Journal: Cell reports. Medicine

Article Title: Safety and immunogenicity of SARS-CoV-2 self-amplifying RNA vaccine expressing an anchored RBD: A randomized, observer-blind phase 1 study.

doi: 10.1016/j.xcrm.2023.101134

Figure Lengend Snippet: Figure 3. SARS-CoV-2 IgG and neutralizing antibody responses (A and B) Serum IgG titers against wild-type SARS- CoV-2 RBD protein (A) and pseudovirus neutralizing antibody titers (ID50) against SARS-CoV-2 variants (B) for non-elderly (left side) and elderly (right side) participants (n = 10 per dose group, and n = 6 for the placebo group). Participants received one injection of VLPCOV-01 (0.3, 1.0, or 3.0 mg), 30 mg BNT162b2, or placebo on day 1 (week 0). Logarithmic values are reported as geometric mean titers for serum IgG and neutralizing antibody against pseudovirus. Bars indicate 95% CIs. (C) The correlation between serum neutralizing antibody titers against pseudovirus Wuhan (wild type) and IgG antibody titers against SARS-CoV-2 RBD protein following booster vaccination. Pearson’s product-moment correlation coefficient and p value were calculated following log trans- formation of source data (r = 0.950, p < 0.001).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-human CD154-FITC (clone: TRAP1) BD Biosciences cat# 555699; RRID: AB_396049 Mouse anti-human CD3-BUV615 (clone: SP34-2) BD Biosciences cat# 751249; RRID: AB_2875266 Mouse anti-human CD4-PE-Cy5.5 (clone: S3.5) Thermo Ficher Scientific cat# MHCD0418; RRID: AB_10376013 Mouse anti-human CD8-BUV563 (clone: RPA-T8) BD Biosciences cat# 612914; RRID: AB_2870200 Mouse anti-human CD27-PE-Cy5 (clone: 1A4CD27) Beckman coulter cat# 6607107 Mouse anti-human CD45RO-BUV805 (clone: UCHL1) BD Biosciences cat# 748367; RRID: AB_2872786 Mouse anti-human IFN-g-BV786 (clone: 4S.B3) BioLegend cat# 502542; RRID: AB_2563882 Mouse anti-human TNF-BV650 (clone: MAb11) BioLegend cat# 502938; RRID: AB_2562741 Rat anti-human IL-13-BV421 (clone: JES10-5A2) BD Biosciences cat# 563580; RRID: AB_2738290 Mouse anti-human IL-21-Ax647 (clone: 3A3-N21) BD Biosciences cat# 560493; RRID: AB_1645421 Mouse anti-human IL-4-PE-Cy7 (clone:8D4-8) BD Biosciences cat# 560672; RRID: AB_1727547 Mouse anti-human IL-17A-BV605 (clone: BL168) BioLegend cat# 512326; RRID: AB_2563887 Rat anti-human IL-2-BUV737 (clone: MQ117H12) BD Biosciences cat# 612836 Mouse anti-human CD107A-BV711 (clone: H4A3) BioLegend cat# 328640; RRID: AB_2565840 Mouse anti-human MIP1b-Alexa700 (clone: D21-1351) BD Biosciences cat# 561278; RRID: AB_10612008 Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG1 (AS35) ACROBiosystems cat# SAD-S35 Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG2 (AS35) ACROBiosystems cat# SAD-S66 Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG3 (AS35) ACROBiosystems cat# SAD-S67 Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG4 (AS35) ACROBiosystems cat# SAD-S68 Mouse anti-Human IgG1 Fc Secondary Antibody, HRP Thermo Fisher Scientific cat# MH1715; RRID: AB_2539710 Mouse Anti-Human IgG2 Fc-BIOT HP6002 SouthernBiotech cat# 9070-08; RRID: AB_2796638 Mouse Anti-Human IgG3 Hinge-BIOT HP6050 SouthernBiotech cat# 9210-08; RRID: AB_2796700 Mouse Anti-Human IgG4 Fc-BIOT HP6025 SouthernBiotech cat# 9200-08; RRID: AB_2796692 Biological samples Human PBMCs This study This study Chemicals, peptides, and recombinant proteins Benzonase Nuclease, Purity >90% MERCK Millipore cat# 70746 (Continued on next page) Cell Reports Medicine 4, 101134, August 15, 2023 e1

