunofluorescence, flow cytometry and transwell assays. The expression of inflammatory cytokines was examined by RT-PCR and ELISA, and the signalling pathways involved were examined by western blot analysis. RESULTS: HMGB3 expression was increased in exosomes derived from silica-exposed macrophages. Exosomal HMGB3 significantly upregulated the expression of inflammatory cytokines, activated the STAT3/MAPK (ERK1/2 and p38)/NF-κB pathways in monocytes/macrophages, and promoted the migration of these cells by CCR2. CONCLUSIONS: Exosomal HMGB3 is a proinflammatory modulator of silica-induced inflammation that promotes the inflammatory response and recruitment of monocytes/macrophages by regulating the activation of the STAT3/MAPK/NF-κB/CCR2 pathways.

The SpyCatcher-SpyTag interaction mediates tunable anti-tumor cytotoxicity of NK cells.

Chimeric antigen receptor (CAR)-modified T and NK cell immunotherapy is a promising approach for cancer treatment. Due to the lack of tunability in anti-tumor activity, conventional CAR therapies have limited efficacy at low tumor antigen densities. To tune the CAR response to tumor cell surface antigens, we have developed a split CAR using the SpyCatcher-SpyTag system. The SpyCatcher serves as the ectodomain to constitute a SpyCatcher-CAR (SpyCAR), while SpyTag is attached to the antibodies that recognize tumor antigens. With dimerization mediated by SpyCatcher and SpyTag, the number and activation level of SpyCARs recruited by tumor antigens depends on the SpyTag number in the "antibody-SpyTag" fusion protein. The results demonstrated that the increasing number of SpyTags effectively enhanced the cytotoxicity of SpyCAR-NK92 cells against target cells. The development of SpyCAR with tunable cytotoxicity provides a novel strategy for CAR-based tumor immunotherapies.

Rigorous simulation model of double-Ronchi shearing interferometry on lithographic tools.

Double-Ronchi shearing interferometry is widely used in wavefront aberration measurements for advanced lithography projection lens systems. A rigorous simulation model of double-Ronchi shearing interferometry on lithographic tools is the precondition for phase-shifting retrieval algorithm design and error analysis. This paper presents a rigorous simulation model of double-Ronchi shearing interferometry considering the vector nature of light. The model is accurate and can be used in the study of double-Ronchi shearing interferometry.

Epithelial TIPE1 Protein Guards against Colitis by Inhibiting TNF-α-Mediated Inflammation.

Intestinal epithelial cells (IECs) at the internal/external interface orchestrate the mucosal immune response, and IEC dysfunction has been linked to multiple inflammatory diseases, including inflammatory bowel disease. In this study, we found that a member of the TNF-α-induced protein 8 (TNFAIP8 or TIPE) family called TIPE1 is indispensable for maintaining epithelial cell barrier integrity and homeostasis under inflammatory conditions. TIPE1-deficient mice, or chimeric mice that were deficient in TIPE1 in their nonhematopoietic cells, were more sensitive to dextran sulfate sodium-induced experimental colitis; however, TIPE1 deficiency had no impact on the development of inflammation-associated and sporadic colorectal cancers. Mechanistically, TIPE1 prevented experimental colitis through modulation of TNF-α-dependent inflammatory response in IECs. Importantly, genetic deletion of both TIPE1 and its related protein TNFAIP8 in mice led to the development of spontaneous chronic colitis, indicating that both of these two TIPE family members play crucial roles in maintaining intestinal homeostasis. Collectively, our findings highlight an important mechanism by which TIPE family proteins maintain intestinal homeostasis and prevent inflammatory disorders in the gut.

Intrinsic and extrinsic factors determining natural killer cell fate: Phenotype and function.

Natural killer (NK) cells are derived from hematopoietic stem cells. They belong to the innate lymphoid cell family, which is an important part of innate immunity. This family plays a role in the body mainly through the release of perforin, granzyme, and various cytokines and is involved in cytotoxicity and cytokine-mediated immune regulation. NK cells involved in normal immune regulation and the tumor microenvironment (TME) can exhibit completely different states. Here, we discuss the growth, development, and function of NK cells in regard to intrinsic and extrinsic factors. Intrinsic factors are those that influence NK cells to promote cell maturation and exert their effector functions under the control of internal metabolism and self-related genes. Extrinsic factors include the metabolism of the TME and the influence of related proteins on the "fate" of NK cells. This review targets the potential of NK cell metabolism, cellular molecules, regulatory genes, and other mechanisms involved in immune regulation. We further discuss immune-mediated tumor therapy, which is the trend of current research.

