In Situ Radical Reaction-Modified Carbon Dot Nanocapsules with Macrophage Escape and Prolonged Imaging.

Carbon dots (CDs) have emerged as an extremely promising platform for biological imaging, owing to their optical properties and low toxicity. However, one of the major challenges in utilizing CDs for in vivo imaging is their high immunogenicity and rapid clearance, which limits their potential. Herein, a novel approach for mitigating these issues is presented through the development of carbon dot nanocapsules (nCDs). Specifically, CDs are encapsulated within a zwitterionic polymer shell composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) to create nCDs with a size of ≈40 nm. Notably, the nCDs exhibit excitation-dependent photoluminescence behavior in the range of 550-600 nm, with tunability based on the excitation wavelength. In confocal imaging, CDs display a strong fluorescence signal after 8 h of incubation with phagocytes, while nCDs show minimal signal, suggesting that nCDs may be capable of evading phagocyte uptake. Furthermore, imaging studies in zebrafish demonstrate that nCDs exhibit a retention time >10 times longer than that of CDs, with fluorescence intensity remaining at 81% after 10 h compared to only 8% for CDs. Taken together, the study presents a novel approach for enhancing the performance of CDs in in vivo imaging applications, offering significant potential for clinical translation.

A novel ALK inhibitor ZYY inhibits Karpas299 cell growth in vitro and in a mouse xenograft model and induces protective autophagy.

In recent years, anaplastic lymphoma kinase (ALK) rearrangement-positive anaplastic large cell lymphoma (ALCL) has rising morbidity and mortality. Unfortunately, no ALK inhibitor has been approved by the FDA for single treatment of ALK rearrangement-positive ALCL. In this study, we investigated the antitumor effect of ZYY, a novel ALK inhibitor, showing a strong growth inhibitory effect on Karpas299 cells in vitro and in vivo. Specifically, ZYY significantly reduced the mRNA and protein expression of ALK and its downstream signaling proteins in Karpas299 cells. Furthermore, ZYY induced G1 phase arrest and promoted apoptosis in Karpas299 cells. Furthermore, we demonstrated that ZYY-induced apoptosis was mainly related to the mitochondria-dependent endogenous pathway. In vitro studies further showed that ZYY induced autophagy in Karpas299 cells, along with increased levels of the autophagy-related proteins, including LC3II and Beclin-1. Moreover, knockdown Beclin-1 and application of autophagy inhibitor chloroquine potentiated ZYY-induced cytotoxicity and apoptosis in vitro, indicating that cytoprotective autophagy might be triggered by ZYY in Karpas299 cells. Taken together, the novel ALK inhibitor ZYY has tremendous potential for treating human ALCL, and a combination of autophagy and ALK inhibition could effectively elicit potent antitumor effects.

PHF14 enhances DNA methylation of SMAD7 gene to promote TGF-ß-driven lung adenocarcinoma metastasis.

Aberrant activation of TGF-ß signaling plays a pivotal role in cancer metastasis and progression. However, molecular mechanisms underlying the dysregulation of TGF-ß pathway remain to be understood. Here, we found that SMAD7, a direct downstream transcriptional target and also a key antagonist of TGF-ß signaling, is transcriptionally suppressed in lung adenocarcinoma (LAD) due to DNA hypermethylation. We further identified that PHF14 binds DNMT3B and serves as a DNA CpG motif reader, recruiting DNMT3B to the SMAD7 gene locus, resulting in DNA methylation and transcriptional suppression of SMAD7. Our in vitro and in vivo experiments showed that PHF14 promotes metastasis through binding DNMT3B to suppress SMAD7 expression. Moreover, our data revealed that PHF14 expression correlates with lowered SMAD7 level and shorter survival of LAD patients, and importantly that SMAD7 methylation level of circulating tumor DNA (ctDNA) can potentially be used for prognosis prediction. Together, our present study illustrates a new epigenetic mechanism, mediated by PHF14 and DNMT3B, in the regulation of SMAD7 transcription and TGF-ß-driven LAD metastasis, and suggests potential opportunities for LAD prognosis.

