Journal: Analytical and Bioanalytical Chemistry

Article Title: Determination of lithium in human serum by isotope dilution atomic absorption spectrometry

doi: 10.1007/s00216-021-03636-6

Figure Lengend Snippet: Results of isotope dilution high-resolution continuum source graphite furnace atomic absorption spectrometry (ID-HR-CS-GFAAS) and certified Li content in human serum certified reference materials (CRMs)

Article Snippet: The samples analyzed consisted of five serum certified reference materials (CRMs): BCR 304 (Joint Research Centre, Belgium), ERM-DA250a and ERM-DA251a (LGC Limited, UK), Seronorm L-1 (SERO AS, Norway), and Seronorm L-2 (SERO AS, Norway).

Techniques: Isotope Dilution

Journal: Microbiology Spectrum

Article Title: Proteomic Identification of Potential Target Proteins of Cathepsin W for Its Development as a Drug Target for Influenza

doi: 10.1128/spectrum.00921-22

Figure Lengend Snippet: Intracellular rather than secreted CTSW exerts its proviral function in IAV entry. (A) HEK293T cells cotransfected with plasmids encoding viral proteins (HA and NA) and wild-type CTSW (CTSW WT) or an enzymatically inactive mutant of CTSW (CTSW mut) were lysed, and the indicated proteins were detected by Western blot analysis using monoclonal mouse anti-HA, polyclonal rabbit anti-NA, polyclonal rabbit anti-GAPDH, and monoclonal mouse anti-CTSW antibodies. (B) HEK293T cells were transfected with the indicated CTSW expression constructs. At 24 h posttransfection, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer, left untreated (−) or treated with PNGase F (+) to remove N -linked oligosaccharides from glycoproteins, and subjected to Western blot analysis to detect CTSW and GAPDH as described in the legend to panel A. (C) HEK293T cells were transfected with plasmids encoding either His-tagged CTSW WT or mut. His-CTSW WT and mut were purified from the cell extracts (whole-cell lysates [WCL]) or the supernatants (sups) of the transfected cells via nickel Sepharose beads and subjected to Western blot analysis for CTSW using a monoclonal mouse anti-CTSW antibody. (D) A549 cells were transfected with a nontargeting (scrambled) siRNA (siSCR) or an siRNA targeting CTSW (siCTSW). At 24 h posttransfection, the transfection medium was changed to DMEM only, and another 24 h later, supernatant containing (siSCR) or lacking (siCTSW) secreted CTSW was harvested and preincubated with A/WSN/1933 (MOI = 10 PFU per cell) for 2 h at 37°C. siRNA-treated cells were then infected with preincubated virus as indicated. At 3 h postinfection, cells were fixed, stained for the viral nucleoprotein (NP) and nuclei, and analyzed by fluorescence microscopy. Mean fluorescence intensity (MFI) in the nuclei (nuclear NP signal) was quantified using ImageJ ( n > 600) and normalized to that of cells treated with siSCR and infected with virus preincubated with secreted CTSW. Mean values from three independent experiments are plotted, error bars represent standard deviations (SDs), and individual data points are shown. Statistical testing was done using the unpaired t test: ***, P < 0.001; ns, not significant.

Article Snippet: Human embryonic kidney (HEK293T) (ATCC) and human lung adenocarcinoma (A549) (ATCC) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% (vol/vol) fetal bovine serum (FBS) (Thermo Fisher Scientific), 100 units/mL penicillin, and 100 μg/mL streptomycin (Thermo Fisher Scientific) at 37°C with 5% CO 2 and controlled humidity.

