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Abstract Small cell lung cancer (SCLC) is an aggressive pulmonary neuroendocrine malignancy featured by cold tumor immune microenvironment (TIME), limited benefit from immunotherapy, and poor survival. The spatial heterogeneity of TIME significantly associated with anti-tumor immunity has not been systemically studied in SCLC. We performed ultra-high-plex Digital Spatial Profiling on 132 tissue microarray cores from 44 treatment-naive limited-stage SCLC tumors. Incorporating single-cell RNA-sequencing data from a local cohort and published SCLC data, we established a spatial proteo-transcriptomic landscape covering over 18,000 genes and 60 key immuno-oncology proteins that participate in signaling pathways affecting tumorigenesis, immune regulation, and cancer metabolism across 3 pathologically defined spatial compartments (pan-CK-positive tumor nest; CD45/CD3-positive tumor stroma; para-tumor). Our study depicted the spatial transcriptomic and proteomic TIME architecture of SCLC, indicating clear intra-tumor heterogeneity dictated via canonical neuroendocrine subtyping markers; revealed the enrichment of innate immune cells and functionally impaired B cells in tumor nest and suggested potentially important immunoregulatory roles of monocytes/macrophages. We identified RE1 silencing factor (REST) as a potential biomarker for SCLC associated with low neuroendocrine features, more active anti-tumor immunity, and prolonged survival.

Abstract Our previous study revealed that PI3K/AKT/mTOR signaling was associated with SCLC radioresistance. SBC2 cells were used as primary radioresistance models, while H446 cells were continuously exposed to ionizing radiation (IR) to develop acquired radioresistance. Cell viability and apoptosis assays were used to investigate synergistic effects of BEZ235/GSK2126458 and IR in vitro, while immunoblotting, metabolite quantitative analysis and bioinformatic analyses were utilized to explore the underlying mechanism. Both genetically engineered mouse models (GEMM) and subcutaneous tumor models were used to confirm the synergistic effect in vivo. Key molecules of PI3K/AKT/mTOR signaling were upregulated after IR, which was correlated with primary radioresistance, and they were more expressed in acquired radioresistant cells. BEZ235/GSK2126458 effectively enhanced the cytotoxic effects of IR. BEZ235/GSK2126458 plus IR elevated γ-H2AX and p-Nrf2 expression, suggesting DNA and oxidative stress damage were intensified. Mechanistically, BEZ235/GSK2126458 plus IR significantly reduced the expression of G6PD protein, the rate-limiting enzyme of the pentose phosphate pathway (PPP). In detail, PI3K/mTOR inhibitors reinforced interaction between G6PD and HSPA8/HSC70, and G6PD was degraded by chaperone-mediated autophagy processes. Their metabolites (NADPH and R-5P) were decreased, and ROS levels were indirectly elevated, both of which exacerbated cell death. PI3K/AKT/mTOR signaling activator, insulin, enhanced SCLC radioresistance, while the synergistic effect of BEZ235/GSK2126458 and IR can be attenuated by N-acetylcysteine, and enhanced by 6-amino niacinamide. GEMM and allograft transplantation assays further confirmed their synergistic effect in vivo. This study provided insights into the connection between PI3K/AKT/mTOR signaling and the PPP underlying radioresistance and provided evidence of mechanisms supporting PI3K/mTOR inhibitors as possible therapeutic strategies to abrogate SCLC radioresistance.

Abstract Oncolytic viruses have recently been proven to be an effective and promising cancer therapeutic strategy, but there is rare data about oncolytic therapy in esophageal squamous cell carcinoma (ESCC), especially oncolytic measles virotherapy. Therefore, this study aimed to explore whether the recombinant measles virus vaccine strain rMV-Hu191 has an oncolytic effect against ESCC cells in vitro and in vivo and elucidate the underlying mechanisms. Our results showed that rMV-Hu191 could efficiently replicate in and kill ESCC cells through caspase-3/GSDME-mediated pyroptosis. Mechanistically, rMV-Hu191 triggers mitochondrial dysfunction to induce pyroptosis, which is mediated by BAK (BCL2 antagonist/killer 1) or BAX (BCL2 associated X). Further analysis revealed that rMV-Hu191 activates inflammatory signaling in ESCC cells, which may enhance the oncolytic efficiency. Moreover, intratumoral injection of rMV-Hu191 induced dramatic tumor regression in an ESCC xenograft model. Collectively, these findings imply that rMV-Hu191 exhibits an antitumor effect through BAK/BAX-dependent caspase-3/GSDME-mediated pyroptosis and provides a potentially promising new therapy for ESCC treatment.

