Journal: Advanced Science

Article Title: Melanoma Derived Exosomes Amplify Radiotherapy Induced Abscopal Effect via IRF7/I‐IFN Axis in Macrophages

doi: 10.1002/advs.202304991

Figure Lengend Snippet: CircPIK3R3/IRF7/I‐IFN axis participates in the combination of radiotherapy and anti‐PD1 mediated abscopal effect. A) The treatment model involving RT, anti‐PD1, and RO8191: C57/BL6 mice were subcutaneously inoculated with 1 × 106 sh‐NC B16F10 cells or sh‐circ‐0011074 B16F10 cells. On day 5, C57/BL6 mice were intravenously injected with 1 × 106 B16F10‐luc cells. Starting from day 6, mice were administered the IFN receptor agonist RO8191 via daily intraperitoneal injections at a dose of 1 mg kg −1 . On day 7, radiotherapy was initiated, with a daily dose of 8 Gy administered for 3 consecutive days. On day 7, mice were also administered anti‐PD1 via intraperitoneal injection every 2 days at a dose of 100 µg/mouse until the endpoint of observation. B,C) Measurement of subcutaneous tumor weight in each group to assess treatment efficacy ( n = 3). D,E) Evaluation of fluorescent intensity in lung metastatic foci using bioluminescence imaging to assess treatment efficacy ( n = 6). F,G) Immunohistochemical examination of CD8 + T cell infiltration in subcutaneous tumors and lung metastatic foci ( n = 3). H,I) Immunofluorescence detection of IRF7 + macrophage infiltration in subcutaneous tumors and lung metastatic foci (n = 3). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by two‐tailed unpaired Student t‐test.

Article Snippet: Anti‐mouse PD‐1 mAb (Bio X Cell, USA) was administered intraperitoneally (200 mg per mouse) every 2 days during treatment.

Techniques: Injection, Imaging, Immunohistochemical staining, Immunofluorescence, Two Tailed Test

Journal: Pathogens

Article Title: Exploration of the Binding Site of Arachidonic Acid in gp63 of Leishmania mexicana and in Orthologous Proteins in Clinically Important Parasites

doi: 10.3390/pathogens13090718

Figure Lengend Snippet: Role of the L. mexicana gp63 protein in COX-like activity. ( A ) The plasmid map highlights a mutated Nde I site in the L. mexicana His-tag-gp63 protein, with a new peptide indicated by a pink arrow produced by a different reading frame. ( B ) Changes in the NdeI site’s reading frame in amino acids are shown for both wild type and mutant proteins. The red box signifies the insertion of two nucleotides, resulting in a 262 amino acid product with a molecular weight of 29 kDa. ( C ) Western blot analysis of proteins from E. coli DH5α transformed with wild-type (pPROEX-gp63) and mutant (pPROEX-gp63fs+2NDeI) plasmids on a 10% SDS-PAGE gel. Plasmid pPROEX served as a control. Recombinant gp63 proteins were detected using a commercial anti-tag histidine antibody (1:1000) in the presence of IPTG. Supernatant = SN; pellet = P. ( D ) Recombinant protein purification from Triton-X100 solubilized pellets of rpLmxPROEX-gp63 and rpLmxPROEX-gp63fs+2NDeI transformed bacteria, followed by Ni2+ resin incubation and analysis via 15 % SDS-PAGE and Western blot. Lanes 1 and 2 correspond to two eluates of the protein and wild recombinant, and in lanes 1′ and 2′, two eluates corresponding to the mutant recombinant protein are shown. ( E ) Assessment of COX-like activity in wild-type and mutant constructs’ total extracts using a COX activity kit, with the vector serving as a negative control. These experiments were carried out in triplicate conducted in three independently performed biological assays. Purified recombinant proteins were concentrated and subjected to dialysis with renaturation buffer. ( F ) COX activity detected in purified recombinant proteins, with analyses conducted in triplicate across two independent experiments.

