Identification of prognosis-related gene features in low-grade glioma based on ssGSEA

Low-grade gliomas (LGG) are commonly seen in clinical practice, and the prognosis is often poor. Therefore, the determination of immune-related risk scores and immune-related targets for predicting prognoses in patients with LGG is crucial. A single-sample gene set enrichment analysis (ssGSEA) was performed on 22 immune gene sets to calculate immune-based prognostic scores. The prognostic value of the 22 immune cells for predicting overall survival (OS) was assessed using the least absolute shrinkage and selection operator (LASSO) and univariate and multivariate Cox analyses. Subsequently, we constructed a validated effector T-cell risk score (TCRS) to identify the immune subtypes and inflammatory immune features of LGG patients. We divided an LGG patient into a high-risk–score group and a low-risk–score group based on the optimal cutoff value. Kaplan–Meier survival curve showed that patients in the low-risk–score group had higher OS. We then identified the differentially expressed genes (DEGs) between the high-risk–score group and low-risk-score group and obtained 799 upregulated genes and 348 downregulated genes. The analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) show that DEGs were mainly concentrated in immune-related processes. In order to further explore the immune-related genes related to prognosis, we constructed a protein–protein interaction (PPI) network using Cytoscape and then identified the 50 most crucial genes. Subsequently, nine DEGs were found to be significantly associated with OS based on univariate and multivariate Cox analyses. It was further confirmed that CD2, SPN, IL18, PTPRC, GZMA, and TLR7 were independent prognostic factors for LGG through batch survival analysis and a nomogram prediction model. In addition, we used an RT-qPCR assay to validate the bioinformatics results. The results showed that CD2, SPN, IL18, PTPRC, GZMA, and TLR7 were highly expressed in LGG. Our study can provide a reference value for the prediction of prognosis in LGG patients and may help in the clinical development of effective therapeutic agents

Immune Evasion Strategies of Pre-Erythrocytic Malaria Parasites

Malaria is a mosquito-borne infectious disease of humans. It begins with a bite from an infected female Anopheles mosquito and leads to the development of the pre-erythrocytic and blood stages. Blood-stage infection is the exclusive cause of clinical symptoms of malaria. In contrast, the pre-erythrocytic stage is clinically asymptomatic and could be an excellent target for preventive therapies. Although the robust host immune responses limit the development of the liver stage, malaria parasites have also evolved strategies to suppress host defenses at the pre-erythrocytic stage. This paper reviews the immune evasion strategies of malaria parasites at the pre-erythrocytic stage, which could provide us with potential targets to design prophylactic strategies against malaria

Divergent roles of amino acid residues inside and outside the BB loop affect human Toll-like receptor (TLR)2/2, TLR2/1 and TLR2/6 responsiveness.

TLR2 specifically recognizes a wide range of ligands by homodimerizing or heterodimerizing with TLR1 or TLR6. However, the molecular basis of the specific signalling transduction induced by TLR2 homodimerization or heterodimerization with TLR1 or TLR6 is largely unknown. In this study, we found three amino acid residues, two (663L and 688N) outside and one (681P) inside the BB loop, which were conserved in all of the TLRs, except for the TLR3 toll/IL-1R(TIR) domain. The responsiveness of human TLR2/2, TLR2/1 or TLR2/6 was completely lost when 663L and 688N were replaced with the corresponding amino acid residues in the TLR3 TIR domain, respectively. However, the response of TLR2 (P681A) to the high concentration of TLR2/TLR6 agonist was almost intact, but the activity of TLR2 (P681A) was greatly reduced when stimulated with the TLR2/1 agonist or the TLR2/2 agonist. Although the surface expression of TLR2 (L663E) was sharply reduced, both the intracellular distribution and the surface expression of all of the other TLR2 mutants were unchanged. The ability of all three TLR2 mutants to recruit MyD88, was consistent with their responsivenesses. Computer modelling indicated that the surface negative charge of all of the TLR2 mutants' BB loops was reduced. Thus, our data demonstrated that the 663L and 688N residues outside of the BB loop were essential for the responsiveness of TLR2/2, TLR2/1 and TLR2/6, but the 681P residue inside of the BB loop exhibited divergent roles in TLR2/2, TLR2/1 and TLR2/6 signalling transduction, thereby providing clues regarding the specific signalling transduction of TLR2/2, TLR2/1 and TLR2/6

Electrostatic charge and conformation of the BB and CD loop were compared between wild-type TLR2 and its mutants.

