Journal: Nature Immunology

Article Title: Influenza vaccination stimulates maturation of the human T follicular helper cell response

doi: 10.1038/s41590-024-01926-6

Figure Lengend Snippet: a , Gating strategy for assessing Jurkat T FH TCR cell line activation in co-culture experiments. b , Frequency of CD69 + Jurkat T cells expressing TCRs T1, T3 and T12 after co-culture with aAPCs infected with influenza PR8 for 24 hours. c , Frequency of CD69 + T1, T3 and T12 Jurkat T cell lines after co-culture with aAPCs transfected with plasmids expressing individual segments of the IAV genome compared to empty vector control for 24 hours. d , Frequency of CD69 + Jurkat T cells expressing TCRs T4, T6, T7, T9 and T10 after co-culture for 24 hours with partially HLA-matched B cells pulsed with influenza protein peptide pools. e , Frequency of CD69 + in T6 Jurkat cells stimulated as in d in the presence of antibodies blocking specific MHC class II molecules. f , Frequency of CD69 + T11 cell line co-culture with aAPCs pulsed with recombinant HA protein, PMA/ionomycin or DMSO control for 24 hours. g , PC1 scores of individual cells from the picked T FH lineages with respect to time. h , Heatmap showing the expressions of genes corresponding to the head and tail PC1 loadings in the twelve T FH clonal lineages.

Article Snippet: To identify the driving peptide motifs that triggered activation in the responding clones, a pool of overlapping oligopeptide sequences spanning M1, M2 and NP proteins of the H1N1 PR8 strain (Mimotopes) was used for the peptide mapping experiments.

Techniques: Activation Assay, Co-Culture Assay, Expressing, Infection, Transfection, Plasmid Preparation, Control, Blocking Assay, Recombinant

Journal: bioRxiv

Article Title: Spatiotemporal development of the human T follicular helper cell response to Influenza vaccination

doi: 10.1101/2023.08.29.555186

Figure Lengend Snippet: A) Gating strategy for assessing Jurkat TFH TCR cell line activation in co-culture experiments. B) Frequency of CD69+ Jurkat T cells expressing TCRs TFH1, TFH3, and TFH12 after co-culture with aAPCs infected with Flu PR8. C) Frequency of CD3+ (top) and CD69+ (bottom) TFH1, TFH3, and TFH12 T cell lines after co-culture with aAPCs transfected with plasmids expressing individual segments of the IAV genome. D) Frequency of CD69+ TFH11 cell line co-culture with aAPCs pulsed with recombinant HA protein, PMA/ionomycin, or unstimulated control. D) Heatmap showing the expressions of genes corresponding to the head and tail PC1 loadings in the picked TFH clonal lineages.

Article Snippet: To identify the driving peptide motifs that triggered activation in the responding clones, a pool of overlapping oligopeptide sequences spanning M1, M2, and NP proteins of the H1N1 PR8 strain (Mimotopes) were used for the peptide mapping experiments.

Techniques: Activation Assay, Co-Culture Assay, Expressing, Infection, Transfection, Recombinant, Control

Journal: Life

Article Title: Biochemical Characterization, Specificity and Inhibition Studies of HTLV-1, HTLV-2, and HTLV-3 Proteases

doi: 10.3390/life11020127

Figure Lengend Snippet: Overall structures of human immunodeficiency virus (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1) viral proteases (PRs). Structures of HIV-1 PR (PDB ID: 5HVP) and HTLV-1 PR (PDB ID: 3LIY) are represented, the inhibitors are bound to the active sites, and the functionally important regions are shown by arrows.

Article Snippet: The oligopeptide substrates representing the natural sequences of HTLV PRs were ordered from BioBasic, while the oligopeptide substrates representing naturally occurring cleavage sites of various viruses [ , ], or the wild-type, the shortened, and the P4, P3, P2, P1, or P1′ variants of HTLV-1 MA/CA cleavage site (KTKVL*VVQPK) were in-house stocks [ ].

Techniques: Virus

Journal: Life

Article Title: Biochemical Characterization, Specificity and Inhibition Studies of HTLV-1, HTLV-2, and HTLV-3 Proteases

doi: 10.3390/life11020127

Figure Lengend Snippet: Purification of untagged HTLV-2 and HTLV-3 PRs. Representative gel images show purified fractions of ( a ) HTLV-2 PR (13.8 kDa) and ( b ) HTLV-3 PR (13.3 kDa). The molecular weight standard is indicated as STD, while the collected fractions are numbered (1–8). Fraction 8 ( a ) and fraction 4 ( b )—having >90% purity—were used for protease assays.

Article Snippet: The oligopeptide substrates representing the natural sequences of HTLV PRs were ordered from BioBasic, while the oligopeptide substrates representing naturally occurring cleavage sites of various viruses [ , ], or the wild-type, the shortened, and the P4, P3, P2, P1, or P1′ variants of HTLV-1 MA/CA cleavage site (KTKVL*VVQPK) were in-house stocks [ ].