Techniques: Injection

Journal: Biosensors

Article Title: Point-of-Care Diagnostic Biosensors to Monitor Anti-SARS-CoV-2 Neutralizing IgG/sIgA Antibodies and Antioxidant Activity in Saliva

doi: 10.3390/bios13020167

Figure Lengend Snippet: Current-potential profiles ( a , c ) and calibration curves ( b , d ) of neutralizing antibodies sIgA and IgG were obtained using the GLEIA−based electrochemical immunosensor. Current−potential responses were tested under different concentrations of ( a ) sIgA 100 ng/mL (blue), 20 ng/mL (grey), 5 ng/mL (orange), 1 ng/mL (red), and 0 ng/mL (black) and ( c ) IgG 62.5 U/mL (blue), 25 U/mL (grey), 6.25 U/mL (orange), 2.5 U/mL (red), and 0 U/mL (black). Calibration curves were indicated for sIgA ( b ) and IgG ( d ) as Michaelis–Menten-type functions by a non-linear curve fitted in the graphing software Origin2022 (OriginLab).

Article Snippet: As positive controls for neutralizing antibodies, the sIgA antibody (E-AB-V1027, Elabscience, Houston, TX, USA), IgG antibody (SPD-M180, Acro Biosystems, Tokyo, Japan), and IgG standard in the IgG ELISA kit (290-84201, FUJIFILM Wako, Osaka, Japan) were used.

Techniques: Software

Journal: Biosensors

Article Title: Point-of-Care Diagnostic Biosensors to Monitor Anti-SARS-CoV-2 Neutralizing IgG/sIgA Antibodies and Antioxidant Activity in Saliva

doi: 10.3390/bios13020167

Figure Lengend Snippet: Comparisons of concentrations of neutralizing antibody IgG ( a ) and sIgA ( b ), antioxidant activity (indicated by luminescence inhibition rate) ( c ), and protein concentration ( d ) using saliva samples from 10 individuals are presented together. All 10 saliva samples were collected 3 weeks after the second dose of the vaccine.

Article Snippet: As positive controls for neutralizing antibodies, the sIgA antibody (E-AB-V1027, Elabscience, Houston, TX, USA), IgG antibody (SPD-M180, Acro Biosystems, Tokyo, Japan), and IgG standard in the IgG ELISA kit (290-84201, FUJIFILM Wako, Osaka, Japan) were used.

Techniques: Antioxidant Activity Assay, Inhibition, Protein Concentration

Journal: Biosensors

Article Title: Point-of-Care Diagnostic Biosensors to Monitor Anti-SARS-CoV-2 Neutralizing IgG/sIgA Antibodies and Antioxidant Activity in Saliva

doi: 10.3390/bios13020167

Figure Lengend Snippet: The four datasets of neutralizing antibodies—IgG and sIgA, antioxidant activity, and protein concentration—measured in 10 samples are depicted individually in a radar chart for each individual. The numbers T1–T10 correspond to the sample numbers 1–10 in . The data are shown as a ratio of the maximum concentration of each measured item.

Article Snippet: As positive controls for neutralizing antibodies, the sIgA antibody (E-AB-V1027, Elabscience, Houston, TX, USA), IgG antibody (SPD-M180, Acro Biosystems, Tokyo, Japan), and IgG standard in the IgG ELISA kit (290-84201, FUJIFILM Wako, Osaka, Japan) were used.