Effects of Dexmedetomidine on Hemodynamics and Anesthesia Effect of Different Doses of General Anesthesia in Patients Undergoing Hepatobiliary Surgery.

In order to provide corresponding anesthesia methods of hepatobiliary diseases and obtain better clinical effect, the hemodynamics and clinical efficacy are evaluated by using different doses of dexamethasone (DEX) during the operation. 97 patients undergoing general anesthesia for hepatobiliary surgery in our hospital from April 2020 to August 2021 are analyzed retrospectively. All patients are divided into G1 (n = 24), G2 (n = 24), G3 (n = 24), and control group (n = 25) according to the random number table method. The G1, G2, and G3 groups are injected with DEX 1.0 ug/kg, DEX 0.8 ug/kg, and DEX 0.5 ug/kg by intravenous infusion pump before induction of general anesthesia, and the control group is injected with 0.9%Nacl solution 10 mL. Intraoperative anesthesia time, postoperative eye opening time, and extubation time of all groups are observed, and the incidences of postoperative adverse reactions are compared. The experimental results show that during general anesthesia in the liver and gallbladder surgery, the patients with high dose of DEX can better maintain the intraoperative hemodynamic parameters, and effectively restrain the postoperative stress reaction.

EML4-ALK G1202R mutation induces EMT and confers resistance to ceritinib in NSCLC cells via activation of STAT3/Slug signaling.

The echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene occurs in approximately 5% of non-small-cell lung cancers (NSCLCs). The development of ALK tyrosine kinase inhibitors (ALK-TKIs) is a major advance in treating NSCLC with the ALK fusion gene. Nevertheless, acquired resistance to ALK-TKIs ultimately limits their use. A prevalent mechanism of drug resistance in kinases occurs through the mutation of G1202R in ALK. However, the mechanisms underlying G1202R resistance to ceritinib are not fully understood. Here, we demonstrated that the expression of EML4-ALK G1202R mutation in A549 cells induced an epithelial-mesenchymal transition (EMT) phenotype and significantly increased the migration and invasion abilities. These phenomena may be due to the upregulation of signal transducer and activator of transcription 3 (STAT3), accompanied by the elevated expression of Slug in EML4-ALK G1202R mutant cells. Furthermore, the combination of ALK and STAT3 inhibitors restored the sensitivity of EML4-ALK G1202R mutant cells to ceritinib. In conclusion, these data indicate that the EML4-ALK G1202R mutation mediates the EMT phenotype by activating the STAT3/Slug signaling pathway, resulting in resistance to ceritinib, and that the combination of STAT3 and ALK inhibitors may overcome ALK mutation-driven drug resistance in the clinic.

The Variation of Duck RIG-I-Mediated Innate Immune Response Induced by Different Virulence Avian Influenza Viruses.

In recent years, the emerging highly pathogenic avian influenza (HPAI) A(H5N8) virus has been reported with features of widely spread, an expanding host range, and cross-species transmission, attracting wide attention. The domestic duck plays a major role in the epidemiological cycle of the HPAI H5N8 virus, but little is known concerning innate immune responses during influenza infection in duck species. In this study, we used two wild-bird-origin viruses, H5N8 and H4N6, to conduct duck infection experiments, and detect the load of the two viruses, and retinoic acid-inducible gene I (RIG-I) and interferon ß (IFN-ß) in the host's natural immune response. Through comparison, it is found that the expression levels of RIG-I and IFN-ß are both fluctuating. The innate immunity starts rapidly within 6 h after infection and is inhibited by the virus to varying degrees. The expression of RIG-I and IFN-ß decreased on 1-2 days post-infection (dpi). The HPAI H5N8 virus has a stronger inhibitory effect on RIG-I than the low pathogenic avian influenza (LPAI) H4N6 virus and is the strongest in the lungs. After infection with HPAI H5N8 virus, 2 dpi, viral RNA replicates in large amounts in the lungs. It has been proven that RIG-I and IFN-ß play an important role in the innate immune response of ducks to HPAI H5N8 virus infection, especially in the lungs. The main battlefield of RIG-I and IFN-ß after infection with the LPAI H4N6 virus is in the rectum. Both viruses have been effectively controlled after 7 dpi. These results will help to understand the transmission mechanisms of avian influenza virus in wild ducks and help effectively prevent and control avian influenza.

