Single-cell combined bioinformatics analysis: construction of immune cluster and risk prognostic model in kidney renal clear cells based on CD8+ T cell-associated genes.

BACKGROUND: Kidney cancer is an immunogenic solid tumor, characterized by high tumor burden and infiltration of CD8+ T cells. Although immunotherapy targeting the PD1/CTLA-4 axis has demonstrated excellent clinical efficacy, clinical outcomes in most patients are poor. METHODS: We used the RNA sequencing data from the GEO database for KIRC GSE121636 and normal kidney tissue GSE131685, and performed single-cell analysis for cluster identification, pathway enrichment, and CD8+ T cell-associated gene identification. Subsequently, the significance of different CD8+ T-cell associated gene subtypes was elucidated by consensus clustering, pathway analysis, mutated gene analysis, and KIRC immune microenvironment analysis in the TCGA-KIRC disease cohort. Single gene analysis identified LAG3 as the most critical CD8+ T-cell-associated gene and its function was verified by cell phenotype and immunohistochemistry in KIRC. RESULTS: In the present study, CD8+ T-cell associated genes in KIRC were screened, including GZMK, CD27, CCL4L2, FXYD2, LAG3, RGS1, CST7, DUSP4, CD8A, and TRBV20-1 and an immunological risk prognostic model was constructed (risk score = - 0.291858656434841*GZMK - 0.192758342489394*FXYD2 + 0.625023643446193*LAG3 + 0.161324477181591*RGS1 - 0.380169045328895*DUSP4 - 0.107221347575037*TRBV20-1). LAG3 was identified and proved as the most critical CD8+ T cell-associated gene in KIRC. CONCLUSION: We proposed and constructed an immunological risk prognostic model for CD8+ T cell-associated genes and identified LAG3 as a pivotal gene for KIRC progression and CD8+ T-cell infiltration. The model comprehensively explained the immune microenvironment and provided novel immune-related therapeutic targets and biomarkers in KIRC.

Single-cell disulfidptosis regulator patterns guide intercellular communication of tumor microenvironment that contribute to kidney renal clear cell carcinoma progression and immunotherapy.

Background: Disulfidptosis, an emerging type of programmed cell death, plays a pivotal role in various cancer types, notably impacting the progression of kidney renal clear cell carcinoma (KIRC) through the tumor microenvironment (TME). However, the specific involvement of disulfidptosis within the TME remains elusive. Methods: Analyzing 41,784 single cells obtained from seven samples of KIRC through single-cell RNA sequencing (scRNA-seq), this study employed nonnegative matrix factorization (NMF) to assess 24 disulfidptosis regulators. Pseudotime analysis, intercellular communication mapping, determination of transcription factor activities (TFs), and metabolic profiling of the TME subgroup in KIRC were conducted using Monocle, CellChat, SCENIC, and scMetabolism. Additionally, public cohorts were utilized to predict prognosis and immune responses within the TME subgroup of KIRC. Results: Through NMF clustering and differential expression marker genes, fibroblasts, macrophages, monocytes, T cells, and B cells were categorized into four to six distinct subgroups. Furthermore, this investigation revealed the correlation between disulfidptosis regulatory factors and the biological traits, as well as the pseudotime trajectories of TME subgroups. Notably, disulfidptosis-mediated TME subgroups (DSTN+CD4T-C1 and FLNA+CD4T-C2) demonstrated significant prognostic value and immune responses in patients with KIRC. Multiple immunohistochemistry (mIHC) assays identified marker expression within both cell clusters. Moreover, CellChat analysis unveiled diverse and extensive interactions between disulfidptosis-mediated TME subgroups and tumor epithelial cells, highlighting the TNFSF12-TNFRSF12A ligand-receptor pair as mediators between DSTN+CD4T-C1, FLNA+CD4T-C2, and epithelial cells. Conclusion: Our study sheds light on the role of disulfidptosis-mediated intercellular communication in regulating the biological characteristics of the TME. These findings offer valuable insights for patients with KIRC, potentially guiding personalized immunotherapy approaches.

Recombinant Toxoplasma gondii Calreticulin protein provides partial protection against acute and chronic toxoplasmosis.