Lnc-PPP2R1B Mediates the Alternative Splicing of PPP2R1B by Interacting and Stabilizing HNRNPLL and Promotes Osteogenesis of MSCs.

Osteogeinc differentiation from mesenchymal stem cells (MSCs) into osteoblasts is a key step for bone tissue engineering in regenerative medicine. The insight into regulatory mechanism of osteogenesis of MSCs facilitates achieving better recovery effect. Long non-coding RNAs are regarded as a family of important moderators in osteogenesis. In this study, we found a novel lncRNA, lnc-PPP2R1B was up-regulated during osteogenesis of MSCs by Illumina HiSeq transcritome sequencing. We demonstrated lnc-PPP2R1B overexpression promoted osteogenesis and knockdown of lnc-PPP2R1B inhibited osteogenesis of MSCs. Mechanically, it physically interacted with and up-regulated heterogeneous nuclear ribonucleoprotein L Like (HNRNPLL), which is a master regulator of activation-induced alternative splicing in T cells. We found lnc-PPP2R1B knockdown or HNRNPLL knockdown decreased transcript-201 of Protein Phosphatase 2A, Regulatory Subunit A, Beta Isoform (PPP2R1B) while increased transcript-203 of PPP2R1B, and did not affect transcript-202/204/206. PPP2R1B is a constant regulatory subunit of protein phosphatase 2 (PP2A), which activates Wnt/ß-catenin pathway by removing phosphorylation and stabilization of ß-catenin and translocation into nucleus. The transcript-201 retained exon 2 and 3, compared to transcript-203. And it was reported the exon 2 and 3 of PPP2R1B were one part of B subunit binding domain on A subunit in PP2A trimer, and therefore retaining exon 2 and 3 promised formation and enzyme function of PP2A. Finally, lnc-PPP2R1B promoted ectopic osteogenesis in vivo. Conclusively, lnc-PPP2R1B mediated alternative splicing of PPP2R1B through retaining exon 2 and 3 by interacting with HNRNPLL and then promoted osteogenesis, which may facilitate an in-depth understanding of function and mechanism of lncRNAs in osteogenesis. Lnc-PPP2R1B interacted with HNRNPLL, and regulated alternative splicing of PPP2R1B through retaining exon 2 and 3, which preserved enzyme function of PP2A and enhanced dephosphorylation and nuclear translocation of ß-catenin, thereby promoting Runx2 and OSX expression and then osteogenesis. And it provided experimental data and potential target for promoting bone formation and bone regeneration.

GmEID1 modulates light signaling through the Evening Complex to control flowering time and yield in soybean.

Soybean (Glycine max) morphogenesis and flowering time are accurately regulated by photoperiod, which determine the yield potential and limit soybean cultivars to a narrow latitudinal range. The E3 and E4 genes, which encode phytochrome A photoreceptors in soybean, promote the expression of the legume-specific flowering repressor E1 to delay floral transition under long-day (LD) conditions. However, the underlying molecular mechanism remains unclear. Here, we show that the diurnal expression pattern of GmEID1 is opposite to that of E1 and targeted mutations in the GmEID1 gene delay soybean flowering regardless of daylength. GmEID1 interacts with J, a key component of circadian Evening Complex (EC), to inhibit E1 transcription. Photoactivated E3/E4 interacts with GmEID1 to inhibit GmEID1-J interaction, promoting J degradation resulting in a negative correlation between daylength and the level of J protein. Notably, targeted mutations in GmEID1 improved soybean adaptability by enhancing yield per plant up to 55.3% compared to WT in field trials performed in a broad latitudinal span of more than 24°. Together, this study reveals a unique mechanism in which E3/E4-GmEID1-EC module controls flowering time and provides an effective strategy to improve soybean adaptability and production for molecular breeding.

Engagement of an optimized lentiviral vector enhances the expression and cytotoxicity of CAR in human NK cells.

