GhCTSF1, a short PPR protein with a conserved role in chloroplast development and photosynthesis, participates in intron splicing of rpoC1 and ycf3-2 transcripts in cotton.

Cotton is one of the most important textile fibers worldwide. As crucial agronomic traits, leaves play an essential role in the growth, disease resistance, fiber quality, and yield of cotton plants. Pentatricopeptide repeat (PPR) proteins are a large family of nuclear-encoded proteins involved in organellar or nuclear RNA metabolism. Using a virus-induced gene silencing assay, we found that cotton plants displayed variegated yellow leaf phenotypes with decreased chlorophyll content when expression of the PPR gene GhCTSF1 was silenced. GhCTSF1 encodes a chloroplast-localized protein that contains only two PPR motifs. Disruption of GhCTSF1 substantially reduces the splicing efficiency of rpoC1 intron 1 and ycf3 intron 2. Loss of function of the GhCTSF1 ortholog EMB1417 causes splicing defects in rpoC1 and ycf3-2, leading to impaired chloroplast structure and decreased photosynthetic rates in Arabidopsis. We also found that GhCTSF1 interacts with two splicing factors, GhCRS2 and GhWTF1. Defects in GhCRS2 and GhWTF1 severely affect intron splicing of rpoC1 and ycf3-2 in cotton, leading to defects in chloroplast development and a reduction in photosynthesis. Our results suggest that GhCTSF1 is specifically required for splicing rpoC1 and ycf3-2 in cooperation with GhCRS2 and GhWTF1.

Simulation and Optimization: A New Direction in Supercritical Technology Based Nanomedicine.

In recent years, nanomedicines prepared using supercritical technology have garnered widespread research attention due to their inherent attributes, including structural stability, high bioavailability, and commendable safety profiles. The preparation of these nanomedicines relies upon drug solubility and mixing efficiency within supercritical fluids (SCFs). Solubility is closely intertwined with operational parameters such as temperature and pressure while mixing efficiency is influenced not only by operational conditions but also by the shape and dimensions of the nozzle. Due to the special conditions of supercriticality, these parameters are difficult to measure directly, thus presenting significant challenges for the preparation and optimization of nanomedicines. Mathematical models can, to a certain extent, prognosticate solubility, while simulation models can visualize mixing efficiency during experimental procedures, offering novel avenues for advancing supercritical nanomedicines. Consequently, within the framework of this endeavor, we embark on an extensive review encompassing the application of mathematical models, artificial intelligence (AI) methodologies, and computational fluid dynamics (CFD) techniques within the medical domain of supercritical technology. We undertake the synthesis and discourse of methodologies for calculating drug solubility in SCFs, as well as the influence of operational conditions and experimental apparatus upon the outcomes of nanomedicine preparation using supercritical technology. Through this comprehensive review, we elucidate the implementation procedures and commonly employed models of diverse methodologies, juxtaposing the merits and demerits of these models. Furthermore, we assert the dependability of employing models to compute drug solubility in SCFs and simulate the experimental processes, with the capability to serve as valuable tools for aiding and optimizing experiments, as well as providing guidance in the selection of appropriate operational conditions. This, in turn, fosters innovative avenues for the development of supercritical pharmaceuticals.

Construction of pseudorabies virus variant attenuated vaccine: codon deoptimization of US3 and UL56 genes based on PRV gE/TK deletion strain.