Techniques: Mutagenesis, Western Blot, Transfection, Expressing, Construct, Radio Immunoprecipitation, Purification, Infection, Virus, Staining, Fluorescence, Microscopy

Journal: Microbiology Spectrum

Article Title: Proteomic Identification of Potential Target Proteins of Cathepsin W for Its Development as a Drug Target for Influenza

doi: 10.1128/spectrum.00921-22

Figure Lengend Snippet: Identification of potential substrates of CTSW by TAILS. (A) Western blot analysis to assess siRNA knockdown efficiency in A549 cells stably overexpressing CTSW wild type, which is resistant to siRNA-mediated knockdown by siCTSW#2 (CTSW WT siRes#2). A nontargeting siRNA (siSCR) served as the negative control. (B) Subcellular fractionation of A549 cells stably overexpressing CTSW (CTSW WT) using the Qproteome cell compartment kit (Qiagen). The cytosolic fraction (F1; C, cytosol) primarily contains cytosolic proteins, the membrane fraction (F2; M, membrane) membrane proteins, the nuclear fraction (F3; N, nucleus) nuclear proteins, and the cytoskeletal fraction (F4; S, cytoskeleton) cytoskeletal proteins. GAPDH served as the marker for F1, calnexin for F2, histone H3 for F3, and actin for F4. (A, B) The indicated proteins were detected using monoclonal mouse anti-CTSW, monoclonal mouse anti-beta-actin, polyclonal rabbit anti-GAPDH, polyclonal rabbit anti-calnexin, and rabbit polyclonal anti-histone H3 antibodies. (C) TAILS (terminal amine isotopic labeling of substrates) workflow to compare the proteomes—more specifically, the proteomes of the membrane fraction—of CTSW WT and CTSW knockdown (KD) cells infected for 90 min with A/WSN/1933 (MOI = 10 PFU per cell). First, the natural and CTSW-generated N-terminal (NH 2 ) and lysine (K) residues are blocked by demethylation, represented by blue (CTSW WT) or green (CTSW KD) dots. The two samples are then mixed and digested with trypsin, which generates internal tryptic peptides with free N termini. These free N termini react with an amine-reactive polymer, whereas the natural and CTSW-generated N termini, which are already blocked, will remain unbound and can be separated from the newly formed internal tryptic peptides by ultrafiltration. The enriched N-terminal peptides, also called the N terminome, are then analyzed and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). (D) Volcano plot displaying significantly altered N-terminal peptides in IAV-infected CTSW KD versus CTSW WT cells (log 2 fold change [FC] of ≤−1 or ≥1, P < 0.01). Peptides that are less abundant in CTSW KD than in CTSW WT cells (direct targets) are shown on the left (blue dots in blue shaded area), and peptides that are more abundant in CTSW KD than in CTSW WT cells (indirect targets) are shown on the right (blue dots in green shaded area). Dark gray dots are significant and do not meet the FC threshold, light gray dots are nonsignificant and do not meet the FC threshold, and white dots are nonsignificant and greater than the upper FC threshold. (E) HEK293T cells cotransfected with constructs encoding tagged target proteins His-HNRNPD, HA-EPN2, Flag-TPP1, and MFGE8-V5 and CTSW or GFP as the negative control were infected for 90 min with A/WSN/1933 (MOI = 10 PFU per cell) or mock treated, and cell lysates were analyzed by Western blotting with tag-specific antibodies, a polyclonal rabbit anti-GAPDH antibody, and a monoclonal mouse anti-CTSW antibody. An asterisk indicates residual MFGE8 staining, as the blot was first stained against the V5 tag and then against CTSW and GAPDH. (F) Cleavage site specificity profiling of CTSW based on TAILS data of all 92 N-terminal peptides that were less abundant in CTSW KD than in CTSW WT cells (left) or of the 19 top-ranked N-terminal peptides (right). The iceLogo plots visualize the amino acid frequencies at P6 to P6′ positions of neo-N termini generated by CTSW with the human Swiss-Prot proteome as the reference set. Dotted lines indicate the cleavage site. Significantly overrepresented amino acids are shown above the x axis, underrepresented residues are shown below the x axis, and amino acids that have not been identified are depicted in salmon pink below the x axis ( P = 0.05).

Article Snippet: Human embryonic kidney (HEK293T) (ATCC) and human lung adenocarcinoma (A549) (ATCC) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% (vol/vol) fetal bovine serum (FBS) (Thermo Fisher Scientific), 100 units/mL penicillin, and 100 μg/mL streptomycin (Thermo Fisher Scientific) at 37°C with 5% CO 2 and controlled humidity.

