A novel fluorescent probe for viscosity and polarity detection in real tobacco root cells and biological imaging.

The disruption of lipid droplet function is associated with the pathogenesis of various diseases. Clarifying the response behavior of lipid droplets to the microenvironment at the cellular level is of great significance. Plant lipids not only exist in phospholipids in cell membranes, but also in aromatic essential oils. Monitoring the level of lipid droplets in plant cells using fluorescent probes provides a simple method for screening lipid-rich varieties. We synthesized a polarity-viscosity responsive coumarin fluorescent probe, Cou-CN, which achieved sensitive detection of polarity and viscosity in dilute solution environments by constructing this simple probe with ICT and TICT properties and verifying it using Gaussian computational simulation. Cou-CN exhibited good lipid droplet illumination effects in HepG2 cells with a correlation coefficient of 0.92 compared to the commercial lipid droplet dye BODIPY. Additionally, co-staining the probe with the lipophilic commercial dye Nile Red in tobacco root stem seedling cells resulted in a high correlation coefficient of 0.9.

c-Myc alone is enough to reprogram fibroblasts into functional macrophages.

BACKGROUND: Macrophage-based cell therapy is promising in solid tumors, but the efficient acquisition of macrophages remains a challenge. Induced pluripotent stem cell (iPSC)-induced macrophages are a valuable source, but time-consuming and costly. The application of reprogramming technologies allows for the generation of macrophages from somatic cells, thereby facilitating the advancement of cell-based therapies for numerous malignant diseases. METHODS: The composition of CD45+ myeloid-like cell complex (MCC) and induced macrophage (iMac) were analyzed by flow cytometry and single-cell RNA sequencing. The engraftment capacity of CD45+ MCC was evaluated by two transplantation assays. Regulation of c-Myc on MafB was evaluated by ChIP-qPCR and promoter reporter and dual luciferase assays. The phenotype and phagocytosis of iMac were explored by flow cytometry and immunofluorescence. Leukemia, breast cancer, and patient-derived tumor xenograft models were used to explore the anti-tumor function of iMac. RESULTS: Here we report on the establishment of a novel methodology allowing for reprogramming fibroblasts into functional macrophages with phagocytic activity by c-Myc overexpression. Fibroblasts with ectopic expression of c-Myc in iPSC medium rapidly generated CD45+ MCC intermediates with engraftment capacity as well as the repopulation of distinct hematopoietic compartments. MCC intermediates were stably maintained in iPSC medium and continuously generated functional and highly pure iMac just by M-CSF cytokine stimulation. Single-cell transcriptomic analysis of MCC intermediates revealed that c-Myc up-regulated the expression of MafB, a major regulator of macrophage differentiation, to promote macrophage differentiation. Characterization of the iMac activity showed NF-κB signaling activation and a pro-inflammatory phenotype. iMac cells displayed significantly increased in vivo persistence and inhibition of tumor progression in leukemia, breast cancer, and patient-derived tumor xenograft models. CONCLUSIONS: Our findings demonstrate that c-Myc alone is enough to reprogram fibroblasts into functional macrophages, supporting that c-Myc reprogramming strategy of fibroblasts can help circumvent long-standing obstacles to gaining "off-the-shelf" macrophages for anti-cancer immunotherapy.

A polar viscosity-sensitive fluorescent probe with large Stokes shifts for simultaneous imaging of lipid droplets and lysosomes in tobacco leaf vein cells and biological systems.

Lipid droplets (LDs) and lysosomes were dynamic organelles present in most eukaryotic cells that were interconnected and worked closely together to ensure the smooth physiological activities of organisms. The interaction between lipid droplets and lysosomes was thought to play a role in the development of certain diseases. In this paper we designed and synthesised a lipid droplet lysosomal probe. The Nap-Lyso-Ph-OH probe was constructed according to the ICT mechanism and exhibited sensitivity to both polarity and viscosity. The probe exhibited low cytotoxicity, a large Stokes shift, excellent selectivity and photostability. The probe was successfully used for labelling and imaging of lipid droplets and lysosomes in cells and zebrafish. Interestingly, we used tobacco seedling cells to explore the ability of Nap-Lyso-Ph-OH for imaging lipid droplet labelling in plant cells.