Toxoplasma gondii, a highly prevalent apicomplexan pathogen, can cause serious or even fatal toxoplasmosis in both animals and humans. Immunoprophylaxis is considered a promising strategy for controlling this disease. Calreticulin (CRT) is known as a pleiotropic protein, which is critical for calcium storage and phagocytosis of apoptotic cells. Our study examined the protective effects of recombinant T. gondii Calreticulin (rTgCRT) as a recombinant subunit vaccine against the T. gondii challenge in mice. Here, rTgCRT was successfully expressed in vitro using prokaryptic expression system. Polyclonal antibody (pAb) has been prepared by immunizing Sprague Dawley rats with rTgCRT. Western blotting showed that rTgCRT and natural TgCRT protein were recognized by serum of T. gondii infected mice and rTgCRT pAb, respectively. T lymphocyte subsets and antibody response were monitored using flow cytometry and enzyme-linked immunosorbent assay (ELISA). The results showed that ISA 201 rTgCRT could stimulate lymphocyte proliferation and induce high levels of total and subclasses of IgG. After the RH strain challenge, a longer survival period was given by the ISA 201 rTgCRT vaccine compared to the control groups; after infection with the PRU strain, we observed a 100% survival rate and a significant reduction in cysts load and size. In the neutralization test, high concentrations of rat-rTgCRT pAb provided 100% protection, while in the passive immunization trial, only weak protection was observed after RH challenge, indicating that rTgCRT pAb needs further modification to improve its activity in vivo. Taken together, these data confirmed that rTgCRT can trigger strong cellular and humoral immune responses against acute and chronic toxoplasmosis.

Preparation and formation mechanism of biomass-based graphite carbon catalyzed by iron nitrate under a low-temperature condition.

Graphite is a widely used industrial material, which experienced a marked shortage caused by the growing demand for electrode anode material and the increased costs for raw material. Graphitic carbon from biomass is a promising approach that will result in low-cost and efficient preparation. Herein, Fe(NO3)3 was selected as the catalyst for pine sawdust, and the effects of temperature and iron content on the graphitization of biochar were investigated. Additionally, the formation mechanism of the graphitic crystallite structure was explored. Results showed that the formation of pyrolysis gas increased with the increase in the amount of catalyst added or pyrolysis temperature. The change in pyrolysis gas, such as H2 and CO, was a critical auxiliary factor reflecting the conversion process. As temperature was increased from 600 °C to 800 °C, the solid products showed high graphitization and low solid yield. Graphite structure mainly formed at 700 °C because of the formation of Fe nanoparticles. The increase in the amount of catalyst could provide more reaction sites and promote the contact between Fe and C, showing that amorphous carbon is dissolved on Fe nanoparticles and precipitated into ordered graphitic carbon. On this basis, a mechanism of "carbon dissolution-precipitation" was proposed to explain the formation of graphite structure, and the whole pyrolysis process included the transformation of the iron element were analyzed.

Plastic leachates lead to long-term toxicity in fungi and promote biodegradation of heterocyclic dye.

The hazardous effects of plastic and plastic leachates on organisms, even bacteria, have attracted widespread attention, but only a limited effort has been devoted to explore the response of fungi to plastic leachate induced by light irradiation. Here, we performed plastic leaching experiments to obtain leachates from polyethylene (PE), polyethylene terephthalate (PET) and polypropylene (PP), and optical properties of plastic leachates were analysed to determine the influence of light conditions and plastic materials on that. The effects of plastic leachates on the production of fungal enzyme and the biodegradation of heterocyclic dye by fungi were evaluated. Results indicated that the UV light greatly enhanced the release of leachates from the three plastics. Both plastic polymers and light irradiation affected the plastic-derived dissolved organic carbon (DOC) and their aromaticity, but the molecular weight of plastic leachates showed no dependency on light irradiation types, and PE was the easiest to photo age and leached more DOC. Plastic leachates had no dose-effect on the production of extracellular enzymes by fungi. PE leachates showed long-term toxicities to fungi, and no manganese peroxidase activities were detected after a 42-day incubation, while that of controls were up to 73.64 ± 8.81 U/L. However, the PE and PP leachates greatly promoted methylene blue degradation by the fungi, but PET leachates relieved the decolouration of methylene blue, probably because of the benzene ring structure in the PET monomer. Fusarium oxysporum had a stronger degradation ability than Phanerochaete chrysosporium. Our results indicate that plastic leachates can influence the production and secretion of fungi ligninolytic extracellular enzymes, and regulate the fungal degradation of heterocyclic dye.