Techniques: Antioxidant Activity Assay, Protein Concentration, Concentration Assay

Journal: Biosensors

Article Title: Point-of-Care Diagnostic Biosensors to Monitor Anti-SARS-CoV-2 Neutralizing IgG/sIgA Antibodies and Antioxidant Activity in Saliva

doi: 10.3390/bios13020167

Figure Lengend Snippet: The results of continuous monitoring of the concentration of neutralizing IgG ( B ) and sIgA ( C ) antibodies, antioxidant activity ( D ), and protein concentration ( E ) in saliva and neutralizing IgG concentration in serum ( A ) before and after 1st, 2nd, and 3rd vaccinations in the same individual over time were compared vertically. The abscissa represents the date and time of sampling. 0: at first vaccination; 1a: 4 days later, 1b: 1 week later, 1c: 2 weeks later, and 1d: 3 weeks later; 2a: 4 days after second vaccination; 2b: 1 week later, 2c: 2 weeks later, 2d: 3 weeks later, 2e: 1 month later, 2f: 2 months later, 2g: 3 months later, 2h: 4 months later, 2i: 5 months later, 2j: 6 months later, and 2k: 8 months later; 3a: 3 days after the third vaccination, 3b: 2 weeks later, and 3c: 1 month later. The units on the vertical axis are as follows: ( A , B ) U/mL, ( C ) ng/mL, ( D ) %, and ( E ) mg/mL.

Article Snippet: As positive controls for neutralizing antibodies, the sIgA antibody (E-AB-V1027, Elabscience, Houston, TX, USA), IgG antibody (SPD-M180, Acro Biosystems, Tokyo, Japan), and IgG standard in the IgG ELISA kit (290-84201, FUJIFILM Wako, Osaka, Japan) were used.

Techniques: Concentration Assay, Antioxidant Activity Assay, Protein Concentration, Sampling

Journal: MedComm

Article Title: Novel aspect of neprilysin in kidney fibrosis via ACSL4‐mediated ferroptosis of tubular epithelial cells

doi: 10.1002/mco2.330

Figure Lengend Snippet: Genetic knockdown of neprilysin improved the fibrotic and ferroptotic phenotype in TGFβ1‐induced TCMK‐1 cells. (A and B) The knockdown efficiency of NEP siRNA (siNEP) in TCMK‐1 cells evaluated by RT‐PCR and Western blot analysis. (C) Representative Western blot images and quantitative analysis of NEP expression in TCMK‐1 cells. (D) RT‐PCR was performed to detect the mRNA level of Fn, α‐SMA, and Col Iα1. (E) RT‐PCR was performed to detect the mRNA level of ACSL4, GPX4, and Ptgs2 (COX2). (F) Western blot analysis was performed to detect protein expression of Fn, α‐SMA, and Col Iα1. (G) Western blot analysis was performed to detect protein expression of ACSL4, GPX4, and COX2. (H) The accumulation of lipid peroxidation in TCMK‐1 cells was analyzed by BODIPY C11 staining (×200, scale bar = 50 μm). NS p > 0.05, versus control. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, versus siNC. ### p < 0.001, ## p < 0.01, # p < 0.05, versus siNC+TGF‐β1. siNC, negative control.

Article Snippet: Primary antibodies against NEP (1:200, 18008‐1‐AP; Proteintech, USA), α‐SMA (1:100, ET1607‐43; Huabio, China), and ACSL4 (1:200, 81196‐1‐RR; Proteintech) were used in the study.

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Staining, Control, Negative Control

Journal: MedComm

Article Title: Novel aspect of neprilysin in kidney fibrosis via ACSL4‐mediated ferroptosis of tubular epithelial cells

doi: 10.1002/mco2.330

Figure Lengend Snippet: Neprilysin aggravated fibrosis by ACSL4‐mediated ferroptosis in TGF‐β1‐induced TCMK‐1 cells. (A and B) The efficiency of NEP overexpression in TCMK‐1 cells evaluated by RT‐PCR and Western blot analysis. (C and D) The overexpression efficiency of ACSL4 in TCMK‐1 cells evaluated by RT‐PCR and Western blot analysis. (E and F) The knockdown efficiency of ACSL4 siRNA in TCMK‐1 cells evaluated by RT‐PCR and Western blot analysis. (G) RT‐PCR was performed to detect the mRNA expression of ACSL4, GPX4, and Ptgs2 (COX2). (H) Western blot analysis was performed to detect protein expression of ACSL4, GPX4, and COX2. (J) RT‐PCR was performed to detect the mRNA expression of Fn, α‐SMA, and Col Iα1. (I) Western blot analysis was performed to detect protein expression of Fn, α‐SMA, and Col Iα1. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, versus oeNC or siNC or oeNC+siNC. #### p < 0.0001, ### p < 0.001, ## p < 0.01, # p < 0.05, versus oeNC+siNC+TGF‐β1. $$$$ p < 0.0001, $$ p < 0.01, $ p < 0.05, versus oeNEP+siNC+TGF‐β1. siNC, silencing negative control; siNEP, NEP siRNA; siACSL4, ACSL4 siRNA; oeNC, overexpression negative control; oeNEP, overexpression of NEP; oeACSL4, overexpression of ACSL4.