lncRNA OR3A4 Promotes the Proliferation and Metastasis of Ovarian Cancer Through KLF6 Pathway.

Aim: Ovarian cancer is a collaborative malignant tumor of the female reproductive system in clinical research. Some clinical studies have shown that OR3A4, which is a cancer-causing lncRNA, plays a major role in promoting the occurrence and development of a variety of tumors. And we also expressed the view that it expressed in ovarian tissue. However, the function of OR3A4 in ovarian cancer remains unclear. Methods and Results: To further verify the function of lncRNA OR3A4 in ovarian cancer, we established the xenograft model in the zebra fish. In this study, cells transformed with OR3A4 shRNA plasmids were transplanted into the zebra fish, and the cell proliferation and migration ability were significantly reduced compared to the empty vector. While knocking out OR3A4, we further downregulated its expression by siRNA of KLF6. Our study found that the knocked out OR3A4 resulted in a decrease in cell proliferation and migration level, which can be found in the downregulated expression of KLF6. We also verify the relationship between OR3A4 and circulating tumor cells in the zebra fish xenograft model, the results indicate that lncRNA OR3A4 may be involved in the resistance of ovarian cancer to complain. Conclusion: lncRNA OR3A4 promotes the proliferation and metastasis of ovarian cancer through the KLF6 pathway.

Construction of irf4a Transgenic Zebrafish Using Tol2 System and Its Potential Application.

PURPOSE: Interferon regulatory factor 4 (IRF4) is identified as a transcriptional factor and plays an important role in the immune response in mammals; however, there are few reports about the function of zebrafish IRF4. METHODS: We first amplified the coding sequence of irf4a from the testis of zebrafish. Besides, the fragments of irf4a, P2A, EGFP, and Tol2 vector were added for homologous recombination. By sequencing, we can get the Tol2-ef1α-irf4a-EGFP recombinant plasmid and it was microinjected into zebrafish embryos. Fluorescence observation was proceeded at days 3 post fertilization; F0 generations expressing green fluorescence in multiple tissues throughout the body were screened as the founder and raised them to sexual maturity. After mating with WT zebrafish to generate F1 offspring, polymerase chain reaction was used to identify whether irf4a was integrated into the zebrafish genome. CONCLUSION: We obtained the systematic overexpressed irf4a transgenic zebrafish with green fluorescence labeled in spine, eyes, heart, brain, and other tissues. The transgenic zebrafish will be used as a tool for the role of IRF4a in the immune response to the inflammation preconditioning in the future study.

Protective Effect of Metformin on Sepsis Myocarditis in Zebrafish.

PURPOSE: We found in previous study that metformin could treat sepsis myocarditis in a mouse model. We employed the zebrafish model organism to investigate the effect of metformin on sepsis myocarditis. METHODS AND RESULTS: Wild-type zebrafish was used to establish a sepsis myocarditis model and combined with image software analysis and cytokine detection, the protective dose of metformin was determined. The results showed that immersion with Escherichia coli could cause 75% mortality in zebrafish and make larvae appear as characteristics of severe sepsis myocarditis. Pretreatment with 10 mM metformin for 3 hours could effectively reduce heart congestion and swelling in zebrafish with sepsis myocarditis and increased the heart rate. It could reduce the mortality and prolong the survival time of zebrafish with sepsis myocarditis; Tg(mpx: EGFP) transgenic zebrafish were adopted to explore the number of neutrophils in zebrafish heart before and after metformin protection, and metformin could maintain the number of neutrophils in zebrafish heart; quantitative real-time reverse transcription-polymerase chain reaction showed that metformin could reduce the expression of pro-inflammatory factors, tumor necrosis factor-α and interleukin (IL)-6, and could promote the anti-inflammatory factor, transforming growth factor-ß and IL-10 expression. CONCLUSION: We established a zebrafish sepsis myocarditis model and applied metformin in advance to provide a protective effect on the zebrafish heart.