Since 2011, pseudorabies based on the pseudorabies virus (PRV) variant has emerged as a serious health issue in pig farms in China. The PRV gE/TK or gE/gI/TK deletion strains protect against emerging PRV variants. However, these variants may cause lethal infections in newborn piglets without PRV antibodies. Previous studies have shown that codon deoptimization of a virulence gene causes virus attenuation. Accordingly, we deoptimized US3-S (US3 gene encoding a short isoform that represents approximately 95% of the total US3 transcription) and UL56 genes (first 10 or all codons) of PRV gE/TK deletion strain (PRVΔTK&gE-AH02) to generate six recombinant PRVs through bacterial artificial chromosome technology. In swine testicular cells, recombinant PRVs with all codon deoptimization of US3-S or UL56 genes were grown to lower titers than the parental virus. Notably, US3-S or UL56 with all codon deoptimization reduced mRNA and protein expressions. Subsequently, the safety and immunogenicity of recombinant PRVs with codon deoptimization of US3-S or UL56 are evaluated as vaccine candidates in mice and piglets. The mice inoculated with recombinant PRVs with codon deoptimization of US3-S or UL56 showed exceptional survival ability without severe clinical signs. All codons deoptimized (US3-S and UL56) significantly decreased virus load and attenuated pathological changes in the brains of the mice. Moreover, the protection efficiency offered by recombinant PRVs with codon deoptimization of US3-S or UL56 showed similar effects to PRVΔTK&gE-AH02. Remarkably, the 1-day-old PRV antibody-negative piglets inoculated with PRVΔTK&gE-US3-ST-CD (a recombinant PRV with all codon deoptimization of US3-S) presented no abnormal clinical symptoms, including fever. The piglets inoculated with PRVΔTK&gE-US3-ST-CD showed a high serum neutralization index against the PRV variant. In conclusion, these results suggest using codon deoptimization to generate innovative live attenuated PRV vaccine candidates.

Bioengineered nanotechnology for nucleic acid delivery.

Nucleic acid-based therapy has emerged as a promising therapeutic approach for treating various diseases, such as genetic disorders, cancers, and viral infections. Diverse nucleic acid delivery systems have been reported, and some, including lipid nanoparticles, have exhibited clinical success. In parallel, bioengineered nucleic acid delivery nanocarriers have also gained significant attention due to their flexible functional design and excellent biocompatibility. In this review, we summarize recent advances in bioengineered nucleic acid delivery nanocarriers, focusing on exosomes, cell membrane-derived nanovesicles, protein nanocages, and virus-like particles. We highlight their unique features, advantages for nucleic acid delivery, and biomedical applications. Furthermore, we discuss the challenges that bioengineered nanocarriers face towards clinical translation and the possible avenues for their further development. This review ultimately underlines the potential of bioengineered nanotechnology for the advancement of nucleic acid therapy.

An unresectable and metastatic intrahepatic cholangiocarcinoma with EML4-ALK rearrangement achieving partial response after first-line treatment with ensartinib: a case report.

Systemic chemotherapies are the primary treatment options for patients with unresectable and metastatic intrahepatic cholangiocarcinoma (ICC), but the effectiveness of current systemic therapies is limited. The development of targeted-therapy has changed the treatment landscape of ICC, and comprehensive genome sequencing of advanced cholangiocarcinoma patients could be beneficial to identify potential targets to guide individualized treatment. Herein, we reported an unresectable and metastatic ICC patient who detected EML4-ALK rearrangement in peripheral blood, which was later confirmed on tissue-based testing, and achieved partial response (PR) after first-line treatment with ensartinib. This case suggests that the liquid biopsy is of clinical value for unresectable or metastatic ICC, and the discovery of rare molecular targets provides new therapeutically approaches for advanced ICC patients.

A Novel Strategy of US3 Codon De-Optimization for Construction of an Attenuated Pseudorabies Virus against High Virulent Chinese Pseudorabies Virus Variant.

In this study, we applied bacterial artificial chromosome (BAC) technology with PRVΔTK/gE/gI as the base material to replace the first, central, and terminal segments of the US3 gene with codon-deoptimized fragments via two-step Red-mediated recombination in E. coli GS1783 cells. The three constructed BACs were co-transfected with gI and part of gE fragments carrying homologous sequences (gI+gE'), respectively, in swine testicular cells. These three recombinant viruses with US3 codon de-optimization ((PRVΔTK&gE-US3deop-1, PRVΔTK&gE-US3deop-2, and PRVΔTK&gE-US3deop-3) were obtained and purified. These three recombinant viruses exhibited similar growth kinetics to the parental AH02LA strain, stably retained the deletion of TK and gE gene fragments, and stably inherited the recoded US3. Mice were inoculated intraperitoneally with the three recombinant viruses or control virus PRVΔTK&gEAH02 at a 107.0 TCID50 dose. Mice immunized with PRVΔTK&gE-US3deop-1 did not develop clinical signs and had a decreased virus load and attenuated pathological changes in the lungs and brain compared to the control group. Moreover, immunized mice were challenged with 100 LD50 of the AH02LA strain, and PRVΔTK&gE-US3deop-1 provided similar protection to that of the control virus PRVΔTK&gEAH02. Finally, PRVΔTK&gE-US3deop-1 was injected intramuscularly into 1-day-old PRV-negative piglets at a dose of 106.0 TCID50. Immunized piglets showed only slight temperature reactions and mild clinical signs. However, high levels of seroneutralizing antibody were produced at 14 and 21 days post-immunization. In addition, the immunization of PRVΔTK&gE-US3deop-1 at a dose of 105.0 TCID50 provided complete clinical protection and prevented virus shedding in piglets challenged by 106.5 TCID50 of the PRV AH02LA variant at 1 week post immunization. Together, these findings suggest that PRVΔTK&gE-US3deop-1 displays great potential as a vaccine candidate.