Histopathology is a critical approach for diagnostic tasks and precision treatment. However, histopathological deep learning tools for auto‐identification remain poorly developed. Meanwhile, the interpretation of the computer vision attention into a cellular process is less efficient in a systematic way. Herein, it is identified that histone acetyltransferase 1 (HAT1) is an aging‐associated gene in the esophagus epithelium by machine learning. An interpretable deep learning model is developed to distinguish morphological changes with varied HAT1 expressions in esophageal squamous carcinoma cells (ESCC). The gradient‐weighted class activation mapping and prediction score analysis reveal that the computer's vision focuses on the nuclear sizes of ESCC. The hypothesized phenotype is verified in HAT1‐knockdown ESCCs. Finally, HAT1 regulating cell senescence by affecting the H3K27 acetylation and E2F transcription factor 7 (E2F7) expression is shown. Herein, the feasibility and benefits of applying histopathological deep learning assistance systems in routine practice scenarios and connecting phenotype and genotype for further genetic research are suggested.

Abstract Necroptotic cell death is mediated by a super-molecular complex called necrosome which consists of receptor-interacting protein kinase 1 and 3 (RIPK1, RIPK3) and mixed-lineage kinase domain-like protein (MLKL). The role of these kinases has been extensively investigated in the regulation of necroptosis. However, whether the protein phosphatase is involved in necroptosis is still largely unknown. Here, we identified protein phosphatase 6 catalytic subunit (PPP6C) promotes TNF-induced necroptosis by genome-wide CRISPR/Cas9 library screening. We found that PPP6C deficiency protects cells from TNF-induced necroptosis in a phosphatase-activity-dependent manner. Mechanistically, PPP6C acts as a TGF-β activated kinase 1 (TAK1) phosphatase to inactivate its kinase activity. Deletion of PPP6C leads to hyperactivation of TAK1 and reduced RIPK1 kinase activity upon TNF stimulation. We further showed that heterozygous deletion of Ppp6c in mouse gastrointestinal tract alleviates necroptosis-related tissue injury and inflammation. Thus, our study identifies PPP6C as an important regulator of necroptosis and highlights a central role of phosphatase in the regulation of necroptosis-related diseases.

BackgroundTo provide a reference for hospital drug selection and rational clinical drug selection based on the evaluation of the safety, nutritional quality, and economy of 27 manufacturers of five varieties (18AA, 18AA-I, 18AA-II, 18AA-IV, 18AA-V) of balanced compound amino acids for injection and (18AA-IIoriginal research).MethodsThe safety of compound amino acids for injection was evaluated by comparing the antioxidant sulfite contents. Based on the amino acid scoring standard mode and the whole egg protein mode as proposed by the Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO) in 1973, we compared the formula. The first limiting amino acid content and the comprehensive quality of the total essential amino acid (EAA) contents of the six formulations were studied. The price/content ratio was used to evaluate their economy.ResultsSimilar variety produced by different manufacturers have the same formula and contents of balanced compound amino acids for injection. Safety: 18AA-IIoriginal research and 18AA-II had the lowest sulfite content. Compared with 18AA-IIoriginal research, the sulfite content of 18AA-I, 18AA, 18AA-V, and 18AA-IV were higher (10 times, 16.67 times, 16.67 times, and 33.33 times, respectively). The lower the sulfite content, the safer the product. Nutritional quality: The proportions of amino acids in the five varieties of compound amino acid injection were all suitable. The order of the first limiting amino acids for the formulations was 18AA-IIoriginal research = 18AA-II>18AA >18AA-I = 18AA-IV>18AA-V. The order of the EAA values for the formulations was 18AA-IIoriginal research = 18AA-II>18AA>18AA-I > 18AA-IV > 18AA-V. The overall effectiveness order was 18AA-IIoriginal research = 18AA-II>18AA > 18AA-I>18AA-IV>18AA-V. Economy: Among the 27 manufacturers, 12 manufacturers had a price/content ratio higher than that of 18AA-II original research manufacturers, and 15 manufacturers had a price/content ratio lower than original research manufacturers.ConclusionThrough its security, effectiveness, and economy of the comprehensive research, we recommended 18AA-II and 18AA-IIoriginal research with high safety, efficacy, and reasonable price as the first choice. 18AA and 18AA-I with better safety and reasonable price, secondary recommendation. 18AA-IV or 18AA-V with poor safety, efficacy, and economy are not recommended.