Adoptive chimeric antigen receptor (CAR)-modified T or NK cells (CAR-T/NK) have emerged as a novel form of disease treatment. Lentiviral vectors (LVs) are commonly employed to engineer NK cells for the efficient expression of CARs. This study reported the influence of single-promoter and dual-promoter LVs on the CAR expression and cytotoxicity of engineered NK cells. We constructed a third-generation NKG2D-based CAR that kills cancer cells by targeting up to eight stress-induced ligands (NKG2DLs). Our results demonstrated that the CAR exhibits both a higher expression level and a higher coexpression concordance with the GFP reporter in HEK-293T or NK92 cells by utilizing the optimized single-promoter pCDHsp rather than the original dual-promoter pCDHdp. After puromycin selection, the pCDHsp produces robust CAR expression and enhanced in vitro cytotoxicity of engineered NK cells. Therefore, infection with a single-promoter pCDHsp lentivector is recommended to prepare CAR-engineered NK cells. This research helps to optimize the production of CAR-NK cells and enhance their functional activity, to provide CAR-NK cell products with better and more uniform quality.

Macrophage derived miR-7219-3p-containing exosomes mediate fibroblast trans-differentiation by targeting SPRY1 in silicosis.

Silicosis is one of the most serious occupational diseases with the main feature of inflammatory cell infiltration, fibroblasts activation, and large deposition of extracellular matrix in the lung. Increasing evidence indicates that macrophage-derived exosomes may play an important role in the development of silicosis by transferring their loaded microRNAs (miRNAs). Hence we carried out high-throughput sequencing to identify the expression of exosomal miRNA from macrophages exposed to silica or not in the previous study. Then we verified that miR-7219-3p was significantly up-regulated in macrophages and their exosomes after silica-exposure, as well as in the silicotic mice model by qRT-PCR, subsequent experiments confirmed that the increase of miR-7219-3p facilitated fibroblast to myofibroblast trans-differentiation (FMT), as well as cell proliferation and migration. Spouty1 (SPRY1), which served as a negative modulator of the Ras/ERK/MAPK signaling pathway, was verified as the target gene of miR-7219-3p, the knockdown or over-expression of SPRY1 apparently promoted or inhibited FMT via the Ras/ERK/MAPK signaling pathway. Furthermore, the inhibition of exosomal miR-7219-3p partially suppressed FMT and silica-induced pulmonary fibrosis in vitro and in vivo. In brief, our results demonstrated that exosomal miR-7219-3p played an important role in FMT and might be a novel therapeutic target of silicosis.

Impact of urban innovation on urban green development in China's Yangtze River Economic Belt: perspectives of scale and network.

Understanding whether and how urban innovation offers a sound solution to the dilemma of urban green development is a crucial response to mitigate the detrimental effect on natural resources and environment for transitioning to sustainable urban development. To address the critical issue, we propose urban green development evaluation index system, and then examine how the urban innovation affects urban green development from the perspectives of government-scale, enterprise-scale, and spatial correlation network, all of which are originally applied in the 108 cities of Yangtze River Economic Belt of China (YREB) during period 2006-2018. The evaluation results show that urban innovation promotes urban green development, and both government-scale and enterprise-scale contribute to the effects. The constructed spatial correlation network of urban innovation illustrates the network structural form and reveals the network property, and further results tell that increasing network density and centrality would promote green development obviously. More specifically, the network density of urban innovation has been tied to the enhancement of urban green development, which is more significant in middle reaches than in lower and upper reaches of YREB. Similarly, optimizing the network's degree centrality and closeness centrality can help facilitate urban green development in whole YREB. Thus, the research findings would provide new insights into the essence and driving forces from various scale and hidden network when exploring and seeking urban green development path.

A chimeric switch-receptor PD1-DAP10-41BB augments NK92-cell activation and killing for human lung Cancer H1299 Cell.