Advances in nanomedicines for lymphatic imaging and therapy.

Lymph nodes play a pivotal role in tumor progression as key components of the lymphatic system. However, the unique physiological structure of lymph nodes has traditionally constrained the drug delivery efficiency. Excitingly, nanomedicines have shown tremendous advantages in lymph node-specific delivery, enabling distinct recognition and diagnosis of lymph nodes, and hence laying the foundation for efficient tumor therapies. In this review, we comprehensively discuss the key factors affecting the specific enrichment of nanomedicines in lymph nodes, and systematically summarize nanomedicines for precise lymph node drug delivery and therapeutic application, including the lymphatic diagnosis and treatment nanodrugs and lymph node specific imaging and identification system. Notably, we delve into the critical challenges and considerations currently facing lymphatic nanomedicines, and futher propose effective strategies to address these issues. This review encapsulates recent findings, clinical applications, and future prospects for designing effective nanocarriers for lymphatic system targeting, with potential implications for improving cancer treatment strategies.

Classification of genotypes based on the VP1 gene of feline calicivirus and study of cross-protection between different genotypes.

Feline calicivirus (FCV) causes upper respiratory tract diseases and even death in cats, thereby acting as a great threat to feline animals. Currently, FCV prevention is mainly achieved through vaccination, but the effectiveness of vaccination is limited. In this study, 105 FCV strain VP1 sequences with clear backgrounds were downloaded from the NCBI and subjected to a maximum likelihood method for systematic evolutionary analysis. Based on the genetic analysis results, FCV-positive sera were prepared using SPF mice and Chinese field cats as target animals, followed by a cross-neutralization assay conducted on the different genotype strains and in vivo challenge tests were carried out to further verify with the strain with best cross-protection effect. The results revealed that FCV was mainly divided into two genotypes: GI and GII. The GI genotype strains are prevalent worldwide, but all GII genotype strains were isolated from Asia, indicating a clear geographical feature. This may form resistance to FCV prevention in Asia. The in vitro neutralization assay conducted using murine serum demonstrated that the cross-protection effect varied among strains. A strain with broad-spectrum neutralization properties, DL39, was screened. This strain could produce neutralizing titers (10 × 23.08-10 × 20.25) against all strains used in this study. The antibody titers against the GI strains were 10 × 23.08-10 × 20.5 and those against the GII strains were 10 × 20.75-10 × 20.25. Preliminary evidence suggested that the antibody titer of the DL39 strain against GI was higher than that against GII. Subsequent cross-neutralization assays with cat serum prepared with the DL39 strain and each strain simultaneously yielded results similar to those described above. In vivo challenge tests revealed that the DL39 strain-immunized cats outperformed the positive controls in all measures. The results of several trials demonstrated that strain DL39 can potentially be used as a vaccine strain. The study attempted to combine the genetic diversity and phylogenetic analysis of FCV with the discovery of potential vaccines, which is crucial for developing highly effective FCV vaccines.

Synthesis of Sulfilimines via Multicomponent Reaction of Arynes, Sulfamides, and Thiosulfonates.