Single-cell RNA transcriptomic analyses of tumor microenvironment of ovarian metastasis in gastric cancer.

PURPOSE: Ovarian metastasis of gastric cancer (GC), commonly referred to as Krukenberg tumors, leads to a poor prognosis. However, the cause of metastasis remains unknown. Here, we present an integrated single-cell RNA sequencing (scRNA-Seq) analysis of the immunological microenvironment of two paired clinical specimens with ovarian metastasis of GC. METHODS: scRNA-Seq was performed to determine the immunological microenvironment in ovarian metastasis of gastric cancer. CellChat was employed to analyze cell-cell communications across different cell types. Functional enrichment analysis was done by enrichKEGG in clusterProfiler. GEPIA2 was used to assess the influence of certain genes and gene signatures on prognosis. RESULTS: The ovarian metastasis tissues exhibit a heterogenous immunological microenvironment compared to the primary tumors. Exhaustion of T and B cells is observed in the ovarian metastasis tissues. Compared to the paired adjacent non-tumoral and primary tumors, the ratio of endothelial cells and fibroblasts is high in the ovarian metastasis tissues. Compared to primary ovarian cancers, we identify a specific group of tumor-associated fibroblasts with MFAP4 and CAPNS1 expression in the ovarian metastatic tissues of GC. We further define metastasis-related-endothelial and metastasis-related-fibroblast signatures and indicate that patients with these high signature scores have a poor prognosis. In addition, the ovarian metastasis tissue has a lower level of intercellular communications compared to the primary tumor. CONCLUSION: Our findings reveal the immunological microenvironment of ovarian metastasis of gastric cancer and will promote the discovery of new therapeutic strategies for ovarian metastasis in gastric cancer.

TRIM24 Cooperates with Ras Mutation to Drive Glioma Progression through snoRNA Recruitment of PHAX and DNA-PKcs.

The factors driving glioma progression remain poorly understood. Here, the epigenetic regulator TRIM24 is identified as a driver of glioma progression, where TRIM24 overexpression promotes HRasV12 anaplastic astrocytoma (AA) progression into epithelioid GBM (Ep-GBM)-like tumors. Co-transfection of TRIM24 with HRasV12 also induces Ep-GBM-like transformation of human neural stem cells (hNSCs) with tumor protein p53 gene (TP53) knockdown. Furthermore, TRIM24 is highly expressed in clinical Ep-GBM specimens. Using single-cell RNA-sequencing (scRNA-Seq), the authors show that TRIM24 overexpression impacts both intratumoral heterogeneity and the tumor microenvironment. Mechanically, HRasV12 activates phosphorylated adaptor for RNA export (PHAX) and upregulates U3 small nucleolar RNAs (U3 snoRNAs) to recruit Ku-dependent DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Overexpressed TRIM24 is also recruited by PHAX to U3 snoRNAs, thereby facilitating DNA-PKcs phosphorylation of TRIM24 at S767/768 residues. Phosphorylated TRIM24 induces epigenome and transcription factor network reprogramming and promotes Ep-GBM-like transformation. Targeting DNA-PKcs with the small molecule inhibitor NU7441 synergizes with temozolomide to reduce Ep-GBM tumorigenicity and prolong animal survival. These findings provide new insights into the epigenetic regulation of Ep-GBM-like transformation and suggest a potential therapeutic strategy for patients with Ep-GBM.

ZBTB7A interferes with the RPL5-P53 feedback loop and reduces endoplasmic reticulum stress-induced apoptosis of pancreatic cancer cells.