Techniques: Western Blot, Knockdown, Stable Transfection, Negative Control, Fractionation, Membrane, Marker, Isotopic Labeling, Infection, Generated, Polymer, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Construct, Staining

Journal: Microbiology Spectrum

Article Title: Proteomic Identification of Potential Target Proteins of Cathepsin W for Its Development as a Drug Target for Influenza

doi: 10.1128/spectrum.00921-22

Figure Lengend Snippet: Integration of TAILS results with data from RNAi screens for IAV host factors. (A, B) List of potential direct targets of CTSW for which an antiviral (A) or a proviral (B) role in IAV infection was predicted by Metascape . (C) Representative images of HEK293T cells incubated with LysoTracker to label acidic organelles (red) in live cells treated with (w/) or without (w/o) 10 nM bafilomycin A1 (BafA1) for 16 h. DAPI was used to counterstain the nuclei (blue). (D) HEK293T cells cotransfected with HA-EPN2- and CTSW- or GFP-encoding plasmids were treated with DMSO or 40 nM bafilomycin A1 (BafA1) 4 h posttransfection for 20 h. Cell lysates were analyzed by Western blotting using polyclonal rabbit anti-GAPDH, monoclonal mouse anti-EPN2, and monoclonal mouse anti-CTSW antibodies. (E) Sequence alignment of epsin 2 (EPN2) and epsin 1 (EPN1) is shown at the top. The unique EPN2 peptide identified by TAILS (terminal amine isotopic labeling of substrates) and the corresponding peptide for EPN1 are boxed. Western blots analyzing HEK293T cells cotransfected with GFP-EPN1- or GFP-EPN2-expressing plasmids and constructs encoding GFP, CTSW WT, or CTSW mut are shown below. In addition to a monoclonal mouse anti-EPN1 antibody, the same antibodies as described in the legend to panel D were used. (F) A549 cells were transfected with a nontargeting siRNA (siSCR) or siRNAs targeting CTSW (siCTSW) or epsin 1 (siEPN1#2, #4, and #6). At 48 h posttransfection, cells were infected with A/WSN/1933 (MOI = 10 PFU per cell) for 1 h at 4°C, followed by 3 h at 37°C. Fixed and permeabilized cells were then stained for the viral nucleoprotein (NP) and nuclei and analyzed by fluorescence microscopy. Mean fluorescence intensity (MFI) in the nuclei (nuclear NP signal) was quantified using ImageJ ( n > 400) and normalized to that of cells treated with siSCR. Mean values from three independent experiments are plotted, error bars represent standard errors of the means (SEMs), and individual data points are shown. (G) Knockdown efficiencies of three siRNAs targeting EPN1 were assessed by reverse transcription-qPCR analysis. GAPDH transcript levels were used for normalization. Shown are mean values from three independent experiments, each performed at least in duplicates. (H) A549-GFP (black symbols) and A549-GFP-EPN2 (pink symbols) cells were infected with WSN-Ren (MOI = 2), and Renilla luciferase expression was monitored with the live-cell substrate EnduRen at the indicated time points. The viral growth curves were measured in three independent experiments, and the area under the curve (AUC) was determined. (F to H) Statistical testing was done using the unpaired t test: ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant.

Article Snippet: Human embryonic kidney (HEK293T) (ATCC) and human lung adenocarcinoma (A549) (ATCC) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% (vol/vol) fetal bovine serum (FBS) (Thermo Fisher Scientific), 100 units/mL penicillin, and 100 μg/mL streptomycin (Thermo Fisher Scientific) at 37°C with 5% CO 2 and controlled humidity.

Techniques: Infection, Incubation, Western Blot, Sequencing, Isotopic Labeling, Expressing, Construct, Transfection, Staining, Fluorescence, Microscopy, Knockdown, Reverse Transcription, Luciferase