In this work, a facile and efficient method for the synthesis of sulfilimines through multicomponent reaction of arynes, sulfamides, and thiosulfonates was developed. A variety of structurally diverse substrates and functional groups were very compatible in the reaction, giving the corresponding sulfilimines in good to high yields. This protocol could be conducted on a gram scale, and the product was easily converted to sulfide and sulfoximine. Mechanism studies revealed that sulfenamide generated in situ is the key intermediate for the reaction.

Selenite affected photosynthesis of Oryza sativa L. exposed to antimonite: Electron transfer, carbon fixation, pigment synthesis via a combined analysis of physiology and transcriptome.

Selenium (Se) is a microelement that can counteract (a)biotic stresses in plants. Excess antimony (Sb) will inhibit plant photosynthesis, which can be alleviated by appropriate doses of Se but the associated mechanisms at the molecular levels have not been fully explored. Here, a rice variety (Yongyou 9) was exposed to selenite [Se(IV), 0.2 and 0.8 mg L-1] alone or combined with antimonite [Sb(III), 5 and 10 mg L-1]. When compared to the 10 mg L-1 Sb treatment alone, addition of Se in a dose-dependent manner 1) reduced the heat dissipation efficiency resulting from the inhibited donors, Sb concentrations in shoots and roots, leaf concentrations of fructose, H2O2 and O2•-; 2) enhanced heat dissipation efficiency resulting from the inhibited accepters value, concentrations of Chl a, sucrose and starch, and the enzyme activity of adenosine diphosphate glucose pyrophosphorylase, sucrose phosphate synthase, and sucrose synthase; but 3) did not alter gas exchange parameters, concentrations of Chl b and total Chl, enzyme activity of soluble acid invertase, and values of maximum P700 signal, photochemical efficiency of PSI and electron transport rate of PSI. Se alleviated the damage caused by Sb to the oxygen-evolving complex and promoted the transfer of electrons from QA to QB. When compared to the 10 mg L-1 Sb treatment alone, addition of Se 1) up-regulated genes correlated to synthesis pathways of Chl, carotenoid, sucrose and glucose; 2) disturbed signal transduction pathway of abscisic acid; and 3) upregulated gene expression correlated to photosynthetic complexes (OsFd1, OsFER1 and OsFER2).

Intensive cycles of neoadjuvant camrelizumab combined with chemotherapy in locally advanced esophageal squamous cell carcinoma: a single-arm, phase II trial.

BACKGROUND: Two cycles of neoadjuvant PD-1 blockade plus chemotherapy induced favorable pathological response and tolerant toxicity in patients with locally advanced esophageal squamous cell carcinoma (ESCC). However, approximately 25% of patients relapsed within 1 year after surgery, indicating that a short course of treatment may not be sufficient. Therefore, exploring the effects of intensive treatment is needed for optimal clinical outcomes. METHODS: Locally advanced ESCC patients were administered three cycles of camrelizumab plus nab-paclitaxel and capecitabine, followed by thoracoscopic esophagectomy. The primary endpoint was pathologic response. Secondary endpoints included safety, feasibility, radiologic response, survival outcomes, and immunologic/genomic correlates of efficacy. RESULTS: Forty-seven patients were enrolled in the study. Forty-two patients received surgery, and R0 resection was achieved in all cases. The complete and major pathological response rates were 33.3% and 64.3%, respectively, and the objective response rate was 80.0%. Three cycles of treatment significantly improved T down-staging compared to two cycles (P = 0.03). The most common treatment-related adverse events were grades 1-2, and no surgical delay was reported. With a median follow-up of 24.3 months, the 1-year disease-free survival and overall survival rates were both 97.6%, and the 2-year disease-free survival and overall survival rates were 92.3% and 97.6%, respectively. Three patients experienced disease recurrence or metastasis ranging from 12.5 to 25.8 months after surgery, and one patient died 6 months after surgery due to cardiovascular disease. Neither programmed death-ligand 1 expression nor tumor mutational burden was associated with pathological response. An increased infiltration of CD56dim natural killer cells in the pretreatment tumor was correlated with better pathological response in the primary tumor. CONCLUSIONS: It seems probable that intensive cycles of neoadjuvant camrelizumab plus nab-paclitaxel and capecitabine increased tumor regression and improved survival outcomes. Randomized controlled trials with larger sample sizes and longer follow-up periods are needed to validate these findings. Trial registration Chinese Clinical Trial Registry, ChiCTR2000029807, Registered February 14, 2020, https://www.chictr.org.cn/showproj.aspx?proj=49459 .