Endoplasmic reticulum (ER) stress is a primary mechanism leading to cell apoptosis, making it of great research interests in cancer management. This study delves into the function of ribosomal protein L5 (RPL5) in ER stress within pancreatic cancer (PCa) cells and investigates its regulatory mechanisms. Bioinformatics predictions pinpointed RPL5 as an ER stress-related gene exhibiting diminished expression in PCa. Indeed, RPL5 was found to be poorly expressed in PCa tissues and cells, with this reduced expression correlating with an unfavorable prognosis. Moreover, RPL5 overexpression led to heightened levels of p-PERK, p-eIF2α, and CHOP, bolstering the proapoptotic effect of Tunicamycin, an ER stress activator, on PCa cells. Additionally, the RPL5 overexpression curbed cell proliferation, migration, and invasion. Tunicamycin enhanced the binding between RPL5 and murine double minute 2 (MDM2), thus suppressing MDM2-mediated ubiquitination and degradation of P53. Consequently, P53 augmentation intensified ER stress, which further enhanced the binding between RPL5 and MDM2 through PERK-dependent eIF2α phosphorylation, thereby establishing a positive feedback loop. Zinc finger and BTB domain containing 7A (ZBTB7A), conspicuously overexpressed in PCa samples, repressed RPL5 transcription, thereby reducing P53 expression. Silencing of ZBTB7A heightened ER stress and subdued the malignant attributes of PCa cells, effects counteracted upon RPL5 silencing. Analogous outcomes were recapitulated in vivo employing a xenograft tumor mouse model, where ZBTB7A silencing dampened the tumorigenic potential of PCa cells, an effect reversed by additional RPL5 silencing. In conclusion, this study suggests that ZBTB7A represses RPL5 transcription, thus impeding the RPL5-P53 feedback loop and mitigating ER-induced apoptosis in PCa cells.

Pumpkin CmoDREB2A enhances salt tolerance of grafted cucumber through interaction with CmoNAC1 to regulate H2O2 and ABA signaling and K+/Na+ homeostasis.

Pumpkin CmoNAC1 enhances salt tolerance in grafted cucumbers. However, the potential interactions with other proteins that may co-regulate salt tolerance alongside CmoNAC1 have yet to be explored. In this study, we identified pumpkin CmoDREB2A as a pivotal transcription factor that interacts synergistically with CmoNAC1 in the co-regulation of salt tolerance. Both transcription factors were observed to bind to each other's promoters, forming a positive regulatory loop of their transcription. Knockout of CmoDREB2A in the root resulted in reduced salt tolerance in grafted cucumbers, whereas overexpression demonstrated the opposite effect. Multiple assays in our study provided evidence of the protein interaction between CmoDREB2A and CmoNAC1. Exploiting this interaction, CmoDREB2A facilitated the binding of CmoNAC1 to the promoters of CmoRBOHD1, CmoNCED6, CmoAKT1;2, and CmoHKT1;1, inducing H2O2 and ABA synthesis and increasing the K+/Na+ ratio in grafted cucumbers under salt stress. Additionally, CmoNAC1 also promoted the binding of CmoDREB2A to CmoHAK5;1/CmoHAK5;2 promoters, further contributing to the K+/Na+ homeostasis. In summary, these findings reveal a crucial mechanism of CmoNAC1 and CmoDREB2A forming a complex enhancing salt tolerance in grafted cucumbers.

KAP1 stabilizes MYCN mRNA and promotes neuroblastoma tumorigenicity by protecting the RNA m6A reader YTHDC1 protein degradation.