Due to moisture in the ore, auxiliary material, and ladle refractory material, the hydrogen element is easily enriched in molten steel. In the metallurgy process, some hydrogen atoms form bubbles and are removed by gravity, whearas others solidify in the strand and remain in the produced steel. When the hydrogen content reaches a certain critical value, the enriched hydrogen atoms congregate to produce a white spot, which greatly reduces the strength and toughness of the steel product, and leads to brittle fracture during its service period. At present, the RH (Ruhrstahl–Heraeus) and VD (vacuum degasser) refining processes are commonly applied in steel plants, which can reduce the hydrogen content to less than 2×10−6. With the demand for high quality steel, the hydrogen content must be further decreased, so hydrogen diffusion in solid steel during the annealing process is gradually attracting increasing attention. In this study, a two-dimensional model was built to investigate the characteristic of dehydrogenation in the bloom annealing process of rail steel. Moreover, the effect of annealing temperature and annealing time on hydrogen diffusion were analyzed, and the annealing parameters were optimized. During the dehydrogenation annealing process, the hydrogen content at the corners and edges of the bloom are found to decrease rapidly, while that in the center of the strand begin to decrease in the later heating stage. As the annealing temperature increases, the starting point of dehydrogenation in the bloom center moves ahead and the maximum dehydrogenation rate increases significantly. With the extension of the soaking period, the central hydrogen content of bloom decreases significantly, but the increase rate of the dehydrogenation gradually decreases. By optimizing the bloom annealing parameters, the hydrogen content in the bloom can be steadily reduced to 0.6×10−6, which fully meets the requirement of high quality steel production.

Glioblastoma (GBM) is the most common and aggressive brain tumor in adults. Overexpression or activation of epidermal growth factor receptor (EGFR) occurs commonly in multiple human cancers and promotes tumorigenesis. However, the underlying molecular mechanism of EGFR aberrant activation and the downstream signaling pathways remains largely unknown. In this study, we report that both SH3-domain kinase binding protein 1 (SH3KBP1) mRNA and protein levels are highly expressed in GBM and its high expression is associated with worse survival of glioma patients. In addition, we provide evidence that SH3KBP1 is prominently expressed in GBM stem cells (GSCs) and have potential to serve as a novel GSCs marker. Moreover, silencing SH3KBP1 dramatically impairs GBM cell proliferation, migration and GSCs self-renewal ability in vitro and xenograft tumors growth in vivo. Most importantly, we found that SH3KBP1 directly interacts with EGFR and may act as an adaptor protein to transduce EGFR signaling. Together, our work uncovers SH3KBP1 as a novel regulator of oncogenic EGFR signaling and also as a potential therapeutic target for GBM patients with EGFR activation.