A Refractory Case of CDKN2A/B Loss Metastatic Intrahepatic Cholangiocarcinoma Achieving a Partial Response After First-Line Treatment with Palbociclib.

Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive and malignant subtype of biliary duct tumors. The poor prognosis of advanced ICC brings great challenges to clinical treatment, and chemotherapy-based therapy remains the standard first-line regimen. In recent years, the development of clinical research on targeted therapy for biliary duct tumors has brought new strategies for clinical treatment, but the targets are limited. Herein, we reported a 68-year-old patient with metastasis ICC harboring CDKN2A/B loss, who achieved a partial response (PR) after the first-line treatment with a cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor called palbociclib, and no obvious side effects were observed. As of the latest follow-up time, the progression-free survival (PFS) had lasted for 20 months. This case reveals the molecular characteristic of ICC patients who respond to palbociclib treatment and illustrates the importance of performing a multiple-gene panel test in ICC patients.

Pmorf0222, a Virulence Factor in Pasteurella multocida, Activates Nuclear Factor Kappa B and Mitogen-Activated Protein Kinase via Toll-Like Receptor 1/2.

Pasteurella multocida primarily causes hemorrhagic septicemia and pneumonia in poultry and livestock. Identification of the relevant virulence factors is therefore essential for understanding its pathogenicity. Pmorf0222, encoding the PM0222 protein, is located on a specific prophage island of the pathogenic strain C48-1 of P. multocida. Its role in the pathogenesis of P. multocida infection is still unknown. The proinflammatory cytokine plays an important role in P. multocida infection; therefore, murine peritoneal exudate macrophages were treated with the purified recombinant PM0222, which induced the secretion of tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß) via the Toll-like receptor 1/2 (TLR1/2)-nuclear factor kappa B (NF-κB)/mitogen-activated protein kinase (MAPK) signaling and inflammasome activation. Additionally, the mutant strain and complemented strain were evaluated in the mouse model with P. multocida infection, and PM0222 was identified as a virulence factor, which was secreted by outer membrane vesicles of P. multocida. Further results revealed that Pmorf0222 affected the synthesis of the capsule, adhesion, serum sensitivity, and biofilm formation. Thus, we identified Pmorf0222 as a novel virulence factor in the C48-1 strain of P. multocida, explaining the high pathogenicity of this pathogenic strain.

Case report: an initially unresectable stage III pulmonary sarcomatoid carcinoma qith EGFR mutation achieving pathological complete response following neoadjuvant therapy with osimertinib plus chemotherapy.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) provide dramatic response to patients with advanced EGFR-mutant non-small cell lung cancer (NSCLC). However, the use of neoadjuvant therapy with EGFR-TKIs in EGFR-mutant NSCLC remains controversial, especially in pulmonary sarcomatoid carcinoma (PSC). One patient with initially unresectable stage III (cT4N0M0) PSC was found to carry EGFR mutation by the next generation sequencing. After neoadjuvant therapy with osimertinib plus chemotherapy, radical resection of the right upper lung lesion was achieved, and the pathological results reached pathological complete response (pCR). To the best of our knowledge, this is the first report of an EGFR-mutant patient with initially unresectable stage III PSC achieved pCR by neoadjuvant therapy with osimertinib plus chemotherapy. Therefore, neoadjuvant therapy with EGFR-TKIs may be a viable option for EGFR-mutant PSC patients.

Case report: Primary hepatocellular carcinoma with portal vein tumor thrombus characterized by active tumor immune microenvironment achieving a complete response following treatment of combined immunotherapy.