BACKGROUND: Neuroblastoma (NB) patients with amplified MYCN often face a grim prognosis and are resistant to existing therapies, yet MYCN protein is considered undruggable. KAP1 (also named TRIM28) plays a crucial role in multiple biological activities. This study aimed to investigate the relationship between KAP1 and MYCN in NB. METHODS: Transcriptome analyses and luciferase reporter assay identified that KAP1 was a downstream target of MYCN. The effects of KAP1 on cancer cell proliferation and colony formation were explored using the loss-of-function assays in vitro and in vivo. RNA stability detection was used to examine the influence of KAP1 on MYCN expression. The mechanisms of KAP1 to maintain MYCN mRNA stabilization were mainly investigated by mass spectrum, immunoprecipitation, RIP-qPCR, and western blotting. In addition, a xenograft mouse model was used to reveal the antitumor effect of STM2457 on NB. RESULTS: Here we identified KAP1 as a critical regulator of MYCN mRNA stability by protecting the RNA N6-methyladenosine (m6A) reader YTHDC1 protein degradation. KAP1 was highly expressed in clinical MYCN-amplified NB and was upregulated by MYCN. Reciprocally, KAP1 knockdown reduced MYCN mRNA stability and inhibited MYCN-amplified NB progression. Mechanistically, KAP1 regulated the stability of MYCN mRNA in an m6A-dependent manner. KAP1 formed a complex with YTHDC1 and RNA m6A writer METTL3 to regulate m6A-modified MYCN mRNA stability. KAP1 depletion decreased YTHDC1 protein stability and promoted MYCN mRNA degradation. Inhibiting MYCN mRNA m6A modification synergized with chemotherapy to restrain tumor progression in MYCN-amplified NB. CONCLUSIONS: Our research demonstrates that KAP1, transcriptionally activated by MYCN, forms a complex with YTHDC1 and METTL3, which in turn maintain the stabilization of MYCN mRNA in an m6A-dependent manner. Targeting m6A modification by STM2457, a small-molecule inhibitor of METTL3, could downregulate MYCN expression and attenuate tumor proliferation. This finding provides a new alternative putative therapeutic strategy for MYCN-amplified NB.

LINC00115 promotes chemoresistant breast cancer stem-like cell stemness and metastasis through SETDB1/PLK3/HIF1α signaling.

BACKGROUND: Cancer stem-like cell is a key barrier for therapeutic resistance and metastasis in various cancers, including breast cancer, yet the underlying mechanisms are still elusive. Through a genome-wide lncRNA expression profiling, we identified that LINC00115 is robustly upregulated in chemoresistant breast cancer stem-like cells (BCSCs). METHODS: LncRNA microarray assay was performed to document abundance changes of lncRNAs in paclitaxel (PTX)-resistant MDA-MB-231 BCSC (ALDH+) and non-BCSC (ALDH-). RNA pull-down and RNA immunoprecipitation (RIP) assays were performed to determine the binding proteins of LINC00115. The clinical significance of the LINC00115 pathway was examined in TNBC metastatic lymph node tissues. The biological function of LINC00115 was investigated through gain- and loss-of-function studies. The molecular mechanism was explored through RNA sequencing, mass spectrometry, and the CRISPR/Cas9-knockout system. The therapeutic potential of LINC00115 was examined through xenograft animal models. RESULTS: LINC00115 functions as a scaffold lncRNA to link SETDB1 and PLK3, leading to enhanced SETDB1 methylation of PLK3 at both K106 and K200 in drug-resistant BCSC. PLK3 methylation decreases PLK3 phosphorylation of HIF1α and thereby increases HIF1α stability. HIF1α, in turn, upregulates ALKBH5 to reduce m6A modification of LINC00115, resulting in attenuated degradation of YTHDF2-dependent m6A-modified RNA and enhanced LINC00115 stability. Thus, this positive feedback loop provokes BCSC phenotypes and enhances chemoresistance and metastasis in triple-negative breast cancer. SETDB1 inhibitor TTD-IN with LINC00115 ASO sensitizes PTX-resistant cell response to chemotherapy in a xenograft animal model. Correlative expression of LINC00115, methylation PLK3, SETDB1, and HIF1α are prognostic for clinical triple-negative breast cancers. CONCLUSIONS: Our findings uncover LINC00115 as a critical regulator of BCSC and highlight targeting LINC00115 and SETDB1 as a potential therapeutic strategy for chemotherapeutic resistant breast cancer.

Myeloid Deletion of Cdc42 Protects Liver From Hepatic Ischemia-Reperfusion Injury via Inhibiting Macrophage-Mediated Inflammation in Mice.

