Transcriptome and metabolome analysis of the developmental changes in Cynanchum thesioides anther.

Cynanchum thesioides, a xerophytic species utilized both as a medicinal herb and a food source, plays a significant role in arid and desert ecosystem management. Its inflorescence is an umbellate cyme, each carrying nearly a thousand flowers; however, its fruiting rate remains remarkably low. The normal development of the anther is a necessary prerequisite for plants to produce seeds. However, our understanding of the anther development process in Cynanchum thesioides remains limited. To better understand the pollen development process in Cynanchum thesioides, the stages of pollen development were determined through paraffin sectioning, and observations were made on the distribution characteristics of polysaccharides and lipid droplets in the pollen development of Cynanchum thesioides using Periodic Acid-Schiff stain (PAS) and 0.5% Sudan Black B tissue staining. Concurrently, the gene expression patterns and metabolite profiles were delineated across various developmental stages of Cynanchum thesioides anthers (T1: microspore stage, T2: tetrad stage, T3: mononuclear stage, and T4: maturation stage). The findings revealed that Cynanchum thesioides pollen is in an aggregate form. Polysaccharides gradually accumulate during maturation and lipid droplets form a surrounding membrane, thereby preventing pollen dispersion. Furthermore, transcriptomic and metabolomic analyses across distinct developmental phases uncovered a plethora of differentially expressed genes and metabolites associated with the flavonoid biosynthesis pathway. Flavonoid levels exhibited dynamic changes concurrent with anther development, aligning with the gene regulatory patterns of the corresponding biosynthetic pathways. The study identified 63 differentially accumulated flavonoid compounds and 21 differentially expressed genes associated with flavonoid biosynthesis. Weighted gene co-expression network analysis revealed six MYB and ten bHLH transcription factors as key candidates involved in flavonoid biosynthesis, with CtbHLH (Cluster-6587.1050) and CtMYB (Cluster-6587.31743) specifically regulating structural genes within the pathway. These findings underscore the pivotal role of flavonoid biosynthesis in anther development of Cynanchum thesioides. In conclusion, this research offers a comprehensive insight into the anther development process in Cynanchum thesioides.

ADT-OH exhibits anti-metastatic activity on triple-negative breast cancer by combinatorial targeting of autophagy and mitochondrial fission.

High basal autophagy and enhanced mitochondrial fission in triple-negative breast cancer (TNBC) cells support cell migration and promote plasticity of cancer cell metabolism. Here, we suggest a novel combination therapy approach for the treatment of TNBC that targets Drp1-mediated mitochondrial fission and autophagy pathways. Hydrogen sulfide (H2S) mediates a myriad of biological processes, including autophagy and mitochondrial function. In this study, we demonstrated that 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), one of the most widely utilized sustained-release H2S donors, effectively suppresses metastasis of TNBC cells in the absence of proliferation inhibition in vitro and in vivo. ADT-OH treatment ameliorated autophagy flux by suppressing autophagosome formation and induced mitochondrial elongation through decreasing expression of dynamin-related protein 1 (Drp1) and increasing expression of mitochondrial fusion protein (Mfn2). At the same time, ADT-OH downregulated mitophagy flux and inhibited mitochondrial function, eventually leading to the inhibition of migration and invasion in TNBC cells. In vivo, intraperitoneal administration of ADT-OH revealed a potent anti-metastatic activity in three different animal models, the MDA-MB-231 orthotopic xenograft model, the 4T1-Luci orthotopic model and the 4T1-Luci tail vein metastasis model. However, ADT-OH has an extremely low water solubility, which is a significant barrier to its effectiveness. Thus, we demonstrated that the solubility of ADT-OH in water can be improved significantly by absorption with hydroxypropyl-ß-cyclodextrin (CD). Remarkably, the obtained CD-ADT-OH demonstrated superior anti-cancer effect to ADT-OH in vivo. Altogether, this study describes a novel regulator of mammalian mitochondrial fission and autophagy, with potential utility as an experimental therapeutic agent for metastatic TNBC.

Camouflaging attenuated Salmonella by cryo-shocked macrophages for tumor-targeted therapy.