Engineered natural killer (NK) cell-based therapies have been potentially broadly applicable and exhibited promising results in clinical trials, particularly in the fight against cancers. NK cell immunotherapy however always remains variable. One major obstacle is the inhibitory pathway including PD1/PDL1, providing tumor cells an escape mechanism from immunosurveillance. In this regard, we rationally designed a chimeric switch-receptor (CSR) PD1-DAP10-41BB, which comprising the ectodomain of PD1 fused to the co-stimulatory receptor DAP10 and 41BB. Therefore, by exchanging the transmembrane and cytoplasmic tail of PD1 with positive costimulatory molecules DAP10 and 41BB signaling domains, the negative PD1/PDL1 signal pathway was thus converted into a positive one. This CSR-expressing NK92 cells showed a typical parental NK92 phenotype and improved cytotoxicity against human lung cancer H1299 cells. Besides, the expression of CSR elicited a significant increase of effector molecules such as perforin and granzymes, which can induce apoptosis of H1299 cells. More importantly, in the solid tumor cell H1299-bearing mice model, the CSR-modified NK92 cells significantly inhibited tumor growth. Collectively, we demonstrated that expression of PD1-DAP10-41BB augmented NK92-cell activation and killing in vitro and in vivo, which provides a considerable avenue of using NK-tailored chimeric receptor engineered NK92 cells to treat a wide range of solid tumors.

In Vitro Nanobody Library Construction by Using Gene Designated-Region Pan-Editing Technology.

Camelid single-domain antibody fragments (nanobodies) are an emerging force in therapeutic biopharmaceuticals and clinical diagnostic reagents in recent years. Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting the large-scale application of nanobodies. In this study, we developed three kinds of gene designated-region pan-editing (GDP) technologies to introduce multiple mutations in complementarity-determining regions (CDRs) of nanobodies in vitro. Including the integration of G-quadruplex fragments in CDRs, which induces the spontaneous multiple mutations in CDRs; however, these mutant sequences are highly similar, resulting in a lack of sequences diversity in the CDRs. We also used CDR-targeting traditional gRNA-guided base-editors, which effectively diversify the CDRs. And most importantly, we developed the self-assembling gRNAs, which are generated by reprogrammed tracrRNA hijacking of endogenous mRNAs as crRNAs. Using base-editors guided by self-assembling gRNAs, we can realize the iteratively diversify the CDRs. And we believe the last GDP technology is highly promising in immunization-free nanobody library construction, and the full development of this novel nanobody discovery platform can realize the synthetic evolution of nanobodies in vitro.

Genetical engineering for NK and T cell immunotherapy with CRISPR/Cas9 technology: Implications and challenges.

Immunotherapy has become one of the most promising strategies in cancer therapies. Among the therapeutic alternatives, genetically engineered NK/T cell therapies have emerged as powerful and innovative therapeutic modalities for cancer patients with precise targeting and impressive efficacy. Nonetheless, this approach still faces multiple challenges, such as immunosuppressive tumor microenvironment, exhaustion of immune effector cells in tumors, off-target effects manufacturing complexity, and poor infiltration of effector cells, all of which need to be overcome for further utilization to cancers. Recently, CRISPR/Cas9 genome editing technology, with the goal of enhancing the efficacy and increasing the availability of engineered effector cell therapies, has shown considerable potential in the novel strategies and options to overcome these limitations. Here we review the current progress of the applications of CRISPR in cancer immunotherapy. Furthermore, we discuss issues related to the NK/T cell applications, gene delivery methods, efficiency, challenges, and implications of CRISPR/Cas9.

A Novel Risk Model Based on Lipid Metabolism-Associated Genes Predicts Prognosis and Indicates Immune Microenvironment in Breast Cancer.

BACKGROUND: Breast cancer (BRCA) is the most common tumor in women, and lipid metabolism involvement has been demonstrated in its tumorigenesis and development. However, the role of lipid metabolism-associated genes (LMAGs) in the immune microenvironment and prognosis of BRCA remains unclear. METHODS: A total of 1076 patients with BRCA were extracted from The Cancer Genome Atlas database and randomly assigned to the training cohort (n = 760) or validation cohort (n = 316). Kaplan-Meier analysis was used to assess differences in survival. Consensus clustering was performed to categorize the patients with BRCA into subtypes. Using multivariate Cox regression analysis, an LMAG-based prognostic risk model was constructed from the training cohort and validated using the validation cohort. The immune microenvironment was evaluated using the ESTIMATE and tumor immune estimation resource algorithms, CIBERSORT, and single sample gene set enrichment analyses. RESULTS: Consensus clustering classified the patients with BRCA into two subgroups with significantly different overall survival rates and immune microenvironments. Better prognosis was associated with high immune infiltration. The prognostic risk model, based on four LMAGs (MED10, PLA2G2D, CYP4F11, and GPS2), successfully stratified the patients into high- and low-risk groups in both the training and validation sets. High risk scores predicted poor prognosis and indicated low immune status. Subgroup analysis suggested that the risk model was an independent predictor of prognosis in BRCA. CONCLUSION: This study demonstrated, for the first time, that LMAG expression plays a crucial role in BRCA. The LMAG-based risk model successfully predicted the prognosis and indicated the immune microenvironment of patients with BRCA. Our study may provide inspiration for further research on BRCA pathomechanisms.