MAY, a novel tubulin inhibitor, induces cell apoptosis in A549 and A549/Taxol cells and inhibits epithelial-mesenchymal transition in A549/Taxol cells.

Non-small-cell lung cancer (NSCLC) is one of the common malignant tumors, and multidrug resistance (MDR) and tumor metastasis limit the anticancer effect of NSCLC. Therefore, it is necessary to develop new anticancer drug that can inhibit MDR and metastasis of NSCLC. In the present study, we found that 5-(2-chlorophenyl)-4-(4-(3,5-dimethoxyphenyl)piperazine-1-carbonyl)-2H-1,2,3- triazole (MAY) displayed strong cytotoxic effect on A549 and taxol-resistant A549 cells (A549/Taxol cells). We further discovered that MAY led to G2/M phase arrest by inhibiting microtubule polymerization in both cells. Then MAY caused apoptosis by the mitochondrial pathway in A549 cells and through the extrinsic pathway in A549/Taxol cells. Interestingly, MAY was not a substrate for P-glycoprotein (P-gp), which was highly expressed in A549/Taxol cells, and MAY inhibited the expression and efflux function of P-gp. Furthermore, MAY inhibited epithelial-mesenchymal transition (EMT) by targeting Twist1 in A549/Taxol cells. In summary, our results suggest that MAY induces apoptosis in A549 and A549/Taxol cells and inhibits EMT in A549/Taxol cells. These findings suggest that MAY could provide a promising method for the treatment of NSCLC, especially for the treatment of resistant NSCLC.

[Preparation of anti-human hemoglobin colloidal gold immunochromatography test strip].

Objective To develop the colloidal gold immunochromatography test strip for qualitatively detecting the hemoglobin (Hb) in human feces. Methods Mouse anti-human Hb monoclonal antibody SPR-5 marked by colloidal gold was coated in glass fiber membrane, and then the mouse anti-human Hb SP-5 monoclonal antibody and goat anti-mouse IgG were immobilized in testing (T) line and control (C) line located in nitrocellulose membranes, respectively. With this double antibody sandwich technique and immunochromatography test, the Hb antigen would react with both antibodies coated in the T line and C line and cause two colour reactions if the detected sample was positive, whereas the antigen-antibody combination and colour reaction only showed up in the C line when the sample was negative. Results The minimum detection limit of this test strip for human Hb was 21 ng/mL and no cross reactions were found in chick Hb, rabbit Hb, sheep Hb, pig Hb and cow Hb. Conclusion The test strips can improve the detection rate of fecal occult blood obviously and avoid false-positive results.

Synthesis and bioevaluation of diarylpyrazoles as antiproliferative agents.

Two series of diarylpyrazoles were designed as potential microtubule targeting agents. Twenty-eight target compounds were synthesized and exhibited potent antiproliferative activity. Compound 15e, displayed potent antiproliferative activity against SGC-7901, KB and HT-1080 cell lines, respectively, and was comparable to the positive control, CA-4. Tubulin polymerization experiments indicated that 15e effectively inhibited the tubulin polymerization, and immunostaining assay revealed that it significantly disrupted tubulin microtubule dynamics. Moreover, cell cycle studies revealed that compound 15e dramatically arrested cell cycle progression at G2/M phase and caused microtubule destabilization. Molecular modeling studies showed that 15e could bind to the colchicine binding site on microtubules.