BACKGROUND & AIMS: Hepatic ischemia-reperfusion injury (HIRI) often occurs in liver surgery, such as partial hepatectomy and liver transplantation, in which myeloid macrophage-mediated inflammation plays a critical role. Cell division cycle 42 (Cdc42) regulates cell migration, cytoskeleton rearrangement, and cell polarity. In this study, we explore the role of myeloid Cdc42 in HIRI. METHODS: Mouse HIRI models were established with 1-hour ischemia followed by 12-hour reperfusion in myeloid Cdc42 knockout (Cdc42mye) and Cdc42flox mice. Myeloid-derived macrophages were traced with RosamTmG fluorescent reporter under LyzCre-mediated excision. The experiments for serum or hepatic enzymic activities, histologic and immunologic analysis, gene expressions, flow cytometry analysis, and cytokine antibody array were performed. RESULTS: Myeloid deletion of Cdc42 significantly alleviated hepatic damages with the reduction of hepatic necrosis and inflammation, and reserved hepatic functions following HIRI in mice. Myeloid Cdc42 deficiency suppressed the infiltration of myeloid macrophages, reduced the secretion of proinflammatory cytokines, restrained M1 polarization, and promoted M2 polarization of myeloid macrophages in livers. In addition, inactivation of Cdc42 promoted M2 polarization via suppressing the phosphorylation of STAT1 and promoting phosphorylation of STAT3 and STAT6 in myeloid macrophages. Furthermore, pretreatment with Cdc42 inhibitor, ML141, also protected mice from hepatic ischemia-reperfusion injury. CONCLUSIONS: Inhibition or deletion of myeloid Cdc42 protects liver from HIRI via restraining the infiltration of myeloid macrophages, suppressing proinflammatory response, and promoting M2 polarization in macrophages.

Transcription factor SlWRKY50 enhances cold tolerance in tomato by activating the jasmonic acid signaling.

Tomato (Solanum lycopersicum) is a cold-sensitive crop but frequently experiences low-temperature stimuli. However, tomato responses to cold stress are still poorly understood. Our previous studies have shown that using wild tomato (Solanum habrochaites) as rootstock can significantly enhance the cold resistance of grafted seedlings, in which a high concentration of jasmonic acids (JAs) in scions exerts an important role, but the mechanism of JA accumulation remains unclear. Herein, we discovered that tomato SlWRKY50, a Group II WRKY transcription factor that is cold inducible, responds to cold stimuli and plays a key role in JA biosynthesis. SlWRKY50 directly bound to the promoter of tomato allene oxide synthase gene (SlAOS), and overexpressing SlWRKY50 improved tomato chilling resistance, which led to higher levels of Fv/Fm, antioxidative enzymes, SlAOS expression, and JA accumulation. SlWRKY50-silenced plants, however, exhibited an opposite trend. Moreover, diethyldithiocarbamate acid (a JA biosynthesis inhibitor) foliar treatment drastically reduced the cold tolerance of SlWRKY50-overexpression plants to wild-type levels. Importantly, SlMYC2, the key regulator of the JA signaling pathway, can control SlWRKY50 expression. Overall, our research indicates that SlWRKY50 promotes cold tolerance by controlling JA biosynthesis and that JA signaling mediates SlWRKY50 expression via transcriptional activation by SlMYC2. Thus, this contributes to the genetic knowledge necessary for developing cold-resistant tomato varieties.

USP7-mediated JUND suppresses RCAN2 transcription and elevates NFATC1 to enhance stem cell property in colorectal cancer.

