Cardiovascular disease (CVD) is the leading cause of death worldwide, especially in developed countries, and atherosclerosis (AS) is the common pathological basis of many cardiovascular diseases (CVDs) such as coronary heart disease (CHD). The role of the gut microbiota in AS has begun to be appreciated in recent years. Trimethylamine N-oxide (TMAO), an important gut microbe-dependent metabolite, is generated from dietary choline, betaine, and L-carnitine. Multiple studies have suggested a correlation between plasma TMAO levels and the risk of AS. However, the mechanism underlying this relationship is still unclear. In this review, we discuss the TMAO-involved mechanisms of atherosclerotic CVD from the perspective of inflammation, inflammation-related immunity, cholesterol metabolism, and atherothrombosis. We also summarize available clinical studies on the role of TMAO in predicting prognostic outcomes, including major adverse cardiovascular events (MACE), in patients presenting with AS. Finally, since TMAO may be a novel therapeutic target for AS, several therapeutic strategies including drugs, dietary, etc. to lower TMAO levels that are currently being explored are also discussed.

HighlightCabZIP63, indirectly activated by CaWRKY40, positively modulates transcription of CabZIP63 and CaWRKY40, enhances the binding of CaWRKY40 to its target promoters, and, therefore, increases resistance to Ralstonia solanacearum and thermotolerance.

Background: Esophageal squamous cell carcinoma (ESCC) is among the most prevalent causes of cancerrelated death in adults. Tumor microenvironment (TME) has been associated with therapeutic failure and lethal outcomes for patients. However, published reports on the heterogeneity and TME in ESCC are scanty. Methods: Five tumor samples and five corresponding non-malignant samples were subjected to scRNA-seq analysis. Bulk RNA sequencing data were retrieved in publicly available databases. Findings: From the scRNA-seq data, a total of 128,688 cells were enrolled for subsequent analyses. Gene expression and CNV status exhibited high heterogeneity of tumor cells. We further identified a list of tumorspecific genes and four malignant signatures, which are potential new markers for ESCC. Metabolic analysis revealed that energy supply-related pathways are pivotal in cancer metabolic reprogramming. Moreover, significant differences were found in stromal and immune cells between the esophagus normal and tumor tissues, which promoted carcinogenesis at both cellular and molecular levels in ESCC. Immune checkpoints, regarded as potential targets for immunotherapy in ESCC were significantly highly expressed in ESCC, including LAG3 and HAVCR2. Eventually, we constructed a cell-to-cell communication atlas based on cancer cells and immune cells and performed the flow cytometry, qRT-PCR, immunofluorescence, and immunohistochemistry analyses to validate the results. Interpretation: This study demonstrates a widespread reprogramming across multiple cellular elements within the TME in ESCC, particularly in transcriptional states, cellular functions, and cell-to-cell interactions. The findings offer an insight into the exploration of TME and heterogeneity in the ESCC and provide new therapeutic targets for its clinical management in the future.

Background Lung adenocarcinoma (LUAD) patients with different American Joint Committee on Cancer stages have different overall 5‐year survival rates. The tumor microenvironment (TME) and intra‐tumor heterogeneity (ITH) have been shown to play a crucial role in the occurrence and development of tumors. However, the TME and ITH in different lesions of LUAD have not been extensively explored. Methods We present a 204,157‐cell catalog of the TME transcriptome in 29 lung samples to systematically explore the TME and ITH in the different stages of LUAD. Traditional RNA sequencing data and complete clinical information were downloaded from publicly available databases. Results Based on these high‐quality cells, we constructed a single‐cell network underlying cellular and molecular features of normal lung, early LUAD, and advanced LUAD cells. In contrast with early malignant cells, we noticed that advanced malignant cells had a remarkably more complex TME and higher ITH level. We also found that compared with other immune cells, more differences in CD8+/CTL T cells, regulatory T cells, and follicular B cells were evident between early and advanced LUAD. Additionally, cell‐cell communication analyses, revealed great diversity between different lesions of LUAD at the single‐cell level. Flow cytometry and qRT‐PCR were used to validate our results. Conclusion Our results revealed the cellular diversity and molecular complexity of cell lineages in different stages of LUAD. We believe our research, which serves as a basic framework and valuable resource, can facilitate exploration of the pathogenesis of LUAD and identify novel therapeutic targets in the future.