Article Snippet: Primary antibodies against NEP (1:200, 18008‐1‐AP; Proteintech, USA), α‐SMA (1:100, ET1607‐43; Huabio, China), and ACSL4 (1:200, 81196‐1‐RR; Proteintech) were used in the study.

Techniques: Over Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Knockdown, Expressing, Negative Control

Journal: Cell metabolism

Article Title: Serine and glycine physiology reversibly modulate retinal and peripheral nerve function.

doi: 10.1016/j.cmet.2024.07.021

Figure Lengend Snippet: Figure 2. Retinal serine is predominately obtained from the circulation (A) % serine M3 label in tissue normalized to % serine (ser) M3 label in plasma after mice were infused with [U-13C3]serine for 2 h (n = 4). (B) Abundance of serine in plasma and tissue relative to levels when serine is infused at 2 nmol/min/g mice (n = 5 for 2 nmol/min/g and n = 4 for 10 nmol/min/g groups). (C) % glycine M2 label in tissue normalized to % glycine (gly) M2 label in plasma after mice were infused with [U-13C2]glycine for 2 h (n = 4). (D) Gene expression of Slc1a4, Slc6a20a, and Slc6a20b per copy of housekeeping gene (36b4) across tissue types (n = 5). (E) Immunofluorescence visualization of SLC1A4 in the mouse retina (SLC1A4, magenta; glutamine synthetase [GS], green; DAPI, blue). ONL, outer nuclear layer; INL, inner nuclear layer; RPE, retinal pigment epithelium. White arrows indicate co-staining of Muller glia. Schematic prepared in BioRender. (F) Immunofluorescence visualization of SLC6A20 in mouse retina (SLC6A20, red; glutamine synthetase [GS], green; DAPI, blue). ONL, outer nuclear layer; INL, inner nuclear layer; RPE, retinal pigment epithelium. Data are mean ± SD (A–C) or mean ± SEM (D) and were analyzed using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-PHGDH antibody produced in rabbit Sigma-Aldrich Cat#: HPA021241; RRID: AB_1855299 GLDC Polyclonal Antibody Invitrogen Cat#: PA5-51701; RRID: AB_2642028 Glutamine Synthetase Monoclonal Antibody (GT1055) Invitrogen Cat#: MA5-27749; RRID: AB_2735204 Anti-ASCT-1 antibody - C-terminal Abcam Cat#: ab189126 SLC6A20 Antibody Affinity Biosciences Cat#: DF9926; RRID: AB_2843120 Anti-ASCT1 (SLC1A4) Antibody Alomone Labs Cat#: ANT-081; RRID: AB_2756719 IRDye 800CW Donkey anti-Rabbit IgG Secondary Antibody LI-COR Cat#: 926-32213; RRID: AB_621848 b-Actin (8H10D10) Mouse mAb Cell Signaling Technology Cat#: 3700; RRID: AB_2242334 Rabbit Anti-Mouse IgG Antibody, HRP conjugate Sigma-Aldrich Cat#: AP160P; RRID: AB_92531 Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 555 Thermo Fisher Cat #: A32794; RRID: AB_2762834 Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 Thermo Fisher Cat #: A32766; RRID: AB_2762823 Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 Thermo Fisher Cat #: A31573; RRID: AB_2536183 Chemicals, peptides, and recombinant proteins [U-13C6]glucose Cambridge Isotope Laboratories Inc. CLM-1396-PK [U-13C3]serine Cambridge Isotope Laboratories Inc. CLM-1574-H-PK [U-13C2]glycine Cambridge Isotope Laboratories Inc. CLM-1017-PK Deposited data Raw data This paper Data S1 Mass spectrometry data (human samples) Green et al.4 https://doi.org/10.1016/ j.molmet.2023.101716 Mass spectrometry data (animal samples) Metabolomics Workbench https://doi.org/10.21228/M86V58 Experimental models: Organisms/strains Mouse: C57/Bl6J Scripps Research In-house breeding colony Mouse: Phgdh floxed/floxed Laboratory of Shigeki Furuya Yang et al.45 Mouse: E2a-Cre Jackson Laboratory 003724 Oligonucleotides See STAR Methods for primers of real-time qPCR Harvard Primer Bank https://pga.mgh.harvard.edu/ primerbank/citation.html Software and algorithms GraphPad Prism 9.5.0 GraphPad Software https://www.graphpad.com/ Adobe Illustrator CS5 Adobe Systems https://www.adobe.com/ products/illustrator.html Image J (v.1.50b) NIH https://imagej.net/ij/ R version 4.1.3 R Core Team https://www.r-project.org Other Control Amino Acid (AA) Diet TD.110839 Envigo Teklad Diets Gly and Ser Deficient AA Diet, R TD.150056 Envigo Teklad Diets 3% Serine AA Diet, Y TD.200661 Envigo Teklad Diets Teklad Global 19% Protein Extruded Rodent Diet 2019 Envigo Teklad Diets Cell Metabolism 36, 1–14.e1–e6, October 1, 2024 e1

