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Background:: Melanoma, a highly malignant skin cancer, is a hot topic in oncology treatment research. Nowadays, tumor immunotherapy, especially immunotherapy combined with other therapies, has attracted more and more attention. Indoleamine 2,3-dioxygenase 2 (IDO2), a ratelimiting enzyme of the tryptophan metabolism pathway in the urine of dogs with immunosuppression, is highly expressed in melanoma tissue. Additionally, IDO2 significantly inhibits the anti-tumor immunity of the body and has become a novel target of melanoma treatment. Nifuroxazide, as an intestinal antibacterial agent, was found to be able to inhibit Stat3 expression and exert an anti-tumor effect. Therefore, the present study aimed to examine the therapeutic effect of a self-designed IDO2-small interfering RNA (siRNA) delivered by attenuated Salmonella combined with nifuroxazide on melanoma- bearing mice, as well as determine its underlying mechanism. Methods:: The effect of nifuroxazide on melanoma was detected by flow cytometry, CCK-8 and colony- forming ability assays, respectively, in vitro. The plasmid of siRNA-IDO2 was constructed, and the mice-bearing melanoma model was established. After the treatment, the tumor growth and survival rate were monitored, and the morphological changes of tumor tissue were detected by HE staining. The expression of related proteins was detected by Western blotting, and the expression of CD4 and CD8 positive T cells in tumor tissue was detected by IHC and IF, and the proportion of CD4 and CD8 positive T cells in spleen was detected by flow cytometry. Results:: The results demonstrated that the combination therapy effectively inhibited the phosphorylation of Stat3 and the expression level of IDO2 in melanoma cells, which effectively inhibited tumor growth and prolonged the survival time of tumor-bearing mice. The mechanistic study revealed that, compared with control groups and monotherapy groups, the combination treatment group reduced the atypia of tumor cells, increased the apoptotic rate, enhanced the infiltration of T lymphocytes in tumor tissue and increased the CD4+ and CD8+ T lymphocytes in the spleen, suggesting that the mechanism may be associated with the inhibition of tumor cell proliferation, the increase of apoptosis and the enhancement of the cellular immunity. Conclusion:: In conclusion, IDO2-siRNA combined with nifuroxazide therapy could serve a significant role in the treatment of melanoma-bearing mice, enhance the tumor immunity and provide an experimental basis for identifying a novel combination method for the treatment of melanoma clinically.

Backgroud: Many dyes or radioactive markers used for sentinel lymph node (SLN) have the shortcomings of false positive and radiation injury. Indocyanine green (ICG) seems to have a lower false positive rate and tissue damage, without a clear field of vision during the operation. Methods: For the shortcomings, we successfully synthesized three anionic pullulan materials, changed the degree of hydrophobic for size controlling (< 50nm) to prepare CHP nanoparticles (NPs) and changed the succinyl degree to prepare CHPC NPs with different negative surface potential. Results: The size of those NPs were less than 50nm under (transmission electron microscope) TEM, with hydrodynamic size of 90.67±2.2nm of CHP, 105.8±1.7nm of CHPC1 and 115.9±2.3nm of CHPC2. Moreover, the Zeta potential of CHP, CHPC1 and CHPC2 were -1.9±0.2mV, -9.6±0.3mV and -19.4±0.7mV. The size of ICG-loading CHP, CHPC1 and CHPC2 NPs increased to 109.4±2.7nm, 113.8±1.2nm and 30.6±3.5 nm, as the zeta potential decreased to -2.7±0.4mV, -12.5±1.6mV and -23.1 ±1.2mV. With the increasing degree of succinyl, the size increased and the zeta potential decreased. At the same time, the higher degree of succinyl drug-loading NPs have lower release and have increased the stability of ICG. We found that the blank-NPs had no significant toxicity to normal cells (HSF), as the ICG@CHP group had larger toxicity than the CHPCs and control. Moreover, the cellular uptake was decreased with the increased degree of succinyl. Conclusion: In this study, we successfully prepared CHPC2 carriers with the maximum negative surface charge, for follow-up research and providing new ideas for SLN.