Photoelectric scanning-based resection method for mobile robot localization.

Indoor localization is a key enabling technology for mobile robot navigation in industrial manufacturing. As a distributed metrology system based on multi-station intersection measurement, the workshop measurement positioning system (wMPS) is gaining increasing attention in mobile robot localization. In this paper, a new, to the best of our knowledge, wMPS-based resection localization method is proposed using a single onmidirectional transmitter mounted on a mobile robot with scanning photoelectric receivers distributed in the work space. Compared to the traditional method that requires multiple stationary transmitters, our new method provides higher flexibility and cost-effectiveness. The position and orientation of the mobile robot are then iteratively optimized with respect to the constraint equations. In order to obtain the optimal solution rapidly, two methods of initial value determination are presented for different numbers of effective receivers. The propagation of the localization uncertainty is also investigated using Monte-Carlo simulations. Moreover, two experiments of automated guided vehicle localization are conducted, and the results demonstrate the high accuracy of the proposed method.

Macrophage-derived exosomes mediate silica-induced pulmonary fibrosis by activating fibroblast in an endoplasmic reticulum stress-dependent manner.

Macrophages play a key role in silicosis, and exosomes are potent mediators of intercellular communication. This suggests that macrophage-derived exosomes have a potential contribution to the pathogenesis of silicosis. To investigate whether macrophage-derived exosomes promote or inhibit lung fibrosis, in vitro, silica-exposed macrophage-derived exosomes (SiO2 -Exos) were collected and cocultured with fibroblasts. The expression of collagen I and α-SMA was evaluated. Furthermore, the endoplasmic reticulum (ER) stress markers BIP, XBP1s and P-eIF2α were assessed after treatment with or without the ER stress inhibitor 4-PBA. In vivo, mice were pre-treated with the exosome secretion inhibitor GW4869 prior to silica exposure. After sacrifice, lung tissues were histologically examined, and the expression of proinflammatory cytokines (TNF-α, IL-1ß and IL-6) in bronchoalveolar lavage fluid (BALF) was measured. The results showed that the expression of collagen I and α-SMA was up-regulated after treatment with SiO2 -Exos, accompanied by increased expression of BIP, XBP1s and P-eIF2α. Pre-treatment with 4-PBA reversed this effect. More importantly, an in vivo study demonstrated that pre-treatment with GW4869 decreased lung fibrosis and the expression of TNF-α, IL-1ß and IL-6 in BALF. These results suggested that SiO2 -Exos are profibrogenic and that the facilitating effect is dependent on ER stress.

Jones pupil decomposition and its lithographic imaging impacts.

The Jones pupil is a full description of imaging properties of projection lenses in optical lithography. The decomposition of the Jones pupil into components with clear physical meanings was studied previously; however, the decomposition method has not been studied systematically. To generalize the existing decomposition method, this work is aimed at finding all the decomposition methods and analyzing the lithographic imaging impacts. In this work, six decomposition methods are proposed, and the lithographic imaging impacts of the Jones components are studied and compared for all the decomposition methods. The results demonstrate that, although the decomposition methods are different, their lithographic impacts are identical. To be specific, apodization has a dominant impact on the critical dimension with a magnitude of 1.3 nm, and the impact of diattenuation is 0.3 nm. In contrast, the impacts of the other Jones components of aberration, birefringence, rotator, and ellipticity are negligible. This work gives a complete understanding of the imaging impacts of the Jones pupil.