The Hippo-YAP pathway responds to diverse environmental cues to manage tissue homeostasis, organ regeneration, tumorigenesis, and immunity. However, how phosphatase(s) directly target Yes-associated protein (YAP) and determine its physiological activity are still inconclusive. Here, we utilized an unbiased phosphatome screening and identified protein phosphatase magnesium-dependent 1A (PPM1A/PP2Cα) as the bona fide and physiological YAP phosphatase. We found that PPM1A was associated with YAP/TAZ in both the cytoplasm and the nucleus to directly eliminate phospho-S127 on YAP, which conferring YAP the nuclear distribution and transcription potency. Accordingly, genetic ablation or depletion of PPM1A in cells, organoids, and mice elicited an enhanced YAP/TAZ cytoplasmic retention and resulted in the diminished cell proliferation, severe gut regeneration defects in colitis, and impeded liver regeneration upon injury. These regeneration defects in murine model were largely rescued via a genetic large tumor suppressor kinase 1 (LATS1) deficiency or the pharmacological inhibition of Hippo-YAP signaling. Therefore, we identify a physiological phosphatase of YAP/TAZ, describe its critical effects in YAP/TAZ cellular distribution, and demonstrate its physiological roles in mammalian organ regeneration.

Shear stress exerted by the blood stream modulates endothelial functions through altering gene expression. KLF2 and KLF4, the mechanosensitive transcription factors, are promoted by laminar flow to maintain endothelial homeostasis. However, how the expression of KLF2/4 is regulated by shear stress is poorly understood. Here, we showed that the activation of PIEZO1 upregulates the expression of KLF2/4 in endothelial cells. Mice with endothelial-specific deletion of Piezo1 exhibit reduced KLF2/4 expression in thoracic aorta and pulmonary vascular endothelial cells. Mechanistically, shear stress activates PIEZO1, which results in a calcium influx and subsequently activation of CaMKII. CaMKII interacts with and activates MEKK3 to promote MEKK3/MEK5/ERK5 signaling and ultimately induce the transcription of KLF2/4. Our data provide the molecular insight into how endothelial cells sense and convert mechanical stimuli into a biological response to promote KLF2/4 expression for the maintenance of endothelial function and homeostasis.

Inflammation can promote cartilage degradation by inducing matrix-degrading enzymes via NF-κB. Here the authors uncover reciprocal inhibition of Yap/Taz and NF-κB signaling though TAK1, and identify Yap activity as critical for maintenance of cartilage integrity in a mouse model of osteoarthritis.

Summary: Intracellular transport of membranous organelles and protein complexes to various destinations is fundamental to signaling transduction and cellular function. The cytoplasmic dynein motor and its regulatory proteins LIS1 and NDE1 are required for transporting a variety of cellular cargos along the microtubule network. In this study, we show that deletion of Lis1 in developing lung endoderm and limb mesenchymal cells causes agenesis of the lungs and limbs. In both mutants, there is increased cell death and decreased fibroblast growth factor (FGF) signaling activity. Mechanistically, LIS1 and its interacting protein NDE1/NDEL1 are associated with FGF receptor-containing vesicles and regulate FGF receptor intracellular trafficking and degradation. Notably, FGF signaling promotes NDE1 tyrosine phosphorylation, which leads to dissociation of LIS1/NDE1 complex. Thus, our studies identify the LIS1/NDE1 complex as an important FGF signaling regulator and provide insights into the bidirectional regulation of cell signaling and transport machinery for endocytosis. : Liu et al. show that Lis1 is required for mouse embryonic lung and limb formation. The LIS1/NDE1/dynein complex regulates FGF receptor trafficking and stability. FGF signaling promotes NDE1 tyrosine phosphorylation, which leads to dissociation of the LIS1/NDE1 complex, suggesting a bidirectional regulation of cell signaling and transport machinery. Keywords: LIS1, NDE1, dynein, FGF signaling, development, endocytosis