Background: Most of the structural modifications to evodiamine (EVO) have focused on the 3- and 10-positions, while structural modifications to the EVO 2-position have not yet been reported. In this study, we investigated the scaffold diversity and bioactivity of EVO from position 2 to gain more insight into the influence of the chemical space around EVO on bioactivity. Objective: The study aims to synthesize two derivatives of EVO with hydroxy groups, 8a and 8b, and to investigate the antitumor activity of EVO derivatives with hydroxy groups compared to EVO. Methods: The synthesized compounds were structurally characterized by 1H NMR, 13C NMR, and mass spectrometry. The effects of compounds 8a, 8b, and EVO on the proliferation of H460, A549, and Eca109 cells in vitro were determined by MTT. The effect of EVO, 8a and 8b on apoptosis of H460 cells was investigated by the annexed V-FITC/propidium iodide (PI) combination assay. The expression of EVO, 8a and 8b on apoptosis-related proteins was examined by Western blot analysis. To simulate the binding ability between small molecules and proteins, molecular docking calculations of EGFRWT and EGFRT790M with 8a and 8b, respectively, were performed using Schrödinger software. Results: In the cytotoxicity assay, compound 8b showed lower IC50 values for the three tumor cell lines (6.69 μM for H460 cells, 20.02 μM for A549 cells, and 16.47 μM for Eca109 cells) compared to compound 8a and EVO, and 8b induced apoptosis by affecting apoptosis-related proteins CRAF, AKT, and ERK in a late apoptotic manner. The molecular docking results showed that 8b has a good binding ability to EGFR upstream of apoptosis-related proteins. Conclusion: These findings suggest that 8b has significantly higher antitumor biological activity than EVO and 8a. This antitumor effect has important implications for the study of EVO derivatives in antitumor models.

Amino acids have a multi-billion-dollar market with rising demand, prompting the development of high-performance microbial factories. However, a general screening strategy applicable to all proteinogenic and non-proteinogenic amino acids is still lacking. Modification of the critical structure of tRNA could decrease the aminoacylation level of tRNA catalyzed by aminoacyl-tRNA synthetases. Involved in a two-substrate sequential reaction, amino acids with increased concentration could elevate the reduced aminoacylation rate caused by specific tRNA modification. Here, we developed a selection system for overproducers of specific amino acids using corresponding engineered tRNAs and reporter genes. As a proof-of-concept, overproducers of five amino acids such as L-tryptophan were screened out by growth-based and/or fluorescence-activated cell sorting (FACS)-based screening from random mutation libraries ofEscherichia coliandCorynebacterium glutamicum, respectively. This study provided a universal strategy that could be applied to screen overproducers of proteinogenic and non-proteinogenic amino acids in amber-stop-codon-recoded or non-recoded hosts.

Background: Exploratory studies have shown that remote ischemic conditioning (RIC) has the potential to lower blood pressure (BP). We investigated whether chronic RIC reduces BP for hypertension. Methods: This is a multicenter, randomized, double-blind, parallel-controlled trial. Patients with an office BP of 130/80 to 160/100 mm Hg and a 24-hour average BP ≥125/75 mm Hg not on antihypertensive medications were recruited. After a 1-week compliance screening phase, they were randomly assigned in a 1:1 ratio to receive RIC or sham RIC twice daily for 4 weeks. The primary efficacy outcome was the change in 24-hour average systolic BP from baseline to 4 weeks. Safety events were assessed over the study period. Results: Ninety-five participants were randomly allocated to the RIC (n=49) and sham RIC (n=46) groups. In the intention-to-treat analysis, the reduction in 24-hour average systolic BP was greater in the RIC group than the sham RIC group (−4.6±9.5 versus −0.9±6.8 mm Hg; baseline-adjusted between-group mean difference: −3.6 mm Hg [95% CI, −6.9 to −0.3 mm Hg]; adjusted P =0.035). The per-protocol analysis showed that 24-hour average systolic BP reduced −5.9±8.6 mm Hg in the RIC group and −0.7±6.7 mm Hg in the sham RIC group (baseline-adjusted between-group mean difference: −5.2 mm Hg [95% CI, −8.5 to −1.9 mm Hg]; adjusted P =0.002). No major adverse events were reported in both groups. Conclusions: RIC is safe in patients with mild hypertension and may lower BP in the absence of antihypertensive medications. However, the effects of RIC on clinical outcomes in these patients require further investigation. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT04915313.