Cancer stem cells (CSCs) encompass a subset of highly aggressive tumor cells that are involved in tumor initiation and progression. This study investigates the function of regulator of calcineurin 2 (RCAN2) in the stem cell property in colorectal cancer (CRC). By analyzing four GEO datasets, we obtained RCAN2 as a stemness-related gene in CRC. RCAN2 was poorly expressed in CRC tissues and cells, especially in CSCs. RCAN2 restoration reduced calcineurin activity and promoted phosphorylation and degradation of nuclear factor of activated T cells 1 (NFATC1) protein, leading to reduced stemness of CSCs. JunD proto-oncogene (JUND), whose protein level was increased in CRC samples and CRC stem cells, bound to RCAN2 and suppressed its transcription. The abundant ubiquitin specific peptidase 7 (USP7) in CSCs enhanced JUND protein stability through deubiquitination modification. Lentivirus-mediated knockdown of USP7 or JUND also blocked the calcineurin-NFATC1 signaling and reduced the protein levels of stemness-related proteins. Moreover, the USP7 knockdown weakened the colony/sphere formation ability as well as the tumorigenicity of CSCs, and it reduced the CSC content in xenograft tumors. However, further restoration of JUND rescued the stemness of the CSCs. Overall, this study demonstrates that USP7-mediated JUND suppresses RCAN2 transcription and activates NFATC1 to enhance stem cell property in CRC. 1. RCAN2 is poorly expressed in CRC tissues and cells and especially in CSCs. 2. RCAN2 reduces stemness of CSCs by blocking calcineurin-NFATC1 signal transduction. 3. JUND binds to RCAN2 promoter to suppresses RCAN2 transcription. 4. USP7 enhances JUND protein stability via deubiquitination modification. 5. Downregulation of USP7 or JUND restores RCAN2 level and suppresses stemness of CSCs.

Targeting TRIM24 promotes neuroblastoma differentiation and decreases tumorigenicity via LSD1/CoREST complex.

PURPOSE: High-risk neuroblastoma (NB) still has an unfavorable prognosis and inducing NB differentiation is a potential strategy in clinical treatment, yet underlying mechanisms are still elusive. Here we identify TRIM24 as an important regulator of NB differentiation. METHODS: Multiple datasets and clinical specimens were analyzed to define the role of TRIM24 in NB. The effects of TRIM24 on differentiation and growth of NB were determined by cell morphology, spheres formation, soft agar assay, and subcutaneous xenograft in nude mice. RNA-Seq and qRT-PCR were used to identify genes and pathways involved. Mass spectrometry and co-immunoprecipitation were used to explore the interaction of proteins. RESULTS: Trim24 is highly expressed in spontaneous NB in TH-MYCN transgenic mice and clinical NB specimens. It is associated with poor NB differentiation and unfavorable prognostic. Knockout of TRIM24 in neuroblastoma cells promotes cell differentiation, reduces cell stemness, and inhibits colony formation in soft agar and subcutaneous xenograft tumor growth in nude mice. Mechanistically, TRIM24 knockout alters genes and pathways related to neural differentiation and development by suppressing LSD1/CoREST complex formation. Besides, TRIM24 knockout activates the retinoic acid pathway. Targeting TRIM24 in combination with retinoic acid (RA) synergistically promotes NB cell differentiation and inhibits cell viability. CONCLUSION: Our findings demonstrate that TRIM24 is critical for NB differentiation and suggest that TRIM24 is a promising therapeutic target in combination with RA in NB differentiation therapy.

Developmental and reproductive toxicity of a recombinant protein subunit COVID-19 vaccine (ZF2001) in rats.

ZF2001, a protein subunit vaccine against coronavirus disease 2019 (COVID-19), contains recombinant tandem repeat of dimeric receptor-binding domain (RBD) protein of the SARS-CoV-2 spike protein with an aluminium-based adjuvant. During the development of this vaccine, two nonclinical studies were conducted to evaluate female fertility, embryo-fetal development, and postnatal developmental toxicity in Sprague‒Dawley rats according to the ICH S5 (R3) guideline. In Study 1 (embryo-fetal developmental toxicity, EFD), 144 virgin female rats were randomly assigned into four groups and received three doses of vaccine (25 µg or 50 µg RBD protein/dose, containing the aluminium-based adjuvant), the aluminium-based adjuvant or a sodium chloride injection administered intramuscularly on days 21 and 7 prior to mating and on gestation day (GD) 6. In Study 2 (pre- and postnatal developmental toxicity, PPND), ZF2001 at a dose of 25 µg RBD protein/dose or sodium chloride injection was administered intramuscularly to female rats (n = 28 per group) 7 days prior to mating and on GD 6, GD 20 and postnatal day (PND) 10. There were no obvious adverse effects in dams, except for local injection site reactions related to the aluminium-based adjuvant (yellow nodular deposits in the interstitial muscle fibres). There were also no effects of ZF2001 on the mating performance, fertility or reproductive performance of parental females, embryo-fetal development, postnatal survival, growth, physical development, reflex ontogeny, behavioural and neurofunctional development, or reproductive performance of the offspring. The strong immune responses associated with binding and neutralising antibodies were both confirmed in dams and fetuses or offspring in these two studies. These results would support clinical trials or the use of ZF2001 in maternal immunisation campaigns, including those involving women with childbearing potential, regardless of pregnancy status.