Antibodies are a type of protein produced by active B cells in response to antigen stimulation. A series of monoclonal antibodies and neutralizing antibodies have been invented and put into clinical use because of their high therapeutic effect and bright developing insight. Patients with cancer, infectious diseases, and autoimmune diseases can all benefit from antibody therapy. However, the targeting aspects and potential mechanisms for treating these diseases differ. In the treatment of patients with infectious diseases such as COVID-19, neutralizing antibodies have been proposed as reliable vaccines against COVID-19, which target the ACE2 protein by preventing virus entry into somatic cells. Monoclonal antibodies can target immune checkpoints (e.g., PD-L1 and CTLA-4), tyrosine kinase and subsequent signaling pathways (e.g., VEGF), and cytokines in cancer patients (e.g. IL-6 and IL-1β). It is debatable whether there is any connection between the use of antibodies in these diseases. It would be fantastic to discover the related points and explain the burden for the limitation of cross-use of these techniques. In this review, we provided a comprehensive overview of the use of antibodies in the treatment of infectious disease and cancer patients. There are also discussions of their mechanisms and history. In addition, we discussed our future outlook on the use of antibodies.

Heart valve disease is a major clinical problem worldwide. Cardiac valve development and homeostasis need to be precisely controlled. Hippo signaling is essential for organ development and tissue homeostasis, while its role in valve formation and morphology maintenance remains unknown. VGLL4 is a transcription cofactor in vertebrates and we found it was mainly expressed in valve interstitial cells at the post-EMT stage and was maintained till the adult stage. Tissue specific knockout of VGLL4 in different cell lineages revealed that only loss of VGLL4 in endothelial cell lineage led to valve malformation with expanded expression of YAP targets. We further semi-knockout YAP in VGLL4 ablated hearts, and found hyper proliferation of arterial valve interstitial cells was significantly constrained. These findings suggest that VGLL4 is important for valve development and manipulation of Hippo components would be a potential therapy for preventing the progression of congenital valve disease.

AIM: To explore the neuroprotective effects of high mobility group box 2 (HMGB2) knockdown on retinal ganglion cells (RGCs) in the retinal ischemia-reperfusion injury (RIRI). METHODS: Oxygen-glucose deprivation (OGD)-injured RGCs from postnatal three-day C57BL/6 mice pups and high intraocular pressure (IOP)-induced RIRI mice were used as cellular and animal models of RIRI. The expression of HMGB2 in the retina of RIRI mice and OGD-injured RGCs was detected through reverse transcription-polymerase chain reaction (RT-qPCR) and Western blotting. The effects of HMGB2 silencing on the morphological changes, RGCs survival, and cell apoptosis in mouse retinal tissues were observed through H&E staining, immunofluorescence staining with RNA-binding protein with multiple splicing (RBPMS) antibody, and TUNEL staining, respectively. RGC viability and apoptosis were examined by CCK-8 and flow cytometry assays. The levels of proteins associated with NOD-like receptor thermal protein domain associated protein 3 (NLRP3)-mediated pyroptosis [NLRP3, Caspase-1, GSDMD-N, interleukin (IL)-1β, IL-18] in vivo and in vitro were measured by Western blotting. RESULTS: HMGB2 protein and NLRP3 were upregulated in the retina of RIRI mice and OGD-injured RGCs (P

Hippo signaling controls organ size and tissue regeneration in many organs, but its roles in chondrocyte differentiation and bone repair remain elusive. Here, we demonstrate that Yap1, an effector of Hippo pathway inhibits skeletal development, postnatal growth, and bone repair. We show that Yap1 regulates chondrocyte differentiation at multiple steps in which it promotes early chondrocyte proliferation but inhibits subsequent chondrocyte maturation both in vitro and in vivo. Mechanistically, we find that Yap1 requires Teads binding for direct regulation of Sox6 expression to promote chondrocyte proliferation. In contrast, Yap1 inhibits chondrocyte maturation by suppression of Col10a1 expression through interaction with Runx2. In addition, Yap1 also governs the initiation of fracture repair by inhibition of cartilaginous callus tissue formation. Taken together, our work provides insights into the mechanism by which Yap1 regulates endochondral ossification, which may help the development of therapeutic treatment for bone regeneration.