<p>A, the conformation of the BB and CD loop in wild-type TLR2, TLR2 (L663E), TLR2 (P681A) and TLR2 (N688A) was compared, and the mutated residues L663E, P681A and N688A were stick represented. B, comparison of the electrostatic surfaces of wild-type TLR2, TLR2 (L663E), TLR2 (P681A) and TLR2 (N688A), the change of positive charge (<i>blue</i>) and negative charge (<i>red</i>) in the BB loop was indicated by a circle, and the mutated residues L663E, P681A and N688A were indicated by an arrow.</p

The ability of all of the TLR2 mutants to recruit MyD88.

<p>HEK293T cells were co-transfected with empty vector pFLAG-CMV8, FLAG-tagged wild-type TLR2 or each of the TLR2 mutants, along with MyD88-HA. At 24 h post-transfection, the cells were stimulated with LTA, Pam3CSK4 or FSL for 30 min. The cells were lysed, and the extracts were immunoprecipitated by anti-FLAG conjugated beads. TLR2 and MyD88 were subsequently detected in the immunoprecipitated proteins by western blot with an anti-FLAG antibody and an anti-HA polyclonal antibody. The quantity of MyD88 in the whole cell lysates was also detected with the anti-HA polyclonal antibody. The experiment was repeated for three times, and one was represented.</p

Responsiveness of human TLR2 (P681A) to TLR2/2, TLR2/1 and TLR2/6 agonists.

<p>HEK293T cells in 24-well plates were transfected with TLR2 (P681A), TK-RL and pBIIx-luc. At 24 h post-transfection, the cells were stimulated with the indicated concentrations of LTA (A), Pam3CSK4 (B) and FSL (C) for 6 h, respectively, and then both firefly and Renilla luciferase activities were determined using a dual-luciferase assay. The experiments were repeated for three times, and all of the data were expressed as the mean ± SD. *<i>P</i><0.05, ** <i>P</i><0.01, ns, not significant.</p

Expression and intracellular distribution of the human TLR2 mutants.

<p>HEK293T cells in 24-well plates were transfected with each indicated mutant and analysed 24 h later. A, the cells were stained with FITC-labelled anti-FLAG and DAPI and observed under confocal fluorescence microscopy. B, the surface expression of wild-type TLR2 (<i>solid line</i>) and each of the TLR2 mutants (<i>thin line</i>) on HEK293 was detected by FACS. C, the expression of wild-type TLR2 (<i>solid line</i>) and each of the TLR2 mutants (<i>thin line</i>) was detected following permeabilisation of the cell membrane. D, the expression of each of the TLR2 mutants was determined by western blot, with β-actin as an internal control. Each experiment was repeated for three times, and one was represented.</p

Responsiveness of human TLR2 (N657E), TLR2 (L663E) and TLR2 (N688A) to TLR2/2, TLR2/1 and TLR2/6 agonists.

<p>HEK293T cells in 24-well plates were transfected with human TLR2 (N657E), TLR2 (L663E) or TLR2 (N688A), and TK-RL and pBIIx-luc. At 24 h post-transfection, the cells were stimulated with the indicated concentrations of LTA (A), Pam3CSK4 (B) and FSL (C) for 6 h, respectively, and then both firefly and Renilla luciferase activities were determined using a dual-luciferase assay. The experiments were repeated for three times, and all of the data were expressed as the mean ± SD. * indicated as <i>p</i><0.05.</p

Conserved amino acid residues in all but not human TLR3 TIR domain and their location in the crystal structure of human TLR2 TIR.

<p>A, All of the TIR domains, including human TLR2, TLR3, TLR1, TLR6, TLR4, TLR5, TLR7, TLR8,TLR9 and TLR10, were aligned. The sequences responsible for the structure of αA, βA, αB, and the amino acid residues specific for human TLR3 TIR and one (657 E) specific for TLR3, but semi-conserved in all other human TLRs TIRs were underlined. The BB loop was indicated as the sequence between βA and αB, and three conserved amino acid residues in all of the TLRs, except for the TLR3 TIR domain, such as L, P and N, were indicated as“*”. The numbers indicate the positions of the amino acid residues in human TLR2. B, Stick representation of all three conserved amino acid Leu (663), Pro (681) and Asn (688), and highlighting BB loop (<i>pink</i>) in the cystal structure of human TLR2 TIR domain.</p