Live bacteria-mediated antitumor therapies mark a pivotal point in cancer immunotherapy. However, the difficulty in reconciling the safety and efficacy of bacterial therapies has limited their application. Improving bacterial tumor-targeted delivery while maintaining biosafety is a critical hurdle for the clinical translation of live microbial therapy for cancer. Here, we developed "dead" yet "functional" Salmonella-loaded macrophages using liquid nitrogen cold shock of an attenuated Salmonella typhimurium VNP20009-contained macrophage cell line. The obtained "dead" macrophages achieve an average loading of approximately 257 live bacteria per 100 cells. The engineered cells maintain an intact cellular structure but lose their original pathogenicity, while intracellular bacteria retain their original biological activity and are delay freed, followed by proliferation. This "Trojan horse"-like bacterial camouflage strategy avoids bacterial immunogenicity-induced neutrophil recruitment and activation in peripheral blood, reduces the clearance of bacteria by neutrophils and enhances bacterial tumor enrichment efficiently after systemic administration. Furthermore, this strategy also strongly activated the tumor microenvironment, including increasing antitumor effector cells (including M1-like macrophages and CD8+ Teffs) and decreasing protumor effector cells (including M2-like macrophages and CD4+ Tregs), and ultimately improved antitumor efficacy in a subcutaneous H22 tumor-bearing mouse model. The cryo-shocked macrophage-mediated bacterial delivery strategy holds promise for expanding the therapeutic applications of living bacteria for cancer.

ADT-OH improves intestinal barrier function and remodels the gut microbiota in DSS-induced colitis.

Owing to the increasing incidence and prevalence of inflammatory bowel disease (IBD) worldwide, effective and safe treatments for IBD are urgently needed. Hydrogen sulfide (H2S) is an endogenous gasotransmitter and plays an important role in inflammation. To date, H2S-releasing agents are viewed as potential anti-inflammatory drugs. The slow-releasing H2S donor 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), known as a potent therapeutic with chemopreventive and cytoprotective properties, has received attention recently. Here, we reported its anti-inflammatory effects on dextran sodium sulfate (DSS)-induced acute (7 days) and chronic (30 days) colitis. We found that ADT-OH effectively reduced the DSS-colitis clinical score and reversed the inflammation-induced shortening of colon length. Moreover, ADT-OH reduced intestinal inflammation by suppressing the nuclear factor kappa-B pathway. In vivo and in vitro results showed that ADT-OH decreased intestinal permeability by increasing the expression of zonula occludens-1 and occludin and blocking increases in myosin II regulatory light chain phosphorylation and epithelial myosin light chain kinase protein expression levels. In addition, ADT-OH restored intestinal microbiota dysbiosis characterized by the significantly increased abundance of Muribaculaceae and Alistipes and markedly decreased abundance of Helicobacter, Mucispirillum, Parasutterella, and Desulfovibrio. Transplanting ADT-OH-modulated microbiota can alleviate DSS-induced colitis and negatively regulate the expression of local and systemic proinflammatory cytokines. Collectively, ADT-OH is safe without any short-term (5 days) or long-term (30 days) toxicological adverse effects and can be used as an alternative therapeutic agent for IBD treatment.

Transcriptome-wide identification of WRKY transcription factors and their expression profiles under different stress in Cynanchum thesioides.

Cynanchum thesioides (Freyn) K. Schum. is an important economic and medicinal plant widely distributed in northern China. WRKY transcription factors (TFs) play important roles in plant growth, development and regulating responses. However, there is no report on the WRKY genes in Cynanchum thesioides. A total of 19 WRKY transcriptome sequences with complete ORFs were identified as WRKY transcriptome sequences by searching for WRKYs in RNA sequencing data. Then, the WRKY genes were classified by phylogenetic and conserved motif analysis of the WRKY family in Cynanchum thesioides and Arabidopsis thaliana. qRT-PCR was used to determine the expression patterns of 19 CtWRKY genes in different tissues and seedlings of Cynanchum thesioides under plant hormone (ABA and ETH) and abiotic stresses (cold and salt). The results showed that 19 CtWRKY genes could be divided into groups I-III according to their structure and phylogenetic characteristics, and group II could be divided into five subgroups. The prediction of CtWRKY gene protein interactions indicates that CtWRKY is involved in many biological processes. In addition, the CtWRKY gene was differentially expressed in different tissues and positively responded to abiotic stress and phytohormone treatment, among which CtWRKY9, CtWRKY18, and CtWRKY19 were significantly induced under various stresses. This study is the first to identify the WRKY gene family in Cynanchum thesioides, and the systematic analysis lays a foundation for further identification of the function of WRKY genes in Cynanchum thesioides.

Combined Cellular Thermometry Reveals That Salmonella typhimurium Warms Macrophages by Inducing a Pyroptosis-like Phenotype.