Techniques: Purification, Molecular Weight

Journal: Life

Article Title: Biochemical Characterization, Specificity and Inhibition Studies of HTLV-1, HTLV-2, and HTLV-3 Proteases

doi: 10.3390/life11020127

Figure Lengend Snippet: Cleavage of HTLV-1 PR/P1 oligopeptide substrate (KGPPVIL*PIQAP) by HTLV-2 PR. Arrows show peaks of substrate and cleavage products in the representative chromatogram. The substrate and product sequences are also shown. The dashed arrow shows a cleavage position, which is indicated by an asterisk. The cleavage position was determined based on the molecular weights of the substrate and cleavage products determined experimentally by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS).

Article Snippet: The oligopeptide substrates representing the natural sequences of HTLV PRs were ordered from BioBasic, while the oligopeptide substrates representing naturally occurring cleavage sites of various viruses [ , ], or the wild-type, the shortened, and the P4, P3, P2, P1, or P1′ variants of HTLV-1 MA/CA cleavage site (KTKVL*VVQPK) were in-house stocks [ ].

Techniques: Mass Spectrometry

Journal: Life

Article Title: Biochemical Characterization, Specificity and Inhibition Studies of HTLV-1, HTLV-2, and HTLV-3 Proteases

doi: 10.3390/life11020127

Figure Lengend Snippet: The effect of NaCl concentration ( a , b ) and temperature ( c , d ) on the activities of HTLV-2 and HTLV-3 PRs. The highest activity was considered to be 100% in each case. Error bars represent SD (n = 2).

Article Snippet: The oligopeptide substrates representing the natural sequences of HTLV PRs were ordered from BioBasic, while the oligopeptide substrates representing naturally occurring cleavage sites of various viruses [ , ], or the wild-type, the shortened, and the P4, P3, P2, P1, or P1′ variants of HTLV-1 MA/CA cleavage site (KTKVL*VVQPK) were in-house stocks [ ].

Techniques: Concentration Assay, Activity Assay

Journal: Life

Article Title: Biochemical Characterization, Specificity and Inhibition Studies of HTLV-1, HTLV-2, and HTLV-3 Proteases

doi: 10.3390/life11020127

Figure Lengend Snippet: Catalytic efficiencies of HTLV PRs on different substrates representing natural cleavage site sequences. Cleavage sites are labelled by asterisks within the sequences. Abbreviations: MA (matrix), CA (capsid), NC (nucleocapsid), and TF1 (trans-frame 1).

Article Snippet: The oligopeptide substrates representing the natural sequences of HTLV PRs were ordered from BioBasic, while the oligopeptide substrates representing naturally occurring cleavage sites of various viruses [ , ], or the wild-type, the shortened, and the P4, P3, P2, P1, or P1′ variants of HTLV-1 MA/CA cleavage site (KTKVL*VVQPK) were in-house stocks [ ].

Techniques: Sequencing

Journal: Life

Article Title: Biochemical Characterization, Specificity and Inhibition Studies of HTLV-1, HTLV-2, and HTLV-3 Proteases

doi: 10.3390/life11020127

Figure Lengend Snippet: Catalytic efficiencies of HTLV PRs on different substrates representing natural cleavage site sequences of viruses other than HTLV. Cleavage sites are labelled by an asterisk within the sequences. Abbreviations: MA (matrix), CA (capsid), NC (nucleocapsid), TF (transframe), RT (reverse-transcriptase), and IN (integrase).

Article Snippet: The oligopeptide substrates representing the natural sequences of HTLV PRs were ordered from BioBasic, while the oligopeptide substrates representing naturally occurring cleavage sites of various viruses [ , ], or the wild-type, the shortened, and the P4, P3, P2, P1, or P1′ variants of HTLV-1 MA/CA cleavage site (KTKVL*VVQPK) were in-house stocks [ ].

Techniques: Reverse Transcription, Sequencing

Journal: Life

Article Title: Biochemical Characterization, Specificity and Inhibition Studies of HTLV-1, HTLV-2, and HTLV-3 Proteases

doi: 10.3390/life11020127

Figure Lengend Snippet: Comparison of the specificity of HTLV-1, HTLV-2, and HTLV-3 PRs using shortened and substituted analogs of HTLV-1 CA/NC oligopeptide substrate. Activity measured on the wild-type KTKVL*VVQPK substrate was considered to be 100%. Only >1% relative activities are plotted. Error bars represent SD (n = 2).

Article Snippet: The oligopeptide substrates representing the natural sequences of HTLV PRs were ordered from BioBasic, while the oligopeptide substrates representing naturally occurring cleavage sites of various viruses [ , ], or the wild-type, the shortened, and the P4, P3, P2, P1, or P1′ variants of HTLV-1 MA/CA cleavage site (KTKVL*VVQPK) were in-house stocks [ ].