Techniques: Tissue, Clinical Proteomics, Gene Expression, Staining, Immunofluorescence

Journal: JCI Insight

Article Title: An IKBKE variant conferring functional cGAS/STING pathway deficiency and susceptibility to recurrent HSV-2 meningitis

doi: 10.1172/jci.insight.173066

Figure Lengend Snippet: ( A – H ) iPSC derived microglia ( A – D ) and neurons ( E – H ) were transfected with IKBKE or control siRNA. ( A and E ) The efficiency of transfection was examined by measuring IKBKE mRNA levels. Cells were infected with HSV-2 for 6 hours, and IFNA2 , IFNB1 , and TNFA mRNA levels were quantified. ( I – L ) iPSC-derived microglia were transfected with siRNA targeting cGAS ( I and J ) or TLR3 ( K and L ). Microglia were infected with HSV-2, and IFNB1 mRNA was quantified ( J and L ). Unpaired t test was used for statistical analysis. ( M ) Graphical illustration of experimental setup for microglia-neuron crosstalk experiments. Created with BioRender.com. ( N ) iPSC-derived neurons were treated with Human Type 1 IFN Neutralizing Antibody Mixture or control IgG (both 1:100) 30 minutes before addition of supernatants from HSV-2–infected microglia or treatment with IFN-β (10 ng/mL). Six hours later, the medium was removed, and the neurons were infected with HSV-2. Supernatants were collected after 16 hours for plaque assay ( O ). Microglia subjected to IKBKE or control knockdown with siRNA were infected with HSV-2. The cells were washed, and the medium was replaced after 1 hour. Supernatants were collected from the microglia after 24 hours and added to the neurons. Six hours later, the medium was removed, and the neurons were infected with HSV-2. Supernatants were collected 16 hours later for plaque assay. Data presented are pooled from 2 independently performed experiments. ( P ) Cells were treated as in panel N , and culture supernatants were analyzed for cell viability. Data presented are from 1 of 2 experiments performed. ( N – P ) Groups were compared with Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. Error bars represent SEM ( A – H ) and SD ( N – P ), and *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Article Snippet: To neutralize type I IFN bioactivity in supernatants from microglia, neurons were cultured with Human Type 1 IFN Neutralizing Antibody Mixture (PBL) 30 minutes before addition of culture supernatants from microglia (1:100).

Techniques: Derivative Assay, Transfection, Control, Infection, Plaque Assay, Knockdown