Objective Hyperbaric oxygen (HBO) is an emerging complementary alternative medical approach in glioma treatment. However, its mode of action is unknown, so this was investigated in the present study. Methods We constructed an intracranial glioma model of congenic C57BL/6J mice. Glioma growth under HBO stimulation was assessed by bioluminescent imaging and magnetic resonance imaging. Flow cytometry assessed direct effects of HBO on reactive oxygen species (ROS) signaling of transplanted glioma cells and organs, and quantified mature T cells and subgroups in tumors, the brain, and blood. Results HBO promoted the growth of transplanted GL261-Luc glioma in the intracranial glioma mouse model. ROS signaling of glioma cells and brain cells was significantly downregulated under HBO stimulation, but thymus ROS levels were significantly upregulated. CD3+ T cells were significantly downregulated, while both Ti/Th cells (CD3+CD4+) and Ts/Tc cells (CD3+CD8+) were inhibited in tumors of the HBO group. The percentage of regulatory T cells in Ti/Th (CD3+CD4+) cells was elevated in the tumors and thymuses of the HBO group. Conclusion HBO induced ROS signaling in the thymus, inhibited CD3+ T cell generation, and facilitated malignant glioma cell growth in vivo in the intracranial glioma mouse model.

An anthracene aromatic unit was introduced into the phenylethynyl structure by a rigid acetylene linkage at the C-9 and C-10 positions via Sonogashira coupling reactions, resulting in a planar and straight-backbone molecule (9,10-bis((4-((3,7-dimethyloctyl)oxy) phenyl) ethynyl) anthracene) (BPEA). Thermogravimetric analysis demonstrated the good thermal stability of the BPEA. Photoluminescence analysis showed that a suitable expanded π-conjugation in the BPEA made its excitation band extend into the visible region, and an intense green emission was observed under blue-light excitation. A bright green light-emitting diode with an efficiency of 18.22 lm/w was fabricated by coating the organic phosphor onto a 460 nm-emitting InGaN chip. All the results indicate that BPEA is a useful green-emitting material which is efficiently excited by blue light, and therefore, that it could be applied in many fields without UV radiation.

Identifying cells of tumor origin is a fundamental question in tumor biology. Answers to this central question will not only advance our understanding of tumor initiation and progression but also have important therapeutic implications. In this study, we aimed to uncover the cells of origin of lung adenocarcinoma, a major subtype of non-small cell lung cancer. To this end, we developed new mouse models of lung adenocarcinoma that enabled selective manipulation of gene activity in surfactant associated protein C (SPC)-expressing cells, including alveolar type II cells and bronchioalveolar stem cells (BASCs) that reside at the bronchioalveolar duct junction (BADJ). Our findings showed that activation of oncogenic Kras alone or in combination with the removal of the tumor suppressor p53 in SPC⁺ cells resulted in development of alveolar tumors. Similarly, sustained EGF signaling in SPC⁺ cells led to alveolar tumors. By contrast, BASCs failed to proliferate or produce tumors under these conditions. Importantly, in a mouse strain in which Kras/p53 activity was selectively altered in type II cells but not BASCs, alveolar tumors developed while BADJs retained normal architecture. These results confirm and extend previous findings and support a model in which lung adenocarcinoma can initiate in alveolar type II cells. Our results establish the foundation for elucidating the molecular mechanisms by which lung cancer initiates and progresses in a specific lung cell type.

The Clinical Assessment Interview for Negative Symptoms (CAINS) was designed in accordance with the recent theory and research in social affective neuroscience and to address the psychometric and conceptual limitations of other instruments assessing negative symptoms. The present study aimed to provide a large-scale validation of the CAINS in China and examine its applicability and validity evidence across the schizophrenia spectrum. Using confirmatory factor analysis, our results replicated the original findings in the US development samples that the CAINS possesses a stable 2-factor structure, namely "motivation/pleasure" and "expression". We also found significant correlations between the CAINS and other negative symptom measures. The CAINS demonstrated good discriminant validity in differentiating negative symptoms in people with schizophrenia, nonpsychotic first-degree relatives and people with social anhedonia. People with schizophrenia exhibited significantly higher CAINS subscale scores than first-degree relatives and healthy controls. In addition, first-degree relatives had higher "motivation/pleasure" scores than healthy controls. The "motivation/pleasure" subscale scores of individuals with social anhedonia were also significantly higher than healthy controls.