Article Snippet: The sample was then treated with 0.5% Triton for 20 min and blocked with 10% fetal bovine serum in PBS for 1 h. Samples were incubated overnight with α-gp63 antibody (CEDARLANE Laboratories Limited, Cat. No. CLP005A, Burlington, Ontario, Canada) at a 1:50 dilution at 4 °C.

Techniques: Activity Assay, Plasmid Preparation, Produced, Mutagenesis, Molecular Weight, Western Blot, Transformation Assay, SDS Page, Control, Recombinant, Protein Purification, Bacteria, Incubation, Construct, Negative Control, Purification

Journal: Pathogens

Article Title: Exploration of the Binding Site of Arachidonic Acid in gp63 of Leishmania mexicana and in Orthologous Proteins in Clinically Important Parasites

doi: 10.3390/pathogens13090718

Figure Lengend Snippet: A BLAST search for sequences like L. mexicana gp63 in different parasitic protozoa.

Article Snippet: The sample was then treated with 0.5% Triton for 20 min and blocked with 10% fetal bovine serum in PBS for 1 h. Samples were incubated overnight with α-gp63 antibody (CEDARLANE Laboratories Limited, Cat. No. CLP005A, Burlington, Ontario, Canada) at a 1:50 dilution at 4 °C.

Techniques:

Journal: Pathogens

Article Title: Exploration of the Binding Site of Arachidonic Acid in gp63 of Leishmania mexicana and in Orthologous Proteins in Clinically Important Parasites

doi: 10.3390/pathogens13090718

Figure Lengend Snippet: Analysis of RNA-seq data repositories from various parasites including L. mexicana (AMA = intracellular amastigotes; AXA = axenic amastigotes; PRO = promastigotes), T. cruzi , E. histolytica , E. dispar , E. invadens , A. castellanii , and N. fowleri . The figure illustrates the presence of transcripts per million (TPM) of mRNA corresponding to the gp63 protein in L. mexicana and orthologous proteins in the analyzed parasites. The TPM value indicates a relative expression level across the samples. Data for this analysis were from AmoebaDB: http://amoebadb.org/amoeba/ (accessed on 28 September 2023 and TriTrypDB: https://tritrypdb.org (accessed on 28 September 2023). Therefore, to establish a possible functional relationship between orthologous proteins with gp63, a multiple alignment was performed. A shows the alignment of the protein sequences of each parasite. The catalytic site region is indicated on a purple background, and the HExxHAxGF motif, which is conserved across the seven proteins analyzed, can be seen. The sequences of the candidate proteins were examined using the Pfam database to determine the presence of functional domains like the L. mexicana gp63 ( B). The results confirmed that all analyzed sequences share the same domain of the M8 peptidase family. This domain was reported to be present in L. mexicana gp63 . This enzyme is found in eukaryotes, including Leishmania and other protozoan parasites. A domain of the Transcription Factor Immunoglobin (TIG) family, called IPT/TIG, was identified in the E. histolytica protein. This domain is characterized by a fold like that of immunoglobulin and is found in tyrosine kinase receptors such as Met and Ron receptors. In addition, this domain is also present in transcription factors involved in DNA binding ( B).

Article Snippet: The sample was then treated with 0.5% Triton for 20 min and blocked with 10% fetal bovine serum in PBS for 1 h. Samples were incubated overnight with α-gp63 antibody (CEDARLANE Laboratories Limited, Cat. No. CLP005A, Burlington, Ontario, Canada) at a 1:50 dilution at 4 °C.

Techniques: RNA Sequencing, Expressing, Functional Assay, Binding Assay

Journal: Pathogens

Article Title: Exploration of the Binding Site of Arachidonic Acid in gp63 of Leishmania mexicana and in Orthologous Proteins in Clinically Important Parasites

doi: 10.3390/pathogens13090718

Figure Lengend Snippet: A phylogenetic tree was constructed for proteins similar to gp63 from L. mexicana . The tree was generated using the complete amino acid sequences of the studied proteins via the neighbor-joining method, employing the phylogenetic inference package (PHYLIP) version 3.5c. The construction utilized the amino acid sequence of gp63 from L. mexicana along with orthologous sequences from various parasites, including L. mexicana (XP_003872886.1), T. cruzi (XP_817808.1), E. histolytica (XP_652632.1), E. dispar (XP_001740726.1), E. invadens (XP_004184102.1), A. castellanii (XP_004337275.1), and N. fowleri (XP_044566011.1).