3,5-Diaryl-1H-pyrazolo[3,4-b]pyridines as potent tubulin polymerization inhibitors: Rational design, synthesis and biological evaluation.

A series of novel 3,5-diaryl-1H-pyrazolo[3,4-b]pyridines as tubulin polymerization inhibitors targeting the colchicine site were designed via ring tethering strategy, which was supported by conformational analysis. The general, chemically unstable and rotational linker, carbanyl group, was locked by 1H-pyrazolo[3,4-b]pyridine to avoid carbonyl reduction and restrict the instability of molecular conformation caused by the rotation of the carbon-carbon single bond beside carbonyl group. All of target compounds were synthesized and evaluated for their antiproliferative activities against three human cancer lines (SGC-7901, A549 and HeLa) by MTT assay. Most of these compounds showed prominent in vitro potency and the most potent compound in this scaffold 13d (SGC-7901: IC50 = 13 nM) could significantly inhibit tubulin polymerization and strongly disrupt cytoskeleton. The results of molecular modeling study revealed that 13d interacts with tubulin by binding to the colchicine site.

Overcoming resistance to mitochondrial apoptosis by BZML-induced mitotic catastrophe is enhanced by inhibition of autophagy in A549/Taxol cells.

OBJECTIVES: Our previous in vitro study showed that 5-(3, 4, 5-trimethoxybenzoyl)-4-methyl-2-(p-tolyl) imidazol (BZML) is a novel colchicine binding site inhibitor with potent anti-cancer activity against apoptosis resistance in A549/Taxol cells through mitotic catastrophe (MC). However, the mechanisms underlying apoptosis resistance in A549/Taxol cells remain unknown. To clarify these mechanisms, in the present study, we investigated the molecular mechanisms of apoptosis and autophagy, which are closely associated with MC in BZML-treated A549 and A549/Taxol cells. METHODS: Xenograft NSCLC models induced by A549 and A549/Taxol cells were used to evaluate the efficacy of BZML in vivo. The activation of the mitochondrial apoptotic pathway was assessed using JC-1 staining, Annexin V-FITC/PI double-staining, a caspase-9 fluorescence metric assay kit and western blot. The different functional forms of autophagy were distinguished by determining the impact of autophagy inhibition on drug sensitivity. RESULTS: Our data showed that BZML also exhibited desirable anti-cancer activity against drug-resistant NSCLC in vivo. Moreover, BZML caused ROS generation and MMP loss followed by the release of cytochrome c from mitochondria to cytosol in both A549 and A549/Taxol cells. However, the ROS-mediated apoptotic pathway involving the mitochondria that is induced by BZML was only fully activated in A549 cells but not in A549/Taxol cells. Importantly, we found that autophagy acted as a non-protective type of autophagy during BZML-induced apoptosis in A549 cells, whereas it acted as a type of cytoprotective autophagy against BZML-induced MC in A549/Taxol cells. CONCLUSIONS: Our data suggest that the anti-apoptosis property of A549/Taxol cells originates from a defect in activation of the mitochondrial apoptotic pathway, and autophagy inhibitors can potentiate BZML-induced MC to overcome resistance to mitochondrial apoptosis.

Conformation impacts on the bioactivities of SMART analogues.

As promising colchicine binding site inhibitors, SMART and its analogues have attracted many research efforts in recent years. A large number of SMART analogues with different B-rings have been reported; however, the effects of B-ring on the bioactivity are still unclear so far. Herein, we speculated that the conformational preference caused by B-rings was crucial for active SMART analogues. Our assumption was supported by the molecular docking studies, molecular dynamic simulation and DFT computations of SMART and its analogues reported by other and our research groups. Moreover, several novel SMART analogues with different conformational preferences were designed and synthesized to disclose the conformation impacts, and the preliminary biological evaluation was in accordance with our assumption.