Techniques: Comparison, Activity Assay

Journal: Life

Article Title: Biochemical Characterization, Specificity and Inhibition Studies of HTLV-1, HTLV-2, and HTLV-3 Proteases

doi: 10.3390/life11020127

Figure Lengend Snippet: Inhibition of HTLV-1, HTLV-2, and HTLV-3 PRs by different inhibitors. ( a ) Comparison of relative activities determined in the presence of HIV-1 PIs applied in a 1-µM final concentration. For control measurements, reaction mixtures contained no inhibitor. Activity determined in the presence of DMSO was considered to be 100%. Error bars represent SD (n = 2). ( b ) For comparison of relative efficacies, Ki values available for BLV 1 , MuLV 3 , HIV-1 2 , and HTLV-1 2 PRs were obtained from the literature. The referred data are comparable as each was obtained from Edans/Dabcyl fluorescent oligopeptide-based measurements. Literature values are shown in the table only for inhibitors where data are available for all four assays. ( c ) Comparison of Ki values determined for IB-268 and IB-269 inhibitors in the case of BLV 1 , HIV-1 2 , HTLV-1 2 , HTLV-2, and HTLV-3 PRs.

Article Snippet: The oligopeptide substrates representing the natural sequences of HTLV PRs were ordered from BioBasic, while the oligopeptide substrates representing naturally occurring cleavage sites of various viruses [ , ], or the wild-type, the shortened, and the P4, P3, P2, P1, or P1′ variants of HTLV-1 MA/CA cleavage site (KTKVL*VVQPK) were in-house stocks [ ].

Techniques: Inhibition, Comparison, Concentration Assay, Control, Activity Assay

Journal: Life

Article Title: Biochemical Characterization, Specificity and Inhibition Studies of HTLV-1, HTLV-2, and HTLV-3 Proteases

doi: 10.3390/life11020127

Figure Lengend Snippet: Sequence alignment of HIV-1 and HTLV PRs. Structure-based alignment of HIV-1 and HTLV-1 PR sequences was performed previously . Consensus active site motif residues are underlined. Sequence numbering is shown for HIV-1 and HTLV PRs. Residue similarity (., :) and identity (*) is shown only for the alignment of HTLV PR sequences. The residues that are identical in HTLV PRs are bold.

Article Snippet: The oligopeptide substrates representing the natural sequences of HTLV PRs were ordered from BioBasic, while the oligopeptide substrates representing naturally occurring cleavage sites of various viruses [ , ], or the wild-type, the shortened, and the P4, P3, P2, P1, or P1′ variants of HTLV-1 MA/CA cleavage site (KTKVL*VVQPK) were in-house stocks [ ].

Techniques: Sequencing, Residue

Journal: Biomolecules

Article Title: Biochemical Characterization of Human Retroviral-Like Aspartic Protease 1 (ASPRV1)

doi: 10.3390/biom10071004

Figure Lengend Snippet: Cleavage site identification in synthetic oligopeptide substrates by HPLC-MS. Oligopeptide substrates—representing wild-type and P2- or P3-modified variants of HIV-1 MA/CA cleavage site—were cleaved by GST-SASP14 PR, by incubating the cleavage reactions at 37 °C overnight. The table shows the m / z values [M + H] + determined by HPLC-ESI-TOF, the calculated values are shown in parentheses. The non-digested substrates were used as blanks, while the fragments were detected only in the digested samples. am and ac denote amide- and acid-terminated peptides, respectively. a denotes digested peptide measured by method 2 (see details in Section 2.10 ).

Article Snippet: Oligopeptide substrates representing the wild-type and modified pro-FLG linker sequences (see later in ) were ordered from BioBasic.

Techniques: Sequencing

Journal: Biomolecules

Article Title: Biochemical Characterization of Human Retroviral-Like Aspartic Protease 1 (ASPRV1)

doi: 10.3390/biom10071004

Figure Lengend Snippet: ASPRV1 is activated by autoproteolysis of the precursor. ( A ) A representative SDS-PAGE gel image shows self-processing of the full-length GST-SASP28 precursor and the release of SASP14. The purified GST-SASP28 was pre-incubated in reaction buffer for 0, 5, 15, 30, and 60 min. Black arrow indicates full-length GST-SASP28 precursor, white arrowheads indicate GST-∆SASP28 and SASP14 as autoproteolytic cleavage fragments, while GST is shown by black arrowhead. ( B ) The relative band intensities were determined via densitometry of the gels. For GST-SASP28 and SASP14, the most intense band was considered to have 100% intensity in the case of each gel. Error bars represent SD ( n = 3). ( C ) The effect of self-processing on enzyme activity was investigated by measuring the hydrolysis of VSQLY↓PIVQ oligopeptide substrate, using an HPLC-based method. Relative activities are plotted as a function of time of pre-incubation for all samples. Error bars represent SD ( n = 2).

Article Snippet: Oligopeptide substrates representing the wild-type and modified pro-FLG linker sequences (see later in ) were ordered from BioBasic.

Techniques: SDS Page, Purification, Incubation, Activity Assay