Article Snippet: The sample was then treated with 0.5% Triton for 20 min and blocked with 10% fetal bovine serum in PBS for 1 h. Samples were incubated overnight with α-gp63 antibody (CEDARLANE Laboratories Limited, Cat. No. CLP005A, Burlington, Ontario, Canada) at a 1:50 dilution at 4 °C.

Techniques: Construct, Generated, Sequencing

Journal: Pathogens

Article Title: Exploration of the Binding Site of Arachidonic Acid in gp63 of Leishmania mexicana and in Orthologous Proteins in Clinically Important Parasites

doi: 10.3390/pathogens13090718

Figure Lengend Snippet: Predicted functions of gp63 homologues in Trypanosomatids and Amoeba species.

Article Snippet: The sample was then treated with 0.5% Triton for 20 min and blocked with 10% fetal bovine serum in PBS for 1 h. Samples were incubated overnight with α-gp63 antibody (CEDARLANE Laboratories Limited, Cat. No. CLP005A, Burlington, Ontario, Canada) at a 1:50 dilution at 4 °C.

Techniques: Activity Assay, Clinical Proteomics, Membrane, Binding Assay

Journal: Pathogens

Article Title: Exploration of the Binding Site of Arachidonic Acid in gp63 of Leishmania mexicana and in Orthologous Proteins in Clinically Important Parasites

doi: 10.3390/pathogens13090718

Figure Lengend Snippet: Comparing the three-dimensional structures of proteins orthologous to L. mexicana gp63. The 3D structures of the analyzed proteins were modeled using the Swiss model. Subsequently, the overlap analysis of these structures was conducted using TopMatch-web. In the visual representation, structurally aligned sequences are highlighted in orange and red to indicate a match, while dissimilar structures are depicted in green or blue. ( A ) Comparative analysis between L. major (PDB ID: LML1) and L. mexicana (XP_003872886.1) is illustrated. ( B ) Comparison between the leishmanolysin (PDB ID: LML1) of L. major and protein XP_817808.1 of T. cruzi is shown. ( C ) Sequences from the genus Entamoeba ( E. histolytica XP_652632.1, E. dispar XP_001740726, and E. invadens XP_004184102.1) are compared with the leishmanolysin (PDB ID: LML1) from L. major . ( D ) Comparative analysis with free-living amoebae, A. castellanii and N. fowleri (XP_004337275.1, XP_044566011.1), respectively, is presented.

Article Snippet: The sample was then treated with 0.5% Triton for 20 min and blocked with 10% fetal bovine serum in PBS for 1 h. Samples were incubated overnight with α-gp63 antibody (CEDARLANE Laboratories Limited, Cat. No. CLP005A, Burlington, Ontario, Canada) at a 1:50 dilution at 4 °C.

Techniques: Comparison

Journal: Pathogens

Article Title: Exploration of the Binding Site of Arachidonic Acid in gp63 of Leishmania mexicana and in Orthologous Proteins in Clinically Important Parasites