Portal vein tumor thrombus (PVTT) is a frequent complication in hepatocellular carcinoma (HCC). HCC patients with PVTT have the characteristics of less treatment tolerance and poor prognosis. Immunotherapy, especially combined immunotherapy, has been successfully used in advanced HCC. However, there are no recognized universally indicators that can predict response or resistance to immunotherapy for HCC. Herein, we reported a 58-year-old HCC patient with PVTT, cirrhosis and chronic viral hepatitis, who achieved complete response (CR) after combined immunotherapy (camrelizumab combined with sorafenib or regorafenib), according to his high enrichment of tumor-infiltrating immune cells and tertiary lymphoid structure (TLS). In this case, we revealed the characteristics of the baseline tumor immune microenvironment (TIME) in a HCC patient who responded well to combined immunotherapy, suggesting that TIME can be used to assist in clinical decision making of immunotherapy for HCC.

Construction of a Novel Infectious Clone of Recombinant Herpesvirus of Turkey Fc-126 Expressing VP2 of IBDV.

The increased virulence of infectious bursal disease virus (IBDV) is a threat to the chicken industry. The construction of novel herpesvirus of turkey-vectored (HVT) vaccines expressing VP2 of virulent IBDV may be a promising vaccine candidate for controlling this serious disease in chickens. We generated a novel infectious clone of HVT Fc-126 by inserting mini-F sequences in lieu of the glycoprotein C (gC) gene. Based on this bacterial artificial chromosome (BAC), a VP2 expression cassette containing the pMCMV IE promoter and a VP2 sequence from the virulent IBDV NJ09 strain was inserted into the noncoding area between the UL55 and UL56 genes to generate the HVT vector VP2 recombinant, named HVT-VP2-09. The recovered vectored mutant HVT-VP2-09 exhibited higher titers (p = 0.0202 at 36 h) or similar growth kinetics to the parental virus HVT Fc-126 (p = 0.1181 at 48 h and p = 0.1296 at 64 h). The high reactivation ability and strong expression of VP2 by HVT-VP2-09 in chicken embryo fibroblasts (CEFs) were confirmed by indirect immunofluorescence (IFA) and Western blotting. The AGP antibodies against IBDV were detected beginning at 3 weeks post-inoculation (P.I.) of HVT-VP2-09 in 1-day-old SPF chickens. Seven of ten chickens immunized with HVT-VP2-09 were protected post-challenge (P.C.) with the virulent IBDV NJ09 strain. In contrast, all chickens in the challenge control group showed typical IBD lesions in bursals, and eight of ten died P.C. In this study, we demonstrated that (i) a novel HVT BAC with the whole genome of the Fc-126 strain was obtained with the insertion of mini-F sequences in lieu of the gC gene; (ii) HVT-VP2-09 harboring the VP2 expression cassette from virulent IBDV exhibited in vitro growth properties similar to those of the parental HVT virus in CEF cells; and (iii) HVT-VP2-09 can provide efficient protection against the IBDV NJ09 strain.

Homologous recombination repair gene mutations as a predictive biomarker for immunotherapy in patients with advanced melanoma.

Background: Nowadays, immunotherapy targeting immune checkpoint receptors is one of the cornerstones of systemic treatment in melanoma. Homologous recombination repair (HRR) is one of the DNA damage response (DDR) pathways, which has been proved to correlate with the efficacy of platinum-based chemotherapy, PARP inhibitor therapy, and immunotherapy in a variety of cancers. However, their predictive value of HRR remained unknown in patients with advanced melanoma. Methods: Data of advanced melanoma patients from an independent cohort (Samstein2018) were used to analyze the correlation with immunogenic markers and the prognostic effect of HRR on immunotherapy, and another four cohorts (pooled cohort: Miao2018, Allen 2015, Hugo2016, and Synder2014) were used for validation. Immune infiltration cell scores analyzed by TCGA-SKCM cohort were used to explore potential mechanisms related to the immune microenvironment. Results: Compared to patients with an HRR wild type (HRRwt), those with HRR mutations (HRRmut) in anti-CTLA-4-treated patients of the Samstein2018 cohort had higher tumor mutation burden (TMB; P = 0.0041) and longer median overall survival (mOS; P = 0.0094). In terms of results validation, it was also confirmed that the mOS (P = 0.0014) of HRRmut patients receiving anti-CTLA-4 therapy was significantly better than that of HRRwt patients in the pooled cohort, and objective response rates (ORR; P = 0.0053) were also found to be significant. However, there was no significant difference in mOS between HRRmut patients who received anti-PD-1/L1 therapy and HRRwt patients in either the discovery (Samstein2018 cohort, P = 0.94) or validation (pooled cohort, P = 0.96) set. Exploratory analysis found that although HRRmut patients showed no significant difference in mOS between anti-CTLA-4 and anti-PD-1/L1 therapy (P = 0.79), the mOS value of the anti-CTLA-4 therapy group (31.7 months) in HRRmut patients was numerically superior to the anti-PD-1/L1 therapy group (27.5 months). In contrast, the mOS of the anti-CTLA-4 therapy group was significantly lower than that of the anti-PD-1/L1 therapy group (12.4 vs. 32.0 months) in HRRwt patients. In addition, transcriptome profiling analysis revealed that the 29 (65.9%)-gene mutation of the HRR pathway associated with reshaping of the immunological microenvironment in melanoma. Conclusions: HRR mutations were associated with a higher TMB level, and better anti-CTLA-4 therapy outcomes. HRR may serve as an independent predictor of anti-CTLA-4 therapy efficacy in patients with advanced melanoma and their clinical value warrants further investigation.