Gut microbial metabolite butyrate improves anticancer therapy by regulating intracellular calcium homeostasis.

BACKGROUND AND AIMS: Gut microbiota are recognized to be important for anticancer therapy, yet the underlying mechanism is not clear. Here, through the analysis of clinical samples, we identify the mechanism by which the gut microbial metabolite butyrate inhibits HCC and then explore new strategies for HCC treatment. APPROACH AND RESULTS: In our study, we demonstrate that gut microbial metabolite butyrate improves anticancer therapy efficacy by regulating intracellular calcium homeostasis. Using liquid chromatography-mass spectrometry analysis, we found that butyrate metabolism is activated in HCC patients compared with healthy individuals. Butyrate levels are lower in the plasma of HCC patients by gas chromatography-mass spectrometry (GC-MS) analysis. Butyrate supplementation or depletion of short-chain Acyl-CoA dehydrogenase (SCAD) gene (ACADS), encoding a key enzyme for butyrate metabolism, significantly inhibits HCC proliferation and metastasis. The profiling analysis of genes upregulated by butyrate supplementation or ACADS knockdown reveals that calcium signaling pathway is activated, leading to dysregulation of intracellular calcium homeostasis and production of reactive oxygen species. Butyrate supplementation improves the therapy efficacy of a tyrosine kinase inhibitor sorafenib. On the basis of these findings, we developed butyrate and sorafenib coencapsulated mPEG-PLGA-PLL nanoparticles coated with anti-GPC3 antibody (BS@PEAL-GPC3) to prolong the retention time of drugs and enhance drug targeting, leading to high anticancer efficacy. BS@PEAL-GPC3 nanoparticles significantly reduce HCC progression. In addition, BS@PEAL-GPC3 nanoparticles display excellent HCC targeting with excellent safety. CONCLUSIONS: In conclusion, our findings provide new insight into the mechanism by which the gut microbial metabolites inhibit HCC progression, suggesting a translatable therapeutics approach to enhance the clinical targeted therapeutic efficacy.

The essential role of jasmonate signaling in Solanum habrochaites rootstock-mediated cold tolerance in tomato grafts.

Tomato (Solanum lycopersicum) is among the most important vegetables across the world, but cold stress usually affects its yield and quality. The wild tomato species Solanum habrochaites is commonly utilized as rootstock for enhancing resistance against abiotic stresses in cultivated tomato, especially cold resistance. However, the underlying molecular mechanism remains unclear. In this research, we confirmed that S. habrochaites rootstock can improve the cold tolerance of cultivated tomato scions, as revealed by growth, physiological, and biochemical indicators. Furthermore, transcriptome profiling indicated significant differences in the scion of homo- and heterografted seedlings, including substantial changes in jasmonic acid (JA) biosynthesis and signaling, which were validated by RT-qPCR analysis. S. habrochaites plants had a high basal level of jasmonate, and cold stress caused a greater amount of active JA-isoleucine in S. habrochaites heterografts. Moreover, exogenous JA enhanced while JA inhibitor decreased the cold tolerance of tomato grafts. The JA biosynthesis-defective mutant spr8 also showed increased sensitivity to cold stress. All of these results demonstrated the significance of JA in the cold tolerance of grafted tomato seedlings with S. habrochaites rootstock, suggesting a future direction for the characterization of the natural variation involved in S. habrochaites rootstock-mediated cold tolerance.

Preclinical Toxicity and Immunogenicity of a COVID-19 Vaccine (ZF2001) in Cynomolgus Monkeys.