The attenuated Salmonella typhimurium VNP20009, enriched in tumors, is known to have antitumor effects and recruit macrophages. Little is known, however, about whether VNP will lead to specific changes in macrophages, e.g., cell temperature. Here, using a real-time wireless multicell thermometry system, we reported for the first time that VNP20009 increases the macrophage temperature by 0.2 °C. Nigericin, recognized as an inducer of pyroptosis, was found to induce macrophage warming. Moreover, the ΔsipD-VNP20009 strain failed to induce macrophage pyroptosis and simultaneously failed to warm macrophages, and the Gsdmd-/- macrophages that were unable to achieve pyroptosis were no longer warmed following VNP20009 induction. These results suggested that the occurrence of macrophage pyroptosis is the key to VNP20009-mediated cell warming. With the aid of a single-cell thermometry system, it was further confirmed that cell warming occurred in pyroptosis-like macrophages. Cellular warming was not detected after the induction of pyroptosis in macrophages with loss of mitochondrial biological function, suggesting a critical role of mitochondria in warming. Moreover, we found that VNP20009 caused local tumor temperature increases. The local tumor warming induced by VNP20009 was significantly reduced after macrophage clearance. Notably, this temperature increase contributed to M1-type polarization. These findings expanded our knowledge of the cellular biological changes induced by the strain on macrophages, as well as the biochemical phenomena accompanying pyroptosis, and provide a reference for the study of biochemical signals transduced to biothermal signals with a combined cell-level temperature detector.

Recombinant Bifidobacterium longum Carrying Endostatin Protein Alleviates Dextran Sodium Sulfate-Induced Colitis and Colon Cancer in Rats.

Bifidobacterium has been widely administrated orally as probiotics to prevent pathogen colonization and modulate the gut microbiome balance. Endostatin is an endogenous inhibitor of angiogenesis and has been shown to inhibit tumor growth, invasion, and metastasis. At present, the combination of endostatin and chemotherapeutic drugs has been regarded as a promising antitumor treatment strategy. In this study, we selected a safe strain of Bifidobacterium longum as a delivery system to transport endostatin to the gastrointestinal tract and explored their combined effect on inflammatory bowel disease (IBD) and colitis-associated cancer. The results indicated that B. longum-Endo relieved dextran sulfate sodium-induced body weight loss, diarrhea, colon shortening, and epithelium damage. Long-term oral administration of B. longum-Endo significantly decreased tumor formation rate, tumor number, and tumor size. Moreover, the effect of B. longum-Endo on gut microbiota dysbiosis was also confirmed by 16S rRNA sequencing analysis. The levels of potentially beneficial bacteria, such as Lactobacillus, Bifidobacterium, Allobaculum, and Parabateroides, were increased in the B. longum-Endo group compared to the model and B. longum groups. Meanwhile, levels of potentially pathogenic bacteria including Desulfovibrio, Helicobacter, and Enterorhabdus were decreased. Taken together, these results suggested that oral administration of recombinant B. longum-Endo strain may be a promising therapeutic strategy for IBD and colitis-associated cancer.

AK2 Promotes the Migration and Invasion of Lung Adenocarcinoma by Activating TGF-ß/Smad Pathway In vitro and In vivo.

Adenylate kinase 2 (AK2) is a wide-spread and highly conserved protein kinase whose main function is to catalyze the exchange of nucleotide phosphate groups. In this study, we showed that AK2 regulated tumor cell metastasis in lung adenocarcinoma. Positive expression of AK2 is related to lung adenocarcinoma progression and poor survival of patients. Knockdown or knockout of AK2 inhibited, while overexpression of AK2 promoted, human lung adenocarcinoma cell migration and invasion ability. Differential proteomics results showed that AK2 might be closely related to epithelial-mesenchymal transition (EMT). Further research indicated that AK2 regulated EMT occurrence through the Smad-dependent classical signaling pathways as measured by western blot and qPCR assays. Additionally, in vivo experiments showed that AK2-knockout in human lung tumor cells reduced their EMT-like features and formed fewer metastatic nodules both in liver and in lung tissues. In conclusion, we uncover a cancer metastasis-promoting role for AK2 and provide a rationale for targeting AK2 as a potential therapeutic approach for lung cancer.

Characterization and Analysis of the Temporal and Spatial Dynamic of Several Enteritis Modeling Methodologies.

Inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis, is a complex disease involving genetic, immune, and microbiological factors. A variety of animal models of IBD have been developed to study the pathogenesis of human IBD, but there is no model that can fully represent the complexity of IBD. In this study, we established two acute enteritis models by oral 3% DSS or intraperitoneal injection of anti-CD3 antibody, and two chronic enteritis models by feeding 3 cycles of 1.5% DSS or 3 months of the high-fat diet, respectively, and then examined the clinical parameters, histological changes, and cytokine expression profiles after the successful establishment of the models. Our results indicated that in 3% DSS-induced acute enteritis, the colorectal injury was significantly higher than that of the small intestine, while in anti-CD3 antibody-induced acute enteritis, the small intestine injury was significantly higher than that of colorectal damage. Besides, in the 1.5% DSS-induced chronic enteritis, the damage was mainly concentrated in the colorectal, while the damage caused by long-term HFD-induced chronic enteritis was more focused on the small intestine. Therefore, our work provides a reference for selecting appropriate models when conducting research on factors related to the pathogenesis of IBD or evaluating the potential diagnosis and treatment possibilities of pharmaceuticals.