Combined analysis of T cell activation and T cell-mediated cytotoxicity by imaging cytometry.

Immunotherapies for the treatment of cancer have spurred the development of new drugs that seek to harness the ability of T cells to recognize and kill malignant cells. There is a substantial need to evaluate how these experimental drugs influence T cell functional outputs in co-culture systems that contain cancerous cells. We describe an imaging cytometry-based platform that can simultaneously quantify activated T cells and the capacity of these T cells to kill cancer cells. Our platform was developed using the Nur77-GFP reporter system because GFP expression provides a direct readout of T cell activation that is induced by T cell antigen receptor (TCR) signaling. We combined the Nur77-GFP reporter system with a cancer cell line that displays a TCR-specific antigen and evaluated the relationship between T cell activation and cancer cell death. We demonstrate that imaging cytometry can be used to quantify the number of activated cytotoxic CD8+ T cells (CTLs) and the capacity of these CTLs to recognize and kill adherent MC38 cancer cells. We tested whether this platform could evaluate heterogenous lymphocyte populations by quantifying the proportion of antigen-specific activated T cells in co-cultures that contain unresponsive lymphocytes. The effects of a SRC family kinase inhibitor on CTL activation and MC38 cell death were also determined. Our findings demonstrate that the Nur77-GFP reporter system can be used to evaluate the effects of diverse treatment conditions on T cell-cancer co-cultures in a microtiter plate-based format by imaging cytometry. We anticipate the combined analysis of T cell activation with T cell-mediated cancer cell death can be used to rapidly assess immuno-oncology drug candidates and T cell-based therapeutics.

A clinical trial of super-stable homogeneous lipiodol-nanoICG formulation-guided precise fluorescent laparoscopic hepatocellular carcinoma resection.

BACKGROUND: Applying traditional fluorescence navigation technologies in hepatocellular carcinoma is severely restricted by high false-positive rates, variable tumor differentiation, and unstable fluorescence performance. RESULTS: In this study, a green, economical and safe nanomedicine formulation technology was developed to construct carrier-free indocyanine green nanoparticles (nanoICG) with a small uniform size and better fluorescent properties without any molecular structure changes compared to the ICG molecule. Subsequently, nanoICG dispersed into lipiodol via a super-stable homogeneous intermixed formulation technology (SHIFT&nanoICG) for transhepatic arterial embolization combined with fluorescent laparoscopic hepatectomy to eliminate the existing shortcomings. A 52-year-old liver cancer patient was recruited for the clinical trial of SHIFT&nanoICG. We demonstrate that SHIFT&nanoICG could accurately identify and mark the lesion with excellent stability, embolism, optical imaging performance, and higher tumor-to-normal tissue ratio, especially in the detection of the microsatellite lesions (0.4 × 0.3 cm), which could not be detected by preoperative imaging, to realize a complete resection of hepatocellular carcinoma under fluorescence laparoscopy in a shorter period (within 2 h) and with less intraoperative blood loss (50 mL). CONCLUSIONS: This simple and effective strategy integrates the diagnosis and treatment of hepatocellular carcinoma, and thus, it has great potential in various clinical applications.