Although the new coronavirus disease 2019 (COVID-19) outbreak occurred in late 2019, it is still endemic worldwide, and has become a global public health problem. Vaccination against SARS-CoV-2 is considered to be the most effective intervention to prevent the spread of COVID-19. ZF2001 is a recombinant protein vaccine based on SARS-CoV-2 receptor-binding domain (RBD) subunit which contains aluminum adjuvant. In order to advance our research on ZF2001 into clinical trial, we investigated the general toxicity and immunogenicity of ZF2001 in cynomolgus monkeys and assessed the possible target organs for vaccine-induced toxicity. In the present research, we observed no significant systemic toxicities and abnormal cardiovascular and respiratory events following four times injections of intramuscular ZF2001 in cynomolgus monkeys. Histological examination revealed recoverable inflammatory changes in quadricep muscle and adjacent lymph node at the vaccine injection site. As expected, the vaccine can produce a strongly specific binding antibody and neutralizing antibodies in cynomolgus monkeys after inoculation. Taken together, our regulatory toxicology research proves the safety and immunogenicity of the ZF2001 vaccine, supporting its entry into large scale clinical trials.

Characteristics of pre-sensitization-related acute antibody-mediated rejection in a rat model of orthotopic liver transplantation.

Background: To establish an animal model of pre-sensitization following liver transplantation either with or without immunosuppressors. To study whether accelerated liver rejection or acute antibody-mediated rejection (AMR) occurred and study the characteristics and potential mechanism in the animal model. Methods: Lewis (LEW) rats were subjected to liver [liver graft of Brown Norway (BN) rat] transplantation 2 weeks after lymphocyte injection (lymphocytes of BN rat; pre-sensitization). At 2 weeks after transplantation, serum samples of recipients were collected for antibody analysis to identify donor-specific alloantibody (DSA) level. The recipients were treated with or without a low dose of immunosuppressor (2 mg/kg). The liver grafts of each group were analyzed by hematoxylin and eosin (HE) stain, Masson stain, CK19, C4d, and CD20 immunohistochemical (IHC) stain, CD3, CD68, and CD86 immunofluorescence and transmission electron microscope (TEM) to study the characteristics of liver rejection. Moreover, cytotoxin-associated genes, M1 macrophages conversion-related proteins, and interleukin-6 (IL-6) signaling pathway proteins were detected by western blotting. Results: High level of DSA and accelerated liver rejection occurred in the pre-sensitized rat models following liver transplant. Accelerated liver graft rejection occurred in the pre-sensitized, post liver transplant rats regardless of whether a low dose immunosuppressor had been applied. Severe injury of the interlobular bile ducts and accelerated fibrosis could be observed. Moreover, evidence of endothelial injury, such as capillary inflammation, was found in the pre-sensitized, post-transplant rats. In addition, C4d deposition and M1 macrophages recruitment were also found in this sensitized followed transplant model, indicating that complement activation might occur in this model. The levels of IL-6, JAK1, STAT3, SHP2, and ERK1-2 were increased in the pre-sensitized, post-transplant rats. Conclusions: Pre-sensitized post liver transplant rats might be potential AMR models for further study.

Ischemic stroke in a patient with EGFR-mutated non-small-cell lung cancer after treatment with ramucirumab.

Vascular endothelial growth factor (VEGF) inhibitors have been widely investigated in the last 10 years, with particular attention paid to their adverse effects because of their efficacy in improving cancer patient survival. Previous research primarily focused on the monoclonal anti-vascular endothelial growth factor antibody bevacizumab and its adverse outcomes. Reports show a higher risk of ischemic stroke, one of the most concerning clinically relevant events, after treatment with bevacizumab. However, few studies have examined the relationship between anti-VEGF receptor 2 monoclonal antibody ramucirumab and its adverse events. This article presents the case of a non-small-cell lung cancer patient who experienced a new ischemic stroke after treatment with ramucirumab. The findings suggest that further studies may be necessary to investigate the relationship between ramucirumab and the risk of ischemic stroke.