As an early type of lung adenocarcinoma, ground glass nodule (GGN) has been detected increasingly and now accounts for most lung cancer outpatients. GGN has a satisfactory prognosis and its characteristics are quite different from solid adenocarcinoma (SADC). We compared the GGN adenocarcinoma (GGN-ADC) with SADC using the single-cell RNA sequencing (scRNA-seq) to fully understand GGNs. The tumor samples of five patients with lung GGN-ADCs and five with SADCs underwent surgery were digested to a single-cell suspension and analyzed using 10× Genomic scRNA-seq techniques. We obtained 60,459 cells and then classified them as eight cell types, including cancer cells, endothelial cells, fibroblasts, T cells, B cells, Nature killer cells, mast cells, and myeloid cells. We provided a comprehensive description of the cancer cells and stromal cells. We found that the signaling pathways related to cell proliferation were downregulated in GGN-ADC cancer cells, and stromal cells had different effects in GGN-ADC and SADC based on the analyses of scRNA-seq results. In GGN-ADC, the signaling pathways of angiogenesis were downregulated, fibroblasts expressed low levels of some collagens, and immune cells were more activated. Furthermore, we used flow cytometry to isolate the cancer cells and T cells in 12 GGN-ADC samples and in an equal number of SADC samples, including CD4+ T and CD8+ T cells, and validated the expression of key molecules by quantitative real-time polymerase chain reaction analyses. Through comprehensive analyses of cell phenotypes in GGNs, we provide deep insights into lung carcinogenesis that will be beneficial in lung cancer prevention and therapy.

CaWRKY40 is a positive regulator of pepper (Capsicum annum) response to Ralstonia solanacearum inoculation (RSI), but the underlying mechanism remains largely unknown. Here, we functionally characterize CaCDPK15 in the defense signaling mediated by CaWRKY40. Pathogen-responsive TGA, W, and ERE boxes were identified in the CaCDPK15 promoter (pCaCDPK15), and pCaCDPK15-driven GUS expression was significantly enhanced in response to RSI and exogenously applied salicylic acid, methyl jasmonate, abscisic acid, and ethephon. Virus-induced gene silencing (VIGS) of CaCDPK15 significantly increased the susceptibility of pepper to RSI and downregulated the immunity-associated markers CaNPR1, CaPR1, and CaDEF1. By contrast, transient CaCDPK15 overexpression significantly activated hypersensitive response associated cell death, upregulated the immunity-associated marker genes, upregulated CaWRKY40 expression, and enriched CaWRKY40 at the promoters of its targets genes. Although CaCDPK15 failed to interact with CaWRKY40, the direct binding of CaWRKY40 to pCaCDPK15 was detected by chromatin immunoprecipitation, which was significantly potentiated by RSI in pepper plants. These combined results suggest that RSI in pepper induces CaCDPK15 and indirectly activates downstream CaWRKY40, which in turn potentiates CaCDPK15 expression. This positive-feedback loop would amplify defense signaling against RSI and efficiently activate strong plant immunity.

SUMMARYMetformin and vitamin D 3 both exhibit a strong antiproliferative action in numerous cancer cell lines, including in human prostate cancer cells. Here we showed that the combination of the two drugs had a much stronger effect on DU145 human prostate cancer cell growth than either drug alone. In this research, cell proliferation was measured by methylthiazol tetrazolium (MTT) assay. Cell apoptosis was determined with Hoechst 33342 staining. Western blotting and cell cycle analyses were used to elucidate potential mechanisms of interaction between the drugs. It is shown that in cultured DU145 cells, vitamin D 3 combined with metformin exhibits synergistic effects on cell proliferation and apoptosis. The underlying antitumor mechanisms may involve altered cycle distribution with a G1/S cell cycle arrest, activation of phospho-AMPK with subsequent inhibition of downstream mTOR signalling pathway, down-regulate c-Myc expression, and reducing the level of anti-apoptotic protein p-Bcl-2. In conclusion, metformin and vitamin D 3 synergistically inhibit DU145 cell growth, indicating a promising clinical therapeutic strategy for the treatment of androgen-independent prostate cancer.

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