doi: 10.3390/pathogens13090718

Figure Lengend Snippet: ( A ) Multiple sequence alignments of proteins like the L. mexicana gp63 protein (XP_003872886.1) alongside counterparts from T. cruzi (XP_817808.1), E. histolytica (XP_652632.1), E. dispar (XP_001740726.1), E. invadens (XP_004184102.1), A. castellanii (XP_004337275.1), and N. fowleri (XP_044566011.1). The alignment, generated using Uniprot online platform, highlights highly conserved or identical residues in red. The green shading indicates identical residues, while light brown and yellow denotes moderately and low conserved residues, respectively. Red letters within blue boxes represent regions within 10 Å of the zinc atom, while the purple background highlights the catalytic site. ( B ) Conserved domains in proteins homologous to gp63. All examined proteins contain the leishmanolysin domain, belonging to the M8 peptidase family, spanning specific regions: 46–570 in L. mexicana , 58–510 in T. cruzi , 27–490 in E. histolytica , 28–490 in E. dispar , 32–423 in E. invadens , 103–406 in A. castellanii , and 222–632 in N. fowleri .

Article Snippet: The sample was then treated with 0.5% Triton for 20 min and blocked with 10% fetal bovine serum in PBS for 1 h. Samples were incubated overnight with α-gp63 antibody (CEDARLANE Laboratories Limited, Cat. No. CLP005A, Burlington, Ontario, Canada) at a 1:50 dilution at 4 °C.

Techniques: Sequencing, Generated

Journal: Pathogens

Article Title: Exploration of the Binding Site of Arachidonic Acid in gp63 of Leishmania mexicana and in Orthologous Proteins in Clinically Important Parasites

doi: 10.3390/pathogens13090718

Figure Lengend Snippet: The binding modes of arachidonic acid (AA) with targets of each parasite are as follows: L. mexicana involves hydrogen bonds with HIS264; T. cruzi shows hydrogen bonds with TYR379 and ARG416; E. histolytica forms hydrogen bonds with HIS206; and E. dispar establishes hydrogen bonds with HIS267 and GLU207. For A. castellanii and N. fowleri , the binding between the gp63-like proteins and AA occurs through hydrophobic bonds. The interaction of the zinc atom with the carboxylic acid of AA is shown in the images with a thick border.

Article Snippet: The sample was then treated with 0.5% Triton for 20 min and blocked with 10% fetal bovine serum in PBS for 1 h. Samples were incubated overnight with α-gp63 antibody (CEDARLANE Laboratories Limited, Cat. No. CLP005A, Burlington, Ontario, Canada) at a 1:50 dilution at 4 °C.

Techniques: Binding Assay

Journal: Pathogens

Article Title: Exploration of the Binding Site of Arachidonic Acid in gp63 of Leishmania mexicana and in Orthologous Proteins in Clinically Important Parasites

doi: 10.3390/pathogens13090718

Figure Lengend Snippet: E. histolytica , E. dispar , and E. invadens exhibit COX-like activity. ( A ) COX activity detection in the genus Entamoeba involved using a commercial kit and exogenous AA in soluble fractions (50 µg protein/25 µL). ( B – D ) Presence of COX-type activity during the encystment process: n ( Ba ), cyst presence was confirmed by calcofluor staining, with cyst purity validated through DIC microscopy analysis ( Bb ). This sample represents the 48-hour encystment process. COX-type activity determination during encystment utilized the commercial kit and exogenous AA, with trophozoite and cyst extracts’ soluble fractions tested using exogenous AA at a final concentration of 20 µM ( C ). In ( Da ), gp63 presence in 48-hour-induced cysts was observed using anti- Leishmania major gp63 monoclonal antibody (CEDARLANE Laboratories Limited, Cat. No. CLP005A, Burlington, Ontario, CA), with the reaction developed through incubation with anti-mouse IgG coupled to FITC. Sample purity was analyzed through DIC microscopy ( Db ). Three independent biological replicates were conducted in triplicate p ≤ 0.01 **, p ≤ 0.001 ***.

Article Snippet: The sample was then treated with 0.5% Triton for 20 min and blocked with 10% fetal bovine serum in PBS for 1 h. Samples were incubated overnight with α-gp63 antibody (CEDARLANE Laboratories Limited, Cat. No. CLP005A, Burlington, Ontario, Canada) at a 1:50 dilution at 4 °C.

Techniques: Activity Assay, Staining, Microscopy